Targeted Therapies in Oncology

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Targeted Therapies in Oncology

Targeted Therapies in Oncology

Edited by

Giuseppe Giaccone

Free University Medical Center Amsterdam, The Netherlands

Jean-Charles Soria

Paris University XI, Institut Gustave Roussy Villejuif, France

Informa Healthcare USA, Inc. 52 Vanderbilt Avenue New York, NY 10017 ª 2007 by Informa Healthcare USA, Inc. Informa Healthcare is an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8493-9371-X (Hardcover) International Standard Book Number-13: 978-0-8493-9371-6 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www. copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe.

Library of Congress Cataloging-in-Publication Data Targeted therapies in oncology / edited by Giuseppe Giaccone, Jean-Charles Soria. p. ; cm. Includes bibliographical references. ISBN-13: 978-0-8493-9371-6 (hardcover : alk. paper) ISBN-10: 0-8493-9371-X (hardcover : alk. paper) 1. Cancer–Treatment. I. Giaccone, Giuseppe. II. Soria, Jean-Charles. [DNLM: 1. Neoplasms–therapy. 2. Gene Therapy. 3. Immunotherapy. QZ 266 T1847 2007] RC270.8.T37 2007 616.990 406–dc22 Visit the Informa Web site at www.informa.com and the Informa Healthcare Web site at www.informahealthcare.com

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Preface

Rapid advances in tumor biology have led to the identification of the molecular circuitry that governs cancer cell proliferation. The better understanding of the key pathways that control tumor progression has enabled the pharmaceutical industry and academia to develop new anti-cancer agents targeting specific molecular events involved in the oncogenic process. The term “targeted therapies” refers to treatment strategies directed against molecular targets considered to be involved in the process of neoplastic transformation. This is not a totally new concept in oncology, since hormonal manipulations have long been applied for the treatment of advanced and local disease in breast, prostate, and thyroid cancers. In the past 30 years, alterations characteristic of neoplastic cells have been described, such as specific translocations, activating mutations, or gene amplifications, which have brought real changes to the nosological classification of cancers. The molecular classification of certain cancers has contributed to the development of a new class of drugs that aims at blocking, with various degrees of specificity, the activity of proteins involved in neoplastic cell development and progression. This book provides a concise and up-to-date panorama of existing targeted therapies and those being developed into valuable anticancer treatments, with an emphasis on the “clinical achievements” obtained with such agents. The biology behind each target has also been discussed. The large number of chapters included in this book reflects the variety of targeted therapies aiming at blocking a wide array of “hallmarks of cancer.” These notably include: signal-transduction inhibitors, anti-angiogenic and vascular-disrupting agents, telomere-targeting compounds, apoptosis modulators, and targeted agents at transversal mechanisms; however, only targeted agents that have already entered the clinical arena have been included. The introduction of these agents has already had a large impact across different tumor-types and in early- as well as advanced-stage cancer. Giuseppe Giaccone Jean-Charles Soria

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Contents

Preface iii Contributors

vii

1. Overview of Existing Therapies

1

Giannis Mountzios and Jean-Charles Soria 2. Signal Transduction Inhibitors, HER Family, EGFR Inhibition, and Clinical Achievements 19

Giuseppe Giaccone and Paolo Zucali 3. HER2 Inhibition and Clinical Achievements

45

Toru Mukohara and Pasi A. Jänne 4. Pan-HER Inhibitors 55

Gérard Milano 5. Inhibiting the Phosphoinositide 3-Kinase/Akt/Mammalian Target of Rapamycin Pathway 65

Janet E. Dancey 6. Farnesyl Transferase Inhibitors in Cancer: Promise, but Limited Proof

85

Laura Fanucchi, Michael P. Fanucchi, and Fadlo R. Khuri 7. Protein Kinase C Inhibitors in the Treatment of Non–Small Cell Lung Cancer

103

Yun Oh, Michael Lahn, Asavari Wagle, and Roy Herbst 8. Signal Transduction Inhibitors: PDGFR and c-KIT Inhibitors

123

Jean-Yves Blay, Jérome Fayette, Laurent Alberti, Severine Tabone-Eglinger, Hiba El Sayadi, Philippe Cassie, Armelle Dufresne, Dominique Ranchère, and Isabelle Ray-Coquard 9. The Insulin-Like Growth Factor 1 Receptor: A Target for Cancer Treatment

Yungan Tao, Jean Bourhis, and Eric Deutsch 10. Aurora Kinase Inhibitors

157

Mitesh J. Borad, Steven L. Warner, and Daniel D. Von Hoff 11. Apoptosis Modulators: p53 Targeting 177

Sunil Chada, Dora Bocangel, Kerstin Menander, and Jack A. Roth 12. Survivin

197

Dario C. Altieri 13. TRAIL Modulators

207

C. H. Mom, I. A. Sloots, S. de Jong, J. A. Gietema, E. G. E. de Vries, and S. Sleijfer 14. VEGF Targeting 223

Lee M. Ellis v

141

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Contents

15. Targeting Angiogenesis with Oral Agents

241

Benjamin Besse and Jean-Pierre Armand 16. Endothelial Cell Propagation Targeting 257

Gordon C. Tucker 17. HIF-1 Inhibitors

283

Giovanni Melillo 18. Antivascular Agents

295

Jane Robertson 19. Matrix Metalloproteinases

315

Stéphane Vignot and Jean-Philippe Spano 20. Src Inhibitors

333

Francisco Cruzalegui 21. Telomerase and Telomere Interacting Agents

349

Jean-François Riou, Anne de Cian, Lionel Guittat, Dennis Gomez, Céline Douarre, Laurent Lacroix, Chantal Trentesaux, and Jean-Louis Mergny 22. Targeting Hsp90: The Cancer Super-Chaperone

Paul Workman and Swee Sharp Index 401

375

Contributors

Laurent Alberti Lyon, France

Department of Médecine, Centre Leon Berard, Laennec,

Dario C. Altieri Department of Cancer Biology and the Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts, U.S.A. Jean-Pierre Armand Department of Medicine, Institut Gustave Roussy, Villejuif, France, and Department of Medicine, Institut Cladius Regaud, Toulouse, France Benjamin Besse Department of Médecine, Paris University XI, Institut Gustave Roussy, Villejuif, France Jean-Yves Blay Department of Médecine, Centre Leon Berard, Laennec, Lyon, France, and Unité de Jour d'Oncologie Médicale Multidisciplinaire Hôpital Edouard Herriot, Place d'Arsonval, Lyon, France Dora Bocangel Departments of Clinical Research and Development, Introgen Therapeutics, Inc., Houston, Texas, U.S.A. Mitesh J. Borad Clinical Translational Research Division, Translational Genomics Research Institute, Phoenix, Arizona, U.S.A. Jean Bourhis Department of Radiation Oncology, Institute Gustave Roussy, Villejuif, France Philippe Cassie Department of Médecine, Centre Leon Berard, Laennec, Lyon, France, and Unité de Jour d'Oncologie Médicale Multidisciplinaire Hôpital Edouard Herriot, Place d'Arsonval, Lyon, France Sunil Chada Departments of Clinical Research and Development, Introgen Therapeutics, Inc., Houston, Texas, U.S.A. Francisco Cruzalegui Division of Cancer Research and Drug Discovery, Institut de Recherches Servier, Croissy-sur-Seine, France Janet E. Dancey Investigational Drug Branch, Cancer Therapy Evaluation Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville, Maryland, U.S.A. Anne de Cian Laboratoire de Biophysique, Muséum National d’Histoire Naturelle, Paris, France S. de Jong Department of Medical Oncology, University Medical Center Groningen, Groningen, The Netherlands Eric Deutsch Department of Radiation Oncology, Institute Gustave Roussy, Villejuif, France E. G. E. de Vries Department of Medical Oncology, University Medical Center Groningen, Groningen, The Netherlands Céline Douarre Laboratoire d’Onco-Pharmacologie, UFR de Pharmacie, Université de Reims Champagne Ardenne, Reims, France

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Contributors

Armelle Dufresne Department of Médecine, Centre Leon Berard, Laennec, Lyon, France, and Unité de Jour d'Oncologie Médicale Multidisciplinaire Hôpital Edouard Herriot, Place d'Arsonval, Lyon, France Hiba El Sayadi

Department of Médecine, Centre Leon Berard, Laennec, Lyon, France

Lee M. Ellis Departments of Surgical Oncology and Cancer Biology, M. D. Anderson Cancer Center, University of Texas, Houston, Texas, U.S.A. Laura Fanucchi Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia, U.S.A. Michael P. Fanucchi Department of Medical Oncology, St. Vincent's Comprehensive Cancer Center, New York Medical College, New York, New York, U.S.A. Jérome Fayette Department of Médecine, Centre Leon Berard, Laennec, Lyon, France, and Unité de Jour d'Oncologie Médicale Multidisciplinaire Hôpital Edouard Herriot, Place d'Arsonval, Lyon, France Giuseppe Giaccone Department of Medical Oncology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands J. A. Gietema Department of Medical Oncology, University Medical Center Groningen, Groningen, The Netherlands Dennis Gomez Laboratoire d’Onco-Pharmacologie, UFR de Pharmacie, Université de Reims Champagne Ardenne, Reims, France Lionel Guittat Paris, France

Laboratoire de Biophysique, Muséum National d’Histoire Naturelle,

Roy Herbst Department of Thoracic/Head and Neck, Medical Oncology, M. D. Anderson Cancer Center, University of Texas, Houston, Texas, U.S.A. Pasi A. Jänne Lowe Center for Thoracic Oncology, Department of Medical Oncology, Dana Farber Cancer Institute, Boston, Massachusetts, U.S.A. Fadlo R. Khuri Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia, U.S.A. Laurent Lacroix Paris, France

Laboratoire de Biophysique, Muséum National d’Histoire Naturelle,

Michael Lahn Oncology Product Development, Eli Lilly and Company, Indianapolis, Indiana, U.S.A. Giovanni Melillo Developmental Therapeutics Program, SAIC Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland, U.S.A. Kerstin Menander Departments of Clinical Research and Development, Introgen Therapeutics, Inc., Houston, Texas, U.S.A. Jean-Louis Mergny Laboratoire de Biophysique, Muséum National d’Histoire Naturelle, Paris, France Gérard Milano Laboratoire d'Oncopharmacologie, du Centre Antoine Lacassagne, Nice, France C. H. Mom Department of Medical Oncology, University Medical Center Groningen, Groningen, The Netherlands Giannis Mountzios Department of Médecine, Institut Gustave Roussy, Villejuif, France

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Contributors

Toru Mukohara Division of Hematology and Oncology, Research Center for Innovative Oncology, National Cancer Center Hospital East, Kashiwa, Japan Yun Oh Department of Thoracic/Head and Neck, Medical Oncology, M. D. Anderson Cancer Center, University of Texas, Houston, Texas, U.S.A. Dominique Ranchère Department of Médecine, Centre Leon Berard, Laennec, Lyon, France Isabelle Ray-Coquard Lyon, France


Jean-François Riou Laboratoire d’Onco-Pharmacologie, UFR de Pharmacie, Université de Reims Champagne Ardenne, Reims, France Jane Robertson Global Oncology Research and Development, AstraZeneca Pharmaceuticals, Aderley Park, Macclesfield, U.K. Jack A. Roth Thoracic and Cardiovascular Surgery, University of Texas, M. D. Anderson Cancer Center, Houston, Texas, U.S.A. Swee Sharp Cancer Research U.K. Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, U.K. S. Sleijfer Department of Medical Oncology, Erasmus University Medical Center, Rotterdam, The Netherlands I. A. Sloots Department of Medical Oncology, University Medical Center Groningen, Groningen, The Netherlands Jean-Charles Soria Department of Médecine, Paris University XI, Institut Gustave Roussy, Villejuif, France Jean-Philippe Spano Service d’Oncologie Médicale, Groupe Hospitalier Pitié Salpétrière, Paris, France Severine Tabone-Eglinger Lyon, France


Yungan Tao Department of Radiation Oncology, Institute Gustave Roussy, Villejuif, France Chantal Trentesaux Laboratoire d’Onco-Pharmacologie, UFR de Pharmacie, Université de Reims Champagne Ardenne, Reims, France Gordon C. Tucker Cancer Drug Discovery Department, Institut de Recherches Servier, Croissy-sur-Seine, France Stéphane Vignot Service d’Oncologie Médicale, Groupe Hospitalier Diaconesses Croix Saint Simon, Paris, France Daniel D. Von Hoff Clinical Translational Research Division, Translational Genomics Research Institute, Phoenix, Arizona, U.S.A. Asavari Wagle Oncology Product Development, Eli Lilly and Company, Indianapolis, Indiana, U.S.A. Steven L. Warner Clinical Translational Research Division, Translational Genomics Research Institute, Phoenix, Arizona, U.S.A. Paul Workman Cancer Research U.K. Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, U.K. Paolo Zucali Department of Medical Oncology and Hematology, Instituto Clinico Humanitas, Milan, Italy

1

Overview of Existing Therapies Giannis Mountzios Department of Médecine, Institut Gustave Roussy, Villejuif, France

Jean-Charles Soria Department of Médecine, Paris University XI, Institut Gustave Roussy, Villejuif, France

INTRODUCTION Definitions Systemic cancer treatment, which followed a continuous progression all along the second half of the twentieth century, seems to be entering a new and exciting era, characterized by a more “sophisticated” selection of targets for cancer therapy, leading to the development of more “intelligent” drugs directed against these specific targets. By definition, molecular targeted therapy refers to every specific treatment strategy directed against well-defined molecular targets considered to be involved in the process of neoplastic transformation. Consequently, every pharmaceutical molecule with specific and unique properties against (or for) a welldefined molecular target implicated in the process of carcinogenesis may be considered as a molecular targeted agent (MTA). The above-mentioned definition does not include within MTA the “classic” cytotoxic agents (alkylating agents, antimetabolites, anticancer antibiotics, vinca alkaloids) or the more recently discovered ones (topo-isomerase inhibitors and taxanes), despite the fact that such agents are also directed against a specific target (e.g., thymidilate synthase or tubulines). Indeed such targets, although specific, do not characterize the process of tumor progression and transformation but participate in physiological process such as DNA synthesis and mitosis. To be more accurate, one could suggest that modern advances in molecular biology have helped to identify specific molecular abnormalities that characterize a certain type of cancer in a unique and repeated manner, allowing thus the development of specialized molecules that can selectively target the specific cancer-cell population and minimize the hazardous effects to normal cells. From “Classic” Chemotherapy to Molecular Targeted Therapy The idea of “attacking” a specific molecular target by interfering with the corresponding pathogenetic mechanism is not a new concept in oncology. Hormonal manipulation for the treatment of local and advanced disease in breast, prostate, and thyroid cancer have long been studied for potential benefits, taking advantage of their unique hormone-depended biological behavior. During the decade 1980–1990, the National Cancer Institute (NCI) established a new research approach, making the hypothesis that developing a solid tumor model in vitro could allow the discovery of potential new therapeutic targets. The choice of an in vitro model, compared to an in vivo model (mouse), was made for reasons of cost, simplicity, rapidity, efficacy, and automatization. The new model was applied in a panel of 60 human cell lines of various histological origin in an 1

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effort to establish a new, disease orientated, therapeutic strategy (1). Although the distinct biological characteristics of cancer cells in vitro were not always applicable in vivo, limiting thus the exploitation of the acquired knowledge in the clinical setting, the study of the biological behavior of cell lines has allowed the discovery of new pathogenetic mechanisms of oncogenesis and the elucidation of the various molecular pathways of signaling cascades that trigger neoplastic transformation. More recently, the sequencing of the human genome has allowed the full identification of the protein kinase complement, or “kinome,” including more than 520 proteins with tyrosine kinase domains, and leading to the generation of MTAs against most tyrosine kinases relevant to oncogenesis (2). When the tyrosine kinase inhibitor imatinib (Gleevec ; Novartis Pharmaceuticals, East Hannover, New Jersey, U.S.A.) became the first MTA proved to possess profound antitumor activity against chronic myeloid leukemia (3) and obtained the U.S. Food and Drug Administration (FDA) approval in 2001 for this disease, this was the first result of this long but fruitful procedure, followed by numerous other molecules targeting a wide variety of human tumors. Classification In a seminal article in 2000, Weinberg and Hanahan described the key-alterations of cancer cells (4); this first description of the six hallmarks of oncogenesis can be used as a framework to classify molecularly targeted agents (Fig. 1). Consequently, one could attempt a first classification according to the molecular mechanism modified by the agent: & & & & & &

Self-sufficiency in growth signals ! Cell-cycle inhibitors and signal transduction modulators Insensitivity to antigrowth signals ! Cell-cycle inhibitors and signal transduction modulators Sustained angiogenesis ! Antiangiogenic and antivascular agents Tissue invasion and metastasis ! Anti-invasive agents Evading apoptosis ! Apoptosis modulators Limitless replicative potential ! Antitelomerase and telomere-interacting agents

Alteration of DNA repairing capacity, as a recognised factor provoking genomic instability, as well as DNA epigenetic modulation, should probably also be added to the above mentioned mechanisms. Moreover, the elucidation of many molecular pathways involved in neoplastic transformation has resulted in numerous new targeted agents affecting diverse and sometimes overlapping mechanisms (heat shock proteins, COX2 interactions, DNA methylation, proteasome function, etc.). OVERVIEW OF THE EXISTING THERAPIES Signal Transduction Inhibitors Background Signal transduction describes the general process by which cells perceive changes in their environment. The more important steps for cellular signal transduction are the following: & & &

Recognition/binding of signal molecules to the cell surface Internalization of the signal Transmission of the signal through the cytoplasm

Overview of Existing Therapies

3

FIGURE 1 The six fundamental mechanisms of carcinogenesis and their corresponding pharmaceutical agents according to their pathogenetic mechanisms. Source: From Ref. 4. & &

Entrance of the signal into the nucleus Signal-dependent modulation of gene activation

Schematically, cell growth signaling can be separated in three distinct parts that are closely connected (Fig. 2). First come the upstream growth factors and their receptors at the cell membrane. Engagement of the receptor by the ligand, which is usually a growth factor, usually triggers the activation of the intracellular domain of the receptor that possesses protein kinase properties. Protein kinases are enzymes that covalently attach phosphates to the side chain of either serine, threonine, or tyrosine residues of specific proteins inside cells. From there, molecular mediators of signal transduction and cytoplasmic messengers form a relay cascade through a “cross talk” involving activation and/or inactivation of intermediate downstream effectors, informing thus the nucleus of the received

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FIGURE 2 Important steps of the cell signaling process (left) and corresponding levels of potential pharmaceutical intervention (right).

stimuli. Activation or inactivation of the intermediate messengers is usually mediated by subsequent phosphorylations or de-phosphorylations of these molecules by specific enzymes, called kinases and phosphatases, respectively. Finally, an effector pathway leads the cell to the eventual division and proliferation. This signal transduction system, comprising growth factors, transmembrane receptor proteins, and cytoplasmic secondary messengers, is often exploited to optimize tumor growth and metastasis in malignancies. Indeed, in cancer cells, key components of these pathways may be altered by oncogenes through overexpression or mutation, leading to dysregulated cell signaling, inhibition of apoptosis, metastasis and cell proliferation (5). The components of these abnormal signaling pathways represent potential selective targets for new anticancer therapies. These potential targets, the majority of which are protein kinases, can be divided into subcategories, according to the level of interaction on the above described process, as follows: ligands (typically growth factors), cellular receptors, intracellular second messengers, and nuclear transcription factors. Agents targeting cellular receptors can be further categorized according to the mechanism of


5

interference into monoclonal antibodies and tyrosine kinase inhibitors of the intracellular domain of the receptor (Fig. 2). Categories Inhibition of the Receptor Function Conceptually, there are three potential approaches to “attack” a membrane receptor (Fig. 2): & & &

Neutralization of the ligand Competitive inhibition of ligand–receptor engagement Inhibition of transduction of the signal from the receptor to secondary cytoplasmic messengers

The first, obvious target for intervention in a signaling cascade is the neutralization of ligands before they can associate with their receptors; this approach has been successfully validated with bevacizumab (Avastin ; Genentech Corp., San Francisco, California, U.S.A.), a humanized monoclonal antibody targeting circulating vascular endothelial growth factor (VEGF) (6). As a second option, direct inhibition of ligand–receptor engagement can be achieved by preventing the binding of the growth factors to their receptors by mimicking the ligand’s structure and interfering thus in the ligand–receptor affinity. The successful example of cetuximab (Erbitux ; ImClone systems, Branchburg, New Jersey, U.S.A.), a chimeric antibody against the epidermal growth factor receptor (EGFR), is a proof of this concept (7). Trastuzumab (Herceptin ; Genentech Corp.) activity against the HER2 receptor is also a clear, successful approach (8). Finally, inhibition of downstream signal transduction by interfering with the kinase activity of the receptors with small-molecule inhibitors (currently registered), like erlotinib (Tarceva ; Genentech Corp.) and geftinib (Iressa ; AstraZeneca Pharmaceuticals, Wilmington, Delaware, U.S.A.) is another effective therapeutic strategy (9). The list of HER-interacting TKI is extremely large (10), with very promising panHER inhibitors, such as lapatinib (Tykerb ; GlaxoSmithKlein Pharmaceuticals, Research Triangle Park, North Carolina, U.S.A.) and HKI-272 (Wyeth Pharmaceuticals, Madison, New Jersey, U.S.A.). Apart from EGFR, various cellular receptors can serve as attractive therapeutic targets, including c-KIT receptor, insulin-growth factor receptor (IGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR). For example, imatinib mesylate has been shown to selectively inhibit a number of protein kinases, including c-KIT and PDGFR. Inhibition of Cytoplasmic Signal Transduction The Ras-Raf/Mitogen-Activated Protein Kinase Pathway. The Ras superfamily of genes encodes small GTP-binding proteins that are responsible for regulation of many cellular processes, including differentiation, cytoskeletal organization, and protein trafficking (Fig. 3) (11). At least 20 members of the Ras protein family are known in mammalian cells and share 30% sequence identity. Ras proteins localize to the inner surface of the cell membrane and act as on/off (Ras-GTP/Ras-GDP) switches controlled by cell surface receptors and transduce signals to the cell nucleus (5). Signaling via the Ras pathway has been elucidated in the last few years. Activated Ras activates Raf, which is a serine-threonine kinase. Raf activates mitogen-activated protein kinase-kinase (MAPKK), also called MEK, which in turn activates mitogen-activated protein kinase (MAPK) or extracellular regulated kinase (ERK). Finally, MAPK activation results in phosphorylation and activation

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FIGURE 3 Simplified illustration of some of the most important signal transduction pathways involved in ongenesis.

of transcriptional factors, such as c-jun, c-myc, and c-fos, provoking cell proliferation (Fig. 3). Oncogenic Ras mutations have been identified in non–small cell, lung, colorectal, pancreatic, bladder, kidney, and thyroid carcinomas (12). Raf mutations have been described in melanoma (13). RAS AND RAF KINASE INHIBITORS. The current approaches for inhibition of Ras include: (a) the inhibition of Ras protein expression through antisense oligonucleotides; (b) the prevention of membrane localization of Ras (farnesyl transferases);


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(c) engineering viruses to kill Ras-transformed cells; and (d) the inhibition of Ras function through inhibition of downstream Ras effectors. Inhibition of the Raf family of serine/threonine kinases, which consists of A-Raf, B-Raf, and C-Raf, is an effective way of proliferative signaling inhibition. Sorafenib (Nexavar ; Bayer Corp., Leverkuzen, Germany) is an orally bioavailable bisarylurea derivative developed initially as an inhibitor of mutant B-Raf, which was found later to inhibit the protein kinase domain of numerous receptors, including VEGFR, PDGFR, KIT, fetal liver (or Fms-like) tyrosine kinase 3 (FLT3), and P38a (14). The clinical use of sorafenib has so far resulted in tumor shrinkage and disease stabilization in renal cell, hepatocellular, colorectal, ovarian, and breast cancers (15). FARNESYL TRANSFERASE INHIBITORS. The enzyme Farnesyltransferase is involved in the posttranslational modification of Ras proteins by linking covalently a farnesylgroup onto Ras. On the basis of the fact that the biochemical procedure of farnesylation is critical for Ras maturation and function, farnesyltransferase inhibitors (FTIs) were originally envisioned as specific and sensitive inhibitors of Ras-mediated neoplastic transformation (16). However, it has become evident in the recent years that FTIs, apart from clear inhibition of Ras farnesylation, exhibit important activity against other polypeptides in addition to Ras that possess the ability to be farnesylated. Three FTIs, tipifarnib, lonafarnib, and BMS-214662, have been extensively tested in the clinic. Tipifarnib seems to exert significant clinical activity, especially in hematological malignancies (17). MEK INHIBITORS. There are two MEK homologues, MEK1 and MEK2, that are ubiquitously expressed in mammals and sequentially phosphorylate ERK1 and ERK2. CI-1040 was the first MEK-targeted agent to enter clinical trials, being a highly potent and selective inhibitor of both MEK isoforms (5). Other inhibitors of MEK kinase [PD0325901 (Pfizer Pharmaceuticals, New York, New York, U.S.A.), AZD6244 (AstraZeneca), MKI-833 (Wyeth)] are currently being evaluated either in preclinical or phase I/II settings.

The PI3K–Akt–Mammalian Target of Rapamycin Pathway. The mammalian target of rapamycin (m-TOR) (Fig. 3) is a serine/threonine kinase that belongs to the family of phosphatidylo-3-inositol kinase-like kinases (PI3K) involved in the regulation of a wide range of growth-related cellular functions including transcription, translation, membrane trafficking, protein degradation, and reorganization of the actin cytoskeleton (18). The best-known function of m-TOR, in the context of cell proliferation, is the regulation of translation initiation, presumably mediated by the activation of the 40S ribosomal protein S6 kinase. m-TOR activity seems to be regulated by PI3K/protein kinase B (PKB or Akt). The PI3K-Akt complex is activated as a result of the ligand-dependent activation of tyrosine kinase receptors, G-protein-coupled receptors, or integrins. On the other hand, phosphatase and tensin homolog (PTEN) is a dephosphorylating agent acting as a negative regulator of PI3K signaling. This phosphorylation stimulates the catalytic activity of Akt, resulting in the phosphorylation of numerous other proteins that affect cell growth, cell-cycle entry, and cell survival (5). PI3K AND AKT INHIBITORS. The main inhibitors of the PI3K-Akt pathway that have entered clinical trials are illustrated in Table 1.

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TABLE 1 Summary of PI3K-AKT Inhibitors Having Entered Clinical Trials Agent UCN-01 PERIFOSINE 17-AAG

Class

Target

Stage of development

Staurosporine Alkyl lipid Geldanamycin

PDK1 AKT HSP90, PDK, AKT

Phase II Phase III Phase III

RAPAMYCIN AND m-TOR INHIBITORS. Rapamycin was the first agent historically described to interact with m-TOR and inhibit the initiation of the translational process leading to slowing or arrest of cells in the G1 phase of the cell cycle. Since then, a number of compounds structurally related to rapamycin have been clinically developed, including tirosel/temsirolimus (Wyeth), RAD001 (Novartis), and AP23573 (Ariad Pharmaceuticals, Inc., Cambridge, Massachusetts, U.S.A.). A summary of the existing clinical trials assessing clinical activity of the various mTOR inhibitors is presented in Table 2. PROTEIN-KINASE C (PKC) INHIBITORS. The PKC family contains a number of serine/threonine specific protein kinases that have been shown to play an important role in a variety of cellular events potentially important for cancer development, including cell growth, cell-cycle progression, differentiation, drug efflux, apoptosis, and tumor angiogenesis (19). Two staurosporine analogues, PKC412 [N-benzoyl-staurosporine] (Novartis) and UCN-01 [7-hydroxy-staurosporin, NCI], which compete for binding to the ATP site on PKCs, have been recently evaluated in clinical trials (20). ISIS-3521, an antisense oligodeoxynucleotide that inhibits PKC-a mRNA, failed to demonstrate activity in a large phase III randomized trial in NSCLC. Enzastaurin (LY317615) is a PKC-b inhibitor (and to a lesser extent a PKC-a, -d, and -« inhibitor, too) with promising activity both in glioblastoma and lung cancer.

Signal Transduction and Activation of Transcription Pathway. One of the most recently recognized oncogenic signaling pathways involves the signal transduction and activation of transcription (STAT) proteins. This family of proteins comprises seven members that have dual roles as cytoplasmic signaling proteins and as nuclear transcription factors that activate a diverse set of genes, including some that are implicated in malignant progression (especially the STATs 3 and 5) (21). Activation of STAT signaling by growth factor receptors is effectuated through a mechanism similar to that described above for cytokine receptors except from the following unique properties: (a) the intrinsic receptor kinase may co-operate with nonreceptor tyrosine kinases (JAK- and SRC-family kinases); (b) once activated, TABLE 2 Summary of the Main Reported Clinical Trials Involving m-TOR Inhibitors Agent Tirosel/temsirolimus Tirosel/temsirolimus Tirosel/temsirolimus Tirosel/temsirolimus AP23573 Tirosel/temsirolimus Tirosel/temsirolimus Source: From Ref. 38.

Tumor

n

ORR (%)

Breast MCL Clioma Melanoma Lung Renal cell Renal cell

109 18 41 33 5 106 209

10 44 5 3 20 7 9


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STAT proteins form activated homo- or hetero-dimers that enter the nucleus; and (c) they bind by themselves to the corresponding promoter gene region of DNA, triggering permanent alterations in the genetic program controlling fundamental biological processes (Fig. 3). STAT

INHIBITORS.

The various approaches of inhibiting STAT signaling

comprise: & & &

&

Blocking of the upstream tyrosine kinases that are responsible for their activation [small molecule inhibitors of JAK (AG490), SRC and EGFR]. Targeting STAT3 and STAT5 directly by antisense oligonucleotides, dominantnegative expression vectors, and small interfering RNA molecules (siRNA). Blocking the dimerization and DNA-binding activity of STATs by short peptides (peptidomimetics), which are able to disrupt STAT signaling and induce tumor-cell apoptosis. Targeting knowing physiological regulators of STAT signaling, such as the suppressor of cytokine signaling (SOCS) family of proteins.

c-KIT Inhibitors. Imatinib mesylate is the first approved anticancer drug that may be considered to be a multitargeted tyrosine kinase inhibitor. Its targets include KIT, PDGFR, and BCR-ABL gene product. BCR-ABL has been linked with chronic myeloid leukemia (CML), while KIT and PDGFR have been associated with gastrointestinal stromal tumors (GIST). Imatinib is currently approved for first-line treatment of CML and GIST. While highly effective for these cancers, a limitation of chronic imatinib therapy is the development of resistance to the drug in a relatively large proportion of patients, for the most part due to acquisition of secondary mutations in the RTK driving the particular tumor (22). Cell-Cycle Inhibitors The cell cycle includes a series of precise, well defined, and coordinated events that include, successively, the post-mitotic G1 phase, the DNA synthesis S phase, the G2 phase, and the mitotic M phase, comprising itself a series of morphological and biochemical steps. Most of the “classic” chemotherapeutic agents, including the antimetabolites, the alkylating agents, the topo-isomerase inhibitors, and the tubulin polymerization/depolymerization inhibitors, exert their antitumor effect by interfering and blocking the cell-cycle process on a unique or multiple levels. Cyclin-dependent kinases (CDKs) are responsible for the phosphorylations required for the activation of the proteins in charge of cell-cycle progression. CDK inhibitors (CDKIs) are CDK counterparts that serve as negative regulators of the cell cycle. CELL-CYCLE INHIBITORS. The development of small molecule inhibitors of CDKs has progressed rapidly, with all of them targeting the ATP-binding site of the kinases and competing with ATP for inhibition. Staurosporine, a metabolite from Streptomyces spp., is a natural ATP-competitive inhibitor of CDKs that was initially identified as a potent inhibitor of protein kinase C (23). One other class of compounds that has been extensively studied is the flavones. Flavopiridol and deschloroflavopiridol are naturally occurring alkaloids showing cytotoxic properties against tumor cell lines. Flavopiridol (Sanofi-Aventis Corp., Paris, France) was

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the first CDK pan-inhibitor (CDK 1, 2, 4, 6, and 9) to enter clinical trials, showing promising results in combination therapy, and enhancing the activity of paclitaxel in human trials (24). Purine analogs, such as roscovitine (Cyclacel Pharmaceuticals, Berkeley Heights, New Jersey, U.S.A.), have also been developed in phase I/II trials. AURORA KINASE INHIBITORS. The aurora kinase family of serine/threonine kinases comprises three members called A, B, and C. Aurora kinases primarily function in early mitosis as they are required for proper centrosome separation and mitotic spindle assembly. Following their unique properties in mitotic spindle formation, they have been considered an attractive target for molecular therapy. A summary of the most important aurora kinase inhibitors is provided in Table 3.

Apoptosis Modulators Apoptosis, defined as programmed cell death, is characterized by cell shrinkage, nuclear condensation, and formation of apoptotic bodies, and may follow one of two alternative pathways: the extrinsic death receptor pathway or the intrinsic mitochondrial pathway (25). In the death receptor pathway, several death domain (DD)-containing members of the tumor necrosis factor (TNF) family of receptors, located at the cell surface, bind to their respective ligands and recruit adaptor proteins, forming a death-inducing signaling complex (DISC) that leads to caspase activation. The mitochondrial pathway is activated in response to stress-associated signals, such as radiation, chemotherapy, or viruses. This appealing idea of inducing apoptosis to “immortal” cancer cells has long-time been exploited in an effort to drive malignant cells to a programmed death pathway. p53 TARGETING. Apoptosis and senescence are the two cornerstones of the “surveillance system” that every organism possesses in order to limit potentially deleterious, uncontrolled cell proliferation. The tumor suppressor protein p53 is a key-factor of this surveillance procedure. Loss of p53 oncosuppressive functions represents a major event during neoplastic transformation, and several therapeutic approaches aiming at the restoration of the control of p53 function in tumor cells have been developed. Furthermore, several mutations of the “wild-type” p53 gene have been described and strongly correlated to oncogenesis in certain types of tumors. Pharmacological methods are under development to either stimulate wild-type p53 protein function, or help mutant p53 proteins to recover wild-type functions. These methods are based on small chemicals (CP-31388, PRIMA-1), peptides (CDB3), or single-chain antibody fragments corresponding to defined p53 domains (26). p53 replacement by means of viral vectors have been tested (RPR/ IGN Adp53), while exploiting p53-deficiency through viral cytolytic infection was also developed (ONYX-015). TABLE 3 The Main Aurora Kinase Inhibitors Having Entered Clinical Trials Drug MLN8054 ZM447439 Hesperadin VX-680

Target

Phase of development

Aurora kinase A Aurora kinase A, B Aurora kinase B Aurora kinase A, B

I I–II I I–II


11

SURVIVIN. Survivin is one of the members of the inhibitor of apoptosis protein (IAP) family of proteins and is differentially overexpressed in many types of human cancer cells, representing therefore an attractive anticancer target either through inactivating the survivin protein or by stopping the production of survivin through inhibition of survivin gene expression. Tetra-O-methyl nordihydroguaiaretic acid (M4N) is a selective inhibitor of survivin transcription that has shown promising results in preclinical trials, including several cell lines and different animal models (27). A methoxyethyl-modified phosphorothiate is currently being developed by Lilly Pharmaceuticals (Indianapolis, Indiana, U.S.A.) as an antisense survivin oligonucleotide (ASO). TUMOR NECROSIS FACTOR-RELATED APOPTOSIS-INDUCING LIGAND. The activation of cell surface “death” receptors by the tumor necrosis factor-related apoptosisinducing ligand (TRAIL) results in direct stimulation of apoptotic signaling pathways (extrinsic stimulation). Molecules that directly activate these receptors, such as agonistic monoclonal antibodies to the TRAIL receptors [HGS-ETR1, HGSETR2, and HGS-TR2J, (Human Genome Sciences, Rockville, Maryland, U.S.A.)] as well as recombinant/modified TRAIL (Apo2L/TRAIL, AMG951; Amgen Corp., Thousand Oaks, California, U.S.A.), are being developed as monotherapies and as part of combination therapies with existing chemotherapeutic drugs and other therapeutic modalities. B-CELL LYMPHOMA 2. The B-cell lymphoma 2 (Bcl-2) family of proteins play a central role in apoptosis by regulating the mitochondrial membrane permeability that mediates the intrinsic pathway of caspase activation. The Bcl-2 proto-oncogene, originally identified at the chromosomal breakpoint t (14;18) (q32;q21) in B-cell follicular lymphomas, is the first recognized and most extensively studied member of the Bcl-2 family. Oblimersen sodium (G3139) is a phosphorothioate complementary to bcl-2 (Genasens) that has been developed in melanoma and chronic lymphocytic leucemia (CLL) patients.

Antiangiogenic and Antivascular Agents When a tumor’s size reaches 2–3 mm of diameter, further tumor growth necessitates vascular supply via a procedure called neoangiogenesis and is induced by a number of factors, among which hypoxia plays a critical role. Angiogenesis is thus a critical step for tumor progression and systemic spread, representing thus an appealing target for effective approach against cancer. VEGF TARGETING. The best characterized and, thus far, the only clinically validated approach in modulating VEGF signaling pathway is the use of bevacizumab (28), which is a recombinant humanized VEGF monoclonal antibody against circulating VEGF-A. Bevacizumab has already been approved by the U.S. FDA as first-line treatment of metastatic colorectal cancer in combination with intravenous fluorouracil-based therapy and has also been shown to be potentially active and associated with better clinical outcomes in combination with paclitaxel and carboplatin as first-line therapy for locally advanced or metastatic nonsquamous non–small cell lung cancer (29). Bevacizumab activity has also been demonstrated in breast cancer patients.

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Using the same principle of ligand sequestration are decoy protein receptors and aptamers. VEGF-Trap is an engineered decoy receptor protein that possesses a 100-fold higher affinity for VEGF than that of the monoclonal antibody. It is currently in phase I trials. Aptamers, on the other hand, are fully synthetic, short nucleic acid sequences of DNA or RNA that are capable of binding with high affinity and target specificity to many diverse types of molecules, such as peptides and proteins, with a mechanism similar to that of the monoclonal antibodies (30). VEGFR TARGETING. An alternate approach to inhibit VEGF function is to interrupt signaling at the receptor level. Ribozymes are antisense RNAs that can bind in a sequence-specific, complementary fashion to their respective target messenger RNA (mRNA), like antisense nucleotides, but at the same time possess the unique property of directly cleaving the target mRNA due to their intrinsic catalytic activity. Angiozyme is a ribozyme directed against the mRNA of VEGFR1 and has been tested in phase I trials (28). In a way similar to EGFR, VEGFR-mediated signaling cascade can also be interrupted by adenosine triphosphate-competitive small-molecule inhibitors of the VEGFR tyrosine kinase activity. Several selected agents exhibiting this kind of activity have been tested in phase I to III clinical trials (Table 4). ENDOTHELIAL CELL PROPAGATION TARGETING. The endothelium is increasingly recognized as a target for biomedical intervention, not only for its accessibility to molecular agents coming from the blood-stream, but also for the active role played by endothelial cell proliferation and propagation to support diseases such as cancer. During neovascularization, endothelial cells need appropriate receptors to interact with the extracellular matrix for migration and survival. Such integrators do exist and constitute a promising target for endothelial propagation targeted therapy: the integrins. Integrins are cell-surface heterodimers of so-called a and b subunits whose association defines the nature of the ligands recognized. Table 5 summarizes the main agents targeting integrins or other endothelial receptors that have entered clinical trials. HYPOXIA INDUCING FACTOR INHIBITION. The most important regulator of the cellular response to reduced oxygen levels identified to date is the hypoxiainducible factor 1(HIF-1). Since overexpression of HIF-1 is a consequence of intratumoral hypoxia, it has been proposed as an appealing target for molecular anticancer therapy. A number of small molecules that inhibit HIF-1 have been

TABLE 4 Most Advanced Agents Exhibiting VEGFR Tyrosine Kinase Inhibitory Activity Drug BAY 439006 PTK/ZK SU11248 AG013706 ZD6474 AEE788 AZD2171

Target Raf, VEGFR, c-KIT, Flt-3, P38a VEGFR PDGFR, VEGFR, c-KIT, Flt-3 VEGFR, PDGFR EGFR, RET, VEGFR EGFR, HER2/neu, VEGFR VEGFR, PDGFR

Clinical phase III/registered III III/registered II II/III I–II II/III

13

Overview of Existing Therapies TABLE 5 Main Endothelial Propagation Targeting Agents Having Entered Clinical Trials Name(s) Vitaxin (MEDI-523) Abegrin (MEDI-522) CNTO 95 Volociximab Eos-200-4 M-200 Angiostatin Endostatin Endostar

Type Humanized monoclonal antibody Humanized monoclonal antibody Human monoclonal antibody Humanized monoclonal antibody Recombinant protein Recombinant protein Recombinant modified endostatin

Target

Clinical status

avb3 integrin

Phase I/II

avb3 integrin

Phase I/II

av integrins

Phase I/II

avb1 integrin

Phase I/II

Integrins? Angiomotin avb1, other receptors avb

Phase I/II Phase I/II Phase I Phase III in lung cancer

identified, some of them initially described as having different molecular targets (rapamycin, topo-isomerase inhibitors, histone deacetylase inhibitors, antimicrotubule agents, and redox pathway inhibitors). ANTIVASCULAR AGENTS. Unlike the inhibition of angiogenesis, which aims at preventing the growth of new blood vessels, vascular targeting aims at the rapid and selective shutdown and/or damage of the established tumor vasculature, leading to secondary tumor cell death. Thalidomide is a non-VEGF-based angiogenesis inhibitor, which, apart from the mechanism of action as an antiangiogenesis agent, acts as well as a modifier of the established tumor microvasculature in diseases such as multiple myeloma. The vascular-targeting compound ZD6126 (AstraZeneca) destabilizes microtubules and selectively disrupts immature tumor endothelial cells, which stops tumor blood flow and induces tumor cell death (31). Combretastatine derivative AVE8062 (Sanofi-Aventis) is another vascular disrupting agent in phase I trials.

Anti-Invasive Agents TARGETING METALLOPROTEINASE. The metalloproteinases (MMP) are a family of zinc-dependent neutral endopeptidases that are capable of degrading essentially all of the components of the extracellular matrix. These proteases, which are synthesized by connective tissue cells, are important for the remodeling of the extracellular matrix that accompanies physiological processes, but also tumor growth, invasion, and metastasis. Several therapeutic approaches concerning MMP inhibition have been developed, including zinc-binding agents, covalent inhibitors, exosite-binding, and allosteric inhibitors (32). Most trials with MMP inhibitors have lead to negative results, including trials with, among others, AG3340 (Agouron/Pfizer Corp., New York, New York, U.S.A.), BAY 129566 (Bayer), and BMS-275291 (Bristol Meyer Squibb, Jacksonville, Florida, U.S.A.). SRC INHIBITORS. The tyrosine kinase pp60src (Src) is the prototypical member of a family of proteins that participate in a broad array of cellular signal transduction processes, including cell growth, differentiation, survival, adhesion, and migration. Abnormal Src family kinase (SFK) signaling has been linked to several disease states, including cancer metastases. Src has thus emerged as a

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molecular target for the discovery of small-molecule inhibitors that regulate Src kinase activity by binding to the ATP pocket within the catalytic domain. AP23451 (Ariad Pharmaceuticals) is a small-molecule inhibitor designed to inhibit Srcdependent bone resorption, and AP23464 is a small-molecule inhibitor designed to inhibit the Src-dependent metastatic spread of cancer (33). Dasatinib (Bristol Meyer Squibb) is on fast-track for registration for imatinib-resistant CML, and AZD0530 (AstraZeneca) is in phase II trials. Bosutinib (Wyeth) is another emerging potent Src inhibitor. Telomerase and Telomere Interacting Agents Telomeres, the extremities of chromosomes, are shortened at each cell division, leading eventually to the senescence of the cell. In order to achieve a great number of cell divisions, cancer cells must acquire the capacity to maintain their telomeres. Therefore, telomeres and telomerase, the enzyme responsible for telomere elongation, represent interesting targets for molecular cancer therapy. A summary of the main agents targeting telomeres or telomerase action is presented in Table 6. Targeted Agents with Transversal Mechanisms CYCLO-OXYGENASE-2 INHIBITORS. The evidence that cyclo-oxygenase (COX) inhibitors and nonsteroidal anti-inflammatory drugs (NSAIDS) could be of benefit against the development and progression of malignancies was originally derived from large epidemiological studies. Nevertheless, recent data indicating that COX isoform 2 (COX2) can play a role in tumor progression in many solid tumors has reinforced the value of COX2 as a cancer therapeutic target. The initial promising results obtained by the first clinical trials with celecoxib and rofecoxib as chemopreventors in colon cancer were followed by an increased risk of severe cardiovascular accidents (34). PROTEASOME INHIBITION. The ubiquitin–proteasome pathway is an important protein degradation system affecting indirectly signal transduction pathways

TABLE 6 Main Therapeutic Strategies for Telomere and Telomerase Targeting Agent AZT SiRNA, ribozymes

NPS, GRN163, S-ODN, 2-5A 5-azacytidine, tricostatine A

Target Catalytic subunit of telomerase (hTERT) hTERT mRNA

Human telomerase RNA component (hTR) Chromatine promoter region, histones

SiRNA, mutated hTR

Telomere structure

Porphyrine and acridine derivatives

Telomeres (G-quartet)

Mechanism of action Inhibition of reverse transcription RNA silencing, block of mRNA by specific binding and degradation Block of mRNA by antisense oligonucleotides Change of chromatide structure that inhibits hTERT transcription Introduction of mutations in telomeric DNA Telomere interacting agents


15

through regulation of cell-cycle proteins. Many of the protein products that can provoke or inhibit neoplastic transformation and growth are temporarily degraded during the cell cycle by the ubiquitin–proteasome pathway, including the tumor suppressor p53, several cyclins and cyclin-dependent kinase inhibitors (CDKIs), and the nuclear factor kB (NF-kB), which is a key factor regulating transcription, angiogenesis, cell adhesion, and apoptosis (5). Taken altogether, it is clear that proteasome inhibitors can act through multiple mechanisms to arrest tumor growth, spread, and angiogenesis. Bortezomib is a proteasome inhibitor that has entered clinical trials with a registration in multiple myeloma and promising results in several solid tumors (35). HEAT SCHOCK PROTEINS. A large body of evidence collected through the past decade has identified the molecular chaperone heat shock protein 90 (Hsp90) as a critical modulator of an extensive network of cellular signaling pathways. Many of the processes overseen by Hsp90 are deregulated in tumor cells, including cellcycle control, gene transcription, and apoptotic signaling. Therefore, Hsp90 inhibition offers the potential of simultaneous disruption of multiple signaling events critical to tumor cell growth and survival. Indeed, small molecule inhibitors of Hsp90 function are actively being evaluated in the clinic as anticancer agents. 17-allylamino-17-demthoxygeldanamycin (17-AAG) was the first pharmacological agent to reach phase I clinical trials, exhibiting prolonged disease stabilization, notably in malignant melanoma (36). HISTONE DEACETYLASE INHIBITORS. Epigenetic silencing of tumor suppressor genes by aberrant DNA methylation and chromatin deacetylation provides interesting targets for chemotherapeutic intervention by inhibitors of these events. 5-Aza-2’-deoxycytidine (decitabine, 5AZA-CdR) is a potent demethylating agent, which can reactivate tumor suppressor genes silenced by excessive DNA methylation. LAQ824 (LAQ) is a novel inhibitor of histone deacetylase (HDAC) that shows antineoplastic activity and can activate genes that produce cell-cycle arrest. Both 5AZA-CdR and LAQ as single agents are currently under clinical investigation in patients with cancer (37).

CONCLUSION This chapter aimed at clarifying the concept of molecular targeted therapy and providing a general overview of MTAs currently under clinical development. Important considerations to be kept in mind regarding their future development are the following: (a) the vast number of MTAs currently under pre-clinical evaluation that could potentially enter the clinical arena in the coming months and years; (b) the remarkable clinical efficacy of some of these agents (HER inhibition, antiangiogenic agents) against different tumor types, including their ability to reverse chemo-resistance to conventional chemotherapy; and (c) the huge impact that MTAs could have in every-day clinical practice of oncology if integrated in earlier stages (locally advanced and resectable disease) or in an earlier setting (first-line therapy). Another exciting and rapidly evolving new area of active research is the integration of MTAs within the established anticancer armamentarium (surgery, radiotherapy, and conventional chemotherapy), in order to optimize their combined action and therapeutic efficacy.

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REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

16. 17. 18. 19. 20.

21. 22. 23. 24. 25.

Monks A, Scudiero D, Skehan P, et al. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J Natl Cancer Inst 1991; 83(11):757–66. Baselga J, Arribas J. Treating cancer’s kinase ‘addiction’. Nat Med 2004; 10(8):786–7. Druker BJ, Talpaz M, Resta DJ, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001; 344(14):1031–7. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100(1):57–70. Adjei AA and Hidalgo M. Intracellular signal transduction pathway proteins as targets for cancer therapy. J Clin Oncol 2005; 23(23):5386–403. Susman E. Bevacizumab adds survival benefit in colorectal cancer. Lancet Oncol 2005; 6(3):136. Meyerhardt JA and Mayer RJ. Systemic therapy for colorectal cancer. N Engl J Med 2005; 352(5):476–87. Brand FX, Ravanel N, Gauchez AS, et al. Prospect for anti-her2 receptor therapy in breast cancer. Anticancer Res 2006; 26(1B):715–22. Anonymous. Erlotinib (Tarceva) for advanced non-small cell lung cancer. Med Lett Drugs Ther 2005; 47(1205):25–6. Baselga J and Arteaga CL. Critical update and emerging trends in epidermal growth factor receptor targeting in cancer. J Clin Oncol 2005; 23(11):2445–59. Adjei AA. Blocking oncogenic Ras signaling for cancer therapy. J Natl Cancer Inst 2001; 93(14):1062–74. Bos JL. Ras oncogenes in human cancer: a review. Cancer Res 1989; 49(17):4682–9. Curtin JA, Fridlyand J, Kageshita T, et al. Distinct sets of genetic alterations in melanoma. N Engl J Med 2005; 353(20):2135–47. Ghobrial IM and Adjei AA. Inhibitors of the ras oncogene as therapeutic targets. Hematol Oncol Clin North Am 2002; 16(5):1065–88. Strumberg D, Richly H, Hilger RA, et al. Phase I clinical and pharmacokinetic study of the Novel Raf kinase and vascular endothelial growth factor receptor inhibitor BAY 43-9006 in patients with advanced refractory solid tumors. J Clin Oncol 2005; 23(5):965–72. Sebti SM and Adjei AA. Farnesyltransferase inhibitors. Semin Oncol 2004; 31(Suppl 1): 28–39. Karp JE, Lancet JE, Kaufmann SH, et al. Clinical and biologic activity of the farnesyltransferase inhibitor R115777 in adults with refractory and relapsed acute leukemias: a phase 1 clinical-laboratory correlative trial. Blood 2001; 97(11):3361–9. Schmelzle T and Hall MN. TOR, a central controller of cell growth. Cell 2000; 103(2):253–62. Gutcher I, Webb PR, and Anderson NG. The isoform-specific regulation of apoptosis by protein kinase C. Cell Mol Life Sci 2003; 60(6):1061–70. Monnerat C, Henriksson R, Chevalier T. Le, et al. Phase I study of PKC412 (N-benzoyl–staurosporine), a novel oral protein kinase C inhibitor, combined with gemcitabine and cisplatin in patients with non-small-cell lung cancer. Ann Oncol 2004; 15(2):316–23. Yu H and Jove R. The STATs of cancer—new molecular targets come of age. Nat Rev Cancer 2004; 4(2):97–105. Verweij J, Casali PG, Zalcberg J, et al. Progression-free survival in gastrointestinal stromal tumours with high-dose imatinib: randomised trial. Lancet 2004; 364(9440):1127–34. Meijer L. Chemical inhibitors of cyclin-dependent kinases. Trends Cell Biol 1996; 6(10):393–7. Carlson BA, Dubay MM, Sausville EA, Brizuela L, and Worland PJ. Flavopiridol induces G1 arrest with inhibition of cyclin-dependent kinase (CDK) 2 and CDK4 in human breast carcinoma cells. Cancer Res 1996; 56(13):2973–8. Green DR. Apoptotic pathways: paper wraps stone blunts scissors. Cell 2000; 102(1):1–4.


26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.

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Vassilev LT, Vu BT, Graves B, et al. In vivo activation of the p53 pathway by smallmolecule antagonists of MDM2. Science 2004; 303(5659):844–8. Lambert JD, Meyers RO, Timmermann BN, and Dorr RT. Tetra-O-methylnordihydroguaiaretic acid inhibits melanoma in vivo. Cancer Lett 2001; 171(1):47–56. Dy GK and Adjei AA. Angiogenesis inhibitors in lung cancer: a promise fulfilled. Clin Lung Cancer 2006; 7(Suppl 4):S145–9. Sandler AB. Advanced non-small-cell lung cancer: new data, therapy choices, and challenging decisions. Oncology (Williston Park) 2006; 20(6):626–8. Rimmele M. Nucleic acid aptamers as tools and drugs: recent developments. Chembiochem 2003; 4(10):963–71. Bozec A, Lassalle S, Gugenheim J, et al. Enhanced tumour antiangiogenic effects when combining gefitinib with the antivascular agent ZD6126. Br J Cancer 2006; 95(6):722–8. Overall CM and Kleifeld O. Towards third generation matrix metalloproteinase inhibitors for cancer therapy. Br J Cancer 2006; 94(7):941–6. Dalgarno D, Stehle T, Narula S, et al. Structural basis of Src tyrosine kinase inhibition with a new class of potent and selective trisubstituted purine-based compounds. Chem Biol Drug Des 2006; 67(1):46–57. Bresalier RS, Sandler RS, Quan H, et al. Cardiovascular events associated with rofecoxib in a colorectal adenoma chemoprevention trial. N Engl J Med 2005; 352(11):1092–102. Rajkumar SV, Richardson PG, Hideshima T, and Anderson KC. Proteasome inhibition as a novel therapeutic target in human cancer. J Clin Oncol 2005; 23(3):630–9. Banerji U, O’Donnell A, Scurr M, et al. Phase I pharmacokinetic and pharmacodynamic study of 17-allylamino, 17-demethoxygeldanamycin in patients with advanced malignancies. J Clin Oncol 2005; 23(18):4152–61. Hurtubise A and Momparler RL. Effect of histone deacetylase inhibitor LAQ824 on antineoplastic action of 5-Aza-2’-deoxycytidine (decitabine) on human breast carcinoma cells. Cancer Chemother Pharmacol 2006; 58(5):618–25. Dancey JE. Therapeutic targets: MTOR and related pathways. Cancer Biol Ther 2006; 5(9):1065–73.

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Signal Transduction Inhibitors, HER Family, EGFR Inhibition, and Clinical Achievements Giuseppe Giaccone Department of Medical Oncology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands

Paolo Zucali Department of Medical Oncology and Hematology, Instituto Clinico Humanitas, Milan, Italy

INTRODUCTION The epidermal growth factor receptor (EGFR) has been extensively investigated as a target for anti-cancer therapy. EGFR is overexpressed in a large number of tumors, including head and neck, colorectal, lung, breast, ovary, prostate, kidney, brain, pancreas, and bladder carcinomas (1). The overexpression of EGFR correlates with poor prognosis and decreased survival in several of these solid tumors (2–4). Moreover, the overexpressing EGFR tumors often produce their own ligands, such as transforming growth factor alpha (TGF-a), leading to the activation of survival pathways via autocrine loops. Signaling through the EGFR axis has been implicated in mediating multiple processes involved in tumor progression and metastasis, including invasion, angiogenesis, proliferation, and inhibition of apoptosis (5). The efficacy of conventional chemotherapy was improved by the anti-EGFR-targeted therapies in both preclinical and clinical studies (1). Although such therapies may lead to partial response or disease stabilization in some patients, the majority of unselected patients do not benefit from anti-EGFR therapy, and those who do eventually develop resistance.

EGFR SIGNALING PATHWAYS EGFR is a member of the ErbB family of trans-membrane tyrosine kinase receptors, which includes ErbB1 (or HER1, or EGFR), ErbB2 (or HER2/neu), ErbB3 (or HER3), and ErbB4 (or HER4). EGFR was the first ErbB family member to be described and remains the best characterized to date (6,7). Epithelial cells and malignant tumors of epithelial origin express EGFR, but EGFR is not expressed on mature hematopoietic cells (7,8). EGFR has six known ligands: epidermal growth factor (EGF), transforming growth factor alpha (TGF-a), amphiregulin, betacellulin, heparin binding EGF, and epiregulin (9). After dimerization and activation, the receptor is internalized, and its degradation or recycling can transiently downregulate signaling mediated by the receptor. Multiple signaling pathways related to cellular proliferation and survival are activated downstream of EGFR, including the ras pathway with the extra-cellular signal-regulated kinase (ERK)/mitogenactivated protein kinase (MAPK), the phosphatidylinositol 3-kinase/Akt (PI3K/ Akt) pathway, and signal transducer and activator of transcription (STAT) 19

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pathways. EGFR activation can also induce cell cycle progression via various mechanisms, including the up-regulation of cyclin D1 (10). EGFR stimulation can significantly increase the activity of c-Src, a signaling intermediate involved in cell cycle progression, motility, angiogenesis, and survival (11) (Fig. 1). EGFR INHIBITORS Small Molecules Tyrosine kinase inhibitors (TKIs) (Table 1) prevent auto-phosphorylation of the EGFR intracellular TK domain by competitive adenosine tri-phosphate (ATP) inhibition at the intracellular catalytic domain. EGFR TKIs fall into two broad classes: reversible inhibitors, such as gefitinib (Iressa ; AstraZeneca, London, U.K.) and erlotinib (Tarceva ; Genentech, South San Francisco, California, U.S.A.), and irreversible inhibitors. Irreversible inhibitors covalently bind specific cysteine residues in the ATP binding site of EGFR. The clinical significance of reversible versus irreversible inhibition is uncertain at this point. The ability to irreversibly bind the tyrosine kinase domain could theoretically produce more sustained antitumor activity. Furthermore, some irreversible inhibitors have been shown to preserve activity against the T790M resistant mutant of EGFR (12). Gefitinib and erlotinib are currently approved in many countries for clinical use in advanced non–small cell lung cancer (NSCLC) patients who failed at least one line of chemotherapy. Erlotinib is also approved for clinical use in pancreatic cancer patients. Mechanistically, gefitinib is able to block MAPK and PI-3K/Akt path-

Ligand

Ras

Raf

Grb/ SOS

R

R

K

K

EGFR

PI3K PTEN

Src

Akt

mTOR MEK

Erk

Proliferation

Bad

STAT

Apoptosis

p70S6K

Cell cycle progression

FIGURE 1 Schematic representation of major components of the EGF receptor pathway.

Small molecule

Small molecule

Monoclonal antibody

AEE788

BMS-599626

Cetuximab (Erbitux )

Irreversible inhibitor Inhibitor of EGFR and VGFR2 Inhibitor of EGFR, HER2, VEGFR2 Pan-inhibitor of all ErbB members Chimeric

EGFRa and ErbB2 TKs EGFR and VEGFR2 TKs EGFR, ErbB2 and VEGFR2 TKs EGFR, ErbB2 and ErbB4 TKs EGFR extracellular domain

Small molecule Small molecule

HKI272 ZD6474 (Zactima )

Irreversible, pan-inhibitor of all ErbB members Irreversible inhibitor

EGFRa, ErbB2 and ErbB4 TKs EGFRa and ErbB2 TKs

Small molecule

Small molecule

Dual reversible inhibitor

EGFR and ErbB2 TKs

Small molecule

Specific reversible inhibitor

EGFR TK

Characteristics Specific reversible inhibitor

Small molecule

Target EGFR TK

Class

Small molecule

Lapatinib (Tykerb , GW572016) Canertinib (CI-1033) EKB 569

Gefitinib (Iressa , ZD1839) Erlotinib (Tarceva , OSI-774)

Agent

TABLE 1 EGFR Inhibitors Route

Drug company

Bristol-Myers Squibb (New York, New York, U.S.A.) Imclone (New York, New York, U.S.A.) Bristol-Myers Squibb Merck KgGA (Darmstadt, Germany)

Oral IV

(Continued)

Novartis (Basel, Switzerland)

Oral

Oral Oral

Oral

Pfizer (New York, New York, U.S.A.) Wyeth Pharmaceuticals (Collegeville, Pennsylvania, U.S.A.) Wyeth Pharmaceuticals Astra-Zeneca

OSIP (Melville, New York, U.S.A.) Genentech (South San Francisco, California, U.S.A.) Roche (Basel, Switzerland) GlaxoSmithKline (Brentford, U.K.)

Astra-Zeneca (London, U.K.)

Oral

Oral

Oral

Oral

EGFR Inhibition and Clinical Achievements

21

Monoclonal antibody

Monoclonal antibody

Monoclonal antibody

Monoclonal antibody

Monoclonal antibody

MDX214

EGF vaccine

2C4

TheraCIMhR3

HuMax-EGFr

EGFR extracellular domain EGFR extracellular domain

Erb2 heterodimerization

Fully human

Humanized

hrEGF bound to protein and alum

Fully human

Fully human

Humanized

Characteristics

IV

IV

IV

ID

IV

IV

IV

Route

EMD (San Diego, California, U.S.A.) Merck KgGA Abgenix (Fremont, California, U.S.A.) Amgen (Thousand Oaks, California, U.S.A.) Medarex (Princeton, New Jersey, U.S.A.) CIMAB (Havana, Cuba) Biocon (Bangalore, India) CancerVax (Carlsbad, California, U.S.A.) YM BioSciences (Mississauga, Ontario) Genentech Roche YM BioSciences (Mississauga, Ontario, Canada) Genmab (Copenhagen, Denmark)

Drug company

Also active on the T790M resistant mutant. Abbreviations: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ID, intradermally; IV, intravenously; TK, tyrosine kinase; VEGFR2, vascular endothelial growth factor.

a

EGFR extracellular domain

Monoclonal antibody

Panitumumab (Vectibix , ABX-EGF)

EGFR extracellular domain EGF

EGFR extracellular domain

Target

Monoclonal antibody

Class

Matuzumab (EMD72000)

Agent

TABLE 1 EGFR Inhibitors (Continued )

22 Giaccone and Zucali

EGFR Inhibition and Clinical Achievements

23

ways, and its treatment is associated with cell-cycle arrest at G1, involving increased expression of the cyclin-dependent kinase (CDK) inhibitor p27KIP1 and decreased expression of CDK2, CDK4, CDK6, cyclin D1, and cyclin D3. Increased characteristics of apoptosis, such as DNA fragmentation, increased Fas protein expression, activation of initiator and effector caspases, and a change in plasma membrane phospholipids packing, were also observed (13,14). However, these effects may be limited and dependent on the tumor type (15). Similar cell-cycle and apoptotic changes have been seen after erlotinib treatment (14,16). Monoclonal Antibodies Monoclonal antibodies (mAb) against EGFR (Table 1) were the first approach used in clinical studies to target EGFR signaling in malignant cells. The mAb therapies competitively inhibit the binding of activating ligands to the extracellular domain of EGFR, inhibiting receptor auto-phosphorylation and, in contrast to the TKIs, inducing its internalization and degradation. Subsequent downstream signaling events are similar to those described for the TKIs gefitinib and erlotinib. Among mAb, cetuximab (Erbitux ; ImClone systems, Inc., Branchburg, New Jersey, U.S.A., licensed to Merck KGaA, Darmstadt, Germany) has achieved approval for use in colorectal cancer (CRC) refractory to irinotecan (Campto ; Pfizer, Capelle aan den Ijssel, The Netherlands) and in locally advanced squamous cell carcinomas of the head and neck (SCCHN) in combination with radiotherapy.

Other EGFR Inhibitors Active immunization could be an attractive alternative to inhibit the EGFR because it would circumvent both the need for multiple infusions and the danger of inducing an immune response typical of the antibodies. Mimotopes are small peptides that mimic a given epitope structurally, but not necessarily by amino acid sequence. The only important prerequisite is that an antibody recognizes the mimotope; for example, antibodies with proven beneficial antitumor properties, such as cetuximab. Riemer and colleagues observed that the epitope-specific immunization is feasible for active anti-EGFR immunotherapy. The in vitro biologic features of mimotope induced antibodies are similar to those of the monoclonal antibody cetuximab (17). Bing Hu and collegues observed that an active antitumor immunity could be induced by dendritic cells pulsed with recombinant ectodomain of mouse EGFR (DC-edMER), which may involve both humoral and cellular immunity, and may provide insight into the treatment of EGFR-positive tumors through the induction of active immunity against EGFR (18). Epidermal growth factor could be another possible target. Ramos and colleagues developed an active specific immunotherapy based on EGF deprivation, observing a correlation between antibody titers, serum EGF levels, and patient survival in a Phase I trial of 43 patients with NSCLC receiving the EGF vaccine (19). The use of antisense oligonucleotides inhibiting EGFR synthesis and the use of antibody-based immunoconjugates represent other interesting strategies. In preclinical studies, both treatments showed a significant inhibition of growth in EGFR positive tumors (20,21).

24

Giaccone and Zucali

CLINICAL TRIALS WITH EGFR INHIBITORS Non–Small Cell Lung Cancer Phase II studies of unselected patients with NSCLC treated with gefitinib and erlotinib as single agents after chemotherapy failure produced response rates and median survivals ranging from 10% to 19% and 6.0 to 8.0 months, respectively (Table 2). On the basis of the encouraging response rate and symptomatic benefit TABLE 2 Randomized Trials of EGFR TKIs in Advanced NSCLC

Study IDEAL 1 2nd/3rd line (Phase II) IDEAL 2 (22) 2nd/3rd line (Phase II) BR.21 (24) 2nd/3rd line (Phase III) ISEL (25) 2nd/3rd line (Phase III) INTACT 1 (26) 1st line (Phase III)

No. of patients

Response rate (%)

Median timeto-progression (months)

Median survival (months)

1-year survival (%)

Gefitinib 250 mg Gefitinib 500 mg

103 105

18.4 19

2.7 2.8

7.6 8.0

35 29

Gefitinb 500 mg Gefitinib 250 mg

102 114

12 10

7.0 6.0

7.0 6.0

27 24

Erlotinib 150 mg placebo

488 243

Gefitinib 250 mg placebo

1129 563

Regimen

Cisplatingemcitabineplacebo Cisplatingemcitabinegefitinib 250 mg Cisplatingemcitabinegefitinib 500 mg INTACT 2 (27) Carboplatin1st line paclitaxel(Phase III) placebo Carboplatinpaclitaxelgefitinib 250 mg Carboplatinpaclitaxelgefitinib 500 mg TALENT (29) Cisplatin1st line gemcitabine(Phase III) placebo Cisplatingemcitabineerlotinib 150 mg TRIBUTE (28) Carboplatin1st line paclitaxel(Phase III) placebo Carboplatinpaclitaxelerlotinib 150 mg

9 4 months. Three patients with osteosarcoma and one with malignant fibrous histiocytoma achieved confirmed partial responses (5%), 46 patients (59%) had stable disease and 29 patients (37%) progressed by the first assessment (111). AP23573 is being studied in patients with, hormone refractory prostate cancer, endometrial cancer, and lymphoproliferative malignancies. Initial studies of combinations of sirolimus and derivatives with standard therapies suggest enhanced toxicity at relatively modest doses may occur. Results from phase I studies evaluating tirosel/temsirolimus with interferon-alpha (112), and 5-fluorouracil (113) showed enhanced toxicities at relatively low doses. Everolimus combined with gemcitabine (114) and with gefitinib (115) demonstrated limiting toxicities at lower doses than the phase II single agent doses. Toxicities of both the standard agent and the mTOR inhibitor appear to occur more frequently. Across these studies, no apparent pharmacokinetic interactions were observed. Enhanced toxicity seen with combinations of mTOR inhibitors and standard cancer agents, may occur via overlapping toxicity of the agents or through alterations in expression of enzymes involved in drug metabolism. Modifications of dose and possibly schedule of administration of mTOR inhibitors may be required to optimize combination regimens. CONCLUSIONS In summary, agents in clinical development that specifically target the PI3K-AktmTOR pathway are currently limited to the rapamycins, which inhibit the downstream kinase mTOR. Multi-targeted inhibitors, such as UCN-01 and perifosine,

PI3K Pathway Inhibitors

77

are also under clinical investigation. Whether modulating downstream targets, such as mTOR rather than PI3K and Akt, will provide a better therapeutic index or whether specifically or broadly modulating multiple signaling pathways will have greater therapeutic effect remain unanswered questions. Initial clinical studies with mTOR inhibitors have shown the agents have anti-tumor activity and may confirm clinical benefit to patients with acceptable toxicity. Achieving a therapeutic index with inhibitors of the upstream components of the PI3K/Akt/mTOR pathway may be problematic. In vitro data show that pathway inhibitors are preferentially cytotoxic in tumor cells that exhibit increased activation of the pathway, suggesting that death of cancer cells without death of normal cells may be possible. To enhance the therapeutic index, identifying patients with tumors that selectively rely on activation of the PI3K/Akt/mTOR pathway for enrollment into clinical trials would be preferable; developing selective inhibitors to mutations in the kinases, such those described in the PI3K catalytic subunit, would be ideal. Inhibition of the pathway in tumor cells that depend upon it for survival might cause apoptosis, but would not kill normal cells that do not grow in these conditions and therefore do not have the same dependence. If a therapeutic index is not achievable when inhibitors are used as single agents, these agents might be valuable as radiation or chemotherapeutic sensitizers at lower doses, where toxicities might be less likely to develop. Given the frequent implication of the PI3K pathway in the pathophysiology of human malignancy, there is every reason to be optimistic that inhibition of the pathway will induce anti-tumor effects in cancer patients. A variety of agents designed to specifically target components of the pathway are in clinical testing and many more are in preclinical evaluation. The major clinical development challenges will be efficiently identifying the appropriate dose, schedule, and combination regimens for patients with susceptible malignancies and monitoring and managing toxicities to optimize therapeutic index. REFERENCES 1. Cantley LC. The phosphoinositide 3-kinase pathway. Science 2002; 296:1655–7. 2. Chang HW, Aoki M, Fruman D, et al. Transformation of chicken cells by the gene encoding the catalytic subunit of PI 3-kinase. Science 1997; 276:1848–50. 3. Vivanco I, Sawyers CL. The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer 2002; 2:489–501. 4. Corvera S, Czech MP. Direct targets of phosphoinositide 3-kinase products in membrane traffic and signal transduction. Trends Cell Biol 1998; 8:442–6. 5. Bellacosa A, Testa JR, Staal SP, Tsichlis PN. A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region. Science 1991; 254:274–7. 6. Staal SP. Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2: amplification of AKT1 in a primary human gastric adenocarcinoma. Proc Natl Acad Sci USA 1987; 84:5034–7. 7. Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev 2004; 18:1926–45. 8. Thomas GV. mTOR and cancer: reason for dancing at the crossroads? Curr Opin Genet Dev 2006; 16:78–84. 9. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 2005; 307:1098–101. 10. Balaraman Y, Limaye AR, Levey AI, Srinivasan S. Glycogen synthase kinase 3beta and Alzheimer’s disease: pathophysiological and therapeutic significance. Cell Mol Life Sci 2006; 63:1226–35.

78

Dancey

11. Chakraborty C. Biochemical and molecular basis of insulin resistance. Curr Protein Pept Sci 2006; 7:113–21. 12. Huang H, Tindall DJ. FOXO factors: a matter of life and death. Future Oncol 2006; 2:83–9. 13. Toker A, Yoeli-Lerner M. Akt signaling and cancer: surviving but not moving on. Cancer Res 2006; 66:3963–6. 14. Yoeli-Lerner M, Toker A. Akt/PKB signaling in cancer: a function in cell motility and invasion. Cell Cycle 2006; 5:603–5. 15. Walsh K. Akt signaling and growth of the heart. Circulation 2006; 113:2032–4. 16. Kim HJ, Hawke N, Baldwin AS. NF-kappaB and IKK as therapeutic targets in cancer. Cell Death Differ 2006; 13:738–47. 17. Manning BD, Cantley LC. Rheb fills a GAP between TSC and TOR. Trends Biochem Sci 2003; 28:573–6. 18. Brugarolas J, Kaelin WG, Jr. Dysregulation of HIF and VEGF is a unifying feature of the familial hamartoma syndromes. Cancer Cell 2004; 6:7–10. 19. Corradetti MN, Inoki K, Bardeesy N, DePinho RA, Guan KL. Regulation of the TSC pathway by LKB1: evidence of a molecular link between tuberous sclerosis complex and Peutz-Jeghers syndrome. Genes Dev 2004; 18:1533–8. 20. Hara K, Maruki Y, Long X, et al. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 2002; 110:177–89. 21. Kim DH, Sarbassov DD, Ali SM, et al. mTOR interacts with raptor to form a nutrientsensitive complex that signals to the cell growth machinery. Cell 2002; 110:163–75. 22. Sarbassov DD, Ali SM, Kim DH, et al. Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol 2004; 14:1296–302. 23. Sarbassov DD, Ali SM, Sengupta S, et al. Prolonged Rapamycin Treatment Inhibits mTORC2 Assembly and Akt/PKB. Mol Cell 2006; 22:159–68. 24. Aoki M, Blazek E, Vogt PK. A role of the kinase mTOR in cellular transformation induced by the oncoproteins P3k and Akt. Proc Natl Acad Sci USA 2001; 98:136–41. 25. Osaki M, Oshimura M, Ito H. PI3K-Akt pathway: its functions and alterations in human cancer. Apoptosis 2004; 9:667–76. 26. Cully M, You H, Levine AJ, Mak TW. Beyond PTEN mutations: the PI3K pathway as an integrator of multiple inputs during tumorigenesis. Nat Rev Cancer 2006; 6: 184–92. 27. Vogt PK, Bader AG, Kang S. PI 3-kinases: hidden potentials revealed. Cell Cycle 2006; 5:946–9. 28. Samuels Y, Ericson K. Oncogenic PI3K and its role in cancer. Curr Opin Oncol 2006; 18:77–82. 29. Shah OJ, Hunter T. Tuberous sclerosis and insulin resistance. Unlikely bedfellows reveal a TORrid affair. Cell Cycle 2005; 4:46–51. 30. Bader AG, Kang S, Zhao L, Vogt PK. Oncogenic PI3K deregulates transcription and translation. Nat Rev Cancer 2005; 5:921–9. 31. Jin W, Wu L, Liang K, Liu B, Lu Y, Fan Z. Roles of the PI-3K and MEK pathways in Ras-mediated chemoresistance in breast cancer cells. Br J Cancer 2003; 89:185–91. 32. Liang K, Jin W, Knuefermann C, et al. Targeting the phosphatidylinositol 3-kinase/ Akt pathway for enhancing breast cancer cells to radiotherapy. Mol Cancer Ther 2003; 2:353–60. 33. Chakravarti A, Loeffler JS, Dyson NJ. Insulin-like growth factor receptor I mediates resistance to anti-epidermal growth factor receptor therapy in primary human glioblastoma cells through continued activation of phosphoinositide 3-kinase signaling. Cancer Res 2002; 62:200–7. 34. Lu Y, Zi X, Zhao Y, Mascarenhas D, Pollak M. Insulin-like growth factor-I receptor signaling and resistance to trastuzumab (Herceptin). J Natl Cancer Inst 2001; 93: 1852–7. 35. Bader AG, Kang S, Vogt PK. Cancer-specific mutations in PIK3CA are oncogenic in vivo. Proc Natl Acad Sci USA 2006; 103:1475–9.


79

36. Granville CA, Memmott RM, Gills JJ, Dennis PA. Handicapping the race to develop inhibitors of the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin pathway. Clin Cancer Res 2006; 12(3 Pt 1):679–89. 37. Powis G, Bonjouklian R, Berggren MM, et al. Wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase. Cancer Res 1994; 54:2419–23. 38. Vlahos CJ, Matter WF, Hui KY, Brown RF. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 1994; 269:5241–8. 39. Hu L, Zaloudek C, Mills GB, Gray J, Jaffe RB. In vivo and in vitro ovarian carcinoma growth inhibition by a phosphatidylinositol 3-kinase inhibitor (LY294002). Clin Cancer Res 2000; 6:880–6. 40. Lemke LE, Paine-Murrieta GD, Taylor CW, Powis G. Wortmannin inhibits the growth of mammary tumors despite the existence of a novel wortmannin-insensitive phosphatidylinositol-3-kinase. Cancer Chemother Pharmacol 1999; 44:491–7. 41. Schultz RM, Merriman RL, Andis SL, et al. In vitro and in vivo antitumor activity of the phosphatidylinositol-3-kinase inhibitor, wortmannin. Anticancer Res 1995; 15:1135–9. 42. Ihle NT, Paine-Murrieta G, Berggren MI, et al. The phosphatidylinositol-3-kinase inhibitor PX-866 overcomes resistance to the epidermal growth factor receptor inhibitor gefitinib in A-549 human non-small cell lung cancer xenografts. Mol Cancer Ther 2005; 4:1349–57. 43. Ihle NT, Williams R, Chow S, et al. Molecular pharmacology and antitumor activity of PX-866, a novel inhibitor of phosphoinositide-3-kinase signaling. Mol Cancer Ther 2004; 3:763–72. 44. Williams R, Baker AF, Ihle NT, Winkler AR, Kirkpatrick L, Powis G. The skin and hair as surrogate tissues for measuring the target effect of inhibitors of phosphoinositide-3-kinase signaling. Cancer Chemother Pharmacol 2006; 58:444–50. 45. Mora A, Komander D, van Aalten DM, Alessi DR. PDK1, the master regulator of AGC kinase signal transduction. Semin Cell Dev Biol 2004; 15:161–70. 46. Kikani CK, Dong LQ, Liu F. “New”-clear functions of PDK1: beyond a master kinase in the cytosol? J Cell Biochem 2005; 96:1157–62. 47. Sato S, Fujita N, Tsuruo T. Interference with PDK1-Akt survival signaling pathway by UCN-01 (7-hydroxystaurosporine). Oncogene 2002; 21:1727–38. 48. Takahashi I, Asano K, Kawamoto I, Tamaoki T, Nakano H. UCN-01 and UCN-02, new selective inhibitors of protein kinase C. I. Screening, producing organism and fermentation. J Antibiot (Tokyo) 1989; 42:564–70. 49. Omura-Minamisawa M, Diccianni MB, Batova A, et al. In vitro sensitivity of T-cell lymphoblastic leukemia to UCN-01 (7-hydroxystaurosporine) is dependent on p16 protein status: a Pediatric Oncology Group study. Cancer Res 2000; 60:6573–6. 50. Graves PR, Yu L, Schwarz JK, et al. The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01. J Biol Chem 2000; 275:5600–5. 51. Monks A, Harris E, Connelly J, Hose C. Enhancement of fludarabine and gemcitabine toxicity by UCN-01 in a variety of human tumor cell lines. Proc Am Assoc Cancer Res 1999; 40(March):Abstract 45. 52. Monks A, Harris ED, Vaigro-Wolff A, Hose CD, Connelly JW, Sausville EA. UCN-01 enhances the in vitro toxicity of clinical agents in human tumor cell lines. Invest New Drugs 2000; 18:95–107. 53. Fuse E, Tanii H, Kurata N, et al. Unpredicted clinical pharmacology of UCN-01 caused by specific binding to human alpha1-acid glycoprotein. Cancer Res 1998; 58:3248–53. 54. Fuse E, Tanii H, Takai K, et al. Altered pharmacokinetics of a novel anticancer drug, UCN-01, caused by specific high affinity binding to alpha1-acid glycoprotein in humans. Cancer Res 1999; 59:1054–60. 55. Sausville EA, Arbuck SG, Messmann R, et al. Phase I trial of 72-hour continuous infusion UCN-01 in patients with refractory neoplasms. J Clin Oncol 2001; 19:2319–33.

80

Dancey

56. Sausville EA, Lush RD, Headlee D, et al. Clinical pharmacology of UCN-01: initial observations and comparison to preclinical models. Cancer Chemother Pharmacol 1998; 42(Suppl):S54–9. 57. Dees EC, Baker SD, O’Reilly S, et al. A phase I and pharmacokinetic study of short infusions of UCN-01 in patients with refractory solid tumors. Clin Cancer Res 2005; 11(2 Pt 1):664–71. 58. Hotte SJ, Oza A, Winquist EW, et al. Phase I trial of UCN-01 in combination with topotecan in patients with advanced solid cancers: a Princess Margaret Hospital Phase II Consortium study. Ann Oncol 2006; 17:334–40. 59. Kortmansky J, Shah MA, Kaubisch A, et al. Phase I trial of the cyclin-dependent kinase inhibitor and protein kinase C inhibitor 7-hydroxystaurosporine in combination with fluorouracil in patients with advanced solid tumors. J Clin Oncol 2005; 23:1875–84. 60. Zhu J, Huang JW, Tseng PH, et al. From the cyclooxygenase-2 inhibitor celecoxib to a novel class of 3-phosphoinositide-dependent protein kinase-1 inhibitors. Cancer Res 2004; 64:4309–18. 61. Tseng PH, Lin HP, Zhu J, et al. Synergistic interactions between imatinib mesylate and the novel phosphoinositide-dependent kinase-1 inhibitor OSU-03012 in overcoming imatinib mesylate resistance. Blood 2005; 105:4021–7. 62. Tseng PH, Wang YC, Weng SC, et al. Overcoming trastuzumab resistance in HER2overexpressing breast cancer cells by using a novel celecoxib-derived phosphoinositide-dependent kinase-1 inhibitor. Mol Pharmacol 2006; 70:1534–41. 63. Ding H, Han C, Zhu J, Chen CS, D’Ambrosio SM. Celecoxib derivatives induce apoptosis via the disruption of mitochondrial membrane potential and activation of caspase 9. Int J Cancer 2005; 113:803–10. 64. Hou CC, Hung SL, Kao SH, Chen TH, Lee HM. Celecoxib induces heme-oxygenase expression in glomerular mesangial cells. Ann N Y Acad Sci 2005; 1042:235–45. 65. Eue I. Growth inhibition of human mammary carcinoma by liposomal hexadecylphosphocholine: participation of activated macrophages in the antitumor mechanism. Int J Cancer 2001; 92:426–33. 66. Wang YZ, Liu XY, Wu LJ, Zhang YB, Hou L, Xu QS. Antiproliferation effects of hexadecylphosphocholine on solid tumour and leukaemia selectively in vitro. Drugs Exp Clin Res 1997; 23:97–102. 67. Guerin PJ, Olliaro P, Sundar S, et al. Visceral leishmaniasis: current status of control, diagnosis, and treatment, and a proposed research and development agenda. Lancet Infect Dis 2002; 2:494–501. 68. Hilgard P, Klenner T, Stekar J, Nossner G, Kutscher B, Engel J. D-21266, a new heterocyclic alkylphospholipid with antitumour activity. Eur J Cancer 1997; 33:442–6. 69. Patel V, Lahusen T, Sy T, Sausville EA, Gutkind JS, Senderowicz AM. Perifosine, a novel alkylphospholipid, induces p21(WAF1) expression in squamous carcinoma cells through a p53-independent pathway, leading to loss in cyclin-dependent kinase activity and cell cycle arrest. Cancer Res 2002; 62:1401–9. 70. Kondapaka S, Singh S, Dasmahapatra G, Sausville E, Roy K. Perifosine-A novel alkyllysophospholipid, modulates the Akt signaling pathway. Proc Am Assoc Cancer Res 2002; 43:2977. 71. Kondapaka SB, Singh SS, Dasmahapatra GP, Sausville EA, Roy KK. Perifosine, a novel alkylphospholipid, inhibits protein kinase B activation. Mol Cancer Ther 2003; 2:1093–103. 72. Ruiter GA, Zerp SF, Bartelink H, van Blitterswijk WJ, Verheij M. Anti-cancer alkyllysophospholipids inhibit the phosphatidylinositol 3-kinase-Akt/PKB survival pathway. Anticancer Drugs 2003; 14:167–73. 73. De Siervi A, Marinissen M, Diggs J, Wang XF, Pages G, Senderowicz A. Transcriptional activation of p21(waf1/cip1) by alkylphospholipids: role of the mitogenactivated protein kinase pathway in the transactivation of the human p21(waf1/cip1) promoter by Sp1. Cancer Res 2004; 64:743–50. 74. Ruiter GA, Zerp SF, Bartelink H, van Blitterswijk WJ, Verheij M. Alkyl-lysophospholipids activate the SAPK/JNK pathway and enhance radiation-induced apoptosis. Cancer Res 1999; 59:2457–63.


81

75. Crul M, Rosing H, de Klerk GJ, et al. Phase I and pharmacological study of daily oral administration of perifosine (D-21266) in patients with advanced solid tumours. Eur J Cancer 2002; 38:1615–21. 76. Van Ummersen L, Binger K, Volkman J, et al. A phase I trial of perifosine (NSC 639966) on a loading dose/maintenance dose schedule in patients with advanced cancer. Clin Cancer Res 2004; 10:7450–6. 77. Argiris A, Cohen E, Karrison T, et al. A phase II trial of perifosine, an oral alkylphospholipid, in recurrent or metastatic head and neck cancer. Cancer Biol Ther 2006; 5:766–70. 78. Ernst DS, Eisenhauer E, Wainman N, et al. Phase II study of perifosine in previously untreated patients with metastatic melanoma. Invest New Drugs 2005; 23:569–75. 79. Knowling M, Blackstein M, Tozer R, et al. A phase II study of perifosine (D-21226) in patients with previously untreated metastatic or locally advanced soft tissue sarcoma: A National Cancer Institute of Canada Clinical Trials Group trial. Invest New Drugs 2006; 24:435–9. 80. Posadas EM, Gulley J, Arlen PM, et al. A phase II study of perifosine in androgen independent prostate cancer. Cancer Biol Ther 2005; 4:1133–7. 81. Douros J, Suffness M. New antitumor substances of natural origin. Cancer Treat Rev 1981; 8:63–87. 82. Huang S, Liu LN, Hosoi H, Dilling MB, Shikata T, Houghton PJ. p53/p21(CIP1) cooperate in enforcing rapamycin-induced G(1) arrest and determine the cellular response to rapamycin. Cancer Res 2001; 61:3373–81. 83. Huang S, Shu L, Easton J, et al. Inhibition of mammalian target of rapamycin activates apoptosis signal-regulating kinase 1 signaling by suppressing protein phosphatase 5 activity. J Biol Chem 2004; 279:36490–6. 84. Guba M, von Breitenbuch P, Steinbauer M, et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med 2002; 8:128–35. 85. Zhong H, Hanrahan C, van der Poel H, Simons JW. Hypoxia-inducible factor 1alpha and 1beta proteins share common signaling pathways in human prostate cancer cells. Biochem Biophys Res Commun 2001; 284:352–6. 86. Zhong H, Chiles K, Feldser D, et al. Modulation of hypoxia-inducible factor 1alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/ AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res 2000; 60:1541–5. 87. Franz DN, Leonard J, Tudor C, et al. Rapamycin causes regression of astrocytomas in tuberous sclerosis complex. Ann Neurol 2006; 59:490–8. 88. Hidalgo M, Eric Rowinsky E, Erlichman C, et al. A phase I and pharmacological study of CCI-779, a rapamycin ester cell cycle inhibitor. Ann Oncol 2000; 11(Suppl 4):133 (Abstract 606O). 89. Hidalgo M, Rowinsky E, Erlichman C, et al. Phase I and pharmacological study of CCI-779, a cell cycle inhibitor. Proceedings of the 11th NCI-EORTC-AACR Symposium on New Drugs in Cancer Therapy. Clin Cancer Res (suppl) 2000; 6:Abstract 413. 90. Raymond E, Alexandre J, Faivre S, et al. Safety and pharmacokinetics of escalated doses of weekly intravenous infusion of CCI-779, a novel mTOR inhibitor, in patients with cancer. J Clin Oncol 2004; 22:2336–47. 91. Chang SM, Kuhn J, Wen P, et al. Phase I/pharmacokinetic study of CCI-779 in patients with recurrent malignant glioma on enzyme-inducing antiepileptic drugs. Invest New Drugs 2004; 22:427–35. 92. Forouzesh B, Buckner J, Adjei A, et al. Phase I, bioavailability, and pharmacokinetic study of oral dosage of CCI-779 administered to patients with advanced solid malignancies. EJC 2002; 38(Suppl 7):Abstract 168. 93. Witzig TE, Geyer SM, Ghobrial I, et al. Phase II trial of single-agent temsirolimus (CCI-779) for relapsed mantle cell lymphoma. J Clin Oncol 2005; 23:5347–56. 94. Oza A, Elit L, Biagi J, et al. A phase II study of temsirolimus (CCI-779) in patients with metastatic and/or recurrent endometrial cancer—NCIC CTG IND.160. Clin Cancer Res 2005; 11(24 (part 2)):9099s.

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Dancey

95. Chan S, Scheulen ME, Johnston S, et al. Phase II study of temsirolimus (CCI-779), a novel inhibitor of mTOR, in heavily pretreated patients with locally advanced or metastatic breast cancer. J Clin Oncol 2005; 23:5314–22. 96. Atkins MB, Hidalgo M, Stadler WM, et al. Randomized phase II study of multiple dose levels of CCI-779, a novel mammalian target of rapamycin kinase inhibitor, in patients with advanced refractory renal cell carcinoma. J Clin Oncol 2004; 22:909–18. 97. Pandya KJ, Levy DE, Hidalgo M, et al. A randomized, phase II ECOG trial of two dose levels of temsirolimus (CCI-779) in patients with extensive stage small cell lung cancer in remission after induction chemotherapy. A preliminary report. Proc Am Soc Clin Oncol 2005; 24:Abstract 2005. 98. Margolin K, Longmate J, Baratta T, et al. CCI-779 in metastatic melanoma: a phase II trial of the California Cancer Consortium. Cancer 2005; 104:1045–8. 99. Chang SM, Wen P, Cloughesy T, et al. Phase II study of CCI-779 in patients with recurrent glioblastoma multiforme. Invest New Drugs 2005; 23:357–61. 100. Galanis E, Buckner JC, Maurer MJ, et al. Phase II trial of temsirolimus (CCI-779) in recurrent glioblastoma multiforme: a North Central Cancer Treatment Group Study. J Clin Oncol 2005; 23:5294–304. 101. Hudes G, Carducci M, Tomczak P, et al. A phase 3, randomized, 3-arm study of temsirolimus (TEMSR) or interferon-alpha (IFN) or the combination of TEMSR þ IFN in the treatment of first-line, poor-risk patients with advanced renal cell carcinoma (adv RCC). Journal of Clinical Oncology, 2006 ASCO Annual Meeting Proceedings Part I 2006; 24(8S (June 20 Supplement)):Abstract LBA4. 102. Kirchner GI, Meier-Wiedenbach I, Manns MP. Clinical pharmacokinetics of everolimus. Clin Pharmacokinet 2004; 43:83–95. 103. O’Donnell A, Faivre S, Judson I, et al. A phase I study of the oral mTOR inhibitor RAD001 as monotherapy to identify the optimal biologically effective dose using toxicity, pharmacokinetic (PK) and pharmacodynamic (PD) endpoints in patients with solid tumours. Proc Am Soc Clin Oncol 2003; 22:Abstract 803. 104. Amato RJ, Misellati A, Khan M, Chiang S. A phase II trial of RAD001 in patients (Pts) with metastatic renal cell carcinoma (MRCC). J Clin Oncol 2006 ASCO Annual Meeting Proceedings Part 1 2006; 24(18S (June 20 Supplement)): abstract 4530. 105. Metcalf CA III, Bohacek R, Rozamus LW, et al. Structure-based design of AP23573, a phosphorus-containing analog of rapamycin for anti-tumor therapy. Proc Am Assoc Cancer Res 45: abstract 2476 106. Mita MM, Rowinsky EK, Goldston ML, et al. Phase I, pharmacokinetic (PK), and pharmacodynamic (PD) study of AP23573, an mTOR Inhibitor, administered IV daily X 5 every other week in patients (pts) with refractory or advanced malignancies. Proc Am Soc Clin Oncol 2004; 23:Abstract No: 3076. 107. Mita MM, Rowinsky EK, Mita AC, et al. Phase I, pharmacokinetic (PK), and pharmacodynamic (PD) study of AP23573, an mTOR Inhibitor, administered IV daily X 5 every other week in patients (pts) with refractory or advanced malignancies. EJC (Suppl) 16th EORTC-NCI-AACR Symp Mol Targets Cancer Ther 2004; 2(8):Abstract 409. 108. Desai AA, Janisch L, Berk LR, et al. A phase I trial of a novel mTOR inhibitor AP23573 administered weekly (wkly) in patients (pts) with refractory or advanced malignancies: a pharmacokinetic (PK) and pharmacodynamic (PD) analysis. Proc Am Soc Clin Oncol 2004; 23:Abstract 3150. 109. Desai AA, Janisch L, Berk LR, et al. A phase 1 trial of weekly (wkly) AP23573, a novel mTOR inhibitor, in patients (pts) with advanced or refractory malignancies: a pharmacokinetic (PK) and pharmadynamic (PD) analysis. EJC (Suppl) 16th EORTC-NCIAACR Symp Mol Targets Cancer Ther 2004; 2:Abstract 390. 110. Chawla SP, Sankhala KK, Chua V, et al. A phase II study of AP23573 (an mTOR inhibitor) in patients (pts) with advanced sarcomas. Proc Am Soc Clin Oncol 2005; 24:Abstract 9068. 111. Chawla SP, Blay J-Y, Staddon AP, et al. A Phase II Trial of AP23573, a Novel mTOR Inhibitor, in Patients (Pts) with Advanced Soft Tissue or Bone Sarcoma. Clin Cancer Res 2005; 11:9166s Abstract C272.


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112. Dutcher JP, Hudes G, Motzer R, et al. Preliminary report of a phase 1 study of intravenous (IV) CCI-779 given in combination with interferon-? (IFN) to patients with advanced renal cell carcinoma (RCC). Proc Am Soc Clin Oncol 2003; 22:Abstract 854. 113. Punt CJ, Boni J, Bruntsch U, Peters M, Thielert C. Phase I and pharmacokinetic study of CCI-779, a novel cytostatic cell-cycle inhibitor, in combination with 5-fluorouracil and leucovorin in patients with advanced solid tumors. Ann Oncol 2003; 14:931–7. 114. Pacey S, Rea D, Steven N, et al. Results of a phase 1 clinical trial investigating a combination of the oral mTOR-inhibitor Everolimus (E, RAD001) and Gemcitabine (GEM) in patients (pts) with advanced cancers. Proc Am Soc Clin Oncol (Post-Meeting Edition) 2004; 22(July 15 Suppl):Abstract 3120. 115. Milton DT, Kris MG, Azzoli CG, et al. Phase I/II trial of Gefitinib and RAD001 (Everolimus) in patients (pts) with advanced non-small cell lung cancer (NSCLC). Proc Am Soc Clin Oncol 2005; 24:Abstract 7104. 116. Ansell SM, Geyer SM, Kurtin PJ, et al. Anti-tumor activity of mTOR inhibitor temsirolimus for relapsed mantle cell lymphoma: a phase II trial in the North Central Cancer Treatment Group. Proc Am Soc Clin Oncol 2006; 24(18S):7532.

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Farnesyl Transferase Inhibitors in Cancer: Promise, but Limited Proof Laura Fanucchi Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia, U.S.A.

Michael P. Fanucchi Department of Medical Oncology, St. Vincent's Comprehensive Cancer Center, New York Medical College, New York, New York, U.S.A.

Fadlo R. Khuri Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia, U.S.A.

INTRODUCTION The transition from elegant regulation of cell growth, proliferation, and apoptosis to malignant transformation hinges on multiple complicated signaling pathways and cascades. There are several key proto-oncogenes that, when upregulated or mutated, become oncogenes and, through their gene products, greatly increase the malignant potential of their parent cell and progeny. There are several classes of oncogene protein products, including receptor tyrosine kinases like epidermal growth factor receptor (EGFR), cytoplasmic tyrosine kinases like Abl, cytoplasmic serine/threonine kinases like Raf kinase, transcription factors like Myc, and regulatory GTPases such as Ras. Advances in chemotherapeutic agents are increasingly aimed at these specific signaling pathways implicated in both carginogenesis and maintenance of malignant growth. This chapter focuses on Ras as a chemotherapeutic target. The biology of Ras and the role of Ras in human tumors is discussed, followed by the rationale for targeting Ras in chemotherapy. Finally, this chapter presents a summary of the preclinical and clinical development of farnesyltransferase inhibitors, one of the classes of Ras-targeting chemotherapeutics currently in active development and testing. BIOLOGY OF Ras The Ras proto-oncogene family encodes several membrane-bound monomeric G-proteins, which are approximately 21 kDa and have 85% sequence homology (1,2). The Ras gene is an important molecular switch in a complicated series of signaling pathways largely devoted to cellular differentiation and proliferation. Mutations in Ras have been identified in 30% of all human cancers (1). These mutations, particularly in codons 12, 13, and 61, lead to constitutively activated proteins in the Ras family (H, K, M, N, and R) (2). The most well-characterized mechanism of Ras activation occurs via a receptor tyrosine kinase, such as EGFR, 85

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which ultimately leads to conformational change in Ras and exchange of GTP for GDP. Ras has low GTPase activity, and GTPase-activating proteins (GAPs) are therefore required to stimulate hydrolysis of GTP. Mutated Ras proteins are constitutively activated via resistance to GAP-mediated hydrolysis (2). There are numerous Ras downstream effectors implicated in oncogenesis. The Raf serine/threonine kinase family initiates a mitogen-activated protein (MAP) kinase phosphorylation cascade, which leads to the activation of transcription factors for genes involved in cell growth and proliferation (1–3). MEKK is another serine/threonine kinase involved in cell survival and apoptosis (2)—it may also be involved in cell regulation (4). Phosphoinositide 30 -kinase (PI3-K) is an important oncogenic Ras effector through several downstream targets, of which the most well characterized are AKT and Rac/Rho. AKT is involved in apoptosis regulation (2,5) and the progression of cells from G1 to S via upregulation of cyclin D1 levels (1). Rac and Rho are integral to the regulation of the active cytoskeleton (1,6). These effectors are summarized in Figure 1. In addition to the above roles in cellular proliferation, cytoskeletal regulation, and apoptosis, activated Ras has been shown to be pivotal in tumor-induced angiogenesis (7). High levels of VEGF, a powerful growth factor that stimulates angiogenesis, have been found in tumors with Ras mutations. In particular, there is a correlation between expression of oncogenic K-Ras2 and high VEGF levels in pancreatic carcinoma and non–small cell lung carcinoma (7). Further adding to the role of activated Ras in angiogenesis is the observation that Ras acts to downregulate negative regulators of angiogenesis such as thrombospondin (7), which has direct effects on endothelial cell migration and survival (7,8).

Ras Activates Downstream Effector Pathways, but Do We Have Effective Inhibitors? Ras-GDP (inactive) Growth factors

Ras mutation Ras-GTP (active)

GAP (accelerator)

Ral-GDS PI-3 kinase

Raf-1

MEKK

Rac/Rho

AKT

MEK/MAPK

Cytoskeletal morphologic changes

Survival

Cell growth and proliferation

FIGURE 1 Ras signaling pathways modulate cytoskeletal morphology, cell survival, and cell death, as well as cell growth and proliferation in cancer cells. Source: From Ref. 3.

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Extensive post-translational modification is necessary for the hydrophilic Ras proteins to associate with the cellular membrane. Each of the Ras proteins has a CAAX motif on the carboxyl end (where C ¼ cysteine, A ¼ any aliphatic residue, and X ¼ any other residue), which is prenylated by either farnesyltransferase or geranylgeranyltransferase I or II (9). Prenylation forms a covalent thioether bond between the prenyl group and the thiol group of cysteine in the CAAX motif (10). In general, the final “X” determines which enzyme catalyzes the prenylation reaction, with farnesyltransferase preferring CAAX motifs that have X ¼ Met, Ser, Ala, or Gln, and geranylgeranyltransferase preferring Leu. Due to differences in enzyme kinetics, however, most Ras proteins will be farnesylated, except in the presence of farnesyltransferase inhibitors, in which case K-Ras and N-Ras will be geranylgeranylated (10). Then, the terminal AAX motif is cleaved via Rce1 or Afc1 proteases. Carboxymethylation follows, and lastly palmitoylation (with the exception of K-RasB) (2). The post-translational modification of Ras has been a focus of recent drug development, a topic that will be further explored in a later section. Ras in Human Tumors As stated earlier, high frequencies of Ras mutations have been identified in many human tumors, particularly lung, pancreatic, and colorectal malignancies. In light of this finding, there have been attempts to elucidate whether the presence of Ras mutations correlates with disease stage and prognosis. A meta-analysis of 43 publications published between 1990 and 2003 looking at the prognostic significance of K-Ras in lung cancers found that 20.9% of studies identified Ras mutations or P21 (the Ras protein) overexpression as negative prognostic factors for survival (11). Seventy-two percent of the studies found no association, and 2.3% found Ras to be associated with improved prognosis. For the 28 aggregable studies, Ras mutation or P21 expression was associated with decreased survival (overall hazard ratio 1.35; 95% confidence intervals 1.16–1.56) (11). It is important to note that though the overall analysis was statistically significant, in subgroup analysis Ras mutations had significant effect on survival only in adenocarcinoma (ADC), not squamous cell carcinoma (SCC). This may reflect the increased frequency of Ras mutations in ADC compared to SCC (23.12 vs. 7.09%) (11), or selection bias as ADC was assessed more frequently in the reviewed papers. Thirty-five percent of sporadic colorectal carcinomas contain K-Ras mutations. To further evaluate this relationship, the RASCAL I and II studies used data on 3439 patients with colorectal cancer from institutions around the world (12,13). Interestingly, these studies did not find an association between K-Ras mutations and tumor stage, but they did find an increased risk of relapse and death (12,13). In addition, the authors found that a specific point mutation in K-Ras, glycine to valine on codon 12, had a statistically significant effect on failure-free survival ( p ¼ 0.004, HR 1.3) and overall survival (p ¼ 0.008, HR 1.29) (12). K-Ras activation has been demonstrated in 75% to 90% of pancreatic cancers (1,14). Studies have shown a correlation between increasing incidence of K-Ras mutations with increasing grade of dysplasia in pancreatic intraductal neoplasias (14,15). To illustrate, one study using immunohistochemical staining found K-Ras expression in 0% of normal pancreatic duct tissue, 33% of hyperplastic ducts, 67% of dysplastic lesions, and 80% of the ADCs (14). In hematologic malignancies, the frequency of Ras mutations ranges from about 32% to 65% in chronic myelomonocytic leukemia (CMML), 25% to 44% in

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AML, and 6% to 18% in acute lymphoblastic leukemia (ALL) (16). The prognostic significance of Ras mutations in hematologic malignancies, however, is not clear, and may be related to Ras activation through unrelated mutations (16). Ras as a Therapeutic Target The rationale for Ras as target of cancer therapy is based largely on the functioning of the products of the Ras proto-oncogene. As described above, Ras proteins are involved in cell proliferation, regulation of the cell cycle and apoptosis, regulation of the active cytoskeleton, and control of angiogenesis—processes that are integral to oncogenesis. There is also support for the role of the Ras oncogene in tumorigenesis. Early research identified human Ras as homologous to the transforming Harvey and Kristen sarcoma viruses in bladder and lung carcinoma cell lines (17). Studies in mice have shown that oncogenic Ras induces oral tumor formation (18) and myeloproliferative states (19,20). Finally, there is evidence that inhibiting Ras activity in malignant cells decreases the survival and proliferation of these cells (2). For example, a dominant negative mutation of H-Ras, N116Y, inhibited growth of human esophageal (21) and pancreatic (22,23) tumor cells in mouse models. Therefore, considering that Ras mutations are so common in human cancers, and that the mutations are important in the development and persistence of malignant transformation, it is reasonable to target Ras in cancer therapy. FARNESYL TRANSFERASE INHIBITORS There are several compounds in preclinical and clinical trials that target various stages of the Ras signaling cascade. The most promising approaches have been: (a) inhibition of Ras expression via antisense oligodeoxynucleotides, (b) interfering with post-translational modification of Ras via farnesyltransferase inhibitors and geranylgeranyltransferase inhibitors, and (c) inhibition of Ras downstream effectors via MEK inhibitors, PI3-K inhibitors, and others (24). In the following sections, this chapter will focus on the rationale, biology, and preclinical and clinical development of farnesyltransferase inhibitors (FTIs). Currently, three FTIs are in clinical development: tipifarnib (R115777, ZarnestraTM; Johnson and Johnson Pharmaceutical Research and Development, LLC, Raritan, New Jersey, U.S.A.), lonafarnib (SCH66336, SarasarTM; Schering-Plough Research Institute, Lafayette, New Jersey, U.S.A.), and BMS214662 (Bristol-Myers Squibb, New York, New York, U.S.A.) (Table 1). Their development, and potential roles in solid and hematologic malignancies, are discussed later in this Chapter. Farnesylation is required for malignant transformation, invasion, and metastasis (9,10,25). Furthermore, though Ras undergoes extensive post-translational modification, farnesylation is the only obligatory step for Ras association with the plasma membrane (which is essential for Ras functioning) (26). Early research showed that oncogenic Ras cannot cause malignant transformation when farnesylation is inhibited by either mutation of the CAAX motif, or through inhibiting synthesis of farnesyl pyrophosphate (27). A peptidomimetic FTI revealed inhibition of v-Ras transformed cell lines, and no inhibition on v-Raf or v-mos transformed cells, thus demonstrating selective inhibition of Ras-dependent malignant transformation (26). Furthermore, this tetrapeptide and others were capable of inhibiting Ras-dependent transformation, but did not interfere with the growth of normal

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Farnesyl Transferase Inhibitors in Cancer TABLE 1 Chemical Structure and Pharmacology of the Three Farnesyl Transferase Inhibitors (FTIs) Currently in Clinical Testing FTI

Pharmacology

Tipifarnib (B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3R115777 -chlorophenyl)-1-methyl-2(1H)-quinolinone TM Nonpeptidomimetic; orally active; competitively inhibits farnesylation of lamin B and Zarnestra K-RasB peptide substrates with IC50 values of 0.86 and 7.9 nM, respectively Lonafarnib [(þ)-4-[2-[4-(8-Chloro-3,10-dibromo-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]SCH66336 pyridin-11(R)-yl)-1-piperidinyl]-2-oxo-ethyl]-1-piperidinecarboxamide SarasarTM Nonpeptidomimetic; tricyclic; orally active; inhibits farnesylation of H-Ras and K-Ras-4B in vitro with IC50 values of 1.9 and 5.2 nM, respectively BMS214662 [(R)-7-cyano-2,3,4,5-tetrahydro-1-(1H-imidazol-4-ylmethyl)-3-(phenylmethyl)-4-(2thienylsulfonyl)-1H-1,4-benzodi-azepine] Imidazole-containing tetrahydrobenzodiazepine; non-thiol; nonpeptide; CAAX-Comptetitive FTI

animal cell lines at effective concentrations (27). A pivotal study on peptidomimetic FTIs in low micromolar concentrations showed greater than 70% growth inhibition in 42 human tumor cell lines (28). Further research in transgenic mice with single (MMTV-v-Ha-Ras) (29) and multiple (30) oncogenes, which developed salivary and mammary gland carcinomas, revealed significant regression of existing tumors in response to FTIs, with minimal toxicity to normal tissues at effective dosages (29,30). Since these initial studies, there are currently several FTIs in preclinical and clinical trials, which are discussed in the following sections.

Mechanisms of Farnesyl Transferase Inhibitors The mechanisms behind the anticancer effects of FTIs are not completely understood. Several lines of evidence suggest that, though they were developed to target Ras oncoproteins, their efficacy as cancer therapeutics may not entirely be due to inhibition of Ras, but rather due to effects on downstream effectors of Ras or on other signaling cascades altogether. For example, in the presence of FTIs, K-Ras and N-Ras are geranylgeranylated and are able to associate with the plasma membrane (2). Also, there are many other proteins that are farnesylated, such as Rho-B, Rho-E, Rheb, and centromere proteins (CENP-E and CENP-F), which suggests that the action of FTIs extend to other signaling pathways besides Ras (2,31). The centromere proteins, CENP-E and CENP-F, are substrates for farnesyltransferase, but not geranylgeranyltransferase I. They are preferentially expressed during mitosis, and may be mediators of the G2 to M transition (32). The FTI SCH 66336 prevented the farnesylation of both centromere proteins in the human tumor cell line DLD-1, and altered the interaction between CENP-E and the microtubules in the lung carcinoma cell line A549 (32). These results, and others, (33) suggest that the action of FTIs may be attributed to cell-cycle effects, such as the accumulation of cells at the G2 to M transition. The antitumor activity of FTIs may be due in part to inhibition of P13K/Akt cell survival pathways, which are downstream effectors of Ras. In addition to their antiapoptotic activity, these proteins appear to be involved in several aspects of Ras-dependent oncogenesis (e.g., via cyclin D1 upregulation leading to cell-cycle progression) (34). Specifically, the FTI lonafarnib interferes with Akt activity in head and neck cells, (35) and with Akt expression in non–small cell lung cancer cells (31).

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Another protein implicated in the anticancer activity of FTIs is RhoB, one of the Rho proteins that is generally involved in regulation of the active cytoskeleton (1,31). RhoB is a target of farnesyltransferase or geranylgeranyltransferase I. When farnesylation is blocked by FTIs, the increase in geranylgeranylated RhoB appears to lead to apoptosis (31). It has also been demonstrated that SCH66336 induces phosphorylation and, therefore, inactivation of eukaryotic translation elongation factor-2, which leads to inhibition of protein synthesis (36). In addition, SCH66337 also inhibits the activation of NF-kB in the presence of tumor necrosis factor, phorbol 12-myristate 13-acetate, cigarette smoke, okadaic acid, and hydrogen peroxide, which are inflammatory and carcinogenic agents that normally activate NF-kB (37). FTIs may also have activity against other prenyl transferases, such as geranylgeranyl transferase I and II (GGT I and GGT II), which have similar active sites (31). Finally, via an unknown mechanism, FTIs appear to be involved in triggering the production of reactive oxygen species, leading to DNA damage (31). Further research into the various mechanisms underlying the anticancer activity of FTIs is warranted to improve their evidence-based use in the clinical setting. Preclinical Data in Solid Tumors Tipifarnib (R115777) R115777 (tipifarnib) is a selective nonpeptidomimetic FTI. It is a competitive inhibitor of the CAAX peptide binding site of farnesyltransferase with a Ki of 0.5 nM, and inhibits the farnesylation of lamin B1 and K-Ras (38). In vitro studies on intact cells showed that cell lines with N-Ras or H-Ras mutations were more sensitive to R115777 than those with K-Ras mutations (39). In pancreatic, colon, and melanoma in vivo mouse xenograft models, R115777 had significant antitumor effects with oral twicedaily dosing (39). Specifically, R115777 demonstrated an antiproliferative effect via apoptosis of host endothelial cells of the pancreatic tumor vasculature, an antiangiogenic effect in colon tumors, and a pro-apoptotic effect in melanoma tumors (39). A study on the in vitro and in vivo activity of R115777 in the MCF-7 estrogen receptor-positive, wild-type Ras, wild-type p53 model of breast cancer revealed significant growth inhibition both in cells and in nude mice (38). Furthermore, these experiments found that, when compared with xenograft data using cisplatin, R115777 appeared to have more of a cytostatic rather than a cytotoxic effect, suggesting that the drug may have greater utility in chemotherapeutic combinations (38). Several preclinical studies have been done investigating the role of R115777 as a cancer therapeutic agent in combination with other drugs. A study on the combination of aminobisphosphonates and R115777 in epidermoid cancer cells showed a strong synergistic effect on growth inhibition and apoptosis in KB and H1355 cells at concentrations feasible in vivo (42). Synergism between R115777 and docetaxel leading to growth inhibition has also been demonstrated in human epidermoid KB, colon HT-29, and breast HCC1937 cancer cell lines (43). Lonafarnib (SCH66336) SCH66336 (lonafarnib) is an orally bioavailable, nonpeptide, competitive, tricyclic farnesyltransferase inhibitor (44). In an HaRas transgenic mouse model, SCH66336 prophylaxis led to delayed tumor onset and reduced tumor burden, and treatment with SCH66336 caused significant tumor regression (44). In a soft agar cloning assay of 70 primary human tumor specimens, SCH66336 showed a concentrationdependent growth inhibition response in 50% (three of six) of breast tumors, 40%


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(6 of 15) of ovarian tumors, and 38% (5 of 13) of non–small cell lung tumor colony forming units (45). SCH66336 showed further preclinical activity in studies that combined it with other chemotherapeutic agents. In an in vitro study of SCH6636 and taxanes against NCI-H460 human lung tumor xenografts in nude mice and against mammary tumors in wap-Ras transgenic mice, the combination increased tumor growth inhibition in the lung cancer model and sensitized the mammary tumors to paclitaxel (46). SCH6636 was also found to act synergistically with cisplatin at clinically reasonable concentrations in A549 non-small cell lung cancer cells and T98G human glioblastoma cells, but not in MCF-7 breast, HCT116 colon, or BxPC-3 pancreatic ADC cells (47). BMS-214662 BMS-214662 is a tetrahydrobenzodiazepine, non-thiol, nonpeptide, highly selective FTI, with a mean IC50 and IC90 of 1.3 and 1.8 nM, respectively, and is more than 1000 times more selective for farnesyltransferase over geranylgeranyltransferase I (48). In preclinical testing, BMS-214662 was demonstrated to inhibit intracellular Ras processing in H-Ras-transformed Rat1 CVLS cells, and in HCT-116 human colon tumor cells (48). Furthermore, in contrast to other classes of FTIs, BMS214662 was shown to have potent apoptotic activity in vitro and in vivo (48,49). In vivo experiments in mice with HCT-116 human colon tumors treated with oral BMS-214662 resulted in the cure of eight out of eight mice at 600 mg/kg/administration, and intravenous administration at 400 mg/kg/injection yielded six out of seven mice cured (48). In mice harboring human lung carcinoma, Calu-1, oral administration of 800 mg/kg/administration cured four of eight mice; and in mice with EJ-1 bladder carcinoma, 600 mg/kg/administration of BMS-214662 cured eight of eight (48). Preclinical Data in Hematologic Malignancies Tipifarnib (R115777) In an in vitro study of the effects of R115777 on normal and leukemic hematopoisis, KG1a, U937, HL60, THP-1, K562, and UT-7 cell lines were used, along with blood or marrow aspirates from patients with acute myelogenous leukemia (AML) with high blast percentages (Table 2) (50). R115777 at concentrations of 10–50 nM inhibited growth of cell lines, AML blasts, and normal cells, but it did not affect AML blast cell adhesion or transmigration, nor did it induce significant apoptosis (50). An in vitro sensitivity study of samples from 52 pediatric cases of AML and 36 pediatric cases of ALL, found that AML cells were significantly more sensitive to tipifarnib in comparison to B-cell precursor ALL (although T-cell ALL was very sensitive), and that, within AML, the most sensitive subtype was French-American-British M5 (51). In addition, R115777 demonstrated in vitro activity at clinically achievable concentrations in myeloid progenitor cells from patients with myelofibrosis with myeloid metaplasia (52). Furthermore, R115777 induced significant dosedependent growth inhibition and apoptosis in multiple myeloma cell lines (53,54) and in fresh and cloned myeloma cells (55). The in vitro activity of R115777 in multiple myeloma cells appears to be independent of Ras mutation status (55,56). R115777 was found to act synergistically with paclitaxel and docetaxel, but not with doxorubicin, 5-fluorouracil, or cisplatin (and others), to inhibit multiple myeloma cell proliferation and to induce apoptosis—including cells resistant to

Phase I Adjei (75) Phase II Sharma (78) Phase I Ryan (82)

Advanced cancers Colorectal Advanced cancers

NSCLC

Colorectal

Phase II Adjei (69)

Phase III Rao (73)

Cancer types

Advanced cancers

Phase author

Phase I Crul (65)

Doses

Toxicity

Outcomes

6/43 SD; MS 7.7 months No difference in progression-free survival 1/7 PR No objective responses No objective responses

Acceptable profile Gastrointestinal fatigue Diarrhea Neutropenia

350 mg bid · 7 days for 3 wk 200 mg continuous 200 mg/m2 over 1 hr for 21 days

3 SD; 1 PR

300 mg bid · 21 days for 4 wk

Myelo-suppression, neuropathy

300 mg bid · 21 days for 4 wk

300 mg bid continuous

Abbreviations: CR, complete response; FTI, farnesyl transferase inhibitor; MS, median survival; NSCLC, non–small cell lung cancer; PR, partial response; SD, stable disease.

