Gene-Based Therapies for Cancer (Current Cancer Research)

Current Cancer Research

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Jack A. Roth Editor

Gene-Based Therapies for Cancer

Editor Jack A. Roth, M.D., F.A.C.S. Professor and Bud Johnson Clinical Distinguished Chair, Department of Thoracic & Cardiovascular Surgery; Professor of Molecular and Cellular Oncology; Director, W.M. Keck Center for Innovative Cancer Therapies; Chief, Section of Thoracic Molecular Oncology; The University of Texas MD Anderson Cancer Center Houston, TX USA [email protected]

ISBN 978-1-4419-6101-3 e-ISBN 978-1-4419-6102-0 DOI 10.1007/978-1-4419-6102-0 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2010931279 © Springer Science+Business Media, LLC 2010 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in ­connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Foreword

Cancer is a disease of dysfunctional genes. Normal cellular genes that regulate cell proliferation develop carcinogen-induced, or rarely, germline mutations that alter the gene product so that it is permanently in an active configuration. Examples include the Kras oncogene and the epidermal growth factor receptor. These oncogenes confer the property of unlimited replication to cancer cells. Genes that normally suppress cell growth or induce programmed cell death, after sensing DNA damage, develop inactivating mutations in the cancer cell. Examples include the p53 tumor suppressor gene and the retinoblastoma gene. Inactivation of these tumor suppressor genes removes essential growth control mechanisms from the cell. Strategies for replacing inactivated tumor suppressor genes or inactivating oncogenes are logical extensions of the gene therapy concept. Investigators have been pursuing these concepts for almost 20 years, but slow progress in developing systemic delivery vehicles for genes and the ability to target tumors, and fragmentary knowledge of the critical genes to target, have limited progress. Recently, significant progress in sequencing the cancer genome, combined with advances in personalized cancer treatment has converged to accelerate the development of cancer gene therapy. Personalized or targeted cancer treatments rely on high throughput technologies, including DNA sequencing, expression arrays, and proteomics to identify critical pathways for cancer cell survival. Small molecule drug libraries are used to identify drugs that may specifically inhibit a pathway. Dramatic regression of metastatic cancers have occurred in a small percentage of cases with lower toxicity than seen with conventional chemotherapy agents, with these drugs indicating proof-ofprinciple for this approach. However, it is clear that additional critical pathways must be targeted as tumor recurrence occurs rapidly. Importantly, research in this field has identified critical targets for gene therapy approaches and raises the possibility of combining targeted small molecules with gene-based therapies. Gene therapy offers the potential of highly selective targeting of multiple critical pathways with minimal toxicity. Additional progress in the development of targeted and less toxic viral and nonviral vectors has also improved the outlook for cancer gene therapy. In this book, translational gene therapy approaches are emphasized. Chapters include discussions of specific gene delivery technologies as well as their application in the treatment of various cancers with extensive discussions of ongoing clinical trials. One approach that is not discussed at length in this book is the v

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use of genes to modify the immune response to cancer as this is more appropriate for reviews of the immunotherapy of cancer. As is evident from numerous positive clinical trials, gene therapy for cancer has established proof-of-principle. The next step will be to enable this technology to be widely applied for the systemic treatment of cancer. Jack A. Roth, M.D.

Contents

1 RNAi: A New Paradigm in Cancer Gene Therapy................................. Edna M. Mora, Selanere L. Mangala, Gabriel Lopez-Berestein, and Anil K. Sood

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2 Gene-Based Therapy for Cancer: Brain Tumors.................................... Hong Jiang and Juan Fueyo

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3 Gene Therapy of Prostate Cancer............................................................ Svend O. Freytag, Hans Stricker, Benjamin Movsas, Mohamed Elshaikh, Ibrahim Aref, Kenneth Barton, Stephen Brown, Farzan Siddiqui, Mei Lu, and Jae Ho Kim

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4 siRNA Versus shRNA for Personalized Cancer Therapy: Mechanisms and Applications................................................. John S. Vorhies, Donald D. Rao, Neil Senzer, and John Nemunaitis 5 Tumor Suppressor Gene Therapy............................................................ Jack A. Roth, John Nemunaitis, Lin Ji, and Rajagopal Ramesh 6 Targeted Oncolytic Adenovirus for Human Cancer Therapy: Gene-Based Therapies for Cancer........................................................... Toshiyoshi Fujiwara 7 Gene Therapy for Malignant Pleural Mesothelioma.............................. Edmund K. Moon, Sunil Singhal, Andrew R. Haas, Daniel H. Sterman, and Steven M. Albelda

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79 95

8 Mesenchymal Stem/Stromal Cells as Cellular Vehicles for Tumor Targeting.................................................................................. 113 Frank Marini, Matus Studeny, Jennifer Dembinski, Keri L. Watson, Shannon Kidd, Erika Spaeth, Zhizong Zeng, Xiaoyang Ling, Ann Klopp, Fredrick Lang, Brett Hall, and Michael Andreeff

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Contents

  9 Retargeting Adenovirus for Cancer Gene Therapy.............................. 141 Erin E. Thacker and David T. Curiel 10 Lentiviruses: Vectors for Cancer Gene Therapy.................................. 155 Yuan Lin, Amar Desai, and Stanton L. Gerson 11 Interleukin-24 Gene Therapy for Melanoma........................................ 181 Nancy Poindexter, Rajagopal Ramesh, Suhendan Ekmekcioglu, Julie Ellerhorst, Kevin Kim, and Elizabeth A. Grimm 12 Herpes Simplex Virus 1 for Cancer Therapy........................................ 203 Richard L. Price, Balveen Kaur, and E. Antonio Chiocca 13 Telomerase as a Target for Cancer Therapeutics.................................. 231 Jerry W. Shay 14 Gene Therapy for Sarcoma..................................................................... 251 Keila E. Torres and Raphael E. Pollock Index.................................................................................................................. 269

Contributors

Steven M. Albelda, M.D. William Maul Massey Professor of Medicine; Vice Chief, Pulmonary, Allergy, and Critical Care Division, University of Pennsylvania Medical Center; Abramson Research Center, Philadelphia PA, USA Michael Andreeff, M.D., Ph.D. Professor of Medicine and Haas Chair in Genetics; Chief, Molecular Hematology and Therapy, The University of Texas MD Anderson Cancer Center, Departments of Leukemia and Stem Cell Transplantation and Cellular Therapy, Houston TX, USA Ibrahim Aref, M.D. Medical Director, Department of Radiation Oncology, Henry Ford Macomb Hospital, Detroit MI, USA Kenneth Barton, Ph.D. Staff Scientist, Department of Radiation Oncology, Henry Ford Health System, Detroit MI, USA Stephen Brown, Ph.D. Staff Scientist, Department of Radiation Oncology, Henry Ford Health System, Detroit MI, USA E. Antonio Chiocca, M.D., Ph.D. Chairman, Department of Neurological Surgery, Dardinger Family Professor of Oncologic Neurosurgey; Physician Director, OSUMC Neuroscience Signature Program; Co-Director, Dardinger Center for Neuro-oncology and Neurosciences; Co-Director, Viral Oncology Program of the Comprehensive Cancer Center; James Cancer Hospital/Solove Research Institute; The Ohio State University Medical Center, Columbus OH, USA David T. Curiel, M.D., Ph.D. Distinguished Professor of Radiation Oncology (Endowed Chair); Director, Cancer Biology Division and Therapeutics Center, Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO, USA ix

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Contributors

Jennifer Dembinski, Ph.D. Department of Stem Cell Transplantation and Cellular Therapy, Section of Molecular Hematology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Amar Desai, B.S. Ph.D. Student, Department of Pharmacology, Case Western Reserve University, Cleveland OH, USA Suhendan Ekmekcioglu Ph.D. Associate Professor, Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Julie Ellerhorst M.D., Ph.D. Associate Professor, Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Mohamed Elshaikh, M.D. Residency Program Director, Department of Radiation Oncology, Henry Ford Health System, Detroit MI, USA Svend O. Freytag, Ph.D. Division Head of Research in Department of Radiation Oncology, Henry Ford Health System, Detroit MI, USA Juan Fueyo, M.D. Associate Professor, Department of Neuro-Oncology, The University of Texas MD Anderson Cancer Center, Houston TX, USA Toshiyoshi Fujiwara, M.D. Professor and Chairman, Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Phamaceutical Sciences and Vice Director of Center for Gene and Cell Therapy of Okayama University Hospital, Okayama, Japan Director of the Board, Oncolys BioPharma, Minato-ku Tokyo, Japan Stanton Gerson, M.D. Director, University Hospitals Ireland Cancer Center and Director, Case Comprehensive Cancer Center; Professor of Medicine, Oncology and environmental Health Sciences; Case Western Reserve University, Cleveland OH, USA Elizabeth A. Grimm, Ph.D. Professor, Department of Experimental Therapeutics; Francis King Black Memorial Professor of Cancer Research; Deputy Head for Research Affairs, Division of Cancer Medicine; and Co-Director, Melanoma Research Program;

Contributors

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The University of Texas MD Anderson Cancer Center, Houston, TX, USA Andrew R. Haas, M.D., Ph.D. Assistant Professor, Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia PA, USA Brett Hall, Ph.D. Team Lead Biomarkers, Division of Janssen Pharmaceutica, Ortho Biotech Oncology R&D, Oncology Biomarkers Beerse, Johnson & Johnson, Beerse, Belgium Lin Ji, Ph.D. Associate Professor, Department of Thoracic and Cardiovascular Surgery Research, The University of Texas MD Anderson Cancer Center, Houston TX, USA Hong Jiang, Ph.D. Assistant Professor, Department of Neuro-Oncology, The University of Texas MD Anderson Cancer Center, Houston TX, USA Balveen Kaur, Ph.D. Assistant Professor, Dardinger Laboratory for Neuro-oncology and Neurosciences, Department of Neurological Surgery, James Comprehensive Cancer Center and The Ohio State University Medical Center, Columbus OH, USA Shannon Kidd, Ph.D. Graduate Research Assistant, Department of Stem Cell Transplantation and Cellular Therapy, Section of Molecular Hematology, The University of Texas MD Anderson Cancer Center, Houston TX, USA Jae Ho Kim, M.D. Chairman Emeritus, Department of Radiation Oncology, Henry Ford Health System, Detroit MI, USA Kevin Kim, M.D. Associate Professor of Medicine, Department of Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Ann Klopp, M.D. Assistant Professor, Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston TX, USA Fredrick Lang, M.D. Professor, Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston TX, USA

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Yuan Lin, Ph.D. Student, Division of Hematology/Oncology, Case Western Reserve University, Cleveland OH, USA Xiaoyang Ling, Ph.D. Instructor, Department of Stem Cell Transplantation and Cellular Therapy, Section of Molecular Hematology, Houston TX, USA Gabriel Lopez-Berestein, M.D. Professor, Department of Experimental Therapeutics, Center for RNA Interference and Non-Coding RNA, Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston TX, USA Mei Lu, Ph.D. Senior Research Biostatician, Department of Biostatistics and Research Epidemiology, Henry Ford Health System, Detroit MI, USA Lingegowdda S. Mangala, Ph.D. Research Scientist, USRA, Division of Life Sciences, NASA Johnson Space Center, Houston TX, USA Frank Marini, Ph.D. Associate Professor, Department of Stem Cell Transplantation and Cellular Therapy, Section of Molecular Hematology, The University of Texas MD Anderson Cancer Center, Houston TX, USA Edmund K. Moon, M.D. Post-doctoral Fellow Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia PA, USA Edna M. Mora, M.D. Department of Surgery, School of Medicine, University of Puerto Rico, San Juan Puerto Rico; University of Puerto Rico Comprehensive Cancer Center, San Juan Puerto Rico; Adjunct Associate Professor, Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston TX, USA Benjamin Movsas, M.D. Chairman, Department of Radiation Oncology, Henry Ford Health System, Detroit MI, USA John Nemunaitis, M.D. Co-founder Gradalis, Inc, Executive Medical Director and Oncologist, Mary Crowley Cancer Research Centers, Texas Oncology PA, Baylor Sammons Cancer Center, Dallas TX, USA