BMS-214662

Lonafarnib

Tipfarnib

FTI

TABLE 2 Summary of Clinical Trials of Farnesyl Transferase Inhibitors as Single Agents in Solid Tumors

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taxanes and R115777 (57). Finally, tipifarnib was found to act synergistically with bortezomib in multiple myeloma and AML cell lines, and the combination appeared to overcome cell adhesion-mediated drug resistance (58). Lonafarnib (SCH66336) In an in vivo mouse model of Bcr/Abl ALL, a Bcr/Abl leukemia that does not respond well to imatinib, SCH66336 reversed early signs of leukemia and significantly prolonged survival (59,60). Based on the rationale that multiple myeloma is IL-6 dependent, and that IL-6 is secreted via the Ras/Raf/MAPK pathway, a trial of lonafarnib in combination with the proteasome inhibitor bortezomib was undertaken in multiple myeloma cell lines (61). The two agents were found to act synergistically to induce apoptosis more rapidly and to downregulate phosphorylated AKT (61). BMS-214662 Most of the preclinical data on BMS-214662 are in nonhematologic malignancies (48); however, the drug also induced apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells from 18 patients with B-CLL (62). Clinical Data in Solid Tumors Tipifarnib (R115777) Phase I studies of orally-administered R115777 in advanced cancers where no standard therapy was available recommended 500 mg orally bid for 5 consecutive days followed by 5 days of rest, (63) 300 mg orally bid for 28 days followed by 1–2 weeks of rest, (64) and 300 mg orally bid in continuous dosing (65). Doselimiting toxicities included grade 3 neuropathy and grade 2 fatigue at 1300 mg bid, (63) grade 4 neutropenia in one of six patients at 300 mg bid, (64) grade 4 myelosuppression in two of four patients at 400 mg bid, and grade 3 neuropathy in one of five patients at 500 mg bid (65). In the continuous dosing trial, three patients with pancreatic, colon, and cervical carcinomas had stable disease, and one with non–small cell lung cancer refractory to platinum therapy had a partial response lasting five months (65). Phase II trials of R115777 as monotherapy were disappointing, revealing minimal clinical activity in metastatic colorectal carcinoma (66), advanced transitional cell carcinoma (67), pancreatic cancer (68), stage IIIB and stage IV non–small cell lung cancer (69), and sensitive relapse small-cell lung cancer (70). In a study of 76 patients with advanced breast cancer comparing continuous and intermittent dosing, however, 10% and 15% of the continuous dosing group and 14% and 9% of the intermittent dosing group had partial responses and stable disease, respectively (71). The toxicity profile was significantly less in the intermittent dosing group (300 mg bid for 21 days followed by 7 rest days) (71). Several phase I trials of R115777 in combination with other agents have also been undertaken. A study of R115777 at 300 mg bid with gemcitabine at 1000 mg/ m2 and cisplatin at 75 mg/m2 in 27 patients with advanced solid tumors, revealed inhibition of protein farnesylation (as demonstrated by accumulation of prelamin A in buccal mucosa cells of patients), one complete response, and eight partial responses (71). The combination of R115777 at 300 mg bid for 14 days every 3 weeks and irinotecan at 350 mg/m2 demonstrated a clinically relevant response in one trial (72).

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A multicenter, double-blind, placebo-controlled phase III trial of R115777 in 368 patients with refractory advanced colorectal cancer did not reveal a statistically significant difference in progression-free survival between the R115777 and placebo groups, although the experimental drug was found to have an acceptable toxicity profile (73). Nor, in a phase III trial, was tipifarnib in combination with gemcitabine found to prolong overall survival in advanced pancreatic cancer compared to gemcitabine alone (74). Lonafarnib (SCH66336) A phase I trial of SCH66336 in 20 patients with metastatic or locally advanced solid tumors for which there was no established curative or life-prolonging therapy, recommended a dosing schedule of 350 mg bid for 7 days out of every 3 weeks (75). Dose-limiting toxicities were gastrointestinal (nausea, vomiting, diarrhea) and fatigue at 400 mg bid. The study also had one partial response in a patient with metastatic non-small cell lung cancer (75). Another study of SCH66336 in 20 patients with solid tumors found similar dose-limiting toxicities with continuous dosing, with the addition of grade 4 neutropenia, grade 3 neurocortical toxicity, and grade 3 fatigue at 300 mg bid. These authors recommended 200 mg bid as maximally tolerated dose for continuous dosing (76). In a phase I trial of lonafarnib in combination with paclitaxel in 24 patients with solid tumors, the maximally tolerated dose was determined to be lonafarnib 100 mg bid and paclitaxel 175 mg/ m2, with dose-limiting toxicities of grade 3 hyperbilirubinemia (lonafarnib 100 mg bid and paclitaxel 175 mg/m2), grade 4 diarrhea and grade 3 peripheral neuropathy (lonafarnib 125 mg bid and paclitaxel 175 mg/m2); and grade 4 neutropenia with fever and grade 4 diarrhea (lonafarnib 150 mg bid and paclitaxel 175 mg/m2) (77). The authors also reported that 6 of 15 previously treated patients had a durable partial response, including two previously treated with taxanes (77). A phase II study of SCH66336 given continuously at 200 mg to 21 patients with metastatic colorectal cancer refractory to 5-fluorouracil and irinotecan, revealed no objective responses, and grade 3 diarrhea in 42% of patients (78). A trial in 19 patients with advanced unresectable or metastatic transitional cell carcinoma at 200 mg lonafarnib given continuously, also revealed no responses, with 9 of the patients having symptomatic progression while on study (79). Phase II trials of lonafarnib in combination with other chemotherapeutic agents have been more promising. In one study of lonafarnib in combination with paclitaxel in 33 patients with taxane-refractory/resistant non–small cell lung cancer, 10% of the 29 evaluable patients had partial responses and 38% had stable disease (80). The treatment regimen of continuous lonafarnib 100 mg orally bid with paclitaxel 175 mg/m2 intravenously over three hours on Day 8 of each 21-day cycle was well tolerated with minimal toxicity (80). In another phase II study, the combination of SCH66336 and gemcitabine was evaluated as a potential second-line treatment in 33 patients with advanced urothelial tract cancer (81). Patients received SCH66336, 150 mg in the morning and 100 mg in the evening, and gemcitabine, 1000 mg/m2 on day 1, 8, and 15 per 28-day cycle, with minimal toxicity, and the authors reported an overall response rate of 32.3% (95% confidence intervals 17–51%), corresponding to 9 partial responses and one complete response (81). BMS-214662 A phase I trial of BMS-214662 in escalating doses from 36 to 225 mg/m2, as single one-hour infusions every 21 days, was completed in 54 patients with advanced


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solid tumors (predominantly pancreatic, lung, and colorectal cancers), and established 200 mg/m2 as the recommended dose for phase II studies (82). The most frequent adverse effects were noted as reversible grade 3 transaminitis, nausea, and vomiting (82). A phase I study of BMS-214662 as weekly one-hour infusions in 27 patients with solid tumors reported grade 3 and 4 neutropenia as the most common dose-limiting toxicity at doses ranging from 28 to 220 mg/m2 for four weeks (83). There were no objective clinical responses documented in either of these phase I trials. Another phase I trial compared weekly one-hour infusions with weekly 24-hr infusions and found that the 24-hr infusion resulted in decreased maximum farnesyltransferase inhibition in peripheral blood mononuclear cells, but increased duration of enzyme inhibition, and no objective therapeutic benefit over the one-hour schedule (84). BMS-214662 was also evaluated in the phase I setting in combination with paclitaxel and carboplatin (85). Thirty patients with solid tumors received BMS214662 on a one-hour weekly infusion schedule following paclitaxel and carboplatin on the first day of a 21-day cycle, with dose-limiting toxicities of neutropenia, thrombocytopenia, nausea, and vomiting, and a MTD of BMS-214662 at 160 mg/ m2, paclitaxel 225 mg/m2 and carboplatin (area under the curve ¼ 6 on day 1), every 21 days (85). The objective responses were one partial response in a patient with taxane-resistant esophageal cancer, and stable disease over more than four cycles in eight other patients (85). Clinical Data in Hematologic Malignancies Tipifarnib (R115777) A phase I trial of orally-administered R115777, at doses from 100 mg bid to 1200 mg bid for up to 21 days, in 35 adults with refractory and relapsed acute leukemia (AML, ALL, CML in blast crisis, or AML in poor prognostic subgroups) revealed dose-limiting toxicity of central neurotoxicity at 1200 mg bid, with additional toxicities of reversible nausea, renal insufficiency, polydipsia, and myelosuppression (Table 3) (86). In this trial, R115777 was demonstrated to accumulate in bone marrow in a dose-dependent fashion. Clinical responses occurred in 29% of patients, including two complete remissions, in a non-dose-dependent fashion at doses from 100 to 900 mg bid (86). A phase I trial of R115777 in escalating doses with a starting dose of 300 mg bid, in 21 patients with myelodysplastic syndrome, revealed a MTD of 400 mg bid (dose-limiting toxicities were grade 4 hematologic toxicity) and 6 objective responses (87). A trial of R115777 at 600 mg bid for four weeks every six weeks, in adults with Philadelphia chromosome-positive CML, myelofibrosis, or multiple myeloma, revealed that out of 22 patients with CML, 7 achieved a complete or partial response (the majority of these were in the chronic phase); of the 8 patients with myelofibrosis and of the 10 patients with multiple myeloma, there were no objective responses (88). The combination of tipifarnib with idarubicin and cytarabine was tested in 33 patients with newly diagnosed AML or high-risk myelodysplastic syndrome and resulted in 67% complete remission and 6% partial response (89). The treatment protocol was idarubicin 12 mg/ m2/d on days 1–3, cytarabine 1.5 g/m2 IV over 24 hr daily on days 1–4 and tipifarnib 300 mg BID for 21 days. Patients achieving CR received consolidation (5 courses) with idarubicin 8 mg/m2/d on days 1–2, cytarabine 0.75 g/m2/d on days 1–3, and tipifarnib 300 mg BID for 14 days every 4–6 weeks. Maintenance was with tipifarnib 300 mg BID for 21 days every 4–6 weeks for 6 months (89).

Diarrhea, nausea

200 mg bid 56–156 mg/m2 over 1 hr for 1 wk; 300 mg/m2 over 24 hr

AML, ALL, high risk MDS

Phþ CML

600 mg bid · 4 wk for 6 wk

MDS

Phase II Kurzrock (90)

Phase I Borthakur (91) Phase I Cortes (92)

Myelosuppression, neurotoxicity, rash (41% required dose reduction) Diarrhea, nausea

600 mg bid · 4 wk for 6 wk

Toxicity Central neurotoxicity, myelosuppression

Phþ CML, myelofibrosis, multiple myeloma

Doses 100–1200 mg bid

Phase I Cortes (88)

Cancer types

AML, ALL, CML in blast crisis

Phase author

Phase I Karp (86)

Outcomes

2/30 CRi; 1/30 CR for 14 weeks

2/13 clinical responses

8/11 PR at 900 mg; 2/8 PR, 1/8 CR at 600 mg; 4/5 PR at 300 mg 7/22 CR or PR for CML; no responses for other cancers 2/28 CR; 1/28 PR

Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CML, chronic myeloid leukemia; CR, complete response; CRi, complete response with incomplete platelet recovery; FTI, farnesyl transferase inhibitor; MDS, myelodysplastic syndrome; MM, multiple myeloma; MS, median survival; Phþ, Philadelphia chromosome positive; PR, partial response; SD, stable disease.

BMS-214662

Lonafarnib

Tipfarnib

FTI

TABLE 3 Summary of Clinical Trials of Farnesyl Transferase Inhibitors as Single Agents in Hematologic Malignancies

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A phase II trial of R115777 in 28 patients with myelodysplastic syndrome treated at 600 mg bid in cycles of four weeks on, two weeks off, resulted in three responses (two had refractory anemia with excess blasts and one had refractory anemia with excess blasts in transformation) (90). The authors found that 600 mg bid was not well-tolerated (myelosuppression, neurotoxicity, rash), and 41% of the patients required dose-reduction (90). Lonafarnib (SCH66336) To date, there has only been one clinical trial of lonafarnib in hematologic malignancies. This phase I trial investigated lonafarnib, at 200 mg bid starting dose, in 13 patients with Philadelphia chromosome-positive CML in the chronic or accelerated phases, and who failed or were intolerant of imatinib therapy (91). Lonafarnib was generally well-tolerated, with diarrhea (84% of patients) and nausea being the most common adverse effects, and the authors noted two clinical responses (91). BMS-214662 A phase I trial of BMS-214662 in refractory or relapsed high-risk myelodysplastic syndromes and acute leukemias was undertaken to determine dose-limiting toxicity and MTD (92). Thirty patients in total were treated at doses of 56, 84, 118, and 156 mg/m2 in weekly one-hour infusions, and 300 mg/m2 given as a 24-hr continuous infusion. Nausea and diarrhea were the most common adverse effects, and dose-limiting toxicity ( grade 3 diarrhea and grade 2 nausea in one patient) was observed at 156 mg/m2. Seventeen percent of patients showed clinical responses, including two with complete remission with incomplete platelet recovery, one with hematologic improvement, and two with morphologic leukemia-free state (92). CONCLUSIONS AND FUTURE DIRECTIONS Despite their development as a Ras-targeting therapy, the farnesyltransferase inhibitors are now known not to inhibit Ras exclusively. Although clinical testing of the farnesyltransferase inhibitors has been largely disappointing in solid tumors, more recent data in hematologic malignancies are more promising. Tipifarnib has advanced farthest in clinical trials, and been most promising in treatment of AML. AML persists as a treatment challenge, with poor long-term survival, low remission rates, and high treatment-related mortality. An active area of research in AML focuses on identification of aberrant signaling pathways that may be targets of specific drug design. Farnesyltransferase inhibitors show potential in this area, especially given their relatively mild toxicity profile. A recent study on tipifarnib sensitivity in pediatric AML and ALL samples compared to normal bone marrow found large interindividual differences in the in vitro sensitivity of AML and ALL blasts to tipifarnib, with greater overall sensitivity in AML (93). The authors also reported a correlation between resistance to tipifarnib and resistance to cytarabine or 6-thioguanine. Additional studies are warranted to further explore potential biomarkers that are predictive of clinical response to tipifarnib in leukemias. Another area of active and future research is the use of farnesyltransferase inhibitors in combinations with other chemotherapeutic and targeted agents. Recent preclinical work on the combination of lonafarnib and bortezomib, a proteasome inhibitor, in mutiple myeloma was based on the hypotheses that the

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two agents would target different signaling pathways, and that lonafarnib would inhibit IL-6, which is a well-known multiple myeloma growth factor (61). The authors demonstrated significant and synergistic myeloma cell death at low, clinically acheiveable concentrations of the drugs. Furthermore, the order of administration of the two drugs was essential, with sequencing bortezomib before lonafarnib being most effective. The combination resulted in rapid caspase activation and downregulation of p-AKT expression, which correlated with rapid induction of cell death. It is possible that p-AKT expression could be used as a biomarker for response and dosing in further studies (61). Along with increasing understanding of the complicated and myriad signaling pathways involved in cell growth, proliferation, and apoptosis, and where these pathways may lead to carcinogenesis, will come better molecular agents to target aberrant signals.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Adjei AA. Blocking oncogenic Ras signaling for cancer therapy. J Natl Cancer Inst 2001; 93:1062–1074. Friday BB, Adjei AA. K-ras as a target for cancer therapy. Biochim Biophys Acta 2005; 1756:127–144. Campbell SL, Khosravi-Far R, Rossman KL, et al. Increasing complexity of Ras signaling. Oncogene 1998; 17 (11 Reviews):1395–1413. Karandikar M, Xu S, Cobb MH. MEKK1 binds raf-1 and the ERK2 cascade components. J Biol Chem 2000; 75:40120–40127. Song G, Ouyang G, Bao S. The activation of Akt/PKB signaling pathway and cell survival. J Cell Mol Med 2005; 9:59–71. Morrison DK, Cutler RE. The complexity of Raf-1 regulation. Curr Opin Cell Biol 1997; 9:174–9. Kranenburg O, Gebbink MF, Voest EE. Stimulation of angiogenesis by Ras proteins. Biochim Biophys Acta 2004; 1654:23–37. Lawler J. Thrombospondin-1 as an endogenous inhibitor of angiogenesis and tumor growth. J Cell Mol Med 2002; 6:1–12. Prendergast GC, Oliff A. Farnesyltransferase inhibitors: antineoplastic properties, mechanisms of action, and clinical prospects. Semin Cancer Biol 2000; 10:443–52. Sebti SM. Protein farnesylation: implications for normal physiology, malignant transformation, and cancer therapy. Cancer Cell 2005; 7:297–300. Mascaux C, Iannino N, Martin B, et al. The role of RAS oncogene in survival of patients with lung cancer: a systematic review of the literature with meta-analysis. Br J Cancer 2005; 2:131–9. Andreyev HJ, Norman AR, Cunningham D, et al. Kirsten ras mutations in patients with colorectal cancer: the “RASCAL II” study. Br J Cancer 2001; 85:692–6. Andreyev HJ, Norman AR, Cunningham D, et al. Kirsten ras mutations in patients with colorectal cancer: the multicenter “RASCAL” study. J Natl Cancer Inst 1998; 90:675–84. Jeong S, Lee DH, Lee JI, et al. Expression of Ki-67, p53, and K-ras in chronic pancreatitis and pancreatic ductal adenocarcinoma. World J Gastroenterol 2005; 11:6765–9. Lohr M, Kloppel G, Maisonneuve P, et al. Frequency of K-ras mutations in pancreatic intraductal neoplasias associated with pancreatic ductal adenocarcinoma and chronic pancreatitis: a meta-analysis. Neoplasia 2005; 7:17–23. Beaupre DM, Kurzrock R. RAS and leukemia: from basic mechanisms to gene-directed therapy. J Clin Oncol 1999; 17:1071–9. Der CJ, Krontiris G, Cooper GM. Transforming genes of human bladder and lung carcinoma cell lines are homologous to the ras genes of Harvey and Kristin sarcoma viruses. Proc Natl Acad Sci 1982; 79:3637–40.


18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

38. 39.

99

Caulin C, Nguyen T, Longley MA, et al. Inducible activation of oncogenic K-ras results in tumor formation in the oral cavity. Cancer Res 2004; 64:5054–8. Braun BS, Tuveson DA, Kong N, et al. Somatic activation of oncogenic Kras in hematopoietic cells initiates a rapidly fatal myeloproliferative disorder. Proc Natl Acad Sci USA 2004; 101:597–602. Chan IT, Kutok JL, Williams IR, et al. Conditional expression of oncogenic K-ras from its endogenous promoter induces a myeloproliferative disease. J Clin Invest 2004; 113:528–38. Senmaru N, Shichinohe T, Takeuchi M, et al. Suppression of Erk activation and in vivo growth in esophageal cancer cells by the dominant negative Ras mutant, N116Y. Int J Cancer 1998; 78:366–71. Shichinohe T, Senmaru N, Furuuchi K, et al. Suppression of pancreatic cancer by the dominant negative ras mutant, N116Y. J Surg Res 1996; 66:125–30. Takeuchi M, Shichinohe T, Senmaru N, et al. The dominant negative H-ras mutant, N116Y, suppresses growth of metastatic human pancreatic cancer cells in the liver of nude mice. Gene Ther 2000; 7:518–26. Adjei AA. Ras signaling pathway proteins as therapeutic targets. Curr Pharm Des 2001; 7:1581–94. Qian Y, Sebti SM, Hamilton AD. Farnesyltransferase as a target for anticancer drug design. Biopolymers 1997; 43:25–41. Kohl NE, Mosser SD, deSolms SJ, et al. Selective inhibition of ras-dependent transformation by a farnesyltransferase inhibitor. Science 1993; 260:1934–7. James GL, Goldstein JL, Brown MS, et al. Benzodiazepine peptidomimetics: potent inhibitors of Ras farnesylation in animal cells. Science 1993; 260:1937–42. Sepp-Lorenzino L, Ma Z, Rands E, et al. A peptidomimetic inhibitor of farnesyl: protein transferase blocks the anchorage-dependent and -independent growth of human tumor cell lines. Cancer Res 1995; 55:5302–9. Kohl NE, Omer CA, Conner MW, et al. Inhibition of farnesyltransferase induces regression of mammary and salivary carcinomas in ras transgenic mice. Nat Med 1995; 1:792–7. Barrington RE, Subler MA, Rands E, et al. A farnesyltransferase inhibitor induces tumor regression in transgenic mice harboring multiple oncogenic mutations by mediating alterations in both cell cycle control and apoptosis. Mol Cell Biol 1998; 18:85–92. Pan J, Yeung SC. Recent advances in understanding the antineoplastic mechanisms of farnesyltransferase inhibitors. Cancer Res 2005; 65:9109–12. Ashar HR, James L, Gray K, et al. Farnesyl transferase inhibitors block the farnesylation of CENP-E and CENP-F and alter the association of CENP-E with the microtubules. J Biol Chem 2000; 275:30451–7. Ashar HR, James L, Gray K, et al. The farnesyl transferase inhibitor SCH 66336 induces a G(2) –> M or G(1) pause in sensitive human tumor cell lines. Exp Cell Res 2001; 262:17–27. Adjei AA. An overview of farnesyltransferase inhibitors and their role in lung cancer therapy. Lung Cancer 2003; 41(Suppl 1):S55–62. Chun K-H, Lee H-Y, Hassan K, et al. Implication of Protein Kinase B/Akt and Bcl-2/ Bcl-XL suppression by the Farnesyl Transferase Inhibitor SCH66336 in apoptosis induction in squamous carcinoma cells. Cancer Res 2003; 63:4796–800. Ren H, Tai S-K, Khuri F, et al. Farnesyltransferase Inhibitor SCH66336 induces rapid phosphorylation of eukaryotic translation elongation factor 2 in head and neck squamous cell carcinoma cells. Cancer Res 2005; 65:5841–7. Takada Y, Khuri FR, Aggarwal BB. Protein Farnesyltransferase Inhibitor (SCH 66336) Abolishes NF-kB activation induced by various carcinogens and inflammatory stimuli leading to suppression of NF-kB-regulated Gene Expression and Up-regulation of apoptosis. J Biol Chem 2004; 279:26287–99. Kelland LR, Smith V, Valenti M, et al. Preclinical antitumor activity and pharmacodynamic studies with the farnesyl protein transferase inhibitor R115777 in human breast cancer. Clin Cancer Res 2001; 7:3544–50. End DW, Smets G, Todd AV, et al. Characterization of the antitumor effects of the selective farnesyl protein transferase inhibitor R115777 in vivo and in vitro. Cancer Res 2001; 61:131–7.

100 40. 41. 42.

43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.

Fanucchi et al.

Lubet RA, Christov K, You M, et al. Effects of the farnesyl transferase inhibitor R115777 (Zarnestra) on mammary carcinogenesis: prevention, therapy, and role of HaRas mutations. Mol Cancer Ther 2006; 5:1073–8. Yao R, Wang Y, Lu Y, et al. Efficacy of the farnesyltransferase inhibitor R115777 in a rat mammary tumor model: role of Ha-ras mutations and use of microarray analysis in identifying potential targets. Carcinogenesis 2006; 27:1420–31. Caraglia M, D'Alessandro AM, Marra M, et al. The farnesyl transferase inhibitor R115777 (Zarnestra) synergistically enhances growth inhibition and apoptosis induced on epidermoid cancer cells by Zoledronic acid (Zometa) and Pamidronate. Oncogene 2004; 23:6900–13. Caraglia M, Giuberti G, Marra M, et al. Docetaxel induces p53-dependent apoptosis and synergizes with farnesyl transferase inhibitor r115777 in human epithelial cancer cells. Front Biosci 2005; 10:2566–75. Liu M, Bryant MS, Chen J, et al. Antitumor activity of SCH 66336, an orally bioavailable tricyclic inhibitor of farnesyl protein transferase, in human tumor xenograft models and wap-ras transgenic mice. Cancer Res 1998; 58:4947–56. Petit T, Izbicka E, Lawrence RA, et al. Activity of SCH 66336, a tricyclic farnesyltransferase inhibitor, against human tumor colony-forming units. Ann Oncol 1999; 10:449–53. Shi B, Yaremko B, Hajian G, et al. The farnesyl protein transferase inhibitor SCH66336 synergizes with taxanes in vitro and enhances their antitumor activity in vivo. Cancer Chemother Pharmacol 2000; 46:387–93. Adjei AA, Davis JN, Bruzek LM, et al. Synergy of the protein farnesyltransferase inhibitor SCH66336 and cisplatin in human cancer cell lines. Clin Cancer Res 2001; 7:1438–45. Rose WC, Lee FY, Fairchild CR, et al. Preclinical antitumor activity of BMS-214662, a highly apoptotic and novel farnesyltransferase inhibitor. Cancer Res 2001; 61:7507–17. Manne V, Lee FY, Bol DK, et al. Apoptotic and cytostatic farnesyltransferase inhibitors have distinct pharmacology and efficacy profiles in tumor models. Cancer Res 2004; 64:3974–80. Liesveld JL, Lancet JE, Rosell KE, et al. Effects of the farnesyl transferase inhibitor R115777 on normal and leukemic hematopoiesis. Leukemia 2003; 17–1806–12. Goemans BF, Zwaan CM, Harlow A, et al. In vitro profiling of the sensitivity of pediatric leukemia cells to tipifarnib: identification of T-cell ALL and FAB M5 AML as the most sensitive subsets. Blood 2005; 106:3532–7. Mesa RA, Tefferi A, Gray LA, et al. In vitro antiproliferative activity of the farnesyltransferase inhibitor R115777 in hematopoietic progenitors from patients with myelofibrosis with myeloid metaplasia. Leukemia 2003; 17:849–55. Le Gouill S, Pellat-Deceunynck C, Harousseau JL, et al. Farnesyl transferase inhibitor R115777 induces apoptosis of human myeloma cells. Leukemia 2002; 16:1664–7. Beaupre DM, McCafferty-Grad J, Bahlis NJ, et al. Farnesyl transferase inhibitors enhance death receptor signals and induce apoptosis in multiple myeloma cells. Leuk Lymphoma 2003; 44:2123–34. Ochiai N, Uchida R, Fuchida S, et al. Effect of farnesyl transferase inhibitor R115777 on the growth of fresh and cloned myeloma cells in vitro. Blood 2003; 102:3349–53. Beaupre DM, Cepero E, Obeng EA, et al. R115777 induces Ras-independent apoptosis of myeloma cells via multiple intrinsic pathways. Mol Cancer Ther 2004; 3:179–86. Zhu K, Gerbino E, Beaupre DM, et al. Farnesyltransferase inhibitor R115777 (Zarnestra, Tipifarnib) synergizes with paclitaxel to induce apoptosis and mitotic arrest and to inhibit tumor growth of multiple myeloma cells. Blood 2005; 105:4759–66. Yanamandra N, Colaco NM, Parquet NA, et al. Tipifarnib and bortezomib are synergistic and overcome cell adhesion-mediated drug resistance in multiple myeloma and acute myeloid leukemia. Clin Cancer Res 2006; 12:591–9. Reichert A, Heisterkamp N, Daley GQ, et al. Treatment of Bcr/Abl-positive acute lymphoblastic leukemia in P190 transgenic mice with the farnesyl transferase inhibitor SCH66336. Blood 2001; 97:1399–403.


60. 61.

62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78.

101

Mishra S, Zhang B, Groffen J, et al. A farnesyltransferase inhibitor increases survival of mice with very advanced stage acute lymphoblastic leukemia/lymphoma caused by P190 Bcr/Abl. Leukemia 2004; 18:23–8. David E, Sun SY, Waller EK, et al. The combination of the farnesyl transferase inhibitor lonafarnib and the proteasome inhibitor bortezomib induces synergistic apoptosis in human myeloma cells that is associated with down-regulation of p-AKT. Blood 2005; 106:4322–9. Marzo I, Perez-Galan P, Giraldo P, et al. Farnesyltransferase inhibitor BMS-214662 induces apoptosis in B-cell chronic lymphocytic leukemia cells. Leukemia 2004; 18:1599–604. Zujewski J, Horak ID, Bol CJ, et al. Phase I and pharmacokinetic study of farnesyl protein transferase inhibitor R115777 in advanced cancer. J Clin Oncol 2000; 8:927–41. Punt CJ, van Maanen L, Bol CJ, et al. Phase I and pharmacokinetic study of the orally administered farnesyl transferase inhibitor R115777 in patients with advanced solid tumors. Anticancer Drugs 2001; 12:193–7. Crul M, de Klerk GJ, Swart M, et al. Phase I clinical and pharmacologic study of chronic oral administration of the farnesyl protein transferase inhibitor R115777 in advanced cancer. J Clin Oncol 2002; 20:2726–35. Whitehead RP, McCoy S, Macdonald JS, et al. Phase II trial of R115777 (NSC #70818) in patients with advanced colorectal cancer: A Southwest Oncology Group study. Invest New Drugs 2006; 24(4):335–41. Rosenberg JE, von der Maase H, Seigne JD, et al. A phase II trial of R115777, an oral farnesyl transferase inhibitor, in patients with advanced urothelial tract transitional cell carcinoma. Cancer 2005; 103:2035–41. Macdonald JS, McCoy S, Whitehead RP, et al. A phase II study of farnesyl transferase inhibitor R115777 in pancreatic cancer: a Southwest oncology group (SWOG 9924) study. Invest New Drugs 2005; 23:485–7. Adjei AA, Mauer A, Bruzek L, et al. Phase II study of the farnesyl transferase inhibitor R115777 in patients with advanced non-small-cell lung cancer. J Clin Oncol 2003; 21:1760–6. Heymach JV, Johnson DH, Khuri FR, et al. Phase II study of the farnesyl transferase inhibitor R115777 in patients with sensitive relapse small-cell lung cancer. Ann Oncol 2004; 15:1187–93. Johnston SR, Hickish T, Ellis P, et al. Phase II study of the efficacy and tolerability of two dosing regimens of the farnesyl transferase inhibitor, R115777, in advanced breast cancer. J Clin Oncol 2003; 21:2492–9. Adjei AA, Croghan GA, Erlichman C, et al. A Phase I trial of the farnesyl protein transferase inhibitor R115777 in combination with gemcitabine and cisplatin in patients with advanced cancer. Clin Cancer Res 2003; 9:2520–6. Rao S, Cunningham D, de Gramont A, et al. Phase III double-blind placebo-controlled study of farnesyl transferase inhibitor R115777 in patients with refractory advanced colorectal cancer. J Clin Oncol 2004; 22:3950–7. Van Cutsem E, van de Velde H, Karasek P, et al. Phase III trial of gemcitabine plus tipifarnib compared with gemcitabine plus placebo in advanced pancreatic cancer. J Clin Oncol 2004; 22:1430–8. Adjei AA, Erlichman C, Davis JN, et al. A Phase I trial of the farnesyl transferase inhibitor SCH66336: evidence for biological and clinical activity. Cancer Res 2000; 60:1871–7. Eskens FA, Awada A, Cutler DL, et al. Phase I and pharmacokinetic study of the oral farnesyl transferase inhibitor SCH 66336 given twice daily to patients with advanced solid tumors. J Clin Oncol 2001; 19:1167–75. Khuri FR, Glisson BS, Kim ES, et al. Phase I study of the farnesyltransferase inhibitor lonafarnib with paclitaxel in solid tumors. Clin Cancer Res 2004; 10:2968–76. Sharma S, Kemeny N, Kelsen DP, et al. A phase II trial of farnesyl protein transferase inhibitor SCH 66336, given by twice-daily oral administration, in patients with metastatic colorectal cancer refractory to 5-fluorouracil and irinotecan. Ann Oncol 2002; 13:1067–71.

102 79. 80. 81.

82. 83. 84. 85. 86. 87. 88. 89.

90. 91.

92. 93.

Fanucchi et al.

Winquist E, Moore MJ, Chi KN, et al. A multinomial Phase II study of lonafarnib (SCH 66336) in patients with refractory urothelial cancer. Urol Oncol 2005; 23:143–9. Kim ES, Kies MS, Fossella FV, et al. Phase II study of the farnesyltransferase inhibitor lonafarnib with paclitaxel in patients with taxane-refractory/resistant nonsmall cell lung carcinoma. Cancer 2005; 104:561–9. Theodore C, Geoffrois L, Vermorken JB, et al. Multicentre EORTC study 16997: feasibility and phase II trial of farnesyl transferase inhibitor & gemcitabine combination in salvage treatment of advanced urothelial tract cancers. Eur J Cancer 2005; 41: 1150–7. Ryan DP, Eder JP, Jr, Puchlaski T, et al. Phase I clinical trial of the farnesyltransferase inhibitor BMS-214662 given as a 1-hour intravenous infusion in patients with advanced solid tumors. Clin Cancer Res 2004; 10:2222–30. Papadimitrakopoulou V, Agelaki S, Tran HT, et al. Phase I study of the farnesyltransferase inhibitor BMS-214662 given weekly in patients with solid tumors. Clin Cancer Res 2005; 11:4151–9. Eder JP, Jr, Ryan DP, Appleman L, et al. Phase I clinical trial of the farnesyltransferase inhibitor BMS-214662 administered as a weekly 24 h continuous intravenous infusion in patients with advanced solid tumors. Cancer Chemother Pharmacol 2006; 58:107–16. Dy GK, Bruzek LM, Croghan GA, et al. A phase I trial of the novel farnesyl protein transferase inhibitor, BMS-214662, in combination with paclitaxel and carboplatin in patients with advanced cancer. Clin Cancer Res 2005; 11:1877–83. Karp JE, Lancet JE, Kaufmann SH, et al. Clinical and biologic activity of the farnesyltransferase inhibitor R115777 in adults with refractory and relapsed acute leukemias: a phase 1 clinical-laboratory correlative trial. Blood 2001; 97:3361–9. Kurzrock R, Kantarjian HM, Cortes JE, et al. Farnesyltransferase inhibitor R115777 in myelodysplastic syndrome: clinical and biologic activities in the phase 1 setting. Blood 2003; 102:4527–34. Cortes J, Albitar M, Thomas D, et al. Efficacy of the farnesyl transferase inhibitor R115777 in chronic myeloid leukemia and other hematologic malignancies. Blood 2003; 101:1692–7. Ravandi-Kashani F, Kantarjian H, Garcia-Manero G, et al. Tipifarnib in combination with idarubicin and cytarabine in patients with newly diagnosed acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS). J Clin Oncol, ASCO Proc 2006; 24:6557. Kurzrock R, Albitar M, Cortes JE, et al. Phase II study of R115777, a farnesyl transferase inhibitor, in myelodysplastic syndrome. J Clin Oncol 2004; 22:1287–92. Borthakur G, Kantarjian H, Daley G, et al. Pilot study of lonafarnib, a farnesyl transferase inhibitor, in patients with chronic myeloid leukemia in the chronic or accelerated phase that is resistant or refractory to imatinib therapy. Cancer 2006; 106:346–52. Cortes J, Faderl S, Estey E, et al. Phase I study of BMS-214662, a farnesyl transferase inhibitor in patients with acute leukemias and high-risk myelodysplastic syndromes. J Clin Oncol 2005; 23:2805–12. Gotlib J. Farnesyltransferase inhibitor therapy in acute myelogenous leukemia. Curr Hematol Rep 2005; 4:77–84.

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Protein Kinase C Inhibitors in the Treatment of Non–Small Cell Lung Cancer Yun Oh Department of Thoracic/Head and Neck, Medical Oncology, M. D. Anderson Cancer Center, University of Texas, Houston, Texas, U.S.A.

Michael Lahn and Asavari Wagle Oncology Product Development, Eli Lilly and Company, Indianapolis, Indiana, U.S.A.

Roy Herbst Department of Thoracic/Head and Neck, Medical Oncology, M. D. Anderson Cancer Center, University of Texas, Houston, Texas, U.S.A.

INTRODUCTION Since its recognition as a critical signaling pathway in cancer, a variety of approaches have been developed to block protein kinase C (PKC) signaling in cancer cells. Here, we review the different PKC inhibitors currently in clinical investigation and their presumed role in the treatment of non–small cell lung cancer (NSCLC). Based on the knowledge of staurosporine, nonselective and selective PKC inhibitors have been developed. Among the nonselective PKC inhibitors, bryostatin 1 has more extensively been evaluated for its use in the treatment of NSCLC. Recently, the selective PKC inhibitor enzastaurin completed a phase II study to evaluate its activity as a second- and third-line treatment for NSCLC. Finally, we discuss the challenges for the future development of PKC inhibitors in NSCLC. PKC is a family of isoenzymes that are products of distinct genes and are activated in response to growth factors, hormones, and neurotransmitters (1,2). PKC isozymes have been classified into three groups: Group A, or classical (PKC-a, -bI, -bII, and -g), which requires a lipid cofactor (e.g., phosphatidyl-serine, PS), Ca2þ, and 1,2-diacylglycerol (DAG) for activation; Group B, or new (PKC-d, -e, -h, -, and -m), which are Ca2þ-independent; and Group C, or atypical (PKC-l and -z). All PKC isozymes are composed of one polypeptide chain divided into an Nterminal regulatory domain and a C-terminal catalytic domain; four regions (C1–C4) are conserved across all isozymes, and five regions (V1–V5) are variable between isozymes (2). The C1 regulatory region contains the pseudosubstrate domain, which is autoinhibitory and masks the catalytic domain. Activation is initiated by removal of the pseudosubstrate so that the catalytic domain is exposed. The Group A isozymes contain an additional Ca2þ activated regulatory binding site that is absent from the other groups. The C3 (ATP binding site) and C4 (substrate binding site) catalytic regions are found in all groups; phosphorylation is an essential step in enzyme activation (2). After its synthesis, PKC undergoes a series of ordered priming and phosphorylation events in which a conserved threonine at the activation loop is trans-phosphorylated by a phosphoinositide-dependent kinase (PDK-1), followed by the phosphorylation of a turn motif and a hydrophobic 103

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FXXFS/TF/Y motif (2). This priming process via phosphorylation results in a catalytically competent, but inactive, mature PKC, maintained in an autoinhibited conformation by the pseudosubstrate in the cytosol. The matured but inactive PKCs are believed to randomly diffuse in the cytosol. Upon stimulation of agonists, the activation of cell surface receptors (for example, growth factor receptors and G-protein-coupled receptors) results in the activation of phospholipase C at the plasma membrane, followed by a second phase of activation via phospholipases at the cytoplasm (3). The hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C generates second messengers (DAG and calcium) and causes the translocation of cytosolic PKC to the plasma membrane. Pharmacologic stimulation of PKC has been shown to induce malignant transformation and proliferation, apoptosis, cell migration, and cell activation (4). The pharmacologic inhibition of PKCs is associated with decreased growth and survival of tumors, reduction of neoplastic properties, promotion of apoptosis, and sensitization of tumor cells to chemotherapeutic agents (4–13). Hence, a number of PKC inhibitors have been developed and their activity in the treatment of cancer has been pursued for the past 20 years. In the following sections, we review the experience of the various inhibitors currently explored in clinical investigation.

INHIBITORS OF PKCS A number of PKC inhibitors were identified in the mid-1980s (14–17), which were subsequently found to be nonspecific for particular PKC isoenzymes. Some of these inhibitors were evaluated in lung cancer cell lines (18) and have also been considered for the treatment of lung cancer. However, in the past decade progress in the basic understanding of PKC biology and the growing understanding of medicinal chemistry have resulted in the identification of selective PKC inhibitors, including inhibitors of novel pharmacological platform, such as antisense oligonucleotides (ASO) (19–21). When comparing the various inhibitors, it becomes apparent that they all possess multiple activities that are not limited to the kinase inhibition of the PKC isoenzymes, but also can have significant impact on cell cycle (22). Therefore, the definition of PKC inhibitors may not be precise, but for the sake of this review we will use the definition of PKC inhibitors for those molecules that were originally designed to inhibit PKC and its isoenzymes. We will first review the PKC inhibitors with a broad activity on multiple PKCs (i.e., nonspecific PKC inhibitors) and then review those PKC inhibitors with a specific PKC-inhibitory profile (i.e., specific PKC inhibitors) (Table 1).

Nonspecific PKC Inhibitors Staurosporine Staurosporine is an indole-carbazole originally isolated from the bacterium Streptomyces staurosporeus in 1977 (23). It was identified as an anticancer agent from an original screen of over 50 alkaloids in 1994 (24). Staurosporine is comprised of a sugar molecule and a heterocyclic indole-carbazole group. Staurosporine is a potent PKC inhibitor acting on the binding pocket of PKC with a broad spectrum of activity on many other protein kinases (14,25,26). Staurosporine is known to induce apoptosis through a mitochondria-mediated pathway (27), causing an oxidative stress through mitochondrially generated reactive oxygen species (ROS)

IV

Oral

Peptide

Acyclic bisindolylmaleimide

Phase III

N/A

Phase III (development stopped)

Phase I-II

Phase I-II

N/A Phase I-II

Clinical phase

DLBCL, glioblastoma NSCLC

N/A

NHL

NHL, CLL

AML

N/A Lymphoma, melanoma

Single agent activity

No MTD fatigue

N/A

MTD established thrombocytopenia, coagulopathy

MTD established myalgias

No MTD as single agent; diarrhea/ nausea in combination with chemotherapy

N/A MTD established Insulin resistance

Drug-related toxicities N/A

Pharmacodynamic markers

N/A

AKT, GSK-3b ribosomal Protein S6

None

Stimulation of various kinases

Decreased GSK3-b phosphorylation in tumor and PBMCs

N/A

None

None

None Cyclin-dependent kinase; broad serine-threonine kinases None Flt-3, PDGFR, c-KIT, tyrosine kinases, multidrug resistance, gene Pgp, ZNF-198 FGF receptor fusion protein

Various Protein Kinases

Other cancer-related target inhibition

Abbreviations: AML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia; DLBCL, diffuse large B-cell; GSK, glucogen synthase kinase; IV, intravenous; MTD, maximum tolerated dose; NHL, non-Hodgkin's lymphoma; NSCLC, non–small cell lung cancer; PBMC, peripheral blood mononuclear cell; Pgp, P-glycoprotein; PKC, protein kinase C.

IV continuous infusion

IV

Oral

N/A IV

Administration

Phosphorothioate antisense oligonucleotide

C-20 (E,E)-octa2-dienoate ester group

Bryostatin

Specific PKC inhibitors Aprinocarsen (Affinitak, LY900003, ISIS 3521) KAI-9803 (KAI-/CS-9803) Enzastaurin (LY317615)

Indolocarbazole

Indolocarbazole Indolocarbazole

Chemical class

Midostaurin (PKC-412, CGP 41252, 4- N Benzoyl-staurosporine)

Unspecific PKC inhibitors Staurosporine UCN-01 (7-hydroxystaurosporine)

PKC inhibitor

TABLE 1 Overview of PKC Inhibitors

PKC Inhibitors in Lung Cancer

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in a variety of cells (28,29). Based on its potent activity, staurosporine was investigated for anticancer activities (30) after it had been successfully synthesized in 1996 (31,32). Staurosporine has not been evaluated as a therapeutic agent, but many PKC inhibitors synthesized subsequently have been derived from staurosporine. By increasing its diverse structural and biological attributes, more selective therapeutic PKC inhibitors have been developed (33,34). UCN-01 7-Hydroxystaurosporine (UCN-01) occurs naturally in a soil bacterium and is a relatively nonspecific PKC inhibitor. Its anticancer activity appears predominantly related to its effect as a check-point-dependent kinase (CDK) inhibitor. It is no longer being developed by pharmaceutical companies, but its clinical activity continues to be investigated by the National Cancer institute (NCI) (35,36). UCN-01 inhibits serine-threonine kinases including the Ca2þ and phospholipid-dependent PKC. UCN-01 is a potent (500 nmol/L), whereas PKC-z was not inhibited by UCN-01 (37,38). At even higher concentrations (>1 mmol/L), UCN-01 inhibits many other kinases. Subsequent studies revealed that it also potently inhibits the DNA damage response to regulatory kinases chk1, possibly chk2 (39–41) and phosphatidylinositide-dependent kinase 1 (PDK-1) (42). UCN-01 mediates distinct effects in vitro/in vivo: cell cycle arrest in G1, abrogation of G2 arrest by inhibiting chk1, induction of apoptosis and potentiation of cytotoxicity of S-phase-active chemotherapeutics (43). UCN-01 was also found to have potent activity in the National Cancer Institute's (NCI) 60 human cancer cell line panel, and has demonstrated antineoplastic activity in a number of preclinical animal models (44,45). Preclinical evidence that UCN-01 has activity in lung-cancer cells is based on a number of studies. One of the first established that UCN-01 can inhibit tumor cell proliferation and oncogene phosphorylation in A549 lung-cancer cell lines (46,47). However, under certain conditions lung-cancer cell lines seem to develop resistance to UCN-01, depending on status of cell cycle and phosphorylation of retinoblastoma (Rb) (44,48,49). On the basis of its novel antineoplastic properties, an initial phase I clinical trial was completed (35,50). Of 47 patients with refractory neoplasms, one patient (2%) with melanoma had a partial response and a second patient (2%) with anaplastic large cell lymphoma had stabilization of disease for >2.5 years. Although a safe dose and treatment schedule could be defined, dose-limiting toxicities included unexpected hyperglycemia, with frequent lesser degrees of hyperglycemia documented at lower doses. Insulin resistance in peripheral tissues has been implicated because circulating insulin and C-peptide levels increased (35), and the mechanism of insulin resistance appears to involve inhibition of AKT activation and subsequent GLUT4 translocation in response to insulin (51). A phase I trial of UCN-01 (70–90 mg/m2 on day 1) and the topoisomerase I inhibitor, topotecan (0.75–1.0 mg/m2 on days 1–5), was also conducted. This combination showed grade 3/4 hematologic toxicity in 16 of the 33 treated patients (48%). Of 22 evaluable patients, 12 had stable disease (54%) and one patient with ovarian cancer had a partial response (5%) (52). Another combination phase I trial of UCN-01 and perifosine has also been conducted in solid tumor patients (communication by University of Maryland, G.C.C., UCN-01 and Perifosine phase I clinical trial for solid tumors, 2006). A recent study completed a


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molecular profiling study to better understand the activity when UCN-01 is combined with cisplatin. Previous nonclinical studies suggested that this combination was particularly active (48,53). However, platinum-associated toxicities were increased in this study and the study was stopped prior to enrolling its targeted patient number. While these observations are consistent with the proposed effect of UCN-01 increasing cisplatin activity, the study also suggests that the therapeutic window of UCN-01 in conjunction with standard chemotherapy may be too narrow for its future development. Other single agent phase II clinical trials are ongoing for patients with lymphoma at the National Cancer Institute (communication by National Cancer Institute, UCN-01 phase II clinical trial for patients with relapsed or refractory lymphomas, 2006) and should reveal whether its single agent use has a place in the treatment of hematologic malignancies. PKC412 PKC412 (40 -N-benzoyl-staurosporine, CGP 41251, or midostaurin) is an oral multitargeted kinase inhibitor (54). It potently inhibits the Flt-3 receptor tyrosine kinase, mutated in approximately one-third of acute myeloid leukemia (AML) patients, as well as multiple other molecular targets thought to be important for the pathogenesis of AML (55,56). PKC412 has been recognized to also inhibit VEGFR-2, PDGFR, c-KIT, MDR, and the zinc-finger-198 (ZNF-198)-fibroblast growth factor receptor 1 fusion tyrosine kinase that is implicated in myeloproliferative disorders (57). In in vitro and in vivo studies, PKC412 inhibits multiple isoforms of the serine/threonine PKC and the Flt-3 kinase. In mice with the mutated form of Flt-3, PKC412 was shown to prolong survival (57). Furthermore, in preclinical studies PKC412 has demonstrated a broad antiproliferative activity against various tumor cell lines, including those that were resistant to several other chemotherapeutic agents. PKC412 suppresses growth of AML cells and neoplastic mast cells (55,57). Its activity appears to be determined by the presence of specific mutations in the ATP binding pocket of Flt-3 (58). PKC412 also appears to be effective in suppressing growth of gastrointestinal stromal tumors (GIST) that become resistant to imatinib, possibly by inhibiting PDGFR and Flt-3 kinase signaling together (56,59). While PKC412 has not been examined extensively in solid tumors, such as lung cancer, recent studies demonstrate that PKC412 can sensitize lung-cancer cell lines to apoptosis (60). This effect is reminiscent of the effect seen with UCN-01 and perhaps both PKC inhibitors share a similar molecular mechanism of sensitizing tumor cells to standard chemotherapy. PKC412 can be safely administered as a chronic oral therapy, and 150 mg/day was identified as an acceptable dose for phase II studies. Steady-state PKC412 plasma levels at the top three dose cohorts (150–300 mg) were 5–10 times the cellular 50% inhibitory concentration (IC50) for PKC412 of 0.2–0.7 mmol/L. The pharmacokinetics and lack of conventional toxicity indicate that pharmacodynamic measures may be additionally needed to optimize the drug dose and schedule (61). Currently, PKC412 is being investigated in phase I/II trials, including patients with mast cell leukemia (communication by Stanford University Cancer Center, PKC412, Midostaurin, phase II clinical trial for Aggressive Systemic Mastocytosis and Mast Cell Leukemia, 2006), AML and myelodysplastic syndrome (61). A phase I trial of PKC412 in conjunction with gemcitabine and cisplatin for front-line therapy of metastatic NSCLC has already demonstrated feasibility, with grade 3 diarrhea or nausea above the maximum tolerated dose (MTD) of 50 mg/day (62).

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Bryostatin Bryostatin (Bryostatin 1) was identified in a series of cyclic macrolides first isolated from the marine bryozoan Bugula neritina (Order Cheilostomata) in the late 1960s (63). This arborescent bryozoan is found in temperate and subtropical environments worldwide, but only B. neritina from California and the Gulf of Mexico is known to contain bryostatins 1, 2 and 3 that are characterized by the C-20 (E,E)octa-2-dienoate ester (16,64). Unfortunately, material is limited due to the fact that Bryostatin 1 needs to be extracted from bryozoans, which contain small amounts of the active forms (to extract one gram of bryostatin, roughly one ton of the raw bryozoans is needed), and the synthesis has proven difficult (65–67). Bryostatin 1 has been investigated as an anticancer agent in a number of preclinical models (68–70). In vitro studies have shown that bryostatin 1 is an effective anticancer agent that may work by modulation of the PKC receptor. In vitro, bryostatins have direct cancer cytotoxic properties as well as tumor-targeting T lymphocyte activation effects. Bryostatins can also act synergistically with other anticancer drugs to achieve potent antileukemic effect and inhibit growth of lung cancer, prostate and non-Hodgkin's lymphoma tumor cells (68,71–76). Unlike most chemotherapy drugs that are myelosuppressive, bryostatin directly enhances maturation of neutrophils by increasing granulocyte colony stimulating factor secretion from bone marrow stromal cells, but does not appear to affect significantly normal myeloproliferation in patients (77). Bryostatin has been evaluated in lung-cancer cell lines (78), and was found to have a biphasic effect characterized by a transient activation of PKCs followed by inhibition of PKC-d (79). Human clinical trials have suggested that bryostatins have a potential synergistic action with other chemotherapeutic agents (68,80–82). Phase I clinical trials have demonstrated a dose-limiting toxicity of myalgias developing at 48 hours (83–86). Responses have been observed in phase II clinical trials with bryostatin as a single agent. These studies included patients with relapsed lowgrade lymphoma and chronic lymphocytic leukemia (CLL) (87). In this study, Bryostatin 1 administration demonstrated one complete response (4%) and 2 partial responses (8%) out of 25 patients. Single agent activity of Bryostatin was also seen in patients with renal cell carcinoma (88), where it was administered as a weekly dose of 35–40 mg/m2. Partial responses were observed in 2 (6%) out of 32 patients and disease stabilization of longer than 6 months were documented for 6 patients (19%). By contrast, no single agent responses have been seen in other cancer types, including melanoma, colorectal, or ovarian cancer (83,89–91). Early phase I and II data suggested that tumor responses to cisplatin or paclitaxel were enhanced by adding bryostatin (80,92). One study examined the combination of bryostatin with cisplatin in patients with NSCLC (93). A larger phase II study investigated the activity of Bryostatin in combination with paclitaxel in patients with NSCLC (94). In this study bryostatin did not add clinical benefit to paclitaxel. Currently, Bryostatin 1 is in clinical evaluation for the treatment of CLL and NHL by the National Cancer Institute in the United States (communication by National Cancer Institute, Bryostatin 1 and Rituximab in Treating Patients With B-Cell Non-Hodgkin's Lymphoma or Chronic Lymphocytic Leukemia, 2006) and by the Cancer Research Campaign in Great Britain (83). In addition, studies are being conducted to determine whether bryostatin-stimulated T lymphocytes show an enhanced immunotherapeutic effect when combined with the treatment of interleukin 2 (70). Finally, the myelostimulatory effect of


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bryostatin seen in treated patients suggest that bryostatin administration could have additional application, such as a myeloprotective agent during chemotherapy or as a stimulant of hematopoiesis between courses of chemotherapy (77,86). Selective Inhibitors Aprinocarsen Aprinocarsen (Affinitak; LY900003, ISIS 3521) belongs to a class of drugs called ASO (95). ASOs are designed to bind to a specific gene's messenger RNA (mRNA), resulting in the mRNA's degradation by the endogenous enzyme RNAse H and an overall reduction of protein synthesis at the mRNA level (96,97). Aprinocarsen is a phosphorothioate ASO, or a first-generation ASO, targeting human PKC-a. Aprinocarsen can reduce PKC-a mRNA and protein expression in various cancer cell lines (98–100). Sensitivity to aprinocarsen correlates with high PKC-a expression. In the glioblastoma cell line A172, which expresses high levels of PKCa, aprinocarsen exposure caused a marked increase of p53 and IGF-BP-3, which was associated with enhanced tumor cell growth arrest (101). In contrast, such an effect was not observed in the breast cancer cell line MCF-7, which does not express high PKC-a levels (5,101). Aprinocarsen has been given to patients in 21 clinical studies of various malignancies (102). Some encouraging results have been seen in single agent studies in patients with non-Hodgkin's lymphoma (103). Single agent phase II study of aprinocarsen as a continuous infusion over 21 days did not demonstrate objective responses in advanced ovarian carcinoma patients, although one (3%) of 36 patients showed reduction in serum CA125 levels and stable disease for 8 months (104). In combination with chemotherapy, however, aprinocarsen was hypothesized to have synergistic activity. In a phase I/II study for metastatic NSCLC patients, aprinocarsen was given 3 days prior to paclitaxel and carboplatin with a median survival of 15.9 months, almost twice as long as historical controls (105). A phase III study in advanced NSCLC comparing carboplatin and paclitaxel in combination with aprinocarsen versus chemotherapy alone, however, failed to show any difference in response or survival (106), prematurely terminating another phase III NSCLC study of cisplatin and gemcitabine with or without aprinocarsen, which also showed no benefit for the aprinocarsen treated patients (107). KAI-/CS-9803 (KAI-9803) KAI Pharmaceuticals is a specialized company that develops a number of isoenzyme-specific PKC inhibitors. In addition to early lead inhibitory candidates targeting PKC-b, -e, and -g, KAI Pharmaceuticals has advanced the PKC-d inhibitor KAI-/CS-9803 to clinical investigation. This agent was derived from combinatorial modification of the substrate consensus sequences of an individual PKC isozyme. This approach has led to the identification of potent and isozymeselective peptide inhibitors of PKC-a, PKC-d and PKC-z (108). Currently, KAI-/ CS-9803 is being pursued for reducing ischemia and reperfusion injury during the treatment of acute myocardial infarction (AMI). Unlike other agents that have been studied for reperfusion injury, KAI-/CS-9803 acts upstream of both the apoptotic and necrotic pathways by inhibiting PKC-d translocation into the mitochondria. KAI-/CS-9803 is not being evaluated in cancer patients, mainly because the drug platform of pure peptides such as KAI-/CS-9803 is unstable as a systemic therapy. However, similar specific peptide inhibitors of PKCs

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conceivably could be injected intratumorally or perfused into isolated organs as part of loco-regional treatment strategies. Enzastaurin Enzastaurin (LY317615) is an oral serine-threonine kinase inhibitor designed to suppress tumor growth through multiple mechanisms (109). Preclinical studies demonstrate inhibition of cell proliferation, increase in apoptosis, and inhibition of angiogenesis (109). Enzastaurin inhibits signaling through the PKC-b and PI3K/ AKT pathways—signaling pathways activated in a wide variety of cancers— which in turn inhibit the phosphorylation of ribosomal protein S6 and glycogen synthase kinase 3 beta (GSK-3b), an enzyme mediator of energy metabolism and neuronal cell development (109). For example, enzastaurin produced in vitro growth inhibition of SCLC and NSCLC cell lines accompanied by modulation of GSK-3b (110). Furthermore, enzastaurin was found to have synergistic growth inhibition when combined with pemetrexed (111). In addition, low mRNA expression of GSK3-b and high expression of the pro-angiogenic chemokine IL-8 have been shown to correlate with in vitro sensitivity of enzastaurin in freshly explanted human tumor cells. This implicates GSK3-b and IL8 as potential predictive markers for enzastaurin response (112). An ovarian cancer study corroborated the use of GSK3-b phosphorylation as a marker for enzastaurin activity in ovarian cancer models (113). This study demonstrated that taxane-resistant ovarian cancer cells may respond to enzastaurin treatment in low concentrations and suggests that enzastaurin should be investigated in women with taxane-resistant ovarian cancer. In the first phase I study of single-agent enzastaurin in cancer patient (study JCAD), 47 patients were enrolled (mean age, 58 years). Patients received at least one dose of enzastaurin, with a median of two cycles (range 1–17 cycles) (114). Prevalent malignancies in this study were lung cancer (n ¼ 10) and head and neck cancers (n ¼ 9). Although no MTD was identified up to 700 mg/day, 525 mg was chosen as the recommended dose, and 12 additional patients were accrued at that level. This level was found to be associated with a steady-state exposure of 1400 nm, a concentration associated with maximum PKC-b inhibition in cell lines. PKC phosphorylation inhibition was confirmed by flow cytometry in peripheral blood mononuclear cells (115). Exposure of enzastaurin and its metabolites increased proportionally with dose up to 240 mg, and appeared to plateau at 525 and 700 mg. Grade 1 chromaturia, fatigue, and other GI toxicities were the most common, while no clinically significant grade 3/4 toxicities occurred. Twenty-one patients (45%) achieved stable disease for 2–16 cycles, including 4 patients with NSCLC (19%). Hence, the recommended phase II dose was declared at 525 mg for the capsule formulation, which subsequently was replaced by a 500 mg tablet formulation. In advanced NSCLC patients who had progressed through one prior therapy, a phase II trial of single-agent enzastaurin 500 mg daily has demonstrated eighteen of fifty-three patients (34%) to have a best response of stable disease, while no patients had a partial or complete response (116). In this interim analysis of 53 patients, 10 (19%) received therapy for 6 cycles, 3 (6%) of whom received enzastaurin beyond 9 cycles of treatment, and one (2%) who received 11 cycles before progression. The most common toxicity was a varying degree of fatigue (n ¼ 21), noted within 1 week of starting treatment, but fatigue was not reported in patients with disease stabilization. Based on this interim analysis, 10.4% of the


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patients were progression-free at 6 months, and the final study analyses are being expected in 2007. Additional evaluations for the application of enzastaurin in NSCLC are ongoing, including studies to determine how to best combine enzastaurin with other agents active in NSCLC. Other clinical studies with enzastaurin are currently being conducted in NHL and glioblastoma. For advanced diffuse large B-cell lymphoma (DLBCL), patients who had progressed through 2 prior therapies were enrolled in a phase 2 multicenter clinical trial of enzastaurin, 500 mg once daily (117). Twenty-two percent of the 55 study participants were free of disease progression for 2 months. A quarter of those patients remained progression free, with continued responses from 1.5 years to more than 3 years in duration. Treatment was well tolerated, with seven reports of grade 3 toxicity including fatigue, thrombocytopenia, headache, motor neuropathy, and edema. Only one patient experienced grade 4 toxicity for hypomagnesemia. For recurrent glioblastoma multiforme and anaplastic glioma, a phase II study of enzastaurin included 92 patients who had progressive disease after more than one prior regimen of chemotherapy (118). Patients' treatment consisted of an oral fixed dose of 500 mg of enzastaurin, administered daily. Treatment was allowed to continue indefinitely depending upon the patient's response to the drug. Tumor shrinkage was evident in patients who received enzastaurin, with a corresponding response rate of 23% and a stabilization rate of 7%. Overall, enzastaurin was well tolerated in this patient population with the most common side effect being thrombocytopenia (16%). Based on these results in NHL and glioblastoma, two phase III trials of enzastaurin have been launched. The NHL phase III trial (PRELUDE—Preventing Relapse in Lymphoma Using Daily Enzastaurin) is a randomized, placebo controlled study of patients in their first remission from DLBCL (communication, Eli Lilly and Company). The study will compare the efficacy, safety and tolerability of enzastaurin, taken orally for up to three years, versus placebo. This study is planned to enroll 459 patients across 100 sites worldwide. The primary end point of this study will be overall disease-free survival. Additionally, Lilly will be assessing any biomarkers relevant to enzastaurin as a basis for correlating patient response to clinical trial outcomes. The glioblastoma phase III trial (STEERING—Study Evaluating Enzastaurin in Recurrent Glioblastoma) was a randomized, open label registration study, which was to enroll 397 patients to compare the efficacy, safety and tolerability of enzastaurin versus CCNU (lomustine). However, the study's planned interim analysis suggested that enzastaurin would not meet its primary end point of improved progression-free survival over CCNU, and the study has been discontinued (communication, Eli Lilly and Company). Other clinical trials combining enzastaurin with standard chemotherapy regimens are underway or have been completed. Preliminary phase I clinical trial experience combining enzastaurin with gemcitabine and cisplatin (119), pemetrexed (120), or capecitabine (121) have demonstrated no added toxicity when enzastaurin is combined with standard chemotherapies generally used in the treatment for NSCLC, breast, and colorectal cancer. Each study showed that enzastaurin was generally well tolerated across all dose levels and studies in combination with chemotherapy. Finally, there were no observations of significant alterations in pharmacokinetics, suggesting that enzastaurin will have no or limited drug–drug interactions with standard chemotherapies. Further data on

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enzastaurin's safety were reported from a review of data gathered from all early phase I and II studies comprising about 130 patients treated with enzastaurin. This review suggested that enzastaurin is generally well tolerated across all doses and for extended durations with few grade 3/4 toxicities (122). According to this analysis and given the severity of the disease and absence of controlled studies for comparisons, no event other than chromaturia or fecal discoloration (due to reddish-orange color of the active ingredient) and possibly fatigue appear to be definitively attributable to enzastaurin. CONSIDERATIONS FOR THE FUTURE DEVELOPMENT OF PKC INHIBITORS IN NSCLC Despite modest improvements in treatment-related toxicities, the efficacy of firstand second-line chemotherapy treatment for metastatic NSCLC has not changed appreciably during the last decade (123). Equally important is the fact that many patients will not benefit from the current chemotherapies (i.e., the response rate is generally between 20% and 30% at best), and other patients will not be eligible to receive chemotherapy due to their co-morbidities or other ineligibility reasons (performance status, etc.). Therefore, some of the most significant therapeutic advances for metastatic NSCLC during the last several years have focused on developing agents that are more selective in their antitumor effect with the hope of reducing toxicities. One approach was directed at targeting the EGFR and VEGFR pathways (124). As a result of this effort, the addition of bevacizumab, a monoclonal antibody against VEGF, to standard front-line chemotherapy with carboplatin and paclitaxel for NSCLC has been shown to improve response rates, time to progression, and overall survival compared to chemotherapy alone (125,126). Based on these results, the U.S. Food and Drug Administration has approved bevacizumab for use in combination with chemotherapy to treat metastatic NSCLC. In contrast to first-line therapy, standard second-line therapy for metastatic NSCLC has been limited to several single-agent chemotherapy drugs, all associated with a similar 10% to 15% rate of tumor response and overall survival (127,128). This situation changed with the arrival of molecular targeted agents; in particular, EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib (129,130). These agents have shown a marked benefit for appropriately selected patients with metastatic NSCLC. This recent experience of bevacizumab and the tyrosine kinase inhibitors suggests that inhibition of pathways associated with tumor proliferation or angiogensis can alter the clinical outcome of patients with NSCLC. It is understandable that many other VEGFR and EGFR pathway antagonists are currently being evaluated with great interest, either separately or in combination for the treatment of NSCLC (131–137). The success of the EGFR and VEGFR pathway antagonists in treatment of metastatic NSCLC has important implications for development of novel targeted therapies such as the PKC inhibitors. The development of EGFR and VEGFR pathway antagonists has been blessed by a clear appreciation of the biology targeted by these therapeutic agents. Bevacizumab, gefitinib, and erlotinib by design have exquisitely well-defined molecular targets that facilitate rigorous testing of their mechanisms of action and pharmacodynamics. Fortuitously, these drugs also have clinically obvious pharmacodynamic end points—hypertension for bevacizumab and skin toxicity for erlotinib and gefitinib. Even despite these clear molecular, biologic, and clinical endpoints of therapy, the results of clinical


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trials with these agents still have been marked by unexpected results and discoveries that have reinvented their therapeutic indications. It appears that PKC inhibitors act on similar and, in some instances, distinct pathways compared to those of the EGFR and VEGFR inhibitors. Hence, it is not unlikely that they will also provide clinical benefit in the treatment of lung cancer. PKC inhibitors can inhibit cell cycle (138), tumor cell proliferation, and neo-angiogensis, and can induce apoptosis (109). This multivalent activity of PKC inhibitors is based on a broader antitumor biology than the EGFR and VEGFR inhibitors. In contrast to the approved VEGFR and EGFR inhibitors, PKC inhibitors are only at the beginning of their discovery path, and their development path will likely be as challenging as the one for the VEGFR and EGFR inhibitors. Several aspects need to be considered for the successful clinical development of PKC inhibitors in NSCLC. Because the earliest PKC inhibitors do not exhibit an isozyme-specific inhibitory profile, it is difficult to apply well-defined pharmacodynamic endpoints to measure their activity in patients. For instance, the activity of UCN-01 was found to be dependent on the phosphorylation status of Rb (48). Similarly, PKC412 was found to be active in AML cells with a particular mutation (58). These two observations raise the question of whether patient selection would have improved the outcome of the first phase I studies. Perhaps, if patients had been appropriately selected, a different trial design with a focus on pharmacokinetic/ pharmacodynamic (PK/PD) assessment would have more appropriately estimated the activity of these PKC inhibitors. Instead, the original development had to rely on establishing the MTD dose level prior to moving into phase II development. In fact, this alternative approach was subsequently pursued for UCN-01, but by that time the interest level for this molecule had dropped and was superseded by other more attractive compounds in cancer therapy (53). Furthermore, the relevance of PKC expression and its isoenzymes or their effect on downstream proteins has not been studied sufficiently in human tumor tissue samples. In comparison to the EGF and VEGF pathway, little is known about the PKC expression and its activation pathways in humans. This lack of comprehensive assessment of PKC and its activation pathways in human lung cancer tissue may have contributed to the failure of specific PKC inhibitors in the clinical investigation, such as aprinocarsen. Assuming that PKC-a expression was correlated with clinical outcome as cell line studies suggested (100,139), inhibition of PKC-a expression with aprinocarsen should have resulted in clinical benefit. This assumption was not confirmed in larger phase III studies (106,107). Whether this was a result of insufficiently blocking PKC-a levels during administration of aprinocarsen (i.e., an unfavorable PK/PD relationship), or whether the expression of PKC-a was only relevant to the tumor growth within a small patient population enrolled on the larger phase III studies (i.e., leading to the underestimation of the required study sample size), cannot be determined today. But, these experiences underscore the need for designing clinical studies with PKC inhibitors differently than in the past, including the use of PK/PD modeling in early studies. They also suggest that integrating strategies to identify patients who may benefit from PKC inhibitor treatment will be valuable tools to determine the activity of PKC inhibitors in NSCLC patients. Some of these lessons have been applied to the development of the PKC-b inhibitor, enzastaurin. Enzastaurin was originally developed as an antiangiogenic agent, based on implication of PKC-b in angiogenesis and its preclinical activity (109). One of the striking differences of enzastaurin compared to the other PKC

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inhibitors investigated clinically is its favorable toxicity/tolerability profile. In the first study in cancer patients, no clinically relevant grade 3/4 toxicities were observed (114). Also, the subsequent safety review of about 130 patients enrolled in the early phase clinical trials indicated few grade 3/4 drug-related toxicities (2/134, or 2%) (122). This favorable tolerability profile has resulted in a more attractive development path for enzastaurin compared to the more toxic nonspecific PKC inhibitors. However, the lack of not having established an MTD has been challenged by many oncologists and casts some doubt as to whether the actual antitumor dose has been or can be achieved (140). Therefore, it was critical for the enzastaurin program to prove that its targeted exposure levels of 1400 nM was associated with significant inhibition of PKC phosphorylation (115). The favorable tolerability/toxicity profile and the evidence of a PK/PD relationship in patients has allowed the enzastaurin development to proceed in a different fashion than the previous PKC inhibitors. While the PK/PD relationship has been generally addressed for enzastaurin, the greatest challenge for the clinical development of enzastaurin remains in identifying the patient population likely to benefit from this treatment. This will require the discovery and validation of predictive biomarkers, which for most biologic agents have been unpredictable and elusive. The process of biomarker discovery and validation in treatment of metastatic NSCLC continues to be hindered by the inaccessibility of tumor tissue for analysis in the majority of the patients. Optimizing the success of molecular targeted therapies in NSCLC will require a concerted effort to overcome inherent hazards and reluctance associated with performing biopsies of lung cancer or metastatic lesions. Alternatively, one should consider the development of high sensitivity assays to detect tumor-related biomarkers in the circulating bloodstream, such as circulation tumor cell fragments or whole cells (141). Although predictive markers of response to enzastaurin have not yet been determined, mechanisms of anticancer activity have been determined in preclinical studies. Enzastaurin treatment in vitro suppresses the phosphorylation of GSK-3bSer9, ribosomal protein S6S240/244, and AKTThr308, and may thus directly inhibit cancer cell growth (109). In in vivo studies, enzastaurin treatment suppresses GSK-3b phosphorylation in both tumor tissue and in PBMCs, suggesting that GSK-3b phosphorylation may serve as a reliable pharmacodynamic marker for enzastaurin activity. Whether or not the presence or degree of GSK-3b or AKT phosphorylation prior to enzastaurin might predict response to treatment has not been reported, and will be explored in upcoming clinical studies. In summary, PKC inhibitors have not yet established themselves as a new anticancer drug treatment. The success of PKC inhibitor therapy for cancer will depend on demonstrating inhibition of specific PKC targets, discovering the critical biologic profile in tumors required for its anticancer activity, and creating tests to predict which patients would benefit from treatment. The first step towards this goal has been the evaluation of potent isozyme-specific PKC inhibitors. Future studies will need to incorporate tumor biopsies, blood, and/or surrogate tissues to evaluate potential predictive markers, such as GSK-3b AKT, ribosomal protein S6, and IL-8. PKC inhibitors will require much broader clinical and scientific investigations before they can be optimally utilized, but they offer the possibility of multi-valent anticancer effects, including induction of apoptosis, cell cycle inhibition, and interruption of angiogenesis, and have already demonstrated promising clinical efficacy.


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REFERENCES 1. Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 1988; 334:661–665. 2. Parker PJ, Murray-Rust J. PKC at a glance. J Cell Sci 2004; 117(Pt 2):131–2. 3. Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. Faseb J 1995; 9:484–96. 4. Basu A. The potential of protein kinase C as a target for anticancer treatment. Pharmacologic Therapeutics 1993; 59:257–80. 5. O'Brian C, Vogel V, Singletary S, Ward N. Elevated protein kinase C expression in human breast tumor biopsies relative to normal breast tissue. Cancer Research 1989; 49:3215–7. 6. Perletti G, Smeraldi C, Porro D, Piccinini F. Involvement of the alpha isoenzyme of protein kinase C in the growth inhibition induced by phorbol esters in MH1C1 hepatoma cells. Biochem Biophys Res Commun 1994; 205:1589–94. 7. Dlugosz A, Cheng C, Williams E, Dharia A, Denning M, Yuspa S. Alterations in murine keratinocyte differentiation induced by activated rasHa genes are mediated by protein kinase C alpha. Cancer Res 1994; 54:6413–20. 8. Yuspa S. The pathogenesis of squamous cell cancer: lessons learned from studies of skin carcinogenesis—thirty-third G. H. A. Clowes Memorial Award Lecture. Cancer Res 1994; 54:1178–89. 9. Ways D, Kukoly C, deVente J, et al. MCF-7 breast cancer cells transfected with protein kinase C-alpha exhibit altered expression of other protein kinase C isoforms and display a more aggressive neoplastic phenotype. J Clin Invest 1995; 95:1906–15. 10. Adesina A, Dooley N, Yong V, Nalbantoglu J. Differential role for protein kinase C-mediated signaling in the proliferation of medulloblastoma cell lines. Int J Oncol 1998; 12:759–68. 11. Henttu P, Vihko P. The protein kinase C activator, phorbol ester, elicits disparate functional responses in androgen-sensitive and androgen-independent human prostatic cancer cells. Biochem Biophys Res Commun 1998; 244:167–71. 12. Shen GX. Selective protein kinase C inhibitors and their applications. Curr Drug Targets Cardiovasc Haematolog Disorders 2003; 3:301–7. 13. Wang H, Chow DA. Protein kinase C expression links natural antibody binding with surveillance of activated and preneoplastic cells. Scand J Immunol 1999; 49:381–90. 14. Ruegg UT, Gillian B. Staurosporine, K-252 and UCN-01: potent but nonspecific inhibitors of protein kinases. Trends Pharmacol Sci 1989; 10:218–20. 15. Tamaoki T, Nomoto H, Takahashi I, Kato Y, Morimoto M, Tomita F. Staurosporine, a potent inhibitor of phospholipid/Caþþdependent protein kinase. Biochem Biophys Res Commun 1986; 135:397–402. 16. Pettit G, Herald C, Doubek D, Herald D, Arnold E, Clardy J. Isolation and structure of bryostatin-1. J Am Chem Soc 1982; 104:6846–8. 17. Yoshida M, Heike Y, Ohno S, Ikekawa T, Wakasugi H. Involvement of PKC betaII in anti-proliferating action of a new antitumor compound gnidimacrin. Int J Cancer 2003; 105:601–6. 18. Courage C, Snowden R, Gescher A. Differential effects of staurosporine analogues on cell cycle, growth and viability in A549 cells. Br J Cancer 1996; 74:1199–205. 19. Derian C, Zhang H-C, Andrade-Gordon P, et al. Inhibition of angiogenesis by a novel, selective inhibitor of Protein Kinase C-beta. Session Type: Poster Session 129-II. Abstract Number 1958. In: Hematology ASo, ed. ASH; 2004: Blood; 2004. 20. Mani S, Rudin CM, Kunkel K, et al. Phase I clinical and pharmacokinetic study of protein kinase C-alpha antisense oligonucleotide ISIS 3521 administered in combination with 5-fluorouracil and leucovorin in patients with advanced cancer. Clin Cancer Res 2002; 8:1042–8. 21. Young LH, Balin BJ, Weis MT. Go 6983: a fast acting protein kinase C inhibitor that attenuates myocardial ischemia/reperfusion injury. Cardiovas Drug Rev 2005; 23:255–72. 22. Senderowicz AM. Cell cycle modulators for the treatment of lung malignancies. Clin Lung Cancer 2003; 5:158–68.

116

Oh et al.

23. Omura S, Sasaki Y, Iwai Y, Takeshima H. Staurosporine, a potentially important gift from a microorganism. J Antibiot 1995; 48:535–48. 24. Funato N, Takayanagi H, Konda Y, et al. Absolute configuration of staurosporine by X-ray analysis. Tetrahedron Lett 1994; 35:1251–4. 25. Casnellie JE. Protein kinase inhibitors: probes for the functions of protein phosphorylation. Adv Pharmacol 1991; 22:167–205. 26. Buchholz RA, Dundore RL, Cumiskey WR, Harris AL, Silver PJ. Protein kinase inhibitors and blood pressure control in spontaneously hypertensive rats. Hypertension 1991; 17:91–100. 27. Yang J, Liu X, Bhalla K, et al. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 1997; 275:1129–32. 28. Cai J, Jones DP. Superoxide in apoptosis. Mitochondrial generation triggered by cytochrome c loss. J Biol Chem 1998; 273:11401–4. 29. Kruman I, Guo Q, Mattson MP. Calcium and reactive oxygen species mediate staurosporine-induced mitochondrial dysfunction and apoptosis in PC12 cells. J Neurosci Res 1998; 51:293–308. 30. Matsura T, Serinkan BF, Jiang J, Kagan VE. Phosphatidylserine peroxidation/externalization during staurosporine-induced apoptosis in HL-60 cells. FEBS Lett 2002; 524:25–30. 31. Link JT, Raghavan S, Gallant M, Danishefsky SJ, Chou TC, Ballas LM. Staurosporine and ent-Staurosporine: the first total syntheses, prospects for a regioselective approach, and activity profiles. J Am Chem Soc 1996; 118:2825–42. 32. Wood JL, Stoltz BM, Goodman SN. Total synthesis of (þ)-RK-286c, (þ)-MLR-52, (þ)Staurosporine, and (þ)-K252a. J Am Chem Soc 1996; 118:10656–7. 33. Traxler P. Protein tyrosine kinase inhibitors in cancer treatment. Expert Opin Ther Patents 1997; 7:571–88. 34. Strawn LM, Shawver LK. Tyrosine kinases in disease: overview of kinase inhibitors as therapeutic agents and current drugs in clinical trials. Expert Opin Invest Drugs 1998; 7:553–73. 35. Sausville EA, Arbuck SG, Messmann R, et al. Phase I trial of 72-hour continuous infusion UCN-01 in patients with refractory neoplasms. J Clin Oncol 2001; 19:2319–33. 36. Sausville EA. Indifferently pursued or unowned drugs: who should lead where companies do not tread?[comment]. J Clin Oncol 2005; 23:1796–8. 37. Seynaeve CM, Kazanietz MG, Blumberg PM, Sausville EA, Worland PJ. Differential inhibition of protein kinase C isozymes by UCN-01, a staurosporine analogue. Mol Pharmacol 1994; 45:1207–14. 38. Shiro A, Kayo N, Katsushige G, Masami O. Effect of UCN-01, a selective inhibitor of protein kinase C, on the cell-cycle distribution of human epidermoid carcinoma, A431 cells. Cancer Chemother Pharmacol 1994; V33:273–80. 39. Yu Q, La Rose J, Zhang H, Takemura H, Kohn KW, Pommier Y. UCN-01 inhibits p53 up-regulation and abrogates g-radiation-induced G2-M checkpoint independently of p53 by targeting both of the checkpoint kinases, Chk2 and Chk1. Cancer Res 2002; 62:5743–8. 40. Graves PR, Yu L, Schwarz JK, et al. The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01. J Biol Chem 2000; 275:5600–5. 41. Busby EC, Leistritz DF, Abraham RT, Karnitz LM, Sarkaria JN. The radiosensitizing agent 7-hydroxystaurosporine (UCN-01) inhibits the DNA damage checkpoint kinase hChk1. Cancer Res 2000; 60:2108–12. 42. Sato S, Fujita N, Tsuruo T. Interference with PDK1-Akt survival signaling pathway by UCN-01 (7-hydroxystaurosporine). Oncogene 2002; 21:1727–38. 43. Senderowicz AM. Cell cycle modulators for the treatment of lung malignancies. Clin Lung Cancer 2003; 5:158–68. 44. Sugiyama K, Akiyama T, Shimizu M, et al. Decrease in susceptibility toward induction of apoptosis and alteration in G1 checkpoint function as determinants of resistance of human lung cancer cells against the antisignaling drug UCN-01 (7-Hydroxystaurosporine). Cancer Res 1999; 59:4406–12.


117

45. Edward AS, Richard DL, Donna H, et al. Clinical pharmacology of UCN-01: initial observations and comparison to preclinical models. Cancer Chemother Pharmacol 1998; V42:S54–9. 46. Kawakami K, Futami H, Takahara J, Yamaguchi K. UCN-01, 7-hydroxyl-staurosporine, inhibits kinase activity of cyclin-dependent kinases and reduces the phosphorylation of the retinoblastoma susceptibility gene product in A549 human lung cancer cell line. Biochem Biophys Res Commun 1996; 219:778–83. 47. Shimizu E, Zhao MR, Nakanishi H, et al. Differing effects of staurosporine and UCN01 on RB protein phosphorylation and expression of lung cancer cell lines. Oncology 1996; 53:494–504. 48. Mack PC, Gandara DR, Bowen C, et al. RB status as a determinant of response to UCN-01 in non-small cell lung carcinoma. Clin Cancer Res 1999; 5:2596–604. 49. Mack PC, Gandara DR, Lau AH, Lara PN, Jr, Edelman MJ, Gumerlock PH. Cell cycledependent potentiation of cisplatin by UCN-01 in non-small-cell lung carcinoma. Cancer Chemother Pharmacol 2003; 51:337–48. 50. Sausville EA. Cyclin-dependent kinase modulators studied at the NCI: pre-clinical and clinical studies. Curr Med Chem Anticancer Agents 2003; 3:47–56. 51. Kondapaka SB, Zarnowski M, Yver DR, Sausville EA, Cushman SW. 7-Hydroxystaurosporine (UCN-01) inhibition of Akt Thr308 but not Ser473 phosphorylation: a basis for decreased insulin-stimulated glucose transport. Clin Cancer Res 2004; 10:7192–8. 52. Hotte SJ, Oza A, Winquist EW, et al. Phase I trial of UCN-01 in combination with topotecan in patients with advanced solid cancers: a Princess Margaret Hospital Phase II Consortium study. Ann Oncol 2006; 17:334–40. 53. Lara PN, Jr, Mack PC, Synold T, et al. The cyclin-dependent kinase inhibitor UCN-01 plus cisplatin in advanced solid tumors: a California cancer consortium phase I pharmacokinetic and molecular correlative trial. Clin Cancer Res 2005; 11:4444–50. 54. Fabbro D, Ruetz S, Bodis S, et al. PKC412—a protein kinase inhibitor with a broad therapeutic potential. Anticancer Drug Des 2000; 15:17–28. 55. Knapper S, Mills KI, Gilkes AF, Austin SJ, Walsh V, Burnett AK. The effects of lestaurtinib (CEP701) and PKC412 on primary AML blasts: the induction of cytotoxicity varies with dependence on FLT3 signaling in both FLT3 mutated and wild type cases. Blood 2006:blood-2006-04-015487. 56. Cools J, Mentens N, Furet P, et al. Prediction of resistance to small molecule flt3 inhibitors: implications for molecularly targeted therapy of acute leukemia. Cancer Res 2004; 64:6385–9. 57. Chen J, DeAngelo DJ, Kutok JL, et al. PKC412 inhibits the zinc finger 198-fibroblast growth factor receptor 1 fusion tyrosine kinase and is active in treatment of stem cell myeloproliferative disorder. PNAS 2004; 101:14479–84. 58. Heidel F, Solem FK, Breitenbuecher F, et al. Clinical resistance to the kinase inhibitor PKC412 in acute myeloid leukemia by mutation of Asn-676 in the FLT3 tyrosine kinase domain. Blood 2006; 107:293–300. 59. Weisberg E, Wright RD, Jiang J, et al. Effects of PKC412, Nilotinib, and Imatinib against GIST-associated PDGFRA mutants with differential imatinib sensitivity. Gastroenterology 2006; 131:1734–42. 60. Hemstrom TH, Joseph B, Schulte G, Lewensohn R, Zhivotovsky B. PKC412 sensitizes U1810 non-small cell lung cancer cells to DNA damage. Exp Cell Res 2005; 305:200–13. 61. Propper DJ, McDonald AC, Man A, et al. Phase I and pharmacokinetic study of PKC412, an inhibitor of protein kinase C. J Clin Oncol 2001; 19:1485–92. 62. Monnerat C, Henriksson R, Le Chevalier T, et al. Phase I study of PKC412 (Nbenzoyl-staurosporine), a novel oral protein kinase C inhibitor, combined with gemcitabine and cisplatin in patients with non-small-cell lung cancer. Ann Oncol 2004; 15:316–23. 63. Proksch P, Edrada R, Ebel R. Drugs from the seas—current status and microbiological implications. Appl Microbiol Biotechnol 2002; 59:125–34. 64. Hale KJ, Hummersone MG, Manaviazar S, Frigerio M. The chemistry and biology of the bryostatin antitumour macrolides. Natural Product Reports 2002; 19:413–53.

118

Oh et al.

65. Evans DA, Carter PH, Carreira EM, Charette AB, Prunet JA, Lautens M. Total synthesis of Bryostatin 2. J Am Chem Soc 1999; 121:7540–52. 66. Manaviazar S, Frigerio M, Bhatia GS, Hummersone MG, Aliev AE, Hale KJ. Enantioselective formal total synthesis of the antitumor macrolide Bryostatin 7. Org Lett 2006; 8:4477–80. 67. Wender PA, Baryza JL, Bennett CE, et al. The practical synthesis of a novel and highly potent analogue of bryostatin. J Am Chem Soc 2002; 124:13648–9. 68. Kortmansky J, Schwartz GK. Bryostatin-1: a novel PKC inhibitor in clinical development. Cancer Invest 2003; 21:924–36. 69. Grant S. Modulation of ara-C induced apoptosis in leukemia by the PKC activator bryostatin 1. Front Biosci 1997; 2:d242–52. 70. Kos FJ, Cornell DL, Lipke AB, Graham LJ, Bear HD. Protective role of IL-2 during activation of T cells with bryostatin 1. Int J Immunopharmacol 2000; 22:645–52. 71. Wang H, Mohammad RM, Werdell J, Shekhar PV. p53 and protein kinase C independent induction of growth arrest and apoptosis by bryostatin 1 in a highly metastatic mammary epithelial cell line: in vitro versus in vivo activity. Int J Mol Med 1998; 1:915–23. 72. Ali S, Aranha O, Li Y, Pettit GR, Sarkar FH, Philip PA. Sensitization of human breast cancer cells to gemcitabine by the protein kinase C modulator bryostatin 1. Cancer Chemother Pharmacol 2003; 52:235–46. 73. Gschwend JE, Fair WR, Powell CT. Bryostatin 1 induces prolonged activation of extracellular regulated protein kinases in and apoptosis of LNCaP human prostate cancer cells overexpressing protein kinase C alpha. Mol Pharmacol 2000; 57:1224–34. 74. Stanwell C, Gescher A, Bradshaw TD, Pettit GR. The role of protein kinase C isoenzymes in the growth inhibition caused by bryostatin 1 in human A549 lung and MCF-7 breast carcinoma cells. Int J Cancer 1994; 56:585–92. 75. Gebbia V, Citarrella P, Miserendino V, et al. The effects of the macrocyclic lactone bryostatin-1 on leukemic cells in vitro. Tumori 1992; 78:167–71. 76. Tuttle TM, Bethke KP, Inge TH, McCrady CW, Pettit GR, Bear HD. Bryostatin 1-activated T cells can traffic and mediate tumor regression. J Surg Res 1992; 52:543–8. 77. Lilly M, Vo K, Le T, Takahashi G. Bryostatin 1 acts synergistically with interleukin-1 alpha to induce secretion of G-CSF and other cytokines from marrow stromal cells. Exp Hematol 1996; 24:613–21. 78. Dale IL, Bradshaw TD, Gescher A, Pettit GR. Comparison of effects of bryostatins 1 and 2 and 12-O-tetradecanoylphorbol-13-acetate on protein kinase C activity in A549 human lung carcinoma cells. Cancer Res 1989; 49:3242–5. 79. Choi SH, Hyman T, Blumberg PM. Differential effect of bryostatin 1 and phorbol 12-myristate 13-acetate on HOP-92 cell proliferation is mediated by down-regulation of protein kinase C delta. Cancer Res 2006; 66:7261–9. 80. Ajani J, Jiang Y, Faust J, et al. A multi-center phase II study of sequential paclitaxel and bryostatin-1 (NSC 339555) in patients with untreated, advanced gastric or gastroesophageal junction adenocarcinoma. Invest New Drugs 2006; 24:353–7. 81. Dowlati A, Lazarus HM, Hartman P, et al. Phase I and correlative study of combination bryostatin 1 and vincristine in relapsed B-cell malignancies. Clin Cancer Res 2003; 9(16 Pt 1):5929–35. 82. Roberts JD, Smith MR, Feldman EJ, Cragg L, Grant S. Phase I study of bryostatin-1 and fludarabine in patients with chronic lymphocytic leukemia and indolent nonHodgkin's lymphoma. Clin Lymph 2002; 3:184–8. 83. Prendiville J, Crowther D, Thatcher N, et al. A phase I study of intravenous bryostatin 1 in patients with advanced cancer. Br J Cancer 1993; 68:418–24. 84. Sun M-K, Alkon DL. Bryostatin-1: pharmacology and therapeutic potential as a CNS drug. CNS Drug Rev 2006; 12:1–8. 85. Cragg LH, Andreeff M, Feldman E, et al. Phase I trial and correlative laboratory studies of bryostatin 1 (NSC 339555) and high-dose 1-B-D-Arabinofuranosylcytosine in patients with refractory acute leukemia. Clin Cancer Res 2002; 8:2123–33. 86. Philip P, Rea D, Thavasu P, et al. Phase I study of bryostatin 1: assessment of Interleukin 6 and tumor necrosis factor induction in vivo. J Natl Cancer Inst 1993; 85:1812–8.


119

87. Varterasian ML, Mohammad RM, Shurafa MS, et al. Phase II trial of bryostatin 1 in patients with relapsed low-grade non-Hodgkin's lymphoma and chronic lymphocytic leukemia. Clin Cancer Res 2000; 6:825–8. 88. Haas NB, Smith M, Lewis N, et al. Weekly bryostatin-1 in metastatic renal cell carcinoma: a phase II study. Clin Cancer Res 2003; 9:109–14. 89. Blackhall FH, Shepherd FA. Angiogenesis inhibitors in the treatment of small cell and non-small cell lung cancer. Hematol Oncol Clin North Am 2004; 18:1121–41, ix. 90. Clamp A, Blackhall F, Vasey P, et al. A phase II trial of bryostatin-1 administered by weekly 24-hour infusion in recurrent epithelial ovarian carcinoma. Br J Cancer 2003; 89:1152–4. 91. Tozer RG, Burdette-Radoux S, Berlanger K, et al. A randomized phase II study of two schedules of bryostatin-1 (NSC339555) in patients with advanced malignant melanoma: A National Cancer Institute of Canada Clinical Trials Group study. Invest New Drugs 2002; 20:407–12. 92. Rosenthal M, Oratz R, Liebes L, Cahr M, Muggia F. Phase I study of bryostatin-1 (NSC 339555) and cisplatin in advanced malignancies. Proc Am Soc Clin Oncol 1999; 17:873a. 93. Lenz HJ, Raghavan D, Doroshow J, Gandara DR. Phase I study of bryostatin-1 in combination with cisplatin in treating patients with metastatic or unresectable solid tumors including non small-cell lung cancer. Clin Lung Cancer 1999; 1:151–2. 94. Winegarden JD, Mauer AM, Gajewski TF, et al. A phase II study of bryostatin-1 and paclitaxel in patients with advanced non-small cell lung cancer. Lung Cancer 2003; 39:191–6. 95. Hanauske AR, Sundell K, Lahn M. The role of protein kinase C-alpha (PKC-alpha) in cancer and its modulation by the novel PKC-alpha-specific inhibitor aprinocarsen. Curr Pharmaceut Design 2004; 10:1923–36. 96. Crooke ST. Antisense strategies. Curr Mol Med 2004; 4:465–87. 97. Crooke ST. Potential roles of antisense technology in cancer chemotherapy. Oncogene 2000; 19:6651–9. 98. Denham D, Franz M, Denham W, et al. Directed antisense therapy confirms the role of protein kinase C-alpha in the tumorigenicity of pancreatic cancer. Surgery 1998; 124:218–23. 99. Dennis J, Dean N, Bennett C, Griffith J, Lang C, Welch D. Human melanoma metastasis is inhibited following ex vivo treatment with an antisense oligonucleotide to protein kinase C alpha. Cancer Lett 1998; 128:65–70. 100. Yazaki T, Ahmad S, Chahlavi A, et al. Treatment of glioblastoma U-87 by systemic administration of an antisense protein kinase C-alpha phosphorothioate oligodeoxynucleotide. Mol Pharmacol 1996; 50:236–42. 101. Shen L, Dean N, Glazer R. Induction of p53-dependent, insulin-like growth factorbinding protein- 3-mediated apoptosis in glioblastoma multiforme cells by a protein kinase C alpha antisense oligonucleotide. Mol Pharmacol 1999; 55:396–402. 102. Lahn M, Sundell K, Moore S. Targeting protein kinase C-alpha (PKC-a) in cancer with the phosphorothioate antisense oligonucleotide aprinocarsen. Ann N Y Acad Sci 2003; 1002:263–70. 103. Rao S, Watkins D, Cunningham D, et al. Phase II study of ISIS 3521, an antisense oligodeoxynucleotide to protein kinase C alpha, in patients with previously treated low-grade non-Hodgkin's lymphoma. Ann Oncol 2004; 15:1413–8. 104. Advani R, Peethambaram P, Lum BL, et al. A Phase II trial of aprinocarsen, an antisense oligonucleotide inhibitor of protein kinase C alpha, administered as a 21-day infusion to patients with advanced ovarian carcinoma. Cancer 2004; 100:321–6. 105. Yuen A, Advani R, Fisher G, et al. A phase I/II trial of ISIS 3521, an antisense inhibitor of protein kinase C alpha, combined with carboplatin and paclitaxel in patients with non-small cell lung cancer. Proc Am Soc Clin Oncol 2001; 19:2000 (Abstract 1802). 106. Lynch T, Raju R, Lind M, et al. Randomized phase III trial of chemotherapy and antisense, oligonucleotide LY9000003 (ISIS 3521) in patients with advanced NSCLC: Initial report. Proc Am Soc Clin Oncol 2003; 22:623 (Abstract 2604).

120

Oh et al.

107. Paz-Ares L, Douillard J-Y, Koralewski P, et al. Phase III study of gemcitabine and cisplatin with or without aprinocarsen, a protein kinase C-alpha antisense oligonucleotide, in patients with advanced-stage non-small-cell lung cancer. J Clin Oncol 2006; 24:1428–34. 108. Li H. Protein kinase C: novel isozyme-selective peptide inhibitors. Expert Opin Therapeut Patents 2006; 16:1183–7. 109. Graff JR, McNulty AM, Hanna KR, et al. The protein kinase C beta-selective inhibitor, Enzastaurin (LY317615.HCl), suppresses signaling through the AKT pathway, induces apoptosis, and suppresses growth of human colon cancer and glioblastoma xenografts. Cancer Res 2005; 65:7462–9. 110. Nakajima E, Helfrich B, Chan D, et al. Enzastaurin a protein kinase C beta-selective inhibitor, inhibits the growth of SCLC and NSCLC cell lines. Abstract No.13138. In: 2006 ASCO Annual Meeting; 2006: J Clin Oncol, 2006 ASCO Annual Meeting Proceedings Part I; 2006. p. 13138. 111. Oberschmidt O, Eismann U, Schulz L, et al. Enzastaurin and pemetrexed exert synergistic antitumor activity in thyroid cancer cell lines in vitro. Int J Clin Pharmacol Ther 2005; 43:603–4. 112. Oberschmidt O, Eismann U, Lahn MM, et al. In vitro chemosensitivity against enzastaurin correlates with gene expression of IL8 and GSK3-beta. Abstract No. 13046. In: 2006 ASCO Annual Meeting Proceedings Part I; 2006: J Clin Oncol; 2006. p. 13046. 113. Meinhold-Heerlein I, Bauerschlag DO, Bräutigam K, et al. Effects of PKC beta inhibitor enzastaurin on parental and chemoresistant ovarian cancer cell lines. Abstract No. 20037. In: J Clin Oncol, 2006 ASCO Annual Meeting Proceedings Part I, Vol 24, No 18S (June 20 Supplement), 2006: 20037; 2006; 2006. 114. Carducci MA, Musib L, Kies MS, et al. Phase I dose escalation and pharmacokinetic study of enzastaurin, an oral protein kinase C beta inhibitor, in patients with advanced cancer. J Clin Oncol 2006; 24:4092–9. 115. Green LJ, Marder P, Ray C, et al. Development and validation of a drug activity biomarker that shows target inhibition in cancer patients receiving enzastaurin, a novel protein kinase C-beta inhibitor. Clin Cancer Res 2006; 12(11 Pt 1):3408–15. 116. Oh Y, Herbst R, Burris H, Cleverly A, Lahn M, Bepler G. A phase II study of enzastaurin as second or third-line treatment of non-small cell lung cancer (NSCLC). Abstract No. 103. In: 18th EORCT-AACR-NCI Symposium on }Molecular Targets and Cancer Therapeutics}; 2006; Prague, Czech Republic: European Journal of Cancer Supplements; 2006. p. 35. 117. Robertson MJ, Kahl BS, Vose JM, et al. Phase II study of enzastaurin, a protein kinase C beta inhibitor, in patients with relapsed or refractory diffuse large B-ce lymphoma. J Clin Oncol 2007; 25(13):1741-6. 118. Fine HA, Kim L, Royce C, et al. Results from phase II trial of enzastaurin (LY317615) in patients with recurrent high grade gliomas. Abstract No. 1504. In: J Clin Oncol, 2005 ASCO Annual Meeting Proceedings; 2005: J Clin Oncol; 2005. p. 1504. 119. Beerepoot L, Rademaker-Lakhai J, Witteveen E, et al. Phase I and pharmacokinetic evaluation of enzastaurin combined with gemcitabine and cisplatin in advanced cancer. Abstract No. 2046. In: J Clin Oncol, 2006 ASCO Annual Meeting Proceedings Part I; 2006: J Clin Oncol; 2006. p. 2046. 120. Hanauske A, Weigang Koehler K, Yilmaz E, et al. Pharmacokinetic interaction and safety of enzastaurin and pemetrexed in patients with advanced or metastatic cancer. Abstract No. 2047. In: J Clin Oncol, 2006 ASCO Annual Meeting Proceedings Part I; 2006: J Clin Oncol; 2006. p. 2047. 121. Leong S, Camidge R, Eckhardt G, et al. A phase I dose-escalation and pharmacokinetic study of enzastaurin combined with capecitabine in patients with advanced cancer. Abstract No 2048. In: J Clin Oncol, 2006 ASCO Annual Meeting Proceedings Part I; 2006: J Clin Oncol; 2006. p. 2048. 122. Watkins V, Hong S, Lin BK. Enzastaurin safety review: data from phase I and phase II trials. Abstract No. 13077. In: 2006 ASCO Annual Meeting Proceedings Part I; 2006: J Clin Oncol; 2006. p. 13077.


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123. Schiller JH, Harrington D, Belani CP, et al. Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med 2002; 346:92–8. 124. Vokes E, Herbst R, Sandler A. Angiogenesis inhibition in the treatment of lung cancer. Clin Adv Hematol Oncol 2006; 4(11 Suppl 23):1–12. 125. Gridelli C, Rossi A, Maione P. New antiangiogenetic agents and non-small cell lung cancer. Crit Rev Oncol Hematol 2006; 60:76–86. 126. Sandler A, Herbst R. Combining targeted agents: blocking the epidermal growth factor and vascular endothelial growth factor pathways. Clin Cancer Res 2006; 12(14 Pt 2):4421s–5s. 127. Zinner RG, Fossella FV, Herbst RS. Pemetrexed in advanced NSCLC: a review of the clinical data. Oncology (Williston Park) 2004; 18(8 Suppl 5):54–62. 128. Massarelli E, Herbst RS. Use of novel second-line targeted therapies in non-small cell lung cancer. Semin Oncol 2006; 33(1 Suppl 1):S9–16. 129. Herbst RS, Fukuoka M, Baselga J. Gefitinib—a novel targeted approach to treating cancer. Nat Rev Cancer 2004; 4:956–65. 130. Herbst RS. Erlotinib (Tarceva): an update on the clinical trial program. Semin Oncol 2003; 30(3 Suppl 7):34–46. 131. Herbst RS, Johnson DH, Mininberg E, et al. Phase I/II trial evaluating the antivascular endothelial growth factor monoclonal antibody bevacizumab in combination with the HER-1/epidermal growth factor receptor tyrosine kinase inhibitor erlotinib for patients with recurrent non-small-cell lung cancer. J Clin Oncol 2005; 23:2544–55. 132. Wedge SR, Ogilvie DJ, Dukes M, et al. ZD6474 inhibits vascular endothelial growth factor signaling, angiogenesis, and tumor growth following oral administration. Cancer Res 2002; 62:4645–55. 133. Konner J, Dupont J. Use of soluble recombinant decoy receptor vascular endothelial growth factor trap (VEGF Trap) to inhibit vascular endothelial growth factor activity. Clin Colorec Cancer 2004; 4(Suppl 2):S81–5. 134. Heymach JV, Johnson BE, Prager D, et al. A phase II trial of ZD6474 plus docetaxel in patients with previously treated NSCLC: follow-up results. J Clin Oncol (2006 ASCO Annual Meeting Proceedings Part I, abstract 7016) 2006; 24(18S). 135. Johnson BE, Ma P, West H, et al. Preliminary phase II safety evaluation of ZD6474, in combination with carboplatin and paclitaxel, as 1st-line treatment in patients with NSCLC. J Clin Oncol (2005 ASCO Annual Meeting Proceedings Vol 23, No 16S, Part I, abstract 7102) 2005; 23(16S). 136. Rugo HS, Herbst RS, Liu G, et al. Phase I trial of the oral antiangiogenesis agent AG-013736 in patients with advanced solid tumors: pharmacokinetic and clinical results. J Clin Oncol 2005; 23:5474–83. 137. Gatzemeier U, Blumenschein G, Fosella F, et al. Phase II trial of single-agent sorafenib in patients with advanced non-small cell lung carcinoma. J Clin Oncol (2006 ASCO Annual Meeting Proceedings Part I, abstract 7002) 2006; 24(18S). 138. Black JD. Protein kinase C-mediated regulation of the cell cycle. Front Biosci 2000; 5: D406–23. 139. Lahn M, Su C, Li S, et al. Expression levels of protein kinase C-alpha in non-small-cell lung cancer. Clin Lung Cancer 2004; 6:184–9. 140. Adjei AA. What is the right dose? The elusive optimal biologic dose in phase I clinical trials. J Clin Oncol 2006; 24:4054–5. 141. Rosell R, Cecere F, Santarpia M, Reguart N, Taron M. Predicting the outcome of chemotherapy for lung cancer. Curr Opin Pharmacol 2006; 6:323–31.

8

Signal Transduction Inhibitors: PDGFR and c-KIT Inhibitors Jean-Yves Blay and Jérome Fayette Department of Médecine, Centre Leon Berard, Laennec, Lyon, France, and Unité de Jour d'Oncologie Médicale Multidisciplinaire Hôpital Edouard Herriot, Place d'Arsonval, Lyon, France

Laurent Alberti, Severine Tabone-Eglinger, and Hiba El Sayadi Department of Médecine, Centre Leon Berard, Laennec, Lyon, France

Philippe Cassie and Armelle Dufresne Department of Médecine, Centre Leon Berard, Laennec, Lyon, France, and Unité de Jour d'Oncologie Médicale Multidisciplinaire Hôpital Edouard Herriot, Place d'Arsonval, Lyon, France

Dominique Ranchère and Isabelle Ray-Coquard Department of Médecine, Centre Leon Berard, Laennec, Lyon, France

INTRODUCTION The term }targeted molecular treatment} refers to treatment strategies directed against molecular targets considered to be involved in the process of neoplastic transformation, as the result of the identification of alterations characteristic of neoplastic cells, such as specific translocations, activating mutations, or gene amplifications. The identification of these alterations has brought considerable changes to the nosological classification of cancers. It has allowed the development and evaluation of a new class of drugs that aim to block, more or less specifically, the activity of these activating proteins. Molecular targeted therapies can be divided into different categories [(1) EJO]: 1. Therapies that target molecular defects directly contributing to the initiation of malignant transformation. 2. Therapies that target later molecular defects involved in tumor progression but not in the onset of malignant transformation. 3. Therapies that target molecular defects with no direct mediating effect on cell transformation. The KIT, PDGFR, and PDGF proteins were the among first described as mutated activated in specific tumor types in humans most likely as the initial causal event of the oncogenic transformation (2–5). Tyrosine kinase inhibitors targeting these mutated activated kinases, namely imatinib (Glivec ), and, more recently, sunitinib (Sutent ), AMN107, nilotinib (Tasigna ), AMG706, valatinib, or masatinib have hence been among the earliest characterized active agents in this family of compounds. In the present chapter, we review the molecular basis and clinical activity of tyrosine kinase inhibitors targeting (KIT) and PDGFR. 123

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KIT AND PDGFR KIT and Stem-Cell Factor The product of the KIT proto-oncogene, KIT protein, is a transmembrane receptor with tyrosine-kinase activity mediated by its physiological ligand, stem-cell factor (SCF). The KIT gene is located on the long arm of chromosome 4 (4q11–q12). KIT is a 145-KD type III transmembrane tyrosine kinase receptor whose extracellular portion binds a ligand known as SCF, also called Steel factor. The intracellular portion of KIT contains the actual enzymatic domain. KIT is similar in structure to other receptor tyrosine kinases (RTK) with oncogenic capabilities, including platelet-derived growth factor receptors (PDGFRs) A and B, CSF1R and FLT3. KIT is expressed by hematopoietic progenitor cells, mast cells, germ cells, melanocytic cells, and the interstitial cells of Cajal (ICC) (2,3). KIT activation normally occurs when two adjacent receptors are brought together binding to ligand dimers. This process, known as homodimerization, is accompanied by structural changes in the receptors, resulting in activation of the kinase domains. The activated kinases then crossphosphorylate tyrosine residues in the opposed homodimer partner. The phosphotyrosines also serve as binding sites for various substrates, many of which are phosphorylated by KIT or by each other. In many cases, these substrates are themselves kinases and serve as effectors of intracellular signal transduction. As for most growth factor RTKs, multiple physiological functions have been ascribed to signal transduction mediated through KIT. These include cell survival, proliferation, differentiation, adhesion, and apoptosis (programmed cell death) (2,3). In addition, KIT function is essential for normal hematopoiesis, melanogenesis, and for the development and function of mast cells in many tissues and differentiation and proliferation of ICCs in the gut. KIT expression has been documented in a wide variety of normal cells of the hematopoietic lineage, and of the neural crest in humans as well as in their malignant counterparts. The constitutional kinase activation of KIT has been involved in the pathophysiology of tumors derived from these cell types, including mastocytosis/mast cell leukemia, germ cell tumors, small-cell lung carcinoma (SCLC), acute myelogenous leukemia (AML), neuroblastoma, melanoma, ovarian carcinoma, and breast carcinoma (2,3). PDGFRs and PDGFs Similarly, the product of PDGFRA and PDGFRB proto-oncogenes, PDGFR-a and PDGFR-b, are transmembrane receptors with tyrosine-kinase activity located on chromosomes 4 (4q11-q13) and 5 (5q31-q32) respectively. PDGFR chains associate as homo or heterodimers upon the binding of their specific ligands. The family of PDGFs includes four different polypeptides: PDGF-A, PDGF-B, PDGF-C and PDGF-D linked with an amino acid disulfide bond forming homo- or heterodimers (4–7) whose genes are located on chromosomes 7, 22, 4 and 11, respectively (4,6). These factors exert their cellular effects through PDGFR-a and PDGFR-b protein tyrosine kinase receptors. PDGFR-a is activated by PDGF-AA, PDGF-AB, PDGF-BB and PDGF-CC, while PDGF-BB and PDGF-DD bind and activate PDGF-b. A heterodimeric PDGFR-a/b complex has also been identified which can be activated by PDGF-AB, PDGF-BB, and PDGF-CC. Ligand binding induces receptor dimerization, activation and autophosphorylation of the tyrosine kinase domains.

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Platelet-derived growth factors are produced by a wide variety of different cell types. Their production is regulated by a large number of cytokines and growth factors, and also by hypoxia. PDGF are mitogens for connective tissue, in particular during wound healing, and play an important role during embryonal development. Overexpression of PDGF through dysregulation of production, or constitutional activation of receptors has been found associated with different types of malignancies, including gastrointestinal stromal tumors (GIST), DFSP, hypereosinophilic syndromes, and subsets of chronic myelomonocytic leukemias. TYROSINE KINASE INHIBITORS OF KIT AND PDGFR Imatinib Imatinib mesylate, a derivative of 2-phenylamino pyrimidine is an orally administered, small molecule selective tyrosine kinase inhibitor. In vitro studies showed this drug to bind to and specifically inhibit the activity of a small number of related tyrosine kinases, in particular Bcr-Abl, the platelet-derived growth factor (PDGF) receptors, the wild-type and mutant c-KIT (SCF receptor), and recently MCSFR/ CSF1R (9–19). Imatinib is a competitive antagonist of ATP binding that blocks the ability of c-KIT to transfer phosphate groups from ATP to tyrosine residues on substrates proteins, which in turn interrupts c-KIT mediated signal transduction. At the molecular level, this is similar to the way imatinib binds with high affinity to the conserved ATP-binding site in the tyrosine kinase domain of Abl and Bcr-Abl. Sunitinib Sunitinib malate (SU11248; Sutent) is an oral multitargeted tyrosine kinase inhibitor with direct antitumor and antiangiogenic activities. Although both sunitinib and imatinib bind within the ATP-binding domain of both KIT and PDGFRs, they are members of different chemical classes and presumably have different binding characteristics and affinities. Sunitinib inhibits multiple receptors for signalling pathways fundamental to tumor growth and survival, including PDGFR-a and -b; KIT, RET, CSF-1R; and FLT3, but also VEGFR-1, -2 and -3 in marked contrast with imatinib or nilotinib (Fig. 1) (20–23). VEGFR kinases are essential for tumor-related angiogenesis, and this property is not shared by imatinib. Sunitinib, at the recommended dose of 50 mg once daily on a 4/2 schedule (4 weeks on treatment followed by 2 weeks off), has shown significant efficacy and acceptable tolerability in prior phase I/II and III trials of advanced GIST (24). Sunitinib was approved by the U.S. Food andDrug Administration (January 2006) and the EMEA (July 2006) for the treatment of GIST after disease progression on or intolerance to imatinib mesylate therapy, as well as for the treatment of advanced renal cell carcinoma (conditional approval for cytokine-refractory renal cell carcinoma in the European Union (EU). Nilotinib Nilotinib is a second-generation oral inhibitor of selected tyrosine kinases including KIT, PDGFR, and Bcr-Abl, designed to inhibit mutated activated tyrosine kinases resistant to imatinib (25–28). In vitro data in human GIST882 cells show reduction of KIT autophosphorilation similar to imatinib, but nilotinib inhibits cell proliferation in imatinib-sensitive and -resistant GIST cell lines (GIST882, GIST430, and GIST48) more potently than imatinib. This may be related to a preferential accumulation of nilotinib intracellular concentrations, which were found higher

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FIGURE 1 Typical aspect of a false progression to imatinib in a patient with advanced GIST, with hypodense lesions becoming more visible after two months of imatinib treatment (top, before treatment; bottom, after treatment). This would be a progression according to RECIST criteria.

than those of imatinib in GIST cell lines. Nilotinib is currently being tested in imatinib (and/or sunitinib) resistant CML and in GIST (26–28). AMG706 AMG706 is a novel, orally bio-available, small-molecule multikinase inhibitor (KIT, PDGFR, VEGFR1-3 and RET) which, like sunitinib, exerts both strong antiangiogenic (VEGFR) and direct antitumor activity on activated tyrosine kinasess on tumor cells (29,30). After a phase 1 clinical trial in patients with advanced


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solid tumor malignancies, this agent is being developed currently for the treatment of GIST failing imatinib, and RET dependent tumors. Valatinib PTK787/ZK222584 is a novel, oral inhibitor of the receptor tyrosine kinases essential for molecular pathogenesis of GIST (KIT, PDGFRs, VEGFR-1, and VEGFR-2) (31,32). Initially developed for the treatment of advanced colorectal cancer in combination with chemotherapy (33), this agent has also been tested for the treatment of advanced GIST (34). TUMORS WITH MUTATIONS OF KIT, PDGFR, PDGF GIST Gastro intestinal stromal tumors are the most frequent sarcoma of the gastrointestinal tract. It arises from precursors of the interstitial cells of Cajal, the pacemaker cell of the gastrointestinal tract (35–38). The incidence of gastrointestinal stromal tumors is 1.45/100,000 persons per year (35). GIST can occur at any site of the GI tract: the most frequent primary sites are gastric (50%) and small bowel (25%). Colorectal, esophageal and peritoneal GIST are less frequent. GIST can be diagnosed at any age, with a median of 60 in large series, and are generally revealed by an abdominal mass, GI bleeding, anemia, or incidentally. Immunohistochemical analysis show that these tumors are CD117þ (95%), CD34þ (70%), smooth muscle actinþ (40%), while PS100þ in 5% of cases and desminþ in 2% of cases (37–39). Eighty five percent of GIST exhibit mutations in the KIT and PDGFRA genes. A mutation in exon 11, 9, 13 or 17 of the KIT gene is observed in 66%, 13%, 1% and 0.6% of the tumors respectively; within the PDGFRA gene, mutations of exon 18 or 12 are observed in 6% and 1% of the cases (35,40–43). Intra-abdominal tumors suspected to be a GIST in which CD117 immuno-staining is negative, should be considered for molecular analysis for KIT or PDGFR alpha mutations in expert laboratories (37,38). GIST occuring during childhood have a lower incidence of KIT and PDGFRA gene mutations. For localized tumors, risk assessment profile is based on the size and mitotic index. Serosal breaching, primary site, and the nature of the mutations in the KIT and PDGFRA genes may also be prognostic factors (40). Treatment of Localized and Advanced GIST: Role of Imatinib The treatment of localized GIST is surgery. In this setting, adjuvant treatment with imatinib is experimental (37,38). For localized tumors, wedge resection of the stomach and segmental resection of the intestine are considered adequate treatments since GIST tend to grow out of the primary organ. Adjacent organs adherent to the mass should be resected en-bloc with the tumor, in order to avoid capsule rupture and intra-abdominal spillage. Though positive resection margins have not been definitely demonstrated to compromise survival, a re-excision should be considered in cases of intramural, intra-lesionally excised tumors, without infiltration of the serosal surface (37,38). Adjuvant imatinib is not a standard option and should only be given in randomized clinical trials. Neo-adjuvant imatinib should only be given in inoperable tumors, or in tumors when function sparing surgery is the goal (37,38). Conversely, for metastatic or relapsing GIST,

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imatinib is currently the standard first-line treatment while the role of surgery is not known (37,38). In 2001, the first phase I clinical studies evaluating imatinib in advanced or metastatic GIST were started, soon followed by phase II and III trials (14–18). Two parallel phase III studies have compared a 400 mg/day imatinib dose to an 800 mg dose: the US–Canada SO033 study, and the EORTC 62005 study, in 756 and 946 patients, respectively, with advanced GIST. The SO033 trial demonstrated no difference between the two arms in terms of response to treatment, progression-free survival and overall survival. Conversely, the 62005 EORTC study that included more patients with slightly longer follow-up, reported a significant progression-free survival gain in the 800 mg arm. At 24 months of follow up, progression-free survival was 55% in the group of patients receiving 800 mg imatinib, versus 40% in the group receiving 400 mg in the 62005 trial (18). No difference in overall survival was observed in either trial. Imatinib treatment yields 60% to 70% objective responses on conventional radiography (CT-scan/MRI), with disease stabilization in 15% to 20% of the patients and 10% to 15% primary resistant tumors. PET-scan may allow for an earlier detection of imatinib efficacy in this disease. Secondary resistance to the treatment (recurrence after initial response) is now being reported in 30% to 50% of the patients, a number of whom will react positively to secondary treatment with more active, broader spectrum tyrosine kinase inhibitors as described hereafter. The one-year survival rate of patients with advanced GIST, that was approximately 35% before imatinib, is currently close to 90%. Interestingly, median progression-free survival is close to 24 months while median overall survival extends beyond 60 months in the B2222 study, the longest follow up so far (M. von Mehren et al., personal communication). Treatment with imatinib is recommended to be started in all patients with advanced GIST regardless of the presence of residual disease. It is not demonstrated that complete surgical removal of the tumor is useful in this setting (37,38). The 400 mg/day dose is the currently recommended first-line treatment in the advanced phase. Imatinib interruption at 1 year is associated with a high risk of relapse, even for patients in complete remission (45). This was demonstrated in the prospectively randomized BFR14 phase III trial comparing treatment interruption versus continuation in GIST patients responding to imatinib (47). Although most (24/26 patients) responded to imatinib reintroduction, the drug should not be discontinued outside of a clinical trial. Therefore, imatinib is given continuously, generally for several years in the majority of advanced GIST patients. At ASCO 2007, the results of the follow-up of this trial testing imatinib interruption after 3 years will be presented. Molecular Alterations of KIT and PDGFR in GIST and Response to Imatinib Molecular biology of KIT mutations remains a crucial element for treatment prognosis and response to imatinib. Heinrich et al. (41,46) demonstrated that patients bearing mutations of exon 11 had higher response rate, progression-free survival, and overall survival than patients with mutations located in exon 9 or any other part of the molecule.