Contributors

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Nancy Poindexter, Ph.D. Associate Professor, Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Raphael E. Pollock, M.D., Ph.D. Professor and Head, Division of Surgery; Senator A.M. Aiken, Jr. Distinguished Chair; Director, Sarcoma Research Center; Chair, Department of Surgical Oncology; The University of Texas MD Anderson Cancer Center, Houston TX, USA Richard L. Price, B.S. M.D., Ph.D. Student, Dardinger Laboratory for Neuro-oncology and Neurosciences, Department of Neurological Surgery, James Comprehensive Cancer Center and The Ohio State University Medical Center, Columbus OH, USA Rajagopal Ramesh, Ph.D. Associate Professor, Department of Thoracic and Cardiovascular Surgery Research, The University of Texas MD Anderson Cancer Center, Houston TX, USA Donald D. Rao, Ph.D. Director of Interference Technology, Gradalis, Inc, Dallas TX, USA Jack A. Roth, M.D., F.A.C.S. Professor and Bud Johnson Clinical Distinguished Chair, Department of Thoracic and Cardiovascular Surgery; Professor of Molecular and Cellular Oncology; Director, W.M. Keck Center for Innovative Cancer Therapies; Chief, Section of Thoracic Molecular Oncology; The University of Texas MD Anderson Cancer Center, Houston TX, USA Neil Senzer, M.D. Gradalis, Inc, Scientific Director, Mary Crowley Cancer Research Centers, Texas Oncology PA; Adjunct Associate Professor, Baylor University Institute of Biomedical Studies, Dallas TX, USA Jerry W. Shay, Ph.D. Professor and Vice Chairman, Department of Cell Biology, Associate Director, Simmons Comprehensive Cancer Center at The University of Texas Southwestern Medical Center, Dallas TX, USA Farzan Siddiqui, M.D., Ph.D. Resident, Department of Radiation Oncology, Henry Ford Health System, Detroit MI, USA

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Contributors

Sunil Singhal, M.D. Assistant Professor, Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia PA, USA Anil K. Sood, M.D. Professor, Departments of Gynecologic Oncology and Cancer Biology; Co-Director, Center for RNA Interference and Non-Coding RNA; Co-Director, Blanton-Davis Ovarian Cancer Research Program, Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston TX, USA Erika Spaeth, B.S. Graduate Research Assistant, Department of Stem Cell Transplantation and Cellular Therapy, Section of Molecular Hematology, The University of Texas MD Anderson Cancer Center, Houston TX, USA Daniel H. Sterman, M.D. Associate Professor, Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia PA, USA Hans Stricker, M.D. Henry Ford Health System, Vattikuti Urology Institute, Detroit MI, USA Matus Studeny, M.D. Clinical Research Director, Boehringer Ingelheim Pharmaceuticals RCV GmbH and Co KG; Division of Medicine and Clinical Development Department; Vienna, Austria Erin E. Thacker, Ph.D. Adjunct Professor at Birmingham-Southern College; Division of Human Gene Therapy, Departments of Medicine, Obstetrics and Gynecology, Pathology, Surgery, University of Alabama at Birmingham, Birmingham AL, USA Keila E. Torres, Ph.D. Consultant, Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston TX, USA John S. Vorhies, B.A. Consultant, Gradalis, Inc, Dallas TX, USA Zhizong Zeng, M.D. Research Scientist, Department of Stem Cell Transplantation and Cellular Therapy, Section of Molecular Hematology, The University of Texas MD Anderson Cancer Center, Houston TX, USA

Chapter 1

RNAi: A New Paradigm in Cancer Gene Therapy Edna M. Mora, Selanere L. Mangala, Gabriel Lopez-Berestein, and Anil K. Sood

Abstract  RNA interference (RNAi) has revolutionized the field of gene therapy and opened up new opportunities for personalized treatments. However, several challenges remain for gene therapy. Therefore, new approaches for gene regulatory therapies are needed to overcome these challenges. In this chapter, we discuss the clinical significance of the RNAi machinery, clinical applications, delivery systems, off-target effects, imaging, and clinical trials. The remarkable advances in the design, delivery, and understanding of RNAi-based therapeutics predict a bright future for their development as therapeutic agents. It is well established that once a gene is identified as an important player in tumor progression or metastasis, siRNA is a feasible alternative to modulate its expression. Moreover, the development of new delivery systems will further advance the efficiency and localization of siRNA delivery to specific tissues and organs. Concurrently, the development of “intelligent probes” to identify siRNA function in addition to localization will further advance the evaluation of new formulations using imaging techniques. Keywords  RNAi • Delivery systems • Clinical applications • RNAi formulations

A.K. Sood (*) Department of Gynecologic Oncology, U.T.M.D. Anderson Cancer Center, 1155 Herman Pressler, Unit 1362, Houston, TX 77030, USA and Center for RNA Interference and Non-Coding RNA, U. T. M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA and Department of Cancer Biology, U.T.M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 173, Houston, TX 77030 USA e-mail: [email protected] J.A. Roth (ed.), Gene-Based Therapies for Cancer, Current Cancer Research, DOI 10.1007/978-1-4419-6102-0_1, © Springer Science+Business Media, LLC 2010

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1 Introduction Genetic dysregulation is a hallmark of cancer development and progression (Hanahan and Weinberg 2000). It is well known that accumulation of genetic changes in normal cells including mutations, changes in gene copy number, transcription of abnormal genes, and changes in protein translation results in phenotypic changes that support the development of malignant changes followed eventually by the growth of metastatic foci. As a result, these genetic changes provide new opportunities as targets for cancer therapy. Gene regulatory therapy is an attractive alternative to other systemic cancer therapies for several reasons. Systemic therapies such as chemotherapy and hormonal therapy not only affect the growth of primary and metastatic tumors, but also affect normal tissues and organs. As a result, secondary effects (e.g., alopecia, nausea, vomiting, etc.) occur at the expense of the patient’s quality of life. In contrast, therapy designed to target cells with specific genetic abnormalities may translate into a “personalized” approach. For example, FUSI is a tumor suppressor gene that is lost in a large number of lung cancer patients. Treatment of lung cancer patients with a FUSI-gene nanoparticle-based therapy assumes that FUSI expression in the tumor is known prior to therapy (Guinn and Mulherkar 2008). However, several challenges remain for gene therapy, including the limited intratumoral spread of viral particles delivered in the blood stream, development of neutralizing antibodies in the plasma, complement activation, development of non-neutralizing antibodies, unwanted infection of irrelevant cells, and phagocytosis by Kupffer cells (Guinn and Mulherkar 2008). Therefore, new approaches for gene regulatory therapies are needed. Traditionally, gene therapy was conceived as the replacement for “damaged” genes. However, RNA interference (RNAi) has broadened this concept to include the regulation of gene expression. In fact, depending on the specific targets (e.g., IL-8, miRNAs), the RNAi pathway can modulate (increase or decrease) gene function. RNAi has revolutionized the field of gene therapy and opened up new opportunities for personalized treatments. For example, Colombo and colleagues (2008) recently reviewed the role of RNAi in the development of Aurora kinases as a group of antineoplastic drugs. SiRNA was instrumental for documenting the role of Aurora kinases in cell proliferation, and identified these kinases as important targets for cancer therapy. At present, there are several groups pursuing the development of Aurora kinase inhibitors as antineoplastic drugs. While many new therapeutic targets have been identified, it may not be possible to target all of these with conventional approaches such as small molecule inhibitors due to several reasons, including: (1) complex protein structure (e.g., p130Cas) that would be difficult to target with a small molecule inhibitor; (2) nonenzymatic functions, (3) multiple structural domains with independent functions, (4) multiple phosphorylation sites that are critical for function, and (5) incompletely known three-dimensional structure. Moreover, most small molecule inhibitors lack specificity

1  RNAi: A New Paradigm in Cancer Gene Therapy

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and can be associated with undesirable side effects (Rix and Superti-Furga 2009). Similarly, while monoclonal antibodies have shown promise against specific targets such as VEGF, their use is limited to either ligands or cell surface receptors. The use of siRNA is preferable compared to other approaches such as antisense oligomers due to evidence suggesting longer duration of target inhibition as well as reduced toxicity. The development of RNAi-based gene therapies is possible due to a better understanding of the biological basis of this complex system. The discovery of RNAi by Fire and colleagues (1998) has been seminal to our understanding of cellular transcriptional and translational regulation. Furthermore, the description of specific gene silencing by intracellular delivery of exogenous siRNA by Tushl and colleagues (Elbashir et al. 2001) opened a new era in targeted therapeutics. This process is highly conserved and prevalent in a wide variety of organisms including plants, worms, and vertebrates. Subsequent studies in mammalian cells have opened new possibilities for the use of RNAi-mediated gene silencing against various diseases, including cancer.

2 Clinical Significance of the RNAi Processing Machinery The RNAi machinery is a complex system requiring a series of steps including processing by the key enzymes, Dicer and Drosha (Fig. 1). Evaluation of Dicer in lung cancer patients showed a correlation between low Dicer levels and poor prognosis (Karube et  al. 2005). Similarly, Muralidhar and coworkers (2007) found a correlation between the levels of Drosha in cervical squamous cell carcinoma. Our group has evaluated the expression of Dicer and Drosha in clinical samples from ovarian cancer patients using quantitative RT-PCR analysis (Merritt et al. 2008a). The levels of Dicer and Drosha mRNA correlated with protein levels, and were decreased in 60 and 51% of ovarian cancer specimens, respectively. Low Dicer expression correlated with advanced tumor stage ( p = 0.007), and low Drosha expression with suboptimal surgical cytoreduction ( p = 0.02). High expression of both proteins correlated with increased median survival ( p 25%) declines in PSA and there were three (19%) objective PSA responses. However, all PSA responses were short-lived (10 ng/mL). The major difference among these three trials is the investigational agent used. The first trial utilized the same first-generation Ad5-CD/ TKrep adenovirus that was used in the locally recurrent setting. The second and third trials used two different (but related) second-generation adenoviruses both of which contained an improved yeast CD/mutant HSV-1 TKSR39 fusion gene in the E1 region and either the adenovirus death protein (ADP) or human sodium iodide symporter (hNIS) gene in the E3 region. ADP increases the cytolytic effects of oncolytic adenoviruses. hNIS was used as a reporter gene to monitor adenovirus spread and persistence in patients using routine nuclear imaging (Barton et al. 2008). To date, a total of 42 patients have been treated up to an adenovirus dose of 5 × 1012 vp. Overall, the gene therapy/radiotherapy combination has been well tolerated. Gene therapy-related adverse events include transient flu-like symptoms

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(25%) and transaminitis (33%), which are likely attributable to the oncolytic adenovirus, and hematologic events [lymphopenia (89%), anemia (44%), neutropenia (22%), thrombocytopenia (19%)], which are likely attributable to the 5-FC + vGCV prodrug therapy. Importantly, the gene therapy did not exacerbate the most common side effects of prostate radiotherapy (genitourinary and gastrointestinal events). Efficacy was assessed using two endpoints (1) prostate biopsy (³6 cores) positivity at 2 years, which is prognostic for the development of distant metastases and disease-specific survival, and (2) freedom from biochemical/clinical failure (FFF). Better-than-expected biopsy results were obtained in men treated with the gene therapy/radiotherapy combination (Table  1). When considering all patients biopsied thus far, 7 of 33 (21%) had a positive 2-year biopsy, which is better-thanexpected for men with intermediate- to high-risk disease. When the results were broken down by prognostic risk group, most of the treatment effect was observed in the intermediate-risk group with 2 of 21 (10%) having a positive 2-year biopsy. By contrast, 5 of 12 (42%) high-risk patients were positive for cancer at 2 years, which was not significantly different than what was expected for this risk group. Although longer follow-up is needed, the estimated 5-year FFF for the intermediaterisk group is 95%, which is slightly better than expected (60–85%). By contrast, the estimated 5-year FFF for the high-risk group is 63%, which is within the range expected (40–65%). Together, the biopsy and PSA results raise the possibility that replication-competent adenovirus-mediated suicide gene therapy may have the potential to improve the outcome of prostate radiotherapy in select (i.e., intermediaterisk) patient groups. It is possible that saturating the prostate gland with the adenovirus through the use of higher adenovirus doses will generate better results in the high-risk group. Table 1  Posttreatment prostate biopsy results Two-year prostate biopsy results All patients Expected for EBRT only Gene therapy + EBRT (n = 33) Intermediate-risk patients Expected for EBRT only Gene therapy + EBRT (n = 21) High-risk patients Expected for EBRT only Gene therapy + EBRT (n = 12)

Prostate biopsy status % positive p Value 46 21

0.03

42 10

0.02

51 42

0.55

Expected biopsy results are from Zelefsky et al. (2008b). Results for 70 Gy are shown. In the gene therapy cohort, biopsies were based on a mean of 9 cores taken at 2 years after external beam radiotherapy (EBRT) and those with adenocarcinoma and severe treatment effects were scored positive. Intermediate-risk: Stage T1/T2 and Gleason score 7 or PSA 10–20  ng/mL. High-risk: Stage ³ T3 or Gleason score ³8 or PSA >20 ng/mL

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3 Vaccine-Based Gene Therapy Strategies Several different vaccine-based strategies have been evaluated clinically. Such strategies have utilized poxvirus (vaccinia and fowlpox) expressing PSA either alone or in combination with costimulatory molecules, and cell-based vaccines expressing granulocyte-macrophage colony stimulating factor (GM-CSF). They have evaluated in the settings of newly-diagnosed, locally recurrent and metastatic disease. Several have generated provocative results in early stage trials justifying progression to phase 3.