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The prognostic value of the nature of mutation of KIT for response to imatinib was later on confirmed on a larger dataset of more than 355 patients of the 62005 study, confirming the better outcome of patients with exon 11 KIT mutations in GIST cells, but also the differential impact of the dose of imatinib on the subset with exon 9 mutation: in this subgroup, median PFS was 6 months with 400 mg/day, versus 18 months in the 800 mg/day arm, with a trend towards improved overall survival (44). If confirmed at the ASCO 2007 conference within the MetaGIST metaanalysis project, which pools together 1640 patients of the US-CDN SO033 study and EORTC 62005 datasets, this observation may guide the upfront dose of imatinib in the future for GIST. Activating mutations of PDGFRA are found in 36% of GIST patients presenting no detectable mutation of KIT. The presence of PDGFRA exon 12, 14 and 18 mutations was observed in 1%, 0.3% and 6% of a series of more 1000 GIST (41–43). Exon 18 point mutation (mutation D842V) was associated with no response to imatinib. Sunitinib and the Management of Resistance in GIST The management of advanced GIST failing imatinib at the dose of 400 mg/day includes several options (37,38): First, it is critical to confirm the progression— distinguishing false progressions frequently observed in the first months of imatinib treatment (Fig. 1) to identify the type of progression, that is, localized or multifocal, and to discuss the clinical case in a multidisciplinary setting to design the appropriate treatment strategy. The first important issue in cases of progression is to check that the patient is indeed taking the medication appropriately. Indeed, the compliance with the treatment may be suboptimal in some patients (75% if the drug is taken by the patient in compliance studies performed in the U.S.A.). It is therefore essential to stress with the patient that the medication be taken appropriately. The issue of compliance will probably be a major concern associated with oral TKI for the long-term treatment of cancer patients, and of course this problem is not limited to KIT and PDGFR inhibitors. In cases of genuine progression at the dose of 400 mg/day, dose escalation up to 800 mg/day is the recommended approach outside of a clinical trial in most up-to-date clinical practice guidelines (47). In cases of focal progression amenable to surgical removal of a progressive metastatic lesion, the role of surgery has not been demonstrated, and actually is not supported by data published so far. However, when complete surgical removal of all lesions is performed by an experienced team, a median progressionfree survival close to 6–8 months is observed in most studies. Additional prospective evaluation of this approach is needed. Surgery is not recommended most often in cases of multifocal progression. When available, if dose escalation up to 800 mg/day fails or is not feasible for toxicity reasons, sunitinib at the dose of 50 mg/day for 4 weeks every 6 weeks is generally recommended. In a phase III trial, sunitinib, given at 50 mg/day for 4 weeks every 6 weeks, demonstrated a significant improvement in PFS over placebo, translating in a 50% reduction in the risk of death compared to placebo (24). Yet, the control arm in this trial was placebo and some investigators now consider that some patients with focal progression on imatinib still benefit from the drug. Imatinib withdrawal probably results in a flare up of sensitive clones in some if not most patients.

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Of note, preliminary results suggest that GIST with mutations of exon 9 or WT KIT may have a longer TTP than patients with exon 11 mutations (48), while in the latter group, the presence of secondary mutations on exon 13 or 14 is associated with a better outcome than the presence of secondary mutations on exon 17 or 18 (48). This points out possible adaptations of doses of imatinib or selection of secondary agents according to the nature of mutations. Interestingly, a phase II trial has recently been reported investigating a continuous daily dosage of 37.5 mg/day, based on the observation that some patients seem to experience disease progression during the 2-week break of the standard dosing schedule. Response and PFS was found to be similar to that of the standard 4/6 weeks schedule (49). Sunitinib is now the standard option of GIST patients failing or intolerant to imatinib. Therapeutic Agents Under Clinical Investigation AMG706 The results of a multi-center single arm phase II study of AMG706 for the treatment of advanced imatinib-resistant GIST were presented at the recent CTOS 2006 meeting. The primary endpoint was objective response per RECIST by independent review and secondary efficacy endpoints including week 8 FDG-PET response (>25% decrease in average SUVmax of target lesions) and Choi response (10% decrease in uni-dimensional tumor size or 15% decrease in tumor density Hounsfield units) by contrast-enhanced CT (50). Patients received AMG706 125 mg/day orally until PD or toxicity. One hundred thirty-eight patients received at least 1 dose of AMG706. Response rate according to RECIST, PET or Choi criteria (50) were 3%, 31%, 38%, respectively. AMG706 therapy induced durable SD (22 weeks) in 30 of the 120 evaluable patients (25%). Estimated median TTP for responders according to Choi criteria is 23 and 15 weeks for non-responders. Treatment-related adverse events that occurred in 20% of the patients were: diarrhea (49%), hypertension (47%), fatigue (30%), headache (25%) and nausea (20%). Thrombo-embolic events occurred in nine patients (7%). AMG706 demonstrated an encouraging clinical benefit rate (PR þ durable SD 22 weeks) of 28% in patients with advanced high-dose imatinib-resistant GIST. Nilotinib A Phase I study of AMN107 alone and in combination with imatinib has been performed in patients with imatinib-resistant gastrointestinal stromal tumors (GIST) as well as sunitinib resistance (for 67% of patients) (51). The results were presented at the recent ESMO 2006 conference (51). Cohorts of imatinib-resistant GIST patients with radiological progressive disease (PD) were treated with AMN107 alone (400 mg p.o. bid) or with escalating doses of AMN107 (200 mg/day, 400 mg/day, or 400 mg bid) in combination with imatinib (400 mg p.o. bid). Thirty-seven patients (15 women and 22 men), median age 50 years (range 24–83) received AMN107 alone (n ¼ 18) or in combination up to 400 mg bid with imatinib (n ¼ 19) for 9–193 days (median 114 days). Serious adverse events reported in 10 patients included nausea, vomiting, abdominal pain, peritonitis, rectal bleeding and anemia. The combination of AMN107 400 mg bid and imatinib 400 mg bid is associated with excessive skin toxicity. Four patients treated with AMN107 400 mg bid and imatinib 400 mg bid required dose reductions because of grade 3 skin rash. Thirtysix patients are evaluable for efficacy. Sixteen patients exhibited PD (44%),

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17 patients (47%) achieved stable disease (SD) lasting 2 to more than 6 months, and 2 patients had partial response (PR) after the first month of treatment. One of these two patients is receiving AMN107 alone and the other AMN107 plus imatinib. Preliminary efficacy data suggest there may be relevant activity of AMN107 alone and in combination with imatinib in imatinib-resistant metastatic GIST patients. Valatinib A small phase II trial of valatinib 1250 mg/day was presented at ASCO 2006 on 15 patients failing imatinib (34). Two (13%) patients achieved PR, eight (53%) had SD for 3 months or longer, and five progressed. The clinical benefit rate (PR þ SD) was 67% (95% CI, 38–86%). The duration of the 2 PR was 290 and 393þ days, and for the 8 SDs from 137 to 498þ days. The median time-to-progression was 8.9 months. This agent needs further investigation in a larger cohort of patients. Masatinib A phase I study of masatinib, an inhibitor of KIT and PDGFR in pretreated GIST and other tumor types, as well as a window phase II study in untreated GIST will be presented at ASCO 2007. Towards Multiple Lines of Therapy in Advanced GIST Tumor control and responses have now been reported with at least four different agents in patients with advanced GIST failing imatinib, including heavily pretreated patients in fourth or fifth line of TKI (Table 1). Long-term survival in patients failing imatinib is therefore not infrequent and it is interesting to note that, while the median PFS of first-line GIST patients failing imatinib is 24 months, median overall survival of the same series extends beyond 60 months in the B2222 trial (the trial with the longest follow-up). Since most patients will experience long-term survival following imatinib resistance, it has been recently stressed in consensus conferences and clinical practice guidelines that continuous inhibition of KIT should be provided even in patients progressing under imatinib 400 mg/day (37,38). TABLE 1 Response and Progression-Free Survival (PFS) in GIST

Imatinib first line 400 mg/day 800 mg/day Imatinib second line 800 mg/day Sunitinib second line 50 mg/day 4/6 wk Nilotinib 800 mg/day or Imatinib þ Nilotinib Valatinib 1250 mg/day AMG706 125 mg/day

n

Response (%)

Median PFS (month)

473 473

50 54

19 24

18

108

3

3

47

207

8

6

24

48

4

5

51

15

13

8.9

34

120

3

4

30

Abbreviation: GIST, gastrointestinal stromal tumors.

References

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Other Diseases Involving PDFR or PDGF Mutations GIST is the paradigm of a solid tumor treated by tyrosine kinase inhibitors specifically targeting the kinase causing neoplastic transformation. In 2007, a growing number of tumor models benefits from this type of therapy. Chronic Myelomonocytic Leukemia A subset of chronic myelomonocytic leukemia (CMML), characterized by a translocation involving PDGF receptor beta fused with the TEL gene, is a rare variant of CMML but has been demonstrated to be efficiently treated with imatinib in several reports (52–59). Darier–Ferrand Dermatofibrosarcoma Protuberans Darier–Ferrand dermatofibrosarcoma Protuberans (DFSP) is a rare connective tissue disorder of the skin associated with a translocation fusing the collagen 1a1 gene with the beta chain of PDGF (60–62). This molecular alteration results in an autocrine loop driving tumor cell proliferation. Although most often treated with surgery only, some patients may experience local or metastatic relapse (63–65), or may present initially with large unresectable primary lesions (66). In this rare condition, imatinib has been demonstrated to induce prolonged responses in more than 50% of the patients (63–66). Clinical trials are ongoing to establish the exact level of activity of this compound. Hypereosinophilic Syndromes In the last decade, the understanding of the molecular pathophysiology of eosinophilic disorders has considerably improved with the identification of recurrent molecular abnormalities. The majority of these genetic lesions result in constitutively activated fusion tyrosine kinases, resulting in an eosinophiliaassociated myeloid disorder. The recent discovery of the cryptic FIP1L1-PDGFRA gene fusion in karyotypically normal patients with systemic mast cell disease with eosinophilia or idiopathic HES, has redefined these diseases as clonal eosinophilias. In these cases, PDGFR inhibitors such as imatinib or nilotinib have been tested with demonstrated antitumor efficacy (67,68). Empirical Use of KIT and PDGFR Tyrosine Kinase Inhibitors Aggressive Fibromatosis Aggressive fibromatosis also known as desmoid tumors (AF/DT) are rare connective tissue tumors with a malignant loco-regional behavior. When local treatments have failed, cytotoxic agents and hormonal treatment have been reported to induce tumor control in some patients, but only few prospective phase II trials have been reported in the literature. Recently, anti-tumor activity of imatinib in AF/DT was reported in 3 phase II trials. In a first series of 15 patients, a response rate of 17% and a 1 year PFS of 37% was reported (69). In a recently reported phase II trial including 40 patients treated with one year imatinib at the dose of 400 mg/day, PFS at one year was found to be 71% with a median treatment duration of imatinib of 9 months (range 0.8–13.8) and a median follow-up of 13.8 months, 13 of the 40 patients had progressed, 2 of the 4 patients who have interrupted treatment after progression have reprogressed after 4 and 6 months of imatinib interruption. Imatinib induces prolonged disease stabilization in the majority of evaluable patients with AF/DT (70). The exact role


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of KIT and PDGFR modulation in imatinib activity is under investigation. Interestingly, mutations in exon 10 of the KIT gene were observed in patients responding to imatinib (71). However, these alterations are neither sufficient nor necessary for response to imatinib. Chordomas Chordoma is a rare (0.1/100,000/year) neoplastic disorder arising from notochordal remnants. Activated PDGF receptor PDGFRB and its ligand PDGFB has been reported in a series of chordoma patients (72). PDGFRB, and to a lesser extent PDGFRA and KIT, were found highly expressed and phosphorylated, suggesting a possible autocrine/paracrine loop. These results may account for the antitumor activity of imatinib reported by the same group in a prospective series of patients (73). Adenoid Cystic Carcinomas Adenoid cystic carcinoma are rare tumors with frequent overexpression of the KIT protein. PDGFR has also been reported expressed in these tumors, using immunohistochemical analysis of protein expression. Although occasional responses were observed in some patients (74–76), the majority of the reported observations found no evidence of tumor control. The molecular basis for response to imatinib in this disease is not known. Gliomas Autocrine PDGFR stimulation has been demonstrated to contribute to the development of brain tumors: gliomas express epithelial growth factor receptors and/or PDGFR, and overexpression of PDGFR-a and PDGF ligands has been documented in glioblastoma. Cell lines and xenograft models exhibit reduced growth when treated with PDGFR tyrosine kinase inhibitors (79,80). Interestingly responses to imatinib have been reported in patients with gliomas and glioblastoma, in particular when combined with hydroxyurea (81–83). Prostate Cancer Prostate cancer is a major cause of mortality in men. PDGFR has been reported to be overexpressed in the majority of prostate metastases to the bone as well as in primary prostate cancer, and has been suggested to contribute to tumor progression in preclinical models of prostate cancers (84,85). Interestingly, in these models, imatinib has been found to delay tumor progression, reduce tumor cell proliferation, and promote apoptosis while reducing significantly phosphorylated PDGF-R phosphorylation (86). CONCLUSION KIT and PDGFR expression using immunohistochemistry (IHC) on tumor cells are neither necessary nor sufficient criteria to predict the efficacy of a KIT tyrosine kinase inhibitor in a clinical setting. Negative phase II trials in KIT positive melanoma, NSCLC, Ewing tumors, sarcomas, and so on, have well demonstrated this point, despite anecdotal responses in some subtypes (87). This is even more critical for PDGFR for which the reproductibility of IHC detection has been questioned. The existence of activating mutations of these kinases may enable more efficient identification of sensitive tumor types (88). Screening for these and

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other mutations is likely to be the most efficient strategy to identify novel nosological entities susceptible to tyrosine kinase inhibition in the future. ACKNOWLEDGMENTS Supported by a grant from the 2003 Emergence Fund of the French Institut National du Cancer (INCa) through the Canceropole CLARA, an unrestricted grant of the Comité de l'Ain de la Ligue Contre Le Cancer, a Grant from the Comité du Rhône de la Ligue Contre Le Cancer, and the CONTICANET Network of Excellence of the 6th Framework Program of the European Commission. SUMMARY KIT, PDGFR, or PDGF are mutated and activated in several different tumor types, including hematological malignancies and solid tumors, most often arising from the connective tissue. The identification of the specific genetic alterations (translocation, deletion, point mutations, amplifications), which most often distinguishes specific nosological entities (GIST, DFSP, HES, CMML), has paved the way for testing inhibitors of these activated tyrosine kinases in clinical settings. As of 2007, GIST, DFSP, CMML, and HES have been clearly demonstrated to be highly sensitive to these targeted therapies in the clinical setting. The impact of these treatments in other diseases, such as gliomas, chordoma, aggressive fibromatosis, and prostate carcinomas, where a contribution of PDGFR and/or KIT is suspected, is currently under investigation. Future clinical trials testing KIT and PDGFR inhibitors will have to be based upon a strong biological rationale—the identification of a constitutional activation of one of the targets contributing to neoplastic transformation or progression—and to be combined with translational research programs designed to evaluate the response of the molecular target in vivo in the clinical setting. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

Blay JY, Le Cesne A, Alberti L, Ray-Coquard I. Targeted cancer therapies. Bull Cancer 2005; 92:E13–8. Longley BJ, Reguera MJ, Ma Y. Classes of c-kit activating mutations: proposed mechanisms of action and implications for disease classification and therapy. Leuk Res 2001; 25:571–576. Vliagoftis H, Worobec AS, Metcalf DD. The proto-oncogene c-kit and c-kit ligand in human disease. J Alleg Clin Immunol 1997; 100:435–40. Betsholtz C, Johnsson A, Heldin CH, et al. cDNA sequence and chromosomal localization of human platelet-derived growth factor A-chain and its expression in tumour cell lines. Nature 1986; 320:695–9. Li X, Ponten A, Aase K, et al. PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor. Nat Cell Biol 2000; 2:302–9. Uutela M, Lauren J, Bergsten E, et al. Chromosomal location, exon structure, and vascular expression patterns of the human PDGFC and PDGFD genes. Circulation 2001; 103:2242–7. Fredriksson L, Li H, Eriksson U. The PDGF family: four gene products form five dimeric isoforms. Cytokine Growth Factor Rev 2004; 15:197–204. Board R, Jayson GC. Platelet-derived growth factor receptor (PDGFR): a target for anticancer therapeutics. Drug Resist Updat 2005; 8:75–83. Druker BJ, Tamura S, Buchdunger E. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 1996; 2:561–6.


10. 11. 12. 13.

14. 15. 16. 17.

18. 19. 20. 21.

22. 23. 24. 25. 26. 27. 28. 29.

135

Buchdunger E, Cioffi CL, Law N. Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. J Pharmacol Exp Ther 2000; 295:139–45. Savage DG, Antman KH. Imatinib mesylate—a new oral targeted therapy. N Engl J Med 2002; 346:683–93. Druker BJ, Talpaz M, Resta DJ, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001; 344:1084–6. Druker BJ, Sawyers CL, Kantarjian H, et al. Activity of a specific inhibitor of the BCRABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N Engl J Med 2001; 344:1038–42. Joensuu H, Roberts PJ, Sarlomo-Rikala M, et al. Effect of the tyrosine kinase inhibitor STI-571 in a patient with a metastatic gastrointestinal stromal tumor. N Engl J Med 2001; 344:1052–6. Van Oosterom A, Judson I, Verwej J, et al. Safety and efficacy of Imatimib (STI-571) in metastatic gastrointestinal stromal tumors: a phase I study. Lancet 2001; 358:1421–3. Demetri GD, von Mehren M, Blanke CD, et al. Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med 2002; 347:472–80. Verweij J, van Oosterom AT, Blay JY, et al. Imatinib (Gleevec) an active agent for gastrointestinal stromal tumors (GIST), but not for other soft tissue sarcoma (STS) subtypes not characterized for KIT and PDGF-R expression, results of EORTC phase II studies. Eur J Cancer 2003; 39:2006–11. Verweij J, Casali P, Zalcberg J, et al. Improved progression free survival in gastrointestinal stromal tumors with high dose imatinib. Results of a randomized phase III study of the EORTC, ISG and AGITG. Lancet 2004; 364:1127–34. Dewar AL, Cambareri AC, Zannettino AC, et al. Macrophage colony-stimulating factor receptor c-fms is a novel target of imatinib. Blood 2005; 105:3127–32. Abrams TJ, Lee LB, Murray LJ, Pryer NK, Cherrington JM. SU11248 inhibits KIT and platelet-derived growth factor receptor beta in preclinical models of human small cell lung cancer. Mol Cancer Ther 2003; 2:471–8. [Abstract-MEDLINE] Mendel DB, Laird AD, Xin X, et al. In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and plateletderived growth factor receptors: determination of a pharmacokinetic/pharmacodynamic relationship. Clin Cancer Res 2003; 9:327–37. Murray LJ, Abrams TJ, Long KR, et al. SU11248 inhibits tumor growth and CSF-1Rdependent osteolysis in an experimental breast cancer bone metastasis model. Clin Exp Metastasis 2003; 20:757–66. O'Farrell AM, Abrams TJ, Yuen HA, et al. SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent activity in vitro and in vivo. Blood 2003; 101:3597–605. Demetri GD, van Oosterom AT, Garrett CR, et al. Efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumour after failure of imatinib: a randomised controlled trial. Lancet 2006; 368:1329–38. Weisberg E, Manley PW, Breitenstein W, et al. Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl. Cancer Cell 2005; 7:129–41. Prenen H, Guetens G, de Boeck G, et al. Cellular uptake of the tyrosine kinase inhibitors imatinib and AMN107 in gastrointestinal stromal tumor cell lines. Pharmacology 2006; 77:11–16. Weisberg E, Wright RD, Jiang J, et al. Effects of PKC412, Nilotinib, and Imatinib against GIST-associated PDGFRA mutants with differential imatinib sensitivity. Gastroenterology 2006; 131:1734–42. Kantarjian H, Giles F, Wunderle L, et al. Nilotinib in imatinib-resistant CML and Philadelphia chromosome-positive ALL. N Engl J Med 2006; 354:2542–51. Polverino A, Coxon A, Starnes C, et al. AMG 706, an oral, multikinase inhibitor that selectively targets vascular endothelial growth factor, platelet-derived growth factor, and kit receptors, potently inhibits angiogenesis and induces regression in tumor xenografts. Cancer Res 2006; 66:8715–21.

136 30.

31.

32. 33. 34. 35. 36. 37. 38.

39. 40. 41. 42. 43. 44.

45.

46. 47.

Blay et al.

Benjamin R, Schöffski P, Hartmann JT, et al. Initial results of a multicenter single arm phase II study of AMG 706, an oral multi-kinase inhibitor, for the treatment of advanced imatinib-resistant gastrointestinal stromal tumors (GIST). Abstract CTOS 2006. Wood JM, Bold G, Buchdunger E, et al. PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration. Cancer Res 2000; 60:2178–89. Thomas AL, Morgan B, Horsfield MA, et al. Phase I study of the safety, tolerability, pharmacokinetics, and pharmacodynamics of PTK787/ZK 222584 administered twice daily in patients with advanced cancer. J Clin Oncol 2005; 23:4162–71. Tyagi P. Vatalanib (PTK787/ZK 222584) in combination with FOLFOX4 versus FOLFOX4 alone as first-line treatment for colorectal cancer: preliminary results from the CONFIRM-1 trial. Clin Colorec Cancer 2005; 5:24–6. Joensuu H, De Braud F, Coco P, et al. A phase II, open-label study of PTK787/ ZK222584 in the treatment of metastatic gastrointestinal stromal tumors (GISTs) Resistant to imatinib mesylate. J Clin Oncol 2006; Proc ASCO 2006; 24(18S):9531. Kindblom LG, Meis KindBlom J, Bumming P, et al. Incidence, prevalence, phenotype and biologic spectrum of gastrointestinal stromal tumors (GIST): a population based study of 600 cases. Ann Oncol 2003; 13:157. Corless CL, Fletcher JA, Heinrich MC. Biology of gastrointestinal stromal tumors. J Clin Oncol 2004; 22:3813–25. Demetri GD, Benjamin RS, Blanke C, et al. Optimal management of patients with gastrointestinal stromal tumors. Expansion and update of NCCN clinical practice guidelines. JNCCN 2004; 2(Suppl. 1):1–35. Blay JY, Bonvalot S, Casali P, et al. GIST consensus meeting panelists. Consensus meeting for the management of gastrointestinal stromal tumors. Report of the GIST Consensus Conference of 20–21 March 2004, under the auspices of ESMO. Ann Oncol 2005; 16:566–78. Fletcher CDM, Berman JJ, Corless CL, et al. Diagnosis of gastrointestinal stromal tumors: a consensus approach. Hum Pathol 2002; 33:459–65. Miettinen M, Lasota J. Gastrointestinal stromal tumors: review on morphology, molecular pathology, prognosis, and differential diagnosis. Arch Pathol Lab Med 2006; 130:1466–78. Heinrich MC, Corless CL, Blanke CD, et al. Molecular correlates of imatinib resistance in gastrointestinal stromal tumors. J Clin Oncol 2006; 24:4764–74. Heinrich MC, Corless CL, Duensing A, et al. PDGFRA activating mutations in gastrointestinal stromal tumors. Science 2003; 299:708–10. Epub 2003 Jan 09. Corless CL, Schroeder A, Griffith D, et al. PDGFRA mutations in gastrointestinal stromal tumors: frequency, spectrum and in vitro sensitivity to imatinib. J Clin Oncol 2005; 23:5357–64. Debiec-Rychter M, Sciot R, Le Cesne A, et al. On behalf of the EORTC soft tissue and bone sarcoma group, The Italian Sarcoma Group and the Australasian GastroIntestinal Trials Group. KIT mutations and dose selection for imatinib in patients with advanced gastrointestinal stromal tumours. Eur J Cancer 2006; [Epub ahead of print] Blay JY, Le Cesne A, Ray-Coquard I, et al. A prospective multicentric randomized phase III study of imatinib in patients with advanced gastrointestinal stromal tumours comparing interruption versus continuation of treatment beyond 1 year. J Clin Oncol, accepted for publication. Heinrich MC, Corless CL, Demetri GD, et al. Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor. J Clin Oncol 2003; 21:4342–9. Zalcberg JR, Verweij J, Casali PG, et al. EORTC Soft Tissue and Bone Sarcoma Group, the Italian Sarcoma Group; Australasian Gastrointestinal Trials Group. Outcome of patients with advanced gastro-intestinal stromal tumours crossing over to a daily imatinib dose of 800 mg after progression on 400 mg. Eur J Cancer 2005; 41:1751–7.


48. 49. 50. 51.

52. 53. 54. 55. 56. 57. 58. 59. 60. 61.

62. 63. 64. 65. 66. 67.

137

Heinrich MC, Maki RG, Corless CL, et al. Sunitinib (SU) response in imatinib-resistant (IM-R) GIST correlates with KIT and PDGFRA mutation status. J Clin Oncol 2006; 24(Suppl):9502. Blay J-Y, George S, Casali PG, et al. Clinical benefit of continuous daily dosing of sunitinib in patients with advanced gastrointestinal stromal tumour (GIST). Proc ESMO 2006. Choi H, Charnsangavej C, de Castro Faria S, et al. CT evaluation of the response of gastrointestinal stromal tumors after imatinib mesylate treatment: a quantitative analysis correlated with FDG PET findings. Am J Roentgenol 2006; 183:1619–28. Von Mehren M, Reichardt P, Casali PG, et al. A phase I study of nilotinib alone and in combination with imatinib (IM) in patients (pts) with imatinib-resistant gastrointestinal stromal tumors (GIST)—study update. J Clin Oncol 2007; Proc ASCO 2007; 25(18S): 10023. Apperley JF, Gardembas M, Melo JV, et al. Response to imatinib mesylate in patients with chronic myeloproliferative diseases with rearrangements of the platelet-derived growth factor receptor beta. N Engl J Med 2002; 347:481–7. Magnusson MK, Meade KE, Nakamura R, Barrett J, Dunbar CE. Activity of STI571 in chronic myelomonocytic leukemia with a platelet-derived growth factor beta receptor fusion oncogene. Blood 2002; 100:1088–91. Gunby RH, Cazzaniga G, Tassi E, et al. Sensitivity to imatinib but low frequency of the TEL/PDGFRbeta fusion protein in chronic myelomonocytic leukemia. Haematologica 2003; 88:408–15. Cortes J, Kantarjian H. Beyond chronic myelogenous leukemia: potential role for imatinib in Philadelphia-negative myeloproliferative disorders. Cancer 2004; 100:2064–78. Onida F, Beran M. Chronic myelomonocytic leukemia: myeloproliferative variant. Curr Hematol Rep 2004; 3:218–26. Krystal GW. Imatinib mesylate (STI571) for myeloid malignancies other than CML. Leuk Res 2004; 28(Suppl 1):S53–9. Cortes J. CMML: a biologically distinct myeloproliferative disease. Curr Hematol Rep 2003; 2:202–8. Cortes J, Giles F, O'Brien S, et al. Results of imatinib mesylate therapy in patients with refractory or recurrent acute myeloid leukemia, high-risk myelodysplastic syndrome, and myeloproliferative disorders. Cancer 2003; 97:2760–6. Simon MP, Pedeutour F, Sirvent N, et al. Deregulation of the platelet-derived growth factor B-chain gene via fusion with collagen gene COL1A1 in dermatofibrosarcoma protuberans and giant-cell fibroblastoma. Nat Genet 1997; 15:95–8. Shimizu A, O'Brien KP, Sjoblom T, et al. The dermatofibrosarcoma protuberansassociated collagen type Ialpha1/platelet-derived growth factor (PDGF) B-chain fusion gene generates a transforming protein that is processed to functional PDGF-BB. Cancer Res 1999; 59:3719–23. Sjoblom T, Shimizu A, O'Brien KP, et al. Growth inhibition of dermatofibrosarcoma protuberans tumors by the platelet-derived growth factor receptor antagonist STI571 through induction of apoptosis. Cancer Res 2001; 61:5778–83. Maki RG, Awan RA, Dixon RH, Jhanwar S, Antonescu CR. Differential sensitivity to imatinib of 2 patients with metastatic sarcoma arising from dermatofibrosarcoma protuberans. Int J Cancer 2002; 100:623–6. Labropoulos SV, Fletcher JA, Oliveira AM, Papadopoulos S, Razis ED. Sustained complete remission of metastatic dermatofibrosarcoma protuberans with imatinib mesylate. Anticancer Drugs 2005; 16:461–6. McArthur GA, Demetri GD, van Oosterom A, et al. Molecular and clinical analysis of locally advanced dermatofibrosarcoma protuberans treated with imatinib: imatinib target exploration consortium study B2225. J Clin Oncol 2005; 23:866–73. Price VE, Fletcher JA, Zielenska M, et al. Imatinib mesylate: an attractive alternative in young children with large, surgically challenging dermatofibrosarcoma protuberans. Pediatr Blood Cancer 2005; 44:511–5. Tefferi A, Pardanani A. Imatinib therapy in clonal eosinophilic disorders, including systemic mastocytosis. Int J Hematol 2004; 79:441–7.

138 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84.

85. 86. 87.

Blay et al.

Stover EH, Chen J, Lee BH, et al. The small molecule tyrosine kinase inhibitor AMN107 inhibits TEL-PDGFRbeta and FIP1L1-PDGFRalpha in vitro and in vivo. Blood 2005; 106:3206–13. Epub 2005 Jul 19. Heinrich MC, McArthur GA, Demetri GD, et al. Clinical and molecular studies of the effect of imatinib on advanced aggressive fibromatosis (desmoid tumor). J Clin Oncol 2006; 24:1195–203. Penel N, Le Cesne A, Bui B, et al. Imatinib for the treatment of aggressive fibromatosis failing local treatment. A phase II trial of the French Sarcoma Group. Proc Am Soc Clin Oncol 2006; 24:9516. Goncalves A, Monges G, Yang Y, et al. Response of a KIT-positive extra-abdominal fibromatosis to imatinib mesylate and KIT genetic analysis. J Natl Cancer Inst 2006; 98:562–3. Tamborini E, Miselli F, Negri T, et al. Molecular and biochemical analyses of plateletderived growth factor receptor (PDGFR) B, PDGFRA, and KIT receptors in chordomas. Clin Cancer Res 2006; 12:6920–8. Casali PG, Messina A, Stacchiotti S, et al. Imatinib mesylate in chordoma. Cancer. 2004; 101:2086–97. Ochel HJ, Gademann G, Rocken C, Wordehoff H. Effects of imatinib mesylate on adenoid cystic carcinomas. Anticancer Res 2005; 25:3659–64. Faivre S, Raymond E, Casiraghi O, Temam S, Berthaud P. Imatinib mesylate can induce objective response in progressing, highly expressing KIT adenoid cystic carcinoma of the salivary glands. J Clin Oncol 2005; 23:6271–3. Pfeffer MR, Talmi Y, Catane R, Symon Z, Yosepovitch A, Levitt M. A phase II study of Imatinib for advanced adenoid cystic carcinoma of head and neck salivary glands. Oral Oncol. 2006; [Epub ahead of print]. Fleming TP, Saxena A, Clark WC, et al. Amplification and/or overexpression of platelet-derived growth factor receptors and epidermal growth factor receptor in human glial tumors. Cancer Res 1992; 52:4550–3. Hermanson M, Funa K, Koopmann J, et al. Association of loss of heterozygosity on chromosome 17p with high platelet-derived growth factor a receptor expression in human malignant gliomas. Cancer Res 1996; 56:164–71. Shamah SM, Stiles CD, Guha A. Dominant-negative mutants of platelet-derived growth factor revert the transformed phenotype of human astrocytoma cells. Mol Cell Biol 1993; 13:7203–12. Puputti M, Tynninen O, Sihto H, et al. Amplification of KIT, PDGFRA, VEGFR2, and EGFR in gliomas. Mol Cancer Res 2006; 4:927–34. Jayawardena S, Sooriabalan D, Indulkar S, Kim HH, Matin A, Maini A. Regression of grade III astrocytoma during the treatment of CML with imatinib mesylate. Am J Ther 2006; 13:458–9. Wen PY, Yung WK, Lamborn KR, et al. Phase I/II study of imatinib mesylate for recurrent malignant gliomas: North American Brain Tumor Consortium Study 99–08. Clin Cancer Res 2006; 12:4899–907. Reardon DA, Egorin MJ, Quinn JA, et al. Phase II study of imatinib mesylate plus hydroxyurea in adults with recurrent glioblastoma multiforme. J Clin Oncol 2005; 23:9359–68. Fudge K, Wang CY, Stearns ME. Immunohistochemistry analysis of platelet-derived growth factor A and B chains and platelet-derived growth factor alpha and beta receptor expression in benign prostatic hyperplasia and Gleason-graded human prostate adenocarcinomas. Mod Pathol 1994; 7:549–54. Chott A, Sun Z, Morganstern D, et al. Tyrosine kinases expressed in vivo by human prostate cancer bone marrow metastases and loss of the type 1 insulin-like growth factor receptor. Am J Pathol 1999; 155:1271–9. Uehara H, Kim SJ, Karashima T, et al. Effects of blocking platelet-derived growth factor-receptor signaling in a mouse model of experimental prostate cancer bone metastases. J Natl Cancer Inst 2003; 95:458–70. Fiorentini G, Rossi S, Lanzanova G, et al. Tyrosine kinase inhibitor imatinib mesylate as anticancer agent for advanced ocular melanoma expressing immunoistochemical


88.

139

C-KIT (CD 117): preliminary results of a compassionate use clinical trial. J Exp Clin Cancer Res 2003; 22(4 Suppl):17–20. Burger H, den Bakker MA, Kros JM, et al. Activating mutations in c-KIT and PDGFRalpha are exclusively found in gastrointestinal stromal tumors and not in other tumors overexpressing these imatinib mesylate target genes. Cancer Biol Ther 2005; 4:1270–4. [Epub 2005 Nov 18.]

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The Insulin-Like Growth Factor 1 Receptor: A Target for Cancer Treatment Yungan Tao, Jean Bourhis, and Eric Deutsch Department of Radiation Oncology, Institute Gustave Roussy, Villejuif, France

INTRODUCTION The success of kinase inhibitors, such as imatinib (Glivec ; Novartis, Basel, Switzerland), targeting tyrosine kinase (TK)–c-abl, and monoclonal antibodies, such as trastuzumab (Herceptin ; Genentech, South San Francisco, California, U.S.A.), interfering with the function of the human epidermal growth factor receptor-2 (HER-2)/Neu receptor, provides strong evidence for the disruption of signal transduction as an effective anticancer approach (1). Many other new agents are in the process of transfer into the clinic or are already approved [Bevacizumab (Avastin ; Genentech), Bortezomib (Velcade ; Millenium, Cambridge, Massachusetts, U.S.A.), Sorafenib (Nexavar ; Bayer, Leverkusen, North Rhine-Westfalia, Germany), Sunitinib (Sutent ; Pfizer, New York, U.S.A.)]. The insulin-like growth factor 1 receptor (IGF-1R) (2), which belongs to the tyrosin kinase(TK) receptor family, appears to be promising as a new target. IGF-1R is a transmembrane TK, consisting of 2a- and 2b-subunits. The extracellular a-subunits are required for ligand binding, while the transmembrane b-subunits contain the TK catalytic site and the ATP-binding site. Two ligands, IGF-1 and IGF-2, bind to IGF-1R (3). The local bioavailability of ligands is subject to complex physiological regulation and is probably abnormally high in many cancers. Ligands can be delivered from remote sites of production through the circulation or can be locally produced. IGF-binding proteins (IGFBPs) (3) and IGFBP proteases have key roles in regulating ligand bioavailability. IGFBPs prolong the half-life of IGFs, which have the potential to increase IGF-1R activation. On the other hand, these proteins have affinity for IGFs comparable to IGF-1R, and there is competition between IGFBPs and IGF-1R for available ligands in tissue microenvironment. This provides a basis for the inhibitory roles of IGFBPs on IGF-1 signaling. There is evidence that certain IGFBPs also have direct, IGF-independent, growth-regulatory actions. The IGF-2R binds IGF-2, but has no TK domain, and appears to act as a negative influence on proliferation by reducing the amount of IGF-2 available for binding to IGF-1R. Certain IGFBP proteases (often produced by neoplastic cells) that cleave IGFBPs can release free ligand and thereby increase IGF-1R activation. Following ligand binding to IGF-1R, its TK activity stimulates signaling through intracellular networks that regulate cell proliferation and cell survival. Pathways Activated by IGF-1R Binding of IGF-1 and IGF-2 to IGF-1R causes its auto-phosphorylation and leads to activation of multiple signaling pathways. There are four insulin receptor substrate (IRS) proteins in mammalian cells, but IRS-1 and IRS-2 are the most prominent in transmitting signals from either the IGF-1R or the insulin receptor (IR). At least two different major signal transduction pathways have been identified for IGF-1R. One activates Ras, Raf, and mitogen-activated protein kinase 141

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(MAPK)/extracellular signal-regulated kinase (ERK), the main mitogen–transduction pathway, and another pathway is responsible for antiapoptotic signal transduction, involving phosphatidylinositide-3-kinase (PI3K–AKT pathways (Fig. 1). Binding of extracellular ligands to IGF-1R, their cell-surface receptors, activates Ras and this initiates Raf activation (4). This leads to activation of the dual-specificity protein kinases MEK1 and MEK2 (MAPK and ERK kinase) and subsequently the MAPK/ERK proteins ERK1 and ERK2. Depending on the cellular context, this pathway mediates diverse biological functions such as cell growth, survival, and differentiation predominantly through the regulation of transcription, metabolism and cytoskeletal rearrangements. The PI3K–AKT pathway (5) is one of the most frequently altered pathways in all sporadic human tumors. The binding of a growth factor (IGF-1) to its TK receptor (IGF-1R) results in the recruitment and activation of the PI3K to the plasma membrane receptor, which in turn phosphorylates the phosphoinositides, increasing the local concentration of PIP3 and PIP2 at the plasma membrane. The PI3K activity is counteracted in the cell by PTEN, a lipid 3-phosphatase which is the second most common sporadically mutated tumor suppressor. This increase in lipid second messengers recruits and activates the PDK and AKT protein kinases at the plasma membrane where AKT is then fully activated by phosphorylation of ser-473 and thr-308. Through the phosphorylation of a diverse set of substrates, AKT regulates four intersecting biological processes: cell survival, cell-cycle progression, cell growth, and IGF-IR

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FIGURE 1 The insulin-like growth factor 1 receptor (IGF-1R) is a tyrosine kinase cell-surface receptor (RTK) that binds to either IGF-1 or IGF-2. Growth factors activate RTKs and activate two key signal-transduction components: (A) the GTPase Ras-Raf; and (B) the lipid kinase PI(3)K.

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cell metabolism. The AKT substrates that mediate some of these biological processes have been identified. AKT controls cell-cycle progression through several substrates. AKT can phosphorylate and inhibit Glycogen synthase kinase-3 (GSK-3) (6) which phosphorylates several cell-cycle regulators such as c-Myc, cyclin D1 and cyclin E and controls a number of critical cell-cycle events. AKT may also enhance the functions of some transcription factors by inactivating GSK-3. AKT controls cell survival through its inactivation of the proapoptotic protein BAD and its activation of the IkB kinase (IKK)–NFkB (nuclear factor-kB) pathway. In addition, the activated AKT protein moves to the cell nucleus where it phosphorylates the FOXO family of forkhead transcription factors, which is a set of highly conserved substrates of AKT, resulting in their removal from the nucleus into the cytoplasm and producing a change in the forkhead transcriptional activity. FOXOs are well established as being critical in the regulation of lifespan and metabolism downstream of AKT (4). PI3K and AKT are implicated in the activation of the mammalian TOR (mTOR) protein kinase by the phosphorylation of the TSC2 (4). The TSC2 tumor suppressor, tuberin, and its obligate binding partner, hamartin (TSC1), are mutated in a familial tumor syndrome called tuberous sclerosis complex (TSC). Of particular interest, recent studies have shown that AKT and mTOR can mediate activation of the hypoxia-inducible factor-1a (HIF-1a) transcription factor which increases expression of the glucose transporter GLUT1 and glycolytic enzymes, ultimately leading to increased glucose uptake. Stimulation of IGF-1R activates the PIK3–AKT–mTOR pathway causing an mTOR-dependent loss in IRS-1 expression leading to feedback downregulation of signaling through the pathway. The mTOR inhibition induces IRS-1 expression and abrogates feedback inhibition of the pathway, resulting in AKT activation in cancer cell lines and in patients treated with the mTOR inhibitors, such as rapamycin, tirosel/temsirolimus (Wyeth, Madison, New Jersey, U.S.A.) (7) or RAD001 (8). Rapamycin enhances basal AKT activity, AKT phosphorylation, and PI3K activity in multiple myeloma cells and prolongs activation of AKT induced by exogenous IGF-1. Rapamycin prevents serine phosphorylation of IRS-1, enhances IRS-1 association with IGF-1 receptors, and prevents IRS-1 degradation. Thus, mTOR inhibitors activate PI3-K/AKT in multiple myeloma cells (7); activation depends on basal IGF-R signaling; and enhanced IRS-1/IGF-1R interactions secondary to inhibited IRS-1 serine phosphorylation may play a role in activation of the cascade. This feedback inhibition could paradoxically reduce the antitumor effects of mTOR inhibitors by enhancing IGF-1 signaling. IGF-1R inhibition could prevent rapamycin-induced AKT activation and sensitize tumor cells to inhibition of mTOR. In contrast, IGF-1 antagonizes the antiproliferative effects of rapamycin in serum-free medium (8). It suggests that feedback downregulation of receptor TK signaling is a frequent event in tumor cells with constitutive mTOR activation. Reversal of this feedback loop by rapamycin may attenuate its therapeutic effects, whereas combination therapy of inhibition of IGF-1R that ablates mTOR function and prevents AKT activation may have improved antitumor activity. Evidence for IGF-1R Involvement in Cancer Many experimental IGF-1R-positive cancers could be stimulated by IGF-1 produced in tissues remote from the cancer or synthesized locally in an autocrine or paracrine manner. There is evidence from experimental systems and studies of

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clinical specimens that neoplastic progression, particularly in prostate cancer, might be associated with increased expression of IGF-1R. However, IGF-1R amplification does not seem to be frequently observed. IGF-1R seems to have a role in regulating proliferation and differentiation even if its expression levels are low; there is evidence that certain IGFBPs have IGF-independent growth-inhibitory or proapoptotic influences, and that neoplastic cells can develop resistance through these mechanisms. Tumor and IGF-1R Expression The IGF-1R has been implicated in promoting oncogenic transformation, growth, and survival of cancer cells. Several studies, both experimental and clinical, have demonstrated that the IGF-1R is overexpressed compared to normal tissues (9,10) (Table 1). Moreover, IGF-1R is ubiquitously expressed in tissues (11). Strong evidence has been provided that IGF-1R is not an absolute requirement for normal growth. IGF-1R has been shown to be crucial for anchorage independent growth (12), which is unique for malignant cells. This property of IGF-1R also implicates the function of this receptor in tumor progression since the degree of anchorage independency reflects the level of malignancy. This means that metastasis has acquired more anchorage independency and more IGF-1R dependency, compared to the primary tumor. There is a great deal of evidence based on in vitro and in vivo studies demonstrating the importance of IGF-1R signaling in mammalian cell transformation TABLE 1 IGF-1R Expression, IGF Level or Polymorphism in Tumors and Correlation with Cancer Risk, Prognosis Tumor type Prostate cancer

Breast cancer

Colorectal cancer

Lung cancer

Gastric cancer

Pancreatic cancer Bladder cancer Sarcoma Adrenal neoplasia Central nerve system

IGF-1R expression

IGF level or polymorphism

Expression in most prostate cancer cell lines, overexpression in PC-3 and DU-45 cells, etc. Higher in estrogen-dependent cell lines, presence of IGF-1R in biopsy specimens Presence on HCT 116 and CoLo-205, and human colon cancer specimens Expression common in SCLC and NSCLC

High circulating IGF-1 levels, 19-CA-repeat allele associated with worse survival, haplotypes Circulating levels of IGF and IGF-BP, 19-CA-repeat allele, A-202 C polymorphism in the IGF-BP 3 A high IGF-I/IGF-BP-3 ratio high risk cancer, CA 17 repeat allele

Overexpression in primary tumor correlated with increased lymph node metastasis Overexpression Expression Expression Overexpression in pheochromocytomas Gliomas meningiomas express receptor

IGF stimulate growth in SCLC and NSCLC cell lines, A-202C polymorphic variation of IGF-BP-3

Haplotypes for IGF-2R

Abbreviations: 1GF-1R, insulin-like growth factor 1 receptor; NSCLC, non–small cell lung cancer; SCLC, small cell lung cancer.


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and the development of tumors (13). Findings based on prostate cancer studies raised the possibility that tumor cell dependency on IGF-1R may be stage-specific. The multistep transformation of the prostate epithelium is initially IGF-1R dependent. IGF-1 has been shown to stimulate the proliferation of human prostate epithelial cells in culture and to be necessary for normal growth and development of the rat and mouse prostate. IGF-1R appears to be abundantly expressed in most prostate cancer cell lines (11). In cell lines PC-3 and DU-45, IGF-1R mRNA has been found to be overexpressed. The concentration of IGF-1R is higher in estrogen-dependent breast cell lines than in estrogen-independent cell lines. There is a positive correlation between the estrogen, progesterone, and PRL receptors and IGF-1R expression. IGF-1R expression, however, is ubiquitous or nearly ubiquitous, and its activation has been demonstrated to be a potent stimulus for growth (11). IGF-1R overexpression is observed in 43.8% of tumors in primary breast cancer patients, although IGF-1R overexpression has no correlation with prognosis or with other clinicopathologic parameters (14). IGF-1R is overexpressed in 62% of primary tumor and lymph node metastasis of gastric cancer when compared with adjacent tumor-free gastric mucosa. IGF-1R overexpression in primary tumor correlates with increased lymph node metastasis (15). IGF-1R is expressed on the human colon cancer cell lines (16), such as HCT116 and CoLo-205, and several human colon cancer specimens. A high IGF-1/IGFBP-3 ratio may increase the risk of colon cancer development. IGF receptor expression is common in lung cancer. Presence of IGF-1R mRNA has been found in all of the cell lines and most primary lung carcinomas (11). IGF-1 is a potent mitogen, stimulating growth 1.6- to 4.2-fold in a panel of small cell lung cancer (SCLC) cell lines and 1.1- to 2.7-fold in a panel of non–small cell lung cancer (NSCLC) cell lines such as NCI-H1299. Significant overexpression of the IGF-1R in human pheochromocytomas suggests IGF system involvement in the pathogenesis of adrenal neoplasia (17). Gastrointestinal neuroendocrine tumors (NET) frequently express IGFs and IGF-1R and apoptosis, and cell-cycle arrest could be induced by the IGF-1R-TK inhibitor, NVP-AEW541, in NET cells. The inhibition of the IGF/IGFR system appears to be a promising novel approach for future treatment strategies of NET disease (18). CIRCULATING LEVEL OF IGFs, POLYMORPHISMS, AND CANCER Circulating levels of IGF-1 are associated with the risk for developing prostate, breast, colorectal, and lung cancer (Table 1). IGF-2 appears to be overexpressed in most tumor cell lines. The presence of millions of genetic variations (polymorphisms) in the human genome may provide extensive biological variations that affect cancer physiologies, treatment outcome, and prognosis. Polymorphisms of genes encoding growth factors may be good candidates for a possible determinant of treatment outcome and prognosis. IGF-1 is a potent mitogen for the prostate cancer cell lines. Men with high levels of serum IGF-1 are at increased risk of developing clinically evident prostate cancer (19). Circulating levels of IGF-1 and IGFBP-3 may predict the risk of developing advanced-stage prostate cancer (20). Men in the highest quartile of IGF-1 level have a five-fold increased risk of advanced-stage prostate cancer than men in the lowest quartile. A known genetic cytosine–adenine (CA) repeat polymorphism in the promoter region of the human IGF-1 gene may be associated

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with circulating IGF-1 levels. The 19-CA-repeat allele is more frequent in prostate cancer patients than controls. Males homozygous for the 19-allele have a significantly increased risk of prostate cancer (21). The >19 repeats of IGF-1 is significantly associated with a worse cancer-specific survival and the presence of >19 repeats of IGF-1 is an independent risk factor for death along with clinical parameters. IGF-1 (CA) repeat may be a novel predictor in prostate cancer patients with bone metastasis (22). Haplotype analysis and two SNPs reveal significant associations with prostate cancer risk (23). Women with 19-CA-repeat allele homozygote and high IGF-1 levels have a much higher risk of breast cancer (24). The polymorphisms in the IGF-1 and IGFBP-3 genes are associated with an increased risk of breast cancer in familial cases carrying the variant alleles (25,26). Women in the highest quantile of circulating levels of IGF and IGFBP have more than twice the risk of developing breast cancer than those in the lowest, although this effect is only apparent at young ages (27). Colorectal cancer is modestly associated with having an IGF-1 genotype other than homozygous for 19 repeats and having the GG IGFBP-3 genotype. IGF-1 and IGFBP-3 genotype are significant effect modifiers of the relationship between risk factors (body mass index, postmenopausal hormone use and physical activity) and colorectal cancer (28). Elevated IGF-1 levels are associated with sporadic colorectal cancer (CRC) risk in hereditary nonpolyposis colorectal cancer (HNPCC). Patients carrying a shorter IGF1 CA-repeat length polymorphism (17 repeats) have higher CRC risk in HNPCC (29). A-202C polymorphic variation of IGFBP-3 gene constitutes a risk factor for NSCLC. The NSCLC risk correlated significantly with AA genotype (30). The dysregulation of IGF axis could now be considered as another important risk factor for NSCLC. IGF polymorphisms are also associated with osteogenic sarcoma (31). In addition to its role in proliferation of cancer cells, the IGF-1R protects cells from apoptosis caused by growth factor deprivation, anchorage independence, or cytotoxic drug treatment. Downregulation of IGF-1R function by antisense and dominant-negative techniques reduces the growth and tumorigenicity of several cancer cell lines in vivo and in vitro, including colon cancer, melanoma, lung cancer, ovarian cancer, glioblastoma, and neuroblastoma, and others. It is also shown that administration of a blocking antibody directed against this receptor slowed the in vivo proliferation of human breast cancer xenografts. IGF-1R is thus an attractive therapeutic target based on the hypothesis that inhibition of IGF-1R function would result in selective apoptosis and growth inhibition of tumor cells. The key role of IGF-1R signaling in cancer is underscored by the fact that IGF-1R deficient cells fail to be transformed by oncogenes such as Ras, EGFR SV40 and E6 (12). Interplay Between IGF-1R and Other Tyrosine Kinase Receptors IGF-1R signaling inteferes with numerous other growth factors or receptors such as epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and so on. Elevated VEGF levels are correlated with the lymph node metastasis, increased progression, and poor prognosis of cancer. VEGF expression is regulated by IGF-1. IGF-1 stimulates VEGF secretion and induces VEGF promoter activation. IGF-1 stimulates PI3K/Akt and Erk/MAPK pathways in


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SCC-9 cells, each contributing to Hif-1a expression and VEGF secretion (32). Increased Hif-1a expression leads to HIF-1 formation and the production of VEGF. IGF-1 regulates VEGF expression and secretion via HIF-1-dependent and independent pathways (33). Androgen ablation in LNCaP cells upregulates the transcriptional activity of VEGF-C and downregulates the IGF-1R pathway (34). Functional inhibition of IGF-1R signaling upregulates VEGF-C mRNA level. IGF-1R and EGFR are overexpressed in pancreatic cancer and their expression patterns through the cell have been shown to correlate with pancreatic cancer grade and prognosis. Membrane-dominant EGFR and cytoplasm-dominant IGF-1R are more frequent in lower-grade tumors and correlated with favorable prognosis in primary invasive ductal pancreatic carcinomas, whereas cytoplasmdominant EGFR and membrane-dominant IGF-1R are more frequent in highergrade tumors and correlated with poor prognosis (35). IGF-1R and Resistance to Targeted Therapies Signaling via IGF-1R has been associated with resistance to anti-EGFR and HER2-based therapies in the experimental system. Anti-EGFR targeting has been demonstrated to enhance apoptosis and reduce both cellular invasion and angiogenic potential in various tumor settings. However, primary resistance to anti-EGFR therapy has been observed as in preclinical studies as well as in clinical practice in tumors such as lung cancer and glioblastoma (36). Resistance to an EGFR TK inhibitor, AG1478, is associated with an up-regulation of IGF-1R levels in a glioblastoma model. Conversely, IGF-1R overexpression is found to correlate with decreased efficacy of EGF-R targeting in clinical trials, suggesting the importance of IGF-1R signaling in EGF-R inhibitors resistance. IGF-1R mediates resistance to anti-EGFR therapy in primary human glioblastoma cells through continued activation of PI3K-AKT signaling (37). Nevertheless, the recent identification of a novel mutation of the EGFR gene in lung tumors in the TK domain (T790M) of EGFR, rendering cells resistant to the EGFR TK inhibitor gefitinib, strongly suggests that tumors cells remain dependent on an active EGFR pathway for their proliferation (38). Interestingly, co-targeting IGF-1R with EGFR greatly enhances both spontaneous and radiation-induced apoptosis of a glioblastoma model. IGF-1R signaling through PI3K-AKT may represent a novel and potentially important mechanism of resistance to anti-EGFR therapy (39). The addition of an anti-IGF-1R strategy to EGFR targeting treatment may be more effective than a single-agent approach (40) and dual EGFR/ IGFR targeting compounds are currently in development. Tyrphostin AG1024 (an inhibitor of IGF-1R) is used with gefitinib for treatment of MDA468, MDA231, SK-BR-3, and MCF-7 breast cancer lines, which express similar levels of IGF-1R but varying levels of EGFR. Gefitinib and AG1024 when used in combination revealed an additive-to-synergistic effect on cell growth inhibition. Overexpression of IGF-1R in SK-BR-3 cells is sufficient to cause a marked enhancement in gefitinib resistance. IGF-1R signaling reduces the antiproliferative effects of gefitinib in several breast cancer cell lines. Similar findings of an involvement of IGF-1R in EGFR resistance mechanism were also found in pancreas and prostate cancer cell lines (41,42). Co-targeting HER2 and IGF-1R improved the efficacy of therapies directed against HER2/erbB2. In two cell lines, MCF7 and BT474 cells, IGF-1R antagonists enhance the effect of HER2 and ER antagonists. While these agents produce

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a small amount of apoptosis individually, their combination causes a dramatic degree of apoptosis, most striking for the IGF-1R/HER2 antagonist combination (43). Hence such combinations may be useful in tumors in which single drugs are inactive. In breast cancer cell models that overexpress HER2, an increased level of IGF-1R signaling appears to interfere with the action of trastuzumab. Thus, strategies that target IGF-1R signaling may prevent or delay development of resistance to trastuzumab (44,45). Trastuzumab inhibited the growth of MCF-7/ HER2-18 cells, which overexpress HER2 and express IGF-1R, only when IGF-1R signaling is minimized. In SKBR3 cells, which overexpress HER2 but express few IGF-1R, trastuzumab reduced proliferation by 42% regardless of IGF-1 concentration. When SKBR3 cells are genetically altered to overexpress IGF-1R and cultured with IGF-1, trastuzumab has no effect on proliferation. However, the addition of IGFBP-3, which decreased IGF-1R signaling, restored trastuzumab-induced growth inhibition. A strong synergistic interaction has been found in combining trastuzumab and reduction of IGF-1R signaling by expression of dominant-negative IGF1R in HER2—overexpressing MCF7her18 breast cancer cells—and this resulted in potentiation of growth inhibition in transfected cancer cells (46). Simultaneously co-targeting TK receptors may be therapeutically useful, and may provide a specific rationale for combining IGF-1R and HER2 targeting strategies in antineoplastic approaches. We recently found that Bcr-Abl expressing cells harboring imatinib (an inhibitor of the SCF-KIT loop) resistance due to Bcr-Abl gene amplification are sensitive to AG1024 (47,48); whether the effect is a direct consequence of IGF-1R or due to an “off target effect” of AG1024 remains to be determined. Several lines of evidences demonstrated that IGF-1R targeting inhibitors are effective against leukemia, multiple myeloma, and lymphoma models (49–52). IGF-1R blockade by ADW742, a small molecule specific for this receptor, alone and in combination with imatinib, on Ewing tumor cell lines has been studied (53). Addition of imatinib to ADW742 synergistically augmented these effects and is especially effective in inhibiting AKT/mTOR phosphorylation and reducing vascular endothelial growth factor expression in cell lines having high IGF-1R activation levels. Combination of ADW742 with imatinib induces a significant reduction of tumor cell growth, mainly by the increase in apoptosis with a pattern depending on IGF-1R activation levels.

MOLECULAR RESPONSE TO IONIZING RADIATION (CHECKPOINT OF CELL CYCLE, DNA DAMAGE SIGNALING) AND IGF-1R ATM plays a central role in cellular response after DNA damage triggering cellcycle arrest, apoptosis, and DNA repair mechanisms. ATM gene is mutated in the ataxia telangiectasia syndrome (AT), a syndrome characterized by an extreme radio-sensitivity. AT cells express low levels of IGF-1R and show decreased IGF1R promoter activity compared with wild-type cells. Complementation of AT cells with the ATM cDNA results in increased IGF-1R promoter activity and elevated IGF-1R levels, whereas expression in wild-type cells of a dominant-negative fragment of ATM specifically reduces IGF-1R expression. These results are consistent with a role for ATM in regulating IGF-1R expression at the level of transcription. When expression of IGF-1R cDNA is forced in AT cells via a heterogonous viral promoter, near normal radio-resistance is conferred on the cells. This


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suggests that IGF-1R may participate in DNA damage signaling especially after irradiation (54). Besides ATM, the interplay between IGF-1R and p53, Brca1, 14-3-3 d, all proteins involved in DNA damage signaling, also suggests the involvement of IGF-1R signaling in radiation response. Transcription of the IGF-1R gene is controlled by a number of tumor suppressors, including the DNA repair protein BRCA1 (55–57). BRCA1 represses the activity of co-transfected IGF-1R promoter reporter constructs in a number of cell lines (58). BRCA1 does not inhibit IGF-1R gene expression and promoter activity in the presence of a mutant p53 whereas expression of wild-type p53 does not prevent BRCA1 action (55). BRCA1 and p53 suppress IGF-1R promoter activity in an additive manner, suggesting that BRCA1 and p53 cooperate in the regulation of IGF-1R gene transcription. Loss-of-function mutation of p53 and/or BRCA1 in familial and/or sporadic breast cancer may result in aberrant regulation of IGF-1R gene expression. IGF-1R gene is a downstream target for p53 action. Wild-type p53 inhibits transcription of the IGF-1R gene, whereas a number of tumor-derived, mutant forms of p53 enhance IGF-1R gene expression (55). The role of p53 in regulating IGF-1R seems to be complex. The mechanism of action of p53 does not involve direct DNA binding to IGF-1R promoter sequences. P53 is capable of suppressing the activity of the IGF-1R promoter as well as lowering the endogenous levels of IGF-1R mRNA. Tumor-derived, mutant p53 significantly stimulated promoter activity. Upon inhibition of wild-type p53 in malignant melanoma cells overexpressing IGF-1R, the cells respond with a drastic IGF-1R downregulation and cell death. The oncoprotein MDM2 protein, which controls p53 degradation, seems to be involved in the control of IGF-1R expression. The MDM2 has recently also been found to associate with certain cell surface receptors and regulate their functions (59). When p53 is inhibited, MDM2 is redistributed and binds to the IGF-1R (60). MDM2 ubiquitinates and targets IGF-1R for degradation in a proteasome-dependent manner, eventually leading to cell death. Thus, a selective IGF-1 inhibition in cancer cells could be achieved by an increase of MDM2 levels by pharmacological modulations. Reciprocally, the IGF-1 system has been shown to influence the activity of MDM2 in a p38 MAPK-dependent manner. 14-3-3 d is originally identified as a p53-inducible gene responsive to DNAdamaging agents. 14-3-3 d induces G2 arrest by sequestering the mitotic initiation complex, cdc2-cyclinB1, within the cytoplasm after DNA damage and allows the repair of damaged DNA. It has been shown that 14-3-3 d is a positive mediator of IGF-1R-induced cell proliferation. Treatment with IGF-1 increased 14-3-3 d mRNA and protein levels (61); this effect occurs via the PI3-K/Akt pathway and is p53 independent. 14-3-3 d positively mediates IGF-1R-induced cell-cycle progression. The interplay between IGF-1R and Brca1, p53, ATM and 14-3-3 d, all proteins involved in DNA damage signaling and repair, suggests potential synergy between drugs targeting IGF-1R and agents targeting DNA. INHIBITION OF IGF-1R AND RADIO-SENSITIVITY Recent studies have identified IGF-1R as a protein capable of inducing radioresistance in breast cancer, while inhibition of IGF-1R function enhances tumor response to classical therapy (i.e., irradiation and chemotherapy) in breast and colon cancer models. IGF-1R is expressed on NSCLC cells and is involved in the radio-sensitivity in lung cancer cells lines (39). Blocking of IGF-1R function is

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effective in potentiating the effects of radiation in NSCLC cell lines. IGF-1R blockade by a recombinant adenovirus expressing truncated IGF-1R enhances chemotherapy and radiation responses and inhibits tumor growth in human gastric cancer xenografts. We have evaluated, in our laboratory, the effects of the tyrosin kinase IGF-1R inhibitor, AG1024 on radiation response and found a marked enhancement in radio-sensitivity and amplification of radiation-induced apoptosis in the human breast cancer cell line MCF-7 (47,48). INHIBITION OF IGF-1R A variety of approaches, including dominant-negative mutants, kinase defective mutants, antisense oligonucleotides, IGF binding proteins, soluble forms of the receptor, antagonistic and/or neutralizing antibodies or small molecule kinase inhibitors have been used to inhibit IGF-1R signaling. Reducing the levels of the ligands (IGF-1 and IGF-2) has given good results in mice which express only IGF-1 in adult life. However in adult humans, IGF-1 and IGF-2 are both expressed and both of them would have to be targeted. Antisense strategies are the first to be used successfully in vitro and in vivo. Antagonistic antibodies and TK inhibitors represent the most probable clinically viable options (62). Humanized monoclonal antibodies such as: EM164 (63,64) (AVE1642) (65), IMC-A12 (41) and CP-751, 871, h7C10 (Table 2) (66), have been successful in inducing apoptosis of cancer cells, and their usefulness is further supported by the observation that antibodies to the IGF-1R, like antisense strategies, downregulate the receptor. The feasibility of inhibiting IGF-1R function with a specific antibody is first demonstrated using a mouse monoclonal antibody (a-IR-3) directed against the a-subunit of IGF-1R. This antibody inhibits the binding of IGF-1 to its receptor, thereby preventing downstream signaling, tumor cell proliferation in vitro, and tumor growth in vivo. Numerous groups have recently described the identification and characterization of antagonistic and/or neutralizing humanized antibodies targeting the extracellular domain of IGF-1R. Although generated by applying TABLE 2

Specific IGF-1R Targeting Compounds

Compounds

Type of targeting

CP-751, 871 EM164 (AVE1642)

Antibody Antibody

IMC-A12

Antibody

h7C10

Antibody

INSM18 PPP

TK inhibitor TK inhibitor

NVP-ADW742, NVP-AEW541 BMS-536924, BMS-554417

TK inhibitor TK inhibitor

Abbreviation: TK, tyrosine kinase.

Company Pfizer, New York, New York, U.S.A. ImmunoGen, Cambridge, Massachusetts, U.S.A.; Sanofi-Aventis, Paris, France ImClone, New York, New York, U.S.A. Pierre Fabre, Boulogne, France; Merck, Whitehouse Station, New Jersey, U.S.A. Insmed, Richmond, Virginia, U.S.A. Karolinska Institute, Stockholm, Sweden Novartis Pharma, Basel, Switzerland Bristol-Myers Squibb, New York, New York, U.S.A.

Phase of development Phase I Preclinical

Phase I Preclinical

Phase I Preclinical Preclinical Preclinical


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different strategies, such potential biopharmaceuticals have been shown to bind specifically to IGF-1R, thereby preventing the activation of IGF-1R-mediated signaling (62). Parallel to the efforts directed at blocking the physical interaction between IGF-1R and its growth factors, drug discovery activities have also been aimed at modulating IGF-1R TK activity by targeting its intracellular kinase domain. The identification of specific low-molecular mass kinase inhibitors of IGF-1R kinase activity has proven to be a major challenge for medicinal chemistry. In theory, a specific inhibitor of IGF-1R TK activity would be the best solution. The problem is that this type of inhibitor will have to distinguish the TK domain of the IGF-1R from the one of the insulin receptor. The two domains are highly homologous, but there are small differences that could be exploited. These kinase inhibitors could be divided into two groups: ATP antagonists such as: NVP-ADW742 (49), NVPAEW541 (67) and BMS-536924 (68), BMS-554417 (69) and non-ATP antagonists such as: picropodophyllin (PPP), AG538 (70) and INSM18. PPP is a cyclolignan derivative developed at the Karolinska Institute and is a selective inhibitor of IGF-1R kinase activity. PPP potently inhibits IGF-1R autophosphorylation (IC50 of 0.04 mM) and is selective against a panel of other receptor TKs without interfering with insulin receptor activity (51,71–73). PPP did not compete with ATP but interfered with phosphorylation in the activation loop of the kinase domain. PPP reduces phosphorylated Akt and induces apoptosis and tumor regression in xenografted mice. IGF-1Rs of PPP treated cells undergo rapid downregulation. This downregulation may be important for the strong apoptotic effect of this compound. PPP treatment of IGF-1R overexpressing cells results in the preferential inhibition of the PI3K/PKB pathway. QUESTIONS REMAINING Recent successes in the development of small-molecule TK inhibitors, blocking antibodies against the IGF-1R, pose challenges to translational scientists seeking to design clinical trials. There are still many questions to be answered. Kinase inhibitors have potential advantages, including convenient oral administration. It is difficult to predict a priori to what extent these agents will be specific for IGF1R during long term in vivo use, where tissue concentrations might vary. As there are no examples of genetic alterations of this pathway in human tumors, it is unclear how tumors should be selected for treatment using this approach. Is activation of the receptor in a tumor likely to predict responsiveness? What effects may the inhibitors have on IGF-1R in normal tissues and even on the insulin receptor (74)? While several small molecules have a much lower affinity for the IGF-1R than the insulin receptor, the relative affinities in patients and on different tissues remain unknown. It is hoped that intermittent therapy with these or similar agents may have minimal effects, perhaps only on tissues that demonstrate a high level of cellular turnover, such as bone marrow and the gastrointestinal tract. These side effects may therefore be similar to those seen with chemotherapy and may be limited in extent and duration; clinical trials will be required to establish this. Regarding the insulin receptor, intermittent therapy may worsen insulin resistance and diabetes, which may be limited and easily treatable. Finally, because of its strong antiapoptotic activity, downregulation of the IGF-1R could be used in combination with other anticancer therapies that cause

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apoptosis of cancer cells. Blockade of the IGF-1R may be an important form of adjunct therapy for cancer patients. It may reduce side effects by lowering the doses of chemotherapeutic agents, perhaps making chemotherapy more effective. Whether the agent used is a humanized antibody, small peptide inhibitor, or small molecule, it is becoming clear that the IGF system plays a critical role in the development and treatment of cancer. Last, sequence of coadministration in the case of IGF-1R targeting drugs and chemotherapy seems to be critical. REFERENCES 1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 11. 12. 13. 14. 15.

16. 17.

Krause DS, Van Etten RA. Tyrosine kinases as targets for cancer therapy. New Engl J Med 2005; 353:172–87. Baserga R. The insulin-like growth factor-I receptor as a target for cancer therapy. Expert Opin Ther Targets 2005; 9:753–68. Pollak MN, Schernhammer ES, Hankinson SE. Insulin-like growth factors and neoplasia. Nature Rev 2004; :505–18. Shaw RJ, Cantley LC. Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature 2006; 441:424–30. Testa JR, Tsichlis N. AKT signaling in normal and malignant cells. Oncogene 2005; 24:7391–3. Frame S, Cohen P. GSK3 takes centre stage more than 20 years after its discovery. Biochem J 2001; 359(Pt 1):1–16. Shi Y, Yan H, Frost P, Gera J, Lichtenstein A. Mammalian target of rapamycin inhibitors activate the AKT kinase in multiple myeloma cells by up-regulating the insulin-like growth factor receptor/insulin receptor substrate-1/phosphatidylinositol 3-kinase cascade. Mol Cancer Ther 2005; 4:1533–40. O'Reilly KE, Rojo F, She QB, et al. mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res 2006; 66:1500–8. Belfiore A, Pandini G, Vella V, Squatrito S, Vigneri R. Insulin/IGF-I hybrid receptors play a major role in IGF-I signaling in thyroid cancer. Biochimie 999; 81:403–7. Xie Y, Skytting B, Nilsson G, Brodin B, Larsson O. Expression of insulin-like growth factor-1 receptor in synovial sarcoma: association with an aggressive phenotype. Cancer Res 1999; 59:3588–91. Khandwala HM, McCutcheon IE, Flyvbjerg A, Friend KE. The effects of insulinlike growth factors on tumorigenesis and neoplastic growth. Endocrine Rev 2000; 21:215–44. Baserga R, Peruzzi F, Reiss K. The IGF-1 receptor in cancer biology. Int J Cancer 2003; 107:873–7. Yakar S, Leroith D, Brodt P. The role of the growth hormone/insulin-like growth factor axis in tumor growth and progression: lessons from animal models. Cytokine Growth Factor Rev 2005; 16:407–20. Shimizu C, Hasegawa T, Tani Y, et al. Expression of insulin-like growth factor 1 receptor in primary breast cancer: immunohistochemical analysis. Human Pathol 2004; 35:1537–42. Jiang Y, Wang L, Gong W, et al. A high expression level of insulin-like growth factor I receptor is associated with increased expression of transcription factor Sp1 and regional lymph node metastasis of human gastric cancer. Clin Exp Metastasis 2004; 21:755–64. Guo YS, Jin GF, Townsend CM, Jr, et al. Insulin-like growth factor-II expression in carcinoma in colon cell lines: implications for autocrine actions. J Am College Surgeons 1995; 181:145–54. Fottner C, Minnemann T, Kalmbach S, Weber MM. Overexpression of the insulinlike growth factor I receptor in human pheochromocytomas. J Mol Endocrinol 006; 36:279–87.


18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

38.

153

Hopfner M, Baradari V, Huether A, Schofl C, cherubl H. The insulin-like growth factor receptor 1 is a promising target for novel treatment approaches in neuroendocrine gastrointestinal tumors. Endocrine-Related Cancer 2006; 13:135–49. Abu-Amero SN, Ali Z, Bennett P, Vaughan JI, Moore GE. Expression of the insulinlike growth factors and their receptors in term placentas: a comparison between normal and IUGR births. Mol Reproduction Development 1998; 49:229–35. Stygar D, Muravitskaya N, Eriksson B, Eriksson H, Sahlin L. Effects of SERM (selective estrogen receptor modulator) treatment on growth and proliferation in the rat uterus. Reprod Biol Endocrinol 2003; 1:40. Tsuchiya N, Wang L, Horikawa Y, et al. CA repeat polymorphism in the insulin-like growth factor-I gene is associated with increased risk of prostate cancer and benign prostatic hyperplasia. Int J Oncol 2005; 26:225–31. Tsuchiya N, Wang L, Suzuki H, et al. Impact of IGF-I and CYP19 gene polymorphisms on the survival of patients with metastatic prostate cancer. J Clin Oncol 2006; 24:1982–9. Cheng I, Stram DO, Penney KL, et al. Common genetic variation in IGF1 and prostate cancer risk in the Multiethnic Cohort. J Natl Cancer Inst 2006; 98:123–34. Yu H, Li BD, Smith M, Shi R, Berkel HJ, Kato I. Polymorphic CA repeats in the IGF-I gene and breast cancer. Breast Cancer Res Treat 2001; 70:117–22. Wagner K, Hemminki K, Israelsson E, et al. Polymorphisms in the IGF-1 and IGFBP 3 promoter and the risk of breast cancer. Breast Cancer Res Treat 2005; 92:133–40. dos Santos Silva I, Johnson N, De Stavola B, et al. The insulin-like growth factor system and mammographic features in premenopausal and postmenopausal women. Cancer Epidemiol Biomarkers Prev 2006; 15:449–55. Fletcher O, Gibson L, Johnson N, et al. Polymorphisms and circulating levels in the insulin-like growth factor system and risk of breast cancer: a systematic review. Cancer Epidemiol Biomarkers Prev 2005; 14:2–19. Morimoto LM, Newcomb PA, White E, Bigler J, Potter JD. Insulin-like growth factor polymorphisms and colorectal cancer risk. Cancer Epidemiol Biomarkers Prev 2005; 14:1204–11. Zecevic M, Amos CI, Gu X, et al. IGF-1 gene polymorphism and risk for hereditary nonpolyposis colorectal cancer. J Natl Cancer Inst 2006; 98:139–43. Moon JW, Chang YS, Ahn CW, et al. Promoter-202 A/C polymorphism of insulin-like growth factor binding protein-3 gene and non-small cell lung cancer risk. Int J Cancer 2006; 118:353–6. Savage SA, Modi WS, Douglas C, Hoover RN, Chanock SJ. Polymorphisms in genes of the insulin-like growth factor family are associated with osteogenic sarcoma. AACR annual meeting 2005; 762. Slomiany MG, Black LA, Kibbey MM, Day TA, Rosenzweig SA. IGF-1 induced vascular endothelial growth factor secretion in head and neck squamous cell carcinoma. Biochem Biophys Res Commun 2006; 342:851–8. Slomiany MG, Rosenzweig A. Hypoxia-inducible factor-1-dependent and -independent regulation of insulin-like growth factor-1-stimulated vascular endothelial growth factor secretion. J Pharmacol Exp Ther 2006; 318:666–75. Li J, Wang E, Rinaldo F, Datta K. Upregulation of VEGF-C by androgen depletion: the involvement of IGF-IR-FOXO pathway. Oncogene 2005; 24:5510–20. Ueda S, Hatsuse K, Tsuda H, et al. Potential crosstalk between insulin-like growth factor receptor type 1 and epidermal growth factor receptor in progression and metastasis of pancreatic cancer. Mod Pathol 2006; 19:788–96. Luo J, Field SJ, Lee JY, Engelman JA, Cantley LC. The p85 regulatory subunit of phosphoinositide 3-kinase down-regulates IRS-1 signaling via the formation of a sequestration complex. J Cell Biol 2005; 170:455–64. Chakravarti A, Loeffler JS, Dyson NJ. Insulin-like growth factor receptor I mediates resistance to anti-epidermal growth factor receptor therapy in primary human glioblastoma cells through continued activation of phosphoinositide 3-kinase signaling. Cancer Res 2002; 62:200–7. Kobayashi S, Ji H, Yuza Y, et al. An alternative inhibitor overcomes resistance caused by a mutation of the epidermal growth factor receptor. Cancer Res 2005; 65:7096–101.

154 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53.

54. 55. 56. 57. 58.

Tao et al.

Cosaceanu D, Carapancea M, Castro J, et al. Modulation of response to radiation of human lung cancer cells following insulin-like growth factor 1 receptor inactivation. Cancer Lett 2005; 222:173–81. Camirand A, Zakikhani M, Young F, Pollak M. Inhibition of insulin-like growth factor1 receptor signaling enhances growth-inhibitory and proapoptotic effects of gefitinib (Iressa) in human breast cancer cells. Breast Cancer Res 2005; 7:R570–9. Tonra JR, Corcoran E, Makhoul G, et al. Synergistic anti-tumor effects of anti-EGFR monoclonal antibody Erbitux combined with antibodies targeting IGF1R or VEGFR-2. AACR annual meeting 2005; 1193. Jones HE, Goddard L, Gee JM, et al. Insulin-like growth factor-I receptor signalling and acquired resistance to gefitinib (ZD1839; Iressa) in human breast and prostate cancer cells. Endocrine-Related Cancer 2004; 11:793–814. Digiovanna MP, Chakraborty. Combinations of HER2, estrogen receptor (ER) and IGFI receptor (IGF1R) inhibitors induce apoptosis in breast cancer cells: dramatic effects of HER2 inhibitors on non-overexpressing cells. AACR annual meeting 2006; 290. Lu Y, Zi X, Zhao Y, Mascarenhas D, Pollak M. Insulin-like growth factor-I receptor signaling and resistance to trastuzumab (Herceptin). J Natl Cancer Inst 2001; 93: 1852–7. Albanell J, Baselga J. Unraveling resistance to trastuzumab (Herceptin): insulin-like growth factor-I receptor, a new suspect. J Natl Cancer Inst 2001; 93:1830–2. Camirand A, Lu Y, Pollak M. Co-targeting HER2/ErbB2 and insulin-like growth factor-1 receptors causes synergistic inhibition of growth in HER2-overexpressing breast cancer cells. ed Sci Monit 2002; 8:BR521–6. Wen B, Deutsch E, Marangoni E, et al. Tyrphostin AG 1024 modulates radiosensitivity in human breast cancer cells. Br J Cancer 2001; 85:2017–21. Deutsch E, Maggiorella L, Wen B, et al. Tyrosine kinase inhibitor AG1024 exerts antileukaemic effects on STI571-resistant Bcr-Abl expressing cells and decreases AKT phosphorylation. Br J Cancer 2004; 91:1735–41. Mitsiades CS, Mitsiades NS, McMullan CJ, et al. Inhibition of the insulin-like growth factor receptor-1 tyrosine kinase activity as a therapeutic strategy for multiple myeloma, other hematologic malignancies, and solid tumors. Cancer Cell 2004; 5:221–30. Mitsiades CS, Mitsiades . Treatment of hematologic malignancies and solid tumors by inhibiting IGF receptor signaling. Expert Rev Anticancer Ther 2005; 5:487–99. Stromberg T, Ekman S, Girnita L, et al. IGF-1 receptor tyrosine kinase inhibition by the cyclolignan PPP induces G2/M-phase accumulation and apoptosis in multiple myeloma cells. Blood 2006; 107:669–78. Menu E, Jernberg-Wiklund H, Stromberg T, et al. Inhibiting the IGF-1 receptor tyrosine kinase with the cyclolignan PPP: an in vitro and in vivo study in the 5T33MM mouse model. Blood 2006; 107:655–60. Martins AS, Mackintosh C, Martin DH, et al. Insulin-like growth factor I receptor pathway inhibition by ADW742, alone or in combination with imatinib, doxorubicin, or vincristine, is a novel therapeutic approach in Ewing tumor. Clin Cancer Res 2006; 12(11 Pt 1):3532–40. Peretz S, Jensen R, Baserga R, Glazer PM. ATM-dependent expression of the insulinlike growth factor-I receptor in a pathway regulating radiation response. Proc Natl Acad Sci USA 2001; 98:1676–81. Werner H, Maor S. The insulin-like growth factor-I receptor gene: a downstream target for oncogene and tumor suppressor action. Trends Endocrinol Metab 2006; 17:236–42. Abramovitch S, Werner H. Functional and physical interactions between BRCA1 and p53 in transcriptional regulation of the IGF-IR gene. Hormone and metabolic research Hormon-und Stoffwechselforschung 2003; 35:758–62. Sarfstein R, Maor S, Reizner N, Abramovitch S, Werner H. Transcriptional regulation of the insulin-like growth factor-I receptor gene in breast cancer. Mol Cell Endocrinol 2006; 252:241–6. Maor SB, Abramovitch S, Erdos MR, Brody LC, Werner H. BRCA1 suppresses insulinlike growth factor-I receptor promoter activity: potential interaction between BRCA1 and Sp1. Mol Genet Metabol 2000; 69:130–6.


59. 60. 61. 62. 63. 64. 65. 66.

67. 68. 69. 70. 71.

72. 73. 74.

155

Larsson O, Girnita A, Girnita L. Role of insulin-like growth factor 1 receptor signalling in cancer. Br J Cancer 2005; 92:2097–101. Girnita L, Girnita A, Larsson O. Mdm2-dependent ubiquitination and degradation of the insulin-like growth factor 1 receptor. Proc Natl Acad Sci USA 2003; 100:8247–52. Zhang Y, Karas M, Zhao H, Yakar S, LeRoith D. 14-3-3sigma mediation of cell cycle progression is p53-independent in response to insulin-like growth factor-I receptor activation. J Biol Chem 2004; 279:34353–60. Hofmann F, Garcia-Echeverria C. Blocking the insulin-like growth factor-I receptor as a strategy for targeting cancer. Drug Discovery Today 2005; 10:1041–7. Maloney EK, McLaughlin JL, Dagdigian NE, et al. An anti-insulin-like growth factor I receptor antibody that is a potent inhibitor of cancer cell proliferation. Cancer Res 2003; 63:5073–83. Sachdev D, Singh R, Fujita-Yamaguchi Y, Yee D. Down-regulation of insulin receptor by antibodies against the type I insulin-like growth factor receptor: implications for anti-insulin-like growth factor therapy in breast cancer. Cancer Res 2006; 66:2391–402. Bladt F, Vrignaud P, Chiron M, et al. Pre-clinical evaluation of the anti-tumor activity of the IGF1R—specific antibody AVE1642. AACR annual meeting 2006; 289–90. Goetsch L, Gonzalez A, Leger O, et al. A recombinant humanized anti-insulin-like growth factor receptor type I antibody (h7C10) enhances the antitumor activity of vinorelbine and anti-epidermal growth factor receptor therapy against human cancer xenografts. Int J Cancer 2005; 113:316–28. Garcia-Echeverria C, Pearson MA, Marti A, et al. In vivo antitumor activity of NVPAEW541-A novel, potent, and selective inhibitor of the IGF-IR kinase. Cancer Cell 2004; 5:231–9. Wittman M, Carboni J, Attar R, et al. Discovery of a (1H-benzoimidazol-2-yl)-1Hpyridin-2-one (BMS-536924) inhibitor of insulin-like growth factor I receptor kinase with in vivo antitumor activity. J Med Chem 2005; 48:5639–43. Haluska P, Carboni JM, Loegering DA, et al. In vitro and in vivo antitumor effects of the dual insulin-like growth factor-I/insulin receptor inhibitor, BMS-554417. Cancer Res 2006; 66:362–71. Blum G, Gazit A, Levitzki A. Development of new insulin-like growth factor-1 receptor kinase inhibitors using catechol mimics. J Biol Chem 2003; 278:40442–54. Vasilcanu D, Girnita A, Girnita L, Vasilcanu R, Axelson M, Larsson O. The cyclolignan PPP induces activation loop-specific inhibition of tyrosine phosphorylation of the insulin-like growth factor-1 receptor. Link to the phosphatidyl inositol-3 kinase/Akt apoptotic pathway. Oncogene 2004; 23:7854–62. Girnita A, Girnita L, del Prete F, Bartolazzi A, Larsson O, Axelson M. Cyclolignans as inhibitors of the insulin-like growth factor-1 receptor and malignant cell growth. Cancer Res 2004; 64:236–42. Girnita A, All-Ericsson C, Economou MA, et al. The insulin-like growth factor-I receptor inhibitor picropodophyllin causes tumor regression and attenuates mechanisms involved in invasion of uveal melanoma cells. Clin Cancer Res 2006; 12:1383–91. LeRoith D, Helman L. The new kid on the block(ade) of the IGF-1 receptor. Cancer Cell 2004; 5:201–2.

10

Aurora Kinase Inhibitors Mitesh J. Borad, Steven L. Warner, and Daniel D. Von Hoff Clinical Translational Research Division, Translational Genomics Research Institute, Phoenix, Arizona, U.S.A.

DISCOVERY OF THE AURORA KINASES In 1993, the first homologue of the Aurora kinase family was reported to be isolated using a genetic screen to find mutations in yeast that confer an increasein-ploidy phenotype (Ipl1) (1). Shortly thereafter, subsequent reports further characterized the function of Ipl1 to be involved in the process of chromosome segregation (2,3). A Drosophila homologue was discovered in 1995, when it was shown that mutations in a gene named aurora led to mitotic arrest in which condensed chromosomes were attached to circular monopolar mitotic spindles (4). This name was chosen due to the localization of the aurora protein to the poles of the mitotic spindle, similar to the way an aurora borealis is observed at one of the poles of the earth. Furthermore, the name polo was already used to describe a gene of related function (5), and, to keep with a similar theme, aurora was named after the northern lights. In 1997, Sen and colleagues (6) showed that a putative serine/threonine kinase encoding gene that had been previously mapped to chromosome 20q13 (7) was amplified in human breast cancer cell lines, and they named this human homologue breast tumor amplified kinase (BTAK). In that same year, Kimura et al. further characterized this human homologue by reporting its cell cycle-dependent expression and spindle pole localization in HeLa cells (8), and Bischoff and colleagues showed its amplification in human colorectal cancers and its ability to transform rodent fibroblasts (9). These key early reports catapulted the aurora family of kinases to be closely studied as important mitotic kinases, contributors to tumorigenesis, and potential therapeutic targets. By 1998 it had become clear that there were at least three homologues of the aurora kinases that, despite having a high degree of amino acid sequence similarity, were quite distinct in function. However, due to the various groups making key discoveries, the nomenclature for this family of kinases became confusing. For the human homologues, it has been suggested that Aurora A, B, and C become the convention (10,11). BIOLOGICAL FUNCTIONS OF THE AURORA KINASES Aurora A Aurora A (also known as Aurora-2/BTAK/AIK1/ARK1/STK15) is a centrosome-associated kinase that functions to establish mitotic spindles by regulating centrosome duplication and separation. The loss of Aurora A function (or expression) leads to cell cycle arrest and monopolar mitotic spindles (12) and is therefore orthologous to the initial aurora discovered in Drosophila. Although it is well established that Aurora A regulates centrosome function and duplication, the mechanisms underlying this control are yet to be fully 157

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understood. It has been shown that similar to other kinases, Aurora A kinase activity is dependent upon the phosphorylation status of a key threonine residue (T288) located on the activation loop of the enzyme. This phosphate group is removed by Protein Phosphatase 1 or 2A (PP1/2A) rendering Aurora A kinase inactive. The kinase that adds the phosphate group to T288 went many years undiscovered until it was recently shown to be Aurora A kinase itself (13). In this manner, Aurora A is held inactive by its close association with PP1/2A and the switch to the active form requires a signaling pathway involving the small GTPase Ran and the microtubule-associated protein TPX2 (14). Prior to mitotic spindle assembly, Ran releases TPX2 from binding to importin a and b, which allows TPX2 to associate with Aurora A and target it to the microtubules of the mitotic spindle (15). In addition to controlling its localization, TPX2 also activates Aurora A by inducing a small conformational change that protects T288 from PP1/2A and thereby promotes the autophosphorylation of Aurora A (16). Once activated, Aurora A phosphorylates microtuble-associated proteins such as the kinesin Eg5 (17), the acidic coiled-coil protein, TACC (18), and TPX2 itself in order to assemble the mitotic spindle. Aurora B Aurora B (also known as Aurora-1/AIK2/ARK2/STK12) localizes to the centromere during the early stages of mitosis (prophase to anaphase), whereupon it relocates to the midbody throughout cytokinesis (19). It functions along with its binding partners and substrates, INCENP and survivin, to ensure proper kinetochore-microtubule attachments (20). Additionally, Aurora B is required for chromosome segregation and cytokinesis. Therefore, inhibition of Aurora B function results in an increase-in-ploidy phenotype making it orthologous to Ipl1 in yeast. In order for chromosomes to properly segregate during mitosis, microtubules from opposing spindle poles must attach to sister chromatids in a process called chromosome biorientation. An increase in mal-oriented chromosomes was noted in Aurora B-inhibited cells and another study showed Aurora B to be required for correcting non-bioriented chromosomes (21,22). The mechanism by which Aurora B contributed to biorientation was recently ascribed to its interaction with the microtubule-destabilizing mitotic centromere-associated kinesin (MCAK) (23). The emerging model for establishing biorientation is that the microtubule destabilizing activity of MCAK disconnects the stochastic attachment of microtubules to the kinetochore until biorientation is achieved, which is detected by Aurora B. Once established, Aurora B phosphorylates MCAK, which inhibits the catalytic activity of MCAK and the destabilization of microtubules at the kinetochore-microtubule interface halts, resulting in stable chromosome biorientation. In addition to its role in detecting proper kinetochore-microtububle attachments, Aurora B functions in the spindle checkpoint and is required for cytokinesis (24–26). The role of Aurora B in the spindle checkpoint is to recruit checkpoint proteins, such as BubR1, Mad2 and Cenp-E to the kinetochores (27). The inhibition of Aurora B function leads to a bypass of the spindle checkpoint and premature exit from mitosis without undergoing cytokinesis. This series of events leads to polyploidization and potentially to continued re-entry into the cell cycle resulting in multinucleated cells.

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Importantly, Aurora B phosphorylates Histone H3 at the Serine 10 position during mitosis. It was initially believed that Aurora B-mediated phospho-Histone H3 was required for chromosome condensation and mitosis; however, this has recently come under question (28,29). The biological significance of Histone H3 phosphorylation by Aurora B is not fully understood. Regardless, it has proven to be a very important pharmacodynamic endpoint in the preclinical and clinical development of Aurora kinase inhibitors. Aurora C Little is known about the function of Aurora C (also known as Aurora-3/AYK1/ AIK3/AIE1/STK13). The earliest reports show the expression of Aurora C to be primarily in meiotically active cells and to be important for processes such as spermatogenesis (30). Subsequently, Aurora C was shown to be expressed during mitosis when its localization was primarily to the centrosome (31). Most recently, reports suggest that Aurora C is a chromosome passenger protein that functions in a very similar, if not redundant, role to that of Aurora B (30). These seemingly conflicting reports in the literature show that the function of Aurora C needs further elucidation. THE AURORA KINASES IN CANCER Of the three human aurora kinases, Aurora A has been the family member most often and most closely associated with cancer; however, it is becoming evident that Aurora B likely contributes to tumorigenesis. An association between Aurora C and cancer has not been identified. Aurora A Aurora A is overexpressed in a variety of tumor types summarized in Table 1. Additionally, the amplification of the Aurora A gene has been associated with its overexpression in many tumor types, including breast, colorectal and liver cancers (6,9,32). The oncogenecity of Aurora A has been demonstrated in at least some tumorigenesis models, in which its overexpression led to the in vitro and in vivo transformation of rodent fibroblast cells and to the formation of multipolar mitotic spindles and genomic instability (33). Similarly, the overexpression of Aurora A in near diploid human breast epithelial cells revealed centrosome abnormalities, as well as induction of aneuploidy (33). The link between Aurora A overexpression and aneuploidy has led to the hypothesis that Aurora A overexpression and hyperactivity may be a major driving force in the acquisition of other genetic alterations required for tumorigenesis in some tumor types (34). In support of this, Aurora A overexpression has been shown to be an early event in rat mammary carcinogenesis (35) and present in high-grade PIN (prostatic intraepithelial neoplasia) lesions, indicating that this may be an early event that leads to the genetic instability seen in prostate carcinogenesis (36). Further implicating a role for Aurora A in tumorigenesis are discoveries showing its connection with genes and proteins typically associated with cancer. For example, BRCA1 activity is at least in part regulated by its binding to and phosphorylation by Aurora A (37). Furthermore, Aurora A plays a role in the regulation of c-Myc and telomerase expression levels (38) and p53 has been shown to be a substrate for Aurora A leading to the degradation and/or the inactivation

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TABLE 1 Reported Abnormalities of Aurora A and Aurora B in Human Tumors Cancer type Aurora A Breast

Specimen type Cell lines Primary

Colorectal

Cell lines Primary

Ovarian

Cell lines Primary

Prostate

Cell lines Xenografts Primary Cell lines Cell lines Cell lines Primary

Neuroblastoma Cervical Gastric

Bladder Pancreatic Laryngeal Non-Hodgkin’s lymphoma Endometrial Hepatocellular

Cell lines Primary Cell lines Primary Primary Primary Primary Primary

Esophageal Oral (tongue)

Primary Primary

Lung Head and neck Glioma

Cell lines Primary Primary Primary

Kidney Melanoma Medulloblastoma Mantle cell lymphoma

Cell lines Cell lines Primary Primary

Aurora B Colorectal Glioma Thyroid Lung Prostate Kidney Melanoma Breast

Primary Cell lines Primary Cell lines Primary Cell lines Primary Primary Cell lines Cell lines Cell lines

Findings

References

Amplification and overexpression Overexpression in 94% and amplification in 12% Amplification and overexpression Amplification and overexpression in >50% Amplification in 10–15%; Overexpression in 67% Overexpression in 50% Amplification and overexpression Overexpression Overexpression in 98% Amplification and overexpression Amplification and overexpression Amplfication in 29%; overexpression in 44% Amplfication in 5–13%; overxpression in 41–50% Overexpression Amplification and overexpression Overexpression Overexpression in 56–93% Overexpression in 68% Overexpression

(6) (33,75)

Amplification in 56% Amplification in 3%; overexpression in 61% Overexpression in 68% Amplification in 36%; overexpression in 100% Amplification and overexpression Overexpression in 69% Overexpression Amplification in 26–31%; overexpression in 60% Amplification and overexpression Amplification and overexpression Overexpression Amplification in 0%; overexpression

(93) (32)

Overexpression Overexpression Overexpression Overexpression Overexpression Overexpression Overexpression in 59% Overexpression Overexpression Overexpression Overexpression

(33) (9,76) (33,77) (78) (33) (79) (36) (33) (33) (80) (81)

(82) (83,84) (85) (12,86) (87–91) (92)

(94) (95) (9) (96) (97) (98) (9) (9) (99) (100)

(9,101) (102) (103) (104) (104) (9) (44) (105) (9) (9) (9)


161

of p53 protein (39,40). Of further clinical significance, Aurora A overexpression induces resistance to tubulin-targeted agents such as paclitaxel by overriding the spindle assembly checkpoint in mitosis (41). Aurora B Although the biology of Aurora B in disease-free cells is arguably better understood than Aurora A, its role in cancer is not. Indeed Aurora B is overexpressed in a variety of tumor types (Table 1); however its amplification and ability to transform cells (oncogenicity) has not been noted. Therefore, some have speculated that its overexpression is possibly a characteristic of rapidly dividing cells with high mitotic indices, rather than an event that drives tumorigenesis. Contrary to this idea, although Aurora B overexpression alone did not transform rodent fibroblast cells, it did potentiate H-Ras(G12V)-induced transformation (42). In endometrial carcinomas the expression of Aurora B was significantly increased in high-grade tumors, and patients with Aurora B-positive carcinoma showed poor prognosis compared with those with Aurora B-negative tumors (43). Furthermore, Aurora B overexpression does correlate with the level of genomic instability within a tumor, suggesting Aurora B may contribute to the process of acquiring necessary genetic alterations required for tumorigenesis (44). THE AURORA KINASES AS THERAPEUTIC TARGETS Due to the strong association of Aurora A with cancer almost from its initial discovery, it was thought to be the more important therapeutic target. The validation of Aurora B as a potential drug target did not come about until it was discovered that small molecules intended to target either Aurora A or both aurora kinases produced a biological response entirely consistent with Aurora B inhibition alone. Aurora A The work validating Aurora A as a potential therapeutic target has been primarily carried out using gene silencing approaches such as siRNA and antisense oligonucleotides in pancreatic cancer cell lines. The first of these reports showed that Aurora A-specific inhibition by an antisense oligonucleotide resulted in cell cycle arrest in the G2/M phase and in the induction of apoptosis (12). The second study further showed that the specific knockdown of Aurora A in pancreatic cancer cells suppressed in vitro cell growth and in vivo tumorigenicity. Furthermore, a synergistic enhancement of the cytotoxicity of taxanes was shown when combined with siRNA-mediated knockdown of Aurora A (45). MLN8054 is a newly disclosed Aurora kinase inhibitor which is reported to show some selectivity towards Aurora A (46). In recent reports, drug-induced phenotypic changes consistent with Aurora A inhibition occurred at low concentrations; however phenotypic markers (such as phospho-Histone H3 levels) shifted toward a signature of an Aurora B-specific inhibitor when higher concentrations of the same drug were administered (47,48). With the available information it is unclear whether the therapeutic index between Aurora A and Aurora B inhibition is great enough to achieve selectivity in patients. Regardless of its ultimate target profile in patients, the emergence of a small molecule with preferential activity towards Aurora A is very exciting.

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Aurora B In contrast to Aurora A, target validation studies using gene silencing approaches targeting Aurora B are limited. However, several reports do characterize the anti-tumor effects of small molecule inhibitors. These small molecule inhibitors, with the exception of a few, were not intended to be Aurora B specific. In fact, in cell-free enzymatic assays most of them have comparable activity against all three Aurora kinases; however, when the compounds were evaluated in cell-based and animal models, the dual inhibitors produced all the phenotypic changes consistent with Aurora B inhibition alone. The reason for this was recently elucidated by showing that when Aurora B is inactivated, the requirement for Aurora A in mitosis is bypassed (49). Therefore, although a dual aurora kinase inhibitor does indeed inhibit the activities of both kinases, it functionally inhibits Aurora B only because the function of Aurora A is not required. Several aurora kinase inhibitors are in development as anticancer agents; four of them are described below. Hesperadin (Boehringer Ingelheim) was discovered from a cell-based screen to identify small molecules having effects on cell proliferation (21). Its target was identified as Aurora B as it was shown to decrease Histone H3 phosphorylation, induce chromosome segregation defects, and inhibit cytokinesis leading to polyploidization. Hesperadin has been a useful chemical tool to further understand the biology of Aurora B; however, it has not been further developed for potential clinical use. ZM447439 (AstraZeneca) is a dual aurora kinase inhibitor that has again been very instrumental in understanding Aurora B biology and validating Aurora B as a potential drug target (27,50). It also decreases phospho-Histone H3 levels, induces cellular polyploidization, inhibits tumor colony formation, and compromises the spindle checkpoint. It is an early analogue of AZD1152, which is an Aurora B-specific inhibitor currently in clinical trial. VX-680 (Vertex and Merck) potently inhibits all three aurora kinases. It inhibits the proliferation of several cancer cell lines resulting in the accumulation of cells with >4N DNA content (51). VX-680 inhibits Histone H3 phosphorylation and shows activity in mouse xenograft models (52). Importantly, VX-680 shows good activity in refractory acute myelogenous leukemia (AML) cells, which is likely due to its cross-inhibitory activity against FLT-3. Furthermore, VX-680 is currently under investigation as a BCR-ABL inhibitor in chronic myelogenous leukemia (CML) due to its potent activity against the imatinib-resistant T315I mutant of BCR-ABL (53). The fourth compound that has played an important role in the validation of Aurora B as a drug target is PHA-680632 (Nerviano). It has shown good activity on a wide range of cancer cell lines and significant tumor growth inhibition in in vivo tumor models (54). PHA-680632 induces a biological response consistent with Aurora B inhibition, such as decreased Histone H3 phosphorylation, endoreduplication and polyploidy. In cell-free evaluations, PHA-680632 is active against all three aurora kinases. CLINICAL DATA ON AURORA KINASE INHIBITORS Aurora kinase inhibitors for which data are available include MK-0457 (VX-680) and AZD1152. Other aurora kinase inhibitors that are actively being studied both preclinically and in clinical trials are listed in Table 2.

þ þ þ þ þ

Merck/Vertex MK-0457 (VX-680) þ Millennium MLN8054 þ

þ þ

þ þ

AT9283 MP529

SNS-314 Resveratrol

Undesignated R763

CYC116 Hesperadin

Astex Supergen

Sunesis N/A

Avalon Rigel

Cyclacel Boehringer Ingelheim

Unknown

Unknown þ

Unknown Unknown

þ þ

þ

Unknown Unknown

Unknown

þ

Unknown

Rebamipide

N/A

Astra Zeneca ZM447439 Astra Zeneca AZD1152

Unknown

þ

þ

SU6668

Sugen/Pfizer

Unknown Unknown

Unknown Unknown

Unknown Unknown

Unknown Unknown

þ

Unknown þ

þ þ

þ þ

þ þ

PHA-680632 PHA-739358

Unknown

þ

þ

Oral Unknown

Unknown Oral/IV

Unknown Oral

Oral Oral

IV Oral

IV IV

Oral

Oral

Unknown IV

Unknown

Aurora Aurora Aurora A inhibition B inhibition C inhibition Formulation

JNJ-7706621

Compound

Johnson and Johnson Nerviano Nerviano

Source

TABLE 2 Aurora Kinase Inhibitors in Development Comments

Preclinical Preclinical

Preclinical

Preclinical

None None

None Natural product, also inhibits Polo-like kinase 1 None None

Also inhibits CDK1, CDK2 and CDK3 Preclinical None Phase II Schedule: days 1, 8, and 15 MTD: unknown DLTs: unknown No longer in Also inhibits several receptor development tyrosine kinases and TANK-binding kinase 1 Clinical use Inhibits Aurora B in Japan through survivin Preclinical None Phase I Schedule: days 1,8, and 15 MTD: 200 mg DLTs: neutropenia Phase II Also inhibits FLT3 Phase I At high doses also inhibits Aurora B Phase I None Preclinical None

Status Preclinical

References

www.cyclacel.com (21)

www.avalonrx.com www.rigel.com

(57,58) www.supergen.com (113) www.sunesis.com (114)

(51,55) (46–48,109–112)

(27,50,108) (56)

(107)

(60)

(54) (59)

(106)


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MK-0457 (VX-680; Merck/Vertex) MK-0457 is the Aurora kinase inhibitor that is furthest along in clinical development. It is currently in phase II trials in colorectal cancer and also in refractory acute myelogenous leukemia, poor risk myelodysplastic syndrome (MDS), B-cell acute lymphocytic leukemia (ALL) or chronic myelogenous leukemia (CML) in blast crisis (55). MK-0457 inhibits all three of the known Aurora kinases A, B and C. The inhibitory constants suggest that in terms of in vitro potency the inhibition is A > C > B. It also inhibits a multitude of other kinases to some degree including FLT-3, which is thought to be important in refractory acute myeloid leukemia and other hematological malignancies and as such may serve as an area of investigation for this compound independent of its Aurora kinase inhibition. Recently, results from a phase I study of this agent in advanced solid malignancies were presented (55). MK-0457 was administered as a continuous 24-hour intravenous for 5 days infusion every 28 days. An accelerated titration scheme was employed for dose escalation whereby 1–2 patients were treated per dose level until a grade 2 toxicity ensued at which time a more standard modified Fibonacci dose escalation scheme was followed. A total of 22 patients were treated in this first-in-man study. Characteristics of these patients were as follows: male:female (8:14), median age: 57 years, ECOG PS 0:1:2 (6:15:1), cancer types were colon (n = 6), lung (n = 3), pancreas (n = 4) and other (n = 5), and median number of prior therapies was three. The starting dose of MK-0457 was 0.5 mg/m2/hr. Virtually no toxicities were observed until a dose of 8 mg/m2/hr was achieved. One of seven patients had a neutropenic fever at 8 mg/m2/hr, one of six patients had a neutropenic fever at 10 mg/m2/hr, and two of four patients treated at 12 mg/m2/hr experienced grade 4 neutropenia. As such, the preclinically predicted toxicity of neutronpenia was the DLT for the drug. The dose of 10 mg/m2/hr was recommended as the dose most appropriate for future phase II investigations. Three patients had stable disease as their best response and two of these (patient with pancreatic cancer treated at the 2 mg/m2/hr dose and patient with non-small cell lung cancer treated at the 4 mg/m2/hr dose) completed 6 cycles of therapy. Pharmacokinetics were dose-related and showed an initial exponential decline in MK-0457 levels shortly after discontinuation of the infusion followed by a slower terminal phase. It was also noted that the levels of MK-0457 achieved during the infusion at all dose levels were higher than the IC50 (1 mM) during in vitro studies and above the effective doses in xenograft studies. The half-life of MK-0457 after completion of the infusion was about 15 hr. Biomarkers studied during this phase I study were the measurement of Histone H3 phosphorylation in skin (expected effect of this Aurora B substrate would be a decrease with administration of drug), Ki-67 measurement in skin (expected effect of this proliferation marker would be a decrease), and cyclin B1 measurement in skin (expected effect of this mitotic arrest marker would be an increase after administration of drug). Unfortunately, these markers did not help to demonstrate the proof-of-concept as they were found at very low levels at baseline and the changes in these markers were not substantial even at the higher dose levels of MK-0457 (8 and 12 mg/m2/hr) at which they were obtained.


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AZD1152 (AstraZeneca) AZD1152 is an aurora kinase inhibitor that is selective for Aurora B (IC50s: Aurora A ¼ 1369 nM, Aurora B ¼ 0.36 nM, Aurora C ¼ 17.0 nM). It did not exhibit any significant activity against any other kinases among a panel of >50 kinases studied. Data on results from the first phase I investigation in advanced solid malignancies was reported recently (56). The study was tiered into three parts: A, B and C. Part A involved weekly dosing on days 1, 8 and 15 of a 21-day cycle; Part B comprised bi-weekly dosing on days 1 and 15 of a 28 day cycle and; Part C was the expansion phase incorporating tumor biopsies. A total of 19 patients were described. The median age of the patients was 59 years (range 40–71 years). The male:female composition was 14:5. Tumor types were colorectal (n = 8), melanoma/schwannoma (n = 3), prostate cancer (n = 2), nasopharyngeal cancer (n = 1), mesothelioma (n = 1), esophageal cancer (n = 1), pancreatic cancer (n = 1), renal cancer (n = 1), and adenoid cystic cancer (n = 1). World Health Organization (WHO) performance statuses of the patients ranged from 0 to 2 (WHO PS 0, =1; WHO PS 1, n = 16, and WHO PS 2, n = 2). An initial accelerated titration scheme, using single patients per dose level, that reverted to the modified Fibonacci approach, drug-related toxicities examining was used for dose escalation. The starting dose for this agent was 100 mg. A flat dosing approach was employed for the purposes of this study. Dose escalation beyond the second dose level of 200 mg used the modified Fibonacci approach and the classic “3þ3” design. Doses studied ranged from 100 to 450 mg. AZD1152 was administered as a 2 hour-intravenous infusion in all cases. Grade 3/4 neutropenia was the DLT associated with AZD1152 in 3 of 6 patients at 450 mg and 2 of 6 patients at 300 mg. Neutropenia was both febrile and resulted in dose delays at both levels. The dose delays were 14 days and as such a schedule that involved dosing at two-week intervals could incorporate what would otherwise be dose-limiting neutropenia. Of note, no lymphopenia or thrombocytopenia ensued as a result of AZD1152 exposure, and as such from these preliminary data it appears that thrombocytopenia, which could also be dose-limiting, will not be encountered. AZD1152 is a pro-drug, which is converted to the active moiety AZD1152hQPA. Pharmacokinetics revealed rapid clearing of the parent drug and rapid detection of the active moiety, AZD1152-hQPA. The plasma exposure was above the IC50s noted in preclinical studies at all the doses tested. Variability in pharmacokinetic parameters was low between subjects. Stable disease greater than 12 weeks was the best response to therapy achieved in five patients. Of these, one with melanoma (at 450 mg that was dose reduced to 300 mg), one with nasopharyngeal carcinoma (at 450 mg that was also dose reduced to 300 mg), and one with adenoid cystic carcinoma (at 300 mg that had no dose reductions) enjoyed stable disease for 32þ, 25 and 35þ weeks, respectively. Part B of this study which employs dosing on days 1 and 15 (2 week intervals) on a 28-day schedule is currently underway. MLN8054 (Millennium) MLN8054 is an orally available, potent and selective inhibitor of Aurora A. A novel approach was utilized in determining activity of the agent in xenograft models which can be described as a growth modulation index whereby differences

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in growth rates of tumors between treated and untreated groups of animals were compared to find a “signal” that the drug may have activity. Although it is shown to be Aurora A selective, this is only the case at lower concentrations from in vitro studies. At higher concentrations the phenotypic changes in treated cells are more consistent with Aurora B inhibition. Given this preclinical information it will be interesting to see the pharmacodynamic data generated from the phase I study in advanced solid tumors that is currently underway (46–48). AT9283 (Astex) AT9283 is an orally available inhibitor of the Aurora A and Aurora B kinases. It has recently entered into phase I clinical trials in imatinib refractory chronic myelogenous leukemia. This molecule was developed using a structure-based approach towards drug discovery (57,58). PHA739358 (Nerviano) This compound is in a phase II clinical trial in patients with CML with T315I mutations. It is being administered on a 28-day schedule on days 1, 8 and 15 by way of a 6-hour intravenous infusion. Other than what is stated, very limited information is available publicly on this compound (54,59). SU6668 (Pfizer) SU6668 was developed as an oral multi-targeted kinase inhibitor. It is felt to inhibit VEGFR2, PDGFR-beta, c-KIT and FGFR-1. Additionally, recent work has shown that it is also a potent inhibitor in vitro of Aurora A and Aurora B kinases as well as the TANK-binding kinase 1 (60). As such, SU6668 is clearly not a selective Aurora kinase inhibitor. SU6668 has been the subject of four separate phase I studies reported thus far. Given that development of this compound had been discontinued in favor of the now approved agent sunitinib and that it has only been found to be an aurora kinase inhibitor in a single in vitro study, we have not described the clinical development of this compound in detail. ISSUES IN FUTURE CLINICAL DEVELOPMENT OF AURORA KINASE INHIBITORS Clinical Indications for Future Development of Aurora Kinase Inhibitors As noted previously MK-0457 (VX-680) is currently in phase II clinical investigations in patients with colorectal cancer. Extensive preclinical information supports the development of this compound in this tumor type. Many other tumor types have been found to have expression of aurora kinases and would be tumor types of interest from the perspective of clinical development (Table 1). Interestingly, preclinical studies have shown a relationship between estrogen levels and aurora kinase expression. From a drug development perspective, it may make sense to look for differences in response between men and women and between premenopausal and menopausal or post-menopausal women (61,62). Formulation Choice in Drug Development Thus far the two agents with the most extensive clinical experience, AZD1152 and MK-0457, have been developed as intravenous drugs. MLN8054 is being


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developed as on oral drug. Oral drugs will maintain an advantage over intravenous agents of equal efficacy only if they are dosed on a chronic schedule. Intermittent administration would likely favor parenteral agents because the variability in absorption would be removed from the variability and unpredictability equation with regards to the dose of the drug. Given that so far doselimiting neutropenia has been the major obstacle in drug development of this class of compounds, it appears that intermittent administration may be the way forward and that parenteral formulations could be the favored choice. Dose Escalation Schemes Accelerated titration employing one patient per dose level or initial dose level increments of 100% (63) followed by the traditional Fibonacci method has been the dose escalation scheme utilized in the development of both MK-0457 and AZD1152. Dose-limiting toxicities have been manageable with this approach and this will likely be the favored approach for the early development of other agents in this class. Novel Clinical Trial Designs Standard clinical trial approaches that employ investigation of the agent in a particular tumor type have historically had low success rates and have failed to uncover those populations of patients within a group as a whole who benefited but were not identified. A more rational approach that is increasing in popularity is treating only those patients who have either overexpression in the target felt to be affected by the drug, mutations in a gene that is felt to render responsiveness to the drug, or gene amplification of the target felt to be affected by the drug (as in the case of FISHþ HER2/neu breast cancer patients), etc. This approach can be concisely described as treating only targetþ patients after selection. In the case of aurora kinase inhibitor development targetþ could be: increased levels of expression of Aurora A, Aurora B, or Aurora C; amplification of Aurora A, Aurora B, or Aurora C or increased expression of aurora kinase substrates such as CENP-A, myosin regulatory light chain, protein phosphatase-1, TPX-2, INCENP, survivin, topoisomerase II alpha, vimentin, MBD-3, MgcRacGAP, desmin, Ajuba, and TACC (51), or mutations in aurora kinase. A major pitfall of this approach would be in tumor settings where Aurora (A, B or C) or an aurora-related target is abnormal but not a primary contributor to disease. In such a case, the target would not prove to be clinically meaningful because it is eclipsed by a more dominant or compensatory pathway which could potentially negate the effects of inhibiting the Aurora pathway. Regardless, a targetþ approach to select candidates for an aurora-based therapy is logical. Instead of a single arm phase II trial, it may make sense to utilize a randomized phase II whereby a “positive signal” may confer more confidence prior to pursuing a larger, more expensive trial (64). Such a trial design may employ two or three arms. In the case of the three arm design this may be: Arm 1: High Dose Aurora Kinase Inhibitor; Arm 2: Low Dose Aurora Kinase Inhibitor; and Arm 3: Standard Treatment or Placebo if appropriate. The determination of significant differences in a predetermined end-point such as time-to-tumor progression, relapse rate, response rate, or progression free survival can serve as the “signal” that would warrant further investigation of the agent in larger trials.

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If agents that are orally available and chronically dosed are advanced enough in clinical development a novel clinical strategy that can be employed is a randomized discontinuation design (65). Combination of Aurora Kinase Inhibitors with Other Agents Aurora kinase inhibitors, like many other cancer therapeutic agents, may find their optimal utility in combination with agents from other classes. In vitro studies have shown AZD1152 to be synergistic with irinotecan, gemcitabine, docetaxel, oxaliplatin, 5-FU and vinorelbine. Of interest in preclinical systems, it was found that administration of gemcitabine or irinotecan prior to the aurora kinase inhibitor inhibited its activity. This suggests that in the clinical setting with these combinations, perhaps the aurora kinase inhibitor should be administered prior to administration of either gemcitabine or irinotecan (66). In addition to traditional cytotoxics, it would be very worthwhile to explore the combination of agents that disrupt pathways that affect closely related or associated pathways. These include the survivin inhibitors, heat shock protein inhibitors (67–69), and KSP/Eg5 inhibitors (70,71). Even from a clinical perspective it makes more sense to use these combinations because the primary dose-limiting toxicity seen thus far with the aurora kinase inhibitors has been neutropenia, which along with the neutropenia of traditional cytotoxics would definitely preclude clinical development in an optimal fashion. Separate Trials in Heavily Pretreated Patients Given that neutropenia has been the major DLT in early clinical investigations, it would seem reasonable that separate phase II investigations ought to be conducted in patients heavily pretreated with cytotoxic chemotherapy versus minimally pretreated patients, as the MTD for the minimally pretreated patients may very well be higher. Development of Novel Pharmacodynamic Assays One of the most difficult aspects of drug development with new agents has been clinically validating the proof-of-concept regarding mechanisms of action and their correlation to clinically meaningful efficacy. Thus far, in patients receiving MK-0457, skin biopsies evaluating Ki-67, cyclin B1, and phosporylation of serine residue 10 in histone H3 have been used as pharmacodynamic tools. Unfortunately, these assays were not helpful in validating aurora kinase inhibition. It is possible that the tissue selected (skin in this case) may not have been appropriate in terms of reflecting events occurring in the tumor environment/microenvironment. Clearly, novel assays and appropriate tissue selection for observation for evidence of activity are required. Given that aurora kinases are expressed in significant proportions only during active cell division (i.e., M phase) it would be prudent to assess for inhibition of enzyme activity by an aurora kinase inhibitor in actively dividing cells. In addition to the skin, the oropharyngeal mucosa, hair follicles, gut, and bone marrow represent sites where collected samples would be replete in proliferating cells. As such, potential assays could include demonstration of polyploidy in buccal smear samples after administration of an Aurora B kinase inhibitor, hair follicle assay demonstrating cells having monopolar spindles after


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administration of an Aurora A kinase inhibitor, and stool samples containing cells shed from the bowel mucosa demonstrating loss of phosphorylation at the T288 autophosphorylation site after administration of an Aurora A kinase inhibitor (72–74). Analysis of cell populations obtained from tumor samples using FACS (Fluorescence Activated Cell Sorting) analysis to demonstrate a shift from predominantly normal 2N populations to 4N, 8N, 16N, etc. populations (i.e., cells that have not undergone cytokinesis) would also validate success of the compound under investigation from a proof-of-concept standpoint. Use of Imaging Traditional imaging modalities such as CT, MRI, or ultrasound would fail to provide conclusive and early evidence that aurora kinase inhibitors were exerting their desired effects. Given that aurora kinase inhibitors may have more cytostatic as opposed to cytotoxic effects, tumor shrinkage will likely be a rare event, and, as such, these traditional imaging modalities, despite being somewhat helpful from a clinical standpoint, will do nothing to build confidence in validation of the concept that disruption of the aurora kinase pathway is biologically meaningful. PET scans have overcome some of the shortfalls of the traditional imaging modalities in terms of providing a “metabolic” perspective of drug effects or disease status. Unfortunately, PET imaging may not be greatly valuable using the most widely used agent, FDG. This primarily stems from the mechanism of Aurora A inhibitors, which do not affect cellular proliferation but tend to affect disruption of cytokinesis. On the other hand FDG PET may be a valuable tool in evaluating Aurora B inhibitors. With these caveats, it is clear that development of novel imaging agents required to assess activity of this class of compounds is in dire need. CONCLUSION Aurora kinase inhibitors have enjoyed a rapid trajectory from the discovery of these enzymes to the first clinical studies of inhibitors of this novel target. The prompt development of these agents has been fueled by the interesting biology of the aurora family of kinases and by their implicated roles in tumor growth. Appropriate use of aurora kinase inhibitors as single agents, and in combination with other agents that affect complementary or related pathways, will be paramount in the successful integration of this class of drugs into the anti-cancer armamentarium. We remain optimistic that inhibitors targeting the aurora kinases will lead to increased disease-free survival of cancer patients. ACKNOWLEDGMENTS The preparation of this review was supported in part by NIH National Cancer Institute Grant CA95031 and the Drug Development Scholar Fund for the Translational Genomics Research Institute and for the Scottsdale Clinical Research Institute (MB). The authors also appreciate the assistance of Susan McCall with manuscript preparation.