3.1 Poxvirus-Based Vaccines Poxvirus-based vaccines expressing PSA were the first to be evaluated clinically. Poxviruses have several advantages, including proven safety track record, potent adjuvant activity, large genome size facilitating the insertion of therapeutic genes, and ease of manipulation. In the first study conducted at the University of Michigan (Sanda et al. 1999), six patients with locally recurrent prostate cancer after surgery were administered intradermally two doses of vaccinia virus expressing PSA (PROSTVAC). At the time of enrollment, patients were on salvage AST for 2–14 months and had undetectable PSA. Adverse events were limited to transient lowgrade flu-like symptoms, fatigue, and vaccination site erythema. To assess efficacy, AST was interrupted and the rise in PSA was monitored during recovery from the castrate state. Typically, patients will show a rise in PSA within 1–2 months after discontinuation of AST as their testosterone levels recover (although this varies with age). The PSA of one patient remained undetectable for over 8 months after the restoration of testosterone levels, indicative of a possible antitumor response. Several patients developed increased anti-PSA antibody titers following treatment. A number of poxvirus-based approaches have been conducted subsequently and some have generated very provocative results. In a study (E7897) conducted by the Eastern Cooperative Oncology Group (ECOG) (Kaufman et al. 2004), 62 patients with locally recurrent prostate cancer were randomly assigned to receive four vaccinations with (1) fowlpox-PSA (rF-PSA), (2) three rF-PSA followed by one vacciniaPSA (rV-PSA), or (3) one rV-PSA followed by three rF-PSA. All prime/boost vaccination regimens were well tolerated. The most frequent treatment-related adverse event was injection site reaction. When considering all patients, 45% remained free of biochemical progression (defined as £50% increase in PSA), and 78% remained free of clinical progression, for 19 months. Of the three vaccination regimens, the third (rV-PSA followed by three rF-PSA) resulted in the best PSA progression-free survival. Although there were no significant increases in anti-PSA antibody titers, about half of the patients showed PSA-specific T cell responses. In an approach being developed at the National Cancer Institute, recombinant poxvirus-expressing PSA and the T cell costimulatory B7.1 molecule has been

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evaluated in the settings of newly-diagnosed disease concomitant with prostate radiotherapy, nonmetastatic hormone-refractory disease in combination with antiandrogen therapy, and metastatic disease in combination with docetaxel chemotherapy (Gulley et al. 2002, 2005, 2008; Arlen et al. 2005, 2006, 2007; Lattouf et al. 2006; Madan et al. 2008; Lechleider et al. 2008; Paola et al. 2006). In the newlydiagnosed setting, 30 patients were randomized 2:1 to receive vaccine plus radiotherapy or radiotherapy alone (Gulley et al. 2005). The vaccination course consisted of a single priming injection of two recombinant vaccinia viruses (rV) expressing either PSA or B7.1 as an admixture, followed by monthly booster vaccinations with recombinant fowlpox virus (rF)-expressing PSA. Vaccinations were accompanied by local injections of GM-CSF and low-dose systemic IL-2. Prostate radiotherapy was given between the fourth and sixth vaccinations. The investigational therapy was well tolerated. There were no treatment-related ³ grade 3 events. Thirteen of 17 (76%) patients who completed the full vaccination course developed ³threefold increase in PSA-specific T cells whereas patients in the control arm did not. Interestingly, some patients developed T cell reactivity to prostate antigens not contained in the vaccine indicating an “antigen cascade” effect. In the setting of nonmetastatic hormone-refractory disease, 42 patients who exhibited a rising PSA following salvage AST were randomized to receive either poxvirus-based PSA + B7.1 vaccination or nilutamide (Arlen et al. 2005). Patients in the vaccination arm also received GM-CSF and IL-2. Patients who subsequently exhibited biochemical, but not clinical, progression were allowed to crossover and receive treatment on the other arm. A number of patients on the vaccination arm exhibited prolonged declines and stabilization of PSA. Median time to treatment failure was 9.9 months on the vaccination arm versus 7.6 months in the nilutamide arm. Interestingly, in patients who crossed over, median time to treatment failure was 25.9 months (from initiation of therapy) in the vaccination-to-nilutamide group versus 15.5 months in the nilutamide-to-vaccination group. Follow-up analysis demonstrated that crossover patients who received the vaccination followed by nilutamide demonstrated improved overall survival versus the nilutamide-to-vaccination group (6.2 vs. 3.7 years, p = 0.045) (Madan et al. 2008). This vaccination approach was also evaluated in the setting of hormone-refractory metastatic disease (Gulley et al. 2002; Arlen et al. 2006). In a phase 2 study, 28 patients were randomized to receive either the vaccine and weekly docetaxel or vaccine alone. Patients on the vaccine only arm were allowed to cross over to the combined therapy arm at the time of disease progression. Patients on the combined therapy arm exhibited a greater decrease in PSA velocity relative to those on the vaccine arm (79% of whom crossed over) or historical controls who received docetaxel only. Median biochemical/clinical progression-free survival was 6.1 months for patients on the vaccine arm who crossed over, 3.2 months for patients who received the combined therapy up front, 1.8 months for patients who received the vaccine only, and 3.7 months for historical controls. As was observed in previous trials, a number of patients developed T cell reactivity to PSA as well as prostate antigens not contained in the vaccine. There appeared to be a benefit of administering the vaccine prior to chemotherapy.

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The preliminary results obtained with the poxvirus PSA/B7.1 vaccine are very encouraging and suggest that immunotherapy may bolster the effectiveness of subsequent chemotherapy. A more potent poxvirus PSA vaccine containing multiple costimulatory molecules (B7.1, ICAM-1, LFA-3) is now being evaluated in the clinic.

3.2 Cell-Based Vaccines GVAX is an “off-the-shelf” allogenic tumor vaccine comprising GM-CSF-secreting human prostate adenocarcinoma cells. The rationale for using GM-CSF as a cancer therapy is well-founded scientifically, and stems from the fact that it has demonstrated the ability to induce durable, tumoricidal, antitumor immune responses in preclinical models (Dranoff et al. 1993). GM-CSF recruits APC to immunization sites, which, in turn, activate CD4+ and CD8+ T lymphocytes by priming them with oligopeptides derived from dying cancer cells. This ultimately results in the destruction of tumor cells by both T- and B-cell mediated mechanisms. In the first trial, eight men were treated with autologous, GM-CSF-secreting tumor vaccine that was generated ex vivo by retroviral transduction of surgically harvested tumor cells (Simons et al. 1999). Patients received up to six intradermal vaccinations every 3 weeks at two dose levels (1 and 5 × 107 cells/vaccination). Patients were challenged with irradiated, untransduced autologous prostate tumor cells prior to after vaccination to examine delayed type hypersensitivity (DTH) response. The treatment was associated with minimal toxicity. Most of the adverse events were expected and included skin reactions (injection site pain, erythema, swelling, pruritis) and flu-like symptoms (mild low-grade fevers, chills, malaise). All patients demonstrated inflammatory cell infiltrates at the vaccination sites. Immune infiltrates largely comprised neutrophils and eosinophils, which increased significantly relative to prevaccination levels. Eighty-eight percent of patients demonstrated positive DTH tests after vaccination versus 25% prior to vaccination. Postvaccination biopsies were characterized by ingress of macrophages, natural killer cells, T cells, and extensive eosinophilia. Eighty percent of CD3+ T cells expressed CD45RO indicating T cell activation. An increase in antibody titers to prostate tumor antigens were observed in 3 (38%) patients. As expected following prostate resection, all patients demonstrated significant declines in serum PSA. However, all patients ultimately progressed. Owing to the difficulty in obtaining a sufficient number of autologous tumor cells to generate patient-specific tumor vaccines, subsequent trials used “off-the-shelf” allogenic tumor vaccines comprising GM-CSF-secreting human prostate adenocarcinoma cells (GVAX). In a phase 2 study, 34 patients with hormone-refractory, metastatic prostate cancer were vaccinated with a primer dose of 5 × 108 GVAX cells (Simons et  al. 2002; Small et  al. 2007; Higano et  al. 2008). Patients were subsequently administered 12 booster vaccinations every 2 weeks at two dose levels (1 or 3 × 108 cells). At 2-year follow-up, 9 of 22 (41%, two were lost to follow-up) in the

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low-dose cohort, and 7 of 10 (70%) in the high-dose cohort, were alive indicating a trend toward increased survival with the higher dose. There was also a trend toward a longer median time to disease progression (140 vs. 85 days) in the high-dose group as determined by bone scan. A second phase 2 study used a second-generation GVAX vaccine genetically engineered to express higher levels of GM-CSF (Small et  al. 2004). Eighty patients, in three cohorts, with hormone-refractory, metastatic prostate cancer received an escalating dose of GVAX over a 24-week period. Six of 19 (32%) patients in the high-dose group exhibited PSA declines following repeat vaccinations. The proportion of patients demonstrating an antibody response at 12 weeks correlated with the vaccine dose. The majority (62%) of patients tested exhibited stable or reduced osteoclastic activity, which is positive indicator in this setting. Based on these encouraging phase 2 results, two randomized phase 3 trials were conducted in men with hormone-refractory, metastatic prostate cancer comparing the safety and efficacy of GVAX versus docetaxel and prednisone (Vital 1), and GVAX plus docetaxel versus docetaxel and prednisone (Vital 2). Enrollment in Vital 1 (n = 626) was completed. In Vital 2, an imbalance in death was noted between the two treatment arms after enrolling 408 patients triggering early termination of the study. Subsequent analyses show no significant toxicities in the GVAX plus docetaxel arm that could explain the imbalance in deaths. Eighty-five percent of the deaths in both treatment arms were due to prostate cancer, and there was no trend in the causes of death in the remaining patients. The study sponsor speculated that the decision to omit concomitant prednisone in the GVAX immunotherapy treatment arm to avoid the immunosuppressive effects of prednisone may have contributed to an unfavorable outcome compared to the combination of chemotherapy and prednisone. After the termination of Vital 2, follow-up analysis of Vital 1 showed that there was less than a 30% chance of meeting the primary endpoint of improving overall survival and, therefore, this study was terminated also.

4 Replication-Competent, Oncolytic Adenoviruses Strictly speaking, strategies utilizing oncolytic viruses lacking a therapeutic gene are not gene therapy. However, such approaches have been evaluated in the settings of locally recurrent and metastatic disease and have generated encouraging results in the localized setting. The first such trial conducted at the Johns Hopkins Medical Institute used a PSA-selective, oncolytic adenovirus (CV706) in which E1A expression, and therefore virus replication, was driven by a minimal PSA promoter (DeWeese et  al. 2001). This vector design restricts virus replication to PSA-expressing (i.e., prostate) tissues. Twenty patients with locally recurrent prostate cancer received an intraprostatic injection of CV706 up to a dose of 1 × 1013 vp. Despite this high adenovirus dose, the treatment was associated with low toxicity. Ninety-eight percent of the adverse events were mild to moderate. A minority of patients exhibited transaminitis and there was no grade 2 hepatotoxicity. Five of 20 (25%) patients exhibited an

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objective PSA response, which occurred at the two highest adenovirus dose levels suggesting a possible dose effect. One objective response lasted 11 months. A secondary, or delayed, peak of CV706 DNA was detected in patient’s circulation approximately 1 week after the adenovirus injection providing suggestive evidence of viral replication in vivo. Another oncolytic adenovirus (CG7870) related to CV706 was evaluated in the setting of hormone-refractory, metastatic disease (Small et  al. 2006). CG7870 is similar to CV706 except that both the E1A and E1B genes are under the transcriptional control of prostate-specific promoters. In contrast to CV706, CG7870 also contained the immune-modulatory genes of the adenovirus E3 region. Twenty-three patients were administered CG7870 intravenously up to a dose of 6 × 1012 vp. The treatment was well tolerated up to a dose of 3 × 1012 vp. Treatment-related adverse events included mild to moderate flu-like symptoms, hypotension, lymphopenia, thrombocytopenia, and transaminitis. At a dose of 6 × 1012 vp, a constellation of adverse events occurred in two patients halting further dosing. These events included D-dimer formation, a hallmark of disseminated intravascular coagulation, transaminitis and thrombocytopenia. Although none of these events constituted a protocol-defined DLT, accrual was, nevertheless, halted after two patients at this dose level. Five of 23 (22%) patients exhibited a 25–49% decline in PSA, most of which occurred at the higher (>6 × 1011 vp) adenovirus dose levels. Secondary, or delayed, DNA peaks were observed in 70% of patients between days 2 and 8 suggestive of viral replication.