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REFERENCES 1. Chan CS, Botstein D. Isolation and characterization of chromosome-gain and increasein-ploidy mutants in yeast. Genetics 1993; 135:677–91. 2. Francisco L, Chan CS. Regulation of yeast chromosome segregation by Ipl1 protein kinase and type 1 protein phosphatase. Cell Mol Biol Res 1994; 40:207–13. 3. Francisco L, Wang W, Chan CS. Type 1 protein phosphatase acts in opposition to IpL1 protein kinase in regulating yeast chromosome segregation. Mol Cell Biol 1994; 14:4731–40. 4. Glover DM, Leibowitz MH, McLean DA, Parry H. Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles. Cell 1995; 81:95–105. 5. Sunkel CE, Glover DM. Polo, a mitotic mutant of Drosophila displaying abnormal spindle poles. J Cell Sci 1988; 89(Pt 1):25–38. 6. Sen S, Zhou H, White RA. A putative serine/threonine kinase encoding gene BTAK on chromosome 20q13 is amplified and overexpressed in human breast cancer cell lines. Oncogene 1997; 14:2195–200. 7. Kimura M, Matsuda Y, Eki T, et al. Assignment of STK6 to human chromosome 20q13.2!q13.3 and a pseudogene STK6P to 1q41!q42. Cytogenet Cell Genet 1997; 79:201–3. 8. Kimura M, Kotani S, Hattori T, et al. Cell cycle-dependent expression and spindle pole localization of a novel human protein kinase, Aik, related to Aurora of Drosophila and yeast Ipl1. J Biol Chem 1997; 272:13766–71. 9. Bischoff JR, Anderson L, Zhu Y, et al. A homologue of Drosophila aurora kinase is oncogenic and amplified in human colorectal cancers. EMBO J 1998; 17:3052–65. 10. Adams RR, Carmena M, Earnshaw WC. Chromosomal passengers and the (aurora) ABCs of mitosis. Trends Cell Biol 2001; 11:49–54. 11. Nigg EA. Mitotic kinases as regulators of cell division and its checkpoints. Nat Rev Mol Cell Biol 2001; 2:21–32. 12. Rojanala S, Han H, Munoz RM, et al. The mitotic serine threonine kinase, Aurora-2, is a potential target for drug development in human pancreatic cancer. Mol Cancer Ther 2004; 3:451–7. 13. Eyers PA, Erikson E, Chen LG, Maller JL. A novel mechanism for activation of the protein kinase Aurora A. Curr Biol 2003; 13:691–7. 14. Tsai MY, Wiese C, Cao K, et al. A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle assembly. Nat Cell Biol 2003; 5:242–8. 15. Kufer TA, Sillje HH, Korner R, Gruss OJ, Meraldi P, Nigg EA. Human TPX2 is required for targeting Aurora-A kinase to the spindle. J Cell Biol 2002; 158:617–23. 16. Bayliss R, Sardon T, Vernos I, Conti E. Structural basis of Aurora-A activation by TPX2 at the mitotic spindle. Mol Cell 2003; 12:851–62. 17. Giet R, Uzbekov R, Cubizolles F, Le Guellec K, Prigent C. The Xenopus laevis aurorarelated protein kinase pEg2 associates with and phosphorylates the kinesin-related protein XlEg5. J Biol Chem 1999; 274:15005–13. 18. Giet R, McLean D, Descamps S, et al. Drosophila Aurora A kinase is required to localize D-TACC to centrosomes and to regulate astral microtubules. J Cell Biol 2002; 156:437–51. 19. Carmena M, Earnshaw WC. The cellular geography of aurora kinases. Nat Rev Mol Cell Biol 2003; 4:842–54. 20. Murata-Hori M, Wang YL. The kinase activity of aurora B is required for kinetochoremicrotubule interactions during mitosis. Curr Biol 2002; 12:894–9. 21. Hauf S, Cole RW, LaTerra S, et al. The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint. J Cell Biol 2003; 161:281–94. 22. Lampson MA, Renduchitala K, Khodjakov A, Kapoor TM. Correcting improper chromosome-spindle attachments during cell division. Nat Cell Biol 2004; 6:232–7. 23. Andrews PD, Ovechkina Y, Morrice N, et al. Aurora B regulates MCAK at the mitotic centromere. Dev Cell 2004; 6:253–68.


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24. Kallio MJ, McCleland ML, Stukenberg PT, Gorbsky GJ. Inhibition of aurora B kinase blocks chromosome segregation, overrides the spindle checkpoint, and perturbs microtubule dynamics in mitosis. Curr Biol 2002; 12:900–5. 25. Severson AF, Hamill DR, Carter JC, Schumacher J, Bowerman B. The aurora-related kinase AIR-2 recruits ZEN-4/CeMKLP1 to the mitotic spindle at metaphase and is required for cytokinesis. Curr Biol 2000; 10:1162–71. 26. Terada Y, Tatsuka M, Suzuki F, Yasuda Y, Fujita S, Otsu M. AIM-1: a mammalian midbody-associated protein required for cytokinesis. EMBO J 1998; 17:667–76. 27. Ditchfield C, Johnson VL, Tighe A, et al. Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores. J Cell Biol 2003; 161:267–80. 28. Giet R, Glover DM. Drosophila aurora B kinase is required for histone H3 phosphorylation and condensin recruitment during chromosome condensation and to organize the central spindle during cytokinesis. J Cell Biol 2001; 152:669–82. 29. Jelinkova L, Kubelka M. Neither Aurora B activity nor histone H3 phosphorylation is essential for chromosome condensation during meiotic maturation of porcine oocytes. Biol Reprod 2006; 74:905–12. 30. Yanai A, Arama E, Kilfin G, Motro B. Ayk1, a novel mammalian gene related to Drosophila aurora centrosome separation kinase, is specifically expressed during meiosis. Oncogene 1997; 14:2943–50. 31. Kimura M, Matsuda Y, Yoshioka T, Okano Y. Cell cycle-dependent expression and centrosome localization of a third human aurora/Ipl1-related protein kinase, AIK3. J Biol Chem 1999; 274:7334–40. 32. Jeng YM, Peng SY, Lin CY, Hsu HC. Overexpression and amplification of Aurora-A in hepatocellular carcinoma. Clin Cancer Res 2004; 10:2065–71. 33. Zhou H, Kuang J, Zhong L, et al. Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation. Nat Genet 1998; 20:189–93. 34. Warner SL, Bearss DJ, Han H, Von Hoff DD. Targeting Aurora-2 kinase in cancer. Mol Cancer Ther 2003; 2:589–95. 35. Goepfert TM, Adigun YE, Zhong L, Gay J, Medina D, Brinkley WR. Centrosome amplification and overexpression of Aurora A are early events in rat mammary carcinogenesis. Cancer Res 2002; 62:4115–22. 36. Buschhorn HM, Klein RR, Chambers SM, et al. Aurora-A over-expression in highgrade PIN lesions and prostate cancer. Prostate 2005; 64:341–6. 37. Ouchi M, Fujiuchi N, Sasai K, et al. BRCA1 phosphorylation by Aurora-A in the regulation of G2 to M transition. J Biol Chem 2004; 279:19643–8. 38. Yang H, Ou CC, Feldman RI, Nicosia SV, Kruk PA, Cheng JQ. Aurora-A kinase regulates telomerase activity through c-Myc in human ovarian and breast epithelial cells. Cancer Res 2004; 64:463–7. 39. Katayama H, Sasai K, Kawai H, et al. Phosphorylation by aurora kinase A induces Mdm2-mediated destabilization and inhibition of p53. Nat Genet 2004; 36:55–62. 40. Liu Q, Kaneko S, Yang L, et al. Aurora-A abrogation of p53 DNA binding and transactivation activity by phosphorylation of serine 215. J Biol Chem 2004; 279:52175–82. 41. Anand S, Penrhyn-Lowe S, Venkitaraman AR. AURORA-A amplification overrides the mitotic spindle assembly checkpoint, inducing resistance to Taxol. Cancer Cell 2003; 3:51–62. 42. Kanda A, Kawai H, Suto S, et al. Aurora-B/AIM-1 kinase activity is involved in Ras-mediated cell transformation. Oncogene 2005; 24:7266–72. 43. Kurai M, Shiozawa T, Shih HC, et al. Expression of Aurora kinases A and B in normal, hyperplastic, and malignant human endometrium: Aurora B as a predictor for poor prognosis in endometrial carcinoma. Hum Pathol 2005; 36:1281–8. 44. Smith SL, Bowers NL, Betticher DC, et al. Overexpression of aurora B kinase (AURKB) in primary non-small cell lung carcinoma is frequent, generally driven from one allele, and correlates with the level of genetic instability. Br J Cancer 2005; 93: 719–29.

172

Borad et al.

45. Hata T, Furukawa T, Sunamura M, et al. RNA interference targeting aurora kinase A suppresses tumor growth and enhances the taxane chemosensitivity in human pancreatic cancer cells. Cancer Res 2005; 65:2899–905. 46. Hoar KM, Wysong DR, Ecsedy JA. MLN8054 selectively inhibits Aurora A over Aurora B in cultured human tumor cells. Clin Cancer Res 2005; 11(24 Suppl):9109s. 47. Burenkova O, Bond S, Connolly K, et al. MLN8054, an orally active small molecule inhibitor, mediates tumor growth inhibition in vivo through Aurora A kinase. Clin Cancer Res 2005; 11(24 Suppl):9082s, Abstract B200. 48. Manfredi MG, Claiborne CF. MLN8054: A selective, oral small molecule inhibitor of Aurora A kinase entering clinical development for cancer. Clin Cancer Res 2005; 11 (24 Suppl):9082s, Abstract B202. 49. Yang H, Burke T, Dempsey J, et al. Mitotic requirement for aurora A kinase is bypassed in the absence of aurora B kinase. FEBS Lett 2005; 579:3385–91. 50. Gadea BB, Ruderman JV. Aurora kinase inhibitor ZM447439 blocks chromosomeinduced spindle assembly, the completion of chromosome condensation, and the establishment of the spindle integrity checkpoint in Xenopus egg extracts. Mol Biol Cell 2005; 16:1305–18. 51. Harrington EA, Bebbington D, Moore J, et al. VX-680, a potent and selective smallmolecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo. Nat Med 2004; 10:262–7. 52. Gizatullin F, Yao Y, Kung V, Harding MW, Loda M, Shapiro GI. The Aurora kinase inhibitor VX-680 induces endoreduplication and apoptosis preferentially in cells with compromised p53-dependent postmitotic checkpoint function. Cancer Res 2006; 66:7668–77. 53. Young MA, Shah NP, Chao LH, et al. Structure of the kinase domain of an imatinibresistant Abl mutant in complex with the Aurora kinase inhibitor VX-680. Cancer Res 2006; 66:1007–14. 54. Soncini C, Carpinelli P, Gianellini L, et al. PHA-680632, a novel Aurora kinase inhibitor with potent antitumoral activity. Clin Cancer Res 2006; 12:4080–9. 55. Rubin EH, Shapiro GI, Stein MN, et al. A phase I clinical and pharmacokinetic (PK) trial of the aurora kinase (AK) inhibito MK-0457 in cancer patients. J Clin Oncol 2006; 24(18S): abstract 3009(18S). 56. Schellens JH, Boss D, Witteveen PO, et al. Phase 1 and pharmacological study of the novel aurora kinase inhibitor AZD1152. J Clin Oncol 2006; 24(18S): abstract 3008. 57. Gallagher N, Lyons JF, Curry JE, et al. Characterization of aurora kinase inhibitors derived from fragment based lead discovery. Proc Am Assoc Cancer Res 2006; 47:4915. 58. Gill AL, Berdinin V, Howard S, et al. A fragment based drug discovery process used to identify novel aurora kinase inhibitors exhibiting in vivo xenograft activity. Proc Am Assoc Cancer Res 2005; 46:4411. 59. PHA-739358 in treating patients with chronic myelogenous leukemia that relapsed after imatinib mesylate or c-able therapy. Clinical Trials.gov Identifier NCT00335868, 2006. 60. Godl K, Gruss OJ, Eickhoff J, et al. Proteomic characterization of the angiogenesis inhibitor SU6668 reveals multiple impacts on cellular kinase signaling. Cancer Res 2005; 65:6919–26. 61. Hontz AE, Li SA, Salisbury JL, Lingle WL, Li JJ. Enrichment of amplified centrosomes and aurora A and B from solely estrogen induced ectopic uterine stem cell tumors (EUTK) in the syrian hamster kidney. Proc Am Assoc Cancer Res 2006; 47:5642. 62. Li JJ, Werosh SJ, Lingle WL, Salisbury JL, Li SA. Estrogen mediates aurora a kinase overexpression, centrosome amplification, chromosomal instability and oncogenesis in female ACI rat mammary glands. Proc Am Assoc Cancer Res 2004; 45:6. 63. Simon R, Freidlin B, Rubinstein L, Arbuck SG, Collins J, Christian MC. Accelerated titration designs for phase I clinical trials in oncology. J Natl Cancer Inst 1997; 89:1138–47.


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64. Yang JC, Haworth L, Sherry RM, et al. A randomized trial of bevacizumab, an antivascular endothelial growth factor antibody, for metastatic renal cancer. N Engl J Med 2003; 349:427–34. 65. Ratain MJ, Eisen T, Stadler WM, et al. Phase II placebo-controlled randomized discontinuation trial of sorafenib in patients with metastatic renal cell carcinoma. J Clin Oncol 2006; 24:2505–12. 66. Nair JS, Tse A, Keen N, Schwartz GK. A novel aurora B kinase inhibitor with potent anticancer activity either as a single agent or in combination with chemotherapy. J Clin Oncol 2004; 22:9568. 67. Lange BM, Rebollo E, Herold A, Gonzalez C. Cdc37 is essential for chromosome segregation and cytokinesis in higher eukaryotes. EMBO J 2002; 21:5364–74. 68. Miyata Y, Nishida E. CK2 controls multiple protein kinases by phosphorylating a kinase-targeting molecular chaperone, Cdc37. Mol Cell Biol 2004; 24:4065–74. 69. Terasawa K, Minami Y. A client-binding site of Cdc37. FEBS J 2005; 272:4684–90. 70. Bergnes G, Brejc K, Belmont L. Mitotic kinesins: prospects for antimitotic drug discovery. Curr Top Med Chem 2005; 5:127–45. 71. Sakowicz R, Finer JT, Beraud C, et al. Antitumor activity of a kinesin inhibitor. Cancer Res 2004; 64:3276–80. 72. Adolphe C, Hetherington R, Ellis T, Wainwright B. Patched1 functions as a gatekeeper by promoting cell cycle progression. Cancer Res 2006; 66:2081–8. 73. Song K, Fendrick AM, Ladabaum U. Fecal DNA testing compared with conventional colorectal cancer screening methods: a decision analysis. Gastroenterology 2004; 126:1270–9. 74. Velagaleti GV, Tapper JK, Rampy BA, Zhang S, Hawkins JC, Lockhart LH. A rapid and noninvasive method for detecting tissue-limited mosaicism: detection of i(12)(p10) in buccal smear from a child with Pallister-Killian syndrome. Genet Test 2003; 7:219–23. 75. Tanaka T, Kimura M, Matsunaga K, Fukada D, Mori H, Okano Y. Centrosomal kinase AIK1 is overexpressed in invasive ductal carcinoma of the breast. Cancer Res 1999; 59:2041–4. 76. Takahashi T, Futamura M, Yoshimi N, et al. Centrosomal kinases, HsAIRK1 and HsAIRK3, are overexpressed in primary colorectal cancers. Jpn J Cancer Res 2000; 91:1007–14. 77. Hu W, Kavanagh JJ, Deaver M, et al. Frequent overexpression of STK15/Aurora-A/ BTAK and chromosomal instability in tumorigenic cell cultures derived from human ovarian cancer. Oncol Res 2005; 15:49–57. 78. Gritsko TM, Coppola D, Paciga JE, et al. Activation and overexpression of centrosome kinase BTAK/Aurora-A in human ovarian cancer. Clin Cancer Res 2003; 9:1420–6. 79. Bar-Shira A, Pinthus JH, Rozovsky U, et al. Multiple genes in human 20q13 chromosomal region are involved in an advanced prostate cancer xenograft. Cancer Res 2002; 62:6803–7. 80. Kamada K, Yamada Y, Hirao T, et al. Amplification/overexpression of Aurora-A in human gastric carcinoma: potential role in differentiated type gastric carcinogenesis. Oncol Rep 2004; 12:593–9. 81. Sakakura C, Hagiwara A, Yasuoka R, et al. Tumour-amplified kinase BTAK is amplified and overexpressed in gastric cancers with possible involvement in aneuploid formation. Br J Cancer 2001; 84:824–31. 82. Fraizer GC, Diaz MF, Lee IL, Grossman HB, Sen S. Aurora-A/STK15/BTAK enhances chromosomal instability in bladder cancer cells. Int J Oncol 2004; 25:1631–9. 83. Sen S, Zhou H, Zhang RD, et al. Amplification/overexpression of a mitotic kinase gene in human bladder cancer. J Natl Cancer Inst 2002; 94:1320–9. 84. Mhawech-Fauceglia P, Fischer G, Beck A, Cheney RT, Herrmann FR. Raf1, Aurora-A/ STK15 and E-cadherin biomarkers expression in patients with pTa/pT1 urothelial bladder carcinoma; a retrospective TMA study of 246 patients with long-term followup. Eur J Surg Oncol 2006; 32:439–44.

174

Borad et al.

85. Han H, Bearss DJ, Browne LW, Calaluce R, Nagle RB, Von Hoff DD. Identification of differentially expressed genes in pancreatic cancer cells using cDNA microarray. Cancer Res 2002; 62:2890–6. 86. Li D, Zhu J, Firozi PF, et al. Overexpression of oncogenic STK15/BTAK/Aurora A kinase in human pancreatic cancer. Clin Cancer Res 2003; 9:991–7. 87. Li FC, Li YH, Zhao X, et al. Deletion of p15 and p16 genes and overexpression of STK15 gene in human laryngeal squamous cell carcinoma. Zhonghua Yi Xue Za Zhi 2003; 83:316–9. 88. Li YH, Li FC, Wang X, et al. Study on STK15 gene abnormality and centrosomal amplification in laryngeal carcinoma. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2004; 21:240–4. 89. Li YH, Li FC, Zhao X, Zhao Z, Sun KL, Sun XH. Expression of STK15 gene and chromosomal instability in laryngeal carcinoma. Yi Chuan Xue Bao 2002; 29:1048–51. 90. Ye Y, Li FC, Wang SY, et al. The investigation of STK15 gene amplification and overexpression in laryngeal squamous cell carcinoma. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2006; 23:326–9. 91. Zhao X, Li FC, Li YH, et al. Mutation of p53 and overexpression of STK15 in laryngeal squamous-cell carcinoma. Zhonghua Zhong Liu Za Zhi 2005; 27:134–7. 92. Hamada M, Yakushijin Y, Ohtsuka M, Kakimoto M, Yasukawa M, Fujita S. Aurora2/ BTAK/STK15 is involved in cell cycle checkpoint and cell survival of aggressive non-Hodgkin’s lymphoma. Br J Haematol 2003; 121:439–47. 93. Moreno-Bueno G, Sanchez-Estevez C, Cassia R, et al. Differential gene expression profile in endometrioid and nonendometrioid endometrial carcinoma: STK15 is frequently overexpressed and amplified in nonendometrioid carcinomas. Cancer Res 2003; 63:5697–702. 94. Tong T, Zhong Y, Kong J, et al. Overexpression of Aurora-A contributes to malignant development of human esophageal squamous cell carcinoma. Clin Cancer Res 2004; 10:7304–10. 95. Tatsuka M, Sato S, Kitajima S, et al. Overexpression of Aurora-A potentiates HRASmediated oncogenic transformation and is implicated in oral carcinogenesis. Oncogene 2005; 24:1122–7. 96. Xu HT, Ma L, Qi FJ, et al. Expression of serine threonine kinase 15 is associated with poor differentiation in lung squamous cell carcinoma and adenocarcinoma. Pathol Int 2006; 56:375–80. 97. Reiter R, Gais P, Jutting U, et al. Aurora kinase A messenger RNA overexpression is correlated with tumor progression and shortened survival in head and neck squamous cell carcinoma. Clin Cancer Res 2006; 12:5136–41. 98. Klein A, Reichardt W, Jung V, Zang KD, Meese E, Urbschat S. Overexpression and amplification of STK15 in human gliomas. Int J Oncol 2004; 25:1789–94. 99. Neben K, Korshunov A, Benner A, et al. Microarray-based screening for molecular markers in medulloblastoma revealed STK15 as independent predictor for survival. Cancer Res 2004; 64:3103–11. 100. Camacho E, Bea S, Salaverria I, et al. Analysis of Aurora-A and hMPS1 mitotic kinases in mantle cell lymphoma. Int J Cancer 2006; 118:357–63. 101. Tatsuka M, Katayama H, Ota T, et al. Multinuclearity and increased ploidy caused by overexpression of the aurora- and Ipl1-like midbody-associated protein mitotic kinase in human cancer cells. Cancer Res 1998; 58:4811–6. 102. Katayama H, Ota T, Jisaki F, et al. Mitotic kinase expression and colorectal cancer progression. J Natl Cancer Inst 1999; 91:1160–2. 103. Araki K, Nozaki K, Ueba T, Tatsuka M, Hashimoto N. High expression of Aurora-B/ Aurora and Ipll-like midbody-associated protein (AIM-1) in astrocytomas. J Neurooncol 2004; 67:53–64. 104. Sorrentino R, Libertini S, Pallante PL, et al. Aurora B overexpression associates with the thyroid carcinoma undifferentiated phenotype and is required for thyroid carcinoma cell proliferation. J Clin Endocrinol Metab 2005; 90:928–35.


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105. Chieffi P, Cozzolino L, Kisslinger A, et al. Aurora B expression directly correlates with prostate cancer malignancy and influences prostate cell proliferation. Prostate 2006; 66:326–33. 106. Emanuel S, Rugg CA, Gruninger RH, et al. The in vitro and in vivo effects of JNJ7706621: a dual inhibitor of cyclin-dependent kinases and aurora kinases. Cancer Res 2005; 65:9038–46. 107. Tarnawski A, Pai R, Chiou SK, Chai J, Chu EC. Rebamipide inhibits gastric cancer growth by targeting survivin and Aurora-B. Biochem Biophys Res Commun 2005; 334:207–12. 108. Girdler F, Gascoigne KE, Eyers PA, et al. Validating Aurora B as an anti-cancer drug target. J Cell Sci 2006; 119(Pt 17):3664–75. 109. Ecsedy J, Hoar KM, Wysong DR, et al. Effect of aurora A inhibition in cultured human tumor cells using the selective small molecule inhibitor MLN8054. Proc Am Assoc Cancer Res 2006; 47:2066. 110. Galvin KM, Huck J, Burenkova O, et al. Preclinical pharmacodynamic studies of aurora A inhibition by MLN8054. J Clin Oncol 2006; 24:13059. 111. Huck J, Zhang M, Burenkova O, Connolly K, Manfredi MG, Meetze K. Preclinical antitumor activity with MLN8054, a small molecule aurora A kinase inhibitor. Proc Am Assoc Cancer Res 2006; 47:4698. 112. Manfredi MG, Ecsedy J, Meetze K, et al. MLN8054, an orally active aurora A kinase small molecule inhibitor in phase I clinical trials. Proc Am Assoc Cancer Res 2006; 47:4724. 113. Warner SL, Bashyam S, Vankayalapati H, et al. Identification of a lead small-molecule inhibitor of the Aurora kinases using a structure-assisted, fragment-based approach. Mol Cancer Ther 2006; 5:1764–73. 114. Breur JG, Reagan-Shaw SR, Ahmad N. Prostate cancer chemopreventive effects of resveratrol are mediated via down modulation of mitosis kinases polo like kinase 1, aurora A, and aurora B. Proc Am Assoc Cancer Res 2005; 46:5198.

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Apoptosis Modulators: p53 Targeting Sunil Chada, Dora Bocangel, and Kerstin Menander Departments of Clinical Research and Development, Introgen Therapeutics, Inc., Houston, Texas, U.S.A.

Jack A. Roth Thoracic and Cardiovascular Surgery, University of Texas, M. D. Anderson Cancer Center, Houston, Texas, U.S.A.

INTRODUCTION Cancer is a disease initiated, driven, and sustained by genomic instability; many pleiotropic and overlapping signaling pathways contribute to oncogenesis and pathologic progression. Despite accumulating multiple genetic, epigenetic, and chromosomal abnormalities, cancer cells can become dependent on a single or a few oncogenic pathways for both maintenance of the malignant phenotype and cell survival; this phenomenon—termed “oncogene addiction”—has been reported in both cultured cell lines and animal models (1). Thus, reversal of only one or a few of these abnormalities can trigger massive apoptosis resulting in inhibition of cancer cell growth. Considerable progress in the treatment of cancer in recent years stems from the development and clinical application of drugs targeted to specific molecular pathways. Examples of these pathway-specific drugs are Gleevec (imatinib mesylate; Novartis Pharmaceutical Corp., East Hannover, New Jersey, U.S.A.) and Tarceva (erlotinib; Genentech, San Francisco, California, U.S.A.), both of which act as selective tyrosine kinase inhibitors. However, clinical success with these pathway-specific agents has been idiosyncratic. Agents that affect not only one, but various, albeit similar, pathways are being developed and have demonstrated improved clinical results. Such is the case of Sorafenib (Nevaxar; Wayne, New Jersey, U.S.A.), which was initially developed as a RAF-RAS kinase-targeted drug. Further studies showed that, in addition to targeting RAF kinase, Sorafenib also inhibits VEGF and PDGF receptor kinases, as well as KIT and FLT-3 kinases, culminating in tumor cell apoptosis and inhibition of angiogenesis. It appears that the combination of Sorafenib's actions on multiple tyrosine kinase pathways induces an enhanced therapeutic response by this drug. Additional drugs which target multiple kinases that have demonstrated clinical promise are sunitinib (Sutent; Pfizer, Inc., New York, New York, U.S.A.), dasatinib (Sprycel; BristolMyers Squibb, New York, New York, U.S.A.), and lapatinib (GlaxoSmithKline, Brentford, London, U.K.). In contrast, agents which have demonstrated specificity against one kinase target do not always show clinical activity; this concept was exemplified by the lack of robust clinical activity of Iressa(gefitinib; Astrazeneca, Mississauga, Ontario, Canada), despite early enthusiasm. This ability to simultaneously target and interfere with multiple cancerpromoting pathways is a property shared by p53. The p53 gene is a critical tumor suppressor that plays a key role maintaining the integrity of cellular DNA. p53 regulates progression through the cell cycle and, in the presence of DNA damage, 177

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functions as a regulatory node to either facilitate DNA repair, or initiate apoptotic cell death when the damage is too extensive (2–5). The primary mode of action of p53 is transcription modulation; p53 activates or represses expression of hundreds of target genes involved in regulation of cell cycle arrest (p21WAF1/CIP1), apoptosis (bax, bcl-2), and/or DNA-repair processes (6–9). Additionally, p53 inhibits neovascularization by regulating expression of several key proteins in the process, including VEGF, BAI1, TSP1 (10,11), and bFGF-binding protein (12,13). A lack of functional p53 protein can, therefore, allow the accumulation of genomic instability, resulting in unregulated proliferation of damaged cells and tumor formation (14–17). Aberrant p53 pathways are present in virtually all cancer cells, either by mutation/deletion of the p53 gene, or by abnormal regulation of p53 gene expression, stability, or function in the absence of p53 gene mutations (Lane). Mutations have been detected in over 50% of human cancers tested, and up to 70% of non–small cell lung cancer (NSCLC) and squamous cell carcinoma of the head and neck (SCCHN) (18–22). Other alterations of this pathway include inactivation or sequestration of the wild-type p53 gene product (e.g., inactivation via overexpression of MDM2) (Fig. 1), inability to activate p53 protein (e.g., via post-translational modifications), and mutations of downstream p53 targets (e.g., inactive enzymes in the apoptotic cascade) (23,24). Importantly, the presence of altered protein function or mutation of the p53 gene has been associated with poor clinical outcomes in patients with several types of cancer (25–30), and the presence

FIGURE 1 p53 regulatory pathway and map of Advexin genome.

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of p53 mutations or disrupted p53 pathways correlates with resistance to chemotherapy and radiation. Advexin (Introgen Therapeutics, Inc., Houston, Texas, U.S.A.) is an adenoviral vector, derived from adenovirus serotype 5 (Ad5), which mediates overexpression of the human wild-type tumor suppressor protein p53 under the control of the CMV promoter (Fig. 1) (31). The E1 region of the parental Ad5 DNA is deleted, thus preventing replication and expression of adenoviral genes. Numerous preclinical studies have demonstrated that transduction of cancer cells with a replication-incompetent adenoviral vector carrying the wild-type p53 gene (Ad-p53; Advexin), increases apoptosis and decreases proliferation of cancer cells with no apparent effect on normal cells (32). These studies have also shown that p53 sensitizes cancer cells to the effects of chemotherapy or radiation therapy and indicate that p53 may have utility both as monotherapy as well as a component of combination regimens. Significantly, increases in apoptosis and decreases in cancer cell proliferation have been demonstrated following administration of Advexin without observable effects on normal cells (33–35). Clinical studies have demonstrated that Advexin is safe and more easily tolerated than chemotherapy or radiation treatment. Initial clinical trials designed to assess the safety and tolerability of Advexin in patients with a variety of cancers had favorable outcomes, with safety profiles that are superior to those of chemotherapy and radiation (36–39). This review summarizes the extensive preclinical and clinical trials data gathered for Advexin in a variety of treatment settings as monotherapy and in combination with chemotherapy, radiation, and surgery. These studies indicate potential applications of Advexin for cancer prevention, initial cancer treatment, therapy of recurrent tumors and the treatment of disease refractory to standard therapies. Therefore, the use of wild type p53 to target genetic defects in tumor cells allows the application of this agent in a broad spectrum of tumors. PRECLINICAL STUDIES Targeting p53 In Vitro Advexin as Monotherapy As predicted from the known actions of p53 as a tumor suppressor, Advexin induces apoptosis and/or inhibits cell proliferation in cancer cell lines from numerous tumor types, while having comparatively little effect on normal cells. Apoptosis induction in tumor cell lines in response to Advexin monotherapy has been shown to be both dose- and time-dependent (40), and correlate with the dose- and time-dependent expression of p53 protein mediated by Advexin (up to 500-fold protein increase within 48 hours). Induction of apoptosis correlates with the changes in expression of various p53-responsive genes involved in apoptosis, angiogenesis, cell cycle progression, and DNA repair and replication [e.g., p21, MDM2 (41)]. Interestingly, the consequences of enhanced p53 expression last longer than the p53 protein levels, suggesting activation of a signaling cascade that leads to irreversible inhibition of proliferation, decreased viability, and/or apoptosis (42). As a general rule, the effects of Advexin on a cell line are not dependent on the p53 mutational status of that cell line. Very few cancer cell lines are resistant to Advexin; of over 100 tumor cell lines evaluated comprising 15 different tumor types, only two have been found to be completely resistant to Advexin (Table 1). Therefore the extremely high rate of response (i.e., cell death,

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TABLE 1 Cell Lines in which Advexin Inhibits Proliferation and/or Increases Apoptosis Indication Squamous cell carcinoma of the head and neck Non–small cell lung cancer

Breast Colorectal Prostate Cervical Osteosarcoma Esophageal Hepatocellular carcinoma Pancreatic Ovarian Glioma Endometrial Bladder Multiple myeloma

Normal cells a b

Cell lines Tu138, Tu167, Tu167, Tu177, Tu182, JSQ3, SQ-20B, FaDu, HN12, MDA 886, SCC97, HN30 H1299, H358, PC14, H322, H23, H226, H226Br, Calu-1, H596, H157, H460, A549 T47D, SK-BR-3, MDA-MB-231, MDA-MB-435, Calc-18, MCF-7, ZR-75-1 SW620, KM12L4, DLD-1, SW480, HT29, WiDr, HCT116, LoVo DU145, PC-3, LNCaP, C4-2 C33A, HT3, HeLa, C4-1, MS751, ME180, CaSki, SiHa Saos-2, U-20S, SA1 T.Tn, ECGI-10, TE8, YES6 HLE, HLF CFPAC-1, BxPC-3, PANC-1, Capan-1, AsPC-1, MIA PaCa-2, Capan2 SK-OV-3, OVCAR-3, OCC-1, OVCA432, OVCA420, OVCA429, OVCA433 U-251, U-251 MG, U-373 MG, A-172, U-87 MG, D54 MG, D54, EFC-2 SPEC-2 ECV304, KOTCC-1/P, KK47 8226, ARH-77, U266, ARP-1, MC-CAR,; HS-Sultan, IM9, MC-CAR,; HS-Sultan, IM9 MRC-9, CCD-16, HUVEC, MJ90, NHME, NHBE, CASMC, NHLF

Responsea 12/12

12/12

7/7 8/8 4/4 8/8 3/3 4/4 2/2 7/7 7/7 6/8b 1/1 3/3 7/7

0/8

Cell death/apoptosis: shown as number of lines affected/tested. Two glioma lines were resistant.

apoptosis) observed in tumor cells indicates that p53 can overcome apoptosis resistance that is a hallmark of tumor cells. Thus the apoptotic pathways downstream from p53 must be intact in the majority of tumor cells and can be activated to promote cell death. Furthermore, although more than 98% of tumor cells tested are sensitive to Advexin, no toxicity was observed in normal cells (Table 1). Advexin in Combination with Other Agents In addition to its effects as a single agent, there is a wealth of evidence that Advexin augments activity of radiation and other cytotoxic anticancer agents. The combinations demonstrate increased anticancer effects whereas Advexin does not cause an increase in toxicity mediated by conventional cytotoxic treatments (43). Advexin has been tested in vitro in combination with cisplatin, doxorubicin, etoposide, SN-38 (a metabolite of irinotecan), melphalan, carmustine (BCNU), fluorouracil, docetaxel, paclitaxel, radiation, and docetaxel plus radiation, in cell lines derived from SCCHN (44), NSCLC (43,45), glioma (46), breast cancer (47), colorectal cancer, prostate cancer, cervical cancer, epidermoid carcinoma and HCC (unpublished data). In the majority of these studies, Advexin increased tumor cell apoptosis in an additive or synergistic fashion when combined with standard

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Apoptosis Modulators: p53 Targeting TABLE 2 In Vitro and In Vivo Effects of Advexin Combined with Radiation or Cytotoxic Anticancer Agents Mechanism of action Forms DNA crosslinks Antimitotic, prevents microtubule disassembly Binds DNA and prevents nucleic acid synthesis Forms inter- and intra-strand Pt crosslinks Interferes with topoisomerase II Inhibits DNA synthesis Induces DNA single-strand breaks Induces DNA single and double-strand breaks

Drug/Agent Cisplatin Docetaxel Doxorubicin 5-Fluorouracil Etoposide Paclitaxel SN-38 Radiation Docetaxel and radiation

In vitro A, Aþ, S A, S A A, Aþ A, S A A, S A, Aþ, S S

In vivo Aþ Aþ, A Aþ Aþ NT NT NT Aþ Sa

a

Three-way combination more effective than two-way combination or single agents. Abbreviations: A, additive; Aþ, greater than additive; NT, not tested; Pt, platinum; S, synergistic.

anticancer modalities (Table 2). Importantly, inhibition of cytotoxic activity of conventional chemotherapy was not observed in any model evaluated. p53 Gene Therapy in Animal Models Advexin as a Single Agent Therapy Numerous studies have shown Advexin to be effective in animal tumor models, including SCCHN, NSCLC, breast, colorectal, prostate, cervical, ovarian, esophageal, bladder, glioma, hepatocellular carcinoma, and osteosarcoma. Early work using ex vivo models demonstrated that Advexin reduced the tumorigenicity of cells from several cancer types (e.g., Ref. 31). In later studies, intratumoral (IT) injection of Advexin into established SQ human tumor xenografts in nude mice resulted in a reduced growth rate or regression of tumors derived from a wide range of tumor types, including SCCHN, NSCLC, colorectal cancer and breast cancer. Advexin is effective against both p53 mutant and p53 wild-type xenograft tumors. As one would expect based on the broad spectrum of Advexin effects in vitro, Advexin appears to be effective in nearly all in vivo cancer models tested, with the possible exception of p53 wild-type gliomas (47,48). Some effects seen with in vivo models were dramatic, such as the complete inhibition of tumor growth after IT Advexin administration into human cervical cancer xenografts in nude mice reported by Hamada et al. (49). Most in vivo efficacy studies have been performed in SQ xenograft models, but Advexin also inhibits growth in disseminated xenograft cancer models (50) and in orthotopic and syngeneic models (31). As observed with in vitro studies, the antitumor effects of Advexin in animal models correlate with exogenous p53 expression, induction of p21 and mdm2 protein expression, and induction of apoptosis and/or decreased proliferation of cells within the tumor. Ohtani et al. (51) using a SQ NSCLC tumor model, demonstrated increased expression of p53, p21, MDM2, Noxa, and p53AIP1, and increased apoptosis, following a single IT injection of Advexin. Advexin in Combination with Other Agents Combination of Advexin with radiation or chemotherapy agents enhances the antitumor effects of Advexin in various animal models of cancer, and is summarized in Table 2. Enhanced efficacy has been demonstrated in SCCHN (unpublished data), NSCLC (43,52–54), breast cancer (55), colon cancer (56,57), prostate cancer

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3500

Tumor size (mm3)

3000

. .

2500

. .

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Control Control+5Gy dl312 dl312+5Gy Adp53 Adp53+5Gy

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1000 .

500 0 0

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20 25

30 35

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Day FIGURE 2 The combination of Advexin and radiation enhances antitumor effects in xenograft colorectal cancer tumors. Established SQ SW620 tumors were treated after reaching a size of 200 mm3 with three daily injections of PBS (control), dl312 (total dose of 2 · 1011 vp, 7.5 · 109 pfu), or Advexin (Adp53, total dose of 2 · 1011 vp, 7.5 · 109 pfu). Tumors were treated with 5 Gy on day 4. Tumors were measured every other day in two orthogonal dimensions, and tumor volume was calculated based on elliptical dimensions. n = 6 animals/group; points, mean values; bars, SE. Advexin combined with radiation resulted in the greatest degree of tumor growth inhibition. Source: Reproduced from Ref. 58.

(58,59), and glioma (47,60,61) animal models. As observed in vitro, the enhanced tumor growth inhibition observed with combination treatments correlates with increased apoptosis in the tumors (54,57,58). Representative data from one of these studies is presented in Figure 2. Nguyen et al. (57) treated established SQ colorectal cancer xenograft tumors in nude mice with radiation alone, or with Advexin with or without radiation. At the conclusion of the experiment, tumors treated with the combination were significantly smaller than those receiving Advexin or radiation alone. In a similar study using SQ NSCLC xenograft tumors in nude mice (43), the combination of docetaxel, radiation, and Advexin was shown to synergistically inhibit tumor growth. This Advexin-mediated increase in sensitivity to radiation or cytotoxic anticancer agents is not dependent on the p53 status of the tumor model tested. For example, Advexin increased sensitivity to radiation in p53-mutant tumor models of colorectal cancer (56), and NSCLC (43), prostate cancer (58) and NSCLC (53) with wild-type p53. These results are consistent with the in vitro data, which show sensitivity to Advexin-mediated growth inhibition in cell lines with wild-type or mutant p53 genes. This data also underscores the restoration of apoptotic competence provided by wt p53. Many (if not all) established tumor cell lines are considered to be resistant to apoptosis and understanding the molecular pathways responsible for apoptotic resistance is an area of intense research. Toxicity Preclinical Studies Effects of Advexin in Normal Cells Because both cancer and normal cells transduced with Advexin express supraphysiological levels of p53 in, there was an initial concern that treatment with


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Advexin might cause toxic effects in both. However, the toxic effects of Advexin appear to be selective for tumor cells. Various studies document that growth of normal cells is unaffected by Advexin transduction at levels that are cytotoxic to tumor cell lines: these include studies on normal human bronchial epithelial (NHBE) cells (35), normal human fibroblasts cultured from SCCHN tumors (33), normal human lung fibroblasts (41,54), normal human esophageal epithelial cells (60), and normal human hematopoietic stem cells and lymphocytes (62). Further investigation at the biochemical level by Sah et al. (41) found that Advexin had no effect on the DNA repair capacity of MRC-9 cells (normal human fibroblasts), nor on levels of two proteins involved in apoptosis (p21 and Bax). Toxicity studies in animal models have shown that Advexin can induce apoptosis in tumors while having little or no effect on normal cells and tissues in vivo. The reasons for these differential effects on normal and cancer cells are not completely clear, but some hypotheses proposed suggest that p53 may be more active at inducing the transcription of downstream genes in transformed cells than in normal cells (63). Additionally, normal cells may have biochemical differences that would make them less sensitive to the effects of supra-physiological levels of p53, such as a block to apoptosis downstream of p53 expression (54). Finally, p53 protein may be less stable and therefore less effective in normal than transformed cells (63). The selectivity index (i.e., ratio of IC50 for Advexin compared to control Ad-luciferase) varies between different tumor types but often exceeds 100 for tumor lines. In contrast, for normal cells, the selectivity index is 1, reflecting lack of toxicity to normal tissue by p53. Toxicity Studies of Advexin on Animal Models A series of preclinical acute toxicity studies have been conducted with Advexin, using seven routes of administration, including SQ, oral, intraperitoneal (IP), IV-portal vein, and IV. Three of the seven are particularly relevant to clinical studies in which Advexin is administered by IT injection to patients: SQ, the route in nontumor-bearing animals that most closely approximates IT administration; oral, since some of the vector may be swallowed when treating cancers such as head and neck, oral, or esophageal; and IV, because biodistribution analyses conducted during phase 1 and 2 clinical trials have shown some systemic distribution following IT administration. Many of these studies were conducted under GLP, with comprehensive analysis of clinical signs, gross pathology, hematology, clinical chemistry, and histopathologic evaluation. Relevant data from these studies, at doses similar to those used in the phase 3 clinical trials, is discussed below. Overall, Advexin as monotherapy was well-tolerated. SQ administration of Advexin induced minimal dissemination, with no toxicity detected even at the maximum dose tested, 3.7 · 1012 vp/kg (100-fold higher on a per weight basis than the human phase 3 clinical trial dose of 3 · 1010 vp/kg). Oral administration, which also results in limited dissemination, gave a toxicity profile indistinguishable from the control; no toxicity was observed up to the maximum Advexin dose tested (8.3 · 1012 vp/kg). Locally, no toxic effects were observed at the injection site when Advexin was administered SQ. In an IT biodistribution study, Advexin caused some edema, hemorrhage, and a few inflammatory cells within the tumor, but no effects were observed in extra-tumoral tissue (unpublished data). Although lymphocyte infiltration occurred in other IT studies, no effects were observed on adjacent normal tissue (64).

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The safety of Advexin in combination with standard anticancer agents was also evaluated as part of preclinical studies evaluating efficacy of repeated IT administration with standard anticancer agents (docetaxel, cyclophosphamide, doxorubicin, cisplatin, fluorouracil, and radiation), in which mortality, histopathology, weight changes, and gross physical changes were measured. The combination of Advexin with standard anticancer agents administered at therapeutic doses did not increase mortality or histopathologic findings in liver, spleen, kidney, heart, or lung. Advexin does not appear to pose a risk of genotoxicity, mutagenicity, or carcinogenicity. No epidemiological evidence linking adenoviruses to human cancer has been reported and Ad5 DNA has not been found in human tumors. With respect to the potential for genotoxicity via integration of Advexin DNA, adenoviruses as a class are not integrating viruses, and a study specifically designed to evaluate integration of Advexin did not detect any integration events (unpublished results). In summary, preclinical studies show that the liver is the primary organ of toxicity of Advexin at high doses and with systemic routes of administration. Advexin is well-tolerated by routes of administration and at doses which approximate those used in clinical trials. Greater toxicity was generally seen with more systemic routes of administration, and the primary target organ for toxicity, the liver, was also the primary target organ for biodistribution. Advexin does not appear to cause DNA damage or otherwise pose a risk of genotoxicity. CLINICAL EXPERIENCE WITH ADVEXIN A total of 28 phase 1, 2, and 3 clinical studies have been conducted using Advexin; 23 have been finalized, completed, or closed, and five are ongoing. Of these, 16 are monotherapy studies and six have combined Advexin with chemotherapy or radiation. Patients in these studies had advanced cancers, most commonly lung cancer or SCCHN, although studies were also performed in patients with prostate cancer, breast cancer, colorectal cancer and other solid tumors. Advexin typically has been administered via intratumoral injection, although 17 patients in clinical studies have been treated intravenously. The majority of patients in these clinical trials have received multiple cycles of Advexin therapy (39), and the results of these have demonstrated the safety, tolerability, and utility of Advexin as monotherapy and in combination with chemotherapy, radiation, and surgery. These studies also identified a number of prognostic indicators that may be used to identify patients most likely to benefit from Advexin therapy. Below, we summarize the clinical experience of Advexin as a monotherapy (SCCHN), and in combination with chemotherapy (LABC) and radiotherapy (NSCLC). Advexin as Monotherapy Advexin has been evaluated as monotherapy for several types of cancer, including recurrent, unresectable, locally advanced SCCHN and radiation-resistant, locally advanced esophageal cancer (65,66). In this cohort of heavily pretreated patients, Advexin monotherapy was well tolerated with evidence of clinical activity. Patients with recurrent, unresectable, locally advanced SCCHN have a poor prognosis and recurrent disease is usually considered incurable. Median overall survival after first relapse in patients with recurrent SCCHN is dismally short


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regardless of the treatment: 6 months if treated with chemotherapy as monotherapy (67–71), and 6–9 months for patients treated with combination therapy with platinum- or taxane-based regimens (72–75). The rationale for use of a p53targeted therapy in treatment of SCCHN stems from loss of p53 function in approximately 70% of patients with SCCHN, which has been associated with tumorigenesis and resistance to radiation and chemotherapy (20–22). We conducted three phase II trials in this patient population; two studies used a higher dose of Advexin (5 · 1011 to 2.5 · 1012 viral particles (vp)/injection), and one used a lower dose (1–4 · 109 vp/injection). Patients had histologically confirmed SCCHN, with cytologically confirmed recurrence, excluding endolaryngeal recurrence, after first-line therapy administered with a curative intent (50 Gy radiotherapy and/or surgery with or without chemotherapy). All lesions in the head and neck region were accessible to intratumoral treatment; any inaccessible lesions had to be separately evaluable and unlikely to impair the patient's ability to complete the study. The total area of all measurable lesions had to be 30 cm2, and the sum of the longest diameter of each measurable lesion had to be 10 cm. In addition, patients had a Karnofsky performance status (KPS) 60%, a life expectancy >12 weeks, and a tumor tissue sample (primary or recurrent tumor) was made available for p53 genotyping. Results from one of the phase II trials (n ¼ 105) showed the overall tumor response rate to Advexin treatment in evaluable patients was 6%, while 20% showed evidence of durable tumor growth control lasting longer than 3 months. Dose response and survival analyses for patients with recurrent/refractory SCCHN treated in the high- and low-dose phase II trials revealed clinical benefit, as defined by durable tumor growth control. In patients who received at least one cycle of treatment, high dose for Advexin provided survival advantage, as compared to treatment with low dose Advexin. This suggests a dose–response effect induced by Advexin in this patient population. Multivariate analyses conducted on studies in SCCHN patients identified a long progression-free interval after initial therapy (12 months) as the major prognostic factor for all efficacy outcomes (65). The size of treated lesions (25 mm) was a favorable prognostic factor for both tumor response and tumor growth control, while prior irradiation of target lesions was a prognostic factor for the latter. Absence of ulcerated and/or necrotic lesions, and baseline tumor-pain identified tumors more suitable for intralesional Advexin treatment and were independent factors for response. Applying these selection criteria, subgroups of patients in these studies were defined; these groups exhibited overall response rates of 20–30% and tumor growth control rates of 50–60%, depending on the degree of selection. In contrast to previous findings regarding the negative impact of induction chemotherapy on subsequent treatments, our studies indicate that prior chemotherapy was a positive prognostic factor (65). Additionally, lesions treated in a prior radiation field had better tumor growth control than untreated lesions. These observations are consistent with p53-therapy induced apoptosis in the presence of DNA damage due to irradiation or chemotherapy, which result in cytotoxicity when p53 function is reactivated (76,77). Taken together, these results provide evidence that Advexin monotherapy can improve outcomes in heavily pretreated SCCHN patients and suggest that Advexin may provide recurrent SCCHN patients with an effective and well tolerated approach to controlling their disease.

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Advexin in Combination Therapy The key role of p53 as a regulator of cell cycle progression and apoptotic pathways following treatment with DNA damaging agents, in conjunction with the favorable safety profile and clinical activity of Advexin monotherapy supported its evaluation in combination with chemotherapy regimens. In this section we summarize the results of trials evaluating the use of Advexin in combination with chemotherapy for treatment of lung and breast cancer. Overall, the results are consistent with preclinical studies and report incorporation of Advexin into chemotherapy regimens with minimal additional toxicity and with improved efficacy. Advexin and Chemotherapy for Treatment of NSCLC Non–small cell lung cancer accounts for nearly 80% of all lung cancers, and one third of patients diagnosed with NSCLC present with locally advanced, unresectable tumors (78). Despite advances in chemotherapy and the recent approval of biologic therapies gefitinib and erlotinib HCl, the 5-year survival rate for all lung cancers is only 15% (American Cancer Society). Two-year survival for patients with advanced disease ranges from 20% (stage III) to 5% (stage IV), and treatments for patients with advanced disease frequently result in severe side effects that may significantly decrease quality of life (79–85). Cisplatin is the most active single agent in NSCLC, and the drug is a mainstay of combination chemotherapy for this disease. Although several other chemotherapy agents have shown evidence of activity in NSCLC, their use has not increased median survival and is associated with significant toxicity (86–91). Mutations in the p53 gene have been detected in approximately 70% of NSCLC samples tested (19,92,93), and preclinical studies demonstrated activity of Advexin in combination with chemotherapy (83). This provided the rationale for evaluation of the toxicity and antitumor activity of Advexin, delivered via computed tomography-guided percutaneous or bronchoscopic injection into NSCLC tumors obstructing the airway (78). The first Advexin study in lung cancer was conducted by Swisher et al. (94), who treated 28 NSCLC patients with intratumoral injections of 106–1011 pfu, and demonstrated wt-p53 transgene expression that was consistent with antitumor activity in a subset of patients. Below, we review two clinical studies evaluating Advexin in this population of patients. A two-arm phase I study was conducted to evaluate the feasibility, safety, humoral immune response and biologic activity of multiple IT injections of Advexin, and to characterize the pharmacokinetics in patients with advanced NSCLC. Fifteen patients, with life expectancy >12 weeks, histologically confirmed NSCLC resistant or refractory to standard therapies, with lesions accessible to repeated injection and measurable disease with p53 mutations, were enrolled (DNA mutation or protein overexpression). Patients in one arm (n ¼ 9) received escalating doses of Advexin monotherapy (1 · 106 to 1 · 1011 plaqueforming units), administered by fine-needle injection using a bronchoscope; the other arm (n ¼ 6) evaluated Advexin (escalating doses ranging from 1 · 109 to 1 · 1011 pfu), administered on day 4 of a 28-day schedule, in combination with intravenous (IV) cisplatin (80 mg/m2 over 2 hours) administered on day 1 (95). Patients received a total of up to 14 courses of study treatment (median ¼ 3, average ¼ 4.2) and were monitored for adverse events and clinical effects. Results of this study support the feasibility and safety of IT Advexin, alone or in combination with Cisplatin, in patients with advanced NSCLC. Of the


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15 patients enrolled, 13 were assessable for efficacy: one patient had a partial response, while 10 patients had stable disease (3 of these lasting >9 months), and two patients had progressive disease. Symptomatic improvement included reduction in dyspnea, cough, and hemoptysis, observed in 4 (26.7%) patients (95). There was no dose-limiting toxicity associated with the study treatment, and no patient was withdrawn from the study due to adverse effects. Of the adverse events (AE) reported, the most common was a transient, self-limited, fever. Patients usually recovered within 48 hours. Hematologic toxicity was limited (1 incidence of leucopenia, and 3 incidences of grade 2–3 anemia). Transient (lasting 20%. The addition of bevacizumab to weekly paclitaxel was well tolerated, and again there was an increase in grade 3 hypertension, which was easily manageable. There was a significant increase of neuropathy in those patients who received paclitaxel þ bevacizumab, and although it is difficult to determine the reason for this increase in neuropathy, it may be due to the fact that these patients received paclitaxel therapy for a longer duration. As with lung cancer, the addition of bevacizumab to chemotherapy is now being studied in the adjuvant as well as the neoadjuvant. Furthermore, the other targeted therapies are being added to

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bevacizumab in the phase II setting in order to determine if phase III trials are warranted. POTENTIAL MECHANISMS OF ACTION OF BEVACIZUMAB The inhibition of VEGF signaling by bevacizumab may affect tumor growth and progression through several mechanisms, including (1) inhibiting the growth of new vessels, (2) regression of newly formed vasculature, (3) altering vascular function and tumor blood flow (normalization of the vasculature to transiently improve the delivery of and increase the efficacy of cytotoxic agents), and (4) direct effects on tumor cells (39–42). Effects on Vessel Numbers Using animal models of tumor growth, investigators have studied the mechanisms by which VEGF-targeted therapy affects the tumor vasculature. The vessel beds of most normal adult tissues are quiescent. In contrast, the immature vessels in tumors (defined as having a decrease in pericyte coverage) are more susceptible to VEGF-targeted therapy. In a mouse glioma xenograft model, disrupting the expression of VEGF in genetically-engineered mice pruned immature vessels (defined as vessels that lack pericytes) and spared pericyte-associated vessels (43). This observation supports the proposed role of VEGF as a maintainer of endothelial survival in newly formed vessels, with the subsequent appearance of pericyte coverage marking the transition to a less labile, non–VEGF-dependent state (44). Pericytes are believed to support the survival of vascular ECs through several mechanisms, including the production of VEGF (45) and angiopoietin-1, and through N-cadherin-mediated cell–cell interaction (13,46). These observations have prompted the idea that the regression of more-mature tumor vessels may require the additional targeting of pericytes through the blockade of platelet-derived growth factor or other factors that mediate pericyte function (47–49). The simultaneous inhibition of VEGF and platelet-derived growth factor receptor signaling in murine tumor xenografts leads to the dissociation of pericytes from vessels, with subsequent greater EC death and regression of pericyte-covered vessels than with the inhibition of VEGF signaling alone (48–50). It is difficult to obtain serial biopsies of tumors in clinical trials, so there are no large clinical trials that have studied the effects of single-agent bevacizumab on vessel counts before and after therapy. In a phase 1 trial, the investigators were able to obtain pre- and post-bevacizumab therapy biopsies in six patients with locally advanced rectal cancer who were treated with chemoradiotherapy plus bevacizumab. In this small study a single treatment with bevacizumab reduced tumor microvascular density by 29–59% (p < 0.05), tumor blood perfusion by 40–44% (p < 0.05), and tumor blood volume by 16–39% (p < 0.05), as assessed by functional computed tomography 12 days after treatment (51). Studies with other anti-VEGF agents have not shown such striking results; hence, it is necessary that such findings be reproduced in larger studies, to confirm that treatment with single agent bevacizumab can lead to the regression of tumor vessels. There is indirect evidence that anti-VEGF therapy is indeed cytostatic, as was first hypothesized. Clinical measures were recently evaluated in a study in patients with mCRC who were treated with bevacizumab but did not have tumor responses by the Response Evaluation Criteria in Solid Tumors (RECIST).

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These patients did, however, have a greater clinical benefit than patients who were treated with chemotherapy alone (52). The hazard ratio for death was 0.60 [95% confidence interval (CI), 0.40–0.90] in patients with a response (complete or partial) and was 0.76 (95% CI, 0.60–0.96) in patients without a response (stable disease, progressive disease, or nonassessable). Similarly, the hazard ratio for death or disease progression was 0.53 (95% CI, 0.38–0.74) in responders and 0.63 (95% CI, 0.49–0.80) in nonresponders. This suggests a cytostatic effect of bevacizumab, which indicates that it inhibits the growth of new blood vessels. Furthermore, the increase in PFS with higher response rates are greater in patients treated with chemotherapy plus bevacizumab than those treated with chemotherapy alone, supporting the cytostatic effects of bevacizumab (53). Effects on Vessel Function Structural irregularities are common in tumor vasculature. Owing to insufficient vascular supply, tumors lack adequate oxygen, which results in greater stabilization and expression of hypoxia-inducible factor 1. Signaling by this factor induces the expression of a large number of genes that are involved in tumor progression and metastasis (54,55), the most important of which is VEGF (56–58). Overexpression of VEGF causes vascular hyperpermeability. As a result, the tumors have high interstitial fluid pressure, sluggish blood flow, and uneven perfusion, with regions of hypoxia and acidosis (59). The abnormal hemodynamic environment of tumors may render them less sensitive to cancer therapies through several mechanisms (59,60). Uneven perfusion impedes the delivery of cytotoxic agents throughout the tumor. The loss of the vessel-to-exterior fluid pressure gradient and colloid osmotic pressure gradient inhibits the delivery of high-molecularweight agents, such as monoclonal antibodies. Some animal studies support the idea that the normalization of tumor vasculature—specifically, vasoconstriction to transiently improve flow dynamics and pruning of immature, functionally abnormal vessel sprouts—may improve the delivery of cytotoxic agents, with subsequent increases in the efficacy of chemotherapy and radiotherapy (61). Treatment with VEGF-targeted agents in murine xenograft models improves the intratumoral delivery of therapeutic agents. One week after the injection of a monoclonal antibody to VEGF (i.e., the murine precursor of bevacizumab), mice with colon adenocarcinoma xenografts had greater tumor uptake of the cytotoxic agent irinotecan (62). In another study, in mice with mammary carcinoma xenografts, a single dose of DC101, an antibody to VEGFR-2, increased tumor penetration by fluorescence-labeled bovine serum albumin protein (63). Subsequent studies in preclinical models by this group showed a window of opportunity for the normalization of blood vessels that occurs shortly after the anti-VEGF therapy has been delivered (3–5 days). After that, it is likely that the tumor vasculature undergoes further vasoconstriction, which I believe is due to the inhibition of EC-derived nitric oxide, a potent vasodilator. In fact, it has been hypothesized that this blockade of the production of nitric oxide, may in part explain the hypertension that is seen in patients who are treated with anti-VEGF therapy. It is clear that anti-VEGF therapy changes vascular function in addition to inhibiting classic angiogenesis. Activation of VEGFR-2 by VEGF leads to downstream signaling that induces the expression of vasodilators. Specifically, VEGF leads to the induction of nitric oxide and prostacyclin. These mediators lead to a

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relaxation of pericytes and vascular smooth muscle cells that regulate vessel tone. Thus, blocking VEGF signaling limits the secretion of these vasodilators, leading to a “relative vasoconstriction” associated with higher levels of VEGF that are released from tumors. This relative vasoconstriction is consistent with the findings in clinical studies that have used computed tomography scanning or magnetic resonance imaging to show changes in tumor blood flow, blood volume, transit time, and permeability. These imaging modalities show that anti-VEGF therapy causes a rapid decrease in tumor blood flow, blood volume, and permeability that occurs before any changes that would be expected in vessel density. As stated above, it is possible that the hypertension that is associated with anti-VEGF therapy may be due to this vasconstrictive effect. In fact, one clinical study has suggested that early hypertension may predict a response to anti-VEGF therapy (64). Obviously, more research is necessary to better understand the effect of antiVEGF therapy on the tumor vascular bed and on vessel tone in normal tissues. Effects on Tumor Cells In addition to its effects on tumor vasculature, anti-VEGF therapy may have direct inhibitory effects on tumor cells. For example, VEGF provides a survival signal for breast carcinoma cells in vitro (39). Treatment of breast cancer cell lines with SEMA3F, an endogenous protein that competitively inhibits the binding of VEGF to NRP-1 and NRP-2, decreased these cells’ capacity for chemotactic migration (65,66). A recent trial of a bevacizumab-containing regimen in patients with inflammatory breast cancer found that VEGFR-2 was present and activated on tumor cells and that treatment with bevacizumab blocked the activation of tumor cell VEGFR-2 (Fig. 3) (67). Studies in colon cancer and pancreatic cancer cell lines have found that VEGFR-1 is present on all cell lines that were studied. The activation of VEGFR-1 led to an increase in tumor cell invasion and migration, which was blocked by treatment with antibodies to VEGFR-1 (40,41). More recent work has shown that the activation of VEGFR-1 on tumor cells leads to an alteration in cell phenotype from an epithelial phenotype, in which cells are thought to be immobile, to a mesenchymal phenotype, in which cells shifts their molecular machinery to increase cell migration and invasion (i.e., epithelial–mesenchymal transition) (42). Adverse Effects of Anti-VEGF Therapy For the most part, the addition of bevacizumab to chemotherapy is well tolerated. However, there does appear to be some class effects of inhibiting the VEGF pathway as well as tumor-specific effects. Hypertension A very consistent yet easily manageable toxicity of anti-VEGF therapy is hypertension. Almost all patients will experience some increase in both diastolic and systolic blood pressures. However, it is estimated that 10–20% of patients will experience grade 3 or 4 hypertension (grade 4 is rare) requiring adjustment of current medications or the addition of agents to existing regimens. There are no clear cut recommendations for treating hypertension induced by anti-VEGF therapy. However, it is this author’s belief that diuretics should not be used, as many of the chemotherapeutic agents used along with bevacizumab therapy may have the potential for nephrotoxicity or are associated with GI toxicity, leading to

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FIGURE 3 Effect of bevacizumab monotherapy on vascular endothelial growth factor receptor (VEGFR-2) activation on breast carcinoma cells. VEGFR-2 was present and activated on breast carcinoma cells from biopsies from patients with locally advanced or inflammatory breast cancer (left panels). After single agent bevacizumab therapy, VEGFR-2 phosphorylation was decreased on breast cancer cells (two phosphorylation sites were investigated). Source: From Ref. 67.

diarrhea and potentially dehydration. At our institution most patients are treated with calcium channel blockers or ace inhibitors. Proteinuria In the phase I and phase II clinical trials, bevacizumab therapy appeared to be associated with proteinuria. The nephrotic syndrome was exceedingly rare but oncologist should be aware of the potential for the development of this syndrome. For the most part, investigators and oncologists utilizing bevacizumab perform a dipstick on the urine to be sure that there is not excessive protein in patients who are receiving bevacizumab. For patients with a positive dipstick for proteinuria, a 24-hour urinary protein excretion should be done in order to determine the amount of protein being lost in the urine. If this protein loss exceeds 2.5 g in 24 hours, then bevacizumab should be discontinued (Bevacizumab product insert). Disease Specific Toxicities One of the interesting findings from the trials in patients with mCRC was the finding that there was a slight increase in the number of patients who suffered from a bowel perforation. In the initial trial comparing IFL bevacizumab, there were six bowel preparations in the bevacizumab containing arm versus only one in the chemotherapy alone arm. This finding of bowel perforation while receiving bevacizumab has been incredibly consistent among clinical trials; currently, the rate appears to occur in 1.5–1.7% of patients receiving bevacizumab for mCRC.

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Recently, the BRITE Study, a registry of patients who receive chemotherapy plus bevacizumab for mCRC has shed some light on the timing and perhaps etiology of this event. The BRITE registry has accumulated data on more than [1,900] patients who have received chemotherapy plus bevacizumab for mCRC in the United States. This registry is also demonstrated a bowel perforation rate of 1.7%, with the majority of perforations occurring within the first three to six months of therapy. Although the numbers are relatively small, it does appear that patients who have an intact primary tumor and had recent sigmoidoscopy or colonoscopy may have a slight (but not statistically significant) increased risk of bowel perforation. There is also a similar registry collecting data from patients receiving chemotherapy plus bevacizumab for mCRC representing other countries throughout the world (BEAT trial). Perhaps with greater numbers of patients on these two registries we will be able to determine potential indicators of those patients more likely to suffer from a bowel perforation. As stated earlier, patients with NSCLCs with predominant squamous cell histology may also be at risk for bleeding. In the phase II and phase III trials, many of these patients actually had a good response to therapy, though one should follow directions in the package insert after the anticipated approval of bevacizumab for patients with NSCLC. CONCLUSIONS In the past, most investigators would have thought that targeting a ligand would not be as efficacious as targeting a receptor. However, at this stage in clinical development of anti-VEGF/anti-VEGF-receptor therapy, it is clear that significant benefit can be achieved by targeting the ligand VEGF and preventing its binding to its receptors. In most situations, anti-VEGF therapy improved the effects of chemotherapy, but the exact mechanism of action of how this occurs has yet to be determined. Many questions remain with respect to the best use of anti-VEGF therapy in malignant diseases. Such questions/issues include continuation of therapy in subsequent lines of therapy, the efficacy and potential long-term toxicity when used in the adjuvant setting, and, most importantly in the mind of this author, the identification of predictive markers for efficacy and toxicity. Collectively, we still have a great deal to learn about the biology of the VEGF ligand/receptor system in both pathology and physiology, and it is imperative that clinicians and basic scientists keep an open line of communication in order to take knowledge from the laboratory to the clinic, and to try to address issues raised by clinical observation in the research laboratory. ACKNOWLEDGMENTS This work was supported, in part, by NIH Grant CA112390 and the Lockton Fund for Pancreatic Cancer Research. REFERENCES 1.

Dvorak HF. Vascular permeability factor/vascular endothelial growth factor: a critical cytokine in tumor angiogenesis and a potential target for diagnosis and therapy. J Clin Oncol 2002; 20:4368–80.

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2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

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Ferrara N, Hillan KJ, Gerber HP, Novotny W. Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov 2004; 3:391–400. Hicklin DJ, Ellis LM. Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol 2005; 23:1011–27. Ferrara N. Vascular endothelial growth factor. Eur J Cancer 1996; 32A:2413–22. Dvorak HF, Brown LF, Detmar M, Dvorak AM. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol 1995; 146:1029–39. LeCouter J, Moritz DR, Li B, et al. Angiogenesis-independent endothelial protection of liver: role of VEGFR-1. Science 2003; 299:890–3. Plouet J, Moukadiri H. Characterization of the receptor to vasculotropin on bovine adrenal cortex-derived capillary endothelial cells. J Biol Chem 1990; 265:22071–4. Paavonen K, Puolakkainen P, Jussila L, Jahkola T, Alitalo K. Vascular endothelial growth factor receptor-3 in lymphangiogenesis in wound healing. Am J Pathol 2000; 156:1499–504. Kendall RL, Thomas KA. Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor. Proc Natl Acad Sci USA 1993; 90:10705–9. Banks RE, Forbes MA, Searles J, et al. Evidence for the existence of a novel pregnancyassociated soluble variant of the vascular endothelial growth factor receptor, Flt-1. Mol Hum Reprod 1998; 4:377–86. Krussel JS, Casan EM, Raga F, et al. Expression of mRNA for vascular endothelial growth factor transmembraneous receptors Flt1 and KDR, and the soluble receptor sflt in cycling human endometrium. Mol Hum Reprod 1999; 5:452–8. Levine RJ, Lam C, Qian C, et al. Soluble endoglin and other circulating antiangiogenic factors in preeclampsia. N Engl J Med 2006; 355:992–1005. Sundberg C, Kowanetz M, Brown LF, Detmar M, Dvorak HF. Stable expression of angiopoietin-1 and other markers by cultured pericytes: phenotypic similarities to a subpopulation of cells in maturing vessels during later stages of angiogenesis in vivo. Lab Invest 2002; 82:387–401. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003; 9:669–76. Kaipainen A, Korhonen J, Mustonen T, et al. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc Natl Acad Sci USA 1995; 92:3566–70. Miao HQ, Klagsbrun M. Neuropilin is a mediator of angiogenesis. Cancer Metastasis Rev 2000; 19:29–37. Soker S, Takashima S, Miao HQ, Neufeld G, Klagsbrun M. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 1998; 92:735–45. Ellis LM. The role of neuropilins in cancer. Mol Cancer Ther 2006; 5:1099–107. Fuh G, Garcia KC, de Vos, AM. The interaction of neuropilin-1 with vascular endothelial growth factor and its receptor flt-1. J Biol Chem 2000; 275:26690–5. Wang L, Zeng H, Wang P, Soker S, Mukhopadhyay D. Neuropilin-1-mediated vascular permeability factor/vascular endothelial growth factor-dependent endothelial cell migration. J Biol Chem 2003; 278:48848–60. Gluzman-Poltorak Z, Cohen T, Herzog Y, Neufeld G. Neuropilin-2 is a receptor for the vascular endothelial growth factor (VEGF) forms VEGF-145 and VEGF-165 [corrected]. J Biol Chem 2000; 275:18040–5. Zachary I. Signaling mechanisms mediating vascular protective actions of vascular endothelial growth factor. Am J Physiol Cell Physiol 2001; 280:C1375–86. Gerber HP, Dixit V, Ferrara N. Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells. J Biol Chem 1998; 273:13313–16. Rafii S, Lyden D, Benezra R, Hattori K, Heissig B. Vascular and haematopoietic stem cells: novel targets for anti-angiogenesis therapy? Nat Rev Cancer 2002; 2:826–35.

238 25. 26. 27. 28. 29. 30. 31.

32. 33. 34.

35. 36.

37.

38. 39. 40. 41. 42. 43.

Ellis

Kim KJ, Li B, Winer J, et al. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature 1993; 362:841–4. Warren RS, Yuan H, Matli MR, Gillett NA, Ferrara N. Regulation by vascular endothelial growth factor of human colon cancer tumorigenesis in a mouse model of experimental liver metastasis. J Clin Invest 1995; 95:1789–97. Gordon MS, Margolin K, Talpaz M, et al. Phase I safety and pharmacokinetic study of recombinant human anti-vascular endothelial growth factor in patients with advanced cancer. J Clin Oncol 2001; 19:843–50. Holash J, Davis S, Papadopoulos N, et al. VEGF-Trap: a VEGF blocker with potent antitumor effects. Proc Natl Acad Sci USA 2002; 99:11393–8. Jayson GC, Mulatero C, Ranson M, et al. Phase I investigation of recombinant antihuman vascular endothelial growth factor antibody in patients with advanced cancer. Eur J Cancer 2005; 41:555–63. Jayson GC, Zweit J, Jackson A, et al. Molecular imaging and biological evaluation of HuMV833 anti-VEGF antibody: implications for trial design of antiangiogenic antibodies. J Natl Cancer Inst 2002; 94:1484–93. Brekken RA, Overholser JP, Stastny VA, Waltenberger J, Minna JD, Thorpe PE. Selective inhibition of vascular endothelial growth factor (VEGF) receptor 2 (KDR/Flk1) activity by a monoclonal anti-VEGF antibody blocks tumor growth in mice. Cancer Res 2000; 60:5117–24. Kabbinavar FF, Schulz J, McCleod M, et al. Addition of bevacizumab to bolus fluorouracil and leucovorin in first-line metastatic colorectal cancer: results of a randomized phase II trial. J Clin Oncol 2005; 23:3697–705. Hurwitz H, Fehrenbacher L, Novotny W, et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 2004; 350:2335–42. Giantonio B, Catalan P, Meropol N. High-dose bevacizumab improves survival when combined with FOLFOX4 in previously treated advanced colorectal cancer: results from the Eastern Cooperative Oncology Group (ECOG) study E3200. In: American Society of Clinical Oncology, Orlando, FL, May 13–17, 2005, pp. 1s. Kabbinavar FF, Hambleton J, Mass RD, Hurwitz HI, Bergsland E, Sarkar S. Combined analysis of efficacy: the addition of bevacizumab to fluorouracil/leucovorin improves survival for patients with metastatic colorectal cancer. J Clin Oncol 2005; 23:3706–12. Cassidy J, Clarke S, Diaz Rubio E, et al. First efficacy and safety results from XELOX1/NO16966, a randomised 2x2 factorial phase III trial of XELOX vs. FOLFOX4 1 bevacizumab or placebo in first-line metastatic colorectal cancer (MCRC). In: American Society of Clinical Oncology, Atlanta, GA, 2006. Johnson DH, Fehrenbacher L, Novotny WF, et al. Randomized phase II trial comparing bevacizumab plus carboplatin and paclitaxel with carboplatin and paclitaxel alone in previously untreated locally advanced or metastatic non-small-cell lung cancer. J Clin Oncol 2004; 22:2184–91. Cobleigh MA, Langmuir VK, Sledge GW, et al. A phase I/II dose-escalation trial of bevacizumab in previously treated metastatic breast cancer. Semin Oncol 2003; 30:117–24. Bachelder RE, Crago A, Chung J, et al. Vascular endothelial growth factor is an autocrine survival factor for neuropilin-expressing breast carcinoma cells. Cancer Res 2001; 61:5736–40. Fan F, Wey JS, McCarty MF, et al. Expression and function of vascular endothelial growth factor receptor-1 on human colorectal cancer cells. Oncogene 2005; 24:2647–53. Wey JS, Fan F, Gray MJ, et al. Vascular endothelial growth factor receptor-1 promotes migration and invasion in pancreatic carcinoma cell lines. Cancer 2005; 104:427–38. Yang AD, Camp ER, Fan F, et al. Vascular endothelial growth factor receptor-1 activation mediates epithelial to mesenchymal transition in human pancreatic carcinoma cells. Cancer Res 2006; 66:46–51. Benjamin LE, Golijanin D, Itin A, Pode D, Keshet E. Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal. J Clin Invest 1999; 103:159–65.

VEGF Targeting

44. 45. 46. 47. 48. 49. 50. 51. 52.

53.

54. 55. 56. 57.

58. 59. 60. 61. 62. 63.

64.

239

Benjamin LE, Hemo I, Keshet E. A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF. Development 1998; 125:1591–8. Reinmuth N, Liu W, Jung YD, et al. Induction of VEGF in perivascular cells defines a potential paracrine mechanism for endothelial cell survival. FASEB J 2001; 15:1239–41. Gerhardt H, Wolburg H, Redies C. N-cadherin mediates pericytic-endothelial interaction during brain angiogenesis in the chicken. Dev Dyn 2000; 218:472–9. Shaheen RM, Tseng WW, Vellagas R, et al. Effects of an antibody to vascular endothelial growth factor receptor-2 on survival, tumor vascularity, and apoptosis in a murine model of colon carcinomatosis. Int J Oncol 2001; 18:221–6. Bergers G, Song S, Meyer-Morse N, Bergsland E, Hanahan D. Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors. J Clin Invest 2003; 111:1287–95. Pietras K, Hanahan D. A multitargeted, metronomic, and maximum-tolerated dose “chemo-switch” regimen is antiangiogenic, producing objective responses and survival benefit in a mouse model of cancer. J Clin Oncol 2005; 23:939–52. Erber R, Thurnher A, Katsen AD, et al. Combined inhibition of VEGF and PDGF signaling enforces tumor vessel regression by interfering with pericyte-mediated endothelial cell survival mechanisms. FASEB J 2004; 18:338–40. Willett CG, Boucher Y, di Tomaso E, et al. Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer. Nat Med 2004; 10:145–7. Mass R, Sarkar S, Holden SN, Hurwitz H. Clinical benefit from bevacizumab (BV) in responding (R) and non-responding (NR) patients (pts) with metastatic colorectal cancer (mCRC). In: 41st Annual Meeting American Society of Clinical Oncology, Orlando, FL, 2005. Grothey A, Hedrick EE, Mass R, et al. Response rate using conventional criteria is a poor surrogate for clinical benefit on progression-free (PFS) and overall survival (OS) in metastatic colorectal cncer (mCRC): A comparative analysis of N9741 and AVF2107. In: 42nd Annual Meeting Amercian Society of Clinical Oncology, Atlanta, GA, 2006. Pennacchietti S, Michieli P, Galluzzo M, Mazzone M, Giordano S, Comoglio PM. Hypoxia promotes invasive growth by transcriptional activation of the met protooncogene. Cancer Cell 2003; 3:347–61. Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003; 3:721–32. Forsythe JA, Jiang BH, Iyer NV, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 1996; 16:4604–13. Jiang BH, Agani F, Passaniti A, Semenza GL. V-SRC induces expression of hypoxiainducible factor 1 (HIF-1) and transcription of genes encoding vascular endothelial growth factor and enolase 1: involvement of HIF-1 in tumor progression. Cancer Res 1997; 57:5328–35. Oosthuyse B, Moons L, Storkebaum E, et al. Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration. Nat Genet 2001; 28:131–8. Yang AD, Bauer TW, Camp ER, et al. Improving delivery of antineoplastic agents with anti-vascular endothelial growth factor therapy. Cancer 2005; 103:1561–70. Jain RK. Barriers to drug delivery in solid tumors. Sci Am 1994; 271:58–65. Jain RK. Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science 2005; 307:58–62. Wildiers H, Guetens G, De Boeck G, et al. Effect of antivascular endothelial growth factor treatment on the intratumoral uptake of CPT-11. Br J Cancer 2003; 88:1979–86. Tong RT, Boucher Y, Kozin SV, Winkler F, Hicklin DJ, Jain RK. Vascular normalization by vascular endothelial growth factor receptor 2 blockade induces a pressure gradient across the vasculature and improves drug penetration in tumors. Cancer Res 2004; 64:3731–6. Friberg G, Kasza K, Vokes EE, Kindler HL. Early hypertension (HTN) as a potential pharmacodynamic (PD) marker for survival in pancreatic cancer (PC) patients (pts)

240

65. 66. 67.

Ellis

treated with bevacizumab (B) and gemcitabine (G). In: 41st Annual Meeting American Society of Clinical Oncology, Orlando, FL, 2005. Nasarre P, Constantin B, Rouhaud L, et al. Semaphorin SEMA3F and VEGF have opposing effects on cell attachment and spreading. Neoplasia 2003; 5:83–92. Nasarre P, Kusy S, Constantin B, et al. Semaphorin SEMA3F has a repulsing activity on breast cancer cells and inhibits E-cadherin-mediated cell adhesion. Neoplasia 2005; 7:180–9. Wedam SB, Low JA, Yang SX, et al. Antiangiogenic and antitumor effects of bevacizumab in patients with inflammatory and locally advanced breast cancer. J Clin Oncol 2006; 24:769–77.

15

Targeting Angiogenesis with Oral Agents Benjamin Besse Department of Médecine, Paris University XI, Institut Gustave Roussy, Villejuif, France

Jean-Pierre Armand Department of Medicine, Institut Gustave Roussy, Villejuif, France, and Department of Medicine, Institut Claudius Regaud, Toulouse, France

INTRODUCTION The advent of molecular targeted therapies through a better understanding of the cancer cell molecular circuitry has revolutionized treatment approaches in oncology. Targeting neovascularization offers tremendous potentialities (1). There is a finely regulated equilibrium between numerous natural antiangiogenic and proangiogenic factors. The tumor angiogenic phenotype is characterized by high microvessel density in the tumor with elevated vascular endothelial growth factor (VEGF) levels and is usually correlated with a worse prognosis. During angiogenesis, endothelial cells are stimulated by various growth factors that bind to membranous receptors, essentially tyrosine kinase receptors (TKRs). The TKRs directly involved in angiogenesis include receptors for VEGF, FGF, PDGF, angiopoïetin-1 (Ang-1) Ang-2, hepatocyte growth factor (HGF), Eph, and receptors belonging to the epithelial growth factor family. These receptors are expressed by endothelial cells or pericytes but not by tumor cells that secrete ligands. VEGF is the most potent inducer of angiogenesis. VEGF-A, commonly referred to as VEGF, can be induced by hypoxia and hypoxia-inducible factor 1 (HIF-1), inactivation of the von Hippel-Lindau (vHL) tumor suppressor gene, and a number of cytokines and growth factors, including platelet-derived growth factor (PDGF), tumor necrosis factor a (TNF-a), and transforming growth factor b (TGF-b). VEGF binds to VEGFR-1 (Flt-1) and VEGFR-2 (KDR), which are the major mediators of the mitogenesis-, angiogenesis-, and permeability-enhancing effects of VEGF. A large number of oral antiangiogenic agents, particularly TKR inhibitors, are currently under clinical development, with marketing approvals already granted for some of them (sunitinib and sorafenib). This review focuses on the most promising and advanced among them, with special emphasis on the VEGFR pathway (Tables 1 and 2). ANTIANGIOGENIC TYROSINE KINASE INHIBITORS IN CLINICAL TRIALS Sorafenib (BAY 43-9006) Sorafenib (Nexavar ; Bayer, West Haven, Connecticut, U.S.A) is a potent inhibitor of RAF-1, a key enzyme in the RAS/RAF/MEK/ERK signaling pathway, and an inhibitor of VEGFR-2 and PDGFR-b involved in angiogenesis (2). Based on phase I data, sorafenib was further investigated at a dose of 400 mg qd (3). Sorafenib activity was first studied in renal cell carcinoma (RCC), given its activity demonstrated in phase I trials. In a phase II study, 202 RCC patients were treated with 241

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TABLE 1 Oral Antiangiogenic Agents Targeting TKRs Agent PTK787/ZK222584 (Vatalanib) SU 11248 (Sunitinib) ZD6474 BAY 43-9006 (Sorafenib) AG 013736 AZD2171 CP-547,632

Targeted TKRs

Phase

VEGFR-1, VEGFR-2, PDGFR VEGFR, PDGFR, Flt-3, c-KIT VEGFR-2, VEGFR-3, EGFR VEGFR-2, PDGFR (and RAFa) VEGFR, PDGFR, c-KIT VEGFR-2 VEGFR-2, EGFR and PDGFR

III FDA approved III ongoing FDA approved II II II

a Not a TKR. Abbreviations: EGFR, epidermal growth factor receptor; FDA, U.S. Food and Drug Administration; MMP, matrix metalloproteinase; PDGFR, platelet-derived growth factor receptor; TKRs, tyrosine kinase receptors; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.

sorafenib and randomization took place at 12 weeks in patients with a stable disease (SD, 32%) while patients with tumor shrinkage >25% (36%) continued to receive sorafenib until disease progression (PD) (4). In the placebo arm, 18% of the patients were free of progression for 24 weeks compared to 50% in the sorafenib arm (p ¼ 0.008). Similarly, progression-free survival (PFS) was worse in the placebo group as compared to the sorafenib group (6 vs. 23 weeks, p ¼ 0.0001). The TARGET phase III trial confirmed the efficacy of sorafenib in 903 patients with RCC who failed prior treatment (5). PFS was 5.5 months in the sorafenib group and 2.8 months in the placebo group (hazard ratio for disease progression in the sorafenib group = 0.44; 95% confidence interval (CI) ¼ 0.35–0.55; p < 0.01), although an objective response was observed in only 10% of the patients in the sorafenib group (2% in the placebo group). In this setting (second-line treatment in RCC), objective response rates are usually just above 5%. The primary end point, the evaluation of overall survival (OS), was complex because crossover to an alternative treatment was subsequently allowed. Immediately before crossover was allowed, 220 deaths (41% of the protocol defined 540 deaths) had occurred: 97 of 451 patients (22%) in the sorafenib group and 123 of 452 patients (27%) in the placebo group died. At a median follow-up of 6.6 months, the median actuarial overall survival was 14.7 months in the placebo group but had not yet been reached in the sorafenib group (hazard ratio, 0.72; 95% CI ¼ 0.54–0.94; p ¼ 0.02). Grade 3/4 toxicities included a hand–foot skin reaction (6%), diarrhea (2%), fatigue (5%), and hypertension (4%). Grade 3/4 lymphopenia, hypophosphatemia, and elevated lipase levels were reported in 12–13% of the patients. Treatment was discontinued in 10% and 8% of the cases in the sorafenib group and the placebo group, respectively, because of skin and gastrointestinal toxicities. Doppler ultrasonography was performed in a subset of 30 patients (6). Changes in vasculature were positively correlated with PFS and OS, as early as three weeks. Sorafenib was approved for first- and second-line treatment of patients with RCC in December 2005 by the FDA. Its value as an adjuvant treatment is currently being investigated in the SORCE trial. Given that activating BRAF mutations are present in up to 80% of human melanomas, sorafenib has been investigated in this disease. In a phase II study in metastatic melanoma, little or no effect was detected when sorafenib was given alone, even in subgroup of patients with activating BRAF mutation (7). Phase II and III trials are ongoing to evaluate sorafenib in combination with tirosel/temsirolimus (Wyeth, Madison, New Jersey, U.S.A.) or carboplatin-paclitaxel. In a phase II study of sorafenib in untreated hepatocellular carcinoma, 46 out of the 137 patients (33%) exhibited a stable disease at week 16

FDA approved

Sunitinib

GIST

Phase II after imatinib Phase II

Phase III vs. imatinib

Phase II after imatinib and sunitinib

Colorectal

Phase II with capecitabine Phase II/III with FOLFOX Phase II after FOLFOX

Phase I/II with irinotecan and cetuximab Phase II with cetuximab Phase I/II with irinotecan and cetuximab

Breast

Phase II

Phase III with paclitaxel vs. paclitaxel þ bevacizumab Phase III with docetaxel Phase III with capecitabine Phase I/II with exemestane Phase II with trastuzumab Phase II after taxanes Phase II with letrozole

Phase I/II with anastrozole

NSCLC

Phase III with docetaxel (second line) Phase III vs. erlotinib Phase III with pemetrexed (second line) Phase II with paclitaxel-carboplatin

Phase II/II with paclitaxel-carboplatin

Phase II with erlotinib

Phase III with paclitaxel-carboplatin Phase II second line

Melanoma

Phase II

Phase I/II with temozolomide

Phase III with paclitaxel-carboplatin Phase I/II with tirosel/temsirolimus

Abbreviations: FDA, U.S. Food and Drug Administration; GIST, gastrointestinal stromal tumor; NSCLC, non–small cell lung cancer; RCC, renal cell carcinoma.

ZD6474

AZD2171

Vatalanib

FDA approved

Sorafenib

RCC

TABLE 2 Current Status of Oral Antiangiogenic Agents Targeting Tyrosine Kinase Receptors Regarding Cancer Type

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whereas 8% had a partial or minor response (8). With a median survival of 9.2 months, and a manageable toxicity, those results compared favorably to anthracyclin-based regimens. Combination trials with cytotoxic agents are ongoing. In stage III or IV non–small cell lung cancer, sorafenib was given alone to 52 pretreated patients (9). Although no partial response was reported, tumor shrinkage or cavitations were observed in 29% of the cases. One patient died of hemoptysis. Median PFS was 2.7 and 5.3 months in patients with SD. The more frequent grade 3/4 events were hand–foot skin reaction (10%) and hypertension (4%). Those encouraging results led to an ongoing phase III trial evaluating sorafenib as a maintenance treatment after a paclitaxel carboplatin combination in the first line setting. Phase I–III trials are also ongoing in head and neck, pancreatic and prostate cancer, as well as in sarcoma. Sunitinib (SU11248) Sunitinib (Sutent ; Pfizer, New York, New York, U.S.A.) inhibits VEGFR1, PDGFR, and c-KIT, a receptor of the pluripotent cell growth factor (SCF) implicated in malignant blood diseases. At higher concentrations, it inhibits FGFR1, another angiogenesis TKR. Sunitinib was synergistic with radiotherapy in murine models attaining tumor responses and sustained tumor control (10). A phase I study recommended a 50 mg/day dose given orally for four weeks every six weeks, and reported adverse effects such as asthenia, sore mouth, oedema and thrombocytopenia (11). In 38 patients, 6 objective responses were observed in three renal cell carcinomas, one neuroendocrine tumor, one stromal tumor and one unknown primary adenocarcinoma patient. In two phase II studies, a total of 169 patients with RCC received sunitinib after failure of a cytokine (12,13). In a pooled analysis, it resulted in 71 (42%) PR and 40 (24%) SD for at least three months (13). The median PFS for all 168 patients was 8.2 months (95% CI 7.8– 10.4). The most common toxicities experienced by patients were fatigue, diarrhea, neutropenia, elevation of lipase, and anemia. On the basis of these results, a phase III trial comparing sunitinib to interferon alpha in untreated RCC patients was conducted (14). Patients with brain metastases, uncontrolled hypertension, or clinically significant cardiovascular events during the preceding 12 months were excluded. Sunitinib was administered orally at a dose of 50 mg once daily, every four out six weeks, as in the phase II studies. Interferon a (INF-a) was given as a subcutaneous injection three times per week at 3 MU per dose during the first week, 6 MU per dose during the second week, and 9 MU per dose thereafter. The median PFS in the sunitinib group was longer than in the group INF-a (11 vs. 5 months, HR = 0.42, 95% CI 0.32–0.54; p < 0.001). The response rate was also higher in the sunitinib group compared with the INF-a group (31% vs. 6%, p < 0.001). Grade 3/4 toxicities were significantly higher among patients in the INF-a group (12% vs. 7%, p < 0.05); in particular, fatigue (12% vs. 7%) and lymphopenia (22 vs. 12%), whereas patients in the sunitinib group had higher rates of diarrhea (5% vs. no cases), hypertension (8% vs. 1%), and hand–foot skin reaction (5% vs. no cases). An alternative schedule (37.5 mg/day continuously) has been evaluated in a phase II trial: incidence of grade 3/4 adverse events might be lower and this regimen is still active (15). Note that in 66 RCC patients treated with sunitinib 50 mg/day (4 weeks out of 6), thyroid function tests were abnormal in 85% of them, consistent with hypothyroidism (16). Among patients with abnormal thyroid function tests, signs and symptoms possibly related to hypothyroidism were found in 84%


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of them, including signs usually linked to sunitinib as fatigue. Thyroid test abnormalities were detected relatively early in the treatment course (median = at cycle 2). The number of patients was too small to correlate thyroid dysfunction and outcome. Thyroid hormone replacement therapy improved symptoms in a subset of patients. Routine evaluation of thyroid function in patients treated by sunitinib may be therefore of value. Sunitinib has also achieved clinical activity in gastrointestinal stromal tumors (GIST), after failure on imatinib. Ninety-seven patients (92 evaluable) enrolled in a phase II study received sunitinib: 8 (8%) PR and 68 SD (70%, 36 of them for more than six months) were observed (17). The efficacy (response rate, OS) of sunitinib was higher in patients with wild-type KIT or exon 9 mutation compare to those with exon 11 mutation (18). Secondary KIT mutations (in exons 13, 14, 17, or 18) were found in 62% of GISTs with a primary KIT exon 11 mutation, in 16% with a primary KIT exon 9 mutation, but not in any of the GISTs lacking a primary KIT/PDGFRA mutation. Secondary kinase mutations of KIT exon 13 and 14 conferred in vivo and in vitro sensitivity to sunitinib compared to KIT exon 17 and 18 mutations. Given that sunitinib had activity against KIT mutants but not against PDGF-R mutants, it has been hypothesized that the activity of sunitinib against GIST could be distinct from its angiogenic effects (19,20). In a randomized trial, sunitinib was compared to a placebo in 312 patients with GIST after failure of imatinib (2:1) (21). Time to tumor progression (primary endpoint) was better for the sunitinib arm (6.3 vs. 1.5 months, p < 0.00001). Patients were switched from the placebo arm to the sunitinib arm in cases of progression, which could explain why OS did not differ between the two arms. PR and SD were similar to that obtained in the previous phase II study, 7% and 58%, respectively, in the sunitinib arm. The most common treatment–related adverse event was fatigue, occurring in 34% of the patients (any grade) in the sunitinib group, and in 22% of the cases in the placebo group. Sunitinib induced grade 3 (G3) fatigue in 5% of the patients, G3 hypertension in 3%, G3/4 neutropenia in 10%, and G3/4 thrombocytopenia in 5% of them. Results from preclinical models suggest that DCE-MRI could predict sunitinib activity, as shown with PTK/ZK (22,23). TEP-FDG could also be a sensitive surrogate marker of sunitinib efficacy in GIST patients. In 75 patients with imatinib-resistant GIST or intolerance to imatinib, the mean SUVmax (maximum standardized uptake value in up to five lesions) significantly decreased after 7 days of sunitinib (50 mg/day) and rebounded after 7–14 days of therapy (p < 0.001 in both cases) (24). Sunitinib efficacy was also evaluated in 64 patients with metastatic breast cancer pretreated with taxanes and anthracyclines (25). Hematologic toxicities were higher in this population: G3 neutropenia was seen in 39% of the patients and thrombocytopenia in 15% of them. In 51 evaluable patients, sunitinib exhibited significant activity: 7 (14%) had a PR and 1 (2%) a SD > 6 months. In non–small cell lung cancer (NSCLC), sunitinib was evaluated in 110 patients, previously treated with 1 (43%), 2 (44%) or 3 (13%) chemotherapy regimens (26). A first cohort of 63 patients was treated at 50 mg/day for four weeks followed by two weeks rest. Given the notable G3/4 toxicities (asthenia in 27%, myalgia in 18%, nausea 10%), a subsequent cohort was treated at 37.5 mg/day continuously. It resulted in a reduction of G3/4 asthenia, myalgia, and nausea (3%, 3%, and 0%, respectively), an increase of G3/4 neutropenia (11 vs. 5% in the first cohort), and a stable incidence of grade 3 hypertension (5%). Lethal hemorrhages were reported in three patients (two pulmonary and one cerebral hemorrhages). In the first cohort, 11% PR and 44% SD have been

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observed (27% of the patients not evaluated). Sunitinib activity is currently being evaluated in consolidation after first-line chemotherapy in stage IIIB-IV NSCLC. Vatalanib (PTK787/ZK 222584) Vatalanib (or PTK/ZK) inhibits the tyrosine kinase activity of VEGFR1, VEGFR2, and the platelet-derived growth factor receptor (PDGF-R) and is administered orally (27). Preclinical studies demonstrated activity through the inhibition of tumor vasculature, when it was administered alone or in combination with chemotherapy or radiotherapy. Phase I studies have evidenced good tolerability and efficacy in various tumors (28). The most commonly reported adverse events were nausea (59%), fatigue (41%), vomiting (35%), dizziness (29%), and headache (24%). Different solid tumor types have been explored with this agent: colon, brain, renal, and lung carcinomas. In a phase I/II study, patients with advanced colorectal cancer were treated every 14 days with oxaliplatin, 5-FU and folinic acid (known as FOLFOX 4) in combination with PTK/ZK (29). No increase was observed in oxaliplatin/5-FU toxicity and PTK/ZK was well tolerated at doses of 1500 mg/day. Similar results were obtained when PTK/ZK was combined with irinotecan-5-FU-folinic acid (30). PTK/ZK (1250 mg/day) combined with FOLFOX 4 was subsequently compared to a placebo in the CONFIRM-1 phase III trial, reported at the 2005 ASCO meeting (31). A total of 1168 with untreated metastatic colorectal cancer were randomized in that trial. Neutropenia, thrombocytopenia and neuropathy did not differ between the two groups. There were more cases of grade 3/4 hypertension (21% vs. 6%), venous thrombosis (7% vs. 4%) and pulmonary embolism (6% vs. 1%) in the PTK/ZK arm but similar grade 3/4 bleeding and arterial thrombosis. According to investigator-based assessment, there was a statistically significant increase in PFS in PTK/ZK-treated patients. However, a central review failed to document any significant difference. The CONFIRM 2 study investigated the same combination as second-line therapy in irinotecan pretreated patients (32). As in the CONFIRM 1 study, bone marrow toxicity was equivalent in the two arms whereas there were more grade 3/4 adverse events in the PTK/ZK arm: hypertension (22% vs. 5%), dizziness (9% vs. 1%) and venous thrombosis (6% vs. 1%). OS and time-to-progression (TTP) did not significantly differ between the two arms (12.1 vs. 11.8 months and 5.6 vs. 4.1 months, respectively). In a meta-analysis of the CONFIRM 1 and 2 studies, PFS was improved in the PTK arm in patients with elevated LDH (33). Two phase I/II trials have investigated PTK/ZK alone or in combination with either temozolomide or lomustine in patients with recurrent glioblastoma multiforme (34,35). As a single agent, 2 out of 55 patients (4%) experienced a PR and 31 (56%) an SD (median duration: 12.1 weeks). Sixty patients were treated with combined therapy that yielded 4 PR and 27 SD with a median TTP ranging from 12.1 to 16.1 weeks. Another study in 45 patients (37 evaluable) demonstrated the efficacy of PTK/ZK in metastatic renal cancer (36). Seven patients (19%) achieved a measurable response (1 partial and 6 minor) with a median TTP of 5.5 months (95% CI = 3.7–7.9 months), 17 (46%) had SD, and 5 (14%) PD. Rapid disease progression (within 3 months) occurred in only 28% of the patients (95% CI = 12.3–43.6%) treated with at least 1000 mg/day compared with an expected rate of 49.7% (95%


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CI = 43.5–55.9%) based on cytokine therapy in a similar patient population. Oneyear OS was 63.7% (95% CI = 41.9–85.5%). In another study, 55 NSCLC patients pretreated with a single line of platin-based chemotherapy received 1250 mg of PTK/ZK once a day and a further 55 patients received the same dose b.i.d. (37). Most frequent adverse events were nausea, vomiting, and dizziness. Best response by RECIST criteria for 55 evaluated patients (52 qd and 3 b.i.d.) include 2 PR (4%), 30 (55%) SD at week 4, hereof 18 (33%) for at least 12 weeks, and 23 (41%) PD. PTK/ZK efficacy has also been demonstrated in GIST resistant to imatinib (38). Studies have attempted to identify surrogate markers of response to PTK/ ZK. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was performed in patients treated with increasing doses of PTK/ZK (23). The bidirectional transfer constant (Ki), reflecting tumor permeability and vascularity, was negatively correlated with the oral PTK/ZK dose and plasma levels at day 2 (p = 0.01 and p = 0.0001, respectively). SU5416 and SU6668 SU5416, the first specific synthetic inhibitor of VEGF-TKR activity, inhibits the growth of human tumors xenografted into mice (mostly slowly growing tumors). However, despite encouraging preliminary results in pilot studies with a good safety profile, SU5416 proved disappointing in several phase II studies in different tumors and its clinical development was stopped. In particular, a randomized, multicenter, international prospective phase III study was conducted in 737 untreated metastatic colorectal cancer patients. Patients received a 5FU/leucovorin combination (Roswell–Park regimen) alone or concomitantly to SU5416. The toxicity was significantly worse in the SU5416 arm (diarrhea, cardiopulmonary arrest and vomiting) and there was no improvement on response rate, TTP and OS (39). SU6668 has a wide spectrum of activity and was first developed to inhibit tyrosine kinase activity of PDGFR, FGFR1 and VEGFR2. Regression of human tumor xenografts was obtained in mice with a complete histological response following rapid apoptosis in tumor microvessels. In two phase I studies, it was not known whether the plasma concentration of SU6668 at the MTD was sufficient to inhibit VEGFR and thus, induce tumor response (40). Interestingly, a recent study indicated that SU6668 has greater affinity for other tyrosine kinases, including other potential cancer drug targets such as TBK1 (also known as NAK or T2K) and two aurora kinases (41). SU6668 activity against angiogenesis could be minor, and the drug development could be reoriented based on these new preclinical data. Vandetanib (ZD6474) ZD6474 is an inhibitor of VEGFR-2, VEGFR-3 and HER1 (EGFR), albeit to a lesser extent. It has a certain degree of activity against other TKRs (PDGFR > VEGFR1 > Tie-2 > FGFR1). A phase I study demonstrated a safe clinical profile but no tumor response (42). The most common adverse events were diarrhea, rash and nausea. Asymptomatic QTc prolongation was noted in 7/77 patients (9%). In 46 previously treated patients with metastatic breast cancer, ZD6474 was neither effective at the 100 mg/day nor at the 300 mg/day dose (23 patients in each arm) (43). The most frequent grade 3/4 toxicity was diarrhea, concerning only 3 out of 22 patients at the 300 mg/day dose. A clinical trial with docetaxel and ZD6474 has been conducted in patients with advanced NSCLC who had progressed after

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first-line platinum-based chemotherapy (44). In this phase II trial, 127 patients were randomized to three arms: docetaxel plus ZD6474 100 mg/day, ZD6474 300 mg/day or a placebo. Common adverse events included diarrhea, rash and asymptomatic QTc prolongation. Results suggested efficacy with a PFS of 19 weeks versus 17 weeks versus 12 weeks, respectively. A confirmatory phase III trial is ongoing. In a Japanese NSCLC population, ZD6474 was given as a single agent at three different doses (100, 200 or 300 mg/day) after one or two platinum based chemotherapy regimens (45). A response rate of 13% was seen irrespectively of the dose level, but the median duration of treatment was longer in the 300 mg/ day arm. Grade 3/4 toxicities were more frequent in the 300 mg/day arm (67% vs. 39% and 29% in the 200 and 100mg/day arms). QTc-related events were reported in 72% of the patients at the level of 300 mg/day, 61% of the patients at 200 mg/day and 29% of the patients at 100 mg/day. ZD6474 (300 mg/day) has been compared to gefitinib (250 mg/day) in 168 Caucasian NSCLC patients after failure of 1–2 line platinum-based chemotherapy. In this randomized phase II trial, median PFS was significantly longer in the ZD6474 arm (11 vs. 8.1 weeks, p = 0.025). In 37 progressive patients in the gefitinib arm, ZD6474 achieved a disease control > 8 weeks in 16 patients, whereas only 7 out of 29 ZD6474 patients that switched to gefitinib achieved a disease control > 8 weeks (46). Axitinib (AG 013736) Axitinib inhibits VEGFR, PDGFR, and c-KIT TKIs. With continuous oral dosing, the main toxicities were similar to those that occurred with oral VEGR TKIs: hypertension, fatigue, diarrhea, stomatitis, nausea and vomiting (47). Hypertension was the most frequent toxicity reported in the first published phase I study, occurring in 22 patients (61%) and was mainly moderate (grade 1/2 in 18 patients). Impressive antitumor activity was demonstrated in 52 patients with metastatic RCC who failed prior cytokine-based therapy (48). Axitinib (5 mg b.i.d.) induced a PR in 46% of the patients and SD in a further 40%. Grade 3/4 adverse events were diarrhea in 8%, hypertension in 15% and fatigue in 8% of the patients. Evaluation of tumor vascularization by CT perfusion or DCE-MRI was performed in a subset of patients (49,50). The accuracy of the information is limited given the sample size, but it may be a valuable alternative for determining tumor activity. In 32 patients refractory or not suitable candidates for iodine (131I), axitinib induced a partial response in 23% of the patients and a stabilization in 47% of them (51). Grade 3 adverse events were fatigue (9%), hypertension (6%), and diarrhea, nausea, and proteinuria in 3% of the cases. Evaluation of axitinib efficacy in combination with gemcitabine is ongoing in pancreatic carcinomas (52). CP-547,632 CP-547,632 is an orally administered inhibitor of the tyrosine kinase activity of VEGFR2. A phase I study demonstrated good tolerability and 6 SD for more than 8 weeks and 1 for more than 6 months in 22 evaluable patients (53). The toxicity profile when combined with the paclitaxel/carboplatin doublet was reported to be safe in NSCLC patients. In a randomized phase II in the first-line setting (chemotherapy with or without CP-547,632), CP-547,632 conferred no apparent improvement in objective response rate when added to carboplatin and paclitaxel (54).


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AZD2171 AZD2171 is a highly potent and orally active inhibitor of VEGFR-2 tyrosine kinase activity that inhibited the growth of various established human tumor xenografts dose-dependently (55). Consistent with inhibition of pathologic angiogenesis in vivo, tumor perfusion, evaluated with [18F]fluoromethane PET, decreased within 24 hours of starting AZD2171 (56). As a once-daily oral therapy for the treatment of cancer, toxicity reported in a phase I trial consisted mainly of grade 3/4 hypertension (21% of the patients), as well as diarrhea, hoarseness and headache (57). Objective responses were reported in 2 out of 83 patients while 50% of them were stabilized. When combined with the EGFR inhibitor gefinitib (250 mg/day), dose-dependent hypertension was also reported (58). This doublet seemed, however, globally safe and may be further investigated in phase II trials. In combination with the paclitaxel–carboplatin combination, AZD2171 can be administered at the full dose of 45 mg/dose in NSCLC patients (59). AZD2171 has also been successfully combined to FOLFOX 4, pemetrexed, irinotecan and docetaxel in another phase I trial (60). FUTURE DEVELOPMENT Combination of Antiangiogenic Agents Antibodies and tyrosine kinase inhibitors that target the VEGF signaling pathway could be associated on the basis that the mechanism, spectrum of activity, and spectrum of toxicity of each agent is partially different. The significant antitumor activity of sunitinib in patients who had progressed on bevacizumab highlights different therapeutic spectrums and strengthens the rational to combine the two agents (61). This concept has been labeled }vertical blockade,} since the same pathway is targeted at two or more different levels by two or more different agents. In a phase I study, sorafenib was combined with bevacizumab (62). A significant increase in expected single-agent toxicity with mostly sorafenib toxicities was observed when adding bevacizumab. The maximal tolerated dose was sorafenib 200 mg b.i.d. and bevacizumab 5 mg/kg every two weeks. Grade 2–4 hypertension and hand–foot skin reaction were frequent and dose limiting (17/38 and 11/38, respectively), grade 3 proteinuria and thrombocytopenia were also dose limiting. A clinical benefit was seen in 34 out of 37 patients (92%) but PR was restricted to patients with ovarian cancer (4/14, 29%) leading to a subsequent phase II trial in this tumor type. Combination with Chemotherapy Bevacizumab activity has been demonstrated in phase III trials in combination with chemotherapy in colorectal cancer, NSCLC, and breast cancer (63–65). It has also demonstrated activity given as a single agent in renal cell carcinoma in a randomized phase II trial (66). However, large phase III trials combining valatinib with chemotherapy have failed to demonstrate an OS benefit, whereas different agents given alone reached this goal (13,33,67). It is striking to note that no trial with EGFR tyrosine kinase inhibitors has been positive when given concurrently with platinum-based chemotherapy, even if EGFR tyrosine kinase inhibitors trials were positive when given as a single agent (68–70). This raises the possibility of a class characteristic and favors the sequential regimen compared to the concomitant regimen when combining platinum-based chemotherapy and kinase inhibitors.

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Optimize the Use of Oral Antiangiogenic Agents Many issues are left unresolved at this time: What is the optimal duration of oral antiangiogenic agents? What is the role of oral antiangiogenic agent in the adjuvant setting and thus their long-term secondary effects? Are usual response criteria adequate to evaluate oral antiangiogenic efficacy? What are the biological or radiological predictive factors of efficacy? One of the key points is the definition of predictive factors to select the patients who will benefit from antiangiogenic treatments, given the toxicity profile of those agents. Most studies have focused on plasma markers, such as VEGF, soluble receptors (sVEGFR), and IL-8. Initial level of sVEGFR-2 and VEGFR were not predictive of a sensitivity to sorafenib (9). VEGF level increases at initiation of the treatment, but its predictive value remains debated (71,72). Circulating levels of endothelial cells (CEC) have not been related to sunitinib efficacy (73). BRAF mutations, frequent in melanoma, have not been correlated to sorafenib activity (7). Few tissue markers have been investigated even if activation of downstream markers may be of interest (8). The area of predictive markers of antiangiogenic therapy efficacy is yet to be fully explored. CONCLUSION During the past several years, rapid progress has been achieved in the understanding of angiogenesis, including signaling pathways and their regulation. This has enabled the development of numerous potentially interesting agents, many of which are oral agents. Angiogenesis-targeting is now a clinical reality accessible to more and more patients, due to formal approval of agents such as bevacizumab, sorafenib, and sutent. Further advances are awaited in the field. In particular, resistance to antiangiogenic treatments is now an established clinical reality. The molecular basis of this resistance needs to be better understood. Mutations of the p53 protein (observed in 50% of human cancers), which lower tumor cell oxygen requirements and thus their dependence on neovascularization, could be debated. The induction of bcl-2, a gene involved in resistance to apoptosis, has also been observed. Cross activation between the different signaling pathways must also be further elucidated. Preclinical and clinical studies show that VEGFR and EGFR inhibitors can be combined to enhance their efficacy (74,75). One of the present challenges is also to determine whether it is best to simply combine these new antiangiogenic agents or to combine them with conventional cytotoxics so that patient survival can be increased significantly. Ongoing clinical trials are applying these concepts with the prospect of using these antiangiogenic therapies in clinical practice. ACKNOWLEDGMENT The authors thank Lorna Saint Ange for editing. REFERENCES 1. 2.

Ferrara N, Kerbel RS. Angiogenesis as a therapeutic target. Nature 2005; 438:967–74. Wilhelm SM, Carter C, Tang L, et al. BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res 2004; 64:7099–109.


3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

251

Strumberg D, Richly H, Hilger RA, et al. Phase I clinical and pharmacokinetic study of the Novel Raf kinase and vascular endothelial growth factor receptor inhibitor BAY 43-9006 in patients with advanced refractory solid tumors. J Clin Oncol 2005; 23:965–72. Ratain MJ, Eisen T, Stadler WM, et al. Phase II placebo-controlled randomized discontinuation trial of sorafenib in patients with metastatic renal cell carcinoma. J Clin Oncol 2006; 24:2505–12. Escudier B, Eisen T, Stadler WM, et al. Sorafenib in advanced clear-cell renal-cell carcinoma. N Engl J Med 2007; 356:125–34. Lamuraglia M, Escudier B, Chami L, et al. To predict progression-free survival and overall survival in metastatic renal cancer treated with sorafenib: pilot study using dynamic contrast-enhanced Doppler ultrasound. Eur J Cancer 2006; 42:2472–9. Eisen T, Ahmad T, Flaherty KT, et al. Sorafenib in advanced melanoma: a phase II randomised discontinuation trial analysis. Br J Cancer 2006; 95:581–6. Abou-Alfa GK, Schwartz L, Ricci S, et al. Phase II study of sorafenib in patients with advanced hepatocellular carcinoma. J Clin Oncol 2006; 24:4293–300. Gatzemeier U, Blumenschein G, Fosella F, et al. Phase II trial of single-agent sorafenib in patients with advanced non-small cell lung carcinoma. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):7002. Schueneman AJ, Himmelfarb E, Geng L, et al. SU11248 maintenance therapy prevents tumor regrowth after fractionated irradiation of murine tumor models. Cancer Res 2003; 63:4009–16. Faivre S, Delbaldo C, Vera K, et al. Safety, pharmacokinetic, and antitumor activity of SU11248, a novel oral multitarget tyrosine kinase inhibitor, in patients with cancer. J Clin Oncol 2006; 24:25–35. Motzer RJ, Michaelson MD, Redman BG, et al. Activity of SU11248, a multitargeted inhibitor of vascular endothelial growth factor receptor and platelet-derived growth factor receptor, in patients with metastatic renal cell carcinoma. J Clin Oncol 2006; 24:16–24. Motzer RJ, Rini BI, Bukowski RM, et al. Sunitinib in patients with metastatic renal cell carcinoma. J Am Med Assoc 2006; 295:2516–24. Motzer RJ, Hutson TE, Tomczak P, et al. Sunitinib versus Interferon Alfa in metastatic renal-cell carcinoma. N Engl J Med 2007; 356:115–24. De Mulder PH, Roigas J, Gillessen S, et al. A phase II study of sunitinib administered in a continuous daily regimen in patients with cytokine-refractory metastatic renal cell carcinoma (mRCC). J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):4529. Rini BI, Tamaskar I, Shaheen P, et al. Hypothyroidism in patients with metastatic renal cell carcinoma treated with sunitinib. J Natl Cancer Inst 2007; 99:81–3. Maki RG, Fletcher JA, Heinrich MC, et al. Results from a continuation trial of SU11248 in patients (pts) with imatinib (IM)-resistant gastrointestinal stromal tumor (GIST). J Clin Oncol (Meeting Abstracts) 2005; 23(16_suppl):9011. Heinrich MC, Maki RG, Corless CL, et al. Sunitinib (SU) response in imatinib-resistant (IM-R) GIST correlates with KIT and PDGFRA mutation status. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):9502. Seandel M, Shia J, Linkov I, Maki RG, Antonescu CR, Dupont J. The activity of sunitinib against gastrointestinal stromal tumor seems to be distinct from its antiangiogenic effects. Clin Cancer Res 2006; 12:6203–4. Prenen H, Cools J, Mentens N, et al. Efficacy of the kinase inhibitor SU11248 against gastrointestinal stromal tumor mutants refractory to imatinib mesylate. Clin Cancer Res 2006; 12:2622–7. Demetri GD, van Oosterom AT, Garrett CR, et al. Efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumor after failure of imatinib: a randomised controlled trial. Lancet 2006; 368:1329–38. Marzola P, Degrassi A, Calderan L, et al. Early antiangiogenic activity of SU11248 evaluated in vivo by dynamic contrast-enhanced magnetic resonance imaging in an experimental model of colon carcinoma. Clin Cancer Res 2005; 11:5827–32. Morgan B, Thomas AL, Drevs J, et al. Dynamic contrast-enhanced magnetic resonance imaging as a biomarker for the pharmacological response of PTK787/ZK 222584, an

252

24.

25.

26. 27.

28. 29.

30.

31.

32.

33.

34.

35.

36. 37.

Besse and Armand

inhibitor of the vascular endothelial growth factor receptor tyrosine kinases, in patients with advanced colorectal cancer and liver metastases: results from two phase I studies. J Clin Oncol 2003; 21:3955–64. van den Abbeele A, Melenevsky Y, de Vries D, et al. Imaging kinase target inhibition with SU11248 by FDG-PET in patients (pts) with imatinib-resistant gastrointestinal stromal tumors (I-R GIST). J Clin Oncol (Meeting Abstracts) 2005; 23(16_suppl): 9006. Miller KD, Burstein HJ, Elias AD, et al. Phase II study of SU11248, a multitargeted receptor tyrosine kinase inhibitor (TKI), in patients (pts) with previously treated metastatic breast cancer (MBC). J Clin Oncol (Meeting Abstracts) 2005; 23(16_suppl):563. Socinski MA, Novello S, Sanchez JM, et al. Efficacy and safety of sunitinib in a multicenter phase II trial of previously treated, advanced non-small cell lung cancer (NSCLC). Ann Oncol 2006; 17:ix–218. Wood JM, Bold G, Buchdunger E, et al. PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration. Cancer Res 2000; 60:2178–89. Thomas AL, Morgan B, Horsfield MA, et al. Phase I study of the safety, tolerability, pharmacokinetics, and pharmacodynamics of PTK787/ZK 222584 administered twice daily in patients with advanced cancer. J Clin Oncol 2005; 23:4162–71. Thomas AL, Trarbach T, Bartel C, et al. A phase IB, open-label dose-escalating study of the oral angiogenesis inhibitor PTK787/ZK 222584 (PTK/ZK), in combination with FOLFOX4 chemotherapy in patients with advanced colorectal cancer. Ann Oncol 2007; 18:782–8. Trarbach T, Schleucher N, Tewes M, et al. Phase I/II study of PTK787/ZK 222584 (PTK/ZK), a novel, oral angiogenesis inhibitor in combination with FOLFIRI as firstline treatment for patients with metastatic colorectal cancer (CRC). J Clin Oncol (Meeting Abstracts) 2005; 23(16_suppl):3605. Hecht JR, Trarbach T, Jaeger E, et al. A randomized, double-blind, placebo-controlled, phase III study in patients (pts) with metastatic adenocarcinoma of the colon or rectum receiving first-line chemotherapy with oxaliplatin/5-fluorouracil/leucovorin and PTK787/ZK 222584 or placebo (CONFIRM-1). J Clin Oncol (Meeting Abstracts) 2005; 23(16_suppl):LBA3. Koehne C, Bajetta E, Lin E, et al. Results of an interim analysis of a multinational randomized, double-blind, phase III study in patients (pts) with previously treated metastatic colorectal cancer (mCRC) receiving FOLFOX4 and PTK787/ZK 222584 (PTK/ZK) or placebo (CONFIRM 2). J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):3508. Major P, Trarbach T, Lenz H, et al. A meta-analysis of two randomized, double-blind, placebo-controlled, phase III studies in patients (pts) with metastatic colorectal cancer (mCRC) receiving FOLFOX4 and PTK/ZK to determine clinical benefit on progressionfree survival (PFS) in high LDH pts. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):3529. Reardon D, Friedman H, Yung WKA, et al. A phase I/II trial of PTK787/ZK 222584 (PTK/ZK), a novel, oral angiogenesis inhibitor, in combination with either temozolomide or lomustine for patients with recurrent glioblastoma multiforme (GBM). J Clin Oncol (Meeting Abstracts) 2004; 22(14_suppl):1513. Conrad C, Friedman H, Reardon D, et al. A phase I/II trial of single-agent PTK 787/ ZK 222584 (PTK/ZK), a novel, oral angiogenesis inhibitor, in patients with recurrent glioblastoma multiforme (GBM). J Clin Oncol (Meeting Abstracts) 2004; 22(14_suppl):1512. George DJ, Michaelson MD, Oh DK, Reitsma D. A phase I study of single-agent PTK 787/ZK 222584 (PTK/ZK) in metastatic renal carcinoma. J Clin Oncol (Meeting Abstracts) 2003; 22:1548. Besse B, Gauler T, Fischer B, et al. Phase II open-label study to investigate efficacy and safety of PTK787/ZK 222584 orally administrated once daily or twice daily at 1250 mg


38.