5 Summary So what have we learned after treating over 1,000 prostate cancer patients? Two general principles have emerged, neither of which is surprising. First, when administered carefully and at the appropriate doses, gene therapy for prostate cancer is generally safe. Overall, it has been associated with little toxicity when delivered intraprostatically or systemically. Although the toxicities vary with respect to the approach taken and agent dose, the most common gene therapyrelated side effects include injection site reaction (pain or swelling), flu-like symptoms, transaminitis, and hematologic events. The vast majority of these events are mild to moderate and transient lasting less than a few days. The hepatic and hematologic events generate no noticeable symptoms to the patient and are self-limiting. As with most cancer therapies, the frequency and severity of gene therapy-related side effects increase with agent dose, and systemic approaches tend to be more toxic than local approaches. Importantly, of the strategies evaluated thus far, there do not appear to be any long-term side effects of gene therapy and none have exacerbated the most common side effects of standard prostate cancer treatments. Second, although gene therapy has clearly exhibited antitumor activity when applied as a single agent, its greatest potential may lie in its apparent ability to improve the effectiveness of standard cancer therapies. When delivered intraprostatically in

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combination with prostate radiotherapy, adenovirus-based approaches have resulted in better-than-expected 2-year biopsy results, at least in select patient groups. It is possible that better local tumor control can be achieved across all prognostic risk groups by saturating the prostate gland with the adenovirus through the use of higher adenovirus doses. Although high-risk patients (Gleason ³8, PSA >20 ng/mL) will still require some form of systemic therapy (i.e., AST) to combat covert extraprostatic disease, here, too, the gene therapy may provide some benefit. Multiple studies have reported a slowing of disease progression (i.e., lengthening of PSADT) following the administration of gene therapy raising the possibility of an antitumor immune effect. Thus, gene therapy appears to have the potential to impact two clinical endpoints (2-year biopsy status and PSADT) that are highly prognostic for disease progression and prostate cancer-specific mortality. In advanced disease settings, poxvirus-based PSA vaccines have been shown to augment the effectiveness of subsequent chemotherapy and may have the potential to extend survival. Although the underlying basis for this effect is unknown, it is welcome news for prostate cancer is generally resistant to chemotherapy. The best available chemotherapeutic drug (Taxotere) extends survival by only 2–3 months. The poxvirus trials add to the growing body of evidence that prostate cancer may be responsive to some forms of immunotherapy. Together, we consider these preliminary results very encouraging and continue to believe that gene therapy will someday earn a place in the management of prostate cancer when the right approach is applied in the right setting. Whether any of these gene therapy strategies is robust enough to extend survival awaits the completion of prospective, randomized, controlled trials. In the meantime, it is important that we gene therapists come “full circle” and take our clinical observations back into the laboratory to better understand the molecular basis for these provocative preliminary findings.

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Kim JH, Kim S, Brown S, and Freytag S. (1994). Selective enhancement by an antiviral agent of the radiation-induced cell killing of human glioma cells transduced with HSV-tk gene. Cancer Res 54: 6053–6056. Kim JH, Kim SH, Kolozsvary A, Brown S, Kim O, Freytag S. (1995). Selective enhancement of radiation response of herpes simplex virus thymidine kinase transduced 9L gliosarcoma cells in vitro and in vivo by antiviral agents. Int J Radiat Oncol Biol Phys 33: 861–868. Kubo H, Gardner T, Wada Y, Koeneman K, Gotoh G. (2003). Phase I dose escalation clinical trial of adenovirus vector carrying osteocalcin promoter-driven herpes simplex virus thymidine kinase in localized and metastatic hormone-refractory prostate cancer. Hum Gene Ther 14: 227–241. Lattouf J, Arlen P, Pinto P, and Gulley J. (2006). A phase I feasibility study of an intraprostatic prostate-specific antigen-based vaccine in patients with prostate cancer with local failure after radiation therapy or clinical progression on androgen-deprivation therapy in the absence of local definitive therapy. Clin Genitourin Cancer 5: 89–92. Lechleider R, Arlen P, Tsang K, Steinberg S, Yokokawa J, Cereda V, et  al. (2008). Safety and immunologic response of a viral vaccine to prostate-specific antigen in combination with radiation therapy when metronomic-dose interleukin 2 is used as an adjuvant. Clin Cancer Res 14: 5284–5291. Madan R, Gulley J, Schlom J, Steinberg S, Liewehr D, Dahut W, et al. (2008). Analysis of overall survival in patients with non-metastatic castration-resistant prostate cancer treated with vaccine, nilutamide, and combination therapy. Clin Cancer Res 14: 4526–4531. Miles B, Shalev M, Aguilar-Cordova E, Timme T, Lee H, Yang G, et al. (2001). Prostate-specific antigen response and systemic T cell activation after in situ gene therapy in prostate cancer patients failing radiotherapy. Hum Gene Ther 12: 1955–1967. Paola R, Plante M, Kaufman H, Petrylak D, Israeli R, Lattime E, et al. (2006). A phase I trial of pox PSA vaccines (PROSTVAC-VF) with B7-1, ICAM-1, and LFA-3 co-stimulatory molecules (TRICOM) in patients with prostate cancer. J Transl Med 4: 1–5. Patel P, Young J, Mautner V, Ashdown D, Bonney S, Pineda R, et al. (2009). A phase I/II clinical trial in localized prostate cancer of an adenovirus expressing nitroreductase with CB1954. Mol Ther 17: 1292–1299. Rogulski K, Kim JH, Kim SH, Freytag S. (1997). Glioma cells transduced with an E. coli CD/ HSV-1 TK fusion gene exhibit enhanced metabolic suicide and radiosensitivity. Hum Gene Ther 8: 73–85. Rogulski K, Wing M, Paielli D, Gilbert J, Kim JH, Freytag, S. (2000). Double suicide gene therapy augments the therapeutic efficacy of an oncolytic adenovirus through enhanced cytotoxicity and radiosensitization. Hum Gene Ther 11: 67–76. Sanda M, Smith D, Charles L, Hwang C, Pienta K, Schlom J, et al. (1999). Recombinant vacciniaPSA (PROSTVAC) can induce a prostate-specific immune response in androgen-modulated human prostate cancer. Urology 53: 260–266. Sandler H, Dunn R, McLaughlin P, Hayman J, Sullivan M, and Taylor J. (2000). Overall survival after prostate-specific-antigen-detected recurrence following conformal radiation therapy. Int J Radiat Oncol Biol Phys 48: 629–633. Satoh T, Teh B, Timme T, Mai W, Gdor Y, Kusaka N, et al. (2004). Enhanced systemic T-cell activation after in situ gene therapy with radiotherapy in prostate cancer patients. Int J Radiat Oncol Biol Phys 59: 562–571. Simons J, Mikhak B, Chang J-F, DeMarzo A, Carducci M, Lim M, et  al. (1999). Induction of immunity to prostate cancer antigens: results of a clinical trial of vaccination with irradiated autologous prostate tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor using ex vivo gene transfer. Cancer Res 59: 5160–5168. Simons J, Nelson W, Nemuniatis J, Centeno A, Dula E, Urba W, et al. (2002). Phase II trials of a GMCSF gene-transduced prostate cancer cell line vaccine (GVAX) in hormone refractory prostate cancer. Proc Am Soc Clin Oncol 22: 172.

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Small E, Higano C, Smith D, Corman J, Centero A, Streidle C, et al. (2004). A phase 2 study of an allogeneic GM-CSF gene-transduced prostate cancer cell line vaccine in patients with metastatic hormone-refractory prostate cancer (HRPC). J Clin Oncol 22: 4565. Small E, Carducci M, Burke J, Rodriguez R, Fong L, van Ummersen L, et al. (2006). A phase I trial of intravenous CG7870, a replication-selective, prostate-specific antigen-targeted oncolytic adenovirus, for the treatment of hormone-refractory, metastatic prostate cancer. Mol Ther 14: 107–117. Small E, Sacks N, Nemunaitis J, Urba W, Dula E, Centeno A, Nelson W, Ando W, Howard C, Borellini F, Nguyen M, Hege K, Simons J. (2007). Granulocyte macrophage colony-stimulating factor-secreting allogenic cellular immunotheraphy for hormone-refractory prostate cancer. Clin Cancer Res 13: 3883–3891. Takamiya Y, Short M, Ezzeddine Z, Moolten F, Breakefield X, and Martuza R. (1992). Gene therapy of malignant brain tumors: a rat glioma line bearing the herpes simplex virus type 1-thymidine kinase gene and wild type retrovirus kills other tumor cells. J Neurosci Res 33: 493–503. Teh B, Aguilar-Cordova E, Kernen K, Chou C, Shalev M, Vlachaki M, et al. (2001). Phase I/II trial evaluating combined radiotherapy and in situ gene therapy with or without hormonal therapy in the treatment of prostate cancer- a preliminary report. Int J Radiat Oncol Biol Phys 51: 605–613. Teh B, Ayala G, Aguilar L, Mai W, Timme T, Vlachaki M, et al. (2004). Phase I-II trial evaluating combined intensity-modulated radiotherapy and in situ gene therapy with or without hormonal therapy in treatment of prostate cancer-interim report on PSA response and biopsy data. Int J Radiat Oncol Biol Phys 58: 1520–1529. Zelefsky M, Eastman J, Sartor O, and Kantoff P. (2008a). Cancer of the prostate, In DeVita V., Lawrence, T., Rosenberg, S. (eds): Cancer: Principles & Practice of Oncology, Philadelphia, JB Lippincott, pp 1392–1452. Zelefsky M, Reuter V, Fuks Z, Scardino P, and Shippy A. (2008b). Influence of local tumor control on distant metastases and cancer related mortality after external beam radiotherapy for prostate cancer. J Urol 179: 1368–1373.

Chapter 4

siRNA Versus shRNA for Personalized Cancer Therapy: Mechanisms and Applications John S. Vorhies, Donald D. Rao, Neil Senzer, and John Nemunaitis

Abstract  RNA interference (RNAi) is a natural process of gene regulation that can be harnessed to knock down gene and protein targets with high specificity and selectivity. Proteomic and genomic approaches to target identification will soon allow investigators to rapidly indentify biorelevant cancer signal transduction network hubs that are more likely to be susceptible to a therapeutically effective targeted attack by RNAi. At present, the principle methods of mediating the RNAi effect involve synthetic small interfering RNA (siRNA) oligomers and DNA vector driven expression of short hairpin RNA (shRNA). Both these methods can achieve robust and specific knockdown, but they have striking mechanistic differences with broad practical implications. shRNA can effectively target knockdown with low copy numbers and longer lasting effects than siRNA. Bifunctional design has similar benefits to standard shRNA but with greatly enhanced potency. Effective delivery and avoidance of unwanted off-target effects remain as challenges to the clinical development of siRNA and shRNA. This chapter compares and contrasts siRNA, shRNA, and bifunctional shRNA as candidates for personalized solid tumor therapeutics. Keywords  RNAi • Personalized • Cancer therapeutic

J. Nemunaitis (*) Gradalis, Inc., Dallas, TX, USA and Mary Crowley Cancer Research Centers, Dallas, TX, USA and Texas Oncology PA, Dallas, TX, USA and Baylor Sammons Cancer Center, Dallas, TX, USA e-mail: [email protected] J.A. Roth (ed.), Gene-Based Therapies for Cancer, Current Cancer Research, DOI 10.1007/978-1-4419-6102-0_4, © Springer Science+Business Media, LLC 2010

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1 Introduction Refinement of our basic understanding of cancer mechanisms is now shifting ­treatment development from traditional, broadly cytotoxic chemotherapy to more selective approaches. Tumors have long been grouped into clinical and histological subtypes, but significant variation in response to treatment still exists within these subtypes. The underlying variation in tumor gene expression patterns may explain the widely divergent responses to treatment regimens most often prescribed by histological type. Molecular subtyping has the potential to further refine patient treatment groups and to improve treatment outcomes, thereby, establishing a “so-called” personalized medicine approach. As already efficient high-throughput methods are accelerated, the possibility of personalized medicine is becoming a reality. The ultimate goal of personalized therapy is to make the drug development process, from target identification to treatment, feasible in a timescale relevant to a single patient. This would allow physicians to consider each patient’s tumor as a subtype of its own, characterizing it and delivering appropriate treatment. RNA interference (RNAi) is an evolutionarily conserved gene-silencing mechanism that occurs endogenously when small sequences of double stranded RNA, termed microRNA, suppress the translation of partially complementary posttranscriptional mRNA. When exogenously induced, it can be a powerful mechanism for targeted knockdown of over- or constitutively expressed molecular targets. It also has significant practical advantages over small molecules and antibodies in terms of production, potency, and specificity. RNAi is young as a potential therapeutic modality and there are several candidate mechanisms for inducing it. The two primary modes for inducing RNAi are through the introduction of chemically synthesized double-stranded oligomers, called small interfering RNA (siRNA) or through the introduction of a DNA vector, which expresses a short hairpin RNA (shRNA) within the target cells. These two modes have important mechanistic advantages and disadvantages relevant in terms of clinical efficacy, durability, off-target effects, and delivery (Rao et al. 2009).

2 Personalized Cancer Therapy Signal transduction networks in cancer are quite robust to random individual gene/ protein target knockout owing to the presence of functional redundancy and a scalefree interaction topology. Random pathway component failure predominantly affects targets with low connectivity within the network, thereby having limited functional impact. Highly connected information-transfer nodes are particularly vulnerable to attack and constitute weak points in the network. This property is exacerbated in cancer because oncogenic change tends to make cells more highly dependent on a specific rewired pathway (Letai 2008). Exploiting the vulnerability that such pathway dependence creates is useful for its lethality to cancer cells and its decreased

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likelihood of perturbing normal cell function. Personalized RNAi-based therapeutics­ are particularly well suited to take advantage of these mechanisms. Our group is currently developing a model for personalized RNAi-based therapy: we harvested tumor and normal cells from cancer patients, comparing expression profiles for malignant versus normal tissue at the mRNA and protein level by microarray and proteomic analysis (Nemunaitis et al. 2007). The resulting expression data was further analyzed by a computational system developed by our team specifically for clinical application including, but not limited to, gene set enrichment analysis and network inference modeling platforms. This allowed us to prioritize overexpressed potential targets based on their probability of being highly connected, nonredundant points in the network. This individualized target fingerprint then served as the template for the design, synthesis, and validation of individualized therapeutic RNAi molecules with knockdown activity against these targets.

3 Mechanisms of RNAi Whether induced by shRNA or siRNA, the RNAi silencing process, as it is currently understood, converges into a final common set of pathways mediated through short (19–23  bp) oligomers of duplex RNA with 2–3 nt 3¢ overhangs on each strand. The two strands of the duplex are termed the guide (antisense) strand, which is complementary to the target mRNA sequence, and the passenger (sense) strand, which may be completely complementary to the guide strand or it may contain mismatches. Figure 1 summarizes the main entry points into this pathway by exogenous RNAi as well as the end-mechanisms of silencing.

3.1 siRNA Effective exogenous induction of RNAi was initially demonstrated by the application of RNA oligomers (Fire et al. 1998). Once in the cytoplasm, siRNA associates with several proteins that make up the RNA-interfering silencing complex (RISC). Depending on various factors, including the duplex mismatching and the nature of the RISC, RNAi can proceed through “cleavage dependent” or “cleavage independent” pathways. The major component of the RISC is the argonaute family of proteins (Ago1, Ago2, Ago3, and Ago4). Within this family, only Ago2 contains the endonuclease activity. The remaining three members of Argonaute family do not have identifiable endonuclease activity, and presumably function through a cleavageindependent manner (Farazi et al. 2008; Paroo et al. 2007). During RISC assembly in the cleavage dependent mechanism, the passenger strand is cleaved by the RNase H like activity of Ago2 and, provided thermodynamically favorable conditions,

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Fig. 1  Schematic of the cytoplasmic siRNA and shRNA mediated RNAi pathways. siRNA and shRNA are introduced in different ways, but they converge on a common set of pathways in the cytoplasm

the two strands of the duplex are separated. The RISC then scans mRNAs for target sites to which it binds and Ago2 cleaves the mRNA at a single site between nucleotides10 and 11 from the 5¢ end of the guide strand, thereby initiating degradation. The RISC can then dissociate and execute multiple rounds of RNAi (Paroo et al. 2007). If there are mismatches in the duplex RNA, a different, cleavage-independent RISC is assembled that lacks Ago2 endonuclease capacity. During the assembly of the cleavage-independent RISC, the passenger strand is induced to unwind and be

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released by an ATP-dependent helicase. RISCs without endonucleolytic activity scan mRNAs and predominantly bind to partially complementary target sites located at the 3¢ UTR, repressing translation through mRNA sequestration in processing bodies (p-bodies). Phosphorylation of Ago 2 on Serine-387 seems to affect its localization to p-bodies. The functional implications of this are still unknown, but it may repr­ esent an important regulatory step in RNAi via p-body sequestration. The exogenously applied RNAi constructs can be designed to participate in either or both pathways (Grimm 2009; Paroo et al. 2007). Synthetic siRNA enters into the RNAi pathway at the stage of RISC assembly, but if the oligomer is longer than 19–23 bp, it requires processing by a multidomain RNase III-related endonuclease called Dicer before being loaded onto the RISC. Dicer preferentially binds to the 5¢ phosphate of 2 nt 3¢ over-hang and cleaves double-stranded RNA into 21 to 22 nucleotide siRNAs. It also forms an integral component of endogenous RNAi, processing pre-microRNA to mature miRNA and transferring the processed products to the RISC (Macrae et al. 2006; Carmell and Hannon 2004)

3.2 shRNA Several years after exogenous RNAi was discovered, it was shown that RNAi could be induced by the in vitro transcription of shRNA using a T7 RNA polymerase or a U6 promoter on a plasmid construct (Yu et al. 2002; Miyagishi and Taira 2002). shRNAs, unlike siRNAs, are synthesized in the nucleus of cells similarly to miRNA. Thus, studies on the biogenesis of miRNAs have provided the groundwork for understanding the synthesis and maturation of shRNA. shRNA is introduced as a DNA vector encoding an hairpin-like stem-loop structure. Once transcribed in the nucleus, if integrated into a miR30 scaffold, the hairpin containing the pre-shRNA-like construct is processed to a pre-shRNA by a complex containing the RNase III enzyme Drosha and the double-stranded RNA binding domain protein DGCR8 (Fig. 1). The complex measures the hairpin and allows precise processing of the long primary transcripts into individual shRNAs with a 2 nt 3¢ overhang. This processed primary transcript or an exogenous pol IIIbased stem-loop structure is then transported to the cytoplasm by Exportin V via a Ran-GTP-dependent mechanism (Grimm 2009). In the cytoplasm, the pre-shRNA undergoes a dicer-mediated endonucleolytic cleavage step, in which the loop of the hairpin is processed off to form a doublestranded siRNA with 2 nt 3¢ overhangs. Dicer interacts with the double-stranded Tat-RNA-binding protein (TRBP) or PACT (PKR-activating protein) to mediate siRNA production from shRNA (Pillai et al. 2007; Paroo et al. 2007). The activity of exogenous siRNA, unlike shRNA, does not depend on the TRBP/PACT/Dicer complex. After this last processing step, the Dicer-containing complex coordinates loading onto the RISC. Once on the RISC, shRNA and siRNA should follow the same pathways.

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3.2.1 Bifunctional shRNA The concept of bifunctional shRNA rests on the hypothesis that an shRNA ­construct can be designed to utilize both cleavage-dependent and cleavage-independent RISCs. The design of the bifunctional shRNA expression unit consists of two stemloop shRNA structures; one stem-loop structure composed of a fully matched passenger and guide strand duplex for cleavage-dependent RISC loading, the second stem-loop structure composed of a mismatched passenger strand (at the position 9–12) and guide strand for cleavage-independent RISC loading (Fig. 2). Simultaneous expression of both cleavage-dependant and cleavage-independent shRNAs in cells should achieve a higher level of efficacy, greater durability, and more rapid onset than either siRNA or standard shRNA. Multitarget shRNA expression systems have been validated using in vitro (Cheng et al. 2009) cancer systems. There also exists evidence to suggest that this type of functional redundancy is active within the endogenous RNAi system as well. Most mRNAs have multiple miRNA target sites, which allow for cooperative downregulation. In vitro data also suggests that miRNA sequences with the same target and even the same sequence

Fig.  2  Schematic of the bifunctional shRNA Vector design and mechanism. A construct that encodes two shRNAs for each targeted mRNA promotes translation repression through both cleavage-dependent and cleavage-independent RISCs

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naturally associate with different RISCs in  vivo (Azuma-Mukai et  al. 2008; Landthaler et al. 2008).

4 SiRNA Versus shRNA 4.1 Comparative Efficacy shRNA generally has higher efficacy than siRNA when directed to the same target and in vitro. McCleary and colleagues tested shRNA and siRNA directed against firefly luciferase in HeLa cells. More effective inhibition was seen with the shRNA. In an Hepatitis-C virus (HCV) model, 19 and 25 bp shRNAs were compared with 19 and 25 bp siRNA directed against the HCV internal ribosomal entry site using a luciferase reporter in the AVA5 cell line with stable expression (Vlassov et  al. 2007). Both the shRNAs were more potent than either of the 19- or 25-bp siRNAs used. Takahashi et al. (2009) compared a luciferase-directed shRNA driven by different promoters to siRNA of the same sequence in melanoma cells and found that shRNA driven by a U6 promoter was at least 100-fold more potent and longer lasting than siRNA. Comparison of siRNA and shRNA in vivo is difficult as equivalency of strand biasing may not be assured. Several studies have used luciferase reporter systems to quantify siRNA versus shRNA potency in vivo. McAnuff and colleagues (2007) found that siRNA and shRNA are equivalent in potency at 10 mg dose; however, on a molar basis, the shRNA was 250-fold more effective than the siRNA. In a murine HCV model siRNA and shRNA constructs were directed against the nonstructural protein 5B viral polymerase coding region fused with a luciferase gene. siRNA resulted in a 75% expression reduction while shRNA produced a 92.8% average reduction over three experiments (McCaffrey et al. 2002).

4.2 Dicer/Drosha Expression in Cancer and RNAi Effector Suitability Low levels of Dicer and Drosha have been found in tumor samples from patients with ovarian cancer and breast cancer (Merritt et al. 2008; Grelier et al. 2009). In one of these studies (Merritt et al. 2008), shRNA was found to be less effective than siRNA in cells with low Dicer expression. These expression findings stand in contrast to other studies including those that have noted either no downregulation or upregulation of both Drosha and Dicer in ovarian tumors (Lin Zhang et al. 2008; Flavin et  al. 2008). Further investigations of Dicer/Drosha expression in human cancers are needed, but these findings raise the possibility that, at least in some cases, the clinical efficacy of shRNA may be affected by the expression patterns of endogenous miRNA processing machinery (Rao et al. in press).

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4.3 Off-Target Effects There are multiple specific and nonspecific mechanisms through which siRNA and shRNA can cause effects other than the intended mRNA suppression. Specific offtarget effects are mediated by partial sequence complementarity of the RNAi construct to mRNAs other than the intended target. Nonspecific off-target effects include a wide variety of immune- and toxicity-related effects that are intrinsic to the RNAi construct itself or its delivery vehicle.

4.3.1 Specific Off-Target Effects In vitro, siRNA creates off-target expression patterns that are unique and consistent for a given sequence. They also appear to be unrelated to target knockdown (Jackson et al. 2003). Complementarity of the mRNA 3¢UTR with nucleotides 2–7 at the 5¢ end of either the siRNA passenger or guide strands has been shown to be a key determinant in directing off-target effects. This is reminiscent of the “seed” region within miRNA, which guides silencing through complementarity with the 3¢UTR of an mRNA (Birmingham et al. 2006). Although sequence optimization to reduce specific off-target effects will benefit both siRNA and shRNA discriminative functionality, unlike shRNA, siRNA oligomers can be chemically modified to reduce direct off-target effects. Various modifications can encourage preferential strand selection, limit the construct’s association with a certain class of RISC, or discourage seed region complementarity based offtarget effects (Behlke 2008). shRNA seems to cause fewer specific off-target effects than siRNA, potentially because of its use of endogenous processing and regulatory mechanisms (Rao et al. in press). The susceptibility of siRNA to cytoplasmic degradation may also lead to more off-target effects. In one study, shRNA and siRNA of the same core sequence directed toward TP53 were applied to HCT-116 colon carcinoma cells in concentrations necessary to achieve comparable levels of target knockdown. Microarray profiling demonstrated a much higher degree of up- and downregulation of off-target transcripts in the siRNA transfected cells (M. Mehaffey, T. Ward, and M. Cleary, in prep.).