39. 40. 41. 42. 43.

44. 45. 46. 47. 48.

49. 50.

51. 52. 53.

54.

253

as second-line monotherapy in patients (pts) with stage IIIB or stage IV non-small-cell lung cancer (NSCLC). Ann Oncol 2006; 17:ix218–19. Joensuu H, De Braud F, Coco P, et al. A phase II, open-label study of PTK787/ ZK222584 in the treatment of metastatic gastrointestinal stromal tumors (GISTs) resistant to imatinib mesylate. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):9531. Longo R, Sarmiento R, Fanelli M, Capaccetti B, Gattuso D, Gasparini G. Antiangiogenic therapy: rationale, challenges and clinical studies. Angiogenesis 2002; 5:237–56. Kuenen BC, Giaccone G, Ruijter R, et al. Dose-finding study of the multitargeted tyrosine kinase inhibitor SU6668 in patients with advanced malignancies. Clin Cancer Res 2005; 11:6240–6. Godl K, Gruss OJ, Eickhoff J, et al. Proteomic Characterization of the angiogenesis inhibitor SU6668 reveals multiple impacts on cellular kinase signaling. Cancer Res 2005; 65:6919–26. Holden SN, Eckhardt SG, Basser R, et al. Clinical evaluation of ZD6474, an orally active inhibitor of VEGF and EGF receptor signaling, in patients with solid, malignant tumors. Ann Oncol 2005; 16:1391–7. Miller KD, Trigo JM, Wheeler C, et al. A multicenter phase II trial of ZD6474, a vascular endothelial growth factor receptor-2 and epidermal growth factor receptor tyrosine kinase inhibitor, in patients with previously treated metastatic breast cancer. Clin Cancer Res 2005; 11:3369–76. Heymach JV, Johnson BE, Prager D, et al. A phase II trial of ZD6474 plus docetaxel in patients with previously treated NSCLC: follow-up results. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):7016. Nakagawa K, Kiura K, Shinkai T, et al. A randomized double-blind phase IIa dosefinding study of ZD6474 in Japanese patients with NSCLC. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):7067. Natale RB, Bodkin D, Govindan R, et al. ZD6474 versus gefitinib in patients with advanced NSCLC: Final results from a two-part, double-blind, randomized phase II trial. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):7000. Rugo HS, Herbst RS, Liu G, et al. Phase I trial of the oral antiangiogenesis agent AG-013736 in patients with advanced solid tumors: pharmacokinetic and clinical results. J Clin Oncol 2005; 23:5474–83. Rini B, Rixe O, Bukowski R, et al. AG-013736, a multi-target tyrosine kinase receptor inhibitor, demonstrates anti-tumor activity in a phase 2 study of cytokine-refractory, metastatic renal cell cancer (RCC). J Clin Oncol (Meeting Abstracts) 2005; 23(16_suppl):4509. Rixe O, Meric JB, Bloch J, et al. Surrogate markers of activity of AG-013736, a multitarget tyrosine kinase receptor inhibitor, in metastatic renal cell cancer (RCC). J Clin Oncol (Meeting Abstracts) 2005; 23(16_suppl):3003. Liu G, Rugo HS, Wilding G, et al. Dynamic contrast-enhanced magnetic resonance imaging as a pharmacodynamic measure of response after acute dosing of AG-013736, an oral angiogenesis inhibitor, in patients with advanced solid tumors: results from a phase I study. J Clin Oncol 2005; 23:5464–73. Kim S, Rosen LS, Cohen EE, et al. A Phase II study of axitinib (AG-013736), a potent inhibitor of VEGFRs, in patients with advanced thyroid cancer. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):5529. Spano J, Moore M, Kim S, et al. A phase I study of axitinib (AG-013736), a potent inhibitor of VEGFRs, in combination with gemcitabine (GEM) in patients (pts) with advanced pancreatic cancer. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):13092. Tolcher A, O'Leary JJ, deBono JS, et al. A phase I and biologic correlative study of an oral vascular endothelial growth factor-2 (VEGFR-2) tyrosine kinase inhibitor, CP—547,632, in patients with advanced solid tumors. J Clin Oncol (Meeting Abstracts) 2003; 21. Cohen RB, Langer CJ, Simon GR, et al. A phase I/randomized phase II, noncomparative, multicenter, open label trial of CP-547,632 in combination with paclitaxel

254

55. 56. 57. 58. 59.

60.

61. 62. 63. 64. 65.

66. 67. 68. 69.

70. 71.

72.

Besse and Armand

and carboplatin or paclitaxel and carboplatin alone as first-line treatment for advanced non-small cell lung cancer (NSCLC). Cancer Chemother Pharmacol 2006; 60:81–9. Wedge SR, Kendrew J, Hennequin LF, et al. AZD2171: a highly potent, orally bioavailable, vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor for the treatment of cancer. Cancer Res 2005; 65:4389–400. Miller KD, Miller M, Mehrotra S, et al. A physiologic imaging pilot study of breast cancer treated with AZD2171. Clin Cancer Res 2006; 12:281–8. Drevs J, Medinger M, Mross K, et al. Phase I clinical evaluation of AZD2171, a highly potent VEGF receptor tyrosine kinase inhibitor, in patients with advanced tumors. J Clin Oncol (Meeting Abstracts) 2005; 23(16_suppl):3002. van Cruijsen H, Voest EE, van Herpen CML, et al. Phase I clinical evaluation of AZD2171 in combination with gefitinib, in patients with advanced tumors. J Clin Oncol (Meeting Abstracts) 2005; 23(16_suppl):3030. Laurie SA, Arnold A, Gauthier I, et al. Final results of a phase I study of daily oral AZD2171, an inhibitor of vascular endothelial growth factor receptors (VEGFR), in combination with carboplatin (C) þ paclitaxel (T) in patients with advanced non-small cell lung cancer (NSCLC): A study of the National Cancer Institute of Canada Clinical Trials Group (NCIC CTG). J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):3054. Lorusso PM, Heath E, Valdivieso M, et al. Phase I evaluation of AZD2171, a highly potent and selective inhibitor of VEGFR signaling, in combination with selected chemotherapy regimens in patients with advanced solid tumors. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):3034. Rini BI, George DJ, Michaelson MD, et al. Efficacy and safety of sunitinib malate (SU11248) in bevacizumab-refractory metastatic renal cell carcinoma (mRCC). J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):4522. Azad NS, Posadas EM, Kwitkowski VE, et al. Increased efficacy and toxicity with combination anti-VEGF therapy using sorafenib and bevacizumab. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):3004. Sandler A, Gray R, Perry MC, et al. Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med 2006; 355:2542–50. Hurwitz H, Fehrenbacher L, Novotny W, et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 2004; 350:2335–42. Miller KD, Wang M, Gralow J, Dickler M. A randomized phase III trial of paclitaxel versus paclitaxel plus bevacizumab as first-line therapy for locally recurrent or metastatic breast cancer: a trial coordinated by the Eastern Cooperative Oncology Group (E2100). Proc San Antonio Breast Cancer Symp 2005:3a. Yang JC, Haworth L, Sherry RM, et al. A randomized trial of Bevacizumab, an antivascular endothelial growth factor antibody, for metastatic renal cancer. N Engl J Med 2003; 349:427–34. Escudier B, Szczylik C, Eisen T, et al. Randomized phase III trial of the Raf kinase and VEGFR inhibitor sorafenib (BAY 43-9006) in patients with advanced renal cell carcinoma (RCC). J Clin Oncol (Meeting Abstracts) 2005; 23(16_suppl):LBA4510. Herbst RS, Prager D, Hermann R, et al. TRIBUTE: a phase III trial of erlotinib hydrochloride (OSI-774) combined with carboplatin and paclitaxel chemotherapy in advanced non-small-cell lung cancer. J Clin Oncol 2005; 23:5892–99. Gatzemeier U, Pluzanska A, Szczesna A, et al. Results of a phase III trial of erlotinib (OSI-774) combined with cisplatin and gemcitabine (GC) chemotherapy in advanced non-small cell lung cancer (NSCLC). J Clin Oncol (Meeting Abstracts) 2004; 22(14_suppl):7010. Shepherd FA, Rodrigues Pereira J, Ciuleanu T, et al. Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med 2005; 353:123–32. DePrimo SE, Friece C, Huang X, et al. Effect of treatment with sunitinib malate, a multitargeted tyrosine kinase inhibitor, on circulating plasma levels of VEGF, soluble VEGF receptors 2 and 3, and soluble KIT in patients with metastatic breast cancer. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):578. Drevs J, Zirrgiebel U, Schmidt-Gersbach CIM, et al. Soluble markers for the assessment of biological activity with PTK787/ZK 222584 (PTK/ZK), a vascular endothelial


73.

74. 75.

255

growth factor receptor (VEGFR) tyrosine kinase inhibitor in patients with advanced colorectal cancer from two phase I trials. Ann Oncol 2005; 16:558–65. Norden-Zfoni A, Manola J, Desai J, et al. Levels of circulating endothelial cells (CECs) and monocytes as pharmacodynamic markers of SU11248 activity in patients (pts) with metastatic imatinib-resistant GIST. J Clin Oncol (Meeting Abstracts) 2005; 23(16_suppl): 9036. Jung YD, Mansfield PF, Akagi M, et al. Effects of combination anti-vascular endothelial growth factor receptor and anti-epidermal growth factor receptor therapies on the growth of gastric cancer in a nude mouse model. Eur J Cancer 2002; 38:1133–40. Herbst RS, Johnson DH, Mininberg E, et al. Phase I/II trial evaluating the anti-vascular endothelial growth factor monoclonal antibody bevacizumab in combination with the HER-1/Epidermal growth factor receptor tyrosine kinase inhibitor erlotinib for patients with recurrent non-small-cell lung cancer. J Clin Oncol 2005; 23:2544–55.

16

Endothelial Cell Propagation Targeting Gordon C. Tucker Cancer Drug Discovery Department, Institut de Recherches Servier, Croissy-sur-Seine, France

INTRODUCTION Endothelial cell (EC) propagation requires a fine adhesive balance during tumor progression. On the one hand, cell-to-cell contacts must be maintained to prevent loss of blood content—at least during the initial phases of tumor angiogenesis, whereas in more advanced stages, immature tumor vessels can be leaky. On the other hand, interactions with the extracellular matrix allow the expansion of microvessels towards and into solid tumor masses. The strength of the adhesive contacts will dictate whether ECs can migrate or not. Too strong an adhesion, whether intercellular or to the matrix, will prevent cell movement. The same goes for total loss of EC–matrix contact, with the additional consequence that this configuration will also trigger anoikis (homelessness in Greek), i.e., a process of active cell death induced to prevent the propagation of most cohesive normal cells into the body (1,2). Therefore, during neovascularization, ECs need appropriate receptors to interact with the extracellular matrix for migration and survival. They also need to shift from a resting state to a motile phenotype. The intracellular cytoskeleton must respond appropriately. Integrators between the outside and inside of the EC membrane would ideally perform this task. Such integrators do exist and constitute the main receptors for the extracellular matrix: the integrins (3). To gain access to tumor nodules, ECs also need the opening of pathways for cell migration. This event is triggered by the production of specific enzymes altering the extracellular matrix and released by ECs or tumor cells; for instance, matrix metalloproteinases, or MMPs (4) and heparanase (5)—clinical development of inhibitors of these enzymes as antiangiogenic and antimetastatic compounds is ongoing (see chapter 7 for an update on MMP inhibitors and comments in Miao et al. (6) for the PI-88 heparanase inhibitor currently in phase II clinical trials in patients with melanoma, liver, prostate, or lung cancers). This happens in the early stage of tumor angiogenesis when the underlying basement membrane is ruptured to allow microvascular cell escape, and also during the propagation stage itself. The ECs are then confronted with a new environment when compared to their quiescent state in the vessel (connective tissue and tumor stroma extracellular matrices vs. basement membranes). In addition to these events initially taking place near small tumor nodules (Fig. 1), the abnormal nature of solid tumors—often described as wounds that do not heal—can also exacerbate the remodeling of the matrix. In some cases, the matrix is altered by tumor-secreted proteases to the point that massive fragments can escape and reach the circulation; thus, bathing the body and potential metastases with new molecular entities. Some of these primary tumor-produced proteins have been shown to alter metastatic growth by blocking angiogenesis 257

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FIGURE 1 Angiogenesis: a balance between pro- and antiangiogenic factors. In response to the tumor-induced production of angiogenic factors, endothelial cells (ECs) converge as microvascular structures towards primary tumors or metastases. During this step, ECs adhere to and migrate on the extracellular matrix components via specific cell surface receptors. In addition, attachment is crucial for the survival of ECs (loss of matrix engagement triggers the process of active cell death). Primary tumors also secrete degrading enzymes remodeling the local matrix (basement membranes and interstitial matrix) or released into the circulation. As a result, some endogenous proteins in the matrix (like collagens) or in the circulation (like plasminogen) are fragmented. Some of these fragments are endowed with antiangiogenic properties that can act on metastases. Antiangiogenic proteins are also naturally present in the interstitial matrix, like thrombospondin. Black arrows point to the sites of intervention for the EC propagation inhibitors discussed in the text. At the matrix level (location 1), the cell surface adhesion receptors called integrins can be antagonized by antibodies (like Vitaxin , Abegrin, CNTO 95, or Volociximab), peptides (like Cilengitide or ATN-161), or peptidomimetics, or modulated at the expression level (as with E7820) in order to prevent EC migration and survival. Another possibility to hinder EC propagation is to administer exogenous peptides with antiangiogenic potential (location 2) to shift the balance towards tumor growth arrest: recombinant forms of the protein fragments mentioned above, like plasminogen-derived angiostatin or collagen XVIII-derived endostatin (and its variant Endostar), or small peptides derived from natural angiogenic inhibitors (like the thrombospondin-derived ABT510 compound).

(7,8). If not produced locally near the tumor, they can be generated in the circulation through the action of tumor-derived proteases released into the vascular compartment (Fig. 1). The first example of such angiogenesis inhibitors is angiostatin, a fragment of circulating plasminogen (9). Again, among other mechanisms, endothelial receptors for the extracellular matrix, like integrins, were shown to participate in the antiangiogenic action of some of these compounds. Integrins are the main focus of the present chapter. The description of the integrin target strategy is followed by a short incursion into the field of large,

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tumor-derived protein fragments endowed with antiangiogenic potential, like angiostatin and endostatin (9–11). Finally, because it also reached clinical trial as an anti-EC propagation compound, an artificial peptidic fragment derived from the endogenous angiogenesis inhibitor thrombospondin (8,12) is described. In all cases, the emphasis is put on clinical achievements. INTEGRINS AS ENDOTHELIAL CELL PROPAGATION TARGETS The Integrin Family Integrins are cell-surface heterodimers of the so-called a and b subunits whose association dictates the nature of the ligands recognized (3). The protein sequences and structures of the a or b subunit types are similar. Only a subset of ab arrangements have been described, amounting to 24 identified members of the integrin family (Fig. 2). Their cellular expression is more or less specific and nonexclusive. For instance, platelets will selectively express the aIIbb3 integrin (responsible for platelet aggregation through recognition of fibrinogen dimers bridging platelets together); leukocytes express a subset of integrins formed

αE

α2β1 = VLA-2 ↔

β7 α4 α1

↔

α5β1 = VLA-5, FNR

α2

FN, endostatin

α3 α5

β1

α6

αL

β4

αD

α7 α8

β6

αIIb

αM αX

↔

β5

β8 β3

FN, VN, Fg OPN, vWF, TSP, tumstatin

Activated

αvβ3 = VNR ↔

α10

αv

β2

Upregulated & activated

αvβ5

α9 α11

Activated

Coll, LN

FN, VN, Fg OPN, vWF, TSP, endostatin

De novo expressed & activated

FIGURE 2 The integrin family. The lines between a and b subunits denote possible heterodimers associations. Solid lines indicate integrins that can be expressed by endothelial cells (ECs). Some have been shown to participate in EC cell propagation during pathological angiogenesis. Additional information on ligands, other names, and regulation during angiogenesis is provided for the integrins targeted in clinical trials, i.e., a2b1, a5b1, avb3, and avb5. Abbreviations: Coll, collagens; Fg, fibrinogen; FN, fibronectin; FNR, fibronectin receptor; LN, laminins; OPN, osteopontin; TSP, thrombospondin; VLA, very late antigen; VN, vitronectin; VNR, vitronectin receptor; vWF, von Willebrand factor.

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around the b2 subunit, thus enabling them to interact with other cell types; but other integrins, like the fibronectin receptor a5b1, are more promiscuous. Considered initially as a mechanical link between the cytoskeleton and the extracellular matrix or other cell types, integrins were subsequently shown to act as classical receptors involved in signal transduction (13). The number of known cytoplasmic partners increased progressively, and complex arrangements of proteins are now depicted for, on the one hand, actin coupling in relation to cell adhesion, spreading, and movement and, on the other hand, for signaling. Functions assumed by the integrins range from the interaction with the environment (cell adhesion and motility) to other cell behaviors such as proliferation, survival or cell death, translating in tissue events linked to tumor progression as diverse as angiogenesis, tumor invasion, and metastasis (14–16). The activity of integrins is further complicated by the existence of distinct conformations, active and inactive (17). For instance, resting platelets express inactive aIIbb3 integrins that can be rapidly activated to engage in aggregation. Activation apparently results from a conformational change from a bent or folded state to an extended configuration of the integrin subunits, an event facilitated by the existence of a hinge between extracellular domains of both integrin subunits (18). Accompanying changes in the proximity of the short cytoplasmic tail domains modulate the intracellular binding of protein partners involved in cytoskeleton coupling and signaling. These changes are brought about by insideout (e.g., growth factor signaling) and outside-in (integrin ligation) signaling (19). In an active state, integrins bind their extracellular ligands with higher affinity. Relevance and Role of Integrins in Tumor Angiogenesis The role of integrins is not limited to angiogenesis during tumor progression but a subset of integrins have been associated with ECs during neovascularization (20,21). In vitro and in situ descriptive work showed constitutive integrin expression on quiescent microvessels (low levels of a1b1, a3b1, a5b1, a6b1, a6b4, and barely detectable levels of a2b1, avb3, avb5). Integrins can be either downregulated (a1b1, a6b4), upregulated (a5b1, a6b1), or de novo expressed (avb3, a4 integrins) on ECs during angiogenesis (16,22,23). The expression of some integrin ligands can also be modulated during tumor progression. For instance, the ligand fibronectin is upregulated along with its receptor, the a5b1 integrin (24). The situation is more complicated for other integrins that recognize many ligands, such as the avb3, and avb5 integrins. In addition, integrin activation can occur through the action of angiogenic factors; for instance, VEGF activates the a2b1, a5b1, avb3, and avb5 integrins (25). This pattern of expression and its modulation during angiogenesis must be considered as a general overview not reflecting the possible disparities from one tissue to another. Moreover, the repertoire of endothelial integrins expressed or activated depends on the pathophysiological context, as well as the progression of the pathology. Thus, at least in mouse models, the avb3 integrin can be de novo expressed during tumor angiogenesis, but not liver regeneration or healing (26), and its pattern of expression varies as a function of time during angiogenesis associated with retinopathy (27). Similar differences may occur during human tumor progression. Changes in pattern of integrin expression do not necessarily mean that integrins participate in angiogenesis. Knock-out experiments demonstrated causation in the process of angiogenesis for some integrins during embryonic development


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(a4, a5, a9, av, and b8) or tumor angiogenesis (for a1) and not mere epigenetic association (21). On the other hand, although the use of specific integrin antagonists confirmed avb3 or avb5 as targets to inhibit during pathological angiogenesis [reviewed in (21)], this conclusion was challenged by experiments conducted with b3 and b3/b5 knock-out animals: tumor angiogenesis was enhanced (28,29). This reveals that the mechanism thought to explain the role of the avb3 and avb5 integrins during angiogenesis is insufficiently understood. Several hypotheses have been advanced to explain the discrepancies between antagonist and knock-out experiments [reviewed in (22,23,30–32)]: compensation for the loss of integrins during development by other angiogenic pathways exacerbated during pathological angiogenesis or integrin-mediated death (33), a process whereby unligated integrins recruit caspase-8 to induce cell death. Whether unligated avb3 integrins exist in vivo is not known, but, if this happens, eradication of the integrin will suppress this pro-apoptotic pathway and enhance angiogenesis. Interestingly, the concept of vascular integrins as survival receptors has evolved to the point of considering them as dependence receptors (34). By acting as biosensors of the local extracellular matrix, some integrins, such as a5b1 and avb3, can transmit life and death signals. It is again a fine balance between the type of ligands encountered; some bound to the matrix scaffold—thus favoring survival—and others soluble after matrix degradation— soluble ligands can promote active cell death directly, independently of anoikis. The latter property may not necessarily be shared by all integrin ligands, especially small antagonists (35). The precise details of av integrin function in cell behavior are thus still not entirely known. However, the antiangiogenic success of preclinical experiments performed with various integrin inhibitors (antibodies, peptides, small heterocyclic peptidomimetics) directed against a2b1, a5b1, avb3, and avb5 convinced clinicians to evaluate integrin antagonists as antiangiogenic drugs. Incidentally, the term antagonist may be a misnomer if these integrins can play some part in angiogenesis as negative regulators—the compounds could act as agonists of negative regulatory functions rather than as antagonists of integrin positive functions like survival. A summary of the rationale for targeting some of the integrins is provided in Table 1. Of all the integrin antagonists tested in animal models, only antibodies and peptides made it to the clinic (Table 2). The host of heterocyclic inhibitors modeled as peptidic mimetics to target the avb3 integrin and described in the literature [examples in (36,37)] prudently stayed away from clinical trials, at least in oncology—one specific anti-avb3 integrin compound (SB-273005; GlaxoSmithKline, King of Prussia, Pennsylvania, U.S.A.) did enter a phase I trial for osteoporosis however, but its development was halted for undisclosed reasons. This may be envisioned as a cautious behavior in the light of the paucity of clinical favorable responses obtained with the first antagonists evaluated. Developing an antiangiogenic compound is a slow process as exemplified by the duration between the first clinical trial of bevacizumab (the Avastin anti-VEGF antibody) in April 1997 and its drug approval in 2004. Combination therapy was crucial to demonstrate the benefit of this compound. This might prove to be crucial for integrin antagonists too, as exemplified by preclinical data (38–40). Finally, when interpreting clinical—or preclinical—data, it must be kept in mind that integrins can be expressed by the tumor cell themselves—thus facilitating their motile or invasive capacities, as during melanoma vertical growth or metastasis—and also by some stromal cells. An example of the importance of the (Text continues on page 266)

Some endothelia, epithelia, and leukocytes, osteoclasts, fibroblasts, platelets, smooth muscle cells

Some endothelia, epithelia, and leukocytes, osteoclasts, platelets, smooth muscle cells

avb5

Normal human tissue distribution (adapted from Refs. 32,33)

avb3

Type

Activated by some angiogenic factors

de novo expressed and activated by some angiogenic factors

Modulation of expression on tumor endothelial cells (16,20–23)

Expressed in some carcinomas and associated with poor prognosis in some tumor types

Expressed in melanoma and some carcinomas and associated with poor prognosis in some tumor types

Expression in human tumor (adapted from Ref. 133)

TABLE 1 Rationale for Integrin Targeting during Endothelial Cell Progression

av subunit null: embryonic (E10) or perinatal lethal, placental or cerebral vascular defects; b3 subunit null: viable and fertile, hemorrhage, enhanced tumor growth av subunit null: see above; b5 subunit null: viable and fertile without obvious defects, enhanced tumor growth in the b3 null context

Mouse knockout studies (adapted from Refs. 3,135)

Antiangiogenic and antitumor activity demonstrated with numerous antibody, peptide, and non peptide antagonists

Antiangiogenic and antitumor activity demonstrated with numerous antibody, peptide, and non peptide antagonists

Effects of antagonists in preclinical models (reviewed in Ref. 21)

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Epithelium, endothelium, fibroblasts, cells of hematopoietic origin, platelets

a2 subunit

Activated by some angiogenic factors but lost in some tumors

Upregulated along with its ligand fibronectin (24) and activated by some angiogenic factors

Overexpressed—but possibly downregulated—in some tumors depending on the progression (e.g., elevated during melanoma invasion and in RCC with worsening clinical grade) Reduced levels in carcinomas (e.g., during tumor progression in the mammary gland) Antiangiogenic and antitumor activity demonstrated with antibody and peptide antagonists

Antiangiogenic activity demonstrated with an antibody antagonist (134) and the E7820 expression modulator (61)

a5 subunit null: embryonic lethal (E10-11) with vascular defects (dilated and disrupted endothelial tubes); b1 subunit null: embryonic lethal (E5.5) (note that b1 binds many a subunits, Fig. 2) a2 subunit null: viable with few immediate obvious developmental defects; no report of effect on tumor growth

Note: De novo expression or activation of the avb3 and avb5 integrins during tumor angiogenesis has been demonstrated in some but not all human cancers. In some cases, expression of av integrins correlates with a poor prognosis. The somewhat surprising results obtained with tumor grafts in b3 and b3/b5-null animals (enhanced tumor growth) blurred the previously straightforward accepted mechanism of action of these integrins during angiogenesis (see text for comments). Nevertheless, preclinical experiments with various antagonists validated the concept of targeting the avb3 and avb5 integrins to reduce tumor angiogenesis, at least in animal models. The same goes for the other two integrins targeted in the clinic, a5b1 and a2b1. However, a5b1 expression is not systematically enhanced in human tumors—it can be down-regulated in some instances. Contrary to avb3 and avb5 integrins, knock-out studies for a5b1 point to a positive regulatory role of this integrin in angiogenesis, but the embryonic lethality of the knock-out animals did not allow to study the effect of the integrin loss during tumor progression. The a2 subunit is generally expressed at low levels on tumor endothelial cells and downregulated in tumors. However, angiogenic factors like vascular endothelial growth factor (VEGF) can activate it, thus enabling some tumors to use it during angiogenesis. Since studies in a2-null animals failed to reveal abnormalities in vessels, and no report has been published on tumor growth in these animals, the most compelling evidence for its participation in tumor angiogenesis comes from the use of inhibitors of the a2b1 integrin (blocking antibodies and the expression modulator E7820) in animal models.

Widely distributed

a5b1


263

Humanized version of the LM609 murine monoclonal antibody

Humanized version of the LM609 murine monoclonal antibody

Cyclic RGD peptide

Human monoclonal antibody

Humanized monoclonal antibody

Peptide Ac-PHSCN-NH2

Abegrin (MEDI-522, etaracizumab)

Cilengitide EMD 121974

CNTO 95

Volociximab Eos-200-4 M-200

ATN–161

Type

Vitaxin (MEDI-523)

Name(s)

Phase I started November 2002—phase II for the treatment of advanced melanoma and androgenindependent prostate cancer Phase I started August 1998—phase II for the treatment of solid tumours and acute myeloid leukaemia Phase I started December 2003—phase I/II in combination with dacarbazine in patients with stage IV melanoma started in April 2005 Phase I started May 2003— various phase II for the treatment of solid tumours Phase I started January 2003—phase II in advanced renal cell cancer, I/II in malignant melanoma

avb3 integrin

a5b1 & avb3 integrins

a5b1 integrin

av integrins

avb3 and avb5 integrins

Phase I started April 1997— phase II for the treatment of solid tumours

Clinical status

avb3 integrin

Target

TABLE 2 Clinical Trial Status of Inhibitors of Endothelial Cell Propagation Described in the Text

Scripps Research Institute; Applied Molecular Evolution; MedImmune, Inc. MedImmune, Inc.

Company

Well tolerated up to 10 mg/ kg for 2 wk, higher dose explored Safe

Good tolerance in phase I

Attenuon

Protein Design Labs

Centocor; Medarex

Safe and potential benefit for Merck KGaA; EMD glioma treatment (Orphan Pharmaceuticals; Drug designation) National Cancer Institute

Safe and potential benefit for metastatic melanoma

Safe but anecdotal response

Outcome

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Recombinant protein

Recombinant protein

Recombinant modified endostatin

Adenoviral construct

Peptide fragment from TSP

Angiostatin

Endostatin

Endostar

Endostatin gene therapy

ABT-510

CD36

Idem

Idem

a5b1, other receptors

Integrins(?), angiomotin

a2 integrin subunit

Phase I started—phase II in progress

Phase I started in May 2005

Phase I started January 2004—phase I/II planned in colorectal cancer Phase I started in 2000— phase II/development hindered by production issues Phase I started in 1999– phase II in 2002 in melanoma and neuroendocrine tumors; clinical studies stopped in February 2005 because of production issues Phase I started in 2001— phase III in lung cancer EntreMed

Safe, but no objective response found

No additional toxicity in combination chemotherapy, Orphan Drug designation for soft tissue sarcoma

Delayed disease progression, approved by the Chinese FDA in non– small-cell lung cancer in combination with chemotherapy Well tolerated

Sun Yat-sen University; Doublle Bioproduct Abbott Laboratories

Yantai MedGenn

EntreMed Safe and long-term stabilization in some patients, slow tumor shrinkage, but considered a “no response” according to traditional nomenclature

Eisai Medical Research, Inc.

Safe up to 100 mg/day (MTD ¼ 200 mg/day)

Note: No objective responses were observed so far with integrin antagonists or modulators in phase I and II clinical trials. With respect to the clinical development of antiangiogenic protein fragments, clinicals trials in the USA for angiostatin and endostatin were halted because of industrial production issues. However, an optimized version of endostatin, Endostar, has been evaluated in China, as well as an adenoviral construct for endostatin gene therapy (see text for details). Abbreviations: FDA, Food and Drug Administration; TSP, thrombospondin.

Aromatic sulfonamide derivative

E7820


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avb3 integrin during local tissue growth is its role during osteoclast recruitment elicited by tumor cells and subsequent bone destruction in skeletal metastases (41). Incidentally, in this context, experiments with b3 knockout animals and avb3 integrin antagonists are reconciled (42), contrary to the angiogenic context as mentioned earlier. Also, since antiangiogenic compounds can affect the tumor response to ionizing radiation (43), integrin antagonists have been evaluated in preclinical models in combination with radioimmunotherapy (44) or external beam radiotherapy (45–47). Such combinations were shown to increase the efficiency of radiotherapy. Clinical Achievements with Integrin Antagonists Compounds in the Clinic The most advanced integrin antagonists in clinical trials are anti-avb3 compounds (Table 2). One is a monoclonal antibody, MEDI-522 or Abegrin, an affinityoptimized version of the former Vitaxin antibody (MEDI-523) (48). Both antibodies are humanized antibodies derived from the LM609 murine antibody. MEDI-523 is claimed to be more stable and has a 7.2-fold greater affinity to avb3 than MEDI-523 (49,50). The second is a small cyclic pentapeptide comprising the three amino acid–long sequence Arg-Gly-Asp (RGD) present in the ligands of its targets, the integrins avb3 and avb5. It is called EMD 121974 or Cilengitide, cyclo(RGDf-N(Me)V) in amino acid letter coding (51). The earliest phase I trial for an integrin antagonist dates back to 1997 and implied the anti-avb3 antibody Vitaxin. A year after, the cyclic peptide Cilengitide entered human clinical trial. Both compounds are still in phase II. The latest antiavb3 compound entering clinical trial for cancer is another antibody, the human monoclonal antibody CNTO 95, that targets the av integrin subunit (52), thus enlarging the potential activity to other av integrins besides avb3 and avb5. ATN161 or Ac-PHSCN-NH2 in amino acid letter coding, a peptide derived from the fibronectin sequence (53–59) also entered clinical trial in 2003. ATN-161 targets the fibronectin receptor a5b1, but it is also claimed to inhibit other integrins, like avb3 and, contrary to the previous antibodies and peptide, to block integrin-dependent signalling and not integrin-dependent adhesion. Later on in 2003–2004, compounds inhibiting the integrin a5b1 or the a2 subunit were introduced into the clinic: the humanized Volociximab monoclonal antibody (60) and the aromatic sulfonamide derivative E7820 (61,62), respectively. An overview of the clinical achievements with each of these compounds follows. VITAXIN (MEDI-523) AND ABEGRIN (MEDI-522) The first published phase I of the anti-avb3 Vitaxin antibody (MEDI-523) involved 17 patients with advanced (stage IV), incurable malignancies refractory to standard therapy who were treated with 6 weekly infusion doses ranging from 0.1 to 4 mg/kg (63). Among the 14 evaluable patients, 7 disease stabilizations and 1 partial response are reported. Limited drug supply at the time did not permit to go higher than 4 mg/kg/wk. The pharmacokinetics were not different from those of other humanized monoclonal antibodies, with a dose-dependent half-life ranging from 14 (0.1 mg/kg) to 138 hours (4 mg/kg). The optimum dose or schedule could not be determined, but Vitaxin treatment appeared safe. The most frequent


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adverse effect reported were infusion-related fever, chills, nausea, and flushing. For three patients receiving continued treatment beyond the 6 weekly doses, no significant adverse effects were noted. Interestingly, with the exception of one patient, and in fact for only one of his biopsies, no increase in bleeding or woundhealing inhibition was observed after biopsies, suggesting that antiangiogenic treatment with Vitaxin does not increase the risk of bleeding. The partial response concerned a patient with a leiomyosarcoma metastatic to the liver and lasted till 93 weeks of treatment. At that time, the measurable lesions remained stable but the disease progressed outside of the liver and treatment was discontinued. A subsequent pilot study (64) conducted in 15 patients with advanced leiomyosarcomas failed to show objective responses or any significant stabilization after administration of Vitaxin (0.25 mg/kg/wk intravenously) for a median of six months and no concurrent therapy. Another pilot trial (65) in nine patients with metastatic cancer for which there was no standard therapy also failed to elicit objective responses after 1–4 administrations of Vitaxin (10, 50, or 200 mg every three weeks). The three patients who received four administrations (50 or 200 mg) had stable disease at day 85 when taken off study. The trial again showed that the treatment was well tolerated and that no immune response to Vitaxin was elicited. During the same trial, the patients received a low dose of 1 mg of 99Tcm-radiolabeled Vitaxin prior to treatment in an attempt to visualize the distribution of the compound in the tumor vasculature. The 99 Tcm labeling appeared unstable in vivo and imaging of the tumor vasculature was unsuccessful, although a tumor was localized in a melanoma patient possibly because the tumor cells expressed the avb3 targeted integrin. Altogether, the anecdotal responses obtained with the Vitaxin antibody may reflect the use of a suboptimal regimen of administration or nonoptimal stability or affinity. MEDI-523 (Abegrin) is supposed to cope with the two latter issues. In an open-label phase I dose escalation study of 25 treatment-refractory patients with solid tumors, no significant toxicities was observed with MEDI-522 administered at 2–10 mg/kg/wk intravenously, only low-grade constitutional or gastrointestinal symptoms and infusion reactions [(50), see also preclinical data mentioned in this reference]. No maximum tolerated dose was identified. The half-life of MEDI-522 was similar to that of MEDI-523 (Vitaxin) and in the order of 59–106 hours. Also, no immune response to the compound was observed. Tumor blood flow was assessed at baseline and after eight weeks of treatment by dynamic computed tomography imaging on a limited number of patients. Of all the parameters studied (mean blood flow, blood volume, mean transit time, and permeability surface), only the mean transit time was significantly altered: according to the authors, the corresponding increase may be interpreted as a biological response in terms of impeding blood flow through small-caliber neovasculature. No complete or partial response was observed among the 25 patients enrolled, as defined by the RECIST criteria. Interestingly, three patients with metastatic renal cancer had prolonged stable disease for at least 34 weeks. Hypophosphatemia episodes were also observed in several patients. These observations point to a possible tropism of the molecule for renal tissues, normal or pathological. The avb3 integrin is expressed in renal tissues but, as commented by the authors of the phase I study, there are no report of integrin antagonists causing or exacerbating hypophosphatemia or other kidney diseases. Whereas the efficiency of MEDI-522 for metastatic renal cancer remains to be evaluated, a more tangible activity is suggested for patients with metastatic

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melanoma treated with the antibody administered in combination with dacarbazine (DTIC, the current standard of care in advanced melanoma). The phase II multicenter non-comparative trial involved 112 patients with stage IV metastatic melanoma randomized between September 2003 and May 2004 in two arms: MEDI-522 (8 mg/kg/wk) with (55 patients) and without (57 patients) DTIC (1000 mg/m2 once every three weeks). Preliminary observations published in abstract form for the 2005 ASCO meeting (66) hinted at efficacy in terms of overall survival. Adverse effects were mainly grade 1/2, with hematological grade 3/4 adverse events occurring in both arms (37% for MEDI-522 alone and 48% for the combination). Two MEDI-522-related deaths were reported, one in each arm (myocardial infarction and pulmonary embolism). Along with the results of this trial presented at ASCO, in a press release of May 14, 2005 (67) MedImmune announced a 12.7-month median survival for the patients treated with MEDI-522 alone, compared to 9.4-month median survival for the combination arm—the figures were compared to the 7.9-month median survival for DTIC alone in a former unrelated trial [Genasense (Genta Inc., Berkeley Heights, New Jersey, U.S.A.) phase III trial, i.e., oblimersen sodium, an antisense therapy against the Bcl-2 antiapoptotic molecule]. Further data are expected after these promising interim results, as well as on a phase II trial conducted by MedImmune in patients with androgen-independent prostate cancer that has metastasized to bone (enrollment was closed in April 2005 and 126 patients are evaluated). As of August 2006, several phases I and II are ongoing with MEDI-522. A phase I translational study aims at identifying the dose for tumor saturation and biological activity in patients with advanced malignant melanoma. Preliminary data indicate that saturation occurs at 8 mg/kg, as revealed in a press release from MedImmune on April 10, 2006 (68). Phase I studies are in progress for patients with irinotecan-refractory advanced colorectal cancer, or with refractory advanced solid tumors or lymphoma. MEDI-522 is also the subject of phase I trials for adults with plaque psoriasis (subcutaneous injection) and to study the activity and progression of joint damage in patients with active rheumatoid arthritis suboptimally responding to methotrexate. Finally, according to the press release from MedImmune (68), preclinical data suggest that the majority of MEDI-522’s antitumor activity may be mediated through antibody-dependent cellular cytotoxicity or ADCC. While still targeted, the therapy with MEDI-522 may not necessarily relate to the blocking of the avb3 integrin function(s). The outcome with small integrin antagonists like Cilengitide may thus be totally different. Cilengitide The first phase I with the small cyclic peptidic integrin avb3 and avb5 antagonist Cilengitide recruited 37 patients with advanced solid tumors refractory to standard treatment (69). Twice-weekly treatment cycles (1-hour intravenous infusion at doses of 30–1600 mg/m2) were given and no dose-limiting toxicity was found. There was no hematological toxicity related to Cilengitide treatment and nonhematological toxicities were mild and limited to grade 2 (nausea, anorexia, vomiting, fatigue, and malaise). Consequently, a maximal tolerated dose could not be defined. Systemic exposure to Cilengitide increased in a dose-proportional manner. A dose of 120 mg/m2 enabled to reach the target plasma concentration (10 mg/mL) inferred from animal studies for efficacy. No partial or complete response was observed and prolonged stable disease occurred in one patient with


269

colorectal carcinoma (for 168 days) and two with renal carcinoma (for 164 days). The plasma half-life of Cilengitide was short (3–5 hours), hence, the treatment schedule may not be optimal (although the authors noted that similar schedules in mice elicited marked tumor responses). Preliminary data from another phase I trial presented at the ASCO 2006 annual meeting (70) revealed that continuous infusion was safe up to at least 18 mg/hr (one death of unknown cause occurred at the 27 mg/hr dose). Cilengitide may prove more efficient in combination therapy, an option considered for further development. Although Cilengitide entered clinical trial in 1998, extended publications on the clinical efficacy of Cilengitide are sparse, with the exception of a case study (71). When standard treatments and surgery failed after several phases of rapid and massive growth of a squamous cell carcinoma that had its origin in the left jaw, the highly vascularized tumor could not be resected due to extensive bleeding during the surgical attempt and the patient was assigned to best supportive care. A combination of Cilengitide (600 mg/m2 over 60 min intravenously) with a cytostatic agent with a mild toxicity profile (30 min infusion of gemcitabine at 1000 mg/m2 on days 1 and 8 every three weeks) for about six months led to a partial remission. Because of gemcitabine-linked hematological toxicity, the treatment was then switched to Cilengitide alone which led to stabilization for 12 months on maintenance therapy with no tendency toward spontaneous bleeding. This case report suggests that Cilengitide may be efficient for highly vascularized tumors. Several phase I, I/II, and II studies have been conducted or are ongoing with Cilengitide in various settings (72,73): in patients with unresectable stage III or stage IV melanoma; in patients with nonmetastatic or asymptomatic, metastatic androgen-independent prostate cancer (74); in patients with advanced solid tumors or lymphoma; as maintenance therapy in patients with acute myeloid leukemia in first complete remission; in patients with HIV-related Kaposi’s sarcoma; in children with refractory primary brain tumors; and in patients with recurrent or progressive malignant glioma or glioblastoma multiforme. The focus on brain tumors also stems from preclinical data strongly supporting the notion that, besides being effective on ECs during angiogenesis, Cilengitide may affect tumor cell survival directly (75), thus inducing a strong response in animal models (76,77). In their review of Cilengitide’s clinical trials referral resource (72), the authors mentioned—see also press release from Merck KGaA on November 22, 2004 (78)—that out of 51 malignant glioma patients enrolled in the multicenter dose-escalation study designed to determine the maximum tolerated dose of Cilengitide, two patients showed complete responses (twice weekly infusions at 360 and 2400 mg/m2) and three partial responses (two at 120 mg/m2 and one at 360 mg/m2). Four patients had stable disease for more than six months. A phase II study was subsequently initiated (October 2004) in patients with recurrent glioblastoma multiforme who are receiving Cilengitide after first line chemotherapy failed. Because the association with radiation therapy may also provide clinical benefit, it is investigated in a phase I/II randomized study of Cilengitide combined with radiation therapy and temozolomide in patients with newly diagnosed glioblastoma multiforme. Finally, Cilengitide has received a positive opinion for orphan designation for the treatment of glioma by the European Medicines Agency (79). It has now the orphan-drug status in both the European Union and in the United States, and could open a much needed new way for the treatment of glioblastoma.

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Less Advanced Integrin Antagonists (CNTO 95, Volociximab, ATN-161, E7820) By recognizing the av subunit, the human monoclonal antibody CNTO 95 targets the avb3 and avb5 integrins. A phase I dose-escalating study in patients with advanced refractory solid tumors (80) showed its good tolerance when administered from 0.1 up to 10 mg/kg (intravenous infusion on days 0, 28, 35 and 42), with manageable infusion-related fever. A partial response was observed in a patient with cutaneous angiosarcoma (ongoing after two months with prolonged treatment at 10 mg/kg) as well as a stable disease in a patient with ovarian carcinosarcoma (a lesion was undetectable on FDG-PET at day 49 after 10 mg/kg treatment). The initial rapid clearance observed at low doses and slower drug clearance at higher doses may be explained by tissue binding and saturation, respectively. However, preclinical studies did not reveal adverse effects due to widespread tissue binding (81). A phase I/II, multi-center, double-blinded, randomized, placebo-controlled study of the safety and efficacy of CNTO 95, alone (infusion at 3, 5 or 10 mg/kg) and in combination with dacarbazine, opened in April 2005, in patients with stage IV melanoma. Progression-free survival is the end-point of the phase II part. Volociximab is a chimeric humanized monoclonal antibody targeting the a5b1 integrin. A phase I study of the antibody (1-hour infusion at 0.5–15 mg/kg on days 1, 15, 22, 29, and 36) in patients with refractory solid tumors showed no dose-limiting toxicity up to 10 mg/kg, the highest dose evaluated in the report (82). Stable disease was observed in 9 patients out of 15 (with five of six patients receiving 10 mg/kg of antibody). Since monocytes express the target, monocyte saturation with the antibody could be assayed as a surrogate marker of integrin targeting and was achieved at 10 mg/kg. This dose was recommended for subsequent clinical trials. Results from several phase II trials were presented at the ASCO 2006 annual meeting (abstract in 83–85 and 86 for the corresponding posters). Volociximab (intravenous infusion at 10 mg/kg once every 2 weeks for up to 52 weeks) was well tolerated in conjunction with DTIC in the metastatic melanoma trial (83). Out of 37 patients, one had a partial response and 27 had stable disease at week 8. The median overall survival was 7.9 months in a population of which 55% were classified as poor prognosis stage M1c. In the phase II open-label study in patients with refractory metastatic clear cell renal cell cancer (84), Volociximab (intravenous infusion at 10 mg/kg once every 2 weeks for up to 52 weeks) was again well tolerated. Out of 40 patients, one partial response and 32 stable disease were observed. Overall survival was not reached after 11 months since the first patient on study. In the phase II study in patients with metastatic adenocarcinoma of the pancreas (85), Volociximab (intravenous infusion at 10 mg/kg once every 2 weeks for up to 2 years) was well tolerated when added to a conventional treatment with gemcitabine. Additional patients are being enrolled in the study at 15 mg/kg. Out of 19 patients, one partial response and 10 stable disease were observed. For patients who met the entry criteria (14 patients), a median overall survival of 8.2 months and a time to progression of 5.4 months have been observed. An open-label multi-center phase II study of Volociximab (infusion once every 2 weeks for up to 52 weeks) and erlotinib hydrochloride (oral Tarceva daily) in 40 previously treated patients with locally advanced (stage IIIb) or metastatic (stage IV) non–small cell lung cancer was launched in 2006. ATN-161, a small peptidic compound recognizing the a5b1 integrin, entered phase I in January 2003. Results from a phase I in patients with advanced solid


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tumors have been published recently (87). Because tumor growth inhibition by the peptide in preclinical studies was not dose-dependent—optimal response at 1–10 mg/kg and no response at 0.2 and 100 mg/kg (54 and introduction in 87)— the clinical doses were chosen to cover a similar range after interspecies dose conversion. Twenty-six patients were enrolled to receive thrice weekly 10-min intravenous infusion of 0.1–16 mg/kg of ATN-161. No dose-limiting toxicities occurred and treatment-related adverse effects were grade 2 or less. The observed half-life was 3–5 hours, but preclinical data indicated that intermittent dosing was efficient. There was no objective response and six patients experienced stable diseases of more than four months. Four patients (adenoid cystic, renal cell, prostate, and ovarian cancer) with prolonged stable disease had well-documented progressive disease prior to study entry. A phase II study is ongoing in advanced renal cell cancer. A phase I/II study combines ATN-161 with chemotherapy in patients with recurrent intracranial malignant glioma: patients will receive the peptide in combination with carboplatin. Finally, E7820 is a first-in-class oral antiangiogenic sulfonamide targeting the a2 integrin subunit at the expression level. Interim results of a phase I in patients with solid tumors have been presented at recent ASCO annual meetings (88,89). The compound was administered orally from 10 to 200 mg/day in a 28-day cycle. Administration revealed an excellent safety profile up to 100 mg/day, the maximum tolerated dose (MTD). At 200 mg/day, two patients experienced hematological toxicity (grade 4 thrombocytopenia and neutropenia). The half-life ranged from 6 to 12 hours. No changes in integrin levels (a2 integrin subunit expression in circulating platelets) were observed at doses less than 40 mg. Moderate decrease (less than 30%) occurred at 70 mg (2 out of 3 patients) and at 100 mg (3 out of 6); at 200 mg the decrease was more pronounced (50%) and lasted beyond day 28 in 3 out of 4 patients. Disease stabilization beyond the fourth cycle occurred in 6 patients out of 30, which lasted more than 6 months. A phase I/II study (not yet open for patient recruitment as of August 2006) will determine the safety and efficacy of E7820 plus cetuximab in colorectal cancer and explore the MTD of the combination in the first part of the study in patients with advanced solid tumors and then explore the efficacy of this combination in patients with colorectal cancer that is inoperable and/or metastatic. Future of Integrin Antagonists in the Clinic Our present knowledge on integrins suggests that they are not targets specific for ECs during tumor progression. Their concomitant presence on tumour cells, and also on other stromal cells besides the endothelium, complicates the mechanistic interpretation of the action of integrin antagonists seen in the clinic. To illustrate the point with nonendothelial stromal cells, it was recently shown that Volociximab can alter the pattern of cytokine secretion by macrophages (90). It could thus exert indirect antiangiogenic effects by modulating the availability of pro-angiogenic cytokines through a5b1 integrin ligation on stromal cells. Monocytes indeed release pro-angiogenic CXC cytokines when plated on fibronectin, the ligand recognized by a5b1. ATN-161 may exert similar effects since it is homologous to the PHSCN sequence that blocks this release (91). Clearly, the mechanism of action of some of the compounds presented may not be restricted to endothelial targeting. The presence of integrins on ECs and tumor cells is also exploited for other clinical applications: to deliver conjugated cytotoxic compounds and as radioactive and imaging agents for diagnosis or treatment. Exploiting the relatively specific

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presence of avb3 integrins on tumors has been the focus of intense research for several years in these fields, especially with RGD monomers or multimers (92,93). Noninvasive selection of patients with avb3-expressing primary or metastatic tumors is now feasible (94,95) and will undoubtedly help identifying patients more likely to respond to anti-avb3 integrin treatment. OTHER ENDOTHELIAL CELL PROPAGATION INHIBITORS The integrins mentioned above, avb3, avb5, and a5b1, have been clearly identified—although not necessarily fully validated, especially in the clinic—as targets for blocking tumor progression. They are also receptors for pathophysiological ligands shown to block angiogenesis in preclinical settings. The corresponding molecules have in common to be fragments of proteins not necessarily associated with angiogenesis or tumor progression. Angiostatin is thus a fragment of plasminogen (9), endostatin is derived from collagen type XVIII (10), and tumstatin is a noncollagenous fragment of collagen IV (96). They can interact with various receptors—usually distinct from those of the parental proteins. In fact, there is now a long list of protein fragments susceptible to block angiogenesis (8). They do not necessarily interact with integrins as their main receptors and their action may not be limited to targeting the ECs or their propagation. Only two tumor-derived antiangiogenic fragments have entered clinical trials: angiostatin and endostatin. Tumstatin, another likely candidate, has not yet entered phase I trials. Thrombospondin also deserves a mention as the first physiological protein identified to inhibit angiogenesis (97). Its antiangiogenic activity was confirmed in knock-out animals and by transfection experiments for overexpression [reviewed in (8,11)]. This large extracellular matrix component could in fact also play a role in promoting angiogenesis depending on its proteolysis; hence, the interest of evaluating fragments. Dissecting the role of thrombospondin in adhesive events and endothelial tube formation led to an interest in its type I repeat domains shown to interact with its receptors, including the cell surface antiangiogenic receptor CD36. A short sequence from the second type I repeat was optimized for antiangiogenic activity. It led to the nonapeptide analogue called ABT-510 which is a potent antiangiogenic agent in vitro and in vivo mimicking the corresponding inhibitory activity of thrombospondin on EC migration, proliferation and tube formation (98,99). It can also promote apoptosis of ECs, a process improved by concomitant administration of metronomic low-dose chemotherapy (100). It is currently in phase II. Tumor-Derived Protein Fragments (Angiostatin and Endostatin) Angiostatin Angiostatin was discovered in 1994 in Judah Folkman’s laboratory (9). A phase I trial of the compound administered daily intravenously showed its safety without limiting toxicity (101). At room temperature, the activity of the compound in solution is rapidly lost and prevents continuous infusion. The feasibility and safety of subcutaneous administration were shown later (102). Twice daily subcutaneous administration in a small number of patients with advanced non–small cell lung cancer and in combination with paclitaxel and carboplatin yielded results similar to those seen with carboplatin/paclitaxel/bevacizumab in a phase III study (103).


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However, financial issues at EntreMed, the company in charge for the development, cast a shadow on the future of the development of this molecule as a recombinant protein (see below with endostatin). Other scenarios are investigated to bathe the body with the protein: producing angiostatin by direct proteolysis of the plasminogen precursor protein (104) or through the administration of adenoviral constructs encoding the gene sequence of angiostatin as a fusion with the sequence of endostatin (105). A phase I trial for evaluating the first scenario validated the approach although no clinical objective responses were observed. Also, antibodies against one of the receptors of angiostatin on ECs, angiomotin (106–108), are presently generated and may mimic the activity of angiostatin. It is hoped that such antibodies would be easier to produce than angiostatin itself (109). A vaccine strategy has also been tried with success against angiomotin (110). Endostatin Endostatin was discovered in 1997, again in Judah Folkman’s laboratory (10). Daily intravenous administration was found to be safe in the first phase I trials in 1999–2001 without redhibitory toxicity (111–113). A combination of two ways of administration—intravenous for 28 days followed by daily subcutaneous dosing— was found to be safe in human (114). In 2002–2003, phase II trials with the compound administered subcutaneously twice a day showed only two minor responses in 41 patients with advanced neuroendocrine tumors (115). Endostatin was associated with minimal toxicity but overall did not result in significant tumor regression in this indication (116). Endostatin development had to face challenges in terms of protein production and administration. The protein is insoluble in physiological fluids and the production in the bacteria Escherichia coli is associated with problems of stability and proper refolding, not to mention reproducibility issues. Production in the yeast, although feasible, led to a soluble form but with a very low yield, hence requiring high costs for development. In 2003, because of high production costs, EntreMed announced discontinuation of the production of endostatin. The stocks in the United States lasted until 2005, and clinical trials had to be stopped, much to the disappointment of clinical investigators (117). Following the creation of the Yantai Medgenn joint-venture between Dr Luo et Yantai R.C. Pharmaceutical Co. (a Chinese company involved in traditional medicine), a new form of endostatin was produced. Nine amino acids were added at the N-terminal to stabilize the protein and confer a correct folding. Between 2001 and 2004, 493 patients with lung carcinoma received the corresponding protein, Endostar, as part of their chemotherapy in a clinical trial. The results were presented at ASCO 2005 (118): 35.4% of patients in the combination arm exhibited reduced tumor size versus 19.5% in the arm receiving chemotherapy alone. Survival at one year was also enhanced (60% surviving patients in the combination arm vs. 30%). Endostar was approved by the Chinese State Food and Drug Administration in October 2005 for the treatment of non–small cell lung cancer in combination with chemotherapy. This event ranked by the Ministry of Science and Technology of the People’s Republic of China as number 7—just after the re-evaluation of the height of mount Everest, i.e., 8844.43 meters instead of the preceding 8848.13 meters announced in 1975—in the top 10 domestic Science and Technology events for 2005 (119). As with angiostatin and to circumvent the difficulties in manufacturing the recombinant protein, attempts are also being made to administer endostatin

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through gene therapy. A phase I trial of an adenovirus vector for endostatin gene transfer, Ad-rhEndo or E10A (120) has been completed in China in patients with advanced solid tumours and the construct appears to be well tolerated (121). Thrombospondin Fragment (ABT-510) ABT-510 has been evaluated as subcutaneous administrations (cycles of continuous infusion or once- or twice-daily administration as a bolus for 28 days) in a phase I in 39 patients with advanced cancer (122). Continuous infusion was inconvenient since it necessitated daily change of the infusion site to prevent skin reactions. The product showed linear, time-dependent pharmacokinetics and a favorable toxicity profile. No tumor regressions were observed, but several cases of prolonged stable disease occurred. A dose of 100 mg twice daily subcutaneously was retained for phase II studies. A phase II study evaluating the activity of ABT510 alone in the first-line treatment of patients with advanced renal cell carcinoma showed that the treatment was well tolerated but it did not provide obvious improvement in efficacy compared to historical controls (123). On the other hand, progression-free survival in patients with advanced soft tissue sarcoma and treated with ABT-510 exceeded the 14% rate reported by the EORTC for active drugs in this pathology (124). Single-agent therapy or combination treatment may thus be beneficial in this pathology. ABT-510 was granted an Orphan Drug designation by the FDA for soft tissue sarcoma—press release from Abbott May 9, 2005 (125). Since combination studies are expected to yield improved effects, several phase I trials of ABT-510 in combination with chemotherapy have been conducted without additional toxicity. A combination with 5-fluorouracil and leucovorin (5FU/LV) was evaluated in 12 patients with advanced solid malignancies, but the authors (126) cautioned that a larger study may reveal unexpected major toxicities, as seen with the tyrosine kinase VEGF receptor inhibitor SU5416 or with the antiVEGF antibody bevacizumab (occurrence of thromboembolic events in combination with 5FU/LV). In combination with gemcitabine and cisplatin in 13 patients with advanced solid tumors, three partial responses were observed (out of 12 patients evaluable) in patients with head and neck cancer, melanoma, or NSCLC (127). A phase I study in combination with bevacizumab is in progress in patients with advanced solid tumors, as well as combination therapy (carboplatin/taxol) in subjects with NSCLC. A phase II study of ABT-510 alone for the treatment of previously treated metastatic melanoma did not show significant clinical efficacy (128). ABT-510 is currently evaluated in phase II studies for the following pathologies: advanced head and neck cancer, locally recurrent or metastatic renal cell cancer, refractory lymphoma and locally advanced or metastatic soft tissue sarcoma. CONCLUSION It is too early to conclude on a possible benefit in a given niche for integrin inhibitors as a class, even with the anti-avb3 compounds that are more advanced in clinical development, such as Abegrin (MEDI-522) and Cilengitide. As with any new targeted therapy, conducting trials with only the agents under investigation in patients with advanced disease may fail to reveal the potential of integrin antagonists. Their evaluation as adjuvant therapy or in combination with cytotoxic


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agents may prove beneficial. Therefore, the outcome of ongoing and additional combination trials of integrin antagonists is eagerly awaited, since the results with monotherapy treatments were, overall, somewhat anecdotal, with no objective responses in clinical trials. Integrins are believed to be good targets for fighting tumor progression, but the precise mechanisms of their participation in this process is not entirely clear. This may not prevent clinical development of the so-called integrin antagonists, but comes as a warning: in some situations—yet to be discovered—the balance may shift from antiangiogenic to protumorigenic activity, as seen with the first generation of matrix metalloproteinases inhibitors. Large, tumor-derived, antiangiogenic protein fragments, such as angiostatin and endostatin, may fare better than integrin antagonists in the clinic because of their multiple cell receptor targets—including integrins. Again, the mechanism of action of such compounds is not entirely elucidated. The possibility that a common cross-b structure present in many antiangiogenic fragments exerts amyloid-induced cell toxicity has been raised (129). A correct folding of these molecules is indeed necessary for activity, something not always achieved during some processes of recombinant protein production. This may be at the heart of the difficulties encountered at the production level for this class of EC propagation inhibitors, a difficulty potentially resolved by turning to gene therapy—although other burdens in clinical development may occur. The list of endogenous inhibitors of angiogenesis has been steadily growing, recently with about 27 candidates (8), including thrombospondin. Determining the minimally active sequence(s) in these molecules is feasible—as exemplified for endostatin (130) or thrombospondin with ABT-510—and may facilitate development, although the full spectrum of activity of the parental protein may be lost if several receptors or targets participate in the activity (131,132). A large variety of protein fragments is thus likely to enter clinical trials in the future as antiangiogenic compounds. REFERENCES 1. Frisch SM, Screaton RA. Anoikis mechanisms. Curr Opin Cell Biol 2001; 13:555–62. 2. Grossmann J. Molecular mechanisms of “detachment-induced apoptosis—anoikis”. Apoptosis 2002; 7:247–60. 3. Hynes RO. Integrins: bidirectional, allosteric signalling machines. Cell 2002; 110:673–87. 4. Rundhaug JE. Matrix metalloproteinases and angiogenesis. J Cell Mol Med 2005; 9:267–85. 5. Vlodavsky I, Friedmann Y. Molecular properties and involvement of heparanase in cancer metastasis and angiogenesis. J Clin Invest 2001; 108:341–7. 6. Miao HQ, Liu H, Navarro E, et al. Development of heparanase inhibitors for anticancer therapy. Curr Med Chem 2006; 13:2101–11. 7. Clamp AR, Jayson GC. The clinical potential of antiangiogenic fragments of extracellular matrix proteins. Br J Cancer 2005; 93:967–72. 8. Nyberg P, Xie L, Kalluri R. Endogenous inhibitors of angiogenesis. Cancer Res 2005; 65:3967–79. 9. O’Reilly MS, Holmgren L, Shing Y, et al. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 1994; 79:315–28. 10. O’Reilly MS, Boehm T, Shing Y, et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 1997; 88:277–85. 11. Ruegg C, Hasmim M, Lejeune FJ, Alghisi GC. Antiangiogenic peptides and proteins: from experimental tools to clinical drugs. Biochim Biophys Acta 2006; 1765:155–77.

276

Tucker

12. Ren B, Yee KO, Lawler J, Khosravi-Far R. Regulation of tumor angiogenesis by thrombospondin-1. Biochim Biophys Acta 2006; 1765:178–88. 13. Miranti CK, Brugge JS. Sensing the environment: a historical perspective on integrin signal transduction. Nat Cell Biol 2002; 4:E83–90. 14. Hood JD, Cheresh DA. Role of integrins in cell invasion and migration. Nature Rev Cancer 2002; 2:91–100. 15. Jin H, Varner J. Integrins: roles in cancer development and as treatment targets. Br J Cancer 2004; 90:561–5. 16. Rüegg C, Mariotti A. Vascular integrins: pleiotropic adhesion and signaling molecules in vascular homeostasis and angiogenesis. Cell Mol Life Sci 2003; 60:1135–57. 17. Ginsberg MH, Partridge A, Shattil SJ. Integrin regulation. Curr Opin Cell Biol 2005; 17:509–16. 18. Luo BH, Springer TA. Integrin structures and conformational signaling. Curr Opin Cell Biol 2006; 18:579–86. 19. Arnaout MA, Mahalingam B, Xiong JP. Integrin structure, allostery, and bidirectional signaling. Annu Rev Cell Dev Biol 2005; 21:381–410. 20. Akalu A, Cretu A, Brooks PC. Targeting integrins for the control of tumour angiogenesis. Expert Opin Investig Drugs 2005; 14:1475–86. 21. Alghisi GC, Ruegg C. Vascular integrins in tumor angiogenesis: mediators and therapeutic targets. Endothelium 2006; 13:113–35. 22. Hodivala-Dilke KM, Reynolds AR, Reynolds LE. Integrins in angiogenesis: multitalented molecules in a balancing act. Cell Tissue Res 2003; 314:131–44. 23. Stupack DG, Cheresh DA. Integrins and angiogenesis. Curr Top Dev Biol 2004; 64:207–38. 24. Kim S, Bell K, Mousa SA, Varner JA. Regulation of angiogenesis in vivo by ligation of integrin alpha5beta1 with the central cell-binding domain of fibronectin. Am J Pathol 2000; 156:1345–62. 25. Byzova TV, Goldman CK, Pampori N, et al. A mechanism for modulation of cellular responses to VEGF: activation of the integrins. Mol Cell 2000; 6:851–60. 26. Hamano Y, Zeisberg M, Sugimoto H, et al. Physiological levels of tumstatin, a fragment of collagen IV alpha3 chain, are generated by MMP-9 proteolysis and suppress angiogenesis via alphaV beta3 integrin. Cancer Cell 2003; 3:589–601. 27. Chavakis E, Riecke B, Lin J, et al. Kinetics of integrin expression in the mouse model of proliferative retinopathy and success of secondary intervention with cyclic RGD peptides. Diabetologia 2002; 45:262–7. 28. Reynolds LE, Wyder L, Lively JC, et al. Enhanced pathological angiogenesis in mice lacking beta3 integrin or beta3 and beta5 integrins. Nat Med 2002; 8:27–34. 29. Taverna D, Moher H, Crowley D, et al. Increased primary tumor growth in mice null for beta3- or beta3/beta5-integrins or selectins. Proc Natl Acad Sci USA 2004; 101:763–8. 30. Carmeliet P. Integrin indecision. Nat Med 2002; 8:14–16. 31. Hynes RO. A reevaluation of integrins as regulators of angiogenesis. Nat Med 2002; 8:918–921. 32. Hynes RO, Lively JC, McCarty JH, et al. The diverse roles of integrins and their ligands in angiogenesis. Cold Spring Harb Symp Quant Biol 2002; 67:143–53. 33. Stupack DG, Puente XS, Boutsaboualoy S, et al. Apoptosis of adherent cells by recruitment of caspase-8 to unligated integrins. J Cell Biol 2001; 155:459–70. 34. Stupack DG. Integrins as a distinct subtype of dependence receptors. Cell Death Differ 2005; 12:1021–30. 35. Maubant S, Saint-Dizier D, Boutillon M, et al. Blockade of avb3 and avb5 integrins by RGD mimetics induces anoikis and not integrin-mediated death in human endothelial cells. Blood 2006; 108:3035–44. 36. Coleman PJ, Duong LT. Ligands to the integrin receptor alphav beta3. Expert Opin Ther Patents 2002; 12:1009–21. 37. Kerr JS, Slee AM, Mousa SA. The alphav integrin antagonists as novel anticancer agents: an update. Expert Opin Investig Drugs 2002; 11:1765–74.


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38. Lode HN, Moehler T, Xiang R, et al. Synergy between an antiangiogenic integrin alphav antagonist and an antibody-cytokine fusion protein eradicates spontaneous tumor metastases. Proc Natl Acad Sci USA 1999; 96:1591–6. 39. Stoeltzing O, Liu W, Reinmuth N, et al. Inhibition of integrin alpha5beta1 function with a small peptide (ATN-161) plus continuous 5-FU infusion reduces colorectal liver metastases and improves survival in mice. Int J Cancer 2003; 104:496–503. 40. Strieth S, Eichhorn ME, Sutter A, et al. Antiangiogenic combination tumor therapy blocking alpha(v)-integrins and VEGF-receptor-2 increases therapeutic effects in vivo. Int J Cancer 2006; 119:423–31. 41. Bakewell SJ, Nestor P, Prasad S, et al. Platelet and osteoclast beta3 integrins are critical for bone metastasis. Proc Natl Acad Sci USA 2003; 100:14205–10. 42. McHugh KP, Hodivala-Dilke K, Zheng MH, et al. Mice lacking beta3 integrins are osteosclerotic because of dysfunctional osteoclasts. J Clin Invest 2000; 105:433–40. 43. Wachsberger P, Burd R, Dicker AP. Tumor response to ionizing radiation combined with antiangiogenesis or vascular targeting agents: exploring mechanisms of interaction. Clin Cancer Res 2003; 9:1957–71. 44. Burke PA, DeNardo SJ, Miers LA, et al. Cilengitide targeting of alpha(v)beta(3) integrin receptor synergizes with radioimmunotherapy to increase efficacy and apoptosis in breast cancer xenografts. Cancer Res 2002; 62:4263–72. 45. Abdollahi A, Griggs DW, Zieher H, et al. Inhibition of alpha(v)beta3 integrin survival signaling enhances antiangiogenic and antitumor effects of radiotherapy. Clin Cancer Res 2005; 11:6270–9. 46. Albert JM, Cao C, Geng L, et al. Integrin alpha v beta 3 antagonist Cilengitide enhances efficacy of radiotherapy in endothelial cell and non-small-cell lung cancer models. Int J Radiat Oncol Biol Phys 2006; 65:1536–43. 47. Cohen-Jonathan EL, Monferran S, Skuli N, et al. Alphav beta3 integrin pathways controls glioma radioresistance through ILK. Proc Am Assoc Cancer Res 2006; 47: abstract 5180. 48. Mikecz K. Vitaxin. Curr Opin Investig Drugs 2000; 1:199–203. 49. Wu H, Beuerlein G, Nie Y, et al. Stepwise in vitro affinity maturation of Vitaxin, an alphavbeta3-specific humanized mAb. Proc Natl Acad Sci USA 1998; 95:6037–42. 50. McNeel DG, Eickhoff J, Lee FT, et al. Phase I trial of a monoclonal antibody specific for alphavbeta3 integrin (MEDI-522) in patients with advanced malignancies, including an assessment of effect on tumor perfusion. Clin Cancer Res 2005; 11:7851–60. 51. Sorbera LA, Graul A, Castañer J. Cilengitide. Drugs Fut 2000; 25:674–8. 52. Trikha M, Zhou Z, Nemeth JA, et al. CNTO 95, a fully human monoclonal antibody that inhibits alphav integrins, has antitumor and antiangiogenic activity in vivo. Int J Cancer 2004; 110:326–35. 53. Livant DL, Brabec RK, Pienta KJ, et al. Anti-invasive, antitumorigenic, and antimetastatic activities of the PHSCN sequence in prostate carcinoma. Cancer Res 2000; 60:309–20. 54. Plunkett ML, Tel-Tsur Z, Bera M, et al. A novel anti-angiogenic/anti-metastatic peptide, ATN-161 (Ac-PHSCN-NH2), which targets multiple fully activated integrins including alpha-5 beta-1 and alpha-v beta-3, leads to increased anti-tumor activity and increased survival in multiple tumor models when combined with chemotherapy. Eur J Cancer Supplements 2002; 38:79 (Abstract 249). 55. Plunkett ML, Mazar AP. Dose and schedule optimization of a novel anti-angiogenic/ anti-metastatic peptide, ATN-161 (Ac-PHSCN-NH2), which targets multiple fully activated integrins including alpha-5 beta-1 and alpha-v beta-3. Eur J Cancer Supplements 2002; 38:82 (Abstract 262). 56. Stoeltzing O, Liu WB, Reinmuth N, et al. Inhibition of integrin alpha(5)beta(1) function with a small peptide (ATN-161) plus continuous 5-FU infusion reduces colorectal liver metastases and improves survival in mice. Int J Cancer 2003; 104:496–503. 57. Rabbani SA, Khalili P, Mazar AP. A non-RGD based integrin binding peptide (ATN161) that targets activated alpha-5 beta-1 and alpha-v beta-3 blocks the development

278

58. 59.

60. 61. 62. 63. 64. 65. 66.

67.

68.

69.

70.

71. 72. 73.

Tucker

of osteolytic skeletal metastases in a xenograft model of breast cancer [abstract]. Clin Cancer Res 2003; 9(suppl.), abstract 6181. Donate F, Guan X, Callahan JA, et al. ATN-161 (Ac-PHSCN-NH2) has potent antiangiogenic activity through multiple mechanisms of action and localizes to newly formed blood vessels in vivo. Proc Am Assoc Cancer Res 2003; 44: abstract 63. Khalili P, Plunkett M, Beck I, et al. Regression of primary breast tumor and blockage of osteolytic skeletal metastasis by a non-RGD based integrin binding peptide (ATN161) targeting a5b1 and avb3 in a xenograft model of breast cancer [abstract]. Proc Am Assoc Cancer Res 2004; 45: abstract 4001. Ramakrishnan V, Johnson DE, Wills M, et al. A function-blocking chimeric antibody, Eos200-4, against alpha5beta1 integrin inhibits angiogenesis in a monkey model [abstract]. Proc Am Assoc Cancer Res 2003; 44:(2nd ed), abstract 2052. Funahashi Y, Sugi NH, Semba T, et al. Sulfonamide derivative, E7820, is a unique angiogenesis inhibitor suppressing an expression of integrin alpha2 subunit on endothelium. Cancer Res 2002; 62:6116–23. Semba T, Funahashi Y, Ono N, et al. An angiogenesis inhibitor E7820 shows broadspectrum tumor growth inhibition in a xenograft model: possible value of integrin alpha2 on platelets as a biological marker. Clin Cancer Res 2004; 10:1430–8. Gutheil JC, Campbell TN, Pierce PR, et al. Targeted antiangiogenic therapy for cancer using Vitaxin: a humanized monoclonal antibody to the integrin avb3. Clin Cancer Res 2000; 6:3056–61. Patel SR, Jenkins J, Papadopolous N, et al. Pilot study of Vitaxin—an angiogenesis inhibitor—in patients with advanced leiomyosarcomas. Cancer 2001; 92:1347–8. Posey JA, Khazaeli MB, DelGrosso A, et al. A pilot trial of vitaxin, a humanized antivitronectin receptor (anti avb3) antibody in patients with metastatic cancer. Cancer Biother Radiopharm 2001, 16:125–32. Hersey P, Sosman J, O’Day S, et al. A phase II, randomized, open-label study evaluating the antitumor activity of MEDI-522, a humanized monoclonal antibody directed against the human alpha v beta 3 (avb3) integrin, dacarbazine (DTIC) in patients with metastatic melanoma (MM). J Clin Oncol, ASCO Annual Meeting Proceedings 2005; 23 (16S), abstract 7507. Preliminary analysis of phase 2 study shows 53 percent of patients with stage IV metastatic melanoma survive one-year mark when treated with Vitaxin—Median survival of study patients is 12.7 months. Press release from MedImmune May 14, 2005. http://phx. corporateir.net/phoenix.zhtml?c¼83037&p¼irolnewsArticle& ID¼709951&highlight= (accessed August 2006). Additional preclinical and clinical data indicate Abegrin may offer three-pronged approach to cancer therapy—MedImmune to initiate phase 3 study with metastatic melanoma patients in 2006. Press release from MedImmune April 10, 2006. http://phx. corporateir.net/phoenix.zhtml?c¼83037&p¼irolinvestornewsArticle&ID¼840777&highlight¼(accessed August 2006). Eskens FALM, Dumez H, Hoekstra R, et al. Phase I and pharmacokinetic study of continuous twice weekly intravenous administration of Cilengitide (EMD 121974), a novel inhibitor of the integrins avb3 and avb5 in patients with advanced solid tumours. Eur J Cancer 2003; 39:917–26. Undevia SD, Janisch L, Stadler WM, et al. A phase I and pharmacokinetic study of continuous infusion EMD 121974 (EMD), an antiangiogenic avb3 and avb5 integrin antagonist, in patients with advanced solid malignancy. J Clin Oncol, ASCO Annual Meeting Proceedings 2006; 24 (18S), abstract 3052. Raguse JD, Gath HJ, Bier J, et al. Cilengitide (EMD 121974) arrests the growth of a heavily pretreated highly vascularised head and neck tumour. Oral Oncol 2004; 40:228–30. Colevas AD, Scharf O, Schoenfeldt M. Clinical trials referral resource. Current clinical trials of cilengitide, an alpha (v) antagonist in clinical development as an anticancer agent. Oncology (Williston Park) 2004; 18:1778, 1781–2, 1784. Clinical trials search results for Cilengitide (open and closed status). http://www. cancer.gov/search/SearchClinicalTrialsAdvanced.aspx. (accessed August 2006).


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74. Beekman KW, Colevas AD, Cooney K, et al. Phase II evaluations of cilengitide in asymptomatic patients with androgen-independent prostate cancer: scientific rationale and study design. Clin Genitourin Cancer 2006; 4:299–302. 75. Taga T, Suzuki A, Gonzalez-Gomez I, et al. av-integrin antagonist EMD 121974 induces apoptosis in brain tumor cells growing on vitronectin and tenascin. Int J Cancer 2002; 98:690–7. 76. MacDonald TJ, Taga T, Shimida H, et al. Preferential susceptibility of brain tumors to the antiangiogenic effects of an av integrin antagonist. Neurosurgery 2001; 48: 151–7. 77. Barnes JW, Packard A, Zimmerman RE, et al. EMD121974 targeting of avb3 integrin inhibits orthotopic glioblastoma multiforme. Proc Am Assoc Cancer Res 2006; 47, abstract 238. 78. Early clinical study results show encouragement in treatment of glioblastoma brain tumors. Press release from Merck KGaA, November 21, 2004. http://www.merck. de/servlet/PB/show/1378880/Cilengitide%2022.11.04_eng.pdf#search¼%22merck% 20kgaa%20glioblastoma%20brain%202004%22 (accessed September 2006). 79. Public summary of positive opinion for orphan designation of cilengitide for the treatment of glioma. European Medicines Agency. Committee for orphan medicinal products. July 8, 2004. http://www.emea.eu.int/pdfs/human/comp/opinion/ 032604en.pdf (accessed September 2006). 80. Jayson GC, Mullamitha S, Ton C, et al. Phase I study of CNTO 95, a fully human monoclonal antibody (mAb) to av integrins, in patients with solid tumors. J Clin Oncol, ASCO Annual Meeting Proceedings 2005; 23 (16S), abstract 3113. 81. Martin PL, Jiao Q, Cornacoff J, et al. Absence of adverse effects in cynomolgus macaques treated with CNTO 95, a fully human anti-alphav integrin monoclonal antibody, despite widespread tissue binding. Clin Cancer Res 2005; 11:6959–65. 82. Ricart A, Liu G, Tolcher A, et al. A phase I dose-escalation study of anti-alpha5beta1 integrin monoclonal antibody (M200) in patients with refractory solid tumors. Eur J Cancer Suppl 2004; 2:52–3 abstract 166. 83. Cranmer LD, Bedikian AY, Ribas A, et al. Phase II study of volociximab (M200), an a5b1 anti-integrin antibody in metastatic melanoma. J Clin Oncol, ASCO Annual Meeting Proceedings 2006; 24 (18S), abstract 8011. 84. Figlin RA, Kondagunta GV, Yazji S, et al. Phase II study of volociximab (M200), an a5b1 anti-integrin antibody in refractory metastatic clear cell renal cell cancer (RCC). J Clin Oncol, ASCO Annual Meeting Proceedings 2006; 24 (18S), abstract 4535. 85. Valle JW, Ramanathan RK, Glynne-Jones R, et al. Phase II study of volociximab (M200), an a5b1 anti-integrin antibody in metastatic adenocarcinoma of the pancreas (MPC). J Clin Oncol, ASCO Annual Meeting Proceedings 2006; 24 (18S), abstract 4111. 86. 2006 publications. ASCO 2006 Poster Presentations. http://www.pdl.com/index.cfm? navId¼159 (accessed August 2006). 87. Cianfrocca ME, Kimmel KA, Gallo J, et al. Phase 1 trial of the antiangiogenic peptide ATN-161 (Ac-PHSCN-NH(2)), a beta integrin antagonist, in patients with solid tumours. Br J Cancer 2006; 94:1621–6. 88. Mita MM, Mita AC, Goldston M, et al. Pharmacokinetics (PK) and pharmacodynamics (PD) of E7820-an oral sulfonamide with novel, alpha-2 integrin mediated antiangiogenic properties: Results of a phase I study [abstract]. J Clin Oncol, ASCO Annual Meeting Proceedings 2005; 23 (16S), abstract 3082. 89. Mita MM, Mita AC, Ricart A, et al. Phase I study of an anti-angiogenic agent with a novel mechanism of action E7820: Safety, pharmacokinetics (PK) and pharmacodynamic (PD) studies in patients (pts) with solid tumors. J Clin Oncol, ASCO Annual Meeting Proceedings 2006; 24 (18S), abstract 3048. 90. Bhaskar V, Wales P, McClellan M, et al. Volociximab (M200), a chimeric anti-integrin alpha5 beta1 monoclonal antibody, inhibits macrophage secretion of pro-angiogenic cytokines. Proc Am Assoc Cancer Res 2006; 47, abstract 3871. 91. White ES, Livant DL, Markwart S, et al. Monocyte-fibronectin interactions, via alpha(5)beta(1) integrin, induce expression of CXC chemokine-dependent angiogenic activity. J Immunol 2001; 167:5362–6.

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Tucker

92. Temming K, Schiffelers RM, Molema G, Kok RJ. RGD-based strategies for selective delivery of therapeutics and imaging agents to the tumour vasculature. Drug Resist Updat 2005; 8:381–402. 93. Chen X. Multimodality imaging of tumor integrin alphavbeta3 expression. Mini Rev Med Chem 2006; 6:227–34. 94. Haubner R, Weber WA, Beer AJ, et al. Noninvasive visualization of the activated alphavbeta3 integrin in cancer patients by positron emission tomography and [18F]Galacto-RGD. PLoS Med 2005; 2:e70, 244–52. 95. Beer AJ, Haubner R, Sarbia M, et al. Positron emission tomography using [18F]Galacto-RGD identifies the level of integrin alpha(v)beta3 expression in man. Clin Cancer Res 2006; 12:3942–9. 96. Maeshima Y, Colorado PC, Torre A, et al. Distinct antitumor properties of a type IV collagen domain derived from basement membrane. J Biol Chem 2000; 275:21340–8. 97. Good DJ, Polverini PJ, Rastinejad F, et al. A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc Natl Acad Sci USA 1990; 87:6624–8. 98. Haviv F, Bradley MF, Kalvin DM, et al. Thrombospondin-1 mimetic peptide inhibitors of angiogenesis and tumor growth: design, synthesis, and optimization of pharmacokinetics and biological activities. J Med Chem 2005; 48:2838–46. 99. Sorbera LA, Bayés M. ABT-510. Drugs Fut 2005; 30:1081–6. 100. Yap R, Veliceasa D, Emmenegger U, et al. Metronomic low-dose chemotherapy boosts CD95-dependent antiangiogenic effect of the thrombospondin peptide ABT-510: a complementation antiangiogenic strategy. Clin Cancer Res 2005; 11:6678–85. 101. DeMoraes ED, Fogler WE, Grant D, et al. Recombinant human angiostatin (RHA): a phase I clinical trial assessing safety, pharmacokinetic (PK) and pharmacodynamics (PD). Proc Am Soc Clin Oncol 2001; 20, abstract 3. 102. Beerepoot LV, Witteveen EO, Groenewegen G, et al. Recombinant human angiostatin by twice-daily subcutaneous injection in advanced cancer: a pharmacokinetic and long-term safety study. Clin Cancer Res 2003; 9:4025–33. 103. Kurup A, Lin C-W, Murry DJ, et al. Recombinant human angiostatin (rhAngiostatin) in combination with paclitaxel and carboplatin in patients with advanced nonsmall-cell lung cancer: a phase II study from Indiana University. Ann Oncol 2006; 17:97–103. 104. Soff GA, Wang H, Cundiff DL, et al. In vivo generation of angiostatin isoforms by administration of a plasminogen activator and a free sulfhydryl donor: a phase I study of an angiostatic cocktail of tissue plasminogen activator and mesna. Clin Cancer Res 2005; 11:6218–25. 105. Jimenez JA, Li X, Raikwar S, et al. Combinatorial therapy of hormone-refractory prostate cancer employing a prostate-restricted replication-competent adenovirus and a replication-deficient adenovirus encoding human endostatin-angiostatin fusion protein. Proc Am Soc Clin Oncol, Prostate Cancer Symposium 2006, abstract 239. 106. Zetter BR. Hold that line. Angiomotin regulates endothelial cell motility. J Cell Biol 2001; 152:F35–6. 107. Troyanovsky B, Levchenko T, Mansson G, Matvijenko O, Holmgren L. Angiomotin: an angiostatin binding protein that regulates endothelial cell migration and tube formation. J Cell Biol 2001; 152:1247–54. 108. Jiang WG, Watkins G, Douglas-Jones A, Holmgren L, Mansel RE. Angiomotin and angiomotin like proteins, their expression and correlation with angiogenesis and clinical outcome in human breast cancer. BMC Cancer 2006; 6:16. 109. Andreasson P, Carlsson R. Targeting angiogenesis with antibodies for the treatment of cancer. IDrugs 2005; 8:730–3. 110. Holmgren L, Ambrosino E, Birot O, et al. A DNA vaccine targeting angiomotin inhibits angiogenesis and suppresses tumor growth. Proc Natl Acad Sci USA 2006; 103;9208–13. 111. Eder JP, Supko JG, Clark JW, et al. Phase I clinical trial of recombinant human endostatin administered as a short intravenous infusion repeated daily. J Clin Oncol 2002; 20:3772–84.


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112. Herbst RS, Hess KR, Tran HT, et al. Phase I study of recombinant human endostatin in patients with advanced solid tumors. J Clin Oncol 2002; 20:3792–803. 113. Thomas JP, Arzoomanian RZ, Alberti D, et al. Phase I pharmacokinetic and pharmacodynamic study of recombinant human endostatin in patients with advanced solid tumors. J Clin Oncol 2003; 21:223–31. 114. Hansma AH, Broxterman HJ, van der Horst I, et al. Recombinant human endostatin administered as a 28-day continuous intravenous infusion, followed by daily subcutaneous injections: a phase I and pharmacokinetic study in patients with advanced cancer. Ann Oncol 2005, 16, 1695–1701. 115. Kulke M, Bergsland E, Ryan DP, et al. A phase II open-label, safety, pharmacokinetic and efficacy study of recombinant human endostatin in patients with advanced neuro-endocrine tumors. ASCO Annual Meeting Proceedings 2003; 22, abstract 958. 116. Kulke MH, Bergsland EK, Ryan DP, et al. Phase II study of recombinant human endostatin in patients with advanced neuroendocrine tumors. J Clin Oncol 2006; 24:3555–61. 117. Whitworth A. Endostatin: are we waiting for Godot? J Natl Cancer Inst 2006; 98:731–3. 118. Sun Y, Wang J, Liu Y, et al. Results of phase III trial of rh-endostatin (YH-16) in advanced non-small cell lung cancer (NSCLC) patients. J Clin Oncol, ASCO Annual Meeting Proceedings 2005; 23 (16S), abstract 7138. 119. China Science and Technology Newsletter number 426. The Ministry of Science and Technology, People’s Republic of China. Top ten domestic S&T events for 2005 unveiled. January 10, 2006. http://www.most.gov.cn/eng/newsletters/2006/t20060113_27845. htm (accessed September 2006). 120. Li L, Liu RY, Huang JL, et al. Adenovirus-mediated intra-tumoral delivery of the human endostatin gene inhibits tumor growth in nasopharyngeal carcinoma. Int J Cancer 2006; 118:2064–71. 121. Passey S. Endostatin gene therapy inhibits tumour growth. Lancet Oncol 2006; 7:199. 122. Hoekstra R, de Vos FY, Eskens FA, et al. Phase I safety, pharmacokinetic, and pharmacodynamic study of the thrombospondin-1-mimetic angiogenesis inhibitor ABT-510 in patients with advanced cancer. J Clin Oncol 2005; 23:5188–97. 123. Ebbinghaus SW, Hussain M, Tannir NM, et al. A randomized phase 2 study of the thrombospondin-mimetic peptide ABT-510 in patients with previously untreated advanced renal cell carcinoma. J Clin Oncol, ASCO Annual Meeting Proceedings 2005; 23 (16S), abstract 4607. 124. Baker LH, Demetri GD, Mendelson DS. A randomized phase 2 study of the thrombospondin-mimetic peptide ABT-510 in patients with advanced soft tissue sarcoma (STS). J Clin Oncol, ASCO Annual Meeting Proceedings 2005; 23 (16S), abstract 9013. 125. Abbott to present new data on its oncology pipeline at the American Society of Clinical Oncology (ASCO) annual meeting. About ABT-510. Press release from Abbott Laboratories, May 9, 2005. http:/www.abbott.com/global/url/pressRelease/en_US/ 60.5:5/Press_Release_0088.htm (accessed September 2006). 126. Hoekstra R, de Vos FY, Eskens FA, et al. Phase I study of the thrombospondin-1mimetic angiogenesis inhibitor ABT-510 with 5-fluorouracil and leucovorin: a safe combination. Eur J Cancer 2006; 42:467–72. 127. Gietema JA, Hoekstra R, de Vos FY, et al. A phase I study assessing the safety and pharmacokinetics of the thrombospondin-1-mimetic angiogenesis inhibitor ABT-510 with gemcitabine and cisplatin in patients with solid tumors. Ann Oncol 2006; 17:1320–7. 128. Markovic S, Suman V, Rao R. A phase II study of ABT-510 for the treatment of metastatic melanoma. J Clin Oncol, ASCO Annual Meeting Proceedings 2005; 24 (18S), abstract 8041. 129. Gebbink MF, Voest EE, Reijerkerk A. Do antiangiogenic protein fragments have amyloid properties? Blood 2004; 104:1601–5. 130. Tjin Tham Sjin RM, Satchi-Fainaro R, Birsner AE, et al. A 27-amino-acid synthetic peptide corresponding to the NH2-terminal zinc-binding domain of endostatin is responsible for its antitumor activity. Cancer Res 2005; 65:3656–63.

282

Tucker

131. Benezra R, Rafii S. Endostatin’s endpoints—deciphering the endostatin antiangiogenic pathway. Cancer Cell 2004; 5:205–6. 132. Wahl ML, Kenan DJ, Gonzalez-Gronow M, Pizzo SV. Angiostatin’s molecular mechanism: aspects of specificity and regulation elucidated. J Cell Biochem 2005; 96:242–61. 133. Mizejewski GJ. Role of integrins in cancer: survey of expression patterns. Proc Soc Exp Biol Med 1999; 222:124–38. 134. Senger DR, Claffey KP, Benes JE, et al. Angiogenesis promoted by vascular endothelial growth factor: regulation through alpha1beta1 and alpha2beta1 integrins. Proc Natl Acad Sci USA 1997; 94:13612–17. 135. Bouvard D, Brakebusch C, Gustafsson E, et al. Functional consequences of integrin gene mutations in mice. Circulation Res 2001; 89:211–23.

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HIF-1 Inhibitors Giovanni Melillo Developmental Therapeutics Program, SAIC Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland, U.S.A.

INTRODUCTION Hypoxia inducible factor 1 (HIF-1) is a transcription factor that controls the response of mammalian cells to oxygen deprivation. HIF-1 has been implicated in a variety of pathophysiological conditions, including development, inflammation and cancer. Thus, its role as a mediator of fundamental biological processes and the potential modulation of its activity for therapeutic purposes have attracted considerable interest (1,2). Several aspects of the involvement of HIF-1 in human cancer should be emphasized: (1) The regulation of HIF-1a by hypoxia, a common feature of solid tumors known to profoundly affect tumor biology, response to treatment, and patients' prognosis; (2) the influence of genetic alterations, e.g., Von Hippel-Lindau tumor supressor (VHL), PTEN, and p53, frequently detected in human cancers, on HIF-1a expression and function; (3) the induction of HIF-1a accumulation by RTK signaling pathways frequently dysregulated in human cancers; (4) the expression of HIF-1 in multiple cellular components that are present in the tumor microenvironment, including stromal infiltrating cells and endothelial cells. These features legitimate HIF-1 as a crucial player in cancer development and progression and as a potential target for the development of novel therapeutics. Indeed, the interest in HIF-1 is documented by the exponentially increasing number of papers published on this topic over the past decade and by the growing number of academic groups and pharmaceutical industries actively engaged in the identification of novel strategies aimed to inhibit HIF-1 in human cancer. However, many questions still remain unanswered regarding the distinct role of HIF in different tumor types and the best way to achieve HIF inhibition in cancer patients. It can be anticipated that over the next few years more inhibitors will be identified and will approach the preclinical and clinical arena for further testing. A rational plan to validate HIF-1 inhibitors in preclinical models and test them in early clinical trials is warranted, so that this exciting and promising avenue for cancer therapy may yield positive results. HIF-1 AS A TARGET FOR CANCER THERAPY Regulation of HIF-1 Expression HIF-1 is a basic helix–loop–helix PAS transcription factor composed of two subunits, a and b. The b-subunit, also known as aryl hydrocarbon receptor nuclear translocator (ARNT), is constitutively expressed in an oxygen independent fashion, and is also involved in other transcriptional pathways, e.g., by dimerizing with the dioxin receptor, AhR [reviewed in (3,4)]. In contrast, the a-subunit, of which two, HIF-1a and HIF-2a, are best characterized, is rapidly degraded under normoxic conditions but accumulates under low oxygen levels. The mechanism by 283

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which the a-subunit is degraded has been elegantly elucidated over the past few years. A family of enzymes, called PHDs, mediates hydroxylation of two proline residues of HIF-a in a reaction that requires O2, Fe2þ, and 2-oxyglutarate. Upon hydroxylation, the a-subunit is recognized by the product of VHL, pVHL, which functions as the recognition component of an E3 ligase that mediates ubiquitylation and proteasomal degradation of HIF-a. As mentioned above, mutations of pVHL, which are frequently detected in patients with clear cell renal carcinoma, cause an accumulation of HIF-a under normoxic conditions due to impairment of its degradation. However, an increasing number of genetic alterations frequently implicated in human cancers have been associated with dysregulation of HIF-a. In addition to gain-of-function mutations, such as v-src and Ras, loss-of-function alterations, including PTEN, p53, TSC, succinate dehydrogenase, fumarate hydratase, and PML, have been implicated in the accumulation of HIF-a under nonhypoxic conditions (3). In addition, growth factor-dependent signaling pathways frequently dysregulated in human cancers, including EGF, IGF, and Her2/ Neu, have also been implicated in the induction of HIF-1a under normoxic conditions by activation of the PI3K/AKT/mTOR and MAPK pathways, further emphasizing the complexity of HIF-1a regulation and its involvement in fundamental processes of cancer progression. HIF-1 and Gene Expression The list of genes and functions that are controlled by HIF is constantly expanding. The impact that HIF may have in human cancer is highlighted by the function of genes that are controlled by HIF and that profoundly affect the behavior of cancer cells. HIF-inducible genes control tumor metabolism, angiogenesis, cell survival, and migration/invasion, all of which are hallmarks of cancer progression (5). HIF plays a crucial role in the induction of angiogenesis, a feature that may have important therapeutic implications for HIF inhibitors. Vascular endothelial growth factor (VEGF), the best characterized angiogenic factor, is transcriptionally induced by HIF via an HRE present in its promoter, although hypoxia may also control VEGF mRNA stability and/or its translation, and HIF-independent pathways have also been identified (6). A critical pathway controlled by HIF is aerobic glycolysis, a key feature of cancer cells, which have high level of glycolysis even in the presence of oxygen. Indeed, HIF-1 induces a coordinate upregulation of genes involved in glucose metabolism and glycolysis. Finally, in the past few years, a critical role of HIF in the control of cell migration and invasion has also been elucidated by induction of genes such as CXCR4 (7) and lysyl oxidase (8), implicated to a different extent in invasion and metastasis. Each of these genes could represent a viable therapeutic target as well as be affected by strategies targeting HIF-1. HIF-1 Expression in Human Cancer A number of experimental models have confirmed that HIF-1 plays a critical role in tumor formation. However, controversial evidence has also been generated depending on the tumor model used, which has led to some early skepticism as to whether HIF is a good target for therapy (9,10). HIF is overexpressed in a variety of human cancers and its expression is associated with poor prognosis and poor response to treatment (9,11). The pattern of HIF-1 staining that is detected in tumor tissue highlights the involvement of different pathways of HIF activation

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in cancer patients. Indeed, expression of HIF can be detected in perinecrotic areas of hypoxia, where the role of oxygen in its regulation is predominant. However, HIF has also been detected in well-oxygenated areas, consistent with its regulation by growth factors and genetic alterations, as well as in stromal infiltrating cells, which raises the question of the contribution of this cellular component to tumor growth and response to therapy. HIF INHIBITORS: MECHANISMS OF ACTION The majority of HIF-1 inhibitors identified so far can be classified as }nonselective,} as they target signaling molecules or pathways that affect multiple cellular functions (9,10,12). With this caveat in mind, in the next sections of this chapter, information is provided regarding the mechanism of action of some of the HIF inhibitors identified so far. In particular, emphasis is placed on compounds that are relevant to the clinical setting, either because they are in clinical development or because they target pathways for which inhibitors are available (Fig. 1). Inhibitors of Signaling Pathways Consistent with the redundant involvement of HIF-1 in multiples RTK-mediated signaling pathways that are dysregulated in human cancers, several novel inhibitors that have approached the preclinical and clinical arena also have the potential or have indeed been shown to inhibit HIF-1 or HIF-dependent functions. This finding raises the question as to how, if at all, RTK inhibitors (RTKI) can be used in the clinic as HIF-1 inhibitors. There are at least two implications of HIF-1 inhibition by RTKI.

FIGURE 1 Potential mechanisms of action of HIF-1 inhibitors. Abbreviations: EGFR, epidermal growth factor receptor; HDAC, histone deacetylase; HIF, hypoxia inducible factor; VHL, Von-Hippel Lidau tumor supressor gene; 2ME2, 2-methoxyestradiol.

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The first is that inhibition of HIF-1 may play a role in the response of patients to therapy with RTKI. This possibility is supported by findings indicating that HIF-1 is downstream of a number of signaling pathways targeted by RTKI, and, in a cell-type specific fashion, HIF-1 may be a critical mediator of these dysregulated pathways. The second implication is that inhibition of HIF-1 may become a valuable biomarker of activity of RTKI, which can be validated in relevant preclinical models to be then incorporated in early clinical trials. Inhibitors of the mTOR Pathway The mTOR pathway has been implicated in the growth factor-dependent induction of HIF-1a translation (13) as well as in HIF-1a degradation (14). It is then conceivable that mTOR inhibitors currently in clinical development might inhibit HIF-1 and have an impact on downstream pathways, including angiogenesis. Indeed, evidence has been provided that tirosel/temsirolimus (Wyeth, Madison, New Jersey, U.S.A.), a novel mTOR inhibitor in clinical development, inhibited hypoxic dependent induction of HIF-1 and VEGF production (15) and rhabdomyosarcoma xenograft growth by an antiangiogenic mechanism dependent on mTOR/HIF-1a/VEGF signaling (16). Importantly, in a mouse model of AKT1-dependent prostate intraepithelial neoplasia HIF-1a targets, including genes encoding most glycolytic enzymes, constituted the dominant transcriptional response to AKT activation and mTOR inhibition (17) and loss of VHL sensitized kidney cancer cells to the mTOR inhibitor tirosel/temsirolimus in vitro and in mouse models (18). Thus, HIF-1a might be a biomarker of response in cancers in which the mTOR pathway is dysregulated and may also represent an attractive biomarker that could facilitate preclinical and early clinical development of mTOR inhibitors. EGFR Inhibitors HIF-1a is induced upon stimulation of the epidermal growth factor receptor (EGFR) pathway (13). Accordingly, EGFR tyrosine kinase inhibitors, including Tarceva and Iressa currently used in the clinic, inhibit VEGF expression by both HIF-1-dependent and independent mechanisms (19), which may also have implications for the induction of apoptosis by these agents (20). Cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor, also inhibits HIF-1a levels in A431 epidermoid carcinoma cells (21). Inhibitors of AKT AKT is a critical signaling molecule, mediating RTK-dependent pathways that may ultimately affect HIF-1 activity. Thus, AKT has been implicated in the mechanism of action of many small molecule inhibitors of HIF-1 described in the literature (22–26). Little evidence has been provided so far that this is a feasible approach in preclinical models, but obviously AKT is an attractive target for cancer therapy and HIF-1 may represent one of many downstream targets affected by AKT inhibition. Other signaling pathways that are frequently dysregulated in human cancers have been implicated in HIF-1a regulation, and inhibitors of these pathways may potentially block HIF-1a accumulation. In particular, evidence has been provided that the Her2/Neu (27), c-KIT (28), and BCR/ABL (29) pathways are implicated in the induction of HIF-1a and VEGF expression in breast cancer, lung cancer, and leukemic cell lines, respectively.

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Inhibitors of Protein Accumulation Inhibition of HIF-1a by the agents described in this section has been associated with biochemical inhibition of targets that are known to be affected by these compounds. Topoisomerase I Poisons Topotecan, a topoisomerase I poison, is a small molecule inhibitor of HIF-1 identified in a cell-based reporter screen of the NCI Diversity set using U251-HRE cells, which express a luciferase reporter gene under control of three copies of a canonical hypoxia responsive element (30). Several camptothecin analogs have since then been identified as HIF-1 inhibitors, and HIF-1 inhibition seems to be a common property of this class of compounds. Topotecan inhibits HIF-1a translation by a mechanism independent from DNA replication–dependent DNA damage and proteasome function (31). Importantly, daily administration of topotecan inhibited HIF-1a expression in xenografts experiments, which was associated with the inhibition of angiogenesis and tumor growth (32). These results have led to the implementation of a clinical trail that is currently ongoing at the NCI (http://www.clinicaltrials.gov/ct/show/NCT00182676), where the effect of topotecan on HIF-1a expression in tumor tissue is being evaluated in patients with metastatic cancers. This pilot study will provide useful information regarding the potential to inhibit HIF-1a expression in tumor tissue using small molecules. Microtubule-Targeting Agents 2-Methoxyestradiol (2ME2), a novel antitumor and antiangiogenic agent, which is currently in clinical development stage, was found to inhibit tumor growth and angiogenesis at concentrations that efficiently disrupt tumor microtubules (MTs) in vivo (33). In addition, 2ME2 downregulated HIF-1a by inhibiting its translation and blocked HIF-1-induced transcriptional activation of VEGF expression. 2ME2/ tubulin interaction was required for HIF-a downregulation. Interestingly, early clinical trials of this compound have shown that 2ME2 is not associated with common toxicities observed with other microtubule-targeting agents; thus, inhibition of HIF-1 and angiogenesis may be an important mechanism contributing to its biological activity. Hsp90 Inhibitors The benzoquinone ansamycin geldanamycin, an Hsp90-specific inhibitor, was found to inhibit HIF-1a protein accumulation by a mechanism involving its degradation in a proteasome-dependent but VHL-independent fashion (34,35). HIF-1a is one of many Hsp90 client proteins and it is unclear how much inhibition of Hsp90 may be associated with downregulation of HIF-1-target functions. However, analogs of geldanamycin, including 17-AAG and 17-DMAG, are currently in clinical trials for cancer therapy and HIF-1 inhibition may be potentially contributing to the therapeutic activity and/or may represent a valuable biomarker of activity of these compounds. Hsp90 has also been implicated in the mechanism of action of other HIF-1 inhibitors, including radicicol (36) and the farnesyltransferase inhibitor SCH66336 (37). Histone Deacetylase Inhibitors Several mechanisms of action have been suggested for the activity of histone deacetylase (HDAC) inhibitors, a class of compounds in clinical development, on

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HIF-1a degradation. In particular, HDACIs may induce the proteasomal degradation of HIF-1a by a mechanism that is independent of VHL and is secondary to a disruption of the HSP70/HSP90 axis function (38). Alternatively, it has been shown that class II HDAC4 and HDAC6 were associated with the HIF-1a protein and may directly affect its degradation (39). These results suggest that inhibitors of class II HDACs might be used to target HIF-1a in human cancers. Whether or not HIF-1a inhibition contributes to the therapeutic activity observed with the administration of HDAC inhibitors remains to be determined. Inhibitors of DNA Binding An attractive strategy for the inhibition of transcription factors is blocking the DNA binding to specific recognition sequences. Pioneer work in this area has been conducted by Peter Dervan and his group, who have designed synthetic polyamides that can specifically target consensus sequences recognized by transcription factors. Indeed, a synthetic polyamide that specifically inhibits HIF-1 DNA binding has been designed and found to inhibit, as postulated, HIF-1 transcriptional activity (40). A limitation of polyamides as therapeutic reagents may be their poor cellular permeability and diffusion in tumor tissue, although they offer significant advantages for their potential increased specificity. Echinomycin, a small molecule that binds DNA in a sequence-specific fashion, has been identified in a cell-free screen aimed to identify small molecule inhibitors of HIF-1 DNA binding. Echinomycin inhibited HIF-DNA binding but not the binding of AP1 or NF-kB to cognate DNA-binding sites, suggesting a relative degree of sequence specificity (41). Since the HRE-binding site may also overlap with an E-box sequence (CACGTG), echinomycin was also found to inhibit binding of myc to the E-box, a feature that might have potential therapeutic implications. Inhibitors of HIF-1 Transcriptional Activity The transcriptional activity of HIF-1 is mediated by two domains, N-TAD and C-TAD. The C-TAD binds to CBP/p300 for maximal transcriptional activity, and is modulated by posttranslational modifications, including hydroxylation of Asn 803. Chetomin, a small molecule that inhibits HIF-1 binding to CBP, was identified in a screen aimed to identify inhibitors of HIF-1 transcriptional activity (42). This molecule was found to be active in in vitro and in vivo models, which provided proof of principle that HIF inhibition is a viable therapeutic strategy. However, the clinical development of chetomin for cancer therapy appears to be hampered by poor pharmacological properties. Interestingly, a recent evidence has emphasized that inhibition of the proteasome function, which blocks HIF-1a protein degradation, is also associated with inhibition of HIF-1 transcriptional activity by a mechanism that involves the TAD of HIF-1 (43). Velcade, an inhibitor of proteasome function, which is approved for therapy of myeloma, is currently being tested in several tumor types, and it will be interesting to see whether HIF-1 inhibition may be part of its therapeutic activity in tumors overexpressing HIF-1a. Miscellaneous HIF-1 inhibitors are continuously discovered and reported in the literature. However, in many cases, a clear mechanism of action of HIF-1 inhibition is neither reported nor identified.

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PX-478 is a potent antitumor agent and is found to be active in many xenograft models (44). PX-478 inhibits HIF-1a protein accumulation by an unknown mechanism of action. Interestingly, its activity in tumor xenografts seems to be associated with HIF-1a levels (44). Inhibition of HIF-1a protein expression following treatment with PX-478 has been demonstrated in animal models, and this agent will soon be tested in clinical trials as HIF-1 inhibitor in solid tumors. YC-1 is a cyclic GMP activator, known for its antiplatelets and vasodilatory effects. YC-1 inhibits HIF-1a protein accumulation by a mechanism that appears to be independent from the activation of cGMP (45). YC-1 was active in animal models and inhibited HIF-1a expression in tumor tissue; thus, it may be soon tested as an anticancer agent. Many different agents have been implicated in HIF-1 inhibition. Among these, thioredoxin inhibitors were originally found to inhibit HIF-1 protein accumulation (46), although recent evidence indicates that these agents may also inhibit transcriptional activity (47). Curcumin, a component of the yellow spice turmeric, inhibits HIF-1a protein accumulation by several mechanisms, including degradation of HIF-1b, which may be potentially associated with inhibition of HIF-1 activity (48). Gene Therapy Genetic approaches to target HIF-1a expression and function are an attractive strategy to inhibit HIF-1 in human cancers and have been tested in animal models with promising activity. In particular, expression of therapeutic genes under control of HRE, adenoviruses engineered to be expressed under hypoxic conditions, and antisense and siRNA approaches have all been tested and found to be somewhat active in different tumor models (49–54). Although targeting the hypoxic tumor is an attractive therapeutic strategy, the issue of delivery is still largely unresolved and currently hampers the potential application of this approach. Natural Products Many natural products have been identified and found to inhibit HIF-1 protein expression and function (55). In most instances, the exact mechanism of action of these compounds has not been elucidated and the activity has only been shown in cell culture and has not been validated in vivo. Natural products may have novel and interesting mechanisms of action in inhibiting HIF-1. Further studies will be required to determine if any of the agents identified so far has the potential to be used as therapeutic agent for cancer therapy. PRECLINICAL DEVELOPMENT AND TRANSLATIONAL END POINTS The development of molecular targeted agents requires a rationally designed plan to validate the activity on the intended target and to implicate this effect in a meaningful therapeutic activity. Unlike cytotoxic agents, whose development has been largely based on efficacy studies in multiple xenograft models, the development of molecular targeted agents requires preclinical models tailored to the specific agent under investigation. Several approaches have been described to validate the activity of HIF-1 inhibitors. Human cancer cell lines engineered to express the luciferase reporter

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gene under control of hypoxia response elements have been established (32). These cell lines have been used in xenograft and orthotopic models to monitor the activity of HIF-1 inhibitors on their target. The advantage of this approach is that luciferase can be measured in a noninvasive fashion giving the opportunity to serially monitor the effect of an agent on a functional basis. More elegant models based on noninvasive imaging of reporter genes may be anticipated in the future, and they should provide a valuable tool for validating the activity of HIF inhibitors. Since inhibition of HIF-1 may be associated with inhibition of angiogenesis and tumor metabolism, tissue endpoints reflecting these activities have been developed to monitor the effect of HIF inhibitors. Evaluation of tissue endpoints, documenting the functional inhibition of the HIF pathway, is essential to validate the activity of HIF inhibitors and to better understand how to use these agents in the clinic. Tissue endpoints can be easily measured in animal models, and careful analysis of these endpoints should be encouraged and warranted for the development of HIF inhibitors. Analysis of HIF-1a protein levels by IHC or western blot, mRNA expression of HIF-1 target genes by real-time PCR, and surrogate markers such as MVD or CAIX, have all been proposed and applied (32,44,45). A potential limitation of translating these pharmacodynamic endpoints to the clinic is that tissue must be acquired from patients, which is not always feasible or applicable. An alternative approach that could overcome these limitations and find a broader application is the use of imaging techniques assessing functional inhibition of HIF-1. Two main strategies have been used so far: 18FDG-PET, which provides an indication of tumor metabolism, and DCE-MRI, which reflects blood flow and angiogenesis (56–58). The rationale for using tumor metabolism as a readout of HIF-1 activity is based on the coordinate transcriptional regulation of glycolytic enzymes by HIF-1, which is consistent with the possibility that inhibition of HIF is associated with a decrease of PET signal. The application of DCEMRI relies on the assumption that inhibition of HIF may be associated with meaningful inhibition of angiogenesis. Again this is largely supported by the direct induction of a number of angiogenic factors by HIF, including, but not limited to, VEGF. However, these techniques are not widely available for application in preclinical models, and further studies are required to fully elucidate the association between inhibition of HIF and functional results. EARLY CLINICAL DEVELOPMENT Many agents have been shown to inhibit HIF-1a protein expression and function in cell culture models. Several of the agents identified have also been shown to inhibit HIF-1 in animal models, which has been, in turn, associated with antitumor and antiangiogenic activities. However, many questions remain to be answered as to the potential application of these results to the clinical setting. As discussed in the previous paragraph, validation of these agents in relevant preclinical models is essential for further clinical development. As therapeutic efficacy cannot be used as a reliable readout of HIF inhibition, more appropriate translational endpoints should be defined and used in preclinical and early clinical trials to validate the activity of HIF-1 inhibitors. Indeed, early clinical trials of HIF-1 inhibitors should emphasize the activity of the investigational agent on HIF-1 expression and/or function according to the known or proposed mechanism of action of the compound. Imaging techniques should also be developed to measure inhibition of

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HIF-1 or HIF-1-target functions, including, but not limited to, angiogenesis and tumor metabolism. Studies on tumor tissue are warranted to validate the activity of HIF-1 inhibitors on meaningful biological endpoints that may then be correlated with clinical benefit. Although the use of HIF-1 inhibitors in the clinical setting is still in its early phase of development, a strong scientific rationale has been provided for testing these agents in clinical trails aimed to validate the activity on HIF-1 and HIF-1target functions. However, it is plausible that HIF-1 inhibitors may have limited activity when used as single agents (59). Importantly, evidence has been provided that HIF-1 may contribute to resistance to chemotherapy (60) and radiation therapy (61,62), further suggesting that HIF-1 inhibitors may find a valuable application in combination with currently available therapeutic strategies. Indeed, combination therapies should be tested in preclinical models and rapidly translated to relevant clinical models. The rational development of combination strategies with conventional therapeutic approaches, i.e., chemotherapy and radiation therapy, as well as with novel molecular targeted therapies is warranted to fully exploit the potential of this novel and exciting area of developmental therapeutics. ACKNOWLEDGMENTS I would like to thank all the members of my laboratory for helpful discussions. My apologies to the many authors whose work describing HIF-1 inhibitors is not referenced, which is solely due to space limitations. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Melillo G. HIF-1: a target for cancer, ischemia and inflammation–too good to be true? Cell Cycle 2004; 3:154–5. Semenza GL. Involvement of hypoxia-inducible factor 1 in pulmonary pathophysiology. Chest 2005; 128:592S–4S. Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003; 3:721–32. Metzen E, Ratcliffe PJ. HIF hydroxylation and cellular oxygen sensing. Biol Chem 2004; 385:223–30. Semenza GL, Shimoda LA, Prabhakar NR. Regulation of gene expression by HIF-1. Novartis Found Symp 2006; 272:2–8. Hirota K, Semenza GL. Regulation of angiogenesis by hypoxia-inducible factor 1. Crit Rev Oncol Hematol 2006; 59:15–26. Schioppa T, Uranchimeg B, Saccani A, et al. Regulation of the chemokine receptor CXCR4 by hypoxia. J Exp Med 2003; 198:1391–402. Erler JT, Bennewith KL, Nicolau M, et al. Lysyl oxidase is essential for hypoxiainduced metastasis. Nature 2006; 440:1222–6. Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003; 3:721–32. Melillo G. Inhibiting hypoxia-inducible factor 1 for cancer therapy. Mol Cancer Res 2006; 4:601–5. Zhong H, De Marzo AM, Laughner E, et al. Overexpression of hypoxia-inducible factor 1a in common human cancers and their metastases. Cancer Res 1999; 59:5830–5. Giaccia A, Siim BG, Johnson RS. HIF-1 as a target for drug development. Nat Rev Drug Discov 2003; 2:803–11. Zhong H, Chiles K, Feldser D, et al. Modulation of hypoxia-inducible factor 1a expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/ AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res 2000; 60:1541–5.

292 14. 15. 16. 17. 18. 19. 20. 21.

22. 23. 24. 25. 26. 27.

28. 29.

30. 31. 32.

Melillo

Hudson CC, Liu M, Chiang GG, et al. Regulation of hypoxia-inducible factor 1a expression and function by the mammalian target of rapamycin. Mol Cell Biol 2002; 22:7004–14. Del Bufalo D, Ciuffreda L, Trisciuoglio D, et al. Antiangiogenic potential of the Mammalian target of rapamycin inhibitor temsirolimus. Cancer Res 2006; 66:5549–54. Wan X, Shen N, Mendoza A, Khanna C, Helman LJ. CCI-779 inhibits rhabdomyosarcoma xenograft growth by an antiangiogenic mechanism linked to the targeting of mTOR/Hif-1a/VEGF signaling. Neoplasia 2006; 8:394–401. Majumder PK, Febbo PG, Bikoff R, et al. mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways. Nat Med 2004; 10:594–601. Thomas GV, Tran C, Mellinghoff IK, et al. Hypoxia-inducible factor determines sensitivity to inhibitors of mTOR in kidney cancer. Nat Med 2006; 12:122–7. Pore N, Jiang Z, Gupta A, Cerniglia G, Kao GD, Maity A. EGFR tyrosine kinase inhibitors decrease VEGF expression by both hypoxia-inducible factor (HIF)-1-independent and HIF-1-dependent mechanisms. Cancer Res 2006; 66:3197–204. Peng X, Karna P, Cao Z, Jiang B, Zhou M, Yang L. Cross-talk between epidermal growth factor receptor and HIF-1 signal pathways increases resistance to apoptosis by upregulating survivin gene expression. J Biol Chem 2006; 281:25903–14. Luwor RB, Lu Y, Li X, Mendelsohn J, Fan Z. The antiepidermal growth factor receptor monoclonal antibody cetuximab/C225 reduces hypoxia-inducible factor-1a, leading to transcriptional inhibition of vascular endothelial growth factor expression. Oncogene 2005; 24:4433–41. Cao Z, Fang J, Xia C, Shi X, Jiang BH. Trans-3,4,5 -trihydroxystibene inhibits hypoxiainducible factor 1a and vascular endothelial growth factor expression in human ovarian cancer cells. Clin Cancer Res 2004; 10:5253–63. Fang J, Cao Z, Chen YC, Reed E, Jiang BH. 9-beta-D-arabinofuranosyl-2-fluoroadenine inhibits expression of vascular endothelial growth factor through hypoxia-inducible factor-1 in human ovarian cancer cells. Mol Pharmacol 2004; 66:178–86. Fang J, Xia C, Cao Z, Zheng JZ, Reed E, Jiang BH. Apigenin inhibits VEGF and HIF-1 expression via PI3K/AKT/p70S6K1 and HDM2/p53 pathways. FASEB J, 2005; 19:342–53. Tan C, de Noronha RG, Roecker AJ, et al. Identification of a novel small-molecule inhibitor of the hypoxia-inducible factor 1 pathway. Cancer Res 2005; 65:605–12. Zhang Q, Tang X, Lu QY, Zhang ZF, Brown J, Le AD. Resveratrol inhibits hypoxiainduced accumulation of hypoxia-inducible factor-1a and VEGF expression in human tongue squamous cell carcinoma and hepatoma cells. Mol Cancer Ther 2005; 4:1465–74. Laughner E, Taghavi P, Chiles K, Mahon PC, Semenza GL. HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1a (HIF-1a) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Mol Cell Biol 2001; 21:3995–4004. Litz J, Krystal GW. Imatinib inhibits c-Kit-induced hypoxia-inducible factor-1a activity and vascular endothelial growth factor expression in small cell lung cancer cells. Mol Cancer Ther 2006; 5:1415–22. Mayerhofer M, Valent P, Sperr WR, Griffin JD, Sillaber C. BCR/ABL induces expression of vascular endothelial growth factor and its transcriptional activator, hypoxia inducible factor-1a, through a pathway involving phosphoinositide 3-kinase and the mammalian target of rapamycin. Blood 2002; 100:3767–75. Rapisarda A, Uranchimeg B, Scudiero DA, et al. Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway. Cancer Res 2002; 62:4316–24. Rapisarda A, Uranchimeg B, Sordet O, Pommier Y, Shoemaker RH, Melillo G. Topoisomerase I-mediated inhibition of hypoxia-inducible factor 1: mechanism and therapeutic implications. Cancer Res 2004; 64:1475–82. Rapisarda A, Zalek J, Hollingshead M, et al. Schedule-dependent inhibition of hypoxia-inducible factor-1a protein accumulation, angiogenesis, and tumor growth by topotecan in U251-HRE glioblastoma xenografts. Cancer Res 2004; 64:6845–8.

HIF-1 Inhibitors

33. 34. 35. 36.

37. 38. 39. 40. 41. 42. 43. 44. 45. 46.

47.

48. 49. 50. 51. 52.

293

Mabjeesh NJ, Escuin D, LaVallee TM, et al. 2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF. Cancer Cell 2003; 3:363–75. Mabjeesh NJ, Post DE, Willard MT, et al. Geldanamycin induces degradation of hypoxia-inducible factor 1a protein via the proteosome pathway in prostate cancer cells. Cancer Res 2002; 62:2478–82. Isaacs JS, Jung YJ, Mimnaugh EG, Martinez A, Cuttitta F, Neckers LM. Hsp90 regulates a von Hippel Lindau-independent hypoxia-inducible factor-1a -degradative pathway. J Biol Chem 2002; 277:29936–44. Hur E, Kim HH, Choi SM, et al. Reduction of hypoxia-induced transcription through the repression of hypoxia-inducible factor-1a/aryl hydrocarbon receptor nuclear translocator DNA binding by the 90-kDa heat-shock protein inhibitor radicicol. Mol Pharmacol 2002; 62:975–82. Han JY, Oh SH, Morgillo F, et al. Hypoxia-inducible factor 1a and antiangiogenic activity of farnesyltransferase inhibitor SCH66336 in human aerodigestive tract cancer. J Natl Cancer Inst 2005; 97:1272–86. Kong X, Lin Z, Liang D, Fath D, Sang N, Caro J. Histone deacetylase inhibitors induce VHL and ubiquitin-independent proteasomal degradation of hypoxia-inducible factor 1a. Mol Cell Biol 2006; 26:2019–28. Qian DZ, Kachhap SK, Collis SJ, et al. Class II histone deacetylases are associated with VHL-independent regulation of hypoxia-inducible factor 1a. Cancer Res 2006; 66:8814–21. Olenyuk BZ, Zhang GJ, Klco JM, Nickols NG, Kaelin WG, Jr., Dervan PB. Inhibition of vascular endothelial growth factor with a sequence-specific hypoxia response element antagonist. Proc Natl Acad Sci USA 2004; 101:16768–73. Kong D, Park EJ, Stephen AG, et al. Echinomycin, a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity. Cancer Res 2005; 65:9047–55. Kung AL, Zabludoff SD, France DS, et al. Small molecule blockade of transcriptional coactivation of the hypoxia-inducible factor pathway. Cancer Cell 2004; 6:33–43. Kaluz S, Kaluzova M, Stanbridge EJ. Proteasomal inhibition attenuates transcriptional activity of hypoxia-inducible factor 1 (HIF-1) via specific effect on the HIF-1a C-terminal activation domain. Mol Cell Biol 2006; 26:5895–907. Welsh S, Williams R, Kirkpatrick L, Paine-Murrieta G, Powis G. Antitumor activity and pharmacodynamic properties of PX-478, an inhibitor of hypoxia-inducible factor1a. Mol Cancer Ther 2004; 3:233–44. Yeo EJ, Chun YS, Cho YS, et al. YC-1: a potential anticancer drug targeting hypoxiainducible factor 1. J Natl Cancer Inst 2003; 95:516–25. Welsh SJ, Williams RR, Birmingham A, Newman DJ, Kirkpatrick DL, Powis G. The thioredoxin redox inhibitors 1-methylpropyl 2-imidazolyl disulfide and pleurotin inhibit hypoxia-induced factor 1a and vascular endothelial growth factor formation. Mol Cancer Ther 2003; 2:235–43. Jones DT, Pugh CW, Wigfield S, Stevens MFG, Harris AL. Novel thioredoxin inhibitors paradoxically increase hypoxia-inducible factor-a expression but decrease functional transcriptional activity, DNA binding, and degradation. Clin Cancer Res 2006; 12:5384–94. Choi H, Chun YS, Kim SW, Kim MS, Park JW. Curcumin inhibits hypoxia-inducible factor-1 by degrading aryl hydrocarbon receptor nuclear translocator: A mechanism of tumor growth inhibition. Mol Pharmacol 2006; 70:1664–71. Dachs GU, Patterson AV, Firth JD, et al. Targeting gene expression to hypoxic tumor cells. Nat Med 1997; 3:515–20. Sun X, Kanwar JR, Leung E, Lehnert K, Wang D, Krissansen GW. Gene transfer of antisense hypoxia inducible factor-1a enhances the therapeutic efficacy of cancer immunotherapy. Gene Ther 2001; 8:638–45. Cuevas Y, Hernandez-Alcoceba R, Aragones J, et al. Specific oncolytic effect of a new hypoxia-inducible factor-dependent replicative adenovirus on von Hippel-Lindaudefective renal cell carcinomas. Cancer Res 2003; 63:6877–84. Binley K, Askham Z, Martin L, et al. Hypoxia-mediated tumour targeting. Gene Ther 2003; 10:540–9.

294 53. 54. 55. 56. 57. 58. 59. 60. 61. 62.

Melillo

Zhang X, Kon T, Wang H, et al. Enhancement of hypoxia-induced tumor cell death in vitro and radiation therapy in vivo by use of small interfering RNA targeted to hypoxia-inducible factor-1a. Cancer Res 2004; 64:8139–42. Chang Q, Qin R, Huang T, Gao J, Feng Y. Effect of antisense hypoxia-inducible factor 1a on progression, metastasis, and chemosensitivity of pancreatic cancer. Pancreas 2006; 32:297–305. Nagle DG, Zhou YD. Natural product-based inhibitors of hypoxia-inducible factor-1 (HIF-1). Curr Drug Targets 2006; 7:355–69. Jordan BF, Runquist M, Raghunand N, et al. Dynamic contrast-enhanced and diffusion MRI show rapid and dramatic changes in tumor microenvironment in response to inhibition of HIF-1a using PX-478. Neoplasia 2005; 7:475–85. Robey IF, Lien AD, Welsh SJ, Baggett BK, Gillies RJ. Hypoxia-inducible factor-1a and the glycolytic phenotype in tumors. Neoplasia 2005; 7:324–30. Jordan BF, Black K, Robey IF, Runquist M, Powis G, Gillies RJ. Metabolite changes in HT-29 xenograft tumors following HIF-1a inhibition with PX-478 as studied by MR spectroscopy in vivo and ex vivo. NMR Biomed 2005; 18:430–9. Li L, Lin X, Shoemaker AR, Albert DH, Fesik SW, Shen Y. Hypoxia-inducible factor-1 inhibition in combination with temozolomide treatment exhibits robust antitumor efficacy in vivo. Clin Cancer Res 2006; 12:4747–54. Brown LM, Cowen RL, Debray C, et al. Reversing hypoxic cell chemoresistance in vitro using genetic and small molecule approaches targeting hypoxia inducible factor. Mol Pharmacol 2005; 69:411–18. Moeller BJ, Cao Y, Li CY, Dewhirst MW. Radiation activates HIF-1 to regulate vascular radiosensitivity in tumors: role of reoxygenation, free radicals, and stress granules. Cancer Cell 2004; 5:429–41. Moeller BJ, Dreher MR, Rabbani ZN, et al. Pleiotropic effects of HIF-1 blockade on tumor radiosensitivity. Cancer Cell 2005; 8:99–110.

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Antivascular Agents Jane Robertson Global Oncology Research and Development, AstraZeneca Pharmaceuticals, Aderley Park, Macclesfield, U.K.

INTRODUCTION All tumors need to generate their own blood supply in order to obtain sufficient oxygen and nutrients to grow beyond a volume of approximately 1 mm3 (1). They achieve this through the complex processes of angiogenesis, whereby endothelial cells proliferate in response to growth factors and invade the basal lamina, resulting in the budding of new vessels from the existing vasculature (2–4). The endothelial cells of tumor blood vessels are attractive targets for drug development because of their pivotal role in cancer cell survival, growth, and metastasis, and because they are more genetically stable, and potentially less likely to develop resistance to therapeutic agents than the tumor cells themselves (4). Furthermore, because endothelial cell targets are different from those targeted by chemotherapy agents, the potential for combination treatment can be exploited. A number of compounds that target components of the angiogenesis pathways are in development [currently, the most important of these is the vascular endothelial growth factor (VEGF) signaling pathway], and these are described in Chapter 6. The present chapter focuses solely on the development of compounds that target and occlude the established tumor vasculature to interrupt the blood supply. Such compounds have previously been termed “antivascular agents” or “vascular targeting agents,” but it has recently been proposed that the most useful term to distinguish their mechanism of action from antiangiogenic compounds is “vascular disrupting agents” (VDAs) (5), and this term is used hereafter in this chapter. Tumor vasculature differs from normal vasculature in a number of ways: tumor vessels are immature, with incomplete pericyte coverage and increased tortuosity, permeability, and fragility compared to normal blood vessels (6,7). The high proliferative rate of tumor vascular endothelial cells relative to normal endothelial cells was initially identified as a possible target for selective cancer therapies over 20 years ago (8–11). Such targeted approaches are needed because cells in areas of tumors that are relatively distant from the supplying blood vessels are subject to a hypoxic and acidic microenvironment. In these conditions, there are local areas of necrosis, and increased likelihood of cellular resistance to radiotherapy and chemotherapy agents. The delivery of anticancer agents is suboptimal, which also renders conventional treatment approaches less effective (12). VDAs act rapidly after administration to initially occlude and then collapse the existing tumor blood vessels, effectively starving areas of tumors of their blood supply. The result is marked ischemia, necrosis, and hemorrhage in tumors (13–17). These effects are more marked in the central areas of tumors. In contrast, a thin, viable rim of tumor cells characteristically remains at the periphery, where the cells are nourished either by diffusion of nutrients from normal adjacent tissue or from host blood vessels in close proximity that are less susceptible to the agents than the tumor vessels (Fig. 1). 295

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Tumor pre-treatment Necrotic tumor cells

Viable tumor cells

VDA

Endothelial cells

Endothelial cells change shape and detach

Loss of endothelial cells from substratum leads to congestion of blood vessel

Tumor necrosis due to lack of nutrient supply

FIGURE 1 Mechanism of action of vascular disrupting agents (VDAs).

The clinical development program for VDAs has focused on combining these compounds with a variety of anticancer therapies including chemotherapy and radiotherapy, since the latter are more effective in the relatively well-oxygenated and nourished periphery of the tumor where cells are dividing quickly (17). For the same reason, combinations of VDAs with antiangiogenic agents are attractive treatment options since the majority of endothelial cell proliferation and angiogenesis occurs in these peripheral areas. The scheduling of the different modalities appears to be important; VDAs appear to be more effective if given following chemotherapy or radiotherapy than if given beforehand (14,18), possibly because if given beforehand, they may impair the delivery of chemotherapy agents, or because they remove some of the most chemo/radioresistant cells from the hypoxic center of the tumor. In preclinical studies, combinations of VDAs and chemotherapy/radiotherapy agents have not been shown to increase the toxicities of either regimen (14,18). Whereas antiangiogenesis agents may be expected to be most effective when started in the early stages of cancer, VDAs may be applicable to later stages and larger sized tumors (Fig. 2). Also, while chronic dosing of antiangiogenic agents may be required for a sustained antiangiogenic effect, VDAs have an acute effect, and intermittent dosing schedules appear effective. The two approaches may therefore be complementary, and combinations of antiangiogenic agents with VDAs are under investigation. APPROACHES TO THE DEVELOPMENT OF VASCULAR DISRUPTING AGENTS There have been two main approaches to the development of VDAs: a surface ligand targeted approach and the development of small molecules of different classes that enter cells to exert their effects. The compounds in these classes that are currently in clinical development are summarized in Figure 3. The surface-ligand targeted approach links molecules that induce endothelial cell damage or thrombosis to specific antigens on the luminal surface of tumor vessel endothelial cells by means of a monoclonal antibody. Selectivity for tumor endothelial cells is achieved by choosing antigens that are overexpressed on

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Inhibition of angiogenesis

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Blood vessel

Angiogenesis inhibitor

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Blood vessel

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FIGURE 2 Differences in the mechanism of action and antitumor effects between antiangiogenesis agents and vascular disrupting agents.

tumor vessels with respect to normal vessels, e.g., VEGF receptors (19), CD105 (endoglin) (20), ab integrins (21) or the fibronectin ED-B domain (22). Most of these agents have proven effective in preclinical models, but have yet to enter clinical trials. More recently, the cell adhesion molecule N-cadherin has been confirmed as a promising target for VDAs, as the antagonist compound ADH-1 (Adherex Technologies, Durham, North Carolina, U.S.A.) has reported positive Phase I results in the clinic (23,24). There are two major classes of small molecule VDAs: tubulin binding agents and flavonoids. The former bind to b-tubulin subunits to prevent polymerization and microtubule formation, thereby destabilizing the microtubule cytoskeleton, causing a change of shape in endothelial cells and their subsequent detachment from the basal lamina and apoptosis (25–27). Flavonoids cause cytokine release that induces vessel collapse. This chapter focuses specifically on the VDAs that are currently in clinical trials with reference to the development challenges they pose, and their emerging safety, tolerability and efficacy profiles. Tubulin-Binding Agents Microtubules are key components of the cytoskeleton and are vital for a number of essential cellular functions; they form the cytoskeleton of endothelial cells and are required for the maintenance of cellular shape, the formation of the mitotic spindle, and movement of organelles, receptors and transporters through the cytoplasm (28). Microtubules are composed of a- and b-tubulin heterodimers and are dynamic structures, growing by polymerization. Inhibition of microtubular function results in poor alignment of chromosomes during mitosis, mitotic arrest and apoptosis (29), and this forms the basis of the mechanism of action of a number of chemotherapy drugs, specifically the vinca alkaloids and taxanes that can be classified into three groups based on their tubulin-binding domains: the Vinca domain, the taxane domain and the colchicine site. At high doses, vinca

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Small molecules Tubulin binding agents Anti-mitotic and anti-vascular effects • TZT-1027 • ABT-751 Primary anti-vascular effect Combretastatin derivatives • CA4DP • AVE-8062 N-acetylcholinol prodrug • ZD6126 Second generation agents • MN-029 • NPI-2358 • Oxi-4503

Surface-ligand targeted agents Flavonoids DMXAA

Liposomes encapsulating cytotoxic effectors • EndoTAGTM -1 Anti N-cadherin • ADH-1 Exherin™

FIGURE 3 Vascular disrupting agents in clinical development.

alkaloids, taxanes and colchicine have all demonstrated antivascular effects in preclinical models because binding to tubulin alters the cytoskeleton and shape of endothelial cells. This shape change increases vascular permeability and ultimately leads to their detachment from the basal lamina, vessel wall collapse and occlusion of tumor blood flow (25–27). Because these compounds have a narrow therapeutic window with associated toxicities, especially in the gastrointestinal tract and peripheral nervous system, the antivascular effect is not achieved at doses that are tolerable for man. Successful attempts have been made to increase the therapeutic window either by enveloping taxanes within lipid complexes that target endothelial cells in order to deliver high doses to the tumor without high systemic exposure, or by generating novel tubulin-binding agents with a similar but more favorable safety profile compared to taxanes and vinca-alkaloids. MBT-1 (EndoTAG; MediGene, San Diego, California, U.S.A.) is an agent that envelopes paclitaxel within a cationic liposome with a high affinity for tumor vascular endothelial cells. Phase I trials are complete (30,31) and the compound is currently in phase II clinical development as a monotherapy in advanced breast cancer and in combination with gemcitabine in pancreatic cancer. Examples of the second approach are the intravenous (i.v.) dolastatin analogue TZT-1027 (Daiichi Sankyo, Ltd., Tokyo, Japan) and the oral sulfonamide ABT-751 (Abbott Laboratories, Abbott Park, Illinois, U.S.A.), both of which have a similar side effect profile to taxanes in terms of ileus and constipation, but ABT-751 caused markedly less leucopenia and TZT-1027 reported less neurotoxicity. TZT-1027 is currently in phase II development in patients with metastatic soft tissue sarcomas having completed a phase I program designed to identify the optimum dose and schedule for use as monotherapy and as a combination agent (32–36). ABT-751 underwent a similar phase I monotherapy and chemotherapycombination development program (37–42) but in its phase II studies the compound is predominantly used as a monotherapy agent in patients with refractory colorectal cancer, renal cancer, breast cancer and non–small cell lung cancer (NSCLC) (43–46). While the mechanism of action of these agents may be considered

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primarily as antimitotic rather than antivascular, antivascular effects have been demonstrated preclinically (47). The remainder of this section focuses on the tubulin-binding agents whose primary effects are antivascular rather than antimitotic. The first agent to exhibit antivascular effects at tolerable doses was combretastatin (48), a natural derivative of the bark of the South African willow tree (Combretum caffrum) that binds to the colchicine site of b-tubulin. A number of synthetic prodrugs of combretastatin were developed to overcome solubility issues. The most advanced in clinical development is Combretastatin A4 di-phosphate [CA4P (Zybrestatin; OxiGENE, Inc., Waltham, Massachusetts, U.S.A.). Others include Combretastatin A1 di-phosphate (Oxi4503; OxiGENE, Inc.) and AVE-8062 (Sanofi-Aventis, Paris, France). ZD6126 (Angiogene Pharmaceuticals, Ltd., Oxford, U.K.), a water-soluble pro-drug of N-acetylcolchinol, also binds to the colchicine site and has a similar mechanism of action but different pharmacokinetic properties than Combretastatin. Second-generation tubulin binding VDAs such as MN-029 (MediciNova, Inc., San Diego, California, U.S.A.), Oxi4503 (OxiGENE, Inc.), and NPI-2358 (Nereus Pharmaceuticals, San Diego, California, U.S.A.) have more recently moved into clinical development. Preclinical Studies In preclinical assays, CA4P, ZD6126 and AVE-8062 have demonstrated very similar antivascular and antitumor effects at doses that are less than one-tenth of the maximum tolerated dose (49). Demonstration of “proof of principle” for such compounds includes the evidence that they disrupt the tubulin cytoskeleton of tumor vascular endothelial cells leading to endothelial detachment and exposure of the basal lamina and extensive endothelial cell apoptosis (25–27). These compounds have also been shown to exhibit antitumor activity in a broad range of tumors in a manner in keeping with their proposed mechanism of action: reduction in blood flow and tumor vascular volume followed by widespread necrosis (50–53). In vitro, these effects occur selectively in tumors; treatment with CA4P or ZD6126 results in rapid changes in the three-dimensional shape of proliferating endothelial cells, but not of resting endothelial cells, and either no or limited effects are seen in normal tissues (50–53). Preclinically, the compounds have been shown to enhance the antitumor effects of chemotherapy agents, including cisplatin (51), paclitaxel (53), doxorubicin (54) and 5-FU (55), as well as radiotherapy (56–58), radioimmunotherapy (59); hyperthermia (60) and antiangiogenic agents (61). Clinical Studies Table 1 summarizes the main Phase I development programs of CA4P, ZD6126 and AVE-8062, the three tubulin-binding agents whose mechanism of action is primarily antivascular and are most advanced in terms of clinical development. Combretastatin A4 Diphosphate The initial phase I development of CA4P consisted of three monotherapy studies to investigate the safety, tolerability and pharmacokinetic profile of the compound and identify an optimum treatment schedule. The first study (62,63) investigated single doses given at 3 weekly intervals in 25 patients with advanced cancers. The study monitored effects on tumor blood flow using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and changes in plasma levels of cell

10 min i.v

10 min i.v

30 min i.v

20 min i.v

CA4P

ZD6126

AVE-8062

DMXAA

3 weekly Weekly Weekly (lower doses up to 3000 mg/m2 explored)

Every 3 wk Weekly for 3 wk out of 4 5 days every 3 wk Every 2 or 3 wk Weekly 5 days every 3 wk Weekly for 3 out of 4 wk 5 days every 3 wk 3 weekly

Schedule 2

3700 mg/m2 3700 mg/m2 1200 mg/m2

60 mg/m 68 mg/m2 52 mg/m2 80 mg/m2 20 mg/m2 1 g/m2/day 20 mg/m2 22 mg/m2 22 mg/m2

MTD

DLTs Tumor pain, acute coronary syndrome Ataxia, syncope, neuropathy Dyspnea, syncope Tumor/abdominal pain Pulmonary embolism, Reduced LVEF QTc prolonged, hepatic enzymes increased hypotension Cerebral ischemia, myocardial ischemia Myocardial ischemia, asymptomatic hypotension, transient cerebral ischemia Confusion, anxiety, visual disturbance, reduced LVEF, urinary incontinence, QTc prolonged Transient, moderate increases in QTc, increases in blood pressure, visual disturbances

Abbreviations: LVEF, left ventricular ejection fraction; NSCLC, non–small cell lung cancer.

Infusion

Compound

1200 mg/m23 weekly þ chemo NSCLC Ovary Prostate

Ovary NSCLC Anaplastic Thyroid 45–63 mg/m2 weekly for 3 wk out of 4 Renal colorectal NSCLC program suspended pending further preclinical evaluation Colorectal Dose range not defined Program temporarily suspended but restarted

Phase II/III dose range and tumors

TABLE 1 Summary of Early Phase I Clinical Development Program for Small Molecule VDAs and Plans for Further Development

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adhesion molecules in patients at the higher-dose levels. Four dose levels were examined from 18–90 mg/m2. The maximum tolerated dose (MTD) was defined as 60 mg/m2. Dose limiting toxicities (DLTs) above this dose were tumor site pain and two episodes of reversible acute coronary syndrome. Other “vascular” adverse effects included hot flushing, headache, abdominal cramping, and changes in blood pressure and heart rate. There were no typical cytotoxic adverse effects similar to those seen with taxanes. Dose and pharmacokinetic related increases in heart rate (HR) and QTc interval were observed in 15 patients. These effects were reversible by 24 hours. None of the patients was considered to have clinically significant increases in QTc interval, defined as any QTc interval >500 ms, a >25% increase in QTc over baseline, or ventricular arrhythmias. Pharmacokinetic analysis showed a short plasma half-life of approximately 30 min. Significant reductions in tumor blood flow by DCE-MRI were observed in six out of seven patients treated at 60 mg/m2. Three patients had prolonged disease stabilization and one patient with anaplastic thyroid cancer had a complete response that was durable for at least 3 years. In another study, 37 patients with solid malignancies received CA4P daily for 5 days repeated every 3 weeks (64). Again, the unique dose limiting toxicity was tumor pain, observed at a dose of 75 mg/m2. Cardiovascular DLTs (syncope and dyspnoea or hypoxia) were observed at this dose and the MTD in this study was defined as 52 mg/m2, a dose at which decreased tumor perfusion was observed by DCE-MRI. Other adverse events included hypotension, ataxia, headache and transient sensory neuropathy. A partial response was observed in a patient with metastatic soft tissue sarcoma, and prolonged disease stabilization was demonstrated in a number of patients. In the third phase I study, CA4P was given weekly for 3 weeks out of four (65). The only drug-related toxicity up to 40 mg/m2 was tumor pain. Dose-limiting toxicities at higher doses were variable: reversible ataxia at 114 mg/m2, vasovagal syncope and motor neuropathy at 88 mg/m2, and fatal ischemia in previously irradiated bowel at 52 mg/m2. Other adverse events included hypertension, hypotension, visual disturbance and dyspnea. Overall, CA4P was considered to be tolerable at doses of 52 or 68 mg/m2 at this dose schedule. Further phase I studies of CA4P focused on combination therapy with a number of agents including radiotherapy, chemotherapy and radioimmunotherapy. In a phase Ib trial, CA4P at a dose of 50 mg/m2 was given in combination with different radiotherapy schedules (66). The most common drug related toxicities were grade 1 hypertension, mild bradycardia and QTc prolongation (13 ms), lymphopenia, and severe tumor pain that often required opioids. One patient had grade 3 postural syncope. There was no increase in the number or severity of reactions to radiotherapy, and no accumulation of toxicity with repeated doses. Tumor blood flow was assessed using perfusion CT and demonstrated a sustained reduction in tumor blood volume. Following a phase I study of CA4P in combination with carboplatin (67), a three-center phase Ib/II trial of CA4P in combination with carboplatin and paclitaxel, both alone and together, was carried out in patients with advanced ovarian cancer and other solid tumors (68,69). The adverse effects in the Phase Ib portion of the study were mild and self-limiting with no cardiac toxicity, and CA4P added no additional toxicity to the chemotherapy alone. Dose-limiting toxicities of hypertension and ataxia occurred at a CA4P dose of 72 mg/m2. Objective tumor responses, defined according to the response evaluation criteria

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for solid tumors (RECIST) (70) were observed in a number of patients. In a subset of patients with advanced ovarian cancer, RECIST or CA-125 responses were observed in 10 out of 15 patients and four others had prolonged disease stabilization. On the basis of these data, the FDA granted orphan drug status for CA4P for the treatment of this disease in May 2006. The Phase II study in patients with platinum-resistant ovarian cancer (CA4P at 63mg/m2, combined with 175 mg/m2 of paclitaxel and carboplatin at AUC 5) is ongoing. Two studies in NSCLC are planned for 2006. The first, a Phase Ib/II study of CA4P plus chemoradiotherapy for the treatment of stage IIIa/IIIb unresectable NSCLC, will initially evaluate the tolerability of the regimen and identify a recommended Phase II dose for CA4P, then proceed as a randomized, open label, study to determine the RECIST response rate and one-year survival benefit in approximately 80 previously untreated patients. The second study is a randomized, double blind, placebo controlled Phase III study of CA4P in combination with carboplatin and paclitaxel in patients with newly diagnosed unresectable Stage IIIb/IV NSCLC, with the primary endpoint of overall survival. Because of the responses seen in advanced anaplastic thyroid cancer in the monotherapy studies, a trial program in this disease setting was also initiated. Results of the first Phase II study were presented at ASCO annual meeting 2006 (71). In this study, patients with metastatic anaplastic thyroid cancer were treated with CA4P as a single infusion at a dose of 45 mg/m2 on days 1, 8 and 15 of each 28-day cycle until disease progression. The treatment was well tolerated with adverse events of tumor pain but no significant cardiac toxicity. No objective responses were observed but approximately one-quarter of patients experienced prolonged disease stabilization (>3 months). A second phase II study of induction chemotherapy (doxorubicin and cisplatin) followed by CA4P and radiotherapy has been initiated in patients with newly diagnosed regionally advanced anaplastic thyroid cancer with primary objectives of objective response rate and overall survival and secondary endpoints of defining clinical predictors of response (e.g., pretreatment tumor microvessel density, changes in sICAM-1 levels, and tumor blood flow). ZD6126 A number of single agent Phase I studies of ZD6126 were carried out to identify an optimum schedule and define the tolerability and PK profile. In the first study, patients with advanced solid tumors received single doses of ZD6126 either once every 21 days or once every 14 days. DCE-MRI was used to assess tumor blood flow before and 6 hours after dosing. Significant reductions in tumor blood flow were documented at doses of 80 mg/m2 and higher (72). For patients who received ZD6126 every 3 weeks, additional DCE-MRI assessments were carried out 24 hours and 21 days after dosing and, although some recovery in tumor blood flow was apparent at 24 hours, reduced blood flow was maintained for up to 3 weeks in some patients (73). The terminal phase half-life was approximately 2 hours. The maximum tolerated dose of ZD6126 was defined as 80 mg/m2 for either dosing schedule. The major dose-limiting toxicity was abdominal pain. ZD6126 was associated with asymptomatic, reversible ECG changes and increased troponin concentrations suggestive of ischemia in some patients (74). In a second study 32 patients received weekly infusions of ZD6126 at doses of 5–28 mg/m2 (75). The pharmacokinetics appeared linear across this dose range. The terminal phase half-life was 1–3 hours. The MTD was identified as 20 mg/m2 with dose-limiting toxicities above this level of pulmonary embolism and reduced

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left ventricular ejection fraction. During the study, three patients with progressive disease experienced grade 3 and 4 thromboembolic events (pulmonary embolism and inferior vena cava thrombosis). Other adverse events included grade 1 and 2 reductions in LVEF, asymptomatic increases in CK-MB, and one patient with a history of ischemic heart disease had an acute myocardial infarct 2 weeks after discontinuing ZD6126. A broad Phase II program was initiated to assess the efficacy and tolerability of ZD6126 administered every 2–3 weeks, in monotherapy and combination regimens. The development of ZD6126 was suspended due to toxicity seen early in the Phase II program, and preclinical studies to understand the cardiovascular effects of the drug were initiated. AVE-8062 AVE-8062, a water-soluble analogue of Combretastatin A4, underwent a similar Phase I development program to the other VDAs described previously. The first Phase I study of AVE-8062 in patients with advanced cancer investigated a weekly schedule for 3 weeks out of 4 (76). Patients received doses ranging from 4.5 to 30 mg/m2 and the 30 mg/m2 cohort was expanded due to a DLT of asymptomatic systolic hypotension without evidence of CPK, troponin I, or ECG changes. Reductions in tumor vascular flow by DCE-MRI were observed at and above the 15.5 mg/m2 dose level. AVE8062 was rapidly eliminated with a t1/2 of 15 minutes although an active metabolite was identified with a t1/2 of 7 hours. In a second Phase I study AVE-8062 was given for five consecutive days every 3 weeks, and a third investigated the 30 min i.v. infusion given every 3 weeks in patients with advanced solid tumors (77). Because of the occurrence of four potentially drug-related vascular events (myocardial ischemia, transient asymptomatic hypotension, transient cerebral ischemia, asymptomatic ventricular tachycardia) in the 5-day and weekly schedule studies without residual clinical deficits, all trials were voluntarily and temporarily interrupted. However, since no vascular events have been observed in the three weekly schedule study up to a dose of 22 mg/m2, this trial was later resumed at that dose, with restricted eligibility criteria and increased cardiovascular monitoring (continuous 24-hours ECG, continual ambulatory blood pressure monitoring, serial CPK, troponin, ECG, ventriculographies and echocardiograms) and the 22 mg/m2 dose was found to be well tolerated (77). Two combination studies are currently open: a phase I study of AVE-8062 in combination with oxaliplatin every 3 weeks, and a study in combination with cisplatin 70 mg/m2 every 3 weeks both in patients with advanced solid tumors. Exclusion criteria for both studies are strict with respect to cardiovascular risk factors and patients are required to remain in the hospital for intensive cardiac monitoring during the study. Second Generation Tubulin Depolymerizing Agents MN-029 (denibulin hydrochloride) was designed to be more potent and with possibly less central nervous system toxicity than first generation VDAs. Preliminary results of the first phase I study of MN-029 given every 3 weeks have been reported (78). At the time of the presentation, the maximum tolerated dose had not been reached but the 180 mg/m2 cohort was expanded to six patients because of a dose-limiting toxicity (DLT) of acute reversible myocardial ischemia in one patient. Adverse events included nausea, vomiting, hypotension, fatigue and diarrhea.

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Tumor blood flow reduction assessed by DCE-MRI was recorded at doses of 120 mg/m2 and higher. A second Phase I study of MN-029 given every 7 days (days 1, 8 and 15), followed by a 2-week recovery period over two treatment cycles is currently open. OXi4503 (combretastatin-1 phosphate) is a second-generation combretastatin analogue that has recently entered clinical development. In the first Phase I dose escalation trial it was given as a weekly i.v. infusion for 3 weeks to identify the maximum tolerated dose and effects on tumor blood flow by DCE-MRI and PET scanning in patients with advanced solid tumors. NPI-2358 is a novel VDA tubulin-binding agent derived from a marine fungus. The first phase I study commenced in April 2006. N-Cadherin as a Target for VDAs N-cadherin (N-cad) is a cell adhesion molecule expressed by both the vascular endothelial cells and tumor cells of invasive tumors. The switching of cadherin between its E- and N-forms appears to increase tumor cell survival and their invasive and metastatic potential. ADH-1 Exherin (Adherex Technologies) is an antagonist of N-cad that has shown tumor vascular disruption and apoptosis in preclinical models. Results from two phase I studies of ADH-1 were reported in 2006 (23,24). The first was a phase I study of weekly doses of i.v. ADH-1 given to patients with N-cad positive solid tumors, to evaluate safety, pharmacokinetics, antitumor activity and effect on tumor vasculature assessed by DCE-MRI. Of the 55 patients screened, 56% were N-cad positive. ADH-1 was given weekly for 3 weeks out of 4 at but the schedule was later amended to weekly ADH-1 without interruption. No patients had experienced greater than grade 2 study drug related AEs at the time of the report. Preliminary evidence of antitumor activity, reductions in tumor blood flow and tumor pain, was reported. The second Phase I study was in patients with refractory solid tumors stratified according to their N-cad expression. ADH-1 was well tolerated and the maximum tolerated dose was not defined. The most commonly reported adverse events were grade 1–2 fatigue, nausea, dysgeusia, and flushing. Four of the 28 subjects in the N-cad positive group demonstrated antitumor activity (one PR, one MR and two patients with prolonged (>7 months) SD, while no antitumor activity was noted in the N-cad negative group (n ¼ 18). The mean terminal Phase t1/2 was 2.2 hours. Further development has concentrated on N-cad positive patients. Two Phase II studies are ongoing in subjects with recurrent N-Cad positive advanced solid tumors. In the first, ADH-1 is administered as a single agent at 600mg/m2 once every 3 weeks to patients with selected solid tumors (renal cell carcinoma, hepatocellular carcinoma, adrenocortical carcinoma, head and neck squamous cell cancer, gastro-oesophageal carcinoma, breast carcinoma or NSCLC). A second study is examining a weekly dosing schedule of 600 mg/m2 of ADH-1 in patients with similar tumor types. Three combination studies have also been initiated with docetaxel, carboplatin, and capetitabine. Flavonoids Flavonoids such as flavone-8-acetic acid (FAA) and its derivative 5,6 dimethylxanthenone-4-acetic acid (DMXAA [AS-1404]; Antisoma, plc, London, U.K.) do not directly bind to tubulin but cause similar levels of tumor endothelial cell damage as tubulin-binding agents (79,80). They induce a number of plasma cytokines including serotonin, nitric oxide, interferon and tumor necrosis factor a (TNF-a), and

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cause the local release of TNF-a from activated macrophages within the tumor itself (81–83). TNF-a has been shown to cause hemorrhagic necrosis in tumors (84) and it appears to be a necessary component for the response to flavonoids since antibodies against TNF-a inhibit FAA-induced vascular collapse in preclinical models (81,82). DMXAA has a higher potency than FAA in animal models and, unlike FAA, has been shown to stimulate human macrophages to produce TNF-a (85,86). In preclinical models, DMXAA has been shown to act synergistically with a number of treatment modalities including radiotherapy (87), chemotherapy (88), bio reductive agents (89), radioimmunotherapy (90), and immunotherapy (91). DMXAA (AS1404)—Clinical Studies The Phase I program consisted of three studies designed to investigate the safety and pharmacokinetic profile of DMXAA, and to identify an optimum dose and schedule for Phase II development. The first study investigated a three weekly schedule (92). Dose-limiting toxicities occurred at 4900 mg/m2 and included confusion, tremor, slurred speech, visual disturbance, anxiety, urinary incontinence and possible left ventricular failure. These were all rapidly reversible on stopping the drug. Transient asymptomatic prolongation of the QTc interval was seen at doses of 2000 mg/m2 and above. Tumor blood flow reduction was demonstrated by DCE-MRI and increases in plasma 5-hydroxyindoleacetic acid (5-HIAA) and plasma nitrate occurred in most patients treated at doses above 850 mg/m2. The second study evaluated a weekly dosing schedule (93). Dose-dependent increases in 5-HIAA were observed at doses of 650 mg/m2 and above. There was one unconfirmed partial response at 1300 mg/m2. The terminal half-life was approximately 8 hours. In both studies the pharmacokinetic profile was non-linear due to saturation of protein binding at higher doses. The maximum tolerated dose from the two studies was established at 3700 mg/m2 due to dose-limiting toxicities of urinary incontinence, visual disturbance and anxiety occurring at 4900 mg/m2. The visual disturbance was transient, rapid in onset and consisted of blurring, flickering and alteration of color discrimination associated with electroretinogram (ERG) abnormalities. A third study (94) was initiated to further investigate the cardiac and ophthalmic adverse events, to define a range of doses that produced an acceptably small effect on the QTc interval, and to identify an optimal dose for combination studies. Patients were allocated randomly to receive one of six sequential doses of DMXAA (300–3000 mg/m2), each given once weekly. Transient, moderate increases in QTc, increases in blood pressure and visual disturbances were observed at the two highest doses. Plasma levels of 5-HIAA increased acutely after treatment, dose dependently, up to 1200 mg/m2, and then plateaued. Doses in the range of 1200 mg/m2 were well tolerated and selected for further studies. Subsequent studies have concentrated on combination therapy. Preliminary results of a phase I/II study in combination with carboplatin/paclitaxel in patients with stage IIIb or IV previously untreated NSCLC have been reported (95). The safety of the combination was initially assessed in one patient who received standard doses of carboplatin and paclitaxel with DMXAA at 600 mg/m2. No toxicities were observed and the patient had a partial response to treatment. Patients were then randomized to receive up to six cycles of carboplatin and paclitaxel alone or with DMXAA at 1200 mg/m2. In a further study of six patients, a dose of 1800 mg/m2 was investigated. The safety profile in the control and DMXAA arms was comparable. No pharmacokinetic interactions were observed. Seventy patients were evaluable for efficacy, of whom 34 received DMXAA plus

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standard chemotherapy while 36 received standard chemotherapy alone. There was an improvement in time-to-tumor progression (132 vs. 115 days for the standard chemotherapy group) and objective tumor response (31.2% vs. 22.2% with standard chemotherapy) assessed by central independent review. The projected 6-month survival rate was 82.0% for patients receiving DMXAA and 54.8% for patients receiving standard chemotherapy, and the projected median survival is currently 12.0 months with DMXAA and 7.6 months for the standard chemotherapy group. An extension study in which additional NSCLC patients are treated at 1800 mg/m2 is ongoing and a Phase III study is planned. A second randomized Phase II study has been carried out in combination with carboplatin and paclitaxel in 70 patients with recurrent platinum-sensitive ovarian cancer with a progression-free interval of more than 6 months after initial response to platinum-based chemotherapy (96). Patients were randomized to receive carboplatin and paclitaxel with or without DMXAA at 1200 mg/m2. The combination appeared tolerable in this disease setting and efficacy assessments are ongoing. A third randomized Phase II study has completed recruitment of patients with hormone-refractory metastatic prostate cancer. Patients were randomized to receive three weekly docetaxel doses with or without DMXAA for up to 10 cycles in the absence of disease progression, and following progression patients will receive weekly DMXAA alone. Preliminary data from the first 64 of 74 patients in the trial have recently been reported (97). There was a PSA response rate of 57% in those receiving AS1404 plus docetaxel chemotherapy compared with 35% in those receiving docetaxel alone, while the proportion of men showing disease progression by PSA was 17% in the AS1404 group compared with 29% with docetaxel alone. The combination of AS1404 with docetaxel was well tolerated, without exacerbation of chemotherapy side effects. OPPORTUNITIES AND CHALLENGES IN THE DEVELOPMENT OF VDAS The development of VDAs has provided an exciting new approach to the treatment of cancer. The preclinical and clinical studies outlined in this chapter illustrate the effectiveness of these agents in multiple tumor types. Since no single approach is likely to be effective alone in the treatment of such a complex disease as cancer, the potential of VDAs to be synergistic with a number of other anticancer measures, including chemotherapy, radiotherapy, immunotherapy, and antiangiogenic agents, is of primary importance. However, the description of the clinical development programs of the most advanced agents also serves to illustrate the new challenges posed by these compounds, some of which are shared by the antiangiogenic compounds and other novel targeted therapies currently in development. The first challenge has been that VDAs, while causing extensive necrosis and hemorrhage within tumors, may not themselves cause tumor shrinkage as single agents, making assessment of objective tumor responses difficult by conventional methods. This has resulted in the implementation of alternative imaging methods, such as DCE-MRI or dynamic contrast CT (DC-CT), to assess tumor blood flow as evidence that the compounds are exerting an antivascular effect in tumors. While such methods provide convincing and often dramatic evidence of tumor blood flow reduction following administration of single doses, there are currently limitations to the applicability of the techniques. In particular, the high degree of intra- and

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inter-patient variability makes interpretation and quantification of absolute levels of reduction difficult, and because the relationship between tumor blood flow reduction and efficacy in terms of progression-free survival is not yet clear, the value of such imaging techniques in guiding the choice of dose for Phase II development is limited. There are also technical challenges, such as identification of the optimum contrast agents suitable for use in humans, and the current limited reproducibility of results between different centers. In most early clinical trials, the advanced imaging techniques have been carried out at only one center to minimize variability, which is an approach possible in Phase I development. However, in order to determine whether demonstration of early tumor blood flow reduction is a true biomarker of efficacy, DCE-MRI changes will need to be evaluated in larger efficacy studies in single tumor types, which will raise the issue of inter-center variability. Another major challenge is the choice of dose and schedule for the VDAs. The development of any i.v. agent requires multiple Phase I studies to define the optimum schedule, but, since the side effect profile for the majority of cytotoxic agents is well understood and clearly dose-related, the strategy has generally been to define the maximum tolerated dose to take into the next phase of development. In contrast, there are significant challenges to defining an optimum dose and schedule for an agent with an unknown adverse event profile, and where the goal is to define an optimum biologically active dose rather than a maximum tolerated dose. None of the agents in development has taken the maximum tolerated dose forward to further studies, but some have identified a lower biologically active dose based on a combination of safety, a pharmacokinetic profile in the range predicted to be effective from preclinical experiments, and evidence of pharmacodynamic effects on tumor blood flow and, in some cases, soluble biomarkers, e.g., HIAA for DMXAA, circulating endothelial cells for ZD6126, and sICAM for CA4P and ADH-1. In most of the clinical studies of individual VDAs, the adverse event profile has not been clearly dose-related or consistent between Phase I studies. VDAs cause a unique adverse event of tumor pain, presumably due to ischemia and necrosis, but this has not been sufficient to guide dosing. These agents are associated with a different spectrum of adverse events compared to conventional cytotoxic agents, with the major class effect occurring in the cardiovascular system, possibly because of drug-induced microvessel vasoconstriction and/or endothelial cell activation leading to the formation of micro-thrombi in the coronary circulation and compromising the blood supply to the cardiac muscle. Most clinical studies of all the VDAs in development have reported adverse events of asymptomatic elevations in troponin or CK-MB indicative of myocardial damage, acute coronary syndrome, QTc prolongation, and reduction in left ventricular ejection fraction. In most cases, the agents have a short half-life, and the effects are quickly reversible; however, a number of cases of pulmonary embolism and myocardial infarction have been reported. For three agents (AVE-8062, ZD6126, and DMXAA) this led to re-evaluation of the clinical programs. In the case of DMXAA, a study was specifically designed to further evaluate the observed cardiac and visual effects; in the case of ZD6126, further preclinical studies have been performed to define ways of preventing and managing the cardiac effects; and for AVE-8062, clinical development was restarted at a lower dose, with a less frequent treatment schedule, and with a number of strict restrictions on cardiovascular eligibility criteria and extensive monitoring of cardiac function and blood pressure. While this is possible in a clinical trial setting, it may prove challenging to provide the necessary close cardiac monitoring for patients once such agents are approved and widely prescribed.

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The choice of dose and schedule for a VDA faces a further challenge when combining these novel agents with conventional chemotherapy agents; in this case the choice of schedule may be limited, for practical as well as scientific reasons, by the need to align with the chemotherapy cycles. The possibility of drug interactions must be taken into account, e.g., the potential of the combination to cause or exacerbate QTc prolongation, the potential for a pharmacokinetic interaction, or the potential for overlapping cardiac toxicities, which may be the case with high cumulative doses of anthracyclines. In summary, this is an exciting new class of agents, some of which are at an advanced stage of clinical development. The results of the Phase III studies of CA4P in ovarian cancer and DMXAA in NSCLC will clarify the potential of VDAs to improve response rates, progression free survival, and overall survival in these diseases compared to conventional chemotherapy alone, and will further define the risk to patients in terms of cardiovascular side effects. Future challenges for the class will be to define which patients are most likely to benefit from the addition of such agents, and which of the current chemotherapies and novel targeted agents in development are most suitable for combination to maximize their antitumor effects. REVIEWS Thorpe PE. Vascular targeting agents as cancer therapeutics. Clin Cancer Res 2004; 10:415–27. Chaplin DJ, Dougherty GJ. Tumour vasculature as a target for cancer therapy. Br J Cancer 1999; 80:57–64. Siemann DW, Chaplin DJ, Horsman MR. Vascular-targeting therapies for treatment of malignant disease. Cancer 2004; 100:2491–9.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Folkmann J. How is blood vessel growth regulated in normal and neoplastic tissue? Cancer Res 1986; 46:467–73. Ellis LM, Liu W, Ahmad SA, et al. Overview of angiogenesis: biologic implications for anti-angiogenic therapy. Semin Oncol 2001; 28:94–104. Marme D. The impact of anti-angiogenic agents on cancer therapy. J Cancer Res Clin Oncol 2003; 129:607–20. Kerbel R, Folkman J. Clinical translation of angiogenesis inhibitors. Nat Rev Cancer 2002; 2:727–39. Siemann DW, Bibby MC, Dark GB, et al. Differentiation and definition of vasculartargeted therapies. Clin Cancer Res 2005; 11:416–20. Eberhard A, Kahlert S, Goede V, et al. Heterogeneity of angiogenesis and blood vessel maturation in human tumors: implications for antiangiogenic tumor therapies. Cancer Res 2000; 60:1388–93. Ruoslahti E. Specialization of tumour vasculature. Nat Rev Cancer 2002; 2:83–90. Hirst DG, Denekamp J, Hobson B. Proliferation kinetics of endothelial and tumour cells in three mouse mammary carcinomas. Cell Tissue Kinet 1982; 15:251–61. Hobson B, Denekamp J. Endothelial proliferation in tumors and normal tissue: continuous labeling studies. Br J Cancer 1984; 49:405–13. Denekamp J, Hobson B. Endothelial cell proliferation in experimental tumours. Br J Cancer 1982; 46:711–20. Denekamp J. Vascular endothelium as the vulnerable element in tumours. Acta Radiol Oncol 1984; 23:217–25. Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res 1989; 49:6449–65.

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13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

24.

25. 26. 27. 28. 29. 30.

31.

32. 33.

309

Arap W, Pasqualini R, Ruoslahti E. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 1998; 279:377–80. Blakey DC, Westwood FR, Walker M, et al. Antitumor activity of the novel vascular targeting agent ZD6126 in a panel of tumor models. Clin Cancer Res 2002; 8(6):1974–83. Grosios K, Holwell SE, McGown AT, et al. In vivo and in vitro evaluation of combretastatin A-4 and its sodium phosphate prodrug. Br J Cancer 1999; 81:1318–27. Dark GG, Hill SA, Prise VE, et al. Combretastatin A-4, an agent that displays potent and selective toxicity toward tumor vasculature. Cancer Res 1997; 57:1829–34. Landuyt W, Ahmed B, Nuyts S, et al. In vivo antitumor effect of vascular targeting agents combined with either ionizing radiation or anti-angiogenesis treatment. Int J Radiat Oncol Biol Phys 2001; 49:443–50. 35. Chaplin DJ, Pettit GR, Hill SA. Anti-vascular approaches to solid tumour therapy: evaluation of combretastatin A4 phosphate. Anticancer Res 1999; 19:189–95. Veenendaal LM, Jin H, Ran S, et al. In vitro and in vivo studies of a VEGF121/ rGelonin chimeric fusion toxin targeting the neovasculature of solid tumors. Proc Natl Acad Sci USA 2002; 99:7866–71. Seon BK. Expression of endoglin (CD105) in tumor blood vessels. Int J Cancer 2002; 99:310–1. Brooks PC, Clark R, Cherish DA. Requirements of vascular integrin avb3 for angiogenesis. Science 1994; 264:569–71. Nilsson F, Kosmehl H, Zardi L, et al. Targeted delivery of tissue factor to the ED-B domain of fibronectin, a marker of angiogenesis, mediates the infarction of solid tumors in mice. Cancer Res 2001; 61:711–16. Sessa C, Perotti A, Maur M, et al. An enriched phase I, pharmacokinetic and pharmacodynamic study of the N-cadherin (NCAD) cyclic competitive binder exherin (ADH-1) in patients with solid tumors. J Clin Oncol 2006; 24: ASCO Annual Meeting Proceedings Abstract 3042. Stewart J, Jonker DJ, Goel R, et al. Final clinical and pharmacokinetic (PK) results from a phase 1 study of the novel N-cadherin (N-cad) antagonist, Exherin (ADH-1), in patients with refractory solid tumors stratified according to N-cad expression. J Clin Oncol 2006; 24; ASCO Annual Meeting Proceedings Abstract 3016. Seed L, Slaughter DP, Limarzi LR. Effect of colchicine on human carcinoma. Surgery 1940; 7:696–709. Baguley BC, Holdaway KH, Thomsen LL, Zhuang L, Zwi LJ. Inhibition of growth of colon 38 adenocarcinoma by vinblastine and colchicine. Evidence for a vascular mechanism. Eur J Cancer 1991; 27:482–7. Hill SA, Sampson LE, Chaplin DJ. Anti-vascular approaches to solid tumor therapy: evaluation of vinblastine and flavone acetic acid. Int J Cancer 1995; 63:119–23. Jordan MA, Wilson L. Microtubules as a target for anticancer drugs. Nat Rev Cancer 2004; 4:253–65. Jordan MA, Wendell K, Gardiner S, et al. Mitotic bock induced in HeLa cells by low concentrations of paclitaxel (Taxol) results in abnormal mitotic exit and apoptotic cell death. Cancer Res 1996; 56:816–25. Ridwelski K, Kettner E, Fahlke J. Phase I study of the anti-neovascular agent MBT-0206 to evaluate safety and efficacy in patients with metastatic or advanced unresectable gastrointestinal cancer (GI cancer). J Clin Oncol 2004; ASCO Annual Meeting Proceedings; 22: Abstract 3163. Bartelheim K, Ognerubov NA, Semiglazov VF, et al. Phase Ib study of the antineovascular agent MBT-0206 to evaluate safety and efficacy in patients with metastatic breast cancer. J Clin Oncol 2004; ASCO Annual Meeting Proceedings; 22: Abstract 3079. Yamamoto N, Andoh M, Kawahara M, et al. Phase I study of TZT-1027, an inhibitor of tubulin polymerization, given weekly · 3 as a 1-hour intravenous infusion in patients with solid tumors. Proc Am Soc Clin Oncol 21: 2002; abstract 420. De Jonge M, Madretsma S, Van der Gaast A, et al. TZT-1027, a novel dolastatin 10 derivative: Phase I and pharmacologic study of day 1 and 8 IV administration every

310

34. 35.

36.

37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52.

Robertson

3 weeks in patients (pts) with advanced solid tumors. Proc Am Soc Clin Oncol 2003; 22: Abstract 613. Schoffski P, Thate B, Beutel G, et al. Phase I evaluation of the 3-weekly administration of TZT-1027 in patients with solid tumors. Proc Am Soc Clin Oncol 2004; 22: Abstract 845. Blagden S, Thomas A, De-Bono J, et al. Phase I study of intravenous TZT-1027 (T) and carboplatin (C), administered on Day 1 (T and C) and Day 8 (T) every three weeks in patients (pts) with advanced solid tumors. Proc Am Soc Clin Oncol 2004; 22: Abstract 3141. A Phase I Study of Intravenous TZT-1027 and Gemcitabine (GEM), Administered on Days 1 and 8 of a Three Week Course in Patients with Advanced Solid Tumors. 17th Annual AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics, Philadelphia, Pennsylvania, USA, abstract B241. Sprague E, Fleming GF, Carr R, et al. Phase I study of 21-day continuous dosing of the oral antimitotic agent ABT-751. Proc Am Soc Clin Oncol 2003; 22: Abstract 518. Kobayashi H, Hande KR, Berlin JD, et al. Phase I results of ABT-751, a novel microtubulin inhibitor, administered daily · 7 every 3 weeks. Proc Am Soc Clin Oncol 2004; 22: Abstract No: 2079. Yee KWL, Hagey A, Verstovsek S, et al. Phase 1 study of ABT-751, a novel microtubule inhibitor, in patients with refractory hematologic malignancies. Clin Cancer Res 2005; 11:6615–24. Fox E, Adamson PC, Hagey A, et al. Phase I trial of oral ABT-751 in pediatric patients. Proc Am Soc Clin Oncol 2005; 23: Abstract 8527. Michels E, Ellard E, Le L, et al. A phase I study of ABT-751 in combination with docetaxel in patients with metastatic hormone-refractory prostate cancer. Proc Am Soc Clin Oncol 2006; 24: Abstract 4651. Messersmith WA, Rudek MA, Laheru D, et al. Phase I study of ABT-751 in combination with CAPIRI (capecitabine and irinotecan) and bevacizumab in patients with advanced colorectal cancer. Proc Am Soc Clin Oncol 2006; 24: Abstract 13553. Dragnev KH, Rigas JR, Disalvo WM, et al. A phase I trial of ABT-751 and carboplatin (C) in patients (pts) with previously treated non-small cell lung cancer (NSCLC) Interim Results. Proc Am Soc Clin Oncol 2006; 24: Abstract 17098. Benson AB, Kindler HL, Jodrell D, et al. Phase 2 study of ABT-751 in patients with refractory metastatic colorectal carcinoma. Proc Am Soc Clin Oncol 2005; 23. Abstract 3537. Hagey AE, Figlin RA, Moldawer N, et al. Preliminary phase 2 results of ABT-751 in subjects with advanced renal cell carcinoma. Proc Am Soc Clin Oncol 2005; 23: Abstract 4603. Washington DK, Storniolo AV, Saleh M, et al. Phase II Results of ABT-751 in Subjects with Taxane-Refractory Breast Cancer: Interim Analysis Interim Results. Proc Am Soc Clin Oncol 2005; 23, abstract 724. Segreti JA, Polakowski JS, Koch KA, et al. Tumor selective antivascular effects of the novel antimitotic compound ABT-751: an in vivo rat regional hemodynamic study. Cancer Chemother Pharmacol 2004; 54:273–81. Tozer GM, Prise VE, Wilson J, et al. Combretastatin A-4 phosphate as a tumor vascular-targeting agent: early effects in tumors and normal tissues. Cancer Res 1999; 59:1626–34. Galbraith SM, Chaplin DJ, Lee F, et al. Effects of combretastatin A4 phosphate on endothelial cell morphology in vitro and relationship to tumour vascular targeting activity in vivo. Anticancer Res 2001; 21:93–102. Kanthou C, Tozer GM. The tumour vascular targeting agent combretastatin A-4 phosphate induces reorganisation of the actin cytoskeleton and early membrabe blebbing in human endothelial cells. Blood 2002; 99:2060–9. Blakey DC, Westwood FR, Walker M, et al. Antitumor activity of the novel vascular targeting agent ZD6126 in a panel of tumor models. Clin Cancer Res 2002; 8:1974–83. Blakey DC, Ashton SE, Westwood FR, Walker M, Ryan AJ. ZD6126: a novel small molecule vascular targeting agent. Int J Radiat Oncol Biol Phys 2002; 54:1497–502.

Antivascular Agents

53. 54. 55. 56. 57. 58. 59. 60. 61. 62.

63. 64. 65. 66. 67. 68. 69.

70. 71. 72.

311

Davies PD, Dougherty GJ, Blakey DC, et al. ZD6126, a new agent causing selective damage of tumour vasculature. Proc Am Assos Cancer Res 2000; 41:2085–90. Nelkin BD, Ball DW. Combretastatin A-4 and doxorubicin combination treatment is effective in a preclinical model of human medullary thyroid carcinoma. Oncol Rep 2001; 8:157–60. Grosios K, Loadman PM, Swaine DJ, et al. Combination chemotherapy with combretastatin A4 phosphate and 5-fluoro-uracil in an experimental murine colon adenocarcinoma. Anticancer Res 2000; 20:229–33. Li L, Rojiani A, Siemann DW. Targetting the tumour vasculature with combretastatin A-4 diphosphate: effects on radiation therapy. Int J Radiat Oncol Biol Phys 1998; 42:899–903. Siemann DW, Rojiani AM. Enhancement of radiation therapy by the novel vascular targeting agent ZD6126. Int J Radiat Oncol Biol Phys 2002; 53:164–71. Horsman MR, Murata R. Vascular targeting effects of ZD6126 in a C3H mouse mammary carcinoma and the enhancement of radiation response. Int J Radiat Oncol Biol Phys 2003; 57:1047–55. Pedley RB, El-Emir E, Flynn A, et al. Eradication of colorectal xenografts by combined radioimmunotherapy and combretastatin A-4 3-O-phosphate. Cancer Res 2001; 61:4716–22. Eikesdal HP, Bjerkvig R, Mella O, et al. Combretastatin A-4 and hyperthermia: a potent combination for the treatment of solid tumours. Radiother Oncol 2001; 60:147–54. Wedge SR, Kendrew J, Ogilvie DJ, et al. Combination of the VEGF receptor tyrosine kinase inhibitor ZD6474 and vascular targeting agent ZD6126 produces an enhanced anti-tumor response. Proc Am Soc Cancer Res 2002; 43:1081–6. Dowlati, A, Robertson K, Cooney M, et al. A phase I pharmacokinetic and translational study of the novel vascular targeting agent combretastatin A-4 phosphate on a singledose intravenous schedule in patients with advanced cancer. Cancer Res 2002; 62:3408–16. Cooney M, Radivoyevitch T, Dowlati A, et al. Cardiovascular safety profile of combretastatin A4 phosphate in a single-dose phase I study in patients with advanced cancer. Clin Cancer Res 2004; 10:96–100. Stevenson JP, Rosen M, Sun W, et al. Phase I trial of the antivascular agent combretastatin A4 phosphate on a 5-day schedule to patients with cancer: magnetic resonance imaging evidence for altered tumor blood flow. J Clin Oncol 2003; 21:4428–38. Rustin GJ, Galbraith SM, Anderson H, et al. Phase I clinical trial of weekly combretastatin A4 phosphate: clinical and pharmacokinetic results. J Clin Oncol 2003; 21:2815–22. Ng QS, Carnell D, Milner J, et al. Phase Ib trial of Combretastatin A4 Phosphate (CA4P) in combination with radiotherapy (RT): Initial clinical results. Proc Am Soc Clin Oncol 2005; 23: Abstract 3117. Bilenker JH, Flaherty KT, Rosen M, et al. Phase I trial of combretastatin A-4 phosphate with carboplatin. Clin Cancer Res 2005; 11:1527–33. Rustin GJ, Nathan PD, Boxall J, et al. A phase Ib trial of combretastatin A- 4 phosphate (CA4P) in combination with carboplatin or paclitaxel chemotherapy in patients with advanced cancer. Proc Am Soc Clin Oncol 2005; 23: Abstract 3103. Rustin GJ, et al. A dose escalation study combining combretastatin A4 phosphate (CA4P) with carboplatin or paclitaxel in patients with advanced cancer. Interim Results. 17th Annual AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics, Philadelphia, Pennsylvania, USA. Abstract No: A12. Therasse P, Arbuck S, Eisenhauer EA. New guidelines to evaluate the response to treatment in solid tumors. J Natl Cancer Inst 2000; 92:205–16. Cooney M, Savvides P, Agarwala P, et al. Phase II study of combretastatin A4 phosphate (CA4P) in patients with advanced anaplastic thyroid carcinoma (ATC). Proc Am Soc Clin Oncol 2006; 24: Abstract 13074. Evelhoch JL, LoRusso PM, He Z, Magnetic resonance imaging measurements of the response of murine and human tumors to the vascular-targeting agent ZD6126. Clin Cancer Res 2004; 10:3650–7.

312 73. 74. 75. 76. 77. 78.

79. 80. 81. 82. 83.

84. 85. 86. 87. 88. 89. 90. 91.

Robertson

DelProposto Z, LoRusso P, Latif Z, et al. MRI effects of the vascular-targeting agent ZD6126 on tumour vasculature. Proc Am Soc Clin Oncol 2002; abstract 440. Gadgeel SM, LoRusso PM, Wozniak AJ, Wheeler C. A dose-escalation study of the novel vascular-targeting agent, ZD6126, in patients with solid tumors. Proc Am Soc Clin Oncol 2002; Abstract 438. Beerepoot LV, Radema SA, Witteveen EO, et al. Phase I clinical evaluation of weekly administration of the novel vascular targeting agent, ZD6126, in patients with solid tumours. J Clin Oncol 2006; 24:1491–8. Tolcher AW, Forero L, Celio P. Phase I, pharmacokinetic, and DCE-MRI correlative study of AVE8062A, an antivascular combretastatin analogue, administered weekly for 3 weeks every 28-days. Proc Am Soc Clin Oncol 2003; 22: Abstract 834. Sessa C, LoRusso P, Tolcher AW. A Pharmacokinetic and DCE-MRI-dynamic Phase I study of the antivascular combretastatin analogue AVE8062A administered every 3 weeks. Proc Am Soc Clin Oncol 2005; Abstract 5827. Ricart AD, Cooney M, Sarantopoulos J, et al. A phase I pharmacokinetic (PK) and pharmacodynamic (PD) study of MN-029, a novel vascular disrupting agent (VDA), in patients (pts) with advanced solid tumors. J Clin Oncol 2006; 24: ASCO Annual Meeting Proceedings Abstract 3096. Smith GP, Calveley SB, Smith MJ, et al. Flavone acetic acid (NSC 347512) induces hemorrhagic necrosis of mouse colon 26 and 38 tumours. Eur J Cancer Clin Oncol 1987; 23:1209–11. Ching LM, Zwain S, Baguley BC. Relationship between tumour endothelial cell apoptosis and tumour blood flow shutdown following treatment with the antivascular agent DMXAA in mice. Br J Cancer 2004; 90:906–10. Mahadevan V, Malik ST, Meager A, et al. Role of tumor necrosis factor in flavone acetic acid-induced tumor vasculature shutdown. Cancer Res 1990; 50:5537–42. Philpott M, Baguley BC, Ching LM. Induction of tumour necrosis factor a by single and repeated doses of the anti-tumor agent 5,6-dimethylxanthenone-4-acetic acid. Cancer Chemother Pharmacol 1995; 36:143–8. Thomsen LL, Baguley BC, Wilson WR. Nitric oxide: its production in host-cellinfiltrated EMT6 spheroids and its role in tumour cell killing by flavone-8-acetic acid and 5,6-dimethylxanthenone-4-acetic acid. Cancer Chemother Pharmacol 1992; 31:151–5. Old LJ. Tumor necrosis factor (TNF). Science 1985; 230:630–2. Ching LM, Joseph WR, Crosier KE, et al. Induction of tumor necrosis factor-a messenger RNA in human and murine cells by the flavone acetic acid analogue 5,6-dimethylxanthenone-4-acetic acid (NSC 640488). Cancer Res 1994; 54:870–2. Philpott M, Joseph WR, Crosier KE, et al. Production of tumour necrosis factor-a by cultured human peripheral blood leucocytes in response to the anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (NSC 640488). Br J Cancer 1997; 76:1586–91. Wilson WR, Li AE, Cowan DS, et al. Enhancement of tumor radiation response by the antivascular agent 5,6-dimethylxanthenone-4-acetic acid. Int J Radiat Oncol Biol Phys 1998; 42:905–8. Siim BG, Lee AE, Shalal-Zwain S, et al. Marked potentiation of the antitumour activity of chemotherapeutic drugs by the antivascular agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Cancer Chemother Pharmacol 2003; 51:43–52. Lash CJ, Li AE, Rutland M, et al. Enhancement of the anti-tumour effects of the antivascular agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) by combination with 5-hydroxytryptamine and bioreductive drugs. Br J Cancer 1998; 78:439–45. Pedley RB, Boden JA, Boden R, et al. Ablation of colorectal xenografts with combined radioimmunotherapy and tumor blood flow-modifying agents. Cancer Res 1996; 56:3293–300. Kanwar JR, Kanwar RK, Pandey S, et al. Vascular attack by 5,6-dimethylxanthenone4-acetic acid combined with B7.1 (CD80)-mediated immunotherapy overcomes immune resistance and leads to the eradication of large tumors and multiple tumor foci. Cancer Res 2001; 61:1948–56.

Antivascular Agents

92. 93. 94. 95. 96. 97.

313

Jameson MB, Thompson PI, Baguley BC, et al. Clinical aspects of a phase I trial of 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a novel antivascular agent. Br J Cancer 2003; 88:1844–50. Rustin GJ, Bradley C, Galbraith S, et al. 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a novel antivascular agent: phase I clinical and pharmacokinetic study. Br J Cancer 2003; 88:1160–7. McKeage M., Fong P, Jeffery M, et al. 5,6-Dimethylxanthenone-4-acetic acid in the treatment of refractory tumors: a phase I safety study of a vascular disrupting agent. Clin Cancer Res 2006; 12:1776–84. McKeage M, AS 1404-201 Study Group Investigators. Phase Ib/II study of DMXAA combined with carboplatin and paclitaxel in non-small cell lung cancer (NSCLC). Proc Am Soc Clin Oncol 2006; 24: Abstract 7102. Gabra H, AS 1404-201 Study Group Investigators. Phase II study of DMXAA combined with carboplatin and paclitaxel in recurrent ovarian cancer. Proc Am Soc Clin Oncol 2006; 24: Abstract 5032. Mainwaring P, et al. Tumor microenvironment. Meeting, Boston MA, 2006.

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Matrix Metalloproteinases Stéphane Vignot Service d’Oncologie Médicale, Groupe Hospitalier Diaconesses Croix Saint Simon, Paris, France

Jean-Philippe Spano Service d’Oncologie Médicale, Groupe Hospitalier Pitié Salpétrière, Paris, France

INTRODUCTION Basic research about cancer was initially focused on tumor cells, and carcinogenesis was then considered as the result of serial mutations (activation of oncogenes and loss of expression of tumor suppressor genes) modifying their phenotype (1). A good understanding of tumor progression led afterwards to a growing interest in stroma (2). Stroma is composed of nontransformed cells in the host and an extracellular matrix (ECM) that supports adhesion of cells and transmits signals through cellsurface adhesion receptors. The ECM contains collagens, noncollageneous glycoproteins, and proteoglycans. The basement membrane is a specialized compound of ECM that separates the epithelial cells from the underlying stroma, providing the first barrier against invasion of carcinomas cells (3). Modifications of stroma are mainly assured by matrix metalloproteinases (MMPs). These proteolytic enzymes are involved in many physiological processes, including ovulation, embryogenesis, and immune regulation. Disruptions in MMPs regulation leads to the pathogenic mechanism of cancer and also other diseases, such as rheumatoid arthritis, multiple sclerosis, aortic aneurysms, arterial restenosis lesions, and bullous skin disorders. This chapter will expose recent data on MMPs, focusing on their role in carcinogenesis, and presenting main clinical implications. THE MMP FAMILY Classification and Structure The MMPs comprise a large family with more than 21 enzymes. The MMPs are endopeptidases that can cleave virtually any component of the ECM. These enzymes were initially named according to their substrate and then divided into collagenases, gelatinases, stromelysins and matrilysins. Actual classification is based on their structure and a sequential numbering system has been adapted (Table 1) (4). All MMPs have a similar domain structure with a “pre” region to target for secretion, a “pro” region to maintain latency and an active catalytic region that contains the zinc-binding active site. The majority of MMPs have additional domains that are important in substrate recognition and in inhibitor binding (5). A subset of MMPs, formally known as membrane type MMPs (MT-MMPs) also contains a transmembrane domain (6). Unlike other MMPs, they are not 315

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TABLE 1 The Matrix Metalloproteinases (MMP) Family MMP designationa MMP-1 MMP-2 MMP-3 MMP-7 MMP-8 MMP-9 MMP-10 MMP-11 MMP-12 MMP-13 MMP-14 MMP-15 MMP-16 MMP-17 MMP-18 MMP-19 MMP-20 MMP-21 MMP-22 MMP-23 MMP-24 MMP-25 MMP-26 MMP-27 MMP-28

Common name(s) Collagenase-1, interstitial collagenase, fibroblast collagenase, tissue collagenase Gelatin-binding Gelatinase A, 72-kDa gelatinase, 72-kDa type IV collagenase, neutrophil gelatinase Stromelysin-1, transin-1, proteoglycanase, procollagenase activating protein Matrilysin, matrin, PUMP1, small uterine metalloproteinase Collagenase-2, neutrophil collagenase, PMN collagenase, granulocyte collagenase Gelatinase B, 92-kDa gelatinase, 92-kDa type IV collagenase Stromelysin-2, transin-2 Stromelysin-3 Metalloelastase, macrophage elastase, macrophage metalloelastase Collagenase-3 MT1-MMP, MT-MMP1 MT2-MMP, MT-MMP2 MT3-MMP, MT-MMP3 MT4-MMP, MT-MMP4 Collagenase-4 (Xenopus; no human homologue known) RASI-1, MMP-18b Enamelysin Homologue of Xenopus XMMP CMMP (chicken; no human homologue known) Cysteine array MMP (CA-MMP), femalysin, MIFR MT5-MMP, MT-MMP5 MT6-MMP, MT-MMP6, leukolysin Endometase, matrilysin-2 Epilysin

a

MMP-4, -5, and -6 have been abandoned. b MMP-19 was initially named MMP-18 but, since this designation was already used for a Xenopus homologue the classification was modified.

secreted but remain attached to cell membrane, which allows control of proteolytic activity in the direct neighborhood of the cell. The secreted MMPs can also localize to the cell surface by binding to integrins or to CD44, or through interactions with cell-surface–associated heparan sulphate proteoglycans collagen type IV or the ECM metalloproteinase inducer (EMMPRIN) (7–9). In addition to the MMPs, a second family of proteins with metalloproteinase activity has been described and named ADAMs (a disintegrin and metalloproteinase) or adamlysin metalloproteinases. These proteins are characterized by a disintegrin region that can mediate cell adhesion and fusion events. Among 30 known ADAMs, 16 have an additional metalloproteinase activity (10,11). In addition, adamlysin metalloproteinases with thrombospondin motifs (ADAM-TS) have been described in the ECM (12,13). Physiological Functions The main activity of MMPs was thought to participate to degradation of structural components of the ECM in order to facilitate cell migration. Cleavage by MMPs of ECM proteins like integrins also affects direct cellular signaling and functions (14) and can generate fragments with new functions [cleavage of laminin-5 and collagen type IV results in exposure of cryptic sites that promote migration

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(15–17)], and cleavage of cell-adhesion molecules allows cell migration and releases other factors that enhance mobility (18,19). MMPs and ADAMs participate in the activation of precursor forms of many growth factors, including transforming growth factor-a (TGFa) (cell-membrane– bound precursor) and TGFb (inactive extracellular complex) (20,21). Growth factor receptors are also MMP substrates such as FGFR 1, c-MET, HER2 and HER4 (22–24). Finally, the MMPs participate in the activation of their own zymogen and of other MMPs, as described below. Regulation MMP activity is regulated at multiple levels. The MMP genes are transcriptionally responsive to many growth factors, cytokines and hormones (25). MMPs are secreted as inactive zymogens (pro-MMPs). They are kept inactive by an interaction between a cysteine-sulfhydryl group in the propeptide domain and the zinc ion bound to the catalytic domain. Activation requires proteolytic removal of the propeptide prodomain to release the catalytically active enzyme. This processing can be achieved outside the cell by other MMPs or by other proteases, especially plasmin (5). However, MMP-11, MMP-28 and the MT-MMPs can also be activated by intracellular furin-like serine proteinases before they reach the cell surface (7). MMP activity is tightly controlled by endogenous inhibitors. The main inhibitor of MMPs in tissue fluids is a2-macroglobulin, an abundant plasma protein that binds to MMPs (26). The a2-macroglobulin–MMP complex that binds to a Scavenger receptor is irreversibly cleared by endocytosis. Thrombospondin-2 acts in a similar way by forming a complex with MMP-2 and facilitating scavengerreceptor–mediated endocytosis (27). Thrombospondin-1 binds to pro-MMP-2 and -9 and directly inhibits their activation (28). Other known molecules involved in the regulation of MMP activity are tissue-inhibitors of metalloproteinases (TIMPs 1–4) which can reversibly inhibit specific MMPs (levels of expression of TIMPs are tissue specific); RECK (REversion-inducing Cysteine-rich protein with Kazal motifs, the only known membrane-bound MMP inhibitor) and a1-PDX (29). IMPLICATION OF MMP IN CARCINOGENESIS: FUNDAMENTAL DATA Mechanisms of Action Originally, MMPs were considered to be important almost exclusively in invasion and metastasis. However, they are involved in other steps of carcinogenesis: regulation of cell growth, apoptosis, angiogenesis or immune response. Although MMPs favor tumor progression according to most data, some results suggest, on the contrary, that MMPs could somehow inhibit tumor progression. This dual regulation emphasizes the complexity of MMPs network in this setting. The main involved mechanisms are summarized in Table 2 and detailed more precisely below. Invasion and Metastasis Metastasis formation requires multiple steps. First, cancer cells have to cross the epithelial basement membrane and invade the surrounding stroma. They enter blood or lymphatic vessels, extravasate and finally establish new proliferating colonies. Experimental evidence for the role of MMPs in metastasis is based on in vitro invasion assays: TIMPs inhibits, and MMP-2, -3, -13 and -14 promote invasion of cell lines through collagen type I or matrigel (experimental ECM secreted by the

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TABLE 2 MMPs and Carcinogenesis Favor carcinogenesis Invasion/ metastasis Angiogenesis Proliferation

Apoptose Immune response

Disfavor carcinogenesis

Destruction of ECM Disruption in chemokine/ Remodeling of ECM chemokine Cleavage of cell receptor receptor signal? Remodeling of ECM Direct action on endothelial cells? Release of growth factors Activation of TGFb Release of (membrane bounded or ECM FasL, TNFa trapped) Disruption in integrins regulation Release of both anti-and proapoptotic factors Cleavage of IL2-R Chemokines disruption Activation IGFb

Abbreviations: ECM, extracellular matrix; FasL, Fas ligand; ILZ-1, interleukin Z-R; MMP, matrix metalloproteinase; TGF, transforming growth factor; TNF, tumor necrosis factor.

Engelbrecht-Holm-Swarm mouse sarcoma cell line) (30,31). In vivo xenograft metastasis assays confirm this result: downregulation of MMP-9 in cancer cells is associated with reduction of the number of metastases formed in the lungs of mice after cancer cell injection (32), and Mmp2- and Mmp9-null mice exhibit less lung metastasis as compared with wild-type mice (33). During the first step of migration, cancer cells must detach from both neighboring cells and the surrounding matrix. MMPs are involved in this process by cleaving CD44 (main receptor of hyaluronan) (19) or E-cadherin. Cleavage of E-cadherin releases in addition a fragment that promotes cell invasion in a paracrine manner in vitro, possibly by binding to and interfering with the function of other full-length E-cadherin molecules (18). This event is also thought to participate in the epithelial to mesenchymal transition, a phenotypic change of cancer cell associated with invasion potency (34–36). Activation of PAR1 by MMP-1 has also been shown to be linked to invasion and tumorigenesis (37). MMPs participate in the late events in the metastatic process, when the cancer cells must enter, survive and exit the blood or lymphatic vessels. For example, MMP-9 seems to be necessary for intravasation (38) and overexpression of MMP-14 (MT-MMP1) increases the number of cancer cells that survive intravenous injection in an experimental metastasis assay (39). On the other hand, implication of MMP activity in extravasation is less clear. Experimentally, TIMP1-overexpressing cancer cells exit the vasculature equally as well as control cells (40). However, metastases are fewer and smaller due to diminished cancer cell growth after extravasation (40). The proliferation at the secondary site, the final step in the establishment of secondary tumors, then probably also involves MMP activity. Regulation of Angiogenesis Angiogenesis is actually known as a crucial aspect of carcinogenesis. The role of MMPs in this process is accessed by animal models where endogenous and synthetic MMP inhibitors reduce tumor angiogenesis (41–43). The first hypothesis would be that MMPs simply degrade ECM and then favor endothelial cell invasion during formation of new vessels (44), but data suggest that MMPs are also involved in direct regulation of endothelial cell migration (45) or regulation of VEGF circulating level (46).


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On the other hand, MMPs may inhibit angiogenesis by releasing fragments with reduce proliferation and/or invasion of endothelial cell (angiostatin and endostatin) (47–50). MMPs and Growth Factors Three mechanisms have been described to explain the impact of MMPs on growthpromoting signals. First, MMPs or ADAMs release the cell-membrane–bound precursors of some growth factors (e.g., TGF-a) (20). Second, some growth factors are sequestered by ECM proteins (e.g., IGFs) and can be released when ECM is degraded by MMPs (51,52). Third, MMPs could disrupt antiproliferation mediated by integrins (53). On the contrary, cancer-cell growth might be negatively regulated by MMPs, for example, by activation of TGF-b (54) or generation of proapoptotic molecules such as Fas ligand (FasL) or TNF-a. Modulation of Apoptosis MMPs or ADAMs actively participate in the apoptotic process by cleaving VEcadherin, PECAM-1 and E-cadherin during apoptosis of endothelial or epithelial cells (55–57). In fact, MMPs have both pro- and antiapoptotic actions. Whereas overexpression of MMP-3 seems to favorite apoptosis in mammary epithelial cells (58), MMP-7 is able to promote cell survival by cleaving proheparin–binding epidermal growth factor (pro-HB-EGF) to generate mature HB-EGF, which stimulates HER4 and then inhibits apoptosis (59). In addition, MMP-7 is involved in the release of membrane-bound FASL, a transmembrane stimulator of the death receptor FAS. Released FASL induces apoptosis of neighboring cells, or decreases cancer-cell apoptosis, depending on the system (60,61). Moreover, the release of IGFs mediated by MMPs could act as a survival factor. This mechanism is thought to explain the results of tumor assay models where overexpression of MMP11 decreases spontaneous apoptosis and of xenograft experiments on Mmp11-null mice where cancer cell injection results in a higher rate of spontaneous apoptosis than in wild-type hosts (62). MMPs and Immune Response MMPs are implicated in the escape mechanisms that are observed in tumors that prevent the immune system from recognizing and attacking cancer cells. IL-2Ra can be cleaved by MMPs (especially MMP-9) that suppress the proliferation of T lymphocytes (63). MMPs also activate TGF-b, an important inhibitor of the T-lymphocyte response against tumors (21,64). MMPs are additionally involved in the regulation of chemoattractant factors (activation of CXCL8, inactivation of CXCL-1, -4, -7, -12, or CCL7). This action leads to a lower infiltration of tumors by neutrophil and macrophages (62,65,66). Interestingly, chemokines and chemokine receptors are thought to be involved in the metastasis process as the biological support of the “seed and soil” theory (67,68). MMPs activity on this system may then inhibit metastasis. Tumor–Stroma Interactions Cancer cells were initially considered the only source of MMPs during previously described phenomena (69). This hypothesis is correct for MMP-7 but most MMPs are in fact synthesized by stromal cells. In situ hybridization experiments

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demonstrate that mRNA of MMP-1, -2, -3, -14 are primarily localized in stromal fibroblasts, especially in proximity to invading cancer cells but not in carcinoma cells in various human tumors (70–72). Cancer cells might stimulate tumor stromal cells to synthesize MMPs in a paracrine manner (7). MMPs that are secreted by stromal cells can still be recruited to the cancer-cell membrane. Indeed, MMP-2 mRNA is expressed by stromal cells of human breast tumors, whereas MMP-2 protein is found on both stromal and cancer-cell membranes (73). Besides interleukins and growth factors, EMMPRIN (CD147) might be a key molecule for the stimulation of MMPs, secretion by stromal cells. This 58-kDa glycoprotein is highly expressed during embryogenesis and was originally designated tumor collagenase stimulating factor (TCSF) after isolation and purification from the plasma membrane of cancer cells and demonstration of its function in stimulating fibroblast synthesis of MMP-1 (74). This action was also observed for fibroblast synthesis of MMP-2 and MMP-3 (75). In vitro, recombinant EMMPRIN or EMMPRIN purified from cancer cells to stimulate fibroblast production of MMPs (75,76) and cancer cell lines transfected with EMMPRIN cDNA exhibits more secondary tumors in xenograft metastasis assays (77). Regulation of EMMPRIN synthesis by cancer cells might be controlled by HER1 pathways (78). Note that this glycoprotein is also thought to be involved in HIV internalization (79,80). After secretion from fibroblasts, MMP-1 is able to bind to EMMPRIN on the tumor cell surface, forming a highly active proteolytic system involved in ECM degradation and activation of other MMPs (81). CLINICAL ASPECTS MMP and Prognosis The expression and activity of MMPs are increased in almost every type of human cancer, and multiple studies show a negative association between MMP levels and prognosis. This correlates with advanced tumor stage, increased invasion and metastasis, and shortened survival. For example, expression of MMP-11 in breast cancer has been associated with malignant disease only, and it is not expressed in normal breast tissue or benign fibroadenomas (82). MMP-11 expression was observed not only in invasive breast carcinomas but also in some in situ carcinomas where other factors indicated a high risk for the development of an invasive phenotype. Several studies have reported an association between MMP-11 expression, lymph node metastasis, and/or shorter disease-free survival in patients with infiltrating ductal carcinoma of the breast (83–86). In addition, MMP-14 was identified as an independent factor of poor overall survival for nonmetastatic breast cancer when adjusted for clinical prognostic factors (87). In addition, increased tumoral expression of MMP-3, -11, and -14 could be independent negative prognostic factors for survival in small cell cancer while decreased tumoral expression of TIMP-1 is significant for response (88). In colon cancer samples, immunohistochemical detection of interstitial MMP-1 and -7 expression are associated with a poor prognosis independent of Dukes’ stage (89). MMP-1 and MMP-7 expression have also been suggested to be of prognostic value in esophageal cancer (90,91). Pancreas cancer patients with MMP-7 carcinoma had a significantly shorter overall survival time than did those with MMP-7-negative carcinoma and MMP-7 was a significant independent prognostic factor for overall survival in multivariate analysis (92).


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Concerning prostate cancer, serum levels of MMP-2 were found to be significantly higher in men with prostate cancer than in those with benign prostate hyperplasia or with no disease (93). Plasma TIMP-1 is also associated with prostate cancer but not with benign prostate hyperplasia or normal prostate tissue (94). Another study reports that tissue expression of activated MMP-2 was associated with Gleason score, with the highest levels found in tumors with the highest Gleason score and in lymph node metastases (95). Downregulation of E-cadherin and upregulation of MMP-9 and MMP-2 in localized pancreatic tumors had significant prognostic value. The ratio of the mean of the expression of MMP-2 and MMP-9 to E-cadherin expression (MMP: E-cadherin ratio) was found higher in patients with recurrent disease compared with patients remaining in remission (96). Expression of MMP-2 is similarly associated with poor prognosis for ovarian cancer (97), and elevation of serum levels of either MMP-2 or MMP-3 or both could be new predictors of recurrence in patients with advanced urothelial carcinoma after complete resection (98). There are few cases in which increased expression of specific MMPs is correlated with a favorable prognosis. In colon cancer, MMP-9 expression by infiltrating macrophages is associated with reduced metastases (99), although MMP-9 expression in tumors was more recently shown as a negative prognostic factor (100). MMP-12 expression by carcinoma cells is associated with increased survival (101). The mechanisms underlined by this observation are not clear. MMP activity may be associated with the generation of antitumor molecules such as proapoptotic factors or to the recruitment of cytotoxic T-cells. On the other hand, MMP activity in these cases may be irrelevant and the MMPs would just be markers of differentiation of the cancer cells or of an immune reaction. Another hypothesis could derive from inappropriate tests to evaluate MMP levels that are usually defined according to immunochemistry or molecular biology (in situ hybridization or RT–PCR) on tumor samples. As for MMPs being secreted as pro-enzymes, the level of synthesis is, however, not correlated with level of activity, which would be best estimated by zymography (but fresh tissue is needed). Some studies found that high levels of TIMP-1 and -2 also correlate with a poor prognosis. This might reflect the fact that the balance between expression of MMPs and TIMPs, although still favoring the MMPs, is at a higher overall level during the increased matrix remodeling that occurs in tumor progression. High-TIMP levels would therefore be associated with tumor progression and a worse prognosis, but would not cause it. Prognostic value of MMP-2/TIMP-2 imbalance has, for example, been proven for hepatocarcinoma (high level of TIMP-2 and low level of MMP-2 correlated with better relapse-free survival) (102). Targeting MMP in Anticancer Therapy Several agents have been developed in this field. Inhibition of MMP synthesis could be the consequence of other therapies, such as tyrosine kinase receptor signaling inhibition, but we will focus here on compounds that block MMPs activity (MMPI: MMP inhibitors). MMPIs have been developed and have been shown in preclinical systems to inhibit local tumor growth and metastasis (70,103). Because of their noncytotoxic effects on the tumor, phase I studies with MMPIs have sought to establish tolerable doses of drug, suitable for protracted administration, that produce serum levels that exceed the inhibitory concentration of targeted MMPs without causing

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unacceptable normal tissue toxicity. Although preclinical models have demonstrated the antitumoral activity of MMPIs, phase II clinical trials, where objective response was primary end point, failed to demonstrate substantial regression of large primary tumors. The development of MMPIs has proceeded rapidly to phase III trial design with the end point of survival. Disappointing results now lead to reconsidering MPPI development. Three main categories of synthetic MMPI have been developed: the collagen peptidomimetics, the collagen nonpeptidomimetics and the tetracycline derivates. Alternative molecules can also be considered. This classification is summarized in Table 3. Collagen Peptidomimetics MMP Inhibitors The peptidomimetic MMP inhibitors mimic the cleavage sites of MMP substrates and include batimastat and marimastat. Batimistat is an inhibitor of MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9, and is effective in reducing the frequency of metastases and improving survival for the treated animals (104), but cannot be administered orally and is no longer tested for the treatment of human cancer. Marimastat, similar to batimistat, is a broad-spectrum peptidomimetic MMPI with inhibitory activity against MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9 (103,105). The principal effect of marimastat is to retard tumor growth and metastatic spread. It does not display cytotoxic activity in cell cultures, and no tumor regression is observed in animal models but a benefit in survival was observed in treated animals (106,107). The molecules have been combined with cytotoxic agents with evidence of activity in animal models (108). Unlike batimistat, marimastat is orally bioavailable (recommended administration twice a day). In clinical trials, musculoskeletal symptoms consisting of pain and tenderness in muscles and joints is the most common toxicity, develops in approximately 30% of patients treated at 10 mg b.i.d. after a median of 3–6 months. Symptoms diminished when the drug was no longer administrated (109,110). This toxicity was observed with other MMPIs and may be mediated by inhibition of the tumor necrosis factor alpha converting enzyme (111). Phase II trials may have exhibited encouraging results in terms of progression-free survival (gliobastoma, pancreas cancer, lung cancer) (112,113), but phase III trials in patients with advanced malignancies have yielded modest results. For example, a trial for advanced pancreatic cancer intended to detect differences in survival between patients treated with various doses of marimastat and gemcitabine failed to detect

TABLE 3 Matrix Metalloproteinase Inhibitors (MMPIs) Category Collagen peptidomimetics MMPI Collagen non-peptidomimetics MMPI

Tetracycline derivates Other molecules

Compound Batimistat Marimastat Prinomastat/AG3340 BAY 12-9566/tanomastat BMS 275291 COL-3 Bisphosphonates AE-941 ( )Epigallocatechin-3-gallate Urokinase-activated recombinant anthrax toxin


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increased survival for the marimastat-treated groups even if the highest dose of marimastat was as effective as the conventional therapy (114). The combination of marimastat with gemcitabine provided no benefit in survival compared to gemcitabine alone (115). Another trial evaluated marimastat against placebo in 369 patients with inoperable gastric cancer who had received no more than one prior chemotherapy regimen. No improvement was detected in the primary end point of the trial, overall survival. Nevertheless, subset analysis suggested an improvement in survival in patients without distant metastases at the time of enrollment, raising the question of the use of MMPIs in order to prevent metastatic spread (116). Collagen Nonpeptidomimetics MMP Inhibitors The nonpeptidomimetic MMPI are synthesized on the basis of the conformation of the MMP active site. This group mainly includes Prinomastat/AG3340, BAY 12-9566/tanomastat and BMS 275291. Preclinical studies of prinomastat have demonstrated reduction in the rate of primary tumor growth and in the number and size of distant metastases in animal tumor models. Furthermore, when prinomastat was administered in combination with a variety of cytotoxic chemotherapeutic agents in these models, antitumor effects were enhanced without an increase in chemotherapy-related toxicity (117,118). Early clinical trials reported dose- and time-dependent musculoskeletal complaints. Prinomastat has been studied in advanced non–small cell lung cancer in combination with platinum-based chemotherapy (gemcitabine and cisplatin) in previously untreated patients and there was no improvement in survival seen with the addition of the MMPI (119). The phase I trial with BAY 12-9566 showed relatively mild adverse effects, and in some cases, disease was stabilized (120). However, all studies with BAY 129566 were terminated when preliminary results from Phase III trials on advanced pancreatic or small cell lung cancers showed significantly poorer survival for groups treated with the drug than for placebo-treated groups (121). In a phase I study, BMS-275291 was well tolerated and dose-limiting toxicities included hypersensitivity (characterized by rash, fever, and dyspnea) and elevations of serum ALT, but not musculoskeletal toxicity (122). BMS-275291 was then thought to be suitable for a prolonged period, during chemotherapy and following completion of treatment without severe musculoskeletal toxicity (123). A randomized phase II/III study was initiated to determine the additional value of BMS-275291 to paclitaxel and carboplatin for advanced non–small cell lung cancer. Final results on 774 patients showed that BMS-275291 added to chemotherapy increases toxicity and does not improve survival in this setting (124). Tetracycline Derivates COL-3, a chemically modified tetracycline is a MMPI that is distinct in its ability to inhibit the activity, activation, and production of MMPs, whereas other MMPIs target only the active enzyme (103). COL-3 inhibits MMP-2 and MMP-9 in vitro and the expression of MMPs in human colon and breast carcinoma cells. It inhibits invasion of various cancer cell lines in vitro, and inhibits tumor growth and metastasis in a rat model (125,126). In a recent phase II trial, COL-3, when administered at 50 mg/day, is both active and reasonably well tolerated (skin toxicity) in the treatment of AIDS-related Kaposi sarcoma, with significant declines

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in MMP-2 and MMP-9 plasma levels from baseline to minimum value with treatment (127). Although formal conclusion is not possible due to the limited nature of a phase II trial, COL-3 warranted further investigation. Other Molecules Another group of drugs might also possess unrecognized activity against the MMPs. The bisphosphonates were originally developed for the treatment of disturbances in calcium homeostasis and for the prevention and palliation of bone metastasis. Their mechanism of action has not been completely elucidated, but they inhibit the enzymatic activity of MMPs (128). Unconventional MMPIs are also being tested in clinical trials: AE-941 (Neovastat), an extract from shark cartilage, inhibits MMPs (129), shows antiangiogenic effects in animal experiments and is now in Phase III clinical trials for the treatment of metastatic non–small cell lung cancer (130). (–)Epigallocatechin3-gallate, a component of green tea, also acts as MMP-2 and -9 inhibitors in vitro (131,132). Finally, cytotoxic agents that are activated by MMPs have been proposed to take advantage of the increased MMP activity in tumors. These include recombinant proteins containing anthrax toxin fused with an MMP cleavage site. These recombinant toxin proteins are activated by MMP cleavage at the cell surface and are internalized by the cell, leading to cell death on in vitro models (133,134). PERSPECTIVES Disappointing results of clinical trials involving MMPIs led to reconsidering the way to use these molecules. The hypothesis was raised that MMPIs would not be expected to have a beneficial effect in patients with active metastatic disease, but they could have a potential role as a component of adjuvant therapy in patients with a carcinoma that has never responded to chemotherapy or ever been completely resected but who are at high risk for relapse. A phase III trial for glioblastoma after completion of surgery and radiotherapy found no improvement in time to progression or overall survival with single agent marimastat or placebo (135). Similarly, marimastat did not prolong survival in patients with small cell cancer who achieved a complete or partial response to initial chemotherapy (136) or in patients with metastatic breast cancer who have responding or stable disease after first line chemotherapy (137). In those studies, marimastat had a negative impact on quality of life due to toxicity, raising the problem of the use of MMPIs as adjuvant therapy for early stage cancer. Phase II studies of MMPIs as adjuvant therapy for operable breast cancer confirm this concern, with high rates of treatment discontinuation (marimastat or BMS-275291) (138,139). Patients developing side effects may, however, have a benefit in survival according to a subset analysis from a double-blind placebo trial of adjuvant treatment by marimastat in patients with unresectable colorectal liver metastases, where no significant effect on survival was observed in all patients (140). BAY 129566 was also evaluated in a randomized phase III as maintenance therapy in patients with advanced ovarian cancer responsive to primary surgery and paclitaxel/platinum containing chemotherapy. The treatment was generally well tolerated and, at the time of the final analysis, there was no evidence of an impact of BAY 12-9566 on progression-free survival or global survival (141). The reasons


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for the disappointing results observed for MMPIs as cancer therapy are not definitively clear. Preclinical data supported this approach, but it is now established that the presence of the target does not mean that the target is relevant. These data suggest that preclinical models may be insufficient. First of all, the complexity of a metastatic process requires targeting multiple steps and biological processes rather than only MMPs. In addition, MMP inhibition may have paradoxical effects that promote rather than inhibit tumor growth and angiogenesis. This dual activity, which has been exposed previously in this chapter and summarized in Table 2, should lead us to consider MMP as a complex network. It has become clear that MMPs do more than degrade structural ECM proteins to promote invasion and metastasis, and a better understanding of the functions of the MMPs would be helpful to determined when and how they could be targeted. Future studies may, for example, focus on downstream effectors of MMPs that are shown to be involved in invasion, such as EMMPRIN or PAR1 (37,77). REFERENCES 1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100:57–70. 2. Bissell MJ, Radisky D. Putting tumours in context. Nat Rev Cancer 2001; 1:46–54. 3. Yurchenco PD, Schittny JC. Molecular architecture of basement membranes. Faseb J 1990;4:1577–90. 4. Egeblad M, Werb Z. New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2002; 2:61–74. 5. Woessner JF. The matrix metalloproteinase family, In: Parks WC, Mecham RP, eds. Matrix Metalloproteinases. San Diego, CA: Academic Press, 1998:1–14. 6. Sato H, Okada Y, Seiki M. Membrane-type matrix metalloproteinases (MT-MMPs) in cell invasion. Thromb Haemost 1997; 78:497–500. 7. Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol 2001; 17:463–516. 8. Yu Q. Stamenkovic I. Localization of matrix metalloproteinase 9 to the cell surface provides a mechanism for CD44-mediated tumor invasion. Genes Dev 1999; 13: 35–48. 9. Brooks PC, Stromblad S, Sanders LC, et al. Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin alpha v beta 3. Cell 1996; 85:683–93. 10. Loechel, F., Gilpin BJ, Engvall E, Albrechtsen R, Wewer UM. Human ADAM 12 (meltrin alpha) is an active metalloprotease. J Biol Chem 1998; 273: 16993–7. 11. Wolfsberg TG, Primakoff P, Myles DG, White JM. ADAM, a novel family of membrane proteins containing a Disintegrin and Metalloprotease domain: multipotential functions in cell-cell and cell-matrix interactions. J Cell Biol 1995; 131:275–8. 12. Kuno K, Kanada N, Nakashima E, Fujiki F, Ichimura F, Matsushima K. Molecular cloning of a gene encoding a new type of metalloproteinase-disintegrin family protein with thrombospondin motifs as an inflammation associated gene. J Biol Chem 1997; 272:556–62. 13. Kuno K, Terashima Y, Matsushima K. ADAMTS-1 is an active metalloproteinase associated with the extracellular matrix. J Biol Chem 1999; 274:18821–6. 14. Streuli C. Extracellular matrix remodelling and cellular differentiation. Curr Opin Cell Biol 1999; 11:634–40. 15. Giannelli G, Falk-Marzillier J, Schiraldi O, Stetler-Stevenson WG, Quaranta V. Induction of cell migration by matrix metalloprotease-2 cleavage of laminin-5. Science 1997; 277:225–8. 16. Xu J, Rodriguez D, Petitclerc E, et al. Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo. J Cell Biol 2001; 154:1069–79.

326

Vignot and Spano

17. Hangai M, Kitaya N, Xu J, et al. Matrix metalloproteinase-9-dependent exposure of a cryptic migratory control site in collagen is required before retinal angiogenesis. Am J Pathol 2002; 161:1429–37. 18. Noe V, Fingleton B, Jacobs K, et al. Release of an invasion promoter E-cadherin fragment by matrilysin and stromelysin-1. J Cell Sci 2001; 114(Pt 1):111–18. 19. Kajita M, Itoh Y, Chiba T, et al. Membrane-type 1 matrix metalloproteinase cleaves CD44 and promotes cell migration. J Cell Biol 2001; 153:893–904. 20. Peschon JJ, Slack JL, Reddy P, et al. An essential role for ectodomain shedding in mammalian development. Science 1998; 282:1281–4. 21. Yu Q, Stamenkovic I. Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis. Genes Dev 2000; 14:163–76. 22. Levi E, Fridman R, Miao HQ, Ma YS, Yayon A, Vlodavsky I. Matrix metalloproteinase 2 releases active soluble ectodomain of fibroblast growth factor receptor 1. Proc Natl Acad Sci U S A 1996; 93:7069–74. 23. Codony-Servat J, Albanell J, Lopez-Talavera JC, Arribas J, Baselga J. Cleavage of the HER2 ectodomain is a pervanadate-activable process that is inhibited by the tissue inhibitor of metalloproteases-1 in breast cancer cells. Cancer Res 1999, 59:1196–201. 24. Nath D, Williamson NJ, Jarvis R, Murphy G. Shedding of c-Met is regulated by crosstalk between a G-protein coupled receptor and the EGF receptor and is mediated by a TIMP-3 sensitive metalloproteinase. J Cell Sci 2001; 114(Pt 6):1213–20. 25. Fini ME, Cook JR, Mohan R, et al. Regulation of MMP gene expression, In: Parks WC, Mecham RP, eds. Matrix Metalloproteinases. San Diego, CA: Academic Press, 1998:299–356. 26. Sottrup-Jensen L, Birkedal-Hansen H. Human fibroblast collagenase-alpha-macroglobulin interactions. Localization of cleavage sites in the bait regions of five mammalian alpha-macroglobulins. J Biol Chem 1989; 264:393–401. 27. Yang Z, Strickland DK, Bornstein P. Extracellular matrix metalloproteinase 2 levels are regulated by the low density lipoprotein-related scavenger receptor and thrombospondin 2. J Biol Chem 2001; 276:8403–8. 28. Bein K, Simons M. Thrombospondin type 1 repeats interact with matrix metalloproteinase 2. Regulation of metalloproteinase activity. J Biol Chem 2000; 275:32167–73. 29. Khatib AM, Siegfried G, Chretien M, Metrakos P, Seidah NG. Proprotein convertases in tumor progression and malignancy: novel targets in cancer therapy. Am J Pathol 2002; 160:1921–35. 30. Deryugina EI, Luo GX, Reisfeld RA, Bourdon MA, Strongin A. Tumor cell invasion through matrigel is regulated by activated matrix metalloproteinase-2. Anticancer Res 1997; 17:3201–10. 31. Ala-Aho R, Johansson N, Baker AH, Kahari VM. Expression of collagenase-3 (MMP13) enhances invasion of human fibrosarcoma HT-1080 cells. Int J Cancer 2002; 97:283–9. 32. Hua J, Muschel RJ. Inhibition of matrix metalloproteinase 9 expression by a ribozyme blocks metastasis in a rat sarcoma model system. Cancer Res 1996; 56:5279–84. 33. Itoh T, Tanioka M, Matsuda H, et al. Experimental metastasis is suppressed in MMP9-deficient mice. Clin Exp Metastasis 1999; 17:177–81. 34. Lochter A, Galosy S, Muschler J, Freedman N, Werb Z, Bissell MJ. Matrix metalloproteinase stromelysin-1 triggers a cascade of molecular alterations that leads to stable epithelial-to-mesenchymal conversion and a premalignant phenotype in mammary epithelial cells. J Cell Biol 1997; 139:1861–72. 35. Birchmeier C, Birchmeier W, Brand-Saberi B. Epithelial-mesenchymal transitions in cancer progression. Acta Anat (Basel) 1996; 156: 217–26. 36. Sternlicht MD, Lochter A, Sympson CJ, et al. The stromal proteinase MMP3/stromelysin-1 promotes mammary carcinogenesis. Cell 1999; 98:137–46. 37. Boire A, Covic L, Agarwal A, Jacques S, Sherifi S, Kuliopulos A. PAR1 is a matrix metalloprotease-1 receptor that promotes invasion and tumorigenesis of breast cancer cells. Cell 2005; 120:303–13.


327

38. Kim J, Yu W, Kovalski K, Ossowski L. Requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay. Cell 1998; 94:353–62. 39. Tsunezuka Y, Kinoh H, Takino T, et al. Expression of membrane-type matrix metalloproteinase 1 (MT1-MMP) in tumor cells enhances pulmonary metastasis in an experimental metastasis assay. Cancer Res 1996; 56:5678–83. 40. Koop S, Khokha R, Schmidt EE, et al. Overexpression of metalloproteinase inhibitor in B16F10 cells does not affect extravasation but reduces tumor growth. Cancer Res 1994; 54:4791–7. 41. Gatto C, Rieppi M, Borsotti P, et al. BAY 12–9566, a novel inhibitor of matrix metalloproteinases with antiangiogenic activity. Clin Cancer Res 1999; 5:3603–7. 42. Martin DC, Sanchez-Sweatman OH, Ho AT, Inderdeo DS, Tsao MS, Khokha R. Transgenic TIMP-1 inhibits simian virus 40 T antigen-induced hepatocarcinogenesis by impairment of hepatocellular proliferation and tumor angiogenesis. Lab Invest 1999; 79:225–34. 43. Oh J, Takahashi R, Kondo S, et al. The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis. Cell 2001; 107:789–800. 44. Zijlstra A, Aimes RT, Zhu D, et al. Collagenolysis-dependent angiogenesis mediated by matrix metalloproteinase-13 (collagenase-3). J Biol Chem 2004; 279:27633–45. 45. Oh J, Seo DW, Diaz T, et al. Tissue inhibitors of metalloproteinase 2 inhibits endothelial cell migration through increased expression of RECK. Cancer Res 2004; 64:9062–9. 46. Bergers G, Brekken R, McMahon G, et al. Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat Cell Biol 2000; 2:737–44. 47. Dong Z, Kumar R, Yang X, and Fidler IJ. Macrophage-derived metalloelastase is responsible for the generation of angiostatin in Lewis lung carcinoma. Cell 1997; 88:801–10. 48. Houghton AM, Grisolano JL, Baumann ML, et al. Macrophage elastase (matrix metalloproteinase-12) suppresses growth of lung metastases. Cancer Res 2006; 66:6149–55. 49. Cornelius LA, Nehring LC, Harding E, et al. Matrix metalloproteinases generate angiostatin: effects on neovascularization. J Immunol 1998; 161:6845–52. 50. Gorrin-Rivas MJ, Arii S, Furutani M, et al. Mouse macrophage metalloelastase gene transfer into a murine melanoma suppresses primary tumor growth by halting angiogenesis. Clin Cancer Res 2000, 6:1647–54. 51. Manes S, Mira E, Barbacid MM, et al. Identification of insulin-like growth factorbinding protein-1 as a potential physiological substrate for human stromelysin-3. J Biol Chem 1997; 272:25706–12. 52. Manes S, Llorente M, Lacalle RA, et al. The matrix metalloproteinase-9 regulates the insulin-like growth factor-triggered autocrine response in DU-145 carcinoma cells. J Biol Chem 1999; 274:6935–45. 53. Agrez M, Chen A, Cone RI, Pytela R, Sheppard D. The alpha v beta 6 integrin promotes proliferation of colon carcinoma cells through a unique region of the beta 6 cytoplasmic domain. J Cell Biol 1994; 127:547–56. 54. Derynck R, Akhurst RJ, Balmain A. TGF-beta signaling in tumor suppression and cancer progression. Nat Genet 2001; 29:117–29. 55. Herren B, Levkau B, Raines EW, Ross R. Cleavage of beta-catenin and plakoglobin and shedding of VE-cadherin during endothelial apoptosis: evidence for a role for caspases and metalloproteinases. Mol Biol Cell 1998; 9:1589–601. 56. Ilan N, Mohsenin A, Cheung L, Madri JA. PECAM-1 shedding during apoptosis generates a membrane-anchored truncated molecule with unique signaling characteristics. Faseb J 2001; 15:362–72. 57. Steinhusen U, Weiske J, Badock V, Tauber R, Bommert K, Huber O. Cleavage and shedding of E-cadherin after induction of apoptosis. J Biol Chem 2001; 276:4972–80. 58. Alexander CM, Howard EW, Bissell MJ, Werb Z. Rescue of mammary epithelial cell apoptosis and entactin degradation by a tissue inhibitor of metalloproteinases-1 transgene. J Cell Biol 1996; 135(6 Pt 1):1669–77.

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Vignot and Spano

59. Yu WH, Woessner JF, Jr., McNeish JD, Stamenkovic I. CD44 anchors the assembly of matrilysin/MMP-7 with heparin-binding epidermal growth factor precursor and ErbB4 and regulates female reproductive organ remodeling. Genes Dev 2002; 16:307–23. 60. Powell WC, Fingleton B, Wilson CL, Boothby M, Matrisian LM. The metalloproteinase matrilysin proteolytically generates active soluble Fas ligand and potentiates epithelial cell apoptosis. Curr Biol 1999; 9:1441–7. 61. Mitsiades N, Yu WH, Poulaki V, Tsokos M, Stamenkovic I. Matrix metalloproteinase7-mediated cleavage of Fas ligand protects tumor cells from chemotherapeutic drug cytotoxicity. Cancer Res 2001; 61:577–81. 62. Boulay A, Masson R, Chenard MP, et al. High cancer cell death in syngeneic tumors developed in host mice deficient for the stromelysin-3 matrix metalloproteinase. Cancer Res 2001; 61:2189–93. 63. Sheu BC, Hsu SM, Ho HN, Lien HC, Huang SC, Lin RH. A novel role of metalloproteinase in cancer-mediated immunosuppression. Cancer Res 2001; 61:237–42. 64. Gorelik L, Flavell RA. Immune-mediated eradication of tumors through the blockade of transforming growth factor-beta signaling in T cells. Nat Med 2001; 7:1118–22. 65. Opdenakker G, Van Damme J. Cytokine-regulated proteases in autoimmune diseases. Immunol Today 1994; 15:103–7. 66. McQuibban GA, Gong JH, Wong JP, Wallace JL, Clark-Lewis I, Overall CM. Matrix metalloproteinase processing of monocyte chemoattractant proteins generates CC chemokine receptor antagonists with anti-inflammatory properties in vivo. Blood 2002; 100:1160–7. 67. Andre F, Cabioglu N, Assi H, Sabourin JC, Delaloge S, Sahin A, et al. Expression of chemokine receptors predicts the site of metastatic relapse in patients with axillary node positive primary breast cancer. Ann Oncol 2006; 17:945–51. 68. Muller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, et al. Involvement of chemokine receptors in breast cancer metastasis. Nature 2001; 410:50–6. 69. Liotta LA. Cancer cell invasion and metastasis. Sci Am 1992; 266:54–9, 62–3. 70. Nelson AR, Fingleton B, Rothenberg ML, Matrisian LM. Matrix metalloproteinases: biologic activity and clinical implications. J Clin Oncol 2000; 18:1135–49. 71. Basset P, Bellocq JP, Wolf C, Stoll I, Hutin P, Limacher JM, et al. A novel metalloproteinase gene specifically expressed in stromal cells of breast carcinomas. Nature 1990; 348:699–704. 72. Heppner KJ, Matrisian LM, Jensen RA, Rodgers WH. Expression of most matrix metalloproteinase family members in breast cancer represents a tumor-induced host response. Am J Pathol 1996; 149:273–82. 73. Polette M, Gilbert N, Stas I, Nawrocki B, Noel A, Remacle A, et al. Gelatinase A expression and localization in human breast cancers. An in situ hybridization study and immunohistochemical detection using confocal microscopy. Virchows Arch 1994; 424:641–5. 74. Biswas C, Zhang Y, DeCastro R, Guo H, Nakamura T, Kataoka H, et al. The human tumor cell-derived collagenase stimulatory factor (renamed EMMPRIN) is a member of the immunoglobulin superfamily. Cancer Res 1995; 55:434–9. 75. Kataoka H, DeCastro R, Zucker S, Biswas C. Tumor cell-derived collagenase-stimulatory factor increases expression of interstitial collagenase, stromelysin, and 72-kDa gelatinase. Cancer Res 1993; 53:3154–8. 76. Guo H, Zucker S, Gordon MK, Toole BP, Biswas C. Stimulation of matrix metalloproteinase production by recombinant extracellular matrix metalloproteinase inducer from transfected Chinese hamster ovary cells. J Biol Chem 1997; 272:24–7. 77. Zucker S, Hymowitz M, Rollo EE, Mann R, Conner CE, Cao J, et al. Tumorigenic potential of extracellular matrix metalloproteinase inducer. Am J Pathol 2001; 158:1921–8. 78. Menashi S, Serova M, Ma L, Vignot S, Mourah S, Calvo F. Regulation of extracellular matrix metalloproteinase inducer and matrix metalloproteinase expression by amphiregulin in transformed human breast epithelial cells. Cancer Res 2003; 63:7575–80.


329

79. Pushkarsky T, Zybarth G, Dubrovsky L, Yurchenko V, Tang H, Guo H, et al. CD147 facilitates HIV-1 infection by interacting with virus-associated cyclophilin A. Proc Natl Acad Sci U S A 2001; 98:6360–5. 80. Yurchenko V, Zybarth G, O’Connor M, Dai WW, Franchin G, Hao T, et al. Active site residues of cyclophilin A are crucial for its signaling activity via CD147. J Biol Chem 2002; 277:22959–65. 81. Guo H, Li R, Zucker S, Toole BP. EMMPRIN (CD147), an inducer of matrix metalloproteinase synthesis, also binds interstitial collagenase to the tumor cell surface. Cancer Res 2000; 60:888–91. 82. Wolf C, Rouyer N, Lutz Y, Adida C, Loriot M, Bellocq JP, et al. Stromelysin 3 belongs to a subgroup of proteinases expressed in breast carcinoma fibroblastic cells and possibly implicated in tumor progression. Proc Natl Acad Sci U S A 1993, 90:1843–7. 83. Chenard MP, O’Siorain L, Shering S, Rouyer N, Lutz Y, Wolf C, et al., High levels of stromelysin-3 correlate with poor prognosis in patients with breast carcinoma. Int J Cancer 1996; 69:448–51. 84. Ahmad A, Hanby A, Dublin E, Poulsom R, Smith P, Barnes D, et al. Stromelysin 3: an independent prognostic factor for relapse-free survival in node-positive breast cancer and demonstration of novel breast carcinoma cell expression. Am J Pathol 1998; 152:721–8. 85. Kawami H, Yoshida K, Ohsaki A, Kuroi K, Nishiyama M, Toge T. Stromelysin-3 mRNA expression and malignancy: comparison with clinicopathological features and type IV collagenase mRNA expression in breast tumors. Anticancer Res 1993; 13:2319–23. 86. Tetu B, Brisson J, Lapointe H, Bernard P. Prognostic significance of stromelysin 3, gelatinase A, and urokinase expression in breast cancer. Hum Pathol 1998; 29:979–85. 87. Tetu B, Brisson J, Wang CS, et al. The influence of MMP-14, TIMP-2 and MMP-2 expression on breast cancer prognosis. Breast Cancer Res 2006; 8: R28. 88. Michael M, Babic B, Khokha R, et al. Expression and prognostic significance of metalloproteinases and their tissue inhibitors in patients with small-cell lung cancer. J Clin Oncol 1999; 17:1802–8. 89. Murray GI, Duncan ME, O’Neil P, Melvin WT, Fothergill JE. Matrix metalloproteinase-1 is associated with poor prognosis in colorectal cancer. Nat Med 1996; 2:461–2. 90. Murray GI, Duncan ME, O’Neil P, McKay JA, Melvin WT, Fothergill JE. Matrix metalloproteinase-1 is associated with poor prognosis in oesophageal cancer. J Pathol 1998, 185:256–61. 91. Adachi Y, Itoh F, Yamamoto H, et al. Matrix metalloproteinase matrilysin (MMP-7) participates in the progression of human gastric and esophageal cancers. Int J Oncol 1998; 13:1031–5. 92. Yamamoto H, Itoh F, Iku S, et al. Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human pancreatic adenocarcinomas: clinicopathologic and prognostic significance of matrilysin expression. J Clin Oncol 2001, 19:1118–27. 93. Gohji K, Fujimoto N, Hara I, Fujii A, et al. Serum matrix metalloproteinase-2 and its density in men with prostate cancer as a new predictor of disease extension. Int J Cancer 1998; 79:96–101. 94. Jung K, Nowak L, Lein M, Priem F, Schnorr D, Loening SA. Matrix metalloproteinases 1 and 3, tissue inhibitor of metalloproteinase-1 and the complex of metalloproteinase1/tissue inhibitor in plasma of patients with prostate cancer. Int J Cancer 1997; 74:220–3. 95. Stearns ME, Wang M. Type IV collagenase (M(r) 72,000) expression in human prostate: benign and malignant tissue. Cancer Res 1993; 53:878–83. 96. Kuniyasu H, Ellis LM, Evans DB, et al. Relative expression of E-cadherin and type IV collagenase genes predicts disease outcome in patients with resectable pancreatic carcinoma. Clin Cancer Res 1999, 5:25–33. 97. Westerlund A, Apaja-Sarkkinen M, Hoyhtya M, Puistola U, Turpeenniemi-Hujanen T. Gelatinase A-immunoreactive protein in ovarian lesions-prognostic value in epithelial ovarian cancer. Gynecol Oncol 1999; 75:91–8.