4.3.2 Nonspecific Off-Target Effects Activation of the innate immune system in the case of exogenous RNAi is likely mediated through cytoplasmic and endosomal mechanisms attuned to recognize exogenous nucleic acids from infectious agents. Introduction of dsRNA longer than 29–30  bp into mammalian cells activates receptors sensitive to exogenous nucleic acids, such as Toll-Like Receptors (TLR), and induces the innate immune system, leading to global degradation of mRNA and upregulation of interferon (IFN)-stimulated gene expression. Though siRNA constructs are less immunogenic

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than longer dsRNA, both siRNA and shRNA can induce a partial IFN response (Robbins et al. 2006). Misinterpreting an immunologic effect of siRNA as a direct effect must be carefully avoided, as naked siRNA has been shown to activate the RNA-sensitive TLR-3 on the surface of vascular endothelial cells, triggering the release of IFN-g and IL-12 that mediate nonspecific antiangiogenic effects in vivo (Kleinman et al. 2008). Activation of TLR 3 is not an issue for shRNA because the construct is presented on a DNA vector. However, TLR 9 is present in the endosome and is activated by unmethylated DNA CpG motifs, necessitating careful plasmid design to avoid immunoactivation (Robbins et al. 2009). Sequence and chemical modification of siRNA (particularly the 2¢ site) can attenuate the immune response (Robbins et al. 2009). shRNA is less likely to induce an inflammatory response through cytoplasmic dsRNA receptors because it is spliced by endogenous mechanisms. In an experiment that compared liposomedelivered siRNA and shRNA in primary CD34+ progenitor-derived hematopoietic cells, it was shown that siRNA induced IFN-alpha and type I IFN genes, while the shRNA of the same sequence did not induce an immune response (Grimm and Kay 2007). Another study showed that modifying an shRNA by integration within an miR-30 scaffold could also decrease the IFN response (Bauer et al. 2009). Over-saturation of nuclear membrane Exportin V and Ago2 by shRNA (particularly at high concentrations) can cause dose-dependent liver injury as a result of downregulation of critical endogenous miRNAs, which rely on the same proteins (Grimm 2009). siRNA avoids this problem and can achieve suppression of a target gene without disrupting endogenous miRNA levels (John et  al. 2007). The oversaturation effect may be promoter related, as stable target gene suppression was subsequently demonstrated at high shRNA doses in a murine model for over one using a pol II promoter system (Grimm 2009). This indicates that selective promoter integration and careful dosing of shRNA is needed to avoid competitive inhibition of the endogenous miRNA biogenesis machinery.

5 Delivery Strategies for Clinical Translation Clinical efficacy of an RNAi cancer therapeutic is limited by the properties of its delivery vehicle. In the case of siRNA, knockdown is directly related to the quantity of the oligomer that enters the tumor cells, whereas in the case of shRNA, the expression vector must reach the nucleus for gene silencing to be achieved. Issues of safety, selective tumor targeting, pharmacokinetics, and pharmacodynamics are also affected by the delivery vehicle. These include resisting host defenses, reaching­ the tumor while avoiding normal tissue, negotiating cell penetration, and, when apropos, endocytosis then endosomal/lysosomal escape and, in the case of shRNA, penetration of the nuclear membrane. Viral vectors have received some clinical attention but concerns over efficient systemic delivery and immunogenicity may limit their clinical utility.

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There are three major classes of nonviral delivery vehicle systems: synthetic polymers, natural/biodegradable polymers, and lipids (Vorhies and Nemunaitis 2009; Vorhies and Nemunaitis 2007; Whitehead et  al. 2009). Hybrids of these can be effective. For instance, there is a cyclodextrin-based cationic polymer which has been used successfully to deliver siRNA targeted to RRM2 in various in vivo cancer models. The same formulation, now called CALAA-01 is currently in Phase I clinical trials (Heidel et al. 2007). Lipid-based nanoparticles are also showing potential for clinical delivery of shRNA and siRNA. Silence Therapeutics has developed a lipid-based delivery vehicle specifically designed for the delivery of siRNA Targeting Protein Kinase N3 endothelial cells. This vehicle, called AtuPLEX, contains a mix of cationic and fusogenic lipids (Aleku et al. 2008). A Phase I trial is currently recruiting to investigate an siRNA therapeutic delivered with AtuPLEX.

6 Conclusions Both siRNA and shRNA have excellent potential for clinical use within the emerging­ paradigm of scale-free biomolecular networks and their inherent integration of evolution and structure. However, several challenges remain that warrant further study at both the preclinical and clinical levels. Mechanisms influencing RNAi silencing efficacy, off-target effects, and delivery are crucial areas for further study, preclinical assessment and clinical translation. The transient effect of siRNA may be more suited to the treatment of infectious disease whereas the heightened potency and temporal and spatial control of shRNA may better suit it for the systemic treatment of malignancy. Finally, bi-shRNA represents an important therapeutic development which, by exploiting the latest advances in our understanding of RNAi mechanisms, may bring us closer to an optimized personalized cancer therapy.

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Chapter 5

Tumor Suppressor Gene Therapy Jack A. Roth, John Nemunaitis, Lin Ji, and Rajagopal Ramesh

Abstract  Recent advances in genetics, molecular biology, and molecular pharmacology have resulted in the development of molecularly targeted therapies. Targeting specific molecular pathways essential for the survival of cancer cells would personalize treatment with the potential to improve outcome and minimize toxicities. In this chapter, we review gene-based targeted therapies for cancer. Discussion focuses on replacement therapies for abnormal p53 function and FUS1 tumor suppressor gene-mediated molecular therapy using nanoparticles for systemic gene delivery. Keywords  Gene • Molecular • Tumor suppressor • Cancer therapy The management of many common cancers has changed through the years with the development of targeted drugs such as angiogenesis and tyrosine kinase inhibitors. However, despite these recent advances, metastatic disease patients receiving frontline treatment with chemotherapy alone or in combination with angiogenesis and tyrosine kinase inhibitors have had only an incremental improvement in survival. The cancer cell has developed six alterations which contribute to malignant growth. These include self sufficiency, insensitivity to growth inhibition (including immune “escape”), independence from programmed cell death, unlimited replicative potential, sustained angiogenesis, and local and metastatic invasiveness (Hanahan and Weinberg 2000). In many instances, these alterations occur because of the inactivation of tumor suppressor genes which may regulate multiple pathways. Targeting a single pathway does not often lead to a robust therapeutic effect because the cancer cell is capable of maintaining its functional characteristics through dynamic feedback loops (Carlson and Doyle 2002; Stelling et al. 2004).

J.A. Roth (*) Anderson Cancer Center, The University of Texas M.D, Houston, TX, USA e-mail: [email protected] J.A. Roth (ed.), Gene-Based Therapies for Cancer, Current Cancer Research, DOI 10.1007/978-1-4419-6102-0_5, © Springer Science+Business Media, LLC 2010

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Cancer cells can survive targeted inhibition of growth factor signaling pathways by virtue of having redundant functional pathways in which different proteins have overlapping functions (Edelman and Gally 2001). Positive and negative feedback controls may allow cancer cells to bypass the growth inhibitory effects of single pathway blockade. Efforts to improve therapeutic outcomes have focused on innovative approaches involving gene replacement. Theoretically, restoration of single tumor suppressor gene function could restore pathway functions that block several of the alterations in malignant cells. The purpose of this chapter is to summarize key tumor suppressor gene replacement strategies.

1 Tumor Suppressor Gene Therapy The genetic basis of cancer is well established. Tobacco smoke, for example, contains more than 100 carcinogens that can damage DNA (Denissenko et al. 1996). Lung cancers have multiple genetic lesions which can be detected in histologically normal bronchial mucosa and premalignant lesions from individuals with history of tobacco consumption. The p53 tumor suppressor gene is mutated in over 50% of lung cancers and other epithelial cancers (Olivier et al. 2009). The p53 tumor suppressor gene appears to contribute to cancer development when it is inactivated and was the initial target of gene therapy approaches to cancer. The p53 protein monitors cellular stress and DNA damage, inducing either growth arrest to facilitate DNA repair or apoptosis (programmed cell death) if DNA damage is extensive (Burns and El-Deiry 1999). When a cell is stressed by oncogene activation, hypoxia, or DNA damage, a functioning p53 pathway may determine whether the cell will receive a signal to arrest at the G1 stage of the cell cycle, whether DNA repair will be attempted, or whether the cell will self-destruct via apoptosis. The observation that expression of a wild-type p53 gene in a cancer cell induces apoptosis provided the rationale for p53 gene therapy replacement to restore a functioning p53 pathway (Fujiwara et al. 1993). One consideration was that if gene therapy could not replace all the damaged genes in a cancer cell, there would be no therapeutic benefit. The observation that restoration of only one of the defective genes is enough to trigger apoptosis suggests that the DNA damage present in a cancer cell may prime it for an apoptotic event. It is important to note that p53 regulates many cell survival pathways and thus restoring a single gene restores the function of multiple pathways. The p53 gene product is a transcription factor that regulates many pathways including apoptosis and cell cycle progression (Raycroft et  al. 1990). p53 also downregulates the prosurvival (or antiapoptotic) genes, including the antiapoptotic genes bcl-2 and bcl-XL, and upregulates the proapoptotic genes bax, bad, bid, puma, and noxa (Adams and Cory 1998). Available transcripts of each of the pro and antiapoptotic genes with bcl2 homology-3 domains interact with one another to form heterodimers, and the relative ratio of proapoptotic to prosurvival proteins in these heterodimers determines the activity of the resulting molecule, thereby

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determining whether the cell lives or undergoes apoptosis. p53 also targets the death-receptor signaling pathway, including DR5 and Fas/CD95, and the apoptosis machinery, including caspase-6, Apaf-1, and PIDD. It also may directly mediate cytochrome c release. Regulation of the p53 pathway at the protein level is mediated by other tumor suppressor genes and by several oncogenes (Burns and El-Deiry 1999). The mdm2 protein normally binds to the N-terminal transactivating domain of p53, which prevents p53 activation and leads to its rapid degradation. In normal cells, mdm2 is inhibited by the expression of p14ARF, a tumor suppressor gene encoded by the same gene locus as p16INK4a expressed as an alternate reading frame (Kamijo et al. 1997). Deletion or mutation of the tumor suppressor gene p14ARF occurs in some cancers and results in increased levels of mdm2, which results in the inactivation of p53 causing inappropriate progression through the cell cycle. The expression of p14ARF is induced by hyperproliferative signals from oncogenes such as ras and myc. The p53 protein can mediate cell cycle arrest. This function is significant, as prolonged tumor stability has often been observed in clinical trials of p53 gene replacement, suggesting that this effect may be predominant over apoptosis in some tumors. p53 is involved in regulating cell cycle checkpoints, and p53 expression can promote cell senescence through its control of cell cycle effectors such as p21CIP1/WAF1. Loss of function in the p53 pathway is the most common alteration identified in human cancers at the present time. About 50% of common epithelial cancers have p53 mutations (Isobe et al. 1994; Martin et al. 1992; Quinlan et al. 1992). In some cancers, loss of p53 also appears to be linked to resistance to conventional DNA damaging therapies, including chemotherapy and ionizing radiation, which require apoptosis to cause cell death.