330

Vignot and Spano

98. Gohji K, Fujimoto N, Komiyama T, et al. Elevation of serum levels of matrix metalloproteinase-2 and -3 as new predictors of recurrence in patients with urothelial carcinoma. Cancer 1996; 78:2379–87. 99. Takeha S, Fujiyama Y, Bamba T, Sorsa T, Nagura H, Ohtani H. Stromal expression of MMP-9 and urokinase receptor is inversely associated with liver metastasis and with infiltrating growth in human colorectal cancer: a novel approach from immune/ inflammatory aspect. Jpn J Cancer Res 1997; 88:72–81. 100. Ogata Y, Matono K, Sasatomi T, et al. The MMP-9 expression determined the efficacy of postoperative adjuvant chemotherapy using oral fluoropyrimidines in stage II or III colorectal cancer. Cancer Chemother Pharmacol 2006; 57:577–83. 101. Yang W, Arii S, Gorrin-Rivas MJ, Mori A, Onodera H, Imamura M. Human macrophage metalloelastase gene expression in colorectal carcinoma and its clinicopathologic significance. Cancer 2001; 91:1277–83. 102. Giannelli G, Bergamini C, Marinosci F, et al. Clinical role of MMP-2/TIMP-2 imbalance in hepatocellular carcinoma. Int J Cancer 2002; 97:425–31. 103. Hidalgo M, Eckhardt SG. Development of matrix metalloproteinase inhibitors in cancer therapy. J Natl Cancer Inst 2001; 93:178–93. 104. Zervox EE, Franz MG, Salhab KF, et al. Matrix metalloproteinase inhibition improves survival in an orthotopic model of human pancreatic cancer. J Gastrointest Surg 2000; 4:614–19. 105. Sledge GW, Jr., Qulali M, Goulet R, Bone EA, Fife R. Effect of matrix metalloproteinase inhibitor batimastat on breast cancer regrowth and metastasis in athymic mice. J Natl Cancer Inst 1995; 87:1546–50. 106. Davies B, Brown PD, East N, Crimmin MJ, Balkwill FR. A synthetic matrix metalloproteinase inhibitor decreases tumor burden and prolongs survival of mice bearing human ovarian carcinoma xenografts. Cancer Res 1993; 53:2087–91. 107. Chirivi RG, Garofalo A, Crimmin MJ, et al. Inhibition of the metastatic spread and growth of B16-BL6 murine melanoma by a synthetic matrix metalloproteinase inhibitor. Int J Cancer 1994; 58:460–4. 108. Anderson IC, Shipp MA, Docherty AJP, Teicher BA. Combination therapy including a gelatinase inhibitor and cytotoxic agent reduces local invasion and metastasis of murine Lewis lung carcinoma. Cancer Res 1996; 56:715–18. 109. Wojtowicz-Praga S, Torri J, Johnson M, et al. Phase I trial of Marimastat, a novel matrix metalloproteinase inhibitor, administered orally to patients with advanced lung cancer. J Clin Oncol 1998; 16:2150–6. 110. Nemunaitis J, Poole C, Primrose J, et al. Combined analysis of studies of the effects of the matrix metalloproteinase inhibitor marimastat on serum tumor markers in advanced cancer: selection of a biologically active and tolerable dose for longer-term studies. Clin Cancer Res 1998; 4:1101–9. 111. Tsuji F, Oki K, Okahara A, et al. Differential effects between marimastat, a TNF-alpha converting enzyme inhibitor, and anti-TNF-alpha antibody on murine models for sepsis and arthritis. Cytokine 2002; 17:294–300. 112. Groves MD, Puduvalli VK, Conrad CA, et al. Phase II trial of temozolomide plus marimastat for recurrent anaplastic gliomas: A relationship among efficacy, joint toxicity and anticonvulsant status. J Neurooncol 2006; 80:83–90. 113. Groves MD, Puduvalli VK, Hess KR, et al. Phase II trial of temozolomide plus the matrix metalloproteinase inhibitor, marimastat, in recurrent and progressive glioblastoma multiforme. J Clin Oncol 2002; 20:1383–8. 114. Bramhall SR, Rosemurgy A, Brown PD, Bowry C, Buckels JA. Marimastat as first-line therapy for patients with unresectable pancreatic cancer: a randomized trial. J Clin Oncol 2001; 19:3447–55. 115. Bramhall SR, Schulz J, Nemunaitis J, Brown PD, Baillet M, Buckels JA. A double-blind placebo-controlled, randomised study comparing gemcitabine and marimastat with gemcitabine and placebo as first line therapy in patients with advanced pancreatic cancer. Br J Cancer 2002; 87:161–7. 116. Bramhall SR, Hallissey MT, Whiting J, et al. Marimastat as maintenance therapy for patients with advanced gastric cancer: a randomised trial. Br J Cancer 2002, 86:1864–70.


331

117. Shalinsky DR, Brekken J, Zou H, et al. Broad antitumor and antiangiogenic activities of AG3340, a potent and selective MMP inhibitor undergoing advanced oncology clinical trials. Ann N Y Acad Sci 1999; 878:236–70. 118. Shalinsky DR, Brekken J, Zou H, et al. Marked antiangiogenic and antitumor efficacy of AG3340 in chemoresistant human non-small cell lung cancer tumors: single agent and combination chemotherapy studies. Clin Cancer Res 1999; 5:1905–17. 119. Bissett D, O’Byrne KJ, von Pawel J, et al. Phase III study of matrix metalloproteinase inhibitor prinomastat in non-small-cell lung cancer. J Clin Oncol 2005; 23:842–9. 120. Hirte H, Goel R, Major P, et al. A phase I dose escalation study of the matrix metalloproteinase inhibitor BAY 12–9566 administered orally in patients with advanced solid tumours. Ann Oncol 2000; 11:1579–84. 121. Moore MJ, Hamm J, Dancey J, et al. Comparison of gemcitabine versus the matrix metalloproteinase inhibitor BAY 12-9566 in patients with advanced or metastatic adenocarcinoma of the pancreas: a phase III trial of the National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol 2003; 21:3296–302. 122. Rizvi NA, Humphrey JS, Ness EA, et al. A phase I study of oral BMS-275291, a novel nonhydroxamate sheddase-sparing matrix metalloproteinase inhibitor, in patients with advanced or metastatic cancer. Clin Cancer Res 2004; 10:1963–70. 123. Douillard JY, Peschel C, Shepherd F, et al. Randomized phase II feasibility study of combining the matrix metalloproteinase inhibitor BMS-275291 with paclitaxel plus carboplatin in advanced non-small cell lung cancer. Lung Cancer 2004; 46:361–8. 124. Leighl NB, Paz-Ares L, Douillard JY, et al. Randomized phase III study of matrix metalloproteinase inhibitor BMS-275291 in combination with paclitaxel and carboplatin in advanced non-small-cell lung cancer: National Cancer Institute of CanadaClinical Trials Group Study BR.18. J Clin Oncol 2005; 23:2831–9. 125. Lokeshwar BL, Schwartz GG, Selzer MG, et al. Inhibition of prostate cancer metastasis in vivo: a comparison of 1,23-dihydroxyvitamin D (calcitriol) and EB1089. Cancer Epidemiol Biomarkers Prev 1999; 8:241–8. 126. Seftor RE, Seftor EA, De Larco JE, et al. Chemically modified tetracyclines inhibit human melanoma cell invasion and metastasis. Clin Exp Metastasis 1998; 16:217–25. 127. Dezube BJ, Krown SE, Lee JY, Bauer KS, Aboulafia DM. Randomized phase II trial of matrix metalloproteinase inhibitor COL-3 in AIDS-related Kaposi’s sarcoma: an AIDS Malignancy Consortium Study. J Clin Oncol 2006; 24:1389–94. 128. Boissier S, Ferreras M, Peyruchaud O, et al. Bisphosphonates inhibit breast and prostate carcinoma cell invasion, an early event in the formation of bone metastases. Cancer Res 2000; 60:2949–54. 129. Gingras D, Renaud A, Mousseau N, Beaulieu E, Kachra Z, Beliveau R. Matrix proteinase inhibition by AE-941, a multifunctional antiangiogenic compound. Anticancer Res 2001; 21:145–55. 130. Falardeau P, Champagne P, Poyet P, Hariton C, Dupont E. Neovastat, a naturally occurring multifunctional antiangiogenic drug, in phase III clinical trials. Semin Oncol 2001; 28:620–5. 131. Garbisa S, Biggin S, Cavallarin N, Sartor L, Benelli R, Albini A. Tumor invasion: molecular shears blunted by green tea. Nat Med 1999; 5:1216. 132. Dell’Aica I, Dona M, Sartor L, Pezzato E, Garbisa S. (-)Epigallocatechin-3-gallate directly inhibits MT1-MMP activity, leading to accumulation of nonactivated MMP-2 at the cell surface. Lab Invest 2002; 82:1685–93. 133. Liu S, Netzel-Arnett S, Birkedal-Hansen H, Leppla SH. Tumor cell-selective cytotoxicity of matrix metalloproteinase-activated anthrax toxin. Cancer Res 2000; 60:6061–7. 134. Abi-Habib RJ, Singh R, Liu S, Bugge TH, Leppla SH, Frankel AE. A urokinaseactivated recombinant anthrax toxin is selectively cytotoxic to many human tumor cell types. Mol Cancer Ther 2006; 5:2556–62. 135. Levin VA, Phuphanich S, Yung WK, et al. Randomized, double-blind, placebocontrolled trial of marimastat in glioblastoma multiforme patients following surgery and irradiation. J Neurooncol 2006; 78:295–302. 136. Shepherd FA, Giaccone G, Seymour L, et al. Prospective, randomized, double-blind, placebo-controlled trial of marimastat after response to first-line chemotherapy in

332

137.

138. 139. 140.

141.

Vignot and Spano

patients with small-cell lung cancer: a trial of the National Cancer Institute of CanadaClinical Trials Group and the European Organization for Research and Treatment of Cancer. J Clin Oncol, 2002; 20:4434–9. Sparano JA, Bernardo P, Stephenson P, et al. Randomized phase III trial of marimastat versus placebo in patients with metastatic breast cancer who have responding or stable disease after first-line chemotherapy: Eastern Cooperative Oncology Group trial E2196. J Clin Oncol 2004; 22:4683–90. Miller KD, Gradishar W, Schuchter L, et al. A randomized phase II pilot trial of adjuvant marimastat in patients with early-stage breast cancer. Ann Oncol 2002; 13:1220–4. Miller KD, Saphner TJ, Waterhouse DM, et al. A randomized phase II feasibility trial of BMS-275291 in patients with early stage breast cancer. Clin Cancer Res 2004; 10:1971–5. King J, Zhao J, Clingan P, Morris D. Randomised double-blind placebo control study of adjuvant treatment with the metalloproteinase inhibitor, Marimastat in patients with inoperable colorectal hepatic metastases: significant survival advantage in patients with musculoskeletal side-effects. Anticancer Res 2003; 23:639–45. Hirte H, Vergote IB, Jeffrey JR, et al. A phase III randomized trial of BAY 12-9566 (tanomastat) as maintenance therapy in patients with advanced ovarian cancer responsive to primary surgery and paclitaxel/platinum containing chemotherapy: a National Cancer Institute of Canada Clinical Trials Group Study. Gynecol Oncol 2006; 102:300–8.

20

Src Inhibitors Francisco Cruzalegui Division of Cancer Research and Drug Discovery, Institut de Recherches Servier, Croissy-sur-Seine, France

INTRODUCTION Many excellent reviews have been published describing in great detail the history of the discovery of the Src family kinases (SFKs), their molecular structure, their, biochemical properties, and their functions in normal tissues. In addition, many papers have described the effects of exogenous expression of viral or mutated forms of c-Src (v-Src) that may or may not represent the biology of deregulated c-Src found in cancer cells. In this review, emphasis is given to evidence found in cancer tissue or cell lines derived from tumors. In addition, small molecule inhibitors targeting Src family kinases are reviewed, from the first broad-spectrum compounds described in the early 1990s to the present day drugs used in the clinic. Src FAMILY KINASES AS TARGETS Link Between Src Family Kinases and Cancer c-Src was the first protein kinase to be identified as selective for tyrosine residues. Its tyrosine kinase activity was initially discovered by studying the 60 kDa protein encoded by the v-Src gene in the chicken Rous sarcoma virus (1,2). The presence of a similar protein in vertebrate cells had already been described (3) and the cellular chicken gene was soon characterized (4). Together, these discoveries led to the awarding of the 1989 Nobel Prize in Physiology and Medicine to Harold Varmus and Michael Bishop. According to the official nomenclature for viral and cellular counterparts, the viral form is known as v-Src and the cellular gene as c-Src (5). The domain structure of c-Src is very highly conserved across species and throughout all the 8 SFKs (c-Src, c-Yes, Fyn, Lyn, Fgr, Lck, Hck, Blk) known today in humans. The C-terminal half is a consensus tyrosine kinase domain containing an autophosphorylation site at Tyr419 (Tyr416 in the chicken sequence). The N-terminal half is more divergent and contains a membrane docking sequence that is myristoylated in all SFKs and additionally palmitoylated in all SFKs except c-Src and Blk (6,7), followed by an SH3 domain and an SH2 domain. In all SFKs, the SH2 domain interacts with a phosphorylated tyrosine at position 530 in the C-terminal end (human c-Src nomenclature). The SH2-P-Tyr530 interaction maintains the kinase in a closed conformation preventing autophosphorylation and substrate phosphorylation (8–10). The kinase responsible for this phosphorylation is called Csk (C-terminal Src kinase) (11). Contrary to c-Src, Csk is a cytoplasmic protein that is recruited to the membrane by docking proteins in order to phosphorylate c-Src. Since c-Src is found to be overactivated in tumors, it is likely that Csk is unable to downregulate c-Src activity in cancer cells. Surprisingly, Csk levels were found to be up to 10 times higher in tumors compared with healthy tissue (12). Whether Csk is inactive or simply uncoupled from c-Src inhibition is unclear. 333

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In the chicken viral forms of c-Src and c-Yes (v-Src and v-Yes), the amino acid sequence is truncated and lacks the tyrosine normally phosphorylated by Csk. Consequently, the mutant v-Src and v-Yes are constitutively active and highly oncogenic. However, since its discovery, mammalian c-Src has very rarely been found mutated in tumors or in healthy tissue. In one case, 12% of analyzed advanced colon carcinomas presented a Gln to stop mutation at codon 531 (13). Since that publication, several groups around the world have tested tumor samples looking for activating mutations with little success (14–18). A group in Japan found that 1 out of 68 endometrial carcinomas contained such a mutation (19). In China, a study reported 1 sample out of 110 examined contained the 531 mutation (20). Consequently, it is now accepted that activating mutations are very rare and that the main mechanisms for elevation of c-Src activity in cancers are overexpression at the RNA level and deregulation of c-Src kinase activity by deficient Tyr530 phosphorylation. Another possibility is that a phosphatase that regulates Tyr530 dephosphorylation is absent in tumors (11). A very large proportion of studies have focused on c-Src, it being usually the most abundant SFK in solid tumors and the one for which most tools have been developed. Consequently, despite nearly 30 years since the discovery of SFKs, little has been done to elucidate selective functions for each of them. The lack of inhibitors targeting a particular SFK has hindered the dissection of any possible selective roles of these kinases. In colon cancer, only c-Src and c-Yes appear to be highy activated compared to healthy tissue (21). c-Yes is strongly activated in colon cancers and in colon cancer cell lines, its activity correlating strongly with worse prognosis of colon cancer (22,23). In melanomas, c-Yes activity is 5–10 times higher than in nontransformed melanocytes although it is not found mutated (24). Furthermore, c-Yes found in melanoma cell lines appears to be further activated by neurotrophins and this activation correlates with increased invasive potential of these cell lines (25). Recently, Lyn was identified as the most prevalent SFK in glioblastomas (26). In B cell chronic lymphocytic leukemia (B-CLL), while all other SFKs are expressed normally, Lyn is overexpressed and constitutively activated. In addition, Lyn is found uniformly distributed all over the cell surface of leukemic lymphocytes, unlike normal B cells where Lyn is localized to lipid rafts. Finally, treatment of B-CLL cells from patients with SFK inhibitors induces apoptosis, suggesting that Lyn activity is necessary for driving survival in this form of leukemia (27). In conclusion, it is possible that in some cancer types, SFKs other than c-Src are critical for tumor survival, growth, or metastasis. Inhibitors that target individual SFKs in such pathologies could help reduce potential side effects of broad-spectrum Src inhibitors, due to inhibition of SFKs involved in lymphocyte functions such as Lck (8). Experimental Validation of SFKs as Targets In the case of the Src family, the transforming ability of v-Src or artificially activated mutants introduced in mammalian cells has often been considered as evidence of an important role of c-Src in cancer. In fact, after all these years, relatively little has been published on validation of endogenous c-Src or other SFK as targets in cancer cells. The induction of DNA synthesis in cultured mouse NIH3T3 mouse fibroblasts following PDGF addition has been a paradigm widely used to examine the role of c-Src downstream of growth factor receptors. Using this model, Sara Courtneidge’s group showed in the 1990s that injection of

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dominant-negative forms of c-Src and Fyn or neutralizing antibodies against c-Src, Fyn and Yes into serum-starved fibroblasts could inhibit PDGF-induced DNA synthesis (28), and that this inhibition could be rescued by c-myc overexpression (29). Later on, using the same system, further papers suggested a pathway leading to c-myc expression, requiring Src family kinases and phosphorylation of the adapter protein Shc and potentially Stat3 (30,31). Finally, further work by Serge Roche’s group revealed a tyrosine kinase cascade in which c-Src phosphorylation of cytoplasmic cAbl leads to the removal of a p53-mediated block for entry into S phase following PDGF stimulation (32). Although these experiments enlightened us on the position of c-Src in a signaling cascade triggered by PDGF, the relevance of PDGF-induced DNA synthesis on starved fibroblasts to cancer is unclear, given that these experiments appear not to have been performed using cultured cancer cells. Still using fibroblasts, Roche et al. also explored a potential role for Src family kinases during mitosis (33). In this case, microinjection of antibodies inactivating c-Src, c-Yes, and Fyn into fibroblasts was carried out in cells syncronized to be in G2. This treatment led to a block of cells at the entry to mitosis. Some years later, these experiments were expanded by microinjection of the same antibodies in cells already in mitosis, resulting in binucleate cells (34). These experiments suggest that Src family kinases play a role in cytokinesis and, although indirectly, suggest that deregulated c-Src in tumor cells could also contribute to progression through mitosis. Experimental evidence of a requirement for c-Src in cell growth and survival of a tumor comes from expression of antisense and dominant-negative constructs in colon carcinoma cells. In colon carcinoma HCT15 cells expression of a dominantnegative c-Src construct inhibits migration of cells in vitro and strongly inhibits peritoneal dissemination in SCID mice. The antimigration effect of DN-Src was shown by a reduced invasion of peritoneal tumors into surrounding tissue and their higher fibrotic nature (35). The same group also showed that treatment of mice with PP2, a selective SFK inhibitor, after intrasplenic injection of HT29 cells, reduced the size of liver metastasis obtained (36). Also in colon carcinoma cells, antisense constructs directed to c-Src reduce the ability of these cells to survive to anoikis in vitro (37). Recently, Gallick et al. used c-Src shRNA expression in the metastatic L3.6pl pancreatic tumor cell line to reduce endogenous c-Src levels. In this context, c-Src knock-down had a clear effect on the metastasis potential of these cells when implanted orthotopically but had no impact on growth of the primary tumor. In contrast, treatment of L3.6pl tumors with Dasatinib, a potent broad Src family inhibitor, had an effect both on primary tumor growth and on metastasis (38). This suggests that in the context of this particular pancreatic cell line, inhibition of c-Src alone is sufficient to stop metastasis, but it may not be sufficient in vivo to trigger tumor regression. Finally, a recent paper reports the effect of inducible overexpression of dominant-negative c-Src in mammary cancer cells. This resulted in a reduction of migration and proliferation rate with an accumulation of cells in G1 and tumor growth in vivo (39). As illustrated by the variety of results obtained in different cell lines, the difficulty of validating endogenous c-Src or other SFKs as targets in preclinical studies is that the signaling context of a particular tumor cell line could determine the response obtained. Selecting a tumor cell line based only on its c-Src activity to test the effect of its inhibition could lead to wrong conclusions. This was shown recently by Johnson et al. in a variety of cell NSCLC and H&N cell lines. The authors showed that although the SFK inhibitor Dasatinib inhibited migration in all

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ReceptorTyr Kinase

Adhesion proteins c-Src

c-Src

Shc rasGAP Integrins

ras

Adherens junctions

c-Raf Akt

STAT3

FAK

E-cadherin

ERK Bad Cyclin D Proliferation

Mcl-1 Bim

Bcl-XL

Survival

Tumor growth

VEGF

Angiogenesis

Modulation of matrix adhesion

Reduced cellcell adhesion

Migration, Invasion

FIGURE 1 Signaling pathways downstream of c-Src. c-Src activity, associated to receptors with tyrosine kinase activity (e.g., EGFR, PDGFR, VEGFR), regulates pathways known to be important in cell proliferation, cancer cell survival, and angiogenesis. The resulting effect is overall tumor growth. The pool of c-Src that associates to adhesion proteins regulates the plasticity of focal adhesions via its regulation of FAK activity and the strength of cell–cell adhesions via the downregulation of E-cadherin at cell–cell junctions. This results in an increased propensity to migrate and invade other tissues. Activation or increased expression of proteins is indicated by an ascending arrow. Inhibition or down-regulation are marked with a descending arrow.

these cell lines, it triggered apoptosis in only few. It was revealed that only in those sensitive cells lines, SFK inhibition lead to Mcl-1 and Bcl-XL degradation (40). The lesson from this paper is that given the variety of signaling pathways in which c-Src is involved, each tumor type will have evolved to position c-Src at different nodes of this network (Fig. 1). The role of c-Src in migration, adhesion and metastasis may be generally maintained, independent of other changes in signaling. c-Src, however, becomes essential for pathways important in growth and survival only in particular contexts. For this reason, caution must be used in drawing conclusions from validation experiments that may be carried out in a “wrong” cellular context in which c-Src only has its “generic” function in promoting migration. While the experiments described above addressed the issue of whether c-Src is required for tumor growth or survival, a different question is whether c-Src can be sufficient to generate tumors. This has been addressed using transgenic expression of wild-type or activated forms of c-Src in mice. Webster et al. (41) expressed activated c-Src (mutation F530) in breast under the control of the MMTV promoter leading to hyperplasias but variable progression to real carcinomas. In skin, however, expression of activated c-Src has different effects depending on the exact cellular layer. Expression in the epidermal spinous layer under the control of the keratin 1 promoter, c-SrcF529 (mouse sequence), only led to hyperplasias that showed enhanced sensitivity to skin tumor promotion in a two-stage tumor

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promotion model (DMBA/TPA treatment) (42). Expression of the wild-type human sequence in the epidermal basal cells, driven by the keratin 5 promoter, led to squamous cell carcinomas, although with slow kinetics (70% of transgenic mice after 1 year) and showed enhanced responsiveness to DMBA/TPA treatment. In the same experiments, expression of activated human c-Src (c-Src530F) led to severe skin defects and death soon after birth (43). Later on, the same group reported that inducible expression of human c-Src530F under the control of an inducible promoter provoked hyperplasias and hyperkeratosis. Remarkably, in this inducible skin model, real squamous cell carcinomas were induced along the periphery of the area of punch biopsies, suggesting that secondary insults may cooperate with Src for triggering carcinogenesis (44). These results suggest that elevated c-Src activity may be sufficient to induce the whole process of carcinogenesis depending on the tissue and the signaling context of the cell. Another issue not addressed in transgenic models yet is the possible need for other Src-family kinases to cooperate with c-Src. As described previously, c-Yes is also highly deregulated in melanoma, colon, and maybe other cancers. It is possible that concomitant activation of c-Src, c-Yes or another signaling pathway may be required for full carcinogenesis. DOWNSTREAM SIGNALING CONSEQUENCES OF SFK DEREGULATION As reviewed recently by Gallick (45), c-Src and other SFKs are connected to a large network of signaling pathways. Understanding the signaling context for c-Src in a particular type of cancer cell not only will allow us to define a functional readout that would reflect Src inhibition (migration, proliferation, apoptosis, etc.) but also it will help us to identify potential biomarkers to monitor a pharmacological response to Src inhibitors in preclinical and clinical studies. However, observations made in a particular tumoral cell line about c-Src’s influence on a signaling cascade may not be generalized to all cell types or pathologies. c-Src associates with receptor tyrosine kinases (RTKs) via an interaction between its SH2 domain and phosphorylated tyrosines on the intracellular domain of the receptor. Downstream of an RTK, c-Src can be linked to the Raf-MEK-ERK cascade either by phosphorylating c-Raf and increasing its activity (46) or via phosphorylation of RasGAP, this reducing its GTPase-enhancing activity and increasing the levels of GTP-bound Ras. A third mechanism by which c-Src can activate the ERK cascade is via its phosphorylation of the adaptor protein Shc, which serves as docking point for the Grb2/SOS resulting in Ras activation (30). Increased ERK activity activates transcription of genes involved in cell proliferation such as c-fos and Cyclin D. ERK activity downstream of c-Src may also regulate the protein level of Mcl-1, an antiapoptosis Bcl-2 family member that can be regulated by ERK. Conversely, the proapoptotic protein Bim is destablized by ERK phosphorylation. High activity of the ERK pathway would then lead to higher turnover of Bim and lower apoptosis (47,48). In response to EGF stimulation, c-Src recruitment by the phosphorylated receptor is rapidly increased. This results in a synergistic phosphorylation of STAT3 on Tyr705, allowing it to dimerize and translocate to the nucleus. STAT3 will then bind to the promoter of genes such as myc, VEGF, and Bcl-XL leading to their increased transcription (49,50). In cancer cells, this pathway can be constitutively activated. In breast cancer cells, in particular, STAT3 is highly phosphorylated in a c-Src-dependent manner (51). Another target of STAT3 is the proto-oncogene c-myc.

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Activation of c-myc expression has mainly been described downstream of PDGF stimulation of serum-starved cells (31). Akt is a Ser/Thr protein kinase activated by lipids and by phosphorylation via PDK1. The lipid ligand involved is generated by PI3K which in turn is activated by survival signals. One of the most important substrates for Akt is the proapoptotic protein Bad. When phosphorylated by Akt, Bad is recruited by 14-3-3 proteins and is prevented from triggering apoptosis. c-Src can increase the activity of PDK1 by direct phosphorylation of tyrosines Y373 and Y376, thus increasing survival signaling (52). In cells in which v-Src or mutated forms have been introduced (such as mouse fibroblasts), v-Src is constitutively found associated with focal adhesions and structures known as podosomes (30). In these cells, the prevailing model is that activated c-Src phosphorylates and activates RhoGAP. This in turn reduces the levels of GTP-loaded Rho and results in actin fiber dissassembly. Endogenous c-Src found in cancer cells, in contrast, is localized to endosomal membranes and very little colocalized with the actin cystoskeleton. In nontransformed cells, c-Src participates in fibronectin receptor signaling since c-Src / fibroblasts show reduced adherence to fibronectin and reduced phosphorylation of proteins involved in cellmatrix adhesion. Upon attachment of cells to fibronectin, c-Src is transiently activated and is localized to focal adhesions. Following this, FAK is autophosphorylated on Tyr397, creating a pTyr attachment point for c-Src. c-Src is then able to phosphorylate FAK on Tyr861 and Tyr925 and increase FAK’s kinase activity. These c-Src-mediated phosphorylated tyrosines allow recruitement of Grb2 and activation of the Ras-ERK cascade. The complex formed by FAK and c-Src will also serve as a scaffold for other signaling proteins such as PI3K. The latter will associate with the SH3 domain of c-Src, rendered available by the activation by FAK. Unfortunately, such a detailed molecular dissection of the role of c-Src in cell-matrix adhesion and migration is lacking in cancer cells where nonmutated but deregulated c-Src exists. Indirect evidence for such c-Src–FAK interaction in cancer cell lines is a constitutive level of SFK-dependent phosphorylation of Tyr861 end Tyr925, and high paxillin phosphorylation (30,53). Src activity has also been shown to be associated with reduced cell–cell adhesions and increased dispersion of tumor cells. E-cadherin, the major adhesion molecule in epithelia, is often expressed at lower levels in metastatic tumors. Elevated c-Src activity correlates inversely with the amount of E-cadherin found at cell junctions (54). Inhibition of SFK activity by the selective inhibitor PP2 leads to E-cadherin accumulation at cell–cell junctions (36,55). The effect of Src on cell–cell adhesion is also present in endothelial cells. In this case, VE-cadherin is directly phosphorylated by c-Src (56,57), leading to dissociation of the cadherin–120 catenin–beta catenin complex. This dissociation could induce an increase in vascular permeability and tumor cell extravasation. Finally, c-Src is also involved in angiogenesis in two ways: first, Src can induce transcription of VEGF in tumor cells (58). Second, VEGFR downstream signaling in endothelial cells is also regulated by c-Src (59). Src INHIBITORS Historical Overview Some of the first reported broad-spectrum ATP-competitor kinase inhibitors were reported to inhibit c-Src, such as Tyrphostins, Genistein, Quercetin, and

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Staurosporin. Given the poor selectivity and high toxicity of these molecules, it was assumed that selectivity could not possibly be achieved by targeting the ATP pocket. For most of the 1980s and early 1990s, little progress was made in the pharmaceutical industry toward selective kinase inhibitors. One of the first compounds to be reported were Pfizer’s pyrazolo-pyrimidines PP1 and related compound PP2 (Fig. 2 and Table 1). These compounds were synthesized with the aim of inhibiting T-cell activation via Src family kinases Lck and FynT. PP1 and PP2 showed potency against Lck (IC50 of 4 nM) and a surprising selectivity for the Src family (60). These days, although less potent against c-Src itself (IC50enz 170 nM), PP2 remains one of the most selective Src inhibitors available and a very useful laboratory tool. For example, in vivo intraperitoneal injection of PP2 reduces invasion of HT29 cells into the liver following intrasplenic injection (36). The next generation of SFK inhibitors appeared in the late 1990s. PD173955 and later PD173956 were shown to inhibit c-Src with an IC50 of 20 nM on pure enzyme assays and at 5 mM in cells. Selectivity was lower than that of PP2, since these compounds are inhibitors of c-Abl, Csk, PDGFR and EGFR. Used at 5 mM, PD173955 was shown to block several cell lines in the G2/M phase of the cell cycle, although an effect on apoptosis was not clearly demonstrated (61). Later, at 1 mM, PD173955 was shown to produce accumulation of cells in G1 and increased the proapoptotic effect of detaching cells from matrix (37). So far, no data have been published on in vivo studies using PD173955 or PD173956. At around the same time, Novartis developed pyrrolo-pyrimidine (ex. CGP77675) and olomouCl

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Wyeth, Pearl River, New York, U.S.A. Ariad, Cambridge, Massachusetts, U.S.A. Novartis, Basel, Switzerland Exelixis, South San Francisco, California, U.S.A. Pfizer, Groton, Connecticut, U.S.A. Pfizer Pfizer

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Non-SFK targets with IC50 3 month stable disease in 21% of patients (abstract 383, AACR-NCI-EORTC, Prague 2006), and 2–10 month stable disease in 23% of patients (abstract 14042, ASCO 2007, Chicago). ACKNOWLEDGMENTS I would like to thank Dr. John Hickman (Servier) and Dr. Serge Roche (CNRS, Montpellier, France) for helpful comments in the preparation of this manuscript. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Hunter T, Sefton BM. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci USA 1980; 77:1311–15. Collett MS, Purchio AF, Erikson RL. Avian sarcoma virus-transforming protein, pp60src shows protein kinase activity specific for tyrosine. Nature 1980; 285:167–9. Oppermann H, Levinson AD, Varmus HE, et al. Uninfected vertebrate cells contain a protein that is closely related to the product of the avian sarcoma virus transforming gene (src). Proc Natl Acad Sci USA 1979; 76:1804–8. Parker RC, Varmus HE, Bishop JM. Cellular homologue (c-src) of the transforming gene of Rous sarcoma virus: isolation, mapping and transcriptional analysis of c-src and flanking regions. Proc Natl Acad Sci USA 1981; 78:5842–6. Coffin JM, Varmus HE, Bishop JM, et al. Proposal for naming host cell-derived inserts in retrovirus genomes. J Virol 1981; 40:953–7. Resh MD. Myristylation and palmitylation of Src family members: the fats of the matter. Cell 1994; 76:411–13. Koegl M, Zlatkine P, Ley S, et al. Palmitoylation of multiple Src-family kinases at a homologuos N-terminal motif. Biochem J 1994; 303:749–53. Thomas SM, Brugge JS. Cellular functions regulated by Src family kinases. Ann Rev Dev Biol 1997; 13:513–609. Harrison SC. Variation on an Src-like theme. Cell 2003; 112:737–40. Huse M, Kuriyan J. The conformational plasticity of protein kinases. Cell 2002; 109:275–82. Bjorge JB, Jaymiw A, Fujita DJ. Selected glimpses into the activation and function of Src kinase. Oncogene 2000; 19:5620–35. Benistant C, Bourgaux JF, Chapuis H, et al. The COOH-terminal Src kinase Csk is a tumor antigen in human carcinoma. Cancer Res 2001; 61:1415–20. Irby RB, Mao W, Coppola D, et al. Activating SRC mutation in a subset of advanced human colon cancers. Nat Genet 1999; 21:187–90. Daigo Y, Furukawa Y, Kawasoe T, et al. Absence of genetic alteration at codon 531 of the human c-src gene in 479 advanced colorectal cancers from Japanese and Caucasian patients. Cancer Res 1999; 59:4222–4. Wang NM, Yeh K-T, Tsai C-H, et al. No evidence of correlation between mutation at codon 531 of src and the risk of colon cancer in Chinese. Cancer Lett 2000; 150:201–4. Nilbert M, Fernebro E. Lack of activating c-SRC mutations at codon 531 in rectal cancer. Cancer Genet Cytogenet 2000; 121:94–5. Laghi L, Bianchi P, Orbetegli O, et al. Lack of mutation at codon 531 of SRC in advanced colorectal cancers from Italian patients. Br J Cancer 2000; 84:196–8. Benistant C, Chapuis H, Mottet N, et al. Deregulation of the cytoplasmic tyrosine kinase cSrc in the absence of a truncating mutation at codon 531 in human bladder carcinoma. Biochem Biophys Res Comm 2000; 273:425–30.

346 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.

41.

Cruzalegui

Sugimura M, Kobayashi K, Sagae S, et al. Mutation of the SRC gene in endometrial carcinoma. Jpn J Cancer Res 2000; 91:395–8. Tan Y-X, Wang H-T, Zhang P, et al. c-Src activating mutation analysis in Chinese patients with colorectal cancer. World J Gastroenterol 2005; 11:2351–3. Park J, Meisler AI, Cartwright CA. c-Yes tyrosine kinase activity in human colon carcinoma. Oncogene 1993; 8:2627–35. Pena SV, Melhem MF, Meisler AI, et al. Elevated c-yes tyrosine kinase activity in premalignant lesions of the colon. Gastroenterology 1995; 108:117–24. Han NM, Curley SA, Gallick GE. Differential activation of pp60(c-src) and pp62(c-yes) in human colorectal carcinoma liver metastases. Clin Cancer Res 1996; 2:1397–404. Loganzo FJr, Dosik JS, Zhao Y, et al. Elevated expression of protein tyrosine kinase c-Yes, but not c-Src, in human malignant melanoma. Oncogene 1993; 8:2637–44. Marchetti D, Parikh N, Sudol M, et al. Stimulation of the protein tyrosine kinase c-Yes but not c-Src by neurotrophins in human brain-metastatic melanoma cells. Oncogene 1998; 16:3253–60. Stettner MR, Wang W, Nabors LB, et al. Lyn kinase activity is the predominant cellular SRC kinase activity in glioblastoma tumor cells. Cancer Res 2005; 65:5535–43. Contri A, Brunati AM, Trentin L, et al. Chronic lymphocytic leukemia B cells contain anomalous Lyn tyrosine kinase, a putative contribution to defective apoptosis. J Clin Invest 2005; 115:369–78. Twamley-Stein GM, Pepperkok R, Ansorge W et al. The Src family tyrosine kinases are required for platelet-derived growth factor-mediated signal transduction in NIH3T3. Proc Natl Acad Sci USA 1993; 90:7696–700. Barone MV, Courtneidge SA. Myc but not Fos rescue of PDGF signalling block caused by kinase-inactive Src. Nature 1995; 378:509–12. Blake RA, Broome MA, Liu X, et al. SU6656, a selective Src family kinase inhibitor, used to probe growth factor signalling. Mol Cell Biol 2000; 20:9018–27. Bowman T, Broome MA, Sinibaldi D, et al. Stat3-mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis. Proc Natl Acad Sci USA 2001; 98:7319–24. Furstoss O, Dorey K, Simon V, et al. c-Abl is an effector of Src for growth factorinduced c-myc expression and DNA synthesis. EMBO J 2002; 21:514–24. Roche S, Fumigalli S, Courtneidge SA. Requirement for Src family protein tyrosine kinases in G2 for fibroblast cell division. Science 1995; 269:1567–9. Tominaga T, Sahai E, Chardin P, et al. Diaphanous-related formins bridge Rho GTPase and Src tyrosine kinase signalling. Mol Cell 2000; 5:13–25. Sakamoto M, Takamura M, Ino Y, et al. Involvement of c-Src in carcinoma cell motility and metastasis. Jpn J Cancer Res 2001; 92:941–6. Nam JS, Ino Y, Sakamoto M, et al. Src family kinase inhibitor PP2 restores the E-cadherin/catenin cell adhesion system in human cancer cells and reduces cancer metastasis. Clin Cancer Res 2002; 8:2430–6. Windham TC, Parikh NU, Siwak DR, et al. Src activation regulates anoikis in human colon tumor cell lines. Oncogene 2002; 21:7797–807. Trevino JG, Summy JM, Lesslie DP, et al. Inhibition of Src expression and activity inhibits tumour progression and metastasis of human pancreatic adenocarcinoma cells in an orthotopic nude mouse model. Am J Pathol 2006; 168:962–72. Gonzalez L, Agullo-Ortuno MT, Garcia-Martinez JM, et al. Role of c-Src in human MCF7 breast cancer cell tumorigenesis. J Biol Chem 2006; 281:20851–64. Johnson FM, Saigal B, Talpaz M, et al. Dasatinib (BMS-354825) tyrosine kinase inhibitor suppresses invasion and induces cell cycle arrest and apoptosis of head and neck squamous cell carcinoma and non-small cell lung cancer cells. Clin Cancer Res 2005; 11:6924–32. Webster MA, Cardiff RD, Muller WJ. Induction of mammary epithelial hyperplasias and mammary tumors in transgenic mice expressing a murine mammary tumor virus/activated c-src fusion gene. Proc Natl Acad Sci USA 1995; 92:7849–53.

Src Inhibitors

42. 43. 44. 45. 46. 47. 48. 49. 50. 51.

52. 53.

54. 55. 56. 57. 58. 59. 60. 61. 62. 63.

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Matsumoto T, Jiang J, Kiguchi K, et al. Overexpression of a constitutively active form of c-src in skin epidermis increases sensitivity to tumor promotion by 12-O-tetradecanoylphorbol-13-acetate. Mol Carcinog 2002; 33:146–55. Matsumoto T, Jiang J, Kiguchi K, et al. Targeted expression of c-Src in epidermal basal cells leads to enhanced skin tumor promotion, malignant progression, and metastasis. Cancer Res 2003; 63:4819–28. Matsumoto T, Kiguchi K, Jiang J, et al. Development of transgenic mice that inducibly express an active form of c-Src in the epidermis. Mol Carcinog 2004; 40:189–200. Summy JM, Gallick GE. Treatment for advanced tumors: SRC reclaims center stage. Clin Cancer Res 2006; 12:1398–401. Marais R, Light Y, Paterson HF, et al. Differential regulation of Raf-1, A-Raf, and B-Raf by oncogenic ras and tyrosine kinases. J Biol Chem 1997; 272:4378–83. Hersey P. Apoptosis and melanoma: how new insights are effecting the development of new therapies for melanoma. Curr Opin Oncol 2006; 18:189–96. Ley R, Ewings KE, Hadfield K, et al. Regulatory phosphorylation of Bim: sorting out the ERK from the JNK. Cell Death Differ 2005; 12:1008–14. Karni R, Jove R, Levitzki A. Inhibition of pp60c-Src reduces Bcl-XL expression and reverses the transformed phenotype of cells overexpressing EGF and HER-2 receptors. Oncogene 1999; 18:4654–62. Wei D, Le X, Zheng L, et al. Stat3 activation regulates the expression of vascular endothelial growth factor and human pancreatic cancer angiogenesis and metastasis. Oncogene 2003; 22:319–29. Laird AD, Li G, Moss KG, et al. Src family kinase activity is required for signal transducer and activator of transcription 3 and focal adhesion kinase phosphorylation and vascular endothelial growth factor signalling in vivo and for anchorage-dependent and independent growth of human tumours. Mol Cancer Ther 2003; 2:461–9. Park J, Hill MM, Hess D, et al. Identification of tyrosine phosphorylation sites on 3-phosphoinositide-dependent protein kinase-1 and their role in regulating kinase activity. J Biol Chem 2001; 276:37459–71. Brunton VG, Avizienyte E, Fincham VJ, et al. Identification of Src-specific phosphorylation site on focal adhesion kinase: dissection of the role of Src SH2 and catalytic functions and their consequences for tumor cell behavior. Cancer Res 2005; 65:1335–42. Avizienyte E, Wyke AW, Jones RJ, et al. Src-induced de-regulation of E-cadherin in colon cancer cells requires integrin signalling. Nat Cell Biol 2002; 4:632–8. Calcagno AM, Fostel JM, Orchekowski RP, et al. Modulation of cell adhesion molecules in various epithelial cell lines after treatment with PP2. Mol Pharm 2005; 2:170–84. Wallez Y, Cand F, Cruzalegui F, et al. Src kinase phosphorylates vascular endothelialcadherin in response to vascular endothelial growth factor: identification of tyrosine 685 as the unique target site. Oncogene 2006; 26:1067–77. Potter MD, Barbero S, Cheresh DA. Tyrosine phosphorylation of V-cadherin prevents binding of p120- and beta-catenin and maintains the cellular mesenchymal state. J Biol Chem 2005; 280:31906–12. Ellis LM, Staley CA, Liu W, et al. Down-regulation of vascular endothelial growth factor in a human colon carcinoma cell line transfected with an antisense expression vector specific for c-src. J Biol Chem 1998; 273:1052–7. Weis SM, Cheresh DA. Pathophysiological consequences of VEGF-induced vascular permeability. Nature 2005; 437:497–504. Hanke JH, Gardner JP, Dow RL, et al. Discovery of a novel, potent, and Src familyselective tyrosine kinase inhibitor. J Biol Chem 1996; 271:695–701. Moasser M, Strethapakdi M, Sachar KS, et al. Inhibition of Src kinases by a selective tyrosine kinase inhibitor causes mitotic arrest. Cancer Res 1999; 59:6145–52. Susa M, Teti A. Tyrosine kinase Src inhibitors: potential therapeutic applications. Drug News Perspect 2000; 13:169–75. Recchia I, Rucci N, Festucia C, et al. Pyrrolopyrimidine c-Src inhibitors reduce growth, adhesion, motility and invasion of prostate cancer cells in vitro. Eur J Cancer 2003; 39:1927–35.

348 64. 65. 66. 67. 68. 69. 70.

71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82.

83. 84.

85.

Cruzalegui

Rucci N, Recchia I, Angelucci A, et al. Inhibition of protein kinase c-Src reduces the incidence of breast cancer metastases and increases survival in mice: implications for therapy. J Pharmacol Exp Ther 2006; 318:161–72. Warmuth M, Damoiseaux R, Liu Y, et al. Src family kinases: potential targets for the treatment of human cancer and leukemia. Curr Pharm Des 2003; 9:2043–59. Kerkela R, Grazette L, Yacobi R, et al. Cardiotoxicity of the therapeutic agent imatinib mesylate. Nat Med 2006; 12:908–16. Wang JYJ. Regulation of cell death by the Abl tyrosine kinase. Oncogene 2000; 19:5643–50. Boschelli F, Weber JM, Lucas J, et al. Abstract. 4206. 93rd Annual meeting of the American Association for Cancer Research, San Francisco CA 2002. Golas JM, Lucas J, Etienne C, et al. SKI-606, a Src/Abl inhibitor with in vivo activity in colon tumour xenograft models. Cancer Res 2005; 65:5358–64. Golas J, Arndt K, Etienne C, et al. SKI-606, a 4-anilino-3-quinolinecarbonitrile dual inhibitor of Src and Abl kinases, is a potent antiproliferative agent against chronic myelogenous leukemia cells in culture and causes regression of K562 xenografts in nude mice. Cancer Res 2003; 63:375–81. Boschelli F, Golas JM, Golas J, et al. 97th Annual meeting of the American Association for Cancer Research, Washigton DC, April 1–5 2006, abstract 4574. Weis S, Cui J, Barnes L, et al. Endothelial barrier disruption by VEGF-mediated Src activity potentiates tumor cell extravasation and metastasis. J Cell Biol 2004; 167:223–9. Weis S, Shintani S, Weber A, et al. Src blockade stabilizes a Flk/cadherin complex, reducing edema and tissue injury following myocardial infarction. J Clin Invest 2004; 113:885–94. Yezhelyev MV, Koehl G, Guba M, et al. Inhibition of Src tyrosine kinase as treatment for human pancreatic cancer growing orthotopically in nude mice. Clin Cancer Res 2004; 10:8028–36. Duxbury MS, Ito H, Zinner MJ, et al. Inhibition of SRC tyrosine kinase impairs inherent and acquired gemcitabine resistance in human pancreatic adenocarcinoma cells. Clin Cancer Res 2004; 10:2307–18. Boyer B, Valles AM, Green T. AACR-NCI-EORTC meeting on Molecular targets and cancer therapeutics. Philadelphia PA, November 14–18, 2005, abstract A239. Eastell R, Hannon R, Gallagher N, et al. 41st Annual meeting of the American Society of Clinical Oncology. Orlando FL. May 13–17, 2005, abstract 3041. Gallagher NJ, Lockton AJ, Macpherson M, et al. 96th Annual meeting of the American Association for Cancer Research, Anaheim CA April 16–20, 2005, abstract 3972. Luo FR, Yang Z, Camuso A, et al. Abstract 4183. 96th Annual meeting of the American Association for Cancer Research, Anaheim CA April 16–20, 2005. Evans TRJ, Morgan JA, Van den Abbeele, et al. Abstract 3034. 41st Annual meeting of the American Society of Clinical Oncology. Orlando FL. May 13–17, 2005. Lesslie DP, Summy JM, Parikh NU, et al. Vascular endothelial growth factor receptor-1 mediates migration of human colorectal carcinoma cells by activation of Src family kinases. Br J Cancer 2006; 94:1710–17. Summy JM, Trevino JG, Lesslie DP, et al. AP23846, a novel and highly potent Src family kinase inhibitor, reduces vascular endothelial growth factor and interleukin-8 expression in human solid tumor cell lines and abrogates downstream angiogenic processes. Mol Cancer Ther 2005; 4:1900–11. Han LY, Landen CN, Trevino JG, et al. Antiangiogenic and antitumor effects of SRC inhibition in ovarian carcinoma. Cancer Res 2006; 66:8633–9. Hennequin LF, Allen J, Breed J, et al. N-(5-Chloro-1,3-benzodioxol-4-y1)-7-[2-(4-methylpiperazin-1-y1)ethoxyl]-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine, a novel, highly selective, orally available, dual specific c-Src/Ab1 kinase inhibitor. J med chem 2006; 49:6465–88. Aftab DT. 18th AACR-NCI-EORTC Symposium on Molecular Targets and Cancer Therapeutics. Prague, Czech Republic. November 7–10, 2006. Abstract 590.

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Telomerase and Telomere Interacting Agents Jean-Fran¸c ois Riou Laboratoire d’Onco-Pharmacologie, UFR de Pharmacie, Université de Reims Champagne Ardenne, Reims, France

Anne de Cian and Lionel Guittat Laboratoire de Biophysique, Muséum National d’Histoire Naturelle, Paris, France

Dennis Gomez and Céline Douarre Laboratoire d’Onco-Pharmacologie, UFR de Pharmacie, Université de Reims Champagne Ardenne, Reims, France

Laurent Lacroix Laboratoire de Biophysique, Muséum National d’Histoire Naturelle, Paris, France

Chantal Trentesaux Laboratoire d’Onco-Pharmacologie, UFR de Pharmacie, Université de Reims Champagne Ardenne, Reims, France

Jean-Louis Mergny Laboratoire de Biophysique, Muséum National d’Histoire Naturelle, Paris, France

INTRODUCTION Researchers have long been investigating novel targets for anticancer drugs. Telomeres and telomerase represent, at least in theory, extremely attractive targets in oncology. However, no such molecule has currently obtained approval, and the most advanced compounds are currently in clinical trials. The next few years will be critical for target validation in the clinic. Independent of this outcome, the extreme ends of chromosomes represent a fascinating field of investigation for cell biologists and oncologists. At their normal state, telomeres protect chromosomal ends from fusion events and provide a means for complete replication of the chromosome. Telomere repeats are added by a specialized enzyme, telomerase, which is overexpressed in most tumor cells. In contrast, telomerase gene expression is repressed in most somatic cells, although limited expression associated with the S phase has been observed in normal cycling cells (1). This differential explains the rationale for telomerase inhibitors. Not only could telomerase become a reliable cancer marker in some pathologies, but it could also be a target for inhibitors, as long-term proliferation of cancer cells requires a telomere-maintenance mechanism. Unlimited proliferative potential, which depends on telomere maintenance, is one of six properties considered hallmarks of cancer cells (2). The validation of telomerase as a potential target for oncology was obtained by the expression of a

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dominant-negative mutant of the catalytic subunit in tumor cells, which results in the inhibition of telomerase activity and reduction in telomere length, as well as cell death and abrogation of tumorigenicity in vivo (3). Less straightforward is the rationale for the development of telomere-interacting agents. Normal cells also have telomeres, thus this target cannot be considered cancer-specific. Nevertheless, it may be proposed that telomeres from normal and cancer cells exhibit differences in structure or accessibility, explaining how a telomere ligand may exhibit selective toxicity. Recent results demonstrated that alterations of the telomere structure is responsible for the genomic instability associated with the tumoral transformation. Some of the initial predictions concerning the mechanism of action of telomere and telomerase agents have been validated, and other new prediction have emerged. Several classes of telomerase inhibitors have been developed and inhibit this enzyme through the targeting of its RNA or catalytic components as well as its DNA substrate, the single-stranded 30 -telomeric overhang. Telomerase inhibitors are chemically diverse and include modified oligonucleotides as well as small molecules, from natural to synthetic origin. This chapter presents the latest view on the mechanism(s) of action of these inhibitors, with an emphasis on a specific class of telomere ligands called Gquadruplex ligands, and discusses their potential use in oncology. Owing to space limitations, we will not consider the use of telomerase as a cancer marker or a prognosis factor [for recent reviews, see (4,5)], and we will restrict our discussion to the therapeutic target aspect of telomerase. Recent excellent reviews (6–10) cover in greater detail some aspects that cannot be detailed here [e.g., Telomerase promoter–mediated suicide gene therapy or telomeric PARP, tankyrases as targets for cancer therapy (11)]. Finally, one should note that telomerase may be involved in other aspects of human health (12), such as aging (13), or genetic diseases such as Dyskeratosis congenita, a rare, inherited bone marrow failure syndrome (14–16). PRESENTATION OF THE TELOMERE The telomeres of human cells range in size from 3 to 15 kb and are composed of tandem repeats of the sequence (50 -TTAGGG-30 ) with a 30 overhang of the G-strand extending 150–400 bases beyond the C-strand (Fig. 1A) (17). Telomere length and structure are critical to cellular proliferation. Normal human cells proliferate for a limited period of time and eventually undergo senescence, a state where cells cease to divide but remain viable. Inactivation of the p53 and Rb tumor suppressor pathways allow cells to bypass senescence, resulting in continued cellular division and further telomere shortening. These cells eventually reach a second proliferative block referred to as crisis, which is characterized by telomere dysfunction and cell death. Cells emerging from crisis maintain stable telomere lengths through the activation of a telomere maintenance mechanism (see below). Since DNA replication machinery is unable to completely replicate the extreme end of linear DNA molecules, telomere sequences are shortened at each round of normal cell division, a situation that leads to the proliferation arrest of normal somatic cells when a critical size is reached. Two mechanisms have been described in immortal and tumor cells to maintain the telomere length:

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BIBR1532

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C1' N

N

N N

N H

H

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H O

N N

O N

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H

N N

N C1'

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OH OH

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DPNS

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TMPI

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N

N

N

C1'

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N+

O

N

O N

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PIPER

TmPyP4

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N+ I-

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O

HO

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BSU1051

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I-

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307A

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N+

NH O N

N H

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12459

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F

N

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BRACO19

N

N HN

MMQ1

O N

N O

HN

N

N

N+ F

N

(C)

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N N O

RHPS4

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Telomestatin

FIGURE 1 Chemical formula of some telomerase inhibitors. (A) Various catalytic inhibitors. (B) Left: Structure of a G-quartet involving four coplanar guanines. Right: Possible conformation of the intramolecular G-quadruplex formed by human telomeric DNA. (C) Chemical formula of some G-quadruplex ligands.

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1. A specialized enzyme called telomerase is able to copy, as a reverse transcriptase, the short GGTTAG motif at the end of the telomere. Telomerase was first identified in ciliates (18). The catalytic subunit of this enzyme, hTERT, uses its RNA subunit (hTR) as a template for adding GGTTAG repeats to the ends of chromosomes [for a recent review, see (19)]. Telomerase is overexpressed in a large number of tumors (about 85%) and is involved in the capping of the telomere end (20) and in the DNA-damage response (21). 2. Telomerase activity is absent in about 15% of tumors and the telomere lengthening is obtained by recombination events between telomeres, known as alternative lengthening of telomere (ALT) (22). Telomere length is heterogeneous, varying from long telomeres (>20 kb) in ALT cells to a shorter size (3–15 kb) in telomerase positive cells. Telomere length reflects a homeostasis between the telomere lengthening mechanisms and the replicating degradation at each round of division, and is controlled by a complex association of telomere-binding proteins that tightly regulates the accessibility of telomerase to the G-overhang. Although identical telomere-binding proteins are qualitatively found in ALT cells, the exact mechanisms involved in the recombination at the telomere are poorly understood. In ALT cells, telomeric sequences and telomeric proteins are associated with large nuclear complexes to form APBs (ALT associated PML bodies), which also contain recombination factors.

OLIGONUCLEOTIDIC INHIBITORS Targeting the RNA Component (hTR) The RNA component of telomerase hTR is absolutely required for telomerase reverse transcription and is therefore a natural target for antitelomerase agents. Unlike hTERT, hTR is present in most normal tissues that do not express telomerase activity. Thus, targeting hTR in normal human somatic cells is not thought to generate toxicity, as these cells are mostly telomerase-negative. Different strategies and chemical modifications (Fig. 1) have successfully been developed to target hTR, starting with antisense oligomers. hTR has several features that makes it a good target for oligonucleotidic inhibitors: (i) hTR is not translated and should remain unprotected by ribosomal machinery, so that RNAs H-independent strategies for targeting hTR should be possible; and (ii) hTR provides a template (nucleotides 46–56; 50 -CUAACCCUAAC-30 ) for reverse transcription that is expected to be highly accessible. These oligonucleotides should be considered as “template agonists” rather than true antisense agents, as their targets are not translated but reverse transcribed. Nevertheless, this class of agents faces the same challenges as antisense agents: cell penetration and chemical stability. Regular DNA is probably too unstable toward nucleases to be considered for in vivo applications, explaining why different groups have tried several modifications to enhance uptake and/or stability. PNAs directed against the template region of hTR were among the first oligomers tested that could efficiently inhibit telomerase (23). Using a scanning approach, the binding determinants within hTR that are needed for the potent inhibition of telomerase by PNAs have been delineated (24). 20 -O-methyl-RNA (20 O-MeRNA) and 20 -O-(2-methoxyethyl) (20 -MOE) RNAs directed against the template region of hTR possess favorable pharmacokinetic properties and inhibit human telomerase with IC50 values of 2–10 nM (25,26). 20 ,50 -Oligoadenylate

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antisense oligomers directed against hTR have been described that could efficiently inhibit telomerase (27) as well as the growth of xenografted tumors (28,29). The most promising analogs may be thiophosphoramidates, which are potent inhibitors (IC50 ¼ 1 nM) that can induce senescence and telomere shortening (30). Two such oligomers, called GRN163 and GRN163L (a lipid palmidate conjugated to GRN163), were tested in vivo against a large panel of xenografted tumors. They exhibited strong antitumor activities on prostate, lymphoma, and myeloma models with short telomeres (31–33). GRN163L effectively inhibited telomerase activity in a dose-dependent fashion in breast cancer cell lines, independently of their genetic background (ERþ, ER, HER2þ, BRCA1 mutants, etc.) (34). Breast cancer cells that exhibited telomerase inhibition also exhibited significant reduction in colony formation and tumorigenicity. Furthermore, GRN163L suppressed tumor growth and lung metastases of MDA-MB-231 cells in vivo (34). GRN163 and GRN163L were also tested in preclinical studies using systemic administration to treat flank xenografts of different human hepatoma cell lines in nude mice. In vivo treatment with GRN163L is also effective in preventing lung metastases in xenograft animal models (35). Breast cancer cells that were treated with GRN163L prior to plating in invasion chambers exhibit significantly diminished invasive potential (36). These assays revealed that GRN163L was superior to the nonlipidated parent compound, GRN163 (37,38): it does not require a lipid carrier to facilitate cellular uptake. GRN163L has recently entered into Phase I/II clinical trials for chronic lymphocytic leukemia. Silencing hTR/hTERT RNA interference (RNAi) has been shown to be an effective method for inhibiting the expression of a given gene in human cells. siRNAs against hTERT have been shown to inhibit telomerase activity in the HT29 immortal human colorectal adenocarcinoma cell line. Moreover, telomere lengths were reduced in cells stably expressing this particular RNA sequence, ultimately leading to a proliferation arrest (39). CATALYTIC INHIBITORS Nucleoside Analogs Nucleoside analogs acting as chain-terminating inhibitors of reverse transcriptases were among the first drugs to be tested for their ability to inhibit telomerase (40). In two immortalized human cell lines, dideoxyguanosine (ddG) caused reproducible, progressive telomere shortening over several weeks of culture, after which point telomeres became stable but remained short (41). Enduring AZT treatment of T-cell leukemia virus I-infected cells, in vitro and in vivo in patients, results in inhibition of telomerase activity, progressive telomere shortening, and increased p14(ARF) expression (42). Azidothymidine also induces apoptosis and inhibits cell growth of human parathyroid cancer cells (43). When tested in combination with 5-fluorouracil (5-FU), the presence of AZT increased 5-FU cytotoxicity, suggesting that the effects of these two drugs are synergistic (44). A very potent and specific nucleoside telomerase inhibitor, 6-thio-20 -deoxyguanosine 50 -triphosphate (TDGTP; IC50 ¼ 0.06 mM), has also been described (45) (Fig. 1A). However, despite these early advances, no clinical or pre-clinical trial is, to our knowledge, scheduled for nucleoside telomerase inhibitors.

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Nonnucleoside Inhibitors A variety of nonnucleoside drugs have also been shown to inhibit telomerase. Examples are epicatechin derivatives, such as epigallocatechin gallate (EGCG), which strongly and directly inhibit telomerase (46–49). The screening of chemical libraries allowed the identification of various molecules as potent inhibitors of telomerase such as DPNS and TNQX (50,51) (Fig. 1A). Long-term cultivation of breast cancer cell line MCF7 with a TNQX concentration that did not cause acute cytotoxicity resulted in progressive telomere attrition followed by an increased incidence of chromosome abnormalities as well as the induction of a senescence phenotype. An independent screen led to the identification of isothiazolone derivatives (52) (IC50 ¼ 1 mM for TMPI). Inhibition of telomerase by TMPI was quenched by dithiothreitol or glutathione, suggesting that these inhibitors act on a cysteine residue. Bisindole derivatives with IC50 values in the submicromolar range were also described (53). Finally, with an IC50 of 93 nM, BIBR1532 (Fig. 1A) is one of the most potent nonnucleoside inhibitors of telomerase (54). BIBR1532 is a mixed-type noncompetitive inhibitor (55). With no evidence of acute cytotoxicity, treatment of cancer cells with this compound led to progressive telomere shortening and, after a characteristic lag, to a proliferation block displaying hallmarks of senescence, which included morphological and proliferative changes, chromosomal aberrations, and altered patterns of gene expression (54). In a mouse xenograft model, pretreatment of tumor cells with this inhibitor led to telomerase inhibition, telomere shortening, and a marked reduction in tumorigenic potential. At higher concentrations, this class of telomerase inhibitor exerts a direct cytotoxic effect on malignant cells of the hematopoietic system, which appears to derive from direct damage of the structure of individual telomeres and is not related to overall telomere shortening (56). Pharmacological telomerase inhibition by BIBR1532 can sensitize drug-resistant and drug-sensitive cells to chemotherapeutic treatment (57). This is an important observation, as BIBR alone has a relatively weak in vivo antitumor activity. Miscellaneous A number of compounds have an effect on telomerase activity or expression of the subunits. Helenalin, a natural sesquiterpene lactone, is a potent and selective inhibitor for human telomerase. In vitro studies indicate that the inhibitory action of this drug on telomerase is selective and direct (58). Using a forward chemical genetics approach, Nakai et al. screened a microbial products library and identified three structurally unrelated antibiotics, chrolactomycin, UCS1025A, and radicicol, as active compounds. Chrolactomycin inhibited human telomerase in a cell-free assay, induced telomere shortening, and a population-doubling–dependent antiproliferative activity (59). On the other hand, a number of molecules, including candidate or validated anticancer drugs, have an effect on telomerase via an indirect mechanism. Telomerase regulation is complex, and a number of pathways may act on telomerase. Here are a few examples: 1. Heat shock protein 90 (Hsp90) is a molecular chaperone whose association is required for stability and function of multiple proteins that promote cancer cell growth and/or survival [for a review, see (60)]. Hsp90 client proteins include telomerase: Hsp90 facilitates the assembly of telomerase and remains


2. 3.

4. 5.

6.

7.

8. 9.

10.

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associated with the functional complex. It is therefore not surprising that Hsp90 inhibitors such as novobiocin or radicicol reduced telomerase activity (61,62). One Hsp90 inhibitor, 17-AAG, is currently in Phase II clinical trial. Telomerase activity has also been reported to be upregulated by protein kinase C (PKC). A PKC inhibitor, bisindolylmaleimide I, inhibited telomerase activity but had no effect on the expressions of telomerase core subunits (61). Imatinib mesylate (Gleevec), a tyrosine kinase inhibitor, demonstrates activity against malignant cells expressing BCR-ABL, c-KIT, and platelet-derived growth factor receptor (PDGF-R). Imatinib mesylate also causes a dosedependent inhibition of telomerase activity in c-KIT-expressing SK-N-MC (Ewing sarcoma), SK-MEL-28 (melanoma), RPMI 8226 (myeloma), MCF-7 (breast cancer), and HSC 536/N (Fanconi anemia) cells. The inhibition of proliferation was associated with a decrease in the S-phase of the cell cycle and an accumulation of cells in the G2/M phase (63). U-73122, an amphiphilic alkylating agent that is commonly used as an inhibitor for phospholipase C, is also a potent and selective inhibitor of human telomerase (64). Interferon-gamma signaling induces growth arrest in many tumors. This compound leads to a posttranscriptional upregulation of the p27 tumor suppressor protein, which inhibits hTERT mRNA expression and telomerase activity (65). This inhibition of the hTERT expression and telomerase activity may be a novel tumor suppressor function of p27 (66). Trichostatin A, a histone deacetylase (HDAC) inhibitor, induces apoptosis in human leukemia cell line U937. The increase in apoptosis was associated with the upregulation in proapoptotic Bax expression and downregulation of antiapoptotic Bcl-2 and Bcl-XL. TSA treatment also markedly inhibited the activity of telomerase in a dose-dependent fashion (67). Cells transfected with dominant-negative hTERT were more likely to undergo apoptosis induced by Trichostatin A than cells transfected with wild-type hTERT (68). Telomerase activity and hTERT expression may also be inhibited by etodolac, a selective COX-2 inhibitor, leading to the conclusion that antitumor effects of etodolac on TMG-L cells are due to inhibition of both angiogenesis and telomerase activity (69). The antiproliferative effects of the tyrosine kinase inhibitor genistein seem to be mediated, at least in part, by its action on hTERT transcriptional activity (70). Pharmacological agents that act on hormonal pathways may interfere with telomerase expression. Estrogens regulate telomerase (71,72) and tamoxifen negatively controls telomerase activity (73). Telomerase has also been proposed as a target for retinoid therapy (74–76). Finally, c-myc seems to be a key factor in the expression level of the catalytic subunit (77). It is therefore not surprising to observe that molecules that inhibit this oncogene also inhibit telomerase (78).

Furthermore, increasing evidence suggests that the role of telomerase is not restricted to a simple lengthening of telomeres. This enzyme, or some of its subunits, could be involved in cell signaling and cell proliferation (the so-called telomerase extracurricular activities). For example, hTERT seems to be involved in mitochondrial apoptosis (79,80). hTERT depletion facilitated the induction of apoptotic cell death by cisplatin, etoposide, mitomycin C, and reactive oxygen

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species, and facilitated the conformational activation of Bax induced by genotoxic agents (80). Therefore, telomerase inhibition could be a promising approach in association with drugs promoting mitochondrial apoptosis.

IMMUNIZATION WITH TELOMERASE Immunotherapeutic strategies that can selectively target tumor cells are actively being sought. The catalytic subunit of telomerase (hTERT) is considered a universal tumor-associated antigen (TAA) due to its higher rate of expression by cancers [for a recent review, see (9)]. It might even be described as a candidate “universal cancer vaccine” (81) and this strategy is currently being tested in clinical trials (phases I/II; a phase III trial is scheduled this year). The rationale of using such vaccines is that TAA will be taken up and processed by antigen presenting cells, loaded onto major histocompatibility complexes (MHC), and recognized by T-lymphocytes (9). Efforts are being made to develop a strategy to elicit a telomerase-specific T-lymphocyte immune response in order to target hTERTþ cancer cells. The telomerase catalytic subunit is capable of triggering antitumor cytotoxic T lymphocyte (CTL) responses. The first 9 amino acid-long hTERT peptide used was p540 (82), also called HR2822. Its sequence is ILAKFLHWL; several other hTERT peptides have been described such as GV1001 (EARPALLTSRLRFIPK); their complete sequences and references may be found in (9). Clinical applications of telomerase vaccinations have been tested recently. A phase I clinical trial was performed with p540; p540-specific CTLs were induced in four out of seven patients and were able to kill tumor cells and partial tumor regression was obtained for one patient (83). Significant results were obtained in the case of non–small cell lung cancer, using a combination of telomerase peptides (GV1001 and HR2822) (84). Immune responses against GV1001 were detected in 13 of 24 evaluable patients, while two patients responded to HR2822. A complete tumor response was observed in one patient. A phase III assay in pancreatic adenocarcinoma is scheduled this year with GV1001. This peptide will be developed as a combination or add-on treatment to gemcitabine, which is currently used as a standard in pancreatic cancer treatment. In theory, hTERT vaccination could harm normal cells expressing the enzyme, especially stem cells and germ cells. Fortunately, hTERT vaccination did not result in a detectable decline in hematopoietic potential despite the expression of hTERT and MHC class I in bone marrow progenitors and stem cells (85). No CTL effect was found in normal telomerase-positive CD34þ hematopoietic cells. Another study did not reveal autoimmune manifestations resulting from vaccination with hTERT (86). In another study, no bone marrow toxicities were observed in long-time survivors with immune responses (84). Not all immunization attempts with telomerase fragments have been successful: recent results suggest that hTERT:p540 is not presented on the surfaces of tumor cells and will not be useful for the immunotherapy of patients with cancer (87). A lack of tumor recognition by hTERT peptide p540-specific CD8þ T cells from melanoma patients has been reported, raising questions as to the use of this peptide for cancer-specific immunotherapy. This reveals inefficient antigen processing by the proteasome (88). Finally, the efficient expansion of hTERT-specific CTLs from donor peripheral blood T lymphocytes remains a major challenge.


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TELOMERE LIGANDS Telomeres and Quadruplexes Telomeric DNA consists of highly repetitive but short sequences characterized by an asymmetry of guanines and cytosines, which are placed on two separate strands. The 30 -terminal region of the G-rich strand of human telomeres is single-stranded and may adopt particular conformations such as T-loops or G-quadruplexes. G-quadruplexes are formed in the presence of monovalent cations and consist of four-stranded structures stabilized by G-quartets. Self-association is favored by self-complementary hydrogen bond donors and acceptors present on both sides of the base, leading to the formation of a cyclic arrangement held by eight hydrogen bonds per quartet (Fig. 1B, left). The presence of a central cation helps maintain the stability of the structure. Potassium, which is very abundant in the intracellular medium, is a very favorable cation for quadruplex formation, explaining why this structure may be very stable under physiological conditions. Different G-quadruplex structures exist, depending on the orientation of the DNA strands and the syn/anti conformation of the guanines. The telomeric G-overhang can fold in several intramolecular quadruplexes that differ by the position of the adjacent loop regions (Fig. 1B, right). Recent data suggest a predominant conformation under physiological conditions (89–91). The existence of G-quadruplexes in vivo was initially established in ciliates using specific antibodies (92,93). Quadruplexes have now been studied in a variety of organisms from Escherichia coli (94) to humans. Concerning human cells, the in vivo existence of G-quadruplexes has been proposed or demonstrated for oncogene promoters and telomeres through the use of specific ligands (see below) (95,96). Optimal telomerase activity requires an unfolded single-stranded substrate, because G-quadruplex formation directly inhibits telomerase elongation in vitro (97). Therefore, ligands that selectively bind to G-quadruplex structures may interfere with telomere conformation and telomere elongation. The number of identified G4 ligands has grown rapidly over a few years. A range of G-quadruplex ligands has been shown to bond quadruplexes in vitro, such as TmPYP4 (98), PIPER (99), amidoanthracene-9,10-diones (such as BSU1051) (100), 2,7-disubstituted amidofluorenones (101), trisubstituted acridines (BRACO19) (102–105), ethidium derivatives (106,107), triazine derivatives (12459) (108), fluoroquinoanthroxazines (109), indoloquinolines (110), dibenzophenanthrolines (MMQ1) (111), bisquinacridines (112), pentacyclic acridinium (RHPS4) (113,114), and 2,6-pyridin-dicarboxamide derivatives (307A and 360A) (96,115). Natural compounds such as telomestatin (116), cryptolepine (117), and meridine (118) also recognize quadruplex DNA and inhibit telomerase activity in the TRAP assay [for a review, see (119)]. The chemical structure of some of these ligands is presented in Figure 1C. Features shared by many of these ligands include a large flat aromatic surface, presence of cationic charges, and ability to adopt a terminal stacking mode. Some of these molecules have also been shown to induce telomere shortening and/or telomere instability triggering apoptosis and/or senescence programs in various cell lines (see below). One of the most interesting demonstrations that telomere is a significant target for these ligands has been provided by a study using a radiolabeled Gquadruplex ligand. The localization of the chromosomal-binding sites of the tritiated pyridine dicarboxamide derivative 360A has been performed in metaphase spreads from cell lines bearing different telomere lengths. The result showed that 360A preferentially binds to the terminal parts of chromosomes in mitosis (96). Binding sites in internal chromosomal regions were also observed but were significantly fewer than those at the end of chromosomes.

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Other Types of Telomere Ligands Minor groove binders may also be used to target double-stranded telomeric repeats (120). A hairpin polyamide–cyclopropanepyrroloindole (CPI) conjugate alkylates its target adenine in the telomere repeats, 50 -CCCTAA-30 , and inhibits the growth of a variety of cancer cell lines (121). The anticancer drug cisplatin, aside from its possible recognition of G-quadruplexes (see above), may also recognize duplex telomeric DNA, as these long-tandem repeats are potential targets for cisplatin and other platinum compounds. Platinum derivatives, including 2,3-dibromosuccinato (2-(methylaminomethyl)pyridine)platinum (II) (122) and cis-dichloropyridine-5-isoquinolinesulfonic acid Pt(II) (123), have been found to represent strong telomerase inhibitors, with IC50 in the mM range. CONSEQUENCES OF TARGETING THE TELOMERE RATHER THAN TELOMERASE Are Telomere Ligands “Simple” Telomerase Inhibitors? There are fundamental differences between the targeting of the telomeric G-overhang using specific ligands and the inhibition of the catalytic activity of telomerase. Telomeres exist in the absence of telomerase activity, and G-quadruplex ligands are expected not only to have an effect against ALT cells but also on normal dividing cells. In contrast, catalytic inhibitors will take the advantage of the very low expression of telomerase activity in normal cells and should not dramatically affect their growth. According to the initial paradigm of the lifespan control by telomerase activity and telomere length, G-quadruplex ligands were first evaluated as telomerase inhibitors to induce telomere shortening and replicative senescence (3). For G-quadruplex ligands, this paradigm is partially true since a functional telomerase inhibition was observed in cell lines treated for several weeks with subtoxic dosages of certain compounds. For example, long-term treatment of the human cancer cells with subtoxic doses of disubstituted triazines or telomestatin induces telomere shortening that correlates with the induction of senescence (108,124–126). However, in some cases, no shortening was observed with G-quadruplex ligands (103,108,127,128). Interestingly, these derivatives were able to downregulate telomerase activity (103,108,113,124). In some cases, this effect may be related to c-myc repression (129,130) or to a modification of hTERT splicing (131). For BRACO19, an important decrease in the nuclear hTERT, together with the formation of cytoplasmic hTERT bound to ubiquitin, may explain the telomerase activity downregulation (132). Other antitumor agents have also been found to downregulate the telomerase activity and hTERT expression has been reported as a marker of the cell proliferation. Thus, the simplest explanation for the ability of the G-quadruplex ligands to downregulate telomerase activity is related to their antiproliferative activity. Direct Effects of G-quadruplex Ligands on Telomeres: Induction of Telomere Dysfunction It was also early observed that G-quadruplex ligands induced a short-term response (apoptosis) that cannot be explained by the sole telomerase inhibition (108,124,128). Telomestatin induced the activation of ATM and Chk2 corresponding to an activation of the DNA damage (124). Subtoxic concentrations of the


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acridine G-quadruplex ligands RHPS4 or BRACO19 could trigger growth arrest in tumor cells after just 15 days of exposure, before any detectable telomere shortening (103,113). 12459 induced apoptosis through the mitochondrial pathway and also provoked the early activation of P53 (133). Short-term and massive apoptosis were also observed from the interference of the telomere capping function of telomerase when hTERT or hTR were modified by mutations. The observation that BRACO19 causes chromosome end-to-end fusion associated with the appearance of p16-associated senescence led researchers to propose that G-quadruplex ligands mostly act to disrupt the telomere structure (134). Such telomeric dysfunction was also observed in cell lines treated with other quadruplex ligands and in cell lines resistant to a triazine derivative with typical images of telophase bridges (125,127,128). These studies suggest that the direct target of these ligands is telomere rather than telomerase activity. The evidence that the antiproliferative effect of G-quadruplex ligands is independent of the presence of telomerase activity also comes from a series of observations: 1. Overexpression of hTERT or a dominant-negative of hTERT in a telomerase positive cell line did not modify the antiproliferative effect of the triazine derivative 12459 (135). 2. All described ligands were also found active in blocking the proliferation of ALT cell lines (108,113,134). 3. On the other hand, the reintroduction of hTERT in ALT cell lines only produces a partial protection against the antiproliferative effects of telomestatin and 307A. In that case, we propose that hTERT acts to protect the integrity of telomeres.

RESISTANCE MECHANISMS; ILLUSTRATION WITH QUADRUPLEX LIGANDS Isolation of Resistant Clones As for most therapeutic agents, the biological effect of G-quadruplex ligands is susceptible to being overcome by the appearance of acquired resistance phenotypes. Cell lines resistant to the triazine derivative 12459 have been selected from parental A549 human lung adenocarcinoma by either progressive adaptation (JFA2) or by EMS mutagenesis (JFD clones) (125,135). Interestingly, these cell lines presented a resistance to the short-term activity of the ligand. However, resistance to the long-term effects of 12459 was only observed in JFA2 cells. The cross resistance to other G4 ligands (telomestatin, BRACO19) is absent for short-term treatment and is only observed for the longterm senescence induction (125,135). These results point out interesting differences between the short- and long-term effects of 12459. Since telomerase was initially established as one of the potential targets of these ligands, we have determined whether hTERT expression, telomerase activity, and telomere length are modified in these resistant models. Both transcriptional enzymatic activity and telomere length were increased in JFA2 and in the majority of the JFD clones (8/15), suggesting that the upregulation of telomerase activity and the telomere length increase could play a role in the resistance to this ligand. Important alterations of hTERT splicing were also observed in these resistant cells, in order to overcome a downregulation of hTERT active transcript induced by 12459 (131). The interpretations of these initial findings become more complex in light of experiments in which an increased telomerase activity was reintroduced into

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the parental cell line (135). In this case, no resistance to 12459 was found, using either short- or long-term treatment by the ligand, in A549 cells overexpressing hTERT. Therefore, the increased telomerase activity appears to be insufficient per se to confer a resistance phenotype. Since these resistant cell lines displayed an increased number of mitotic alterations (anaphase bridges) that transduced important telomere capping alterations, we propose that hTERT overexpression may serve to stabilize or protect the telomere extremities following changes induced by the resistance acquisition. Indeed, the transfection of a dominant-negative hTERT in JFD18 cells partially restores the sensitivity to the short-term treatment with 12459 (135). The upregulation of hTERT expression and the increase of telomere length were also reported in a HCT116 resistant cell line established against the cyclin kinase inhibitor flavopiridol (136). In addition, an overexpression of POT1 mRNA was also described for this cell line, suggesting that an alteration of the shelterin complex (see below) might be associated with the resistance phenotype. Although the potential link between these telomere alterations and the mechanisms underlying the flavopiridol resistance remains unclear, the G-quadruplex ligand BRACO19 presents a synergistic long-term effect with flavopiridol to overcome the resistance. The flavopiridol-resistant cell line is also hypersensitive to BRACO19 alone. However, the very poor permeability of BRACO19 currently limits its biopharmaceutical potential (137). Altogether, these studies, together with the observation that G4 ligands are also active against telomerase negative cell lines, strongly suggest that other factors than telomerase are involved in the mechanism of action of these ligands. Resistance and Apoptosis For most of the G4 ligands studied so far, an apoptotic cell death could be achieved after several cell cycles in tumor-derived cell lines. The triazine ligand 12459 activates the mitochondrial cell death pathway through an alteration of the Bax/Bcl-2 balance, which leads to caspase 3 activation. At short-term, it could be noticed that apoptosis predominates over the appearance of senescent cells for this ligand (133). Some of the JFD clones selected for resistance to 12459 also present an overexpression of the Bcl-2 protein. In addition, A549 cells transfected by Bcl-2 display a resistance to the apoptotic action of 12459 (133). However, the Bcl-2 overexpression is not sufficient to confer resistance to the long-term effect of 12459. Thus, 12459-directed senescence is uncoupled from apoptosis, a result that fits well with the differences observed between JFA2 and JFD clones for long-term and short-term resistance studies. CONSEQUENCES OF TELOMERE TARGETING The Single-Strand G-Overhang is Altered and Degraded Telomestatin induces an important telomere degradation in some cell lines that arises earlier than expected as a result of telomerase inhibition (124,135). These data, together with reports that G-quadruplex ligands also impaired the growth of ALT cell lines lacking telomerase activity, have suggested that additional mechanisms may explain the biological activity of the ligands in tumor cell lines. The G-rich 30 extension (G-overhang) has been implicated in the structure of the telomere extremities to create the T-loop that protects chromosome ends from fusion and their degradation has been associated with the onset of the replicative senescence and more recently to the deprotection of telomeres though inactivation


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of proteins from the shelterin complex (138) (Fig. 2). The hypothesis that G-quadruplex ligands preferentially act to modify the G-overhang conformation or induce its degradation has emerged and has been experimentally evaluated. By using two techniques [T-OLA (139) and a hybridization assay (135,139)], in vitro experiments concluded that telomestatin remains tightly and specifically attached to the G-overhang from treated cells and prevents hybridization of the oligonucleotide probe (140). These observations, together with in vivo DMS protection experiments, represent good evidence for the existence of G-quadruplexes at the telomeric G-overhang when treated by a G-quadruplex ligand. Further experiments established that a real (but partial) degradation of the G-overhang occurred after a longer telomestatin treatment in A549 cells (8–12 days); it was associated with the growth arrest of the cells. Interestingly, in other cell lines models (EcR293 and HT1080), telomestatin is able to induce at short-term (48–72 h) a rapid degradation of the G-overhang, suggesting that an active nucleolytic process is triggered by the ligand. This is also the case for 12459 in A549 cells (128,133) but not for 307A or 360A in A549 and T98G cells, where only a limited G-overhang degradation occurs (128). This suggests that the cellular response to the Gquadruplex stabilization at the telomeric G-overhang varies greatly, depending on the nature of the ligands or the cell line, possibly through a different activation of the DNA damage machinery. Telomere-Binding Proteins are Deregulated The proteins that protect telomeres were identified during the past decade and compose a complex now called shelterin (138). This complex is composed of six proteins, three of which bind directly to the telomeric repeats: TRF1, TRF2, and POT1. TRF1 and TRF2 present a Myb-type DNA-binding domain that allowed the

FIGURE 2 The shelterin complex. (A) Schematic organization of the telomere with the 30 G-overhang. (B) Representation of the shelterin complex that binds and protects the telomere ends. Source: From Ref. 138.

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recognition of 50 -YTAGGGTTR-30 in duplex DNA (141), while POT1 presents two OB-folded domains and displays a strong preference for the single-stranded 50 -(T)TAGGGTTAG-30 sequence (142). The three other proteins are TIN2 (that binds TRF1 and TRF2), TPP1 (that binds TIN2 and POT1), and Rap1 that binds TRF2. Thus, shelterin appears to make the connection between the duplex telomeric DNA and the 30 -G-overhang. Shelterin also associates with several proteins involved in recombination alrepair (Mre11/Rad50/Nbs1,ERCC1/ XPF, WRN, BLM, DNA-PK, PARP-2, and TANK). When either TRF2 or POT1 are inactivated (143,144), the overall amount of the single-stranded G-overhang is diminished by 30–50% and a specific DNA damage response is induced at most telomere ends (145). After TRF2, TIN2, or POT1 inactivation or when telomeres become critically short, 53BP1, g-H2AX, the Mre11 complex, and phosporylated ATM accumulate at chromosome ends (143,146,147). The structures formed by these DNA damage factors are referred to as telomere dysfunction induced foci (TIFs) (148). These studies are consistent with the view that telomere ends are arranged in a peculiar structure in order to protect their integrity. The protein complex shelterin is able to actively change its architecture and to control the detection by DNA damage factors. Interestingly, the inactivation of TRF2 and POT1 cause cellular effects analogous to those reported with G-quadruplex ligands, such as chromosomal instability and loss of the telomeric G-overhang, followed by the appearance of apoptotic and/or senescent cells. Thus, the effect of G-quadruplex ligands has been investigated on the binding of POT1 to the telomeric G-overhang in vitro and in human cells using a GFPPOT1 fusion protein. G-quadruplex stabilization by telomestatin dramatically impairs the binding of POT1 to the telomeric G-overhang in vitro and in some cell lines (HT1080) (149). Interestingly, telomestatin also displaces the telomere localization of TRF2 in tumor cell lines (including HT1080), but not in normal or immortalized cell lines (including EcR293) (149,150). An extensive telomeric repeat fragment (TRF) decrease is also observed in telomestatin-treated tumor cells-explaining the TRF2 decrease at telomeric ends. Accordingly, telomestatin treatment (24-hour) also induced the formation of telomeric phosphorylated g-H2AX foci, which corresponds to TIFs in HT1080 cells, suggesting the induction of an early DNA damage response associated with telomeres triggered by the ligand (Gomez and Riou, unpublished data; Fig. 3). The antitumor response to the G-quadruplex ligand BRACO19 paralleled the loss of the nuclear hTERT protein expression (132). A cytoplasmic hTERT expression that colocalized with ubiquitin was observed in immunostaining of xenograft tissues, suggesting an enhanced destruction of hTERT due to BRACO19 treatment. Since the telomerase complex binds and acts at the telomeric G-overhang, these results are in good agreement with the notion that G-quadruplex ligands will impair or inactivate the function of single-stranded telomere-binding proteins. Efforts are now being made to determine the effect of G-quadruplex ligands on other components of the shelterin complex and associated factors, such as WRN or BLM helicases, that cooperate with POT1 for telomere sequence unwinding (151). QUADRUPLEX RESOLVASES Several proteins have been described to act as G-quadruplex resolvases. Human RecQ helicases, Bloom’s and Werner’s syndrome DNA helicases (152,153,154),

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3’ G4 ligand G4

5’

3’

G4 resolvase? 5’

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Processing by nucleases

5’

3’ G-overhang degradation

Tumor cells

T-loop instability TRF loss POT1 and TRF2 release

Normal or immortalized cells

POT1 release

ALT cells

?

FIGURE 3 G-quadruplex ligands induce telomere dysfunction. Model to explain the differential effects of G-quadruplex ligands in normal and tumor cells to induce G-overhang degradation and/ or TRF loss. G-quadruplex stabilization using a ligand is expected to induce a G-overhang degradation that might be processed after signalization of damage. G4 resolvase might modulate the effect of the ligand. As a consequence, G-overhang degradation induces the release of POT1 from telomeres (normal or immortalized cells). In tumor cells, G-overhang degradation induces a further t-loop instability followed by an important TRF loss associated with the release of TRF2 from telomeres. In ALT cells, the consequences of the G-overhang degradation are unknown.

have been shown to unwind intra- and intermolecular G-quadruplex formed by telomeric sequences or other G-rich sequences. BLM has been proposed to maintain telomeres in ALT cells through recombination mechanisms (155). WRN belongs to the DNA polymerase d replication complex and defective WRN cells are unable to fully replicate telomeres due to a defect in lagging strand synthesis (156). The DEXH helicase encoded by DHX36 was also identified as the major source of G-quadruplex resolvase in HeLa extracts (157). Furthermore, Rtel, a murine gene encoding a DNA helicase homologue to dog-1 is required for telomere elongation (158). Dog-1 was initially identified in the nematode Caenorhabditis elegans for maintaining the stability of long poly dG–poly dC runs (159). Its putative role might be to resolve G-quadruplex associated with the poly dG

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repeats formed during replication, in order to maintain genome integrity. More recently, a study in ciliates has demonstrated that the G-quadruplex formation at telomeres is regulated through the cell cycle. TEBP a–b proteins tether the telomere to the nuclear matrix and stabilize G-quadruplex formation (93). During S phase, the phosphorylation of TEBPb results in the release of the telomeric DNA and the dissociation of G-quartets. Then, the chromosome ends are accessible for replication and extension of the G-overhang by telomerase. These observations and the redundancy of G-quadruplex resolvases indicate that mammalian cells require mechanisms for the removal of G-quadruplex during replication. G-quadruplex ligands from the trisubstituted acridine series were also found to inhibit in vitro unwinding by the RecQ helicases, BLM and WRN (160). Therefore, these ligands should also disrupt telomere synthesis. In addition, their action against other G-quadruplex resolvases during replication might provoke DNA synthesis defects in other G-rich regions of the genome. It is not clear whether such effect would contribute to an antitumor activity. A possible way to determine this is to examine the effect of the ligands in cells defective for these resolvases. CONCLUDING REMARKS Because of the unique character of the telomerase target, in vivo assays and clinical trials involving telomerase inhibitors will require careful consideration compared to those looking at conventional anticancer cytotoxic drugs. Some authors actually consider that telomerase inhibitors should only be used in complement to (or in combination with) a direct cytotoxic agent. According to the initial paradigm for telomerase inhibitors (161), a long delay is expected between the start of the treatment and the proliferation arrest, making these agents alone inefficient against tumors with long telomeres. This drawback has been verified for some inhibitors such as BIBR1532 (54). However, this limitation has been challenged by the observation that telomerase also plays an important role in capping or cell survival, making it an attractive target for immediate effects, even if the initial telomere length is long. Pharmacological strategies that aim at inhibition of telomerase in cancer cells should take into account not only overall telomere shortening, but also rapid induction of a high level telomere dysfunction (162). Another current field of interest is in relation to the possible existence of cancer stem cells. At least in breast cancer, these stem cells are telomerase positive (163): telomerase inhibitors could target these rare cells as, the rest of the tumor. Apart from these “classical” telomerase inhibitors, at least two strategies involving telomerase have been tested or will be tested soon in clinical trial. The telomerase immunization approach is by far the most advanced, since phase III trials are scheduled for 2006. Concerning G-quadruplex ligands, in order to design effective therapies, the end point is (1) to achieve antitumor activity and (2) to keep the toxicity at a low value. The first criterion was recently obtained for the BRACO19 derivative that showed antitumor activity as a single agent in human xenografts (132). Since several other G-quadruplex ligands were reported to exhibit selectivity for tumor cell lines instead of normal progenitors, primary astrocytes, or normal cell lines in culture (124,134), the second criterion to achieve a therapeutic index seems possible. Owing to the presence of G-quadruplexes in other parts of the genome, including oncogene’s promoters, a strategy to develop G4 promoter ligands was


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initiated (130,164,165) and led to the selection of a ligand called CX-3543 (Cylene Pharmaceuticals) that entered a Phase I clinical trial in 2005. Some ligands display interesting in vitro selectivity for the mixed parallel/antiparallel G-quadruplex structure adopted by the c-myc G-quadruplex, as compared to the telomeric Gquadruplex (166). Recent refinements of the structure adopted in solution by the human G-quadruplex raised the possibility that this agent could also target telomeres and thus transform the initial “telomere targeted strategy” to a “genome targeted G-quadruplex strategy.” ACKNOWLEDGMENTS We wish to thank all the members of the laboratories for fruitful discussions to conceive this chapter, E. Mandine, F. Boussin, A. Londono-Vallejo, K. Shin-ya, M.P. Teulade-Fichou, and M.F. O’Donohue for scientific collaborations. This work was supported by the “Association pour la Recherche contre le Cancer”, grant nos 3365 (to J.L.M.) and 3644 (to J.F.R.), by an European Union FP6 grant “MolCancerMed” (LSHC-CT-2004-502943; to J.L.M.) and by the Ligue Nationale Contre le Cancer, Equipe labellisée 2006 (to J.F.R.). REFERENCES 1. Masutomi K, Yu EY, Khurts S, et al. Telomerase maintains telomere structure in normal human cells. Cell 2003; 114(2):241–53. 2. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100(1):57–70. 3. Hahn WC, Stewart SA, Brooks MW, et al. Inhibition of telomerase limits the growth of human cancer cells. Nat Med 1999; 5(10):1164–70. 4. Weise JM, Gunes, C. Telomeres and telomerase. A survey about methods and recent advances in cancer diagnostic and therapy. Histol Histopathol 2006; 21(11):1249–61. 5. Ulaner GA. Telomere maintenance in clinical medicine. Am J Med 2004; 117(4):262–9. 6. Shay JW, Wright WE. Telomerase therapeutics for cancer: challenges and new directions. Nat Rev Drug Discov 2006; 5(7):577–84. 7. Olaussen KA, Dubrana K, Domont J, Spano JP, Sabatier L, Soria JC. Telomeres and telomerase as targets for anticancer drug development. Crit Rev Oncol Hematol 2006; 57(3):191–214. 8. Pendino F, Tarkanyi I, Dudognon C, et al. Telomeres and telomerase: pharmacological targets for new anticancer strategies? Curr Cancer Drug Targets 2006; 6(2):147–80. 9. Huo LF, Tang JW, Huang JJ, et al. Cancer immunotherapy targeting the telomerase reverse transcriptase. Cell Mol Immunol 2006; 3(1):1–11. 10. Kelland LR. Overcoming the immortality of tumour cells by telomere and telomerase based cancer therapeutics—current status and future prospects. Eur J Cancer 2005; 41(7):971–9. 11. Seimiya H. The telomeric PARP, tankyrases, as targets for cancer therapy. Br J Cancer 2006; 94(3):341–5. 12. Blasco MA. Telomeres and human disease: ageing, cancer and beyond. Nat Rev 2005; 6(8):611–22. 13. von Zglinicki T, Martin-Ruiz CM. Telomeres as biomarkers for ageing and age-related diseases. Curr Mol Med 2005; 5(2):197–203. 14. Mitchell JR, Wood E, Collins K. A telomerase component is defective in the human disease dyskeratosis congenita. Nature 1999; 402(6761):551–5. 15. Marrone A, Walne A, Dokal I. Dyskeratosis congenita: telomerase, telomeres and anticipation. Curr Opin Genet Develop 2005; 15(3):249–57. 16. Goldman F, Bouarich R, Kulkarni S, et al. The effect of TERC haploinsufficiency on the inheritance of telomere length. Proc Natl Acad Sci USA 2005; 102(47):17119–24.

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Riou et al.

17. Makarov VL, Hirose Y, Langmore JP. Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening. Cell 1997; 88(5):657–66. 18. Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 1985; 43(2 Pt 1):405–13. 19. Autexier C, Lue NF. The structure and function of telomerase reverse transcriptase. Annu Rev Biochem 2006; 75:493–517. 20. Kim NW, Piatyszek MA, Prowse KR, et al. Specific association of human telomerase activity with immortal cells and cancer. Science 1994; 266(5193):2011–5. 21. Masutomi K, Possemato R, Wong JMY, et al. The telomerase reverse transcriptase regulates chromatin state and DNA damage responses. Proc Natl Acad Sci USA 2005; 102(23):8222–7. 22. Bryan TM, Englezou A, Gupta J, Bacchetti S, Reddel RR. Telomere elongation in immortal human cells without detectable telomerase activity. EMBO J 1995; 14(17):4240–8. 23. Norton JC, Piatyszek MA, Wright WE, Shay JW, Corey DR. Inhibition of human telomerase activity by peptide nucleic acids. Nat Biotechnol 1996; 14(5):615–19. 24. Hamilton SE, Pitts AE, Katipally RR, et al. Identification of determinants for inhibitor binding within the RNA active site of human telomerase using PNA scanning. Biochemistry 1997; 36(39):11873–80. 25. Pitts AE, Corey DR. Inhibition of human telomerase by 2'-O-methyl-RNA. Proc Natl Acad Sci USA 1998; 95(20):11549–54. 26. Elayadi AN, Demieville A, Wancewicz EV, Monia BP, Corey DR. Inhibition of telomerase by 2'-O-(2-methoxyethyl) RNA oligomers: effect of length, phosphorothioate substitution and time inside cells. Nucleic Acids Res 2001; 29(8):1683–9. 27. Kondo S, Kondo Y, Li G, Silverman RH, Cowell JK. Targeted therapy of human malignant glioma in a mouse model by 2-5A antisense directed against telomerase RNA. Oncogene 1998; 16:3323–30. 28. Mukai S, Kondo Y, Koga S, Komata T, Barna BP, Kondo S. 2-5A antisense telomerase RNA therapy for intracranial malignant gliomas. Cancer Res 2000; 60(16):4461–7. 29. Koga S, Kondo Y, Komata T, Kondo, S. Treatment of bladder cancer cells in vitro and in vivo with 2-5A antisense telomerase RNA. Gene Ther 2001; 8(8):654–8. 30. Pruzan R, Pongracz K, Gietzen K, Wallweber G, Gryaznov S. Allosteric inhibitors of telomerase: oligonucleotide N3' ‡ P5' phosphoramidates. Nucleic Acids Res 2002; 30(2):559–68. 31. Akiyama M, Hideshima T, Shammas MA, et al. Effects of oligonucleotide N3' ‡ P5' thio-phosphoramidate (GRN163) targeting telomerase RNA in human multiple myeloma cells. Cancer Res 2003; 63(19):6187–94. 32. Asai A, Oshima Y, Yamamoto Y, et al. A novel telomerase template antagonist (GRN163) as a potential anticancer agent. Cancer Res 2003; 63(14):3931–9. 33. Wang ES, Wu KD, Chin AC, et al. Telomerase inhibition with an oligonucleotide telomerase template antagonist: in vitro and in vivo studies in multiple myeloma and lymphoma. Blood 2004; 103(1):258–66. 34. Hochreiter AE, Xiao H, Goldblatt EM, et al. Telomerase template antagonist GRN163L disrupts telomere maintenance, tumor growth, and metastasis of breast cancer. Clin Cancer Res 2006; 12(10):3184–92. 35. Dikmen ZG, Gellert GC, Jackson S, et al. In vivo inhibition of lung cancer by GRN163L: A novel human telomerase inhibitor. Cancer Res 2005; 65(17):7866–73. 36. Gellert GC, Dikmen ZG, Wright WE, Gryaznov S, Shay JW. Effects of a novel telomerase inhibitor, GRN163L, in human breast cancer. Breast Cancer Res Treat 2006; 96(1):73–81. 37. Herbert BS, Gellert GC, Hochreiter A, et al. Lipid modification of GRN163, an N3' ‡ P5' thio-phosphoramidate oligonucleotide, enhances the potency of telomerase inhibition. Oncogene 2005; 24(33):5262–8. 38. Djojosubroto MW, Chin AC, Go N, et al. Telomerase antagonists GRN163 and GRN163L inhibit tumor growth and increase chemosensitivity of human hepatoma. Hepatology 2005; 42(5):1127–36.


367

39. de Souza Nascimento P, Alves G, Fiedler W. Telomerase inhibition by an siRNA directed against hTERT leads to telomere attrition in HT29 cells. Oncol Rep 2006; 16(2):423–8. 40. Strahl C, Blackburn EH. The Effects of Nucleoside Analogs on Telomerase and Telomeres in Tetrahymena. Nucleic Acids Res 1994; 22(6):893–900. 41. Strahl C, Blackburn EH. Effects of reverse transcriptase inhibitors on telomere length and telomerase activity in two immortalized human cell lines. Mol Cell Biol 1996; 16(1):53–65. 42. Datta A, Bellon M, Sinha-Datta U, et al. Persistent inhibition of telomerase reprograms adult T-cell leukemia to p53-dependent senescence. Blood 2006; 108(3):1021–9. 43. Falchetti A, Franchi A, Bordi C, et al. Azidothymidine induces apoptosis and inhibits cell growth and telomerase activity of human parathyroid cancer cells in culture. J Bone Miner Res 2005; 20(3):410–8. 44. Brown T, Sigurdson E, Rogatko A, Broccoli D. Telomerase inhibition using azidothymidine in the HT-29 colon cancer cell line. Ann Surg Oncol 2003; 10(1):910–5. 45. Fletcher TM, Cathers BE, Ravikumar KS, Mamiya BM, Kerwin SM. Inhibition of human telomerase by 7-deaza-2 '-deoxyguanosine nucleoside triphosphate analogs: potent inhibition by 6-thio-7-deaza-2 '-deoxyguanosine 5 '-triphosphate. Bioorg Chem 2001; 29(1):36–55. 46. Naasani I, Seimiya H, Tsuruo T. Telomerase inhibition, telomere shortening and senescence of cancer cells by tea catechins. Biochem Biophys Res Commun 1998; 249:391–6. 47. Seimiya H, Ohhara T, Suzuki T, et al. Telomere shortening and growth inhibition of human cancer cells by novel synthetic telomerase inhibitors MST-312, MST-295, and MST-199. Mol Cancer Ther 2002; 1(9):657–65. 48. Naasani I, Ohhashi F, Ohhara T, et al. Blocking telomerase by dietary polyphenols is a major mechanism for limiting the growth of human cancer cells in vitro and in vivo. Cancer Res 2003; 63(4):824–30. 49. Yokoyama M, Noguchi M, Nakao Y, Pater A, Iwasaka T. The tea polyphenol, ()-epigallocatechin gallate effects on growth, apoptosis, and telomerase activity in cervical cell lines. Gynecol Oncol 2004; 92(1):197–204. 50. Kim JH, Lee GE, Lee JE, Chung IK. Potent inhibition of human telomerase by nitrostyrene derivatives. Mol Pharmacol 2003; 63(5):1117–24. 51. Kim JH, Lee GE, Kim SW, Chung IK. Identification of a quinoxaline derivative that is a potent telomerase inhibitor leading to cellular senescence of human cancer cells. Biochem J 2003; 373:523–9. 52. Hayakawa N, Nozawa K, Ogawa A, et al. Isothiazolone derivatives selectively inhibit telomerase from human and rat cancer cells in vitro. J Med Chem 1999; 38(35):11501–7. 53. Sasaki S, Ehara T, Sakata I, et al. Development of novel telomerase inhibitors based on a bisindole unit. Bioorg Med Chem Lett 2001; 11(4):583–5. 54. Damm K, Hemmann U, Garin-Chesa P, et al. A highly selective telomerase inhibitor limiting human cancer cell proliferation. EMBO J 2001; 20:6958–68. 55. Pascolo E, Wenz C, Lingner J, et al. Mechanism of human telomerase inhibition by BIBR1532, a synthetic, non-nucleosidic drug candidate. J Biol Chem 2002; 277(18):15566–72. 56. El Daly H, Kull M, Zimmermann S, Pantic M, Waller CF, Martens UM. Selective cytotoxicity and telomere damage in leukemia cells using the telomerase inhibitor BIBR1532. Blood 2005; 105(4):1742–9. 57. Ward RJ, Autexier C. Pharmacological telomerase inhibition can sensitize drugresistant and drug-sensitive cells to chemotherapeutic treatment. Mol Pharmacol 2005; 68(3):779–86. 58. Huang PR, Yeh YM, Wang TCV. Potent inhibition of human telomerase by helenalin. Cancer Lett 2005; 227(2):169–74. 59. Nakai R, Ishida H, Asai A, et al. Telomerase inhibitors identified by a forward chemical genetics approach using a yeast strain with shortened telomere length. Chem Biol 2006; 13(2):183–90.

368

Riou et al.

60. Neckers L. Using natural product inhibitors to validate hsp90 as a molecular target in cancer. Curr Top Med Chem 2006; 6(11):1163–71. 61. Chang JT, Lu YC, Chen YJ, et al. hTERT phosphorylation by PKC is essential for telomerase holoprotein integrity and enzyme activity in head neck cancer cells. Br J Cancer 2006; 94(6):870–8. 62. Compton SA, Elmore LW, Haydu K, Jackson-Cook CK, Holt SE. Induction of nitric oxide synthase-dependent telomere shortening after functional inhibition of Hsp90 in human tumor cells. Mol Cell Biol 2006; 26(4):1452–62. 63. Uziel O, Fenig E, Nordenberg J, et al. Imatinib mesylate (Gleevec) downregulates telomerase activity and inhibits proliferation in telomerase-expressing cell lines. Br J Cancer 2005; 92(10):1881–91. 64. Chen YJ, Sheng WY, Huang PR, Wang TC. Potent inhibition of human telomerase by U-73122. J Biomed Sci 2006; 13(5):667–74. 65. Lindkvist A, Ivarsson K, Jernberg-Wiklund H, Paulsson-Karlsson Y. Interferoninduced sensitization to apoptosis is associated with repressed transcriptional activity of the hTERT promoter in multiple myeloma. Biochem Biophys Res Commun 2006; 341(4):1141–8. 66. Lee SH, Kim JW, Oh SH, et al. IFN-gamma/IRF-1-induced p27kip1 down-regulates telomerase activity and human telomerase reverse transcriptase expression in human cervical cancer. FEBS Lett 2005; 579(5):1027–33. 67. Woo HJ, Lee SJ, Choi BT, Park YM, Choi YH. Induction of apoptosis and inhibition of telomerase activity by trichostatin A, a histone deacetylase inhibitor, in human leukemic U937 cells. Exp Mol Pathol 2007 Feb; 82(1):77–84. 68. Wu P, Meng L, Wang H, et al. Role of hTERT in apoptosis of cervical cancer induced by histone deacetylase inhibitor. Biochem Biophys Res Commun 2005; 335(1):36–44. 69. Hasegawa K, Ohashi Y, Ishikawa K, et al. Expression of cyclooxygenase-2 in uterine endometrial cancer and anti-tumor effects of a selective COX-2 inhibitor. Int J Oncol 2005; 26(5):1419–28. 70. Ouchi H, Ishiguro H, Ikeda N, Hori M, Kubota Y, Uemura H. Genistein induces cell growth inhibition in prostate cancer through the suppression of telomerase activity. Int J Urol 2005; 12(1):73–80. 71. Kyo S, Takakura M, Kanaya T, et al. Estrogen activates telomerase. Cancer Res 1999; 59(23):5917–21. 72. Misiti S, Nanni S, Fontemaggi G, et al. Induction of hTERT expression and telomerase activity by estrogens in human ovary epithelium cells. Mol Cell Biol 2000; 20(11):3764–71. 73. Nanni S, Narducci M, DellaPietra L, et al. Signaling through estrogen receptors modulates telomerase activity in human prostate cancer. J Clin Invest 2002; 110(2):219–27. 74. Pendino F, Dudognon C, Delhommeau F, et al. Retinoic acid receptor alpha and retinoid-X receptor-specific agonists synergistically target telomerase expression and induce tumor cell death. Oncogene 2003; 22(57):9142–50. 75. Pendino F, Flexor M, Delhommeau F, Buet D, Lanotte M, Segal-Bendirdjian E. Retinoids down-regulate telomerase and telomere length in a pathway distinct from leukemia cell differentiation. Proc Natl Acad Sci USA 2001; 98(12):6662–7. 76. Pendino F, Sahraoui T, Lanotte M, Segal-Bendirdjian E. A novel mechanism of retinoic acid resistance in acute promyelocytic leukemia cells through a defective pathway in telomerase regulation. Leukemia 2002; 16(5):826–32. 77. Flores I, Evan G, Blasco MA. Genetic analysis of myc and telomerase interactions in vivo. Mol Cell Biol 2006; 26(16):6130–8. 78. Guo QL, Lin SS, You QD, et al. Inhibition of human telomerase reverse transcriptase gene expression by gambogic acid in human hepatoma SMMC-7721 cells. Life Sci 2006; 78(11):1238–45. 79. Del Bufalo D, Rizzo A, Trisciuoglio D, et al. Involvement of hTERT in apoptosis induced by interference with Bcl-2 expression and function. Cell Death Differ 2005; 12(11):1429–38.


369

80. Massard C, Zermati Y, Pauleau AL, et al. hTERT: a novel endogenous inhibitor of the mitochondrial cell death pathway. Oncogene 2006; 25(33):4505–14. 81. Gozlan, M. Telomerase reverse transcriptase: candidate for “universal cancer vaccine”?. Lancet 2000; 355(9212):1337. 82. Vonderheide RH, Hahn WC, Schultze JL, Nadler LM. The telomerase catalytic subunit is a widely expressed tumor-associated antigen recognized by cytotoxic T lymphocytes. Immunity 1999; 10(6):673–9. 83. Vonderheide RH, Domchek SM, Schultze JL, et al. Vaccination of cancer patients against telomerase induces functional antitumor CD8+ T lymphocytes. Clin Cancer Res 2004; 10(3):828–39. 84. Brunsvig PF, Aamdal S, Gjertsen MK, et al. Telomerase peptide vaccination: a phase I/II study in patients with non–small cell lung cancer. Cancer Immunol Immunother 2006; 55(12):1553–64. 85. Danet-Desnoyers GAH, Luongo JL, Bonnet DA, Domchek SM, Vonderheide RH. Telomerase vaccination has no detectable effect on SCID-repopulating and colonyforming activities in the bone marrow of cancer patients. Exp Hematol 2005; 33(11):1275–80. 86. Nair SK, Heiser A, Boczkowski D, et al. Induction of cytotoxic T cell responses and tumor immunity against unrelated tumors using telomerase reverse transcriptase RNA transfected dendritic cells. Nat Med 2000; 6(9):1011–7. 87. Parkhurst MR, Riley JP, Igarashi T, Li Y, Robbins PF, Rosenberg SA. Immunization of patients with the hTERT:540-548 peptide induces peptide-reactive T lymphocytes that do not recognize tumors endogenously expressing telomerase. Clin Cancer Res 2004; 10(14):4688–98. 88. Ayyoub M, Migliaccio M, Guillaume P, et al. Lack of tumor recognition by hTERT peptide 540-548-specific CD8(+) T cells from melanoma patients reveals inefficient antigen processing. Eur J Immunol 2001; 31(9):2642–51. 89. Ambrus A, Chen D, Dai J, Bialis T, Jones RA, Yang D. Human telomeric sequence forms a hybrid-type intramolecular G-quadruplex structure with mixed parallel/ antiparallel strands in potassium solution. Nucleic Acids Res 2006; 34(9):2723–35. 90. Luu KN, Phan AT, Kuryavyi V, Lacroix L, Patel DJ. Structure of the human telomere in K+ solution: an intramolecular (3 + 1) G-quadruplex scaffold. J Am Chem Soc 2006; 128(30):9963–70. 91. Xu Y, Noguchi Y, Sugiyama H. The new models of the human telomere d[AGGG (TTAGGG)3] in K+ solution. Bioorg Med Chem 2006; 14(16):5584–91. 92. Schaffitzel C, Berger I, Postberg J, Hanes J, Lipps HJ, Plückthun A. In vitro generated antibodies specific for telomeric guanine quadruplex DNA react with Stylonychia lemnae macronuclei. Proc Natl Acad Sci USA 2001; 98:8572–7. 93. Paeschke K, Simonsson T, Postberg J, Rhodes D, Lipps H. Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo. Nat Struct Mol Biol 2005; 12(10):847–54. 94. Duquette ML, Handa P, Vincent JA, Taylor AF, Maizels N. Intracellular transcription of G-rich DNAs induces formation of G-loops, novel structures containing G4 DNA. Genes Dev 2004; 18(13):1618–29. 95. Chang CC, Kuo IC, Ling IF, et al. Detection of quadruplex DNA structures in human telomeres by a fluorescent carbazole derivative. Anal Chem 2004; 76(15):4490–4. 96. Granotier C, Pennarun G, Riou L, et al. Preferential binding of a G-quadruplex ligand to human chromosome ends. Nucl Acid Res 2005; 33(13):4182–90. 97. Zahler AM, Williamson JR, Cech TR, Prescott DM. Prescott DM. Inhibition of telomerase by G-quadruplex DNA structures. Nature 1991; 350(6320):718–20. 98. Han H, Langley DR, Rangan A, Hurley LH. Selective interactions of cationic porphyrins with G-quadruplex structures. J Am Chem Soc 2001; 123(37):8902–13. 99. Fedoroff OY, Salazar M, Han H, Chemeris VV, Kerwin SM, Hurley LH. NMR-based model of a telomerase inhibiting compound bound to G-quadruplex DNA. J Med Chem 1998; 37(36):12367–74. 100. Perry PJ, Reszka AP, Wood AA, et al. Human telomerase inhibition by regioisomeric disubstituted amidoanthracene-9,10-diones. J Med Chem 1998; 41(24):4873–84.

370

Riou et al.

101. Perry PJ, Read MA, Davies RT, et al. 2,7-disubstituted amidofluorenone derivatives as inhibitors of human telomerase. J Med Chem 1999; 42(14):2679–84. 102. Read M, Harrison RJ, Romagnoli B, et al. Structure-based design of selective and potent G quadruplex-mediated telomerase inhibitors. Proc Natl Acad Sci USA 2001; 98(9):4844–9. 103. Gowan SM, Harrison JR, Patterson L, et al. A G-quadruplex-interactive potent smallmolecule inhibitor of telomerase exhibiting in vitro and in vivo antitumor activity. Mol Pharmacol 2002; 61(5):1154–62. 104. Harrison RJ, Cuesta J, Chessari G, et al. Trisubstituted acridine derivatives as potent and selective telomerase inhibitors. J Med Chem 2003; 46(21):4463–76. 105. Moore MJB, Schultes CM, Cuesta J, et al. Trisubstituted acridines as G-quadruplex telomere targeting agents. Effects of extensions of the 3,6-and 9-side chains on quadruplex binding, telomerase activity, and cell proliferation. J Med Chem 2006; 49(2):582–99. 106. Koeppel F, Riou JF, Laoui A, et al. Ethidium derivatives bind to G-quartets, inhibit telomerase and act as fluorescent probes for quadruplexes. Nucleic Acids Res 2001; 29(5):1087–96. 107. Rosu F, Pauw ED, Guittat L, et al. Selective interaction of ethidium derivatives with quadruplexes: an equilibrium dialysis and electrospray ionization mass spectrometry analysis. J Med Chem 2003; 42(35):10361–71. 108. Riou JF, Guittat L, Mailliet P, et al. Cell senescence and telomere shortening induced by a new series of specific G-quadruplex DNA ligands. Proc Natl Acad Sci USA 2002; 99:2672–7. 109. Kim MY, Duan W, Gleason-Guzman M, Hurley LH. Design, synthesis, and biological evaluation of a series of fluoroquinoanthroxazines with contrasting dual mechanisms of action against topoisomerase II and G-quadruplexes. J Med Chem 2003; 46(4):571–83. 110. Caprio V, Guyen B, Opoku-Boahen Y, et al. A novel inhibitor of human telomerase derived from 10H-indolo[3,2-b]quinoline. Bioorg Med Chem Lett 2000; 10(18):2063–6. 111. Mergny JL, Lacroix L, Teulade-Fichou MP, et al. Telomerase inhibitors based on quadruplex ligands selected by a fluorescent assay. Proc Natl Acad Sci USA 2001; 98:3062–7. 112. Teulade-Fichou MP, Carrasco C, Guittat L, et al. Selective recognition of G-quadruplex telomeric DNA by a bis(Quinacridine) macrocycle. J Am Chem Soc 2003; 125(16):4732–40. 113. Gowan S, Heald R, Stevens M, Kelland, L. Potent inhibition of telomerase by smallmolecule pentacyclic acridines capable of interacting with G-quadruplexes. Mol Pharmacol 2001; 60(5):981–8. 114. Cookson JC, Dai FP, Smith V, et al. Pharmacodynamics of the G-quadruplex-stabilizing telomerase inhibitor 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl] acridinium methosulfate (RHPS4) in vitro: Activity in human tumor cells correlates with telomere length and can be enhanced, or antagonized, with cytotoxic agents. Mol Pharmacol 2005; 68(6):1551–8. 115. Lemarteleur T, Gomez D, Paterski R, Mandine E, Mailliet P, Riou J-F. Stabilization of the c-myc gene promoter quadruplex by specific ligands inhibitors of telomerase. Biochem Biophys Res Com 2004; 323:802–8. 116. Kim MY, Vankayalapati H, Shin-ya K, Wierzba K, Hurley LH. Telomestatin, a potent telomerase inhibitor that interacts quite specifically with the human telomeric intramolecular G-quadruplex. J Am Chem Soc 2002; 124:2098–9. 117. Guittat L, Alberti P, Rosu F, et al. Interactions of cryptolepine and neocryptolepine with unusual DNA structures. Biochimie 2003; 85(5):535–47. 118. Guittat L, DeCian A, Rosu F, et al. Ascididemin and meridine stabilise G-quadruplexes and inhibit telomerase in vitro. Biochim Biophys Acta—General Subjects 2005; 1724(3):375–84. 119. Cuesta J, Read M, Neidle S. The design of G-quadruplex ligands as telomerase inhibitors. Mini Rev Med Chem 2003; 3(1):11–21. 120. Maeshima K, Janssen S, Laemmli UK. Specific targeting of insect and vertebrate telomeres with pyrrole and imidazole polyamides. EMBO J 2001; 20(12):3218–28.


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121. Takahashi R, Bando T, Sugiyama H. Specific alkylation of human telomere repeats by hairpin pyrrole-imidazole polyamide. Bioorg Med Chem 2003; 11(12):2503–9. 122. Furuta M, Nozawa K, Takemura M, et al. A novel platinum compound inhibits telomerase activity in vitro and reduces telomere length in a human hepatoma cell line. Int J Cancer 2003; 104(6):709–15. 123. Colangelo D, Ghiglia AL, Viano I, Cavigiolio G, Osella D. Cis-[Pt(Cl)(2)(Pyridine) (5-SO3H-isoquinoline)] complex, a selective inhibitor of telomerase enzyme. Biometals 2003; 16(4):553–60. 124. Tauchi T, Shinya K, Sashida G, et al. Activity of a novel G-quadruplex-interactive telomerase inhibitor, telomestatin (SOT-095), against human leukemia cells: involvement of ATM-dependent DNA damage response pathways. Oncogene 2003; 22(34):5338–47. 125. Gomez D, Aouali N, Renaud A, et al. Resistance to senescence induction and telomere shortening by a G-quadruplex ligand inhibitor of telomerase. Cancer Res 2003; 63(19):6149–53. 126. Shammas MA, Reis RJS, Akiyama M, et al. Telomerase inhibition and cell growth arrest by G-quadruplex interactive agent in multiple myeloma. Mol Cancer Ther 2003; 2(9):825–33. 127. Leonetti C, Amodei S, DAngelo C, et al. Biological activity of the G-quadruplex ligand RHPS4 (3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate) is associated with telomere capping alteration. Mol Pharmacol 2004; 66(5):1138–46. 128. Pennarun G, Granotier C, Gauthier LR, et al. Apoptosis related to telomere instability and cell cycle alterations in human glioma cells treated by new highly selective G-quadruplex ligands. Oncogene 2005; 24(18):2917–28. 129. Izbicka E, Wheelhouse RT, Raymond E, et al. Effects of cationic porphyrins as G-quadruplex interactive agents in human tumor cells. Cancer Res 1999; 59(3):639–44. 130. Rangan A, Fedoroff OY, Hurley LH. Induction of duplex to G-quadruplex transition in the c-myc promoter region by a small molecule. J Biol Chem 2001; 276(7):4640–6. 131. Gomez D, Lemarteleur T, Lacroix L, Mailliet P, Mergny JL, Riou JF. Telomerase down regulation induced by the G-quadruplex ligand 12459 in A549 cells is mediated by hTERT RNA alternative splicing. Nucleic Acids Res 2004; 32(1):371–9. 132. Burger AM, Dai FP, Schultes CM, et al. The G-quadruplex-interactive molecule BRACO-19 inhibits tumor growth, consistent with telomere targeting and interference with telomerase function. Cancer Res 2005; 65(4):1489–96. 133. Douarre C, Gomez D, Morjani H, et al. Overexpression of Bcl-2 is associated with apoptotic resistance to the G-quadruplex ligand 12459 but is not sufficient to confer resistance to long-term senescence. Nucleic Acids Res 2005; 33(7):2192–203. 134. Incles CM, Schultes CM, Kempski H, Koehler H, Kelland LR, Neidle S. A G-quadruplex telomere targeting agent produces p16-associated senescence and chromosomal fusions in human prostate cancer cells. Mol Cancer Ther 2004; 3(10):1201–6. 135. Gomez D, Aouali N, Londono-Vallejo A, et al. Resistance to the short term antiproliferative activity of the G-quadruplex ligand 12459 is associated with telomerase overexpression and telomere capping alteration. J Biol Chem 2003; 278(50):50554–62. 136. Incles CM, Schultes CM, Kelland LR, Neidle S. Acquired cellular resistance to flavopiridol in a human colon carcinoma cell line involves up-regulation of the telomerase catalytic subunit and telomere elongation. Sensitivity of resistant cells to combination treatment with a telomerase inhibitor. Mol Pharmacol 2003; 64(5):1101–8. 137. Taetz S, Baldes C, Murdter TE, et al. Biopharmaceutical characterization of the telomerase inhibitor BRACO19. Pharm Res 2006; 23(5):1031–7. 138. de Lange T. Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev 2005; 19(18):2100–10. 139. Cimino-Reale G, Pascale E, Battiloro E, Starace G, Verna R, D0 Ambrosio E. The length of telomeric G-rich strand 3'-overhang measured by oligonucleotide ligation assay. Nucleic Acids Res 2001; 29(7):E35. 140. Gomez D, Paterski R, Lemarteleur T, Shinya K, Mergny JL, Riou JF. Interaction of telomestatin with the telomeric single-strand overhang. J Biol Chem 2004; 279(40):41487–94.