2 Gene Replacement by p53 in Laboratory Studies The findings described above suggest that expressing a wild-type p53 gene in cancer cells defective in p53 function could mediate either apoptosis or cell growth arrest, both of which would be of therapeutic benefit to a cancer patient. Initial studies showed that restoration of functional p53 using a retroviral vector suppressed the growth of some, but not all, human lung cancer cell lines (Cai et  al. 1993). The first published study of p53 gene therapy showed suppression of tumor growth in an orthotopic human lung cancer model using a retroviral expression vector (Fujiwara et al. 1994a). This was the first study to show that restoring the function of a single tumor suppressor gene could result in the regression of human cancer cells in vivo. Because of the limitations inherent in the use of retroviruses, subsequent studies of p53 gene replacement in lung cancer made use of an adenoviral vector (Ad-p53) (Zhang et al. 1994). The original adenoviral vector was a serotype five replicationdefective vector with a deleted E1 region, which has been used in all p53 clinical trials. Ad-p53 also induced apoptosis in cancer cells with nonfunctional p53 without

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significantly affecting the proliferation of normal cells (Wang et  al. 1995). Subsequent studies with Ad-p53 demonstrated inhibition of tumor growth in a mouse model of human orthotopic lung cancer (Georges et al. 1993) and induction of apoptosis and suppression of proliferation in various other cancer cell lines and in  vivo mouse xenograft tumor models (Bouvet et  al. 1998; Nielsen et  al. 1997; Spitz et al. 1996). Bystander killing (killing of nontransduced cells by transduced cells), now known to be an important phenomenon for the success of gene therapy, appears to involve regulation of angiogenesis (Dameron et al. 1994; Miyashita and Reed 1995), immune upregulation (Carroll et  al. 2001; Molinier-Frenkel et  al. 2000; Yen et  al. 2000), and secretion of soluble proapoptotic proteins (OwenSchaub et al. 1995).

3 Clinical Trials of p53 Gene Replacement Initial preclinical studies used a retrovirus to deliver the p53 gene. The first clinical trial for p53 gene-replacement utilized a replication-defective retroviral vector expressing wild-type p53 driven by a beta-actin promoter (Roth et al. 1996). The retrovirus vector was injected directly into tumors of nine patients with unresectable NSCLC that had progressed after conventional therapy. Three of the nine patients showed evidence of tumor regression. There was no vector-related toxicity, demonstrating the feasibility, safety, and antitumor activity of p53 gene therapy. Following the initial p53 retrovirus vector clinical trial, p53 clinical trials were conducted with the adenovirus p53 vector because of the ease of production, high transduction efficiency, and stability of adenovirus vectors. A phase I trial enrolled 28 NSCLC patients whose cancers had not responded to conventional treatments. Successful gene transfer was demonstrated in 80% of evaluable patients (Swisher et al. 1999). Expression of p53 was detected in 46% of patients, apoptosis was seen in all but one of the patients expressing the gene, and, importantly, no significant toxicity was observed. More than a 50% reduction in tumor size was observed in two patients, with one patient remaining free of tumor for more than a year after concluding therapy and another experiencing nearly complete regression of a chemotherapyand radiation-resistant upper lobe endobronchial tumor. Additional studies in patients with head and neck cancer evaluated Ad-p53 gene transfer as a clinically feasible strategy and showed successful gene transfer and gene expression, low toxicity, and evidence of durable tumor regression (Fig.  1). Clayman and coworkers (1998) reported 33 patients with refractory head and neck cancer treated with adenovirus p53. Li-Fraumeni syndrome (LFS) is an autosomal dominant genetic disease of the p53 gene that dramatically increases the risk of developing multiple primary cancers of differing histologies. The majority of LFS families contain a germ line mutation in the p53 tumor suppressor gene. Senzer and coworkers (2007) described the treatment of a refractory, progressive LFS embryonal carcinoma with adenovirus p53 gene therapy thus targeting the underlying molecular defect of LFS. Treatment with adenovirus p53 resulted in a complete and durable remission of the injected lesion

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Fig.  1  Durable complete response following adenovirus p53 monotherapy in a patient with refractory head and neck squamous cell carcinoma

by FDG-PET scans with symptomatic improvement (Fig.  2). This very striking response offers direct proof-of-principle for p53 gene replacement. A recent study investigated the role of p53 biomarkers that may predict the efficacy of normal p53 delivered by gene therapy in patients (Nemunaitis et  al. 2009). Tumor p53 biomarkers, including p53 protein expression determined by immunohistochemistry, and p53 mutations were evaluated in 116 patients including 29 treated with methotrexate in a Phase III randomized, controlled trial. Profiles favorable for p53 gene therapy efficacy were hypothesized to have either normal p53 gene sequences or low level p53 protein expression while unfavorable p53 inhibitor profiles were predicted to have high level expression of mutated p53 that can inhibit normal p53 protein function. A greater than threefold statistically significant increase in tumor responses was observed for patients with favorable p53 efficacy profiles compared to those with unfavorable p53 inhibitor profiles. In the Phase III trial, there was a greater than twofold statistically significant increased time to progression and survival following p53 gene therapy in patients with favorable p53 profiles compared to unfavorable p53 inhibitor profiles. In contrast, the biomarker profiles predictive of p53 gene therapy efficacy did not predict methotrexate response, time to progression or survival outcomes. Thus tumor p53 biomarker profiles may be useful for predicting p53 gene therapy efficacy in recurrent SCCHN. A novel application of adenovirus p53 is the treatment of localized precancerous lesions. Oral leukoplakia is a precancerous lesion of squamous cell carcinoma. Adenovirus p53 inhibited cell proliferation and induced apoptosis in an oral dysplastic keratinocyte cell line. Twenty-two patients with dysplastic oral leukoplakia were treated with introlesional injections of adenovirus p53 with 16 patients showing a clinical response and 5 patients showing complete regression (Li et al. 2009).

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Fig.  2  Treatment of embryonal cell ovarian carcinoma with injection of adenovirus p53 in a patient with Li-Fraumeni syndrome

4 Gene Replacement in Combination with DNA Damaging Agents Many cancers are resistant to chemotherapy and radiation therapy. The p53 gene mediates the detection of damage to DNA and either directs repair or induces apoptosis. p53 is often mutated or nonfunctional in radiation- and chemotherapy-resistant tumors. Preclinical studies of p53 gene therapy combined with cisplatin in cultured NSCLC cells and in human xenografts in nude mice showed that sequential

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administration of cisplatin and p53 gene therapy resulted in enhanced expression of the p53 gene product (Fujiwara et al. 1994b; Nguyen et al. 1996). Studies of Ad-p53 gene transfer combined with radiation therapy indicated that delivery of Ad-p53 increases the sensitivity of p53-deficient tumor cells to external beam radiation (Spitz et al. 1996). Due to Ad-p53’s low toxicity (less than a 5% incidence of serious adverse events) in initial trials, therapeutic strategies combining Ad-p53 gene replacement and conventional DNA damaging therapies were begun (Yver et al. 1999).

5 Clinical Trials of Tumor Suppressor Gene Replacement Combined with Chemotherapy Twenty-four NSCLC patients with tumors previously unresponsive to conventional treatment were enrolled in a phase I trial of intratumor injection of p53 combined with cisplatin (Nemunaitis et  al. 2000). Seventy-five percent of the patients had previously experienced tumor progression on cisplatin- or carboplatin-containing regimens. Up to six monthly courses of intravenous cisplatin, each followed 3 days later by intratumoral injection of Ad-p53, resulted in 17 patients remaining stable for at least 2 months, 2 patients achieving partial responses, 4 patients continuing to exhibit progressive disease, and 1 patient unevaluable due to progressive disease. Seventy-nine percent of tumor biopsies showed an increase in the number of apoptotic cells, 7% showed a decrease in apoptosis, and 14% showed no change. A phase II clinical trial evaluated two comparable metastatic lesions in each NSCLC patient enrolled in the study (Schuler et  al. 2001). All patients received chemotherapy, either three cycles of carboplatin plus paclitaxel or three cycles of cisplatin plus vinorelbine, and then Ad-p53 was injected directly into one lesion. Ad-p53 treatment resulted in minimal vector-related toxicity and no overall increase in chemotherapy-related adverse events. Patients receiving carboplatin plus paclitaxel, the combination of drugs that provided the greatest benefit on its own, did not realize additional benefit from Ad-p53 gene transfer which would be expected as Ad-p53 was injected in only one lesion in each patient. However, patients treated with the less-successful cisplatin and vinorelbine regimen experienced significantly greater mean local tumor regression, as measured by size, in the Ad-p53-injected lesion than in the control lesion.

6 Clinical Trials of p53 Gene Replacement Combined with Radiation Therapy Preclinical studies suggesting that p53 gene replacement might confer radiation sensitivity to some tumors (Broaddus et al. 1999; Feinmesser et al. 1999; Jasty et al. 1998; Sakakura et al. 1996; Spitz et al. 1996) led to a phase II clinical trial of p53

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gene transfer in conjunction with radiation therapy (Swisher et al. 2000). Patients with a poor performance status who could not undergo surgery and would be at high risk for combined chemotherapy and radiation received 60 Gy over 6 weeks with Ad-p53 injected on days 1, 18, and 32. Nineteen patients with localized NSCLC were treated, resulting in a complete response in 1 patient (5%), partial response in 11 patients (58%), stable disease in 3 patients (16%), and progressive disease in 2 patients (11%). Two patients (11%) were not evaluable due to tumor progression or early death. Three months after the completion of therapy, biopsies revealed no viable tumor in 12 patients (63%) and viable tumor in 3 (16%). Tumors of four patients (21%) were not biopsied because of tumor progression, early death, or weakness. The 1-year progression-free survival rate was 45.5%. Among 13 evaluable patients after 1 year, 5 (39%) had a complete response and 3 (23%) had a partial response or disease stabilization. Most treatment failures were caused by metastatic disease without local progression. Biopsies of the tumor were performed before and after treatment so that detailed studies of gene expression were possible. Ad-p53 vector-specific DNA was detected in biopsy specimens from 9 of 12 patients with paired biopsies (day 18 and 19). The ratio of copies of Ad-p53 vector DNA to copies of actin DNA was 0.15 or higher in eight of nine patients (range, 0.05–3.85), with four patients having a ratio >0.5. For 11 patients with adequate samples for both vector DNA and mRNA analysis, 8 showed a postinjection increase in mRNA expression associated with detectable vector DNA. Postinjection increases in p53 mRNA were detected in 11 of 12 paired biopsies obtained 24 h after Ad-p53 injection, with 10 of 11 increasing threefold or more. Preinjection biopsy specimens that were shown by immunohistochemistry to be negative for p53 protein expression were stained for p53 protein expression after Ad-p53 injection. Staining results confirmed that the p53 protein was expressed in the posttreatment samples in the nuclei of cancer cells. For p21 (CDKN1A) mRNA, increases of statistical significance were noted 24 h after Ad-p53 injection and during treatment, as compared with the pretreatment biopsy. MDM2 mRNA levels were higher during treatment than before treatment. Levels of FAS mRNA did not change significantly during treatment. BAK mRNA expression increased significantly 24 h after the injection of Ad-p53 and thus appeared to be the marker most acutely upregulated by Ad-p53 injection. The safety profile for intratumoral injection of Ad-p53 has been excellent. The most frequently reported adverse events related to treatment with Ad-p53 injection were fever and chills, asthenia, injection site pain, nausea, and vomiting. The vast majority of these events were mild to moderate. To date, no maximum tolerated dose for Ad-p53 injection has been established. Beginning in 1998, a similar Adenovirus p53 expressing vector was tested in China in clinical trials under the name Gendicine. A multicenter, randomized clinical trial was conducted in which Ad-p53 was administered to 135 patients with head and neck squamous cell carcinoma (Peng 2005). Of the enrolled patients, 77% had late-stage III to IV cancer and had failed in either radio- or chemotherapy or were not eligible for surgery. The majority (85%) of the patients had nasopharyngeal cancer. One group received gene therapy in combination with radiotherapy (GTRT)

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and the other group received radiotherapy alone (RT). In the GTRT group, the complete response (CR) rate determined by computed tomography was 64% with 29% partial regression (PR). The response rate in the RT group was 19% of the patients showing CR and 60% PR. There is a significant difference (p 150) by several investigators demonstrating anticancer activity for IL-24 protein are the studies by Kreis et al. (Kreis et al. 2007) who reported IL-24 does not have anti-cancer activity against cancer cells and by Sainz-Perez et al. (2006), who reported high IL-24 expression promotes the survival of chronic lymphocytic leukemia (CLL). The lack of anticancer activity reported for IL-24-mediated survival activity in CLL remains unclear. A consensus among a majority of investigators (>25) studying IL-24 is that

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the results reported by Kreis et al. (2007) are most likely do to their use of reagents from different sources of variable quality. Similarly, two recent studies testing IL-24 on hematopoietic malignancies (Qian et al. 2008) and B-lymphoblastic leukemia (Dong et al. 2008) have shown antitumor activity both in vitro and in vivo thereby negating the findings of Sainz-Perez et al. (2006). From these reports, it is evident that IL-24 exhibits antitumor activity in a majority of human cancer cell lines. Studies investigating the cytokine properties of IL-24 protein showed that it functions as a pro-Th1 cytokine unlike IL-10 which is Th-2 type cytokine (discussed in detail below) (Caudell et al. 2002; Mumm et al. 2006). Apart from the antitumor and cytokine properties of IL-24, we have previously demonstrated that IL-24 also has potent anti-angiogenic and anti-metastatic activities both in vitro and in vivo (Inoue et al. 2005, 2006; Nishikawa et al. 2004; Ramesh et al. 2004a, b). Anti-angiogenic activity was demonstrated for both the intracellular and extracellular forms of the IL-24 protein (Inoue et al. 2005, 2006, Nishikawa et al. 2004; Ramesh et al. 2004a, b). The anti-angiogenic activity exerted by the extracellular IL-24 protein was shown to be receptor-mediated (Ramesh et  al. 2003). Hence, IL-24 is a unique cytokine that undergoes protein modifications akin to classical tumor suppressor proteins and functions as a tumor suppressor/cytokine.

1.2 Cytokine Properties At least five cellular genes encoding secreted proteins with 20–30% homology to IL-10 have been found: IL-19 (Gallagher et  al. 2000), IL-20 (Blumberg et  al. 2001), IL-22 (Xie et al. 2000), IL-26 (Knappe et al. 2000), and mda-7, which was renamed IL-24 with the approval of the HUGO Gene Nomenclature Committee (Caudell et  al. 2002). Four of these genes (IL-10, IL-19, IL-20, and IL-24) are encoded within a 195-kilobase cytokine cluster on chromosome 1q31/32. All of these cytokines are secreted. The human IL-10 protein has only 23% homology to IL-24. IL-20 is the most similar to IL-24, with 33% protein homology. The relatively low amino acid sequence homology among these structurally related homodimeric IL-10 family members suggests that they have quite different biological functions. Message for IL-19, IL-22, IL-24, and IL-26 are expressed by antigen- or mitogen-stimulated human peripheral blood mononuclear cells (PBMCs) (Caudell et al. 2002; Gallagher et al. 2000; Knappe et al. 2000; Poindexter et al. 2005; Xie et  al. 2000). IL-24, as well as IL-19, expression by macrophages is strongly induced by stimulation with lipopolysaccharide (LPS) (Gallagher et  al. 2000). The stimulation of T cells with anti-CD3 causes the expression of IL-22 (Xie et  al. 2000). IL-26 has been identified in viral-transformed T cells, in normal PBMCs, and in some T cell lines (Knappe et al. 2000; Sheikh et al. 2004). All of the IL-10 family members use the class II family of receptors and signal through STAT1, STAT3, or both (Blumberg et al. 2001; Gallagher et al. 2000; Kotenko 2002; Sheikh et al. 2004; Wang et al. 2002; Xie et al. 2000). Descriptive and functional studies of the IL-24 receptor with keratinocytes or transfected cells have shown that

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IL-24 activates STAT3 (Blumberg et  al. 2001; Kotenko 2002; Xie et  al. 2000). IL-19 stimulates human macrophages to produce both IL-6 and tumor necrosis factor (TNF)a (Liao et al. 2002). Similarly, the stimulation of PBMCs with IL-24 also leads to the production of IL-6 and TNFa (Caudell et al. 2002). More recent reports suggest that the IL-10 family of cytokines is involved in the regulation of inflammatory and immune responses (Chada et al. 2004b; Kotenko2002). IL-24 protein is endogenously expressed in normal human skin melanocytes, implying that IL-24 is involved in skin biology (Ekmekcioglu et  al. 2001). The detection of receptors for IL-24 on human keratinocytes suggests that this molecule may be involved in human wound healing as well (Wang et al. 2002). The rat homolog of IL-24, c49a, has been reported to be expressed in proliferating (Poindexter et al. 2007) fibroblasts during wound repair in a rat model (Soo et al. 1999). The relationship of IL-24 to IL-10 and its predicted cytokine features led us to study IL-24 expression in immune cells. As we describe in our publication (Poindexter et al. 2005), we found IL-24 protein to be expressed by human PBMCs as a result of mitogen or antigen stimulation. Our publications (Caudell et al. 2002; Poindexter et al. 2005) provide evidence that IL-24 is part of a cytokine network expressed during the activation of the human cellular immune response. Specifically, IL-24 is known to stimulate human PBMCs to secrete TNFa, IL-6, IL-1b, and granulocyte-macrophage colony stimulating factor (GM-CSF).

2 Expression of IL-24 in Melanocytes and Melanoma Our study (Ekmekcioglu et al. 2001) was the first to evaluate IL-24 expression in human tumors using immunohistochemical analysis. We demonstrated that IL-24 protein was expressed in normal melanocytes and early-stage melanomas; however, IL-24 expression decreased in more advanced melanomas and was completely absent in metastatic lesions, which is consistent with the tumor suppressor role of IL-24. Ellerhorst et  al. evaluated IL-24 protein expression during melanoma disease progression by using the immunohistochemical analysis of clinical tumor biopsy samples from stage III to IV melanomas (n = 82, 41 primary melanomas and 41 metastases) (Ellerhorst et  al. 2002). To determine whether IL-24 loss occurred with tumor progression (from superficial to invasive stages), the study compared IL-24 expression by tumor cells in the epidermis or superficial dermis with that of cells in the deep dermis (Fig. 1). The percentage of IL-24-positive cells and the intensity of the staining decreased significantly with tumor depth (p = 0.003 and p = 0.008, respectively). On the basis of these results, Ellerhorst et al. hypothesized that a similar pattern would be observed when comparing primary melanoma lesions with their corresponding metastases. Indeed, the examination of 24 pairs of primary tumors and their metastases showed significantly lower number (p = 0.001) and intensity (p = 0.001) scores for IL-24 staining in the metastases, relative to the primary tumors.

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Superficial

IL-24 Immunostaining

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++

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Negative

Fig. 1  Loss of IL-24 Immunoreactivity in the invasive from of primary melanoma. The superficial portion of the tumor shows significant cytoplasmic IL-24 expression, which diminishes with invasion into the deep dermis (×40 magnification). Taken from Ellerhorst et  al. (2002) PMID 11844832

3 Re-expression of the Tumor Suppressor Preclinical studies evaluating the activity of IL-24 in tumor models have identified a complex interplay between direct tumor cell death (induced by the intracellular expression of IL-24) and complementary anti-tumor mechanisms (the so-called “bystander effect,” induced by the engagement of the IL-24 receptors). To analyze the mechanism by which IL-24 kills tumor cells, we developed mutants engineered to express IL-24 in the cytosol, nucleus, or endoplasmic reticulum, coupled with glycosylation inhibitors to alter intracellular trafficking. We observed that IL-24 appears to kill tumor cells via two distinct mechanisms. When IL-24 is expressed within a cell, a stress-response signal from the endoplasmic reticulum (ER) via caspases 7 and 12 leads to mitochondrial disruption and apoptosis (Sieger et al. 2004). Alternatively, when IL-24 protein is delivered to a cell bearing IL-24 receptors, ligand binding causes transient phosphorylation of STAT3 and initiates an apoptotic caspase cascade. Also, the mechanism of apoptosis varies depending upon the cell type studied. In a specific apoptosis pathway study that targets human ovarian tumor cell, activation of the extrinsic pathway was demonstrated by increased cellsurface Fas expression and cleavage of Bid and caspase-8, while activation of the intrinsic pathway was demonstrated by the disruption of mitochondrial potential;

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and the activation of downstream capase-9 and caspase- 3 via cytochrome C release (Shanker et al. 2007). In cells that lack IL-24 receptors, IL-24 gene transfer causes apoptosis via the intracellular ER pathway, and exogenous IL-24 protein does not have any effect. In contrast, in cells expressing IL-24 receptors, ligand engagement activates alternate signaling pathways (Chada et  al. 2004b). In a recent study by Sauane et al., it was confirmed that IL-24 protein induces the bystander antitumor effect through an ER stress mechanism mediated by a robust activation of its own protein expression (Sauane et al. 2008). This study reported that exogenous IL-24 protein induces growth inhibition and apoptosis only in cancer cells through a mechanism identical to Ad-mda-7/IL-24 infection. They demonstrated that exogenous IL-24-mediated IL-24 receptor upregulation is essential for the IL-24-induced apoptotic effect. Blocking mda-7/IL-24 expression by RNA interference inhibits extracellular IL-24-mediated apoptosis (Sauane et al. 2008). This complex mechanism utilizing both intrinsic and extrinsic pathways of IL-24 induced cell death is summarized in Fig. 2. Therefore, having purified functional IL-24 protein provides insights into the mechanism by which this molecule exerts the bystander antitumor effects and supports alternative therapeutic approaches in addition to adenovirus delivery. We have found that preferential utilization of extrinsic versus intrinsic pathways by IL-24 is cancer-cell dependent. Treatment with Ad-mda-7/IL-24 results in

IL-20R2 Ad-mda7/IL-24

IL-24 Receptor Subunits

IL-22R1 IL-20R1

Bystander Effect via Secreted IL-24

PKR UPR stress response Caspase 12

IL-20R Type I IL-20R Type II

? NFkB STAT3

IRFs IFNb≠

iNOS Ø

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PERK [Ca2+] release

eIF2a Bcl family

Effector mitochondria Caspases ?

APOPTOSIS

≠BAX G1arrest

Nucleus

NFkB

STAT3

OTHER?

Nucleus

TUMOR CELLS Normal Cells NO CELL DEATH

Fig. 2  Mechanism of IL-24 induced cell death. The gene transfer of IL-24 results in direct and indirect (bystander protein) effects on normal and cancer cells

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apoptosis in melanoma tumor cells, but not in melanocytes. In melanoma, IL-24 expression induces the upregulation of growth arrest and DNA damage (GADD) and pro-apoptotic (BAX) proteins, with a concomitant downregulation of iNOS via the modulation of interferon regulatory factors (Ekmekcioglu et al. 2003; Lebedeva et al. 2002). Selective inhibition of p38 mitogen-activated protein kinase (MAPK) phosphorylation, which induces GADD, abrogates IL-24-induced apoptosis in melanoma and NSCLC cells (Mhashilkar et al. 2003; Sarkar et al. 2002). The treatment of melanoma tumor cell lines with exogenous IL-24 protein results in growth arrest and apoptotic cell death (Chada et al. 2004b). The co-administration of neutralizing antibodies against IL-24 or individual receptor subunits (anti-IL-20R1 or anti-IL-22R1) blocked tumor cell killing, thus demonstrating the specificity of the ligand-receptor interaction. IL-24 protein treatment of melanoma cells also increased the expression of BAX and p21 prior to apoptotic death. A second more novel pathway for melanoma apoptosis has been described involving the induction of endogenous IFN-b followed by IRF regulation and TRAIL/FasL system activation (Ekmekcioglu et  al. 2008). Thus, exogenous IL-24 protein can reverse the cancerous phenotype of melanoma cells and warranted its evaluation as a therapeutic approach in melanoma and other solid tumors.

4 Clinical Experience with Ad mda-7/IL-24 4.1 Metastatic Melanoma Melanoma is the most malignant of skin cancers. The incidence of melanoma in the United States is increasing at an annual rate of 3.1% and these rates have doubled in all socioeconomic status groups over the past 10-year period. Early, localized disease (radial growth phase, RGP) is effectively treated with excision, with a high cure rate (~80%). However, melanoma quickly transitions from RGP to the vertical growth phase (VGP), which leads to disseminated disease that is almost universally fatal. The median survival rate with current chemotherapy regimens for metastatic malignant melanoma has not improved significantly over the last several decades (Anderson et al. 1995). Dacarbazine (DTIC) is the only cytotoxic chemotherapeutic drug approved by the U.S. Food and Drug Administration for the treatment of metastatic melanoma. However, recent phase III studies using the Response Evaluation Criteria in Solid Tumors (RECIST) showed that the response rate of dacarbazine is less than 10% and the median progression-free survival (PFS) is only 1.5 months (Bedikian et  al. 2006, Middleton et  al. 2000). The other classes of drugs with activity between 10 and 15% are the nitrosoureas, vinca alkaloids, and cisplatin (Anderson et al. 1995). Interleukin-2 based regimens can result in long-term benefit in a small fraction of patients, but the majority of patients do not respond. Many types of combination chemotherapy regimens explored in the treatment of metastatic melanoma during the past 25 years have produced response rates of 30–40% (Chapman et al. 1999; Luikart et al. 1984). These regimens, however, were limited

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by the short duration of responses and low rates of complete response. Although the integration of combination chemotherapy with the cytokines interleukin-2 (IL-2) and interferon-alpha (IFN-a), that is, “biochemotherapy,” has resulted in an overall response rate of more than 60%, with 10–20% complete responses, several phase III trials failed