372

Riou et al.

141. Court R, Chapman L, Fairall L, Rhodes D. How the human telomeric proteins TRF1 and TRF2 recognize telomeric DNA: a view from high-resolution crystal structures. EMBO Rep 2005; 6(1):39–45. 142. Lei M, Podell ER, Cech TR. Structure of human POT1 bound to telomeric singlestranded DNA provides a model for chromosome end-protection. Nat Struct Mol Biol 2004; 11(12):1223–9. 143. Hockemeyer D, Sfeir AJ, Shay JW, Wright WE, deLange T. POT1 protects telomeres from a transient DNA damage response and determines how human chromosomes end. EMBO J 2005; 24(14):2667–78. 144. Zhu XD, Niedernhofer L, Kuster B, Mann M, Hoeijmakers JHJ, de Lange T. ERCC1/ XPF removes the 3' overhang from uncapped telomeres and represses formation of telomeric DNA-containing double minute chromosomes. Mol Cell 2003; 12(6):1489–98. 145. Celli GB, deLange T. DNA processing is not required for ATM-mediated telomere damage response after TRF2 deletion. Nat Cell Biol 2005; 7(7):712–8. 146. Takai H, Smogorzewska A, de Lange T. DNA damage foci at dysfunctional telomeres. Curr Biol 2003; 13(17):1549–56. 147. Kim S, Beausejour C, Davalos AR, Kaminker P, Heo SJ, Campisi J. TIN2 mediates functions of TRF2 at human telomeres. J Biol Chem 2004; 279(42):43799–804. 148. d’Adda di Fagagna F, Reaper PM, Clay-Farrace L, et al. A DNA damage checkpoint response in telomere-initiated senescence. Nature 2003; 426(6963):194–8. 149. Gomez D, O’Donohue MF, Wenner T, et al. The G-quadruplex ligand telomestatin inhibits POT1 binding to telomeric sequences in vitro and induces GFP-POT1 dissociation from telomeres in human cells. Cancer Res 2006; 66(14):6908–12. 150. Tahara H, Shinya K, Seimiya H, Yamada H, Tsuruo T, Ide T. G-quadruplex stabilization by telomestatin induces TRF2 protein dissociation from telomeres and anaphase bridge formation accompanied by loss of the 3' telomeric overhang in cancer cells. Oncogene 2006; 25(13):1955–66. 151. Opresko PL, Mason PA, Podell ER, et al. POT1 stimulates RecQ helicases WRN and BLM to unwind telomeric DNA substrates. J Biol Chem 2005; 280(37):32069–80. 152. Sun H, Karow JK, Hickson ID, Maizels N. The Bloom's syndrome helicase unwinds G4 DNA. J Biol Chem 1998; 273(42):27587–92. 153. Mohaghegh P, Karow JK, Brosh RM, Jr, Bohr VA, Hickson ID. The Bloom's and Werner's syndrome proteins are DNA structure-specific helicases. Nucleic Acids Res 2001; 29(13):2843–9. 154. Fry M, Loeb LA. Human Werner syndrome DNA helicase unwinds tetrahelical structures of the fragile X syndrome repeat sequence d(CGG)n. J Biol Chem 1999; 274:12797–802. 155. Stavropoulos DJ, Bradshaw PS, Li X, et al. The Bloom syndrome helicase BLM interacts with TRF2 in ALT cells and promotes telomeric DNA synthesis. Hum Mol Genet 2002; 11(25):3135–44. 156. Crabbe L, Verdun RE, Haggblom CI, Karlseder J. Defective telomere lagging strand synthesis in cells lacking WRN helicase activity. Science 2004; 306(5703):1951–3. 157. Vaughn JP, Creacy SD, Routh ED, et al. The DEXH protein product of the DHX36 gene is the major source of tetramolecular quadruplex G4-DNA resolving activity in HeLa cell lysates. J Biol Chem 2005; 280(46):38117–20. 158. Ding H, Schertzer M, Wu XL, et al. Regulation of murine telomere length by Rtel: An essential gene encoding a helicase-like protein. Cell 2004; 117(7):873–86. 159. Cheung I, Schertzer M, Rose A, Lansdorp PM. Disruption of dog-1 in Caenorhabditis elegans triggers deletions uptstream of guanine-rich DNA. Nat Genet 2002; 31:405–9. 160. Li J-L, Harrison RJ, Reszka AP, et al. Inhibition of the Bloom's and Werner's Syndrome Helicases by G-Quadruplex Interacting Ligands. J Med Chem 2001; 40:15194–202. 161. Herbert BS, Pitts AE, Baker SI, et al. Inhibition of human telomerase in immortal human cells leads to progressive telomere shortening and cell death. Proc Natl Acad Sci USA 1999; 96(25):14276–81. 162. Pantic M, Zimmerman S, Waller CF, Martens UM. The level of telomere dysfunction determines the efficacy of telomerase-based therapeutics in a lung cancer cell line. Int J Oncol 2005; 26(5):1227–32.


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163. Ponti D, Costa A, Zaffaroni N, et al. Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res 2005; 65(13):5506– 11. 164. Siddiqui-Jain A, Grand CL, Bearss DJ, Hurley LH. Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription. Proc Natl Acad Sci USA 2002; 99(18):11593–8. 165. Sun DY, Guo KX, Rusche JJ, Hurley LH. Facilitation of a structural transition in the polypurine/polypyrimidine tract within the proximal promoter region of the human VEGF gene by the presence of potassium and G-quadruplex-interactive agents. Nucleic Acids Res 2005; 33(18):6070–80. 166. Rezler EM, Seenisamy J, Bashyam S, et al. Telomestatin and diseleno sapphyrin bind selectively to two different forms of the human telomeric G-quadruplex structure. J Med Chem Soc 2005; 127(26):9439–47.

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Targeting Hsp90: The Cancer Super-Chaperone Paul Workman and Swee Sharp Cancer Research U.K. Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, U.K.

INTRODUCTION: SETTING THE CONTEXT The dramatic increase in our understanding of how cancer cells subvert normal cellular signaling systems to drive malignant progression (1) has led to the equally dramatic switch in drug discovery and development from the dominance of cytotoxic agents to a new era of targeted molecular cancer therapeutics (2). Our ability to discover and develop new drugs targeted to the underpinning molecular pathology of particular cancers has been accelerated by the integrated application of a powerful toolkit of new technologies and approaches, including genomics and proteomics; high-throughput screening (HTS) of chemical libraries; structure-based design; chemical biology; animal models; and molecular biomarkers for selecting patients, demonstrating proof of concept for target modulation, and enabling rational decisions in clinical trials (3). The selection of new drug targets (4) is frequently based on our increasing identification and understanding of cancer genes (5–8). These can be categorized broadly into oncogenes, tumor suppressor genes, and genome fidelity genes (1) which generally conspire together to hijack biochemical pathways and biological networks to produce the hallmark traits of malignancy (9). Clearly, the products of individual genetically or epigenetically deregulated cancer genes, or other proteins in the pathway that they subvert, can be targeted for cancer therapy (10). However, it is becoming clear that the elements of certain }cancer support systems,} which are not themselves bonafide oncogenes in the classical sense, can also be targeted for effective cancer treatment. Examples of these are the chromatin modifying enzymes, particularly histone deacetylases (11), and components of the protein quality control machinery, specifically the proteasome (12) and the molecular chaperone Hsp90 (13–15), which is the subject of this present chapter. Hsp90: AN UNUSUAL DRUG TARGET Until relatively recently, Hsp90 would not have been near the top of anyone's list as an obvious cancer drug target. As mentioned above, it is not a cancer gene, per se, and is not mutated in malignancy. Moreover, it is widely expressed in healthy cells and plays and important role within them, particularly under stressful conditions (15). Thus, interference with this protective molecular chaperone might have been expected to result in considerable toxicity. However, just as interest in the biology of Hsp90 has grown impressively over the past few years, so has the enthusiasm to develop inhibitors of the chaperone as molecular cancer

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therapeutics (14,16–18). Indeed, research into the translational and pharmacological aspects of Hsp90 have not only developed in parallel, but in fact have been very closely interactive with, and mutually beneficial to, the more fundamental investigations, as this review illustrates. A Pubmed search on }Hsp90} carried out in February 2007 yielded nearly 3300 hits—a remarkable tally for a protein with fairly humble origins. ORIGINS Heat shock proteins, known as Hsps, were first discovered over 40 years ago as proteins that exhibited increased expression in cells in response to elevated temperature (19,20). Many Hsps are molecular chaperones that have an important function in the correct folding of polypeptide chains, a process which can be challenging in the crowded molecular environment of the cell (21–23). Chaperones, including Hsp90, protect the cell against the effects of protein misfolding and aggregation. They carry out this role under normal conditions and also in reaction to cellular stresses, particularly in the adaptive response (24–26). Hsps are numbered according to their respective apparent molecular weights (26). The family of related Hsp90 proteins are a group of abundant 90KDa molecular chaperones that contribute as much as 1–2% of cellular protein, even under normal conditions (15,27–29). The family is made up of Hsp90a and Hsp90b, which predominate in the cytoplasm, Grp94, located in the endoplasmic reticulum, and TRAP1, in the mitochondria. An additional related protein, Hsp90N, has been identified, but its significance and precise role have not been elucidated in detail (30). STRUCTURE AND FUNCTION OF THE Hsp90 SUPER-CHAPERONE The function of Hsp90 is to regulate the conformation, activation, stability, and function of so-called }client} proteins (14–16). All four of the best known Hsp90 family members mentioned above are closely related in sequence and function. However, Hsp90a and Hsp90b are the most studied and best understood, and this review focuses mainly on them. They are present as functional dimers in the cell. The molecular structure of Hsp90 has been elucidated in considerable detail, with X-ray crystallographic studies being particularly informative (31–33). Individual subunits contain three functional domains. These are the N-terminal adenosine triphosphate (ATP)-binding domain, a middle domain that is implicated in client protein binding, and a C-terminal dimerization domain containing the tetratricopeptide repeat (TPR) binding motifs (31). A variety of evidence has shown that ATP binding, ATP hydrolysis, and ATP/adenosine diphosphate (ADP) nucleotide exchange are essential for Hsp90 function (34–38). Together with a highly integrated and orchestrated set of interactions with co-chaperones and other accessory proteins that join with Hsp90 to form a highly dynamic super-chaperone complex, the processes of nucleotide exchange and ATP hydrolysis drive a cycle of conformational changes, which in turn modulate the further interactions with client proteins (31). The recent X-ray crystal structure of the full-length yeast Hsp90 dimer in a complex with a nonhydrolyzable ATP analogue and the co-chaperone p23/Sba1 has been extremely important in supporting what was previously a controversial }molecular clamp} mechanism that is coupled to the ATPase cycle and which involves the closure of

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the so-called }lid} segment and transient dimerisation of the N-terminal domain in the ATP-bound conformational state (39,40). The new structure has revealed the intricate architecture of the }closed} conformational state of the molecular chaperone (32). The structure also sheds light on the extensive series of interactions between the various protein chains. The detailed conformational changes that occur in the N-terminal domain upon ATP binding are defined, as is the structural basis for how the closed state is stabilized by p23/Sba1. Very surprisingly, the closed Hsp90 conformation would not allow client proteins to be enclosed within the Hsp90 dimer in the expected fashion, but rather a bipartite binding surface is provided, the formation and disruption of which is coupled to the ATPase cycle of the chaperone. The structure clearly confirms that the ATPase-coupled molecular clamp model is correct and demonstrates that the proposed dimerization-coupled }split} ATPase mechanism (39,40) that is seen with other members of the GHKL class of ATPases, including the MutL mismatch repair proteins and type II topoisomerases (41, see later), also operates in the case of Hsp90. In addition, the recent X-ray crystallography work gives us a structural basis upon which we can begin to answer the critical question of how client protein activation is achieved. Some insight into this question has been provided by the recent electron microscopic reconstruction of the complex between yeast Hsp90, the kinase cochaperone Cdc37, and the kinase client Cdk4 (33). From this it appears that the two lobes of the Cdk4 kinase are in fact interacting with different domains of the Hsp90. In this way, the conformation of the kinase client can be coupled directly to changes in the relative positioning of the chaperone domains with which it interacts. The precise details of how the coupled chaperone-client interactions result in client protein activation remain to be defined, and this is now a key challenge for the field. The individual roles of the various co-chaperones in the ATPase-driven chaperone cycle are becoming clearer. Client proteins interact first off with the Hsp70/Hsp40/Hip complex (13,31). Hsp70 is then linked to Hsp90 by the adapter Hop/p60, which binds to its respective C-terminal domain via its TPR domain. The binding of Hop/p60 is restricted to ADP-bound Hsp90 in the open conformation, which has a high affinity for hydrophobic substrates. When Hsp90 exchanges ADP for ATP, this brings about the conformational change and transient dimerisation of the N-terminal domains, as discussed above. Hsp70/Hsp40/Hip and Hop then dissociate from the complex, facilitating ATPdependent association with other co-chaperones, such as Cdc37, p23, and immunophilins, thus forming the so-called }mature} complex. Cdc37 loads kinase clients, whereas p23 stabilizes the ATP-bound form of Hsp90, thereby prolonging the period in which the client protein activation can take place (32). Examples of client protein activation are the changes that allow steroid hormone receptors to bind ligand or kinases like AKT to be phosphorylated as part of signal transduction cascades. Prevention of ATP binding to N-terminal Hsp90 (e.g., by inhibitors, see later) blocks the mature complex formation and, instead of client protein activation, promotes degradation of the client via the ubiquitin proteasome pathway (see later). In some instances this has been shown to involve recruitment to the immature Hsp90 complex of the ubiquitin ligase CHIP, another TPR domain protein that can bind to both Hsp90 and Hsp70 (42,43). The action of the Hsp90 activating protein AHA1 (44) is mediated via binding to the middle domain of the chaperone; this then promotes a

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conformational switch in the middle segment catalytic loop that releases the catalytic Arg380 residue (yeast Hsp90 numbering) and facilitates its interaction with ATP in the N-terminal domain (45). In addition to the precise mechanisms by which Hsp90 brings about client protein activation (see above), another outstanding conundrum is how particular clients are recognized while other related proteins are not. Little is understood about the determinants of Hsp90 interaction with its client proteins. In one study, a loop within the N lobe of the kinase domain of ERBB2 was identified as important for Hsp90 binding (46). The amino acid sequence of this loop affects the electrostatic and hydrophobic nature of the surface of the kinase, which appears in turn to influence the interaction with Hsp90. A point mutation in the ERBB2 loop region was found to disrupt Hsp90 binding. These considerations appear to explain the differences in the interaction with Hsp90 of different members the ERBB2 family. Also of interest, the nascent, immature forms of ERBB2 and ERBB1 (EGFR) are both dependent on Hsp90 chaperoning, but whereas the mature ERBB2 retains this dependence, the mature ERBB1 becomes Hsp90-independent. Another recent study of a large number of kinases has again indicated that surface features, rather than the contiguous amino acid sequence, are important in defining recognition of kinases by Hsp90 (46). Furthermore, the authors suggested, based on a comparison of the mitogen-activated protein kinase and the phosphatidylinositol 3-kinase pathways, that the selectivity of Hsp90 towards particular kinases has a functional basis, with Hsp90 regulating kinases that act as hubs that integrate signals from multiple inputs (47). There is much more work to do to understand the rules that govern what makes a client protein for Hsp90. Hsp90 CLIENTS AND OTHER INTERACTIONS To be accepted as a bonafide Hsp90 client, a protein is generally expected to fulfil the following criteria: & & &

Bind to Hsp90 Be depleted in cells after treatment with an Hsp90 inhibitor Undergo degradation by the ubiquitin-proteasome pathway upon Hsp90 inhibition

Although Hsp90 is generally considered to be a relatively selective molecular chaperone, acting as it does at a relatively late stage in the maturation and activation of a relatively small group of client proteins, its customer list nevertheless continues to grow. The current client count is in excess of 100 proteins (15,28). An updated list of }Hsp90 interactors,} for which inclusion requires biochemical evidence of binding, is maintained as part of a very useful website (48). Featuring strongly among the clientele are protein kinases and transcription factors. Greater numbers of proteins have been shown to exhibit evidence of some type of interaction with Hsp90 based on global profiling or genetic approaches (47,49–52). For example, one study identified a network of 198 putative physical interactions and 451 genetic and chemical genetic interactions (49). On the basis of this study, as well as being labeled with terms such as the }super-chaperone} and }cancer chaperone,} Hsp90 has also been referred to as a }master regulator} (16,49). This is not only because of the sheer number of interactions, above and beyond those physical and functional interactions with bona fide clients and co-chaperones, but also in view of the diverse range of biological processes that Hsp90 influences. These involve not only linking a large

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array of homeostatic control mechanisms to the status of the external environment of the cell organism, for example integrating signal transduction pathways with stress responses, but also impacting on chromatin remodeling and gene transcription (15,49,52,53).

Hsp90 INHIBITORY NATURAL PRODUCTS: CHEMICAL BIOLOGY IN ACTION The natural products geldanamycin (Fig. 1) and radicicol (Fig. 2) were the first Hsp90 inhibitors to be discovered and have proved to be extraordinarily invaluable as chemical tools for probing the biology of Hsp90 and demonstrating proof of concept for the molecular chaperone as a therapeutic target. Furthermore, geldanamycin analogues were the first Hsp90 inhibitors to progress to clinical trials (see later). In 1992, Whitesell et al. (54) reported that the benzoquinone ansamycins related to geldanamycin and herbimycin A showed potent antitumor activity that was distinct from their reported inhibition of oncogenic kinases such as SRC. Having defined the 17-position (located on the quinone ring) as one that allows chemical manipulation without compromising antitumor activity, Whitesell et al. carried out a seminal experiment in which they coupled geldanamycin to beads via a linker/spacer group and used this to fish out the main binding protein from cell extracts (55). The main binding protein was identified as Hsp90 by the use of specific antibodies (55) and subsequently by micro-sequencing of protein bound to radio-labeled affinity-tagged geldanamycin (56). As part of the early series of experiments, geldanamycin was shown to inhibit the association of Hsp90 with the v-SRC tyrosine kinase oncoprotein (55). This association between the chaperone and the viral oncogene product had been demonstrated using virally transformed cells as early as 1981 (57–59). Subsequent reports that geldanamycin associated with kinases such as C-RAF (60) and other signaling proteins such as steroid hormone receptors (59) stimulated interest in Hsp90 as a potentially important drug target. This was supported by the recognition that

O R

O O

Cl H H OH N

O N H OH

O

N OH H

O NH2

O

O

OH

O

O

NH2 O

17-DMAG R = NHCH2 CH2N (CH3)2 17-AAG R = NHCH2CH = CH2

O

IPI-504

Geldanamycin R = OCH3 FIGURE 1 Chemical structures of the benzoquinone ansamycin class of Hsp90 inhibitors.

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O

O

O

H

O

O

O

H

HO

O

H

HO Cl

H

H

O

Cl OH

Radicicol

H

HO

O

OH

O

X= N

O

Cl

O

OH

N

Cycloproparadicicol

KF58333 (E-isomer) H HO

OH O

O

Cl HN

O HO Cl

O O

HO O Pochonin D

O OMe

O Radester

FIGURE 2 Chemical structures of the radicicol class of Hsp90 inhibitors.

Hsp90 was expressed in much higher levels in many tumor cells as compared to their normal counterparts, and in some cases this expression is of prognostic significance (28,60,61,62). In other important experiments, treatment of cells with geldanamycin and the related natural product herbimycin A was shown to cause client protein destabilization and degradation, usually via the ubiquitin-proteasome pathway (63). Also of tremendous importance was the use of a combination of biochemical methods and X-ray crystallography to demonstrate that geldanamycin binds at an N-terminal ATP site on Hsp90 (34,35,56). Previously, Csermely et al. had suggested that Hsp90 bound ATP, leading to a conformational change in the chaperone (64). A similar series of experiments to those done with geldanamycin was also carried out with the chemically different natural product radicicol (65–67). Following multiple demonstrations of the bioactivity of geldanamycin and radicicol using in vitro screens, derivatives of the agents have shown anticancer activity in a variety of animal models (14,17). Subsequently, the geldanamycin analogue 17-AAG (tanespimycin, KOS953) (Fig. 1) was progressed to clinical trials, followed by other analogues (68,69; see next section). Radicicol derivatives have not progressed to the clinic but their mode of binding to Hsp90 is very similar to the pyrazole/isoxazole class of inhibitors which are now undergoing development (70; see later). Thus it can be seen that the natural products geldanamycin, radicicol and related compounds have been invaluation both as chemical tools to interrogate Hsp90 biology and also to demonstrate proof of concept in cancer models, leading to clinical trials of 17-AAG. In the following sections, we provide concise summaries of the different classes of Hsp90 inhibitory chemotypes, starting with further information on the natural products and moving onto the more recent synthetic small molecule classes. The current status of the various Hsp90 inhibitors is listed in Tables 1 and 2.

Peptidomimetic Non-peptidic

Interaction between Hsp90 and survivin

Shepherdin 5-aminoimidazole4-carboxamide

SAHA and others

Abbreviations: NECA, 5-N-ethylcarboxamideadenosine; SAHA, suberoylanilide hydroxamic acid.

Various

Bristol-Myers Squibb (New York, New York, U.S.A.) Merck (Whitehouse Station, New Jersey, U.S.A.) Ref (103) Ref (105)

Paclitaxel (Taxol )

Taxane

Geldanamycin dimer

Geldanamycin dimer

Ref (100) Ref (104) NeuTec (Manchester, U.K.)

Radicicol oxime derivatives

Radicicol

Vernalis (Great Abington, Cambridge, U.K.); Novartis (Hanover, New Jersey, U.S.A.); The Institute of Cancer Research (Sutton, Surrey, U.K.) Kyowa Hakko Kogyo (Chiyoda-ku, Tokyo, Japan) Conforma Therapeutics

Novobiocin analogues NECA Mycograb

CCT018159, VER-49009

Pyrazole

Coumarin Adenosine analogue Antibody

CNF2024

Infinity Pharmaceuticals (Cambridge, Massachusetts, U.S.A.) Conforma Therapeutics

Kosan Biosciences; National Cancer Institute

17-DMAG (alvespimycin, KOS1022) IP-504

Company/institution Kosan Biosciences; (Hayward, California, U.S.A.); National Cancer Institute; (Bethesada, Maryland, U.S.A.); Conforma Therapeutics (San Diego, California, U.S.A.)

17-AAG (tanespimycin, KOS953, CNF1010)

Drug or lead compound

Purine

Hydroquinone form of 17-AAG

Benzoquinone ansamycin

Chemical class

Histone deacetylase

C-terminal Grp94 Other inhibitors

N-terminal ATPbinding pocket

Mode of Hsp90 binding

TABLE 1 The Current Status of Hsp90 Inhibitors Current status

Preclinical research Preclinical research

Preclinical research Preclinical research Phase II clinical trial for invasive candidiasis Phase I combination clinical trial with 17-AAG Phase I clinical trials

Preclinical research

Preclinical research

Phase I clinical trials in multiple myeloma, with or without bortezomib (Velcade ) Phase II in melanoma and breast cancer with trastuzumab (Herceptin ) CNF1010 is in Phase I clinical trials in solid tumors Phase I clinical trials in hematologic cancers and solid tumors/breast cancer with trastuzumab Phase I clinical trials in multiple myeloma and gastrointestinal stromal tumors Phase I clinical trials in solid tumors Preclinical discovery


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TABLE 2 Other Hsp90 Inhibitors that are Being Developed by Pharmaceutical Companies Pharmaceutical company

Compound

Development stage/current status

Astex Therapeutics (Milton Road, Cambridge, U.K.) Serenex (Durham, North Carolina, U.S.A.)

AT-13387

Lead compound selected; preclinical development Preclinical development Lead compound; target client specific Preclinical research Phase I clinical trials in solid tumors Lead optimization Preclinical research Preclinical research

ArQule (Woburn, Massachusetts, U.S.A.) Abraxis (New York, New York, U.S.A.) Biotica (Saffron Walden, Essex, U.K.) TopoTarget (Kobenhavn, Denmark) Locus (Blue Bell, Pennsylvania, U.S.A.)

SNX-5422 SNX-4862 ARQ-250RP ABI-010 – – –

GELDANAMYCIN ANALOGUES Early work focused mainly on geldanamycin (Fig. 1) and the related benzoquinone-ansamycin natural product, herbimycin A. In addition to work reviewed in the previous section, early studies in Japan revealed the bioactivity of these agents in several systems (71). Natural product inhibitors of Hsp90 are now well known to show up as hits in a variety of cellular screening assays. Some of these early screens demonstrated that the benzoquinone ansamycins could reverse oncogenic transformation by v-SRC (72). Though the effects were originally attributed to the relative inhibitory activity on tyrosine kinases like SRC, the observations did support the exploration of the benzoquinone ansamycins as potential antitumor agents. The activity in v-SRC transformation reversion assays was subsequently reinterpreted in the light of the knowledge that v-SRC is a client protein that is highly dependent on Hsp90, and which is rapidly degraded in cells exposed to Hsp90 inhibitors. As mentioned above, Hsp90 is a member of the small group of GHKL-type ATPases, including the MutL mismatch repair proteins and type II topoisomerases (41). X-ray crystallography studies revealed that the N-terminal domain adopts a so-called Bergerat fold. This is characterized by a two-layer a/b sandwich, in which the a helices define a deep nucleotide-binding pocket that extends all the way from the external protein surface to the buried face of the highly twisted b-sheet (31). The shape of the nucleotide binding site is very unusual compared to other ATP-binding proteins, including other classes of ATPases and kinases (41). As a result, the bound nucleotide is forced to adopt a bent topology (35). X-ray crystallography studies of geldanamycin bound to the N-terminal domain of Hsp90 (Fig. 3) showed, importantly, that the antibiotic adopts a folded or C-shape conformation as a result of the switch to a cis-amide bond, as distinct from the trans-amide bond that is adopted in solution, resulting in accurate mimicry of the unusual bent configuration of the natural nucleotide (67). The unusual ATP site topography forms the basis of the high selectivity of Hsp90 inhibitors. Much of the bound water found in Mg2þ-ADP complexes with Hsp90 is displaced by geldanamycin, although key water molecules are retained and participate in the anchoring network of hydrogen bonding interactions in the ATP pocket (67). The benzoquinone ring of geldanamycin is located at the top of the deep nucleotide binding pocket, whereas the ansa ring is oriented deep in the bottom of the ATP site. The X-ray co-crystal structure provides a basis for the rational design of inhibitory analogues based of geldanamycin. Certain features


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FIGURE 3 Pymol diagram showing the binding interactions between the adenosine triphosphate (ATP) binding site of yeast N-terminal domain of Hsp90 and geldanamycin. Hydrogen bonds are shown as dotted lines, water molecules are black colored balls, nitrogen atoms are in black, and oxygen atoms are in light grey. This representation is based on the structure published by Roe et al. Source: From Ref. 67.

are essential for activity, including the 7-carbamate group. Geldanamycin proved to be too toxic to give a therapeutic index in animal models (73). From a series of analogues, 17-AAG was identified as promising (74,75). 17-AAG has similar activity to geldanamycin against cancer cells in vitro (76) and causes cell cycle arrest and apoptosis in human colon cancer cell lines (77). It has also shown good antitumor effect at well tolerated doses in animal models (78). In addition, it exhibits strong anti-angiogenic properties (79). Complications with 17-AAG include its limited water solubility, leading to the use of a cumbersome formulation in the clinic, as well as variable metabolism by polymorphic cytochrome P450 CYP3A4 and also by NQO1/ DT-diaphorase, together with susceptibility to efflux by P-glycoprotein and lack of oral bioavailability (80,81). The analogue 17-DMAG (alvespimycin, KOS1022) (Fig. 1) is more water soluble and orally bioavailable while retaining antitumor activity (82). This agent has followed 17-AAG into the clinic. A remaining concern about the geldanamycin series is the presence of the quinone that participates in the target binding (Fig. 3) (69). Quinones are known to undergo redox cycling and to cause hepatotoxicity, which is seen with 17-AAG in the clinic (see later). Our discovery that 17-AAG is much more potent in tumor cells containing high levels of NQO1 led us to suggest that this reductase may metabolize 17-AAG to a more potent Hsp90-inhibitory reduction product, most likely the hydroquinone (80). Subsequently, Guo et al. confirmed that the 17-AAG hydroquinone is a somewhat more potent Hsp90 inhibitor (83). Recognizing that the hydroquinone would be more soluble than 17-AAG, this agent, which is known as IPI-504 (Fig. 1), is now being developed as a drug in its own right for intravenous administration (84) and has recently entered clinical trials.

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RADICICOL ANALOGUES As referred to above, radicicol (Fig. 2) behaves in many ways very similarly to the geldanamycins. However, radicicol achieves its nucleotide mimicry in a different way (69). It is oriented in the opposite sense to the benzoquinone ansamycin in the N-terminal nucleotide pocket. Thus, the aromatic resorcinol ring points down into the base of the deep pocket, whereas the macrocycle containing the conjugated bond system and the epoxide group binds at the top near the surface. Of note is the network of hydrogen bonds, including key water molecules, that anchor the resorcinol unit at the bottom of the ATP site. As with geldanamycin, radicicol adopts a folded structure, but this is less pronounced than with the benzoquinone ansamycin, being more of an L-shape than a C-shape, and is the conformation also adopted by the unliganded radicicol. Radicicol has a more potent binding affinity for Hsp90 (i.e., it has a lower dissociation constant Kd), at least under the conditions and short term incubations used in the experimental determination (69). This is apparently due to differences in enthalpy/entropy factors. Lacking the quinone, radicicol is not susceptible to metabolism to NQO1. It does, however, still contain structural features that are not desirable in a drug, including the presence of potentially reactive electrophilic sites. Presumably because of stability, metabolic liability, and potential toxicity issues, radicicol itself did not show promising activity at non-toxic doses in animal models. On the other hand, radicicol oximes (e.g., KF58333) (Fig. 2) do show activity in human tumor xenografts in vivo (85). Despite this animal model activity, no radicicol analogue has progressed to clinical trial, possibly due to toxicity to the eye (17,18). However, the key resorcinol unit present in radicicol features as an important structural element within the pyrazole/isoxazole scaffold class of Hsp90 inhibitors (70; see later). Given its modest complexity and amenability to fairly short syntheses, a range of variants have been prepared, including cycloproparadicicol, pochonin D, and also radester (Fig. 2), which is a hybrid comprising part structures of radicicol (resorcinol unit) and geldanamycin (benzoquinone). (For more details, see references 16 and 86.) A series of simplified ring and conformational analogues of radicicol were synthesized and showed a degree tolerance for different macrocyclic ring sizes and quite a high level of potency in certain examples (87). PURINE-SCAFFOLD INHIBITORS Given, on the one hand, the combined promise and limitations exhibited by the natural product-based Hsp90 inhibitors and, on the other hand, the technologies that are available for HTS and structure-based design, it was logical that projects would be initiated to discover synthetic small molecule classes. The first of these series to emerge have been the purine-scaffold inhibitors designed by Chiosis et al. on the basis of molecular modeling (88) using the published crystal structures of Hsp90 (34,35,67). The initial agents were exemplified by the first compound PU3 (89) and the more potent analogue PU24FCl (90) (for chemical structures, see Fig. 4). Contrary to the rigid docking predictions, X-ray co-crystal structures showed that the trimethoxyphenyl ring did not bind in the expected fashion but induces a conformational change that opens up a new hydrophobic pocket in Hsp90a (91).

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FIGURE 4 Chemical structures of purine-scaffold Hsp90 inhibitors.

Analogue PU24FCl showed activity in a human tumor xenograft at quite high doses (90). A more recent report has disclosed a number of water soluble 8-arylsulfanyl, 8-arylsulfoxyl, and 8-arylsulfonyl adenyl derivatives based on the purine-scaffold; PU-H58 (Fig. 4) has emerged to be the most potent in vitro and showed good activity in animal models (92). Optimization based on detailed structure-activity relationships has resulted in compounds that show therapeutic activity by the oral route at fairly high doses (93), which may be needed to overcome rapid clearance. The purine analog CNF2024 (structure not disclosed) is now in Phase I clinical trials (Table 1) (94). PYRAZOLE-SCAFFOLD INHIBITORS The fertile pyrazole-scaffold compounds (70) were discovered by HTS using the yeast Hsp90 enzyme and malachite green as a readout for inorganic phosphate release upon ATP hydrolysis (95,96). This screen identified the 3,4-diarylpyrazole resorcinol hit CCT018159 (Fig. 5), which had similar single digit micromolar potency on the Hsp90 protein target as the clinical agent 17-AAG, although the cellular activity was lower (97). More detailed biological evaluation showed that the activity of CCT018159 on human cancer cells was not affected by NQO1 or P-glycoprotein, representing significant advantages over 17-AAG (97). Protein crystallography studies were initiated very soon after hit identification, defining the principal ligand-protein interactions, thus allowing a structurebased approach to be taken. As referred to earlier, the resorcinol unit in the pyrazole series binds in the identical way to the equivalent structural motif in radicicol (96,97). In the same way as with radicicol, the resorcinol unit anchors the compounds into the base of the nucleotide binding pocket via a network of HO

O

HO

Cl

O

N

H N

O

HO

HO N

N H

CCT018159

N H

O

VER-49009 CCT0129397

FIGURE 5 Chemical structures of pyrazole-scaffold Hsp90 inhibitors.

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hydrogen bonds with the protein, both direct and water-mediated. The ethyl group on the resorcinol ring of CCT018159 binds into a hydrophobic pocket, as do other lipophilic substituents in that position, such as chloro (96). Structure-based design, carried out in collaboration between our own group and Vernalis Ltd, led to a major gain in potency by the addition of a hydrogen bond donor at C5 of the pyrazole ring (98). A series of potent C5 amides were generated that benefited from the predicted additional protein-ligand interaction. These are exemplified by VER-49009 (CCT0129397) (Fig. 5). Structurally related compounds were found in an independent screen, but the particular compounds disclosed had little activity in cells, probably due to poor membrane penetration as a result of the presence of a carboxylate residue (99). Other studies have shown that pyrazole-scaffold can be replaced by alternative heterocyclic rings, such as isoxazole and triazole (70). The progress with the pyrazole resorcinols has nicely illustrated the combined power of HTS coupled with X-ray crystallography-driven structure-based design. NOVOBIOCIN ANALOGUES The coumarin-based antibiotics bind to bacterial DNA gyrase, an ATPase closely related to Hsp90. The coumarin antibiotic novobiocin (Fig. 6) was reported to bind to a proposed ATP site in the C-terminus of Hsp90 (100,101). X-ray crystallography studies are not informative on the C-terminus. More potent analogues have been identified from a library of novobiocin derivatives, of which compound A4 (Fig. 6) was the most active (102). OTHER Hsp90 INHIBITORS Various companies and academic groups are now developing a range of small molecule Hsp90 inhibitors (Tables 1 and 2). The peptidomimetic shepherdin was designed by modeling the interface between Hsp90 and the antiapoptotic and mitotic regulator client protein survivin (103). It has been reported to interact with the ATP pocket of Hsp90, to destabilize client proteins and to induce massive apoptosis, with antitumor activity also seen in vivo at well tolerated doses (103). Anti-leukemic activity has been described (104). The non-peptidic small molecule 5-aminoimidazole-4-carboxamide was designed using a combined structure-activity and dynamics-based computational chemistry strategy and shown to be active (105).

OH

O

O

OH

O

NH O

O

MeO

O

HO

OH Novobiocin

FIGURE 6 Chemical structures of novobiocin and A4.

O

O OMe

O H 2N

O

H N

O OH A4

O


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Based on experience with other drug target classes and the potential for distinct effects on different client proteins, the identification of isoform-selective inhibitors of particular Hsp90 family members is of interest. The adenosine analogue 5-N-ethylcarboxamideadenosine (NECA) (Fig. 7) is reported to be selective for Grp94 over Hsp90 (106). A cell-impermeable geldanamycin derivative was reported to exhibit antiinvasive activity, potentially via effects on Hsp90a, which can be secreted extracellularly where it interacts with matrix metalloproteinase 2 (107,108). A human recombinant antibody directed against fungal Hsp90 has been developed as an antifungal agent and shows promising activity in that setting (Table 1) (109). It has also been considered as a potential cancer treatment (110). The cytotoxic anticancer agents cisplatin and paclitaxel (Taxol ; BristolMeyers Squibb, New York, New York, U.S.A.) have been reported to bind weakly to Hsp90, but the significance of this is unclear (see ref 14). Histone deacetylase (HDAC) inhibitors such as depsipeptide FK228 and LAQ824 (Fig. 8) have been reported to inhibit Hsp90 based on the molecular signature of client protein depletion and induction of Hsp70 (see later) in cancer models and patients (111–113). HDAC inhibitors increase the acetylation of Hsp90 and an acetylation site in the middle domain of Hsp90 has been identified which regulates chaperone function (114). This appears to be mediated by HDAC6. Other post-translational modifications are known to occur on Hsp90 that may also affect its activity. A synergistic interaction between HDAC and Hsp90 inhibitors (LBH589 and 17-AAG, respectively) was reported in leukemic cells (115). However, we have observed an antagonistic interaction between the HDAC inhibitor trichostatin A and 17-AAG in a human ovarian cancer cell line; this antagonism was consistent with effects on total cellular acetylation, which was reduced by 17AAG, and also with changes in the expression of chromatin-modifying enzymes and other chromatin-associated proteins (116). These interactions are complex and potentially context-dependent. They likely relate to the functional link, mentioned earlier in this chapter, between Hsp90, chromatin regulation, and gene transcription. THERAPEUTIC SELECTIVITY It is important to consider how the therapeutic selectivity of Hsp90 inhibitors for tumor versus normal cells may be achieved. This may be related to one or more of the following factors: & &

Simultaneous combinatorial depletion of multiple oncogenic client proteins (117) Combinatorial effects on all of the hallmark traits of cancer (9,117) NH2 N

N N

O

N O

N H HO NECA

OH FIGURE 7 Chemical structure of an inhibitor of the Hsp90 isoform Grp94.

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Me H N

O OH

O N H

N

OH

HN

S

S

NH Me

O Me

HN

O

HN Me

LAQ824

O O

Me

O

FK228

FIGURE 8 Chemical structures of inhibitors of histone deacetylase. & & &

&

&

&

Exploitation of }oncogene addiction} (117), whereby cancer cells are more dependent upon, or addicted to, the primary oncogenic drivers of the disease The greater dependence of mutated oncogenic client proteins on Hsp90, as compared to the wild type counterpart (e.g., as in B-RAF) (118,119) The stressed state of malignant cells due to the expression of overexpressed and mutated oncogenes and microenvironmental factors such as hypoxia, nutrient deprivation, and so on Related to the stressed state, the predominance within cancer cells of Hsp90 in a super-chaperone complex that is hypersensitive to Hsp90 inhibition, as distinct from the uncomplexed, more resistant form that is seen in normal cells (120) The triggering of toxicity via the unfolded protein response, especially in those cancers where protein secretion is important, or simply due to the high load of overexpressed and mutated kinases in malignant cells The accumulation of Hsp90 inhibitors in malignant versus normal cells, perhaps related to the super-chaperone complex (120)

Combinatorial activity on multiple oncogenic proteins is believed by the present authors to be very important for the anticancer activity and selectivity of Hsp90 inhibitors (117). Examples of Hsp90 client proteins that regulate the cancer hallmark traits and that are depleted by Hsp90 inhibitors include several kinases (ERBB2, B-RAF, C-RAF, CDK4), hormone receptors (androgen and Oestrogen receptors), other transcription factors (p53 and HIF1a), and additional proteins (catalytic subunit of telomerase hTERT) (Fig. 9). It is clear that further studies are required to elucidate the mechanistic basis for the selectivity of Hsp90 drugs. This may also help to develop biomarkers that would predict for sensitivity of individual patients (121). CLINICAL TRIALS The first-in-class Hsp90 inhibitor 17-AAG has undergone a series of Phase I clinical trials (68,69) and is now in Phase II evaluation as a single agent, as well as in combination studies (Table 1). In the Phase I trials, 17-AAG was shown to give good pharmacokinetic exposures, consistent with activity in preclinical models, and the molecular signature of Hsp90 inhibition was demonstrated in peripheral blood mononuclear cells


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FIGURE 9 Examples of Hsp90-dependent client proteins involved in the six hallmark traits of malignancy. As a result of combinatorial depletion of multiple Hsp90 client proteins, all of the hallmark traits are modulated, leading to a powerful anticancer effect.

(PBMCs) and tumor biopsies (122–125). In the Phase I study at our own institution, we used the molecular signature of target modulation that we validated in a human tumor xenograft model (126). This comprised depletion of the kinase client proteins C-RAF and CDK4 and induction of the co-chaperone Hsp70. Toxicities observed consisted of liver transaminitis, diarrhea, nausea, vomiting, anorexia, fatigue, and anemia. There may have been some contribution to the side-effects from the rather cumbersome vehicle that contained egg phospholipid and dimethyl sulphoxide (DMSO). The DMSO gave rise to an unpleasant odor. Various schedules were evaluated, including weekly, twice weekly (days 1, 4), daily · 5 (21 day cycle), and daily · 3 (14 day cycle). The toxicity seen was both dose- and schedule-dependent, with more hepatotoxicity seen upon daily administration. Pharmacokinetic-pharmacodynamic (PK-PD) data obtained from PBMCs and a limited number of tumor biopsies (122) were compatible with the preclinical PK-PD data from many human cancer cell lines in vitro and from human tumor xenografts in immunosuppressed mice (122). In our own trial, the weekly dose of 450 mg/m2 was limited by the formulation. The PK-PD data indicated that optimal Hsp90 inhibition may have been obtained with a twice weekly schedule, but this was not practical (122). The PK-PD data also suggested that antitumor activity may have been expected (122). During the initial Phase I studies, there were no complete or partial responses as defined by the Response Evaluation Criteria in Solid Tumors (RECIST) system. This was probably not surprising, given that 17-AAG is more cytostatic than pro-apoptotic when tested against cancer cells in culture, probably related to the induction of antiapoptotic co-chaperones such as Hsp70 and Hsp27 (77). In our study, we saw two cases of prolonged stable disease in patients with heavily pretreated, drug resistant, metastatic, malignant melanoma (122). Activity was also seen in renal cancer and in prostate cancer, as determined by decreases in circulating prostate tumor antigen (PSA) and computed tomography (CT) scans in patients with androgenindependent disease (69). Phase I studies have been initiated in pediatric cancer (127). Various Phase II studies are now underway using once weekly (300–450 mg/m2) or twice weekly (220 mg/m2) schedules in patients with melanoma,

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prostate, breast, thyroid, renal, and ovarian cancers, together with mantle cell lymphoma and malignant mast cell neoplasm (69,127). Improved formulations of 17-AAG are being evaluated in the clinic. A cremphor-based formulation has been developed by Kosan Pharmaceuticals (KOS953) (Table 1). Following a Phase I trial of KOS953 across tumor types, a Phase II study in trastuzumab (Herceptin ; Genentech, Inc., South San Francisco, California, U.S.A.) refractory ERBB2 amplified breast cancer is underway (69,128). The trastuzumab was continued with the 17-AAG. The more frequent side-effects were fatigue and gastrointestinal toxicities, with minimal liver toxicity, and no bone marrow suppression. An ERBB2-positive breast cancer patient had a confirmed partial response according to RECIST criteria and three further breast cancer patients had tumor regressions in the range of 21–25%. A further Phase I trial of KOS953 in combination with the proteasome inhibitor bortezomib (Velcade ) is ongoing in multiple myeloma patients. Initial results appear promising with 6 of 12 patients with bortezomib-refractory cancer showing a response to the combined treatment (69). A second improved formulation of 17-AAG involving an oil-in-water emulsion has been developed by Conforma Therapeutics (now Biogen Idec) (129). The pharmacokinetic properties were similar to those in the egg phospholipid/ DMSO formulation initially developed by the U.S. National Cancer Institute, and no toxicities appeared to be associated with the new formulation. A decrease in the expression of the ERBB2 extracellular domain in plasma, determined as a pharmacodynamic endpoint, was observed at doses >83 mg/m2. Three minor responses were recorded at these dose levels in melanoma, gastric, and duodenal cancers. A series of Phase Ib combination studies are underway with the original formulation of 17-AAG and involving either solid tumors or hematological malignancies (69,127). Drugs that are being combined with 17-AAG include docetaxel (Taxotere ), paclitaxel (Taxol), irinotecan, cytarabine, cisplatin/gemcitabine (Gemzar ), bortezomib (Velcade), and imitanib (Glivec ). The combination of 17-AAG and taxanes has appeared very promising in preclinical studies (130,131). In a Phase I study of 17-AAG plus docetaxel, patients were treated using a weekly or three weekly schedule (69). Toxicity to date has involved fatigue and neutropenia. A partial response by RECIST criteria was observed, with minor responses in prostate, lung, melanoma, and urethral cancers. There does not appear to be any pharmacokinetic interaction. The more water soluble 17-AAG analogue, 17-DMAG has followed the parent drug into the clinic (Table 1). 17-DMAG has the advantages of greater aqueous solubility and oral bioavailability. Both intravenous and oral studies are underway (69,127). The highly water soluble hydroquinone derivative of 17-AAG, IPI-504, is being developed by Infinity Pharmaceuticals and has entered Phase I studies (132).The first non-ansamycin Hsp90 inhibitor to enter clinical trials is the purine-scaffold compound CNF2024 from Conforma Therapeutics. It will be interesting to see whether the non-benzoquinone ansamycins are devoid of liver toxicity, as metabolism of the quinone is one hypothesis to explain the hepatotoxicity. Hsp90 inhibitors from a variety of other companies are in preclinical development (Tables 1 and 2) and it can be anticipated that a number of these will enter clinical trials in the coming months and years. Biomarkers are essential for rational modern drug development (133). A variety of pharmacodynamic biomarkers have been or can be explored in the


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clinical trials. The initial studies mainly looked at the expression of the kinase clients C-RAF and CDK4 in PBMCs in leukocytes. In addition, induction of Hsp70 was also measured. Western immunoblotting was generally used. Although quantitation is difficult with western blotting, the use of the molecular biomarker signature proved very informative (122). ELISA methodology has been developed, allowing accurate quantitation in PBMCs and tumor tissue (134). In addition, it may be possible to monitor Hsp70 in the plasma (134). ERBB2 extracellular domain and insulin-like growth factor binding protein 2 show promise as biomarkers that can be monitored in the circulation by ELISA (135). In addition to the range of client proteins and co-chaperones that are candidate biomarkers for normal tissue and tumor studies based on a large literature of preclinical work, we have used non-biased global profiling methods successfully to identify mRNAs and proteins that exhibit increased or decreased expression in cells following treatment with Hsp90 inhibitors (44,116,136). Of particular potential interest was our identification of elevations in the expression of Hsp90 isoforms—the targets of the drug treatment—as well as the induction of the Hsp90-activating protein AHA1 (44,136). Induction of these proteins could impact sensitivity as well as provide pharmacodynamic biomarkers. Similarly, induction of the anti-apoptotic chaperones, such as Hsp70 isoforms and Hsp27, may also influence drug sensitivity. Our recent proteomic analysis identified interesting protein expression changes that may be of biological significance or provide further potential biomarkers (116). Of note was the altered expression of a group of chromatin-modifying enzymes and other chromatin-associated proteins. One of these was the protein arginine methyl transferase 5 (PRMT5), which exhibited decreased expression in human cancer cells after Hsp90 inhibition. Follow up studies showed that PRMT5 was bound to Hsp90 and is a potential client protein (116). The alteration in chromatin-modifying enzyme levels may be biologically relevant with respect to histone modification and the control of gene expression. Indeed, as mentioned earlier, treatment of cells with 17-AAG caused a decrease in protein acetylation in human ovarian cancer cells and antagonized the increase in protein acetylation caused by the HDAC inhibitor trichostatin A (116). Furthermore, these agents exhibited an antagonistic effect on proliferation. In contrast, however, a synergistic interaction has been seen with the combination of HDAC and Hsp90 inhibitors in leukemic cells, suggesting context-dependence that requires further analysis. Minimally invasive methods are of great interest as an alternative source of biomarker endpoints (137). In collaborative studies we have identified an unusual metabolite signature by magnetic resonance spectroscopy (MRS) in cell and tumors following treatment with 17-AAG (138). This signature involves choline metabolism in the cell. Preliminary studies have been carried out to determine the feasibility of monitoring changes in choline uptake and metabolism by positron emission tomography (PET) (139). Also of potential clinical application is the use of a 68Ga-labelled F(ab')2 fragment of trastuzumab to detect the rapid degradation of ERBB2 using PET (140). WHAT CLIENT PROTEINS ARE IMPORTANT IN WHICH DISEASES? It is a considerable advantage of Hsp90 inhibitors that they do exert combinatorial effects on numerous oncogenic client proteins, signaling pathways and hallmark traits (117). This results in a powerful all out attack on cancer cells on many fronts.

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In addition, it is likely that the simultaneous combinatorial effects will decrease the likelihood of drug resistance developing. At the same time, however, the fact that so many oncogenic players are affected makes it difficult to ascertain how any particular group of patients will respond or whether any particular client proteins will dominant the response in a particular tumor type or patient. Some of the main reasons for expecting that differential effects may occur are the observations that: (i) some client proteins are more important than others in driving particular cancers; and (ii) various client proteins show differential responses to Hsp90 inhibition in terms of both dose-response and time-dependence. For example, ERBB2 is depleted at much lower Hsp90 inhibitor concentrations and after much shorter exposures compared to most other kinases. So, how may we expect these client protein differences to play out in terms of tumor response in the clinic? &

&

&

&

& &

&

&

Clearly, in those patients with ERBB2-positive breast cancers, the depletion of this receptor tyrosine kinase is likely to dominate the response. This likely explains the activity that is being seen in the clinic in this setting. In hormone-refractory prostate cancer, effects on both ERBB2 and the androgen receptor are likely to be very important and this is a disease setting in which Hsp90 inhibitors should be evaluated. The RAS-RAF-MEK-ERK pathway is very important in melanoma. Effects on mutant B-RAF and wild type C-RAF may well be important in the responses seen in this cancer. Since mutant EGFR receptors are very sensitive to Hsp90 inhibitors, activity in non–small cell lung cancers (NSCLCs) harboring these mutations may be anticipated. Mutations of KIT in gastrointestinal stromal tumors (GIST) may lead to activity in these sarcomas. In cancers where the PI3 kinase pathway is important, effects on players in this pathway, such as phospho-AKT, may play a key role, as in ovarian cancer, glioblastoma, and other malignancies. Renal cell cancers are driven by loss of VHL, which causes stabilization of HIF1a, leading to increased expression of VEGF and other key downstream gene products. Depletion of HIF1a, which is a client protein for Hsp90, is likely to be important in this disease. In addition, HIF1a is stabilized by hypoxia in many cancers, and depletion of this could contribute to the powerful anti-angiogenic effects of Hsp90 inhibitors. In cancers where protein trafficking and the unfolded protein response are especially important, as in multiple myeloma, we can expect the effects of Hsp90 inhibitors of this pathway to the very important (141).

The above are just a few examples of tumor types in which therapeutic activity may be mediated by effects on particular client proteins. It is likely that other examples will emerge as we gain greater knowledge of the molecular pathology of human cancers and of the molecular pharmacology of Hsp90 inhibitors. CONCLUSIONS AND FUTURE PERSPECTIVES The Hsp90 story is a fascinating one and remains incomplete. There is no doubt that our knowledge of the complex and multi-faceted role of Hsp90 in cancer


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and normal cells will continue to grow very rapidly over the next few years. Equally well, as many Hsp90 inhibitors enter the clinic over the same time frame, we will learn more about how best to use these intriguing agents, and, through them, we will discover much about the role of Hsp90 in human biology and pathology. The tolerability and early signs of clinical activity with the geldanamycinbased agents is encouraging many academic investigators and companies to get involved in this area. It should be emphasized, however, that, until an Hsp90 inhibitor gains regulatory approval in a human cancer, the ultimate proof of concept for Hsp90 as an important therapeutic target will remain unproven. It will be interesting to see whether such therapeutic activity will be revealed with single agent Hsp90 inhibitors or will require these drugs to be used in combination with other targeted molecular cancer therapeutics or cytotoxic agents. What is clear is that the natural product Hsp90 inhibitors have been extraordinarily valuable in helping to probe the biology of Hsp90 in normal and tumor cells and to pave the way for the development of synthetic small molecule inhibitors. Complementing this chemical biology approach, various other drug discovery technologies are also proving important, particularly the combinations of HTS and structural-based design. In addition, the discovery and application of various molecular biomarkers of drug effects has also been extremely valuable. Pharmacodynamic biomarkers are now readily available and the increasing emphasis will be on non-invasive methodologies, such as PET and MRS/MRI, as well as the discovery and validation of biomarkers that will be predictive of which patients will respond. As both intravenous and oral Hsp90 inhibitors of various chemotypes enter the clinic, there are some key questions that need to be addressed. For example, which Hsp90 isoforms should be inhibited and which ones should be spared? Will a particular isoform be more important for anticancer activity and others for toxic side-effects? These questions can be addressed using both chemical tools and RNA interference. Based on experience with kinases and other drug targets, it might be envisioned that the initial drugs will be pan-Hsp90, whereas follow up drugs might explore the impact of isoform selectivity with respect to activity and toxicity. Drugs that interfere with features of Hsp90 other than nucleotide binding and hydrolysis, such as co-chaperone interactions, may provide differing biological effects. It is clear that the next few years will be exciting as the role of Hsp90 inhibitors in cancer treatment is defined. But, in addition, Hsp90 inhibitors are likely to be evaluated in diseases other than cancer. Clearly there is potential in conditions where protein folding defects are involved in the disease pathology, including Huntingdon's, Alzheimer's, and prion-related diseases (142). Recent studies have shown that the downregulation of the Hsp90 co-chaperone AHA1 rescues the misfolding of the common disease variant of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) (143). The results suggest AHA1 as a potential target in the treatment of cystic fibrosis. Thus targeting Hsp90 and other components of the chaperone may provide a general framework for correction of misfolding disease. The activity of the proteasome inhibitor bortezomib and of Hsp90 inhibitors in cancer provides validation for the modulation of protein quality control and protein folding as a viable therapeutic approach. Success will stimulate interest in modulating other targets in these pathways for cancer and other conditions.

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ACKNOWLEDGMENTS The work of the authors' laboratory was funded by Cancer Research U.K. grant numbers CA309/A2187 and C309/A8274. Paul Workman is a Cancer Research U.K. Life Fellow. We thank our colleagues in the Signal Transduction and Molecular Pharmacology Team and Chaperone Project Team and also our many collaborators for valuable discussion. We also thank Dr. Chrisostomos Prodromou for Figure 3 and Pam Stevens for help with preparation of the manuscript. REFERENCES 1. Vogelstein B, Kinzler KW. Cancer genes and the pathways they control. Nature Med 2005; 11:261–3. 2. Workman P. Genomics and the second golden era of cancer drug development. Mol BioSyst 2005; 1:17–26. 3. Collins I, Workman P. New approaches to molecular cancer therapeutics. Nature Chem Biol 2006; 2:689–700. 4. Workman P. Drugging the cancer kinome: progress and challenges in developing personalized molecular cancer therapeutics. Cold Spring Harbor Symp Quant Biol 2005; 70:1–18. 5. Futreal PA, Coin L, Marshall M, et al. A concensus of human cancer genes. Nat Rev Cancer 2004; 4:177–83. 6. Futreal PA, Wooster R, Stratton MR. Somatic mutations in human cancer: insights from resequencing the protein kinase gene family. Cold Spring Harb Symp Quant Biol 2005; 70:43–9. 7. http://www.sanger.ac.uk/genetics/cgp/census (accessed 11 February 2007). 8. Sjoblom T, Jones S, Wood LD, et al. The consensus coding sequences of human breast and colorectal cancers. Science 2006; 314(5797):268–74. 9. Hanahan D, Weinberg RQA. The hallmarks of cancer. Cell 2000; 100:57–70. 10. Benson JD, Chen YN, Cornell-Kennon SA, et al. Validating cancer drug targets. Nature 2006; 441:451–6. 11. Marks PA, Breslow R. Dimethyl sulfoxide to vorinostate: development of this histone deacteylase inhibitor as an anticancer drug. Nat Biotechnol 2007; 25:84–90. 12. Adams J, Kauffman M. Development of the proteasome inhibitors Velcade (Bortezomib). Cancer Inve 2004; 22:304–11. 13. Powers M, Workman P. Targeting of multiple signalling pathways by heat shock protein 90 molecular chaperone inhibitors. Endo Rel Cancer 2006; Suppl 1:S125–35. 14. Sharp S, Workman P. Inhibitors of Hsp90 molecular chaperone: Current status. Adv Cancer Res 2006; 95:323–48. 15. Whitesell L, Lindquist SL. HSP90 and the chaperoning of cancer. Nat Rev Cancer 2005; 5(10):761–72. 16. McDonald E, Workman P, Jones K. Inhibitors of the Hsp90 molecular chaperons: Attacking the master regulator in cancer. Curr Top Med Chem 2006; 17:1091–109. 17. Chiosis G, Radina A, Moulick K. Emerging Hsp90 inhibitors: from discovery to clinic. Anticancer Agents Med Chem 2006; 6:1–8. 18. Janin YL. Heat shock protein inhibitors. A text book example of medicinal chemistry? J Med Chem 2005; 48:7503–12. 19. Ritossa F. A new puffing pattern induced by temperature shock and DNP in Drosophila. Experientia 1962; 19:571–73. 20. Ritossa F. Discovery of the heat shock response. Cell Stress Chaperones 1996; 1:97–8. 21. Bukau B, Deuerling E, Pfund C, et al. Getting newly synthesised proteins into shape. Cell 2000; 101:119–22. 22. Hartl U, Hayer-Hartl M. Molecular chaperones in the cytosol: from nascent chain to folded proteins. Science 2002; 295:1852–8. 23. Young JC, Agashe VR, Siegers K, et al. Pathways of chaperone-mediated protein folding in the cytosis. Nat Rev Mol Cell Biol 2004; 5:781–91.


395

24. Leppa S, Sistoned L. Heat shock response—pathophysiological implications. Ann Med 1997; 29:73–8. 25. Jolly C, Morimoto RI. Role of the heat shock response and molecular chaperones in oncogenesis. J Natl Cancer Inst 2007; 92:1564–72. 26. Takaymam S, Reed JC, Honma S. Heat shock proteins regulators of apoptosis. Oncogene 2003; 22:9041–7. 27. Csermely R, Schnaider T, Soti C, et al. The 90-Kda molecular chaperones family: structure, function, and clinical applications. A comprehensive review. Pharmacol Ther 1998; 79:129–68. 28. Maloney A, Workman P. Hsp90 as a new therapeutic target for cancer therapy: the story unfolds. Expert Opin Biolog Ther 2002; 2:3–24. 29. Wegele H, Muller L, Buchner J. Hsp70 and Hsp90—a real team for protein folding. Rev Physiol Biochem Pharmacol 2004; 151:1–44. 30. Grammatikakis N, Vultur A, Ramana CV, et al. The role of Hsp90N, a new member of the Hsp90 family, in signal transduction and neoplastic transformation. J Biol Chem. 2002; 277(10):8312–20. 31. Pearl LH, Prodromou C. Structure and mechanism of Hsp90 molecular chaperone machinery. Annu Rev Biochem 2006; 75:271–94. 32. Ali MM, Roe SM, Vaughan CK, et al. Crystal structure of an HSP90-nucleotide-p23/ Sba1 closed chaperone complex. Nature 2006; 440:1013–7. 33. Vaughan CK, Gohlke U, Sobott F, et al. Structure of an Hsp90-Cdc37-Cdk4 complex. Mol Cell 2006; 23:697–707. 34. Stebbins CE, Russo AA, Schneider C, et al. Crystal structure of an Hsp90-geldanamycin complex: targeting of a protein chaperone by an antitumor agent. Cell 1997; 89(2): 239–50. 35. Prodromou C, Roe SM, O'Brien R, et al. Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone. Cell 1997; 90: 65–75. 36. Csermely P, Kajtar J, Hollosi M, et al. ATP induces a conformational change of the 90kDa heat shock protein (hsp90). J Biol Chem 1993; 268(3):1901–7. 37. Panaretou B, Prodromou C, Roe SM, et al. ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo. EMBO J 1998; 17(16): 4829–36. 38. Grenert JP, Johnson BD, Toft DO. The importance of ATP binding and hydrolysis by hsp90 in formation and function of protein heterocomplexes. J Biol Chem 1999; 274(25):17525–33. 39. Chadli A, Bouhouche I, Sullivan W, et al. Dimerization and N-terminal domain proximity underlie the function of the molecular chaperone heat shock protein 90. Proc Natl Acad Sci USA 2000; 97(23):12524–9. 40. Prodromou C, Panaretou B, Chohan S, et al. The ATPase cycle of Hsp90 drives a molecular 'clamp' via transient dimerization of the N-terminal domains. EMBO J 2000; 19(16):4383–92. 41. Chene P. ATPases as drug targets: learning from their structure. Nat Rev Drug Discov 2002; 1(9):665–73. 42. Connell P, Ballinger CA, Jiang J, et al. The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins. Nat Cell Biol 2001; 3:93–6. 43. Demand J, Alberti S, Patterson C, et al. Cooperation of a ubiquitin domain protein and an E3 ubiquitin ligase during chaperone/proteasome coupling. Curr Biol 2001; 11:1569–77. 44. Panaretou B, Siligardi G, Meyer P, et al. Activation of the ATPase activity of hsp90 by the stress-regulated cochaperone aha1. Mol Cell 2002; 10:1307–18. 45. Meyer P, Prodromou C, Liao C, et al. Structural basis for recruitment of the ATPase activator Aha1 to the Hsp90 chaperone machinery. EMBO J 2004; 23(6):1402–10. 46. Xu W, Xitong Y, Xiang Z, et al. Surface charge and hydrobicity determine ErB2 binding to the Hsp90 chaperone complex. Nat Struc Mol Biol 2005; 12:120–6. 47. Citri A, Harari D, Shokat G, et al. Hsp90 recognizes a common surface on client kinases. J Biol Chem 2006; 281(20):14361–9.

396

Workman and Sharp

48. http://www.picard.ch/downloads/Hsp90 interactors.pdf (accessed 11 February 2007). 49. Zhao R, Davey M, Hsu YC, et al. Navigating the chaperone network: an integrative map of physical and genetic interactions mediated by the hsp90 chaperone. Cell 2005; 120:715–27. 50. Falsone SF, Gesslbauer B, Tirk F, et al. A proteomic snapshot of the human heat shock protein 90 interactome. FEBS Lett 2005; 579(28):6350–454. 51. Millson SH, Truman AW, King V, et al. A two-hybrid screen of the yeast proteome for Hsp90 interactors uncovers a novel Hsp90 chaperone requirement in the activity of a stress-activated mitogen-activated protein kinase, Slt2p (Mpk1p). Eukaryot Cell 2005; 4:849–60. 52. Uffenbeck SR, Krebs JE. The role of chromatin structure in regulating stress-induced transcription in Saccharomyces cerevisiae. Biochem Cell Biol 2006; 84(4):477–89. 53. Sangster TA. Hsp90 and chromatin: where is the link? Cell Cycle 2003; 2(3):166–8. 54. Whitesell L, Shifrin SD, Scwab G, et al. Benzoquinonoid ansamycins possess selective tumoricidal activity unrelated to src kinase inhibition. Cancer Res 1992; 52(7):1721–8. 55. Whitesell L, Mimnaugh EG, De Costa B, et al. Inhibition of heat shock protein HSP90pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation. Proc Natl Acad Sci USA 1994; 91(18):8324–8. 56. Grenert JP, Sullivan WP, Fadden P, et al. The amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an ATP/ADP switch domain that regulates hsp90 conformation. J Biol Chem 1997; 272(38):23843–50. 57. Oppermann H, Levinson AD, Levintow L, et al. Two cellular proteins that immunoprecipitate with the transforming protein of Rous sarcoma virus. Virology 1981; 113(2):736–51. 58. Xu Y, Linquist S. Heat-shock protein hsp90 governs the activity of pp60v-src kinase. Proc Natl Acad Sci USA 1993; 90(15):7074–8. 59. Ziemiecki A, Catelli MG, Joab I, et al. Association of the heat shock protein hsp90 with steroid hormone receptors and tyrosine kinase oncogene products. Biochem Biophys Res Commun 1986; 138(3):1298–307. 60. Stancato LF, Silverstein AM, Owens-Grillo JK, et al. The hsp90-binding antibiotic geldanamycin decreases Raf levels and epidermal growth factor signaling without disrupting formation of signaling complexes or reducing the specific enzymatic activity of Raf kinase. J Biol Chem 1997; 272(7):4013–20. 61. Ferrarini M, Heltai S, Zocchi MR, et al. Unusual expression and localization of heatshock proteins in human tumor cells. Int J Cancer 1992; 51(4):613–9. 62. Jaatella M. Escaping cell death: survival proteins in cancer. Exp Cell Res 1999; 248: 30–43. 63. Mimnaugh EG, Chavany C, Neckers L. Polyubiquitination and proteasomal degradation of the p185c-erbB-2 receptor protein-tyrosine kinase induced by geldanamycin. J Biol Chem 1996; 271(37):22796–801. 64. Csermely P, Kajtar J, Hollosi M, et al. ATP induces a conformational change of the 90-kDa heat shock protein (hsp90). J Biol Chem 1993; 268(3):1901–7. 65. Schulte TW, Akinaga S, Soga S, et al. Antibiotic radicicol binds to the N-terminal domain of Hsp90 and shares important biologic activities with geldanamycin. Cell Stress Chaperones 1998; 3(2):100–8. 66. Sharma SV, Agatsuma T, Nakano H. Targeting of the protein chaperone, HSP90, by the transformation suppressing agent, radicicol. Oncogene 1998; 16(20):2639–45. 67. Roe SM, Prodromou C, O'Brien R, et al. Structural basis for inhibition of the Hsp90 molecular chaperone by the antitumor antibiotics radicicol and geldanamycin. J Med Chem 1999; 42(2):260–6. 68. Pacey S, Banerji U, Judson I, et al. Hsp90 inhibitors in the clinic. Handb Exp Pharmacol 2006; (172):331–58. 69. Solit DB, Rosen N. Hsp90: a novel target for cancer therapy. Curr Top Med Chem 2006; 6(11):1205–14. 70. McDonald E, Jones K, Brough PA, et al. Discovery and development of pyrazolescaffold Hsp90 inhibitors. Curr Top Med Chem 2006; 6(11):1193–203.


397

71. Uehara Y. Natural product origins of Hsp90 inhibitors. Curr Cancer Drug Targets 2003; 3:325–30. 72. Fukazawa H, Uehara Y, Murakami Y, et al. Labeling of v-Src and BCR-ABL tyrosine kinase with [14C] herbimycin A and its use in the elucidation of the kinase inactivation mechanism. FEBS Letts 1994; 340:155–8. 73. Supko JG, Hickman RL, Grever MR, et al. Preclinical pharmacologic evaluation of geldanamycin as an antitumor agent. Cancer Chemother Pharmacol 1995; 36(4):305–15. 74. Schnur RC, Corman ML, Gallaschun RJ, et al. Inhibition of the oncogene product p185erbB-2 in vitro and in vivo by geldanamycin and dihydrogeldanamycin derivatives. J Med Chem 1995; 38(19):3806–12. 75. Schnur RC, Corman ML, Gallaschun RJ, et al. erbB-2 oncogene inhibition by geldanamycin derivatives: synthesis, mechanism of action, and structure-activity relationships. J Med Chem 1995; 38(19):3813–20. 76. Schulte TW, Neckers LM. The benzoquinone ansamycin 17-allylamino-17-demethoxygeldanamycin binds to HSP90 and shares important biologic activities with geldanamycin. Cancer Chemother Pharmacol 1998; 42(4):273–9. 77. Hostein I, Robertson D, DiStefano F, et al. Inhibition of signal transduction by the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin results in cytostasis and apoptosis. Cancer Res 2001; 61:4003–9. 78. Solit DB, Zheng FF, Drobnjak M, et al. 17-Allylamino-17-demethoxygeldanamycin induces the degradation of androgen receptor and HER-2/neu and inhibits the growth of prostate cancer xenografts. Clin Cancer Res 2002; 8:986–93. 79. Sanderson S, Valenti M, Gowan S, et al. Benzoquinone ansamycin heat shock protein 90 inhibitors modulate multiple functions required for tumor angiogenesis. Mol Cancer Ther 2006; 5:522–32. 80. Kelland LR, Sharp SY, Rogers PM, et al. DT-Diaphorase expression and tumor cell sensitivity to 17-allylamino, 17-demethoxygeldanamycin, an inhibitor of heat shock protein 90. J Natl Cancer Inst 1999; 91(22):1940–9. 81. Egorin MJ, Zuhowski EG, Rosen DM, et al. Plasma pharmacokinetics and tissue distribution of 17-(allylamino)-17-demethoxygeldanamycin(NSC 330507) in CD2F1 mice1. Cancer Chemother Pharmacol 2001; 47:291–302. 82. Burger AM, Fiebig HH, Stinson SF, Sausville EA. 17-(Allylamino)-17-demethoxygeldanamycin activity in human melanoma models. Anticancer Drugs 2004; 15:377–87. 83. Guo W, Reigan P, Siegel D, et al. Formation of 17-allylamino-demethoxygeldanamycin(17-AAG) hydroquinone by NAD(P)H:quinone oxidoreductase 1: role of 17-AAG hydroquinone in heat shock protein 90 inhibition. Cancer Res 2005; b65:10006–15. 84. Sydor JR, Normant E, Pien CS, et al. Development of 17-allylamino-17-demethoxygeldanamycin hydroquinone hydrochloride(IPI-504), an anti-cancer agent directed against Hsp90. Proc Natl Acad Sci USA 2006; 103:17408–13. 85. Agatsuma T, Ogawa H, Akasaka K, et al. Halohydrin and oxime derivatives of radicicol: synthesis and antitumor activities. Bioorg Med Chem 2002; 10:3445–54. 86. Hadden MK, Lubhors DJ, Blass BSJ. Geldanamycin in radicicol and chimeric inhibitory of the Hsp90 N-terminal ATP site. Curr Top Med Chem 2006; 6:1173–82. 87. Proisy N, Sharp SY, Boxall K, et al. Inhibition of Hsp90 with synthetic macrolactones: synthesis and structural and biological evaluation of ring and conformational analogs of radicicol. Chem Biol 2006; 13(11):1203–15. 88. Choisis G. Discovery and development of purine-scaffold Hsp90 inhibitors. Curr Top Med Chem 2006; 6:11832–901. 89. Chiosis G, Timaul MN, Lucas B. A small molecule designed to bind to the adenine nucleotide pocket of Hsp90 causes Her2 degradation and the growth arrest and differentiation of breast cancer cells. Chem Biol 2001; 8:289–99. 90. Vilenchik M, Solit D, Basso A, et al. Targeting wide-range oncogenic transformation via PU24FCl, a specific inhibitor of tumor Hsp90. Chem Biol 2004; 11:787–97. 91. Wright L, Barril X, Dymock B, et al. Structure-activity relationships in purine-based inhibitor binding to HSP90 isoforms. Chem Biol 2004; 11:775–85. 92. He H, Zatorska D, Kim J, et al. Identification of potent water soluble purine-scaffold inhibitors of the heat shock protein 90. J Med Chem 2006; 49:381–90.

398

Workman and Sharp

93. Biamonte MA, Shi J, Hong K, et al. Orally active purine-based inhibitors of the heat shock protein 90. J Med Chem 2006; 49:817–28. 94. Chiosis G, Caldas LE, Solit, D. Heat shock protein-90 inhibitors: a chronicle from geldanamycin to today's agents. Curr Opin Investig Drugs 2006; 7:534–41. 95. Rowlands MG, Newbatt YM, Prodromou C, et al. High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity. Anal Biochem 2004; 327: 176–83. 96. Cheung KM, Matthews, TP, James K, et al. The identification, synthesis, protein crystal structure and in vitro biochemical evaluation of a new 3,4-diarylpyrazole class of Hsp90 inhibitors. Bioorg Med Chem Lett 2005; 15:3338–43. 97. Sharp SY, Boxall K, Rowlands M, et al. In vitro biological characterisation of a novel, synthetic diaryl pyrazole resorcinol class of heat shock protein 90 inhibitors. Cancer Res 2007; 67:2206–16. 98. Dymock BW, Barril X, Brough PA, et al. Novel, potent small-molecule inhibitors of the molecular chaperone Hsp90 discovered through structure-based design. J Med Chem 2005; 48:4212–5. 99. Kreusch A, Han S, Brinker A, et al. Crystal structures of human HSP90alpha-complexed with dihydroxyphenylpyrazoles. Bioorg Med Chem Lett 2005; 15(5):1475–8. 100. Marcu MG, Chadli A, Bouhouche I, et al. The heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized ATP-binding domain in the carboxyl terminus of the chaperone. J Biol Chem 2000; a275:37181–6. 101. Marcu MG, Schulte TW, Neckers, L. Novobiocin and related coumarins and depletion of heat shock protein 90-dependent signaling proteins. J Natl Cancer Inst 2000; b92: 242–8. 102. Yu XM, Shen G, Neckers L, et al. Hsp90 inhibitors identified from a library of novobiocin analogues. J Am Chem Soc 2005; 127:12778–9. 103. Plescia J, Salz W, Xia F, et al. Rational design of shepherdin, a novel anticancer agent. Cancer Cell 2005; 7(5):457–68. 104. Gyurkocza B, Plescia J, Raskett CM, et al. Antileukemic activity of shepherdin and molecular diversity of hsp90 inhibitors. J Natl Cancer Inst 2006; 98(15):1068–77. 105. Meli M, Pennati M, Curto M, et al. Small-molecule targeting of heat shock protein 90 chaperone function: rational identification of a new anticancer lead. J Med Chem 2006; 49(26):7721–30. 106. Soldano KL, Jivan A, Nicchitta CV, et al. Stucture of the N-terminal domain of GRP94: basis for ligand specificity and regulation. J Biol Chem 2003; 48:48330–8. 107. Tsutsumi S, Scroggins B, Tian, Z, et al. Cell-impermeable derivatives of the heat shock protein 90 inhibitor geldanamycin display low cytotoxicity but strongly inhibit tumor cell invasion in vitro. Clin Cancer Res 2005; 11(24 Suppl):9149s. 108. Eustace BK, Sakurai T, Stewart JK, et al. Functional proteomic screens reveal an essential extracellular role for hsp90 alpha in cancer cell invasiveness. Nat Cell Biol 2004; 6:507–14. 109. Matthews RC, Rigg G, Hodgetts S, et al. Preclinical assessment of the efficacy of mycograb, a human recombinant antibody against fungal HSP90. Antimicrob Agents Chemother 2003; 47:2208–16. 110. Matthews RC, Burnie JP. Recombinant antibodies: a natural partner in combinatorial antifungal therapy. Vaccine 2004; 22:865–71. 111. Fuino L, Bali P, Wittmann S, et al. Histone deacetylase inhibitor LAQ824 downregulates Her-2 and sensitizes human breast cancer cells to trastuzumab, taxotere, gemcitabine, and epothilone B. Mol Cancer Ther 2003; 2:971–84. 112. Kristeleit R, Tandy D, Atadja P, et al. Effects of the histone deacetylase inhibitor (HDACi) LAQ824 on histone acetylation, Hsp70 and c-Raf in peripheral blood lymphocytes from patients with advanced solid tumours enrolled in a phase I clinical trial. Proc American Assoc Clin Onc 2004; 22:3032. 113. Yu X, Guo ZS, Marcu MG, et al. Modulation of p53, ErbB1, ErbB2, and Raf-1 expression in lung cancer cells by depsipeptide FR901228. J Natl Cancer Inst 2000; 94: 504–13.


399

114. Scroggins BT, Robzyk K, Wang D, et al. An acetylation site in the middle domain of Hsp90 regulates chaperone function. Mol Cell 2007; 25(1):151–9. 115. George P, Bali P, Annavarapu S, et al. Combination of the histone deacetylase inhibitor LBH589 and the hsp90 inhibitor 17-AAG is highly active against human CML-BC cells and AML cells with activating mutation of FLT-3. Blood 2005; 105: 1768–76. 116. Maloney A, Clarke P, Naaby-Hansen S, et al. Gene expression microarray and proteomic profiling of human ovarian cancer cells following treatment with the HSP90 molecular chaperone inhibitor 17AAG. Cancer Res 2007; 67:3239–53. 117. Workman P. Combinatorial attack on multistep oncogenesis by inhibiting the Hsp90 molecular chaperone. Cancer Letts 2004; 206:149–57. 118. da Rocha Dias S, Friedlos F, Light Y, et al. Activated B-RAF is an Hsp90 client protein that is targeted by the anticancer drug 17-allylamino-17-demethoxygeldanamycin. Cancer Res 2005; 65(23):10686–91. 119. Grbovic OM, Basso AD, Sawai A, et al. V600E B-Raf requires the Hsp90 chaperone for stability and is degraded in response to Hsp90 inhibitors. Proc Natl Acad Sci USA 2006; 103(1):57–62. 120. Kamal A, Thao L, Sensintaffar J, et al. A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors. Nature 2003; 425:407–10. 121. Maloney A, Clarke PA, Workman P. Genes and proteins governing the cellular sensitivity to HSP90 inhibitors: a mechanistic perspective. Curr Cancer Drug Targets 2003; 5:331–41. 122. Banerji U, O'Donnell A, Scurr M, et al. Phase I pharmacokinetic and pharmacodynamic study of 17-allylamino, 17-demethoxygeldanamycin in patients with advanced malignancies. J Clin Oncol 2005; 23:4152–61. 123. Goetz, MP, Toft D, Reid J, et al. Phase I trial of 17-allylamino-17-demethoxygeldanamycin in patients with advanced cancer. J Clin Oncol 2005; 23:1078–87. 124. Grem JL, Morrison G, Guo XD, et al. Phase I and pharmacologic study of 17-(allylamino)-17-demethoxygeldanamycin in adult patients with solid tumors. J Clin Oncol 2005; 23(9):1885–93. 125. Ramanathan RK, Trump DL, Eiseman JL, et al. Phase I pharmacokinetic-pharmacodynamic study of 17-(allylamino)-17-demethoxygeldanamycin (17AAG, NSC 330507), a novel inhibitor of heat shock protein 90, in patients with refractory advanced cancers. Clin Can Res 2005; 11:3385–91. 126. Banerji U, Walton M, Raynaud F, et al. Pharmacokinetic-pharmacodynamic relationships for the heat shock protein 90 molecular chaperone inhibitor 17-allylamino, 17-demethoxygeldanamycin in human ovarian cancer xenograft models. Clin Cancer Res 2005; 11:7023–32. 127. http://nci.nih.gov/clinicaltrials (accessed 14 February 2007). 128. Modi S, Stopeck A, Gordon MS, et al. Phase I trial of KOS-953, a heat shock protein 90 inhibitor, and trastuzumab (T). J Clin Oncol (Meeting Abstracts) 2006; 24:501. 129. Dragovich T, Gordon M, Saif W, et al. Phase I study of CNF1010 (lipid formulation of 17-(allylamino)-17-demethoxygeldnamycin: 17AAG). Clin Can Res 2005; 11(24 Suppl): 9117s. 130. Solit DB, Basso AD, Olshen AB, et al. Inhibition of heat shock protein 90 function down-regulates Akt kinase and sensitizes tumors to Taxol. Cancer Res 2003; 63: 2139–44. 131. Sain N, Krishnan B, Ormerod MG, et al. Potentiation of paclitaxel activity by the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin in human ovarian carcinoma cell lines with high levels of activated AKT. Mol Cancer Ther 2006; 5: 1197–208. 132. Demetri GD, George S, Abbeele VD, et al. Inhibition of heat shock protein 90 (Hsp90) with the novel agent IPI-504 to overcome resistance to tyrosine kinase inhibitiors (TKIs) in metastatic GIST: Results of a phase I trial. ASCO meeting:2007; abstract 12. 133. Sarker D, Workman P. Pharmacodynamic biomarkers for molecular cancer therapeutics. Adv Cancer Res 2007; 96, 213–68.

400

Workman and Sharp

134. Hardcastle A, Boxall K, Richards J, et al. Solid-phase immunoassays in mechanismbased drug discovery: their application in the development of inhibitors of the molecular chaperone heat-shock protein 90. Assay Drug Dev Technol 2005; 3:273–85. 135. Zhang H, Chung D, Yang YC, et al. Identification of new biomarkers for clinical trials of Hsp90 inhibitors. Mol Cancer Ther 2006; 5:1256–64. 136. Clarke PA, Hostein I, Banerji U, et al. Gene expression profiling of human colon cancer cells following inhibition of signal transduction by 17-allylamino-17-demethoxygeldanamycin, an inhibitor of the hsp90 molecular chaperone. Oncogene 2000; 19: 4125–33. 137. Workman P, Aboagye EO, Chung YL, et al. Minimally invasive pharmacokinetic and pharmacodynamic technologies in hypothesis-testing clinical trials of innovative therapies. J Natl Cancer Inst 2006; 98:580–98. 138. Chung YL, Troy H, Banerji U, et al. Magnetic resonance spectroscopic pharmacodynamic markers of the heat shock protein 90 inhibitor 17-allylamino, 17-demethoxygeldanamycin (17AAG) in human colon cancer models. J Natl Cancer Inst 2003; 95: 1624–33. 139. Liu D, Hutchinson OC, Osman S, et al. Use of radiolabelled choline as a pharmacodynamic marker for the signal transduction inhibitor geldanmycin. Br J Cancer 2002; 23(87):783–9. 140. Smith-Jones PM, Solit DB, Akhurst T, et al. Imaging the pharmacodynamics of HER2 degradation in response to Hsp90 inhibitors. Nat Biotechnol 2004; 22:701–6. 141. Davenport EL, Moore HE, Dunlop AS et al. Heat shock protein inhibition is associated with activation of the unfolded protein response (UPR) pathway in myeloma plasma cells. Blood 2007; May 24 (Epub ahead of print). 142. Barral JM, Broadley SA, Schaffar G et al. Roles of molecular chaperones in protein misfolding diseases. Semin Cell Dev Biol 2004; 15(1):17–29. 143. Wang X, Venable J, LaPointe P, et al. Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis. Cell 2006; 127:803–15.

Index

Aggressive fibromatosis, 132–133 Agonistic antibodies, 211–214 AKT, inhibitors of, 72–73, 286 Alkylphospholipid (APL), 72 AMG706, 126–127 Amphiregulin, 19 Angiogenesis, 258 regulation of, 318–319 survivin regulation of, 198, 200 targeting with oral agents, 241–255. See also under Targeting tumor angiogenesis, integrins role in, 260–266 Angiopoietin-1 (Ang-1), 198 Angiostatin, 258, 265, 272–273 Aniliquinazoline, 26 Antiangiogenic agents, 11–13 endothelial cell propagation targeting, 12 hypoxia inducing factor inhibition, 12 VEGF targeting, 11–12 VEGFR targeting, 12 tyrosine kinase inhibitory activity, 12 Antiangiogenic tyrosine kinase inhibitors in clinical trials, 241–249 Axitinib (AG 013736), 248 AZD2171, 243, 248–249 CP-547,632, 248 Sorafenib (BAY 43-9006), 241–244 SU5416 and SU6668, 247 Sunitinib, 243–246 Vatalanib, 243, 247–248 Antibody-dependent cellular cytotoxicity (ADCC), 46 Anticancer therapy, targeting MMP in, 321–322 Anti-invasive agents, 13–14 SRC inhibitors, 13–14 targeting metalloproteinase, 13 Antisense survivin oligonucleotide (ASO), 11 Antisense therapy, 199–200, 214 Antivascular agents, 13–14, 295–313 Anti-VEGF therapies, 226–232 adverse effects of, 234–236 disease specific toxicities, 235–236 hypertension, 234–235

12459 G-quadruplex ligand, 351 17-AAG, 383, 388–390 17-Allylamino, 17-demethoxygeldanamycin (17-AAG), 215 2C3 Antibody, 226 2-Cyano-3, 12-dioxoolean-1,9-dien-28-oic acid (CDDO), 215 2-Methoxyestradiol (2ME2), 287 307A G-quadruplex ligand, 351 5,6 Dimethylxanthenone- 4-acetic acid. See DMXAA 5-Fluorouracil (5FU), 227, 353 5-Hydroxyindoleacetic acid (5-HIAA), 305 7-Hydroxystaurosporine, 106

A4 inhibitors, 386 AbegrinTM (MEDI-522, etaracizumab), 264, 266–272 ABT-510, thrombospondin fragment, 265, 274 ABT-751, 298 Adamlysin metalloproteinases with thrombospondin motifs (ADAMTS), 316 Adenoid cystic carcinomas, 133 Adenomatous polyposis coli (APC) protein, 197 Adenovirus, 150, 184, 274, 289 serotype 5, 179 Advexin animal models, toxicity studies on, 183–184 clinical experience with, 184–189 in combination therapy, 186: locally advanced breast cancer treatment, 187–188; NSCLC treatment, 186–187, 189; and radiation therapy, 189 as monotherapy, 184–185 genome map, 178 p53 targeting, 179–181 in combination therapy, 180–181 effects on normal cells, 182–183 as monotherapy, 179–180 AEE788, 57–58

401

402 [Anti-VEGF therapies adverse effects of] proteinuria, 235 pivotal trials evaluating, 226–232 AP23994, 339, 340 Apoptosis, 214, 360 apoptosis modulators, 10–11, 177–190, 319 Bcl-2, 11 p53 targeting, 10. See also p53 survivin, 11 toxicity preclinical studies, 182–184: advexin effects. See Advexin TRAIL, 11 Aprinocarsen, 109 Aryl hydrocarbon receptor nuclear translocator (ARNT), 283 ATN–161, 264, 270–271 Aurora A, 157–158 biological functions, 157–158 in cancer, 159–161 as therapeutic targets, 161 Aurora B, 158–159 biological functions, 158–159 in cancer, 161 as therapeutic targets, 162 Aurora C, 159 biological functions, 159 Aurora kinase inhibitors, 10, 157–169. See also individual entries biological functions, 157–159 aurora A, 157–158 aurora B, 158–159 aurora C, 159 in cancer, 159–161 clinical data, 162–166 AT9283, 166 AZD1152, 165 MK-0457, 164 MLN8054, 165–166 PHA739358, 166 SU6668, 166 discovery, 157 future clinical development, 166–169 clinical indications, 166 combination with other agents, 168 dose escalation schemes, 167 drug development, 166–167 heavily pretreated patients,168 imaging, use of, 169 novel clinical trial designs, 167–168 novel pharmacodynamic assays, 168–169 as therapeutic targets, 161–162

Index AVE-8062, 298–299, 303 Axitinib (AG 013736), 248 AZD0530, 339, 340, 342 AZD2171, 243, 248–249 Azidothymidine, 353

B cell chronic lymphocytic leukemia (B-CLL), 334 Batimistat, 322 BAY 12-9566/tanomastat, 322 B-cell non-hodgkin’s lymphoma, 108 Bcl-2 protein, 214 Bcl-XLprotein, 214, 336 Benzoquinone ansamycin class of Hsp90 inhibitors. chemical structures of, 379 Bergerat fold, 382 Betacellulin, 19 Bevacizumab, 5, 11, 225 action of, mechanisms, 232–236 tumor cells, effects on, 234 vessel function, effects on, 233–234 vessel numbers, effects on, 232–233 for advanced stage disease, 228 anti-VEGF therapy, adverse effects of, 234 Capecitabine þ Bevacizumab, 231 and chemotherapy for non–small cell lung cancer, 230–231 in combination with chemotherapy for mCRC, 227–230 in metastatic breast carcinoma, 231–232 Paclitaxel Bevacizumab, 231 BIBR1532, 351 BMS 275291, 322 BMS-214662 in hematologic malignancies, 93, 97 in solid tumors, 91, 94–95 BMS-354825 (Dasatinib, Sprycel), 339, 340, 342 BMS-599626, 60–61 chemical structure, 61 Bortezomib, 215 BRACO19, 351 Breast cancer gefitinib, 33 HER2 in, 45–46 HER2 measurement, 45–46 pathogenesis, 45 pathophysiology of, 45 trastuzumab, 47–51

Index Bryostatin, 108 BSU1051, 351 Bugula neritina, 108

CA4DP, 298–299 Cancer therapy cancer vaccine/immunotherapy, survivin in, 200–201 HIF-1 inhibitors as target for, 283–285. See also HIF-1 inhibitors Carboplatin, 301 Carcinogenesis, fundamental mechanisms, 3 Catalytic inhibitors, 353–356 chrolactomycin, 354 Imatinib mesylate (Gleevec), 355 nonnucleoside inhibitors, 354 nucleoside analogs, 353 radicicol, 354 Tamoxifen, 355 Trichostatin A, 355 U-73122, 355 UCS1025A, 354 CCT018159, 385 Celecoxib, 72 Cell signaling process, 4 potential pharmaceutical intervention, 4 Cell-cycle inhibitors, 9–10 aurora kinase inhibitors, 10. See also separate entry Cellular FLICE-like inhibitory protein (cFLIP), 207 Cetuximab, 5 colorectal cancer, 28 head and neck cancer, 30–31 CGP76030, 339, 340–341 Chemotherapy, 25 locally advanced breast cancer treatment, 187–188 NSCLC treatment, 186–187 Chordomas, 133 Chrolactomycin, 354 Chronic lymphocytic leucemia (CLL), 11 Chronic myelogenous leukemia (CML), 132, 162 CI-1033, 58–60 chemical structure, 59 Cilengitide, 268–269 Cilengitide EMD 121974, 264 in combination therapy, 269 Cisplatin, 111

403 Classic chemotherapy, 1–2 Client proteins, 389, 391–392 CNTO 95, 264, 270–271 COL-3, 322 Collagen non-peptidomimetics MMPI, 322, 323 BAY 12-9566/tanomastat, 322 BMS 275291, 322 prinomastat/AG3340, 322 Collagen peptidomimetics MMP inhibitors, 322–323 Batimistat, 322 Marimastat, 322 Colorectal cancer (CRC), 23, 28–29 cetuximab trials, 28 Combretastatin A4 diphosphate (CA4DP), 299–302 Cowden’s syndrome, 69 CP-547,632, 248 Cross talk, 3 c-Src protein, 333–334 c-Src shRNA expression, 335 c-SrcF529, 336 Cyclin-dependent kinase inhibitors (CDKIs), 15 Cyclin-dependent kinases (CDKs), 9 Cyclo-oxygenase-2 inhibitors, 14 Cytochrome c, 209 Cytoplasmic signal transduction, 5–9 Cytotoxic T lymphocytes (CTL), 209, 356

Darier–Ferrand dermatofibrosarcoma protuberans, 132 Dasatinib, 14, 339, 342–343 Death receptors DR4, 208–210 DR5, 208–210 Death-inducing signaling complex (DISC), 208–209 Denosumab, 210 Deschloroflavopiridol, 9 Desmoid tumors, 132–133 Dideoxyguanosine (ddG), 353 Dimethyl sulphoxide (DMSO), 389 DMXAA (AS1404), 304 clinical studies, 305–306 DNA binding, inhibitors of, 288 Docetaxel, 59 Doppler ultrasonography, 242 DPNS, 351

404 DR4 (death receptor 4), 208–213 DR5 (death receptor 5), 208–213 Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), 299

E7820, 265, 270–271 Echinomycin, 288 ECM metalloproteinase inducer (EMMPRIN), 316, 320 ECOG 3200 trial, 227 EGCG, 351 EGFR (Epidermal growth factor receptor), 19–36, 286 EGFR signaling pathways, 19–20 major components representation, 20 inhibitors, 20–23 clinical trials, 24–34: breast cancer, 33; colorectal cancer, 28–29; esophageal cancer, 32; glioblastoma, 32; head and neck cancer, 29–32; non–small cell lung cancer, 24–28; pancreatic cancer, 32 response predictors, 34–36: biological features, 34–36; clinical features, 34 small molecules, 20–23: EGFR TKIs classes, 20–23; mimotopes, 23; monoclonal antibodies, 23 ligands of, 19 signal transduction inhibitors, 19–36 Endostar, 265 Endostatin gene therapy, 265, 272, 273–274 Endothelial cell propagation targets integrins as, 259–266. See also Integrins Enzastaurin, 110–112 Epidermal growth factor receptor. See EGFR Epigallocatechin gallate (EGCG), 354 Epiregulin, 19 ErbB receptor, 55–56 human solid tumors, expression in, 56 Erlotinib, 5, 20 glioblastoma, 32 head and neck cancer, 31 non–small cell lung cancer, 25 Esophageal cancer, 32 Everolimus, 74 Extracellular regulated kinase (ERK), 5, 19

Index Farnesyl transferase inhibitors, 85–98 hematologic malignancies clinical data, 95–97 preclinical data, 91–93 mechanisms, 89–90 Ras. See separate entry solid tumors clinical data, 93–95 preclinical data, 90–91 Fas-associated death domain (FADD), 208 Flavone-8-acetic acid (FAA), 304 Flavonoids, 297, 304–306 DMXAA, 304–306. See also separate entry Flavopiridol, 9 Fluorescence in situ hybridization (FISH), 35–36 Fluorouracil, 227 FOLFOX [Folinic Acid (leucovorin), 5-FU, Oxaliplatin], 227 Fyn, 335

Gastrointestinal stromal tumors. See GIST Gefitinib, 5, 20 breast cancer, 33 colorectal cancer, 29 esophageal cancer, 32 glioblastoma, 32 head and neck cancer, 31 non–small cell lung cancer, 25 Geldanamycin, 379, 380–383 Gemcitabine, 111 Gene therapy/expression, 289 HIF-1 and, 284 to target survivin, 201 Genistein, 338 GIST (Gastrointestinal stromal tumors), 107, 245, 392 chronic myelomonocytic leukemia, 132 Darier–Ferrand dermatofibrosarcoma protuberans, 132 hypereosinophilic syndromes, 132 molecular alterations, 128 sunitinib and resistance management, 129 multiple lines of therapy in, 131 mutations of KIT, PDGFR, PDGF, 127–134 imatinib, role of, 127–128 therapeutic agents, 130–131 AMG706, 130 masatinib, 131

Index [GIST (Gastrointestinal stromal tumors) therapeutic agents] nilotinib, 130–131 valatinib, 131 Glioblastoma, 32 Gliomas, 133 G-quadruplexes, 357 effects on telemeres, 358–359 inducing telomere dysfunction, 363 G-quartet, 351 G-rich 3’extension (G-overhang), 360 GRN163 oligomer, 353 GRN163L oligomer, 353 Growth factors, MMPs and, 319 GSK3 (Glycogen synthase kinase-3 beta), 66, 143 GTPase-activating protein (GAP), 68, 86

hAGP (Human alpha-1 acid glycoprotein), 72 Head and neck cancer, 29–32 recurrent/metastatic (R/M) disease, 29–30 squamous cell carcinoma, 29 Heat shock protein 90. See Hsp90 Helenalin, 354 Hematologic malignancies BMS-214662, 93, 97 lonafarnib, 93, 97 tipifarnib, 91–93, 95–97 Heparin binding EGF, 19 HER2 inhibition, 45–51 action mechanism, 46–47 breast cancer, 45–46 clinical achievement in, 47–51: lapatinib, 51; trastuzumab, 47–51. See also separate entry measurement, 45–46 pathogenesis, 45 pathophysiology in, 45 resistance mechanism, 47 Herbimycin A, 379 Heterogeneous tumors, 197 HGS-ETR1, 212 HGS-ETR2, 212–213 HIF inhibitors. See also HIF-1 inhibitors 2-Methoxyestradiol (2ME2), 287 of AKT, 286 of DNA binding, 288 early clinical development, 290–291 EGFR inhibitors, 286

405 [HIF inhibitors] gene therapy, 289 HDAC inhibitors, 287–288 of HIF-1 transcriptional activity, 288 Hsp90 inhibitors, 287 mechanisms of action, 285–289 microtubule-targeting agents, 287 of mTOR pathway, 286 natural products, 289 of protein accumulation, 287 of signaling pathways, 285–286 preclinical development and translational end points, 289–290 topoisomerase I poisons, 287 HIF-1 (Hypoxiainducible factor ), 12 HIF-1 inhibitors, 283–294 gene expression and, 284 as a target for cancer therapy, 283–285 HIF-1 expression: in human cancer, 284–285; regulation, 283–284 HIF-1a (Hypoxia-inducible factor-1a), 143 Histone deacetylase (HDAC) inhibitors, 15, 214, 287–288, 387 chemical structures of inhibitors of, 388 HKI-272, 61 HR2822, 356 Hsp90 inhibitors, 202, 215, 287, 354 benzoquinone ansamycin class of, 379 current status of, 381 Hsp90 isoform Grp94 chemical structure, 387 radicicol class of, 380 Hsp90 super-chaperone, 375–394 A4 inhibitors, 386 clinical trials, 388–391 co-chaperones, 377 function of, 376–378 ADP nucleotide exchange in, 376 ATP binding in, 376 ATPase hydrolysis in, 376 geldanamycin analogues, 382–383 Hsp90 clients and other interactions, 378–379 Hsp90 inhibitory natural products, 379–382 novobiocin analogues, 386 origins, 376 purine-scaffold inhibitors, 384–385 pyrazole-scaffold inhibitors, 385–386 radicicol analogues, 384 structure of, 376–378

406 [Hsp90 super-chaperone] therapeutic selectivity, 387–388 hTR telomerase, RNA component of, 352–353 Human alpha-1 acid glycoprotein (hAGP), 72 Human epidermal growth factor receptor (HER), 19–36, 216. See also EGFR HER2 inhibition. See also separate entry pan-HER inhibitors. See separate entry HuMV833 antibody, 226 Hypereosinophilic syndromes, 132 Hypertension, 234–235 Hypoxia inducible factor. See HIF inhibitors

IGF-1R, 141–152 Imatinib mesylate (Gleevec), 355 Imatinib, 125 Immune response and MMPs, 319 Immunization with telomerase, 356 Immunotherapy, survivin-directed, 200–201 Inhibitors, EGFR, 21–22. See also EGFR inhibitor of apoptosis (IAP) gene family, 11, 197 Insulin-like growth factor 1 receptor (IGF1R), 141–152 activated pathways, 141–143 circulating levels, polymorphisms and cancer, 145–148 targeted therapies, resistance to, 147–148 tyrosine kinase receptors, 146–147 expression in tumors, 144–145 inhibition, 150–151 involvement in cancer, evidence for, 143–144 ionizing radiation, 148–149 radio-sensitivity and inhibition, 149–150 targeting compounds, 150 Integrins, 258 a1b1, 260 a2b1, 260 a3b1, 260 a4, 261 a5b1, 260, 272 a5, 261 a6b1, 260 a6b4, 260 b8, 261 a9, 261

Index [Integrins] avb3, 260, 272 avb5, 260, 272 av, 261 Cilengitide, 268–269 clinical trial status, 264–265 AbegrinTM (MEDI-522, etaracizumab), 264 ABT-510, 265 Angiostatin, 265 ATN–161, 264 Cilengitide EMD 121974, 264 CNTO 95, 264 E7820, 265 Endostar, 265 Endostatin gene therapy, 265 Vitaxin (MEDI-523), 264 Volociximab Eos-200-4 M-200, 264 endostatin, 272 as endothelial cell propagation targets, 259–266 rationale for, 262–263: a2 subunit, 263; a5b1, 263; avb3, 262; avb5, 262 integrin antagonists, future, 271–272 integrin family, 259–260 less advanced integrin antagonists ATN-161, 270–271 CNTO 95, 270–271 E7820, 270–271 Volociximab, 270–271 plasminogen, 272 thrombospondin, 272 thrombospondin fragment (ABT-510), 274 in tumor angiogenesis, 260–266 Irinotecan, 28, 227

KAI-/CS-9803, 109–110 Karnofsky performance status (KPS), 185 Kinase inhibitors targeting (KIT), 123 empirical use, 132–133 adenoid cystic carcinomas, 133 aggressive fibromatosis, 132–133 chordomas, 133 gliomas, 133 prostate cancer, 133 and stem-cell factor, 124 tumors with mutations, 127–130 GIST. See separate entry tyrosine kinase inhibitors, 125–127

Index [Kinase inhibitors targeting (KIT) tyrosine kinase inhibitors] AMG706, 126–127 imatinib, 125 nilotinib, 125–126 sunitinib, 125 valatinib, 127

Lapatinib, 51 dual-HER inhibitors, 56–57 for trastuzumab resistance, 51 Leishmaniasis, 72 Lesions, premalignant, 211 Leucovorin (LCV), 227 Locally advanced breast cancer (LABC), 187 neoadjuvant advexin and chemotherapy, 187–188 Lonafarnib in hematologic malignancies, 93, 97 in solid tumors, 90–91, 94 Lyn, 334

M(4)N, 201 mAb (Monoclonal antibodies), 23 Marimastat, 322 Matrix metalloproteinases (MMPs) in carcinogenesis, implication of, 315–332 angiogenesis regulation, 318–319 fundamental data, 317–320 invasion and metastasis, 317–318 mechanisms of action, 317 clinical aspects, 320–324 collagen non-peptidomimetics MMPI, 322–323. See also separate entry collagen peptidomimetics MMP inhibitors, 322–323. See also separate entry ECM metalloproteinase inducer (EMMPRIN), 316 and growth factors, 319 immune response and, 319 membrane type MMPs (MT-MMPs), 315 MMP family, 315–317 classification and structure, 315–316 MMP-1, 320 MMP-11 expression, 320 MMP-2, 321 MMP-7, 320

407 [Matrix metalloproteinases (MMPs)] physiological functions, 316–317 plasma TIMP-1, 321 and prognosis, 320–321 regulation, 317 targeting MMP in anticancer therapy, 321–322 tetracycline derivates, 322, 323–324 tumor–stroma interactions, 319–320 MBT-1, 298 Mcl-1, 336 mCRC chemotherapy, 227–230 Metastasis, 317–318 metastatic breast carcinoma, Bevacizumab in, 231–232 metastatic colorectal cancer (mCRC), 226 Microtubule-targeting agents, 287 Miltefosine, 72 Mimotopes, 23 Mitogen-activated protein kinase (MAPK), 19, 73, 142 Mitogen-activated protein kinase-kinase (MAPKK), 5 Mitotic centromere-associated kinesin (MCAK), 158 MMQ1, 351 MN-029 (denibulin hydrochloride), 298, 303 mTOR (mammalian targetof rapamycin) inhibitors, 8, 73–76 mTOR pathway, inhibitors of, 286 Myelodysplastic syndrome, 164

Natural products, inhibiting HIF-1, 289 N-cadherin (N-cad) as a target for VDAs, 304 Neoangiogenesis, 11 Neovascularization, 257 Neuropilin-1 (NRP-1), 224–225 Neuropilin-2 (NRP-2), 224–225 Nilotinib, 125 Nonnucleoside inhibitors, 354 Non–small cell lung cancer (NSCLC), 20, 298, 392 advexin and chemotherapy, 186–187 advexin and radiation therapy, 189 chemotherapy for, 230–231 protein kinase C inhibitors. See separate entry Novobiocin analogues, 386 NPI-2358, 299

408 Oblimersen, 214 Oligonucleotidic inhibitors, 352–353 hTR telomerase, RNA component of, 352–353 silencing hTR/hTERT, 353 Oncogenes, 70 addiction, 177 Oral agents angiogenesis targeting with, 241–255. See also under Targeting Osteoprotegerin (OPG), 208, 210 Oxi4503, 299

p53 targeting, 10 gene therapy in animal models, 181–182 in vitro, 179–181 advexin monotherapy, 179–180 advexin with other agents, 180–181 regulatory pathway, 178 Paclitaxel, 301 Pan-HER inhibitors, 55–62 dual-HER inhibitors, 56 AEE788, 57–58 lapatinib, 56–57 ErbB receptors and ligands, 55–56 limits and prospects, 61–62 natural inhibitors, 58 BMS-599626, 60–61: chemical structure, 61 CI-1033, 58–60: chemical structure, 59 HKI-272, 61 PD173955/56, 340 Perifosine, 72 Peripheral blood mononuclear cells (PBMCs), 388–389 Peroxisome proliferator-activated receptor g(PPARg), 215 Peutz-Jeghers syndrome, 69 Phosphatidylinositol 3-kinase (PI3K), 19 inhibitors, 70–71 Phosphoinositide 3-kinase/Akt/ mTOR pathway, 65–77 in normal and cancer cells, 65–70 oncogenes and tumor suppressor genes, 70 targeting agents, 70–76 AKT Inhibitors, 72–73: Perifosine, 72–73 mTOR inhibitors, 73–76 PDK1 Inhibitors, 71–72 PI3K Inhibitors, 70–71

Index Picropodophyllin, 151 PIPER, 351 Placenta growth factor (PlGF), 223 Plasminogen, 272 Platelet-derived growth factor receptor (PDGFR), 123–134 empirical use, 132–133 adenoid cystic carcinomas, 133 aggressive fibromatosis, 132–133 chordomas, 133 gliomas, 133 prostate cancer, 133 and PDGFs, 124–125 tyrosine kinase inhibitors, 125–127 AMG706, 126–127 imatinib, 125 nilotinib, 125–126 sunitinib, 125 valatinib, 127 PP2, 339, 340 Premalignant lesions, TRAIL pathway in, 211 Prinomastat/AG3340, 322 Prognosis, MMPs and, 320–321 Progression-free survival (PFS), 242 Proheparin–binding epidermal growth factor (pro-HB-EGF), 319 Prostate cancer, 133 Protein accumulation, inhibitors of, 287 Protein arginine methyl transferase 5 (PRMT5), 391 Protein kinase C inhibitors non–small cell lung cancer treatment, 103–114 future development considerations, 112–114 nonspecific PKC inhibitors, 104–109: bryostatin, 108–109; PKC412, 107; staurosporin, 104–106; UCN-01, 106–107 selective inhibitors, 109: aprinocarsen, 109; KAI-/CS-9803, 109–110; enzastaurin, 110–112 Protein kinase C isoforms (PKC), 71, 107 Proteinuria, 235 Purine-scaffold Hsp90 inhibitors, 384–386 CCT018159, 385 VER-49009 (CCT0129397), 385 PX-478, 289 PX-866, 71

Index Quadruplex resolvases, 362–364 Quadruplexes, telomeres and, 357 G-quadruplexes, 357 Quercetin, 338

Radicicol analogues, 354, 379, 384 of Hsp90 inhibitors, 380 Rapamycin, 143 Ras, 85–88 biology, 85–87 in human tumors, 87–88 K-Ras mutations, 87 signaling pathways, 86 as therapeutic target, 88 Receptor tyrosine kinases (RTKs), 337 Recombinant human (rh) TRAIL, 207, 211–214 safety, in vivo, 211 Renal cell carcinoma (RCC), 241–244 Response evaluation criteria for solid tumors (RECIST), 232, 301, 389 RHPS4, 351 RhTRAIL. See Recombinant human (rh) TRAIL Rituximab, 108

SCCHN advexin, 184–186 Serine, 473, 67 Shelterin, 361 Shepherdin, 202 Signal transduction inhibitors, 2–9 categories, 5 cytoplasmic signal transduction inhibition, 5–9 C-kit inhibitors, 9 farnesyl transferase inhibitors, 7 MEK inhibitors, 7 PI3K and AKT inhibitors, 7–8 protein-kinase C (PKC) inhibitors, 8 rapamycin and m-TOR inhibitors, 8 rapamycin pathway, 7 Ras and Raf kinase inhibitors, 6–7 STAT inhibitors, 9 transcription pathway activation, 8–9 PDGFR. See Platelet derived growth factor receptor receptor function inhibition, 5 Single-strand G-overhang, 360–361

409 Sirolimus, 73–74 SKI606 (Bosutinib), 339, 340, 341–342 Small interfering RNA (siRNA), 200 Small molecules, to target survivin, 201–202 Solid tumors BMS-214662, 91 lonafarnib, 90–91, 94 tipifarnib, 90, 93–94 Sorafenib (BAY 43-9006), 241–244 Sprycel, 339, 340, 342 Squamous cell carcinoma, 29 Src family kinases (SFKs) cancer and, link between, 333–334 c-Src protein, 333 c-Src, 334–338 deregulation, downstream signaling consequences, 337–338 as targets, 333–337 experimental validation of, 334–337 v-Src, 334–338 Src inhibitors, 338–344. See also Src family kinases (SFKs) AP23994, 339 AZD0530, 339 BMS 354825 (Dasatinib), 339 CGP76030, 339 compounds in preclinical development, 344 historical overview, 338–341 molecular structure of, 339 molecules in clinical trials, 341–343 AZD0530, 342 BMS-354825 (Dasatinib, Sprycel), 342–343 SKI-606, 341–342 PD173955/56, 340 PP2, 339 PP2, 340 SKI606 (Bosutinib), 339 Sprycel, 339 SU6656, 340 STAT (Signal transducer and activator of transcription), 8, 19 Staurosporin, 9, 71, 104–106, 339 STK11 (Serine threonine kinase 11), 66 SU5416 and SU6668, 247 SU6656, 340–341 Sunitinib (SU11248), 125, 244–246 in GIST, 245 resistance management in GIST, 129 Surface-ligand targeted approach, to VDAs development, 296–298

410 Survivin, 11, 197–205 in angiogenesis regulation, 198, 200 biology of, 197–198 in ancillary aspects of tumor, 198 as an apoptosis inhibitor, 198 in mitosis of tumoe cells, 198 future prospects, 202–203 survivin-based therapeutics, 199–202 antisense, 199–200 as anticancer agents, 198 cancer vaccine/immunotherapy, 200–201 conceptual advantages, 198 gene therapy, 201 rationale of, 198–199 shepherdin, 202 small molecules, 201–202 targeting effect on mitosis, 199 tumor-specific expression of, 197–198 unique features, 197

Tamoxifen, 355 Targeted cancer therapy, 197 Targeted molecular treatment, 123 Targeting angiogenesis with oral agents, 241–255 antiangiogenic tyrosine kinase inhibitors in clinical trials, 241–249. See also separate entry future development, 249–250 TDG-TP, 351 Telomerase and telomere interacting agents, 349–365 12459, 351 307A, 351 and quadruplexes, 357 BIBR1532, 351 BRACO19, 351 BSU1051, 351 catalytic inhibitors, 353–356. See also separate entry DPNS, 351 EGCG, 351 G-quartet, 351 immunization with, 356 MMQ1, 351 oligonucleotidic inhibitors, 352–353. See also separate entry PIPER, 351 presentation, 350–352

Index [Telomerase and telomere interacting agents] quadruplex resolvases, 362–364. See also separate entry resistance mechanisms, 359–360 apoptosis and, 360 isolation of resistant clones, 359–360 RHPS4, 351 TDG-TP, 351 telomerase vaccinations, 356 telomere ligands, 357–358 telomere targeting versus telomerase targeting, 358–359 G-quadruplex ligands effects on, 358–359 telomere targeting, consequences of, 360–362 single-strand G-overhang: alteration and degradation, 360–361 telomere-binding proteins: deregulation, 361–362 telomestatin, 351 therapeutic strategies, 14 TMPI, 351 TmPyP4, 351 TNQX, 351 Telomestatin, 351 Tirosel/temsirolimus, 8, 73–76, 143, 242, 243, 286 Tetracycline derivates, 322, 323–324 COL-3, 322 Tetra-O-methyl nordihydroguaiaretic acid (M(4)N), 201 TGF-a (Transforming growth factor alpha), 19 Therapies, existing, 1–15 antiangiogenic agents, 11–13. See also separate entry anti-invasive agents, 13–14 antivascular agents, 13 apoptosis modulators, 10–11. See also Apoptosis cell-cycle inhibitors, 9–10. See also separate entry classic chemotherapy, 1–2 classisfication, 2 definitions, 1 molecular targeted therapy, 1–2 signal transduction inhibitors, 2–9. See also separate entry telomerase and telomere interacting agents, 14 transversal mechanisms, 14

411

Index [Therapies, existing transversal mechanisms] cyclo-oxygenase-2 inhibitors, 14 heat shock proteins, 15 histone deacetylase inhibitors, 15 proteasome inhibition, 14–15 Threonine 308, 67 Thrombospondin, 272 ABT-510, 274 Tipifarnib in hematologic malignancies, 91–93, 95–97 in solid tumors, 90, 93–94 Tissue-inhibitors of metalloproteinases (TIMPs), 317 TMPI, 351 TmPyP4, 351 TNQX, 351 Topoisomerase I poisons, 287 TRAIL (Tumor necrosis factor-related apoptosis inducing ligand) modulators, 11, 207–221 in lymphocyte proliferation, 209 in premalignant lesions, 211 physiological role, 209–210 signaling pathway, 207–209 targeting of, 211–214 therapeutic implications, 211–216. See also Recombinant human (rh) TRAIL potential of combination therapies, 214–216 TRAIL death receptors agents targeting, 212 TRAIL receptors, role of, 210–211 Trastuzumab, 5 for adjuvant breast cancer, 48–50 in metastatic breast cancer, 47–48 in neoadjuvant therapy, 50–51 Trichostatin A, 355 Tuberous sclerosis complex (TSC), 67–68 Tubulin binding agents, 297–303 ABT-751, 298 AVE-8062, 298, 303 CA4DP, 298 clinical studies, 299–303 MN-029, 298 NPI-2358, 298 Oxi-4503, 298 preclinical studies, 299 combretastatin A4 diphosphate, 299–302 second generation tubulin depolymerizing agents, 303–304 MN-029 (denibulin hydrochloride), 303

[Tubulin binding agents second generation tubulin depolymerizing agents] N-cadherin as target for VDAs, 304 TZT-1027, 298 ZD6126, 298, 302–303 Tumor necrosis factor-related apoptosis inducing ligand. See TRAIL Tumor suppressor genes, 70 Tumor-derived protein fragments angiostatin, 272–273 endostatin, 272–274 Tumor–stroma interactions, 319–320 Tyr419, 333–334 Tyrosine kinase inhibitors (TKIs), 20 Tyrphostins, 338 TZT-1027, 298

U-73122, 355 UCN-01, 71, 106–107 UCS1025A, 354

Vaccination, survivin-based, 201 Valatinib, 127 Vandetanib (ZD6474), 243, 247–248 Vascular disrupting agents (VDAs), 295 in clinical development, 298 development approaches to, 296–306: surface-ligand targeted approach, 296 opportunities and challenges in, 306–308: choice of dose and schedule, 307; tumor shrinkage, 306 flavonoids, 297, 304–306. See also separate entry mechanism of action of, 266 tubulin binding agents, 297–303. See also separate entry Vascular endothelial growth factor. See VEGF Vasculature, tumor normal vasculature versus, 295 Vasoconstriction, 233–234 Vatalanib (PTK787/ZK 222584), 243, 246–247 VEGF receptors, 5, 11–12, 26, 198, 223–224, 284 VEGF targeting, 223–240. See also Bevacizumab

412 [VEGF targeting] anti-VEGF therapies. See Anti-VEGF therapies functions of, 225 NRP-1 and NRP-2, 224–225 VEGF ligand family, 223 VEGF ligand, agents targeting, 225–226 2C3, 226 HuMV833, 226 VEGF-Trap, 226 VEGF receptors, 223–224 VEGFR1 (Flt-1), 223–224 VEGFR2, 224, 235 VEGFR3, 224 VER-49009 (CCT0129397), 385 Vessel numbers, 232–233 Vitaxin (MEDI-523), 264, 266–272 99 Tcm labeling, 267 Volociximab, 270–271

Index [Volociximab] Volociximab Eos-200-4 M-200, 264 Von Hippel-Lindau tumor supressor (VHL), 283

Wortmannin, 71

YC-1, 289 YM155, 201

ZD6126, 298–299, 302–303 ZD6474, 243, 247–248

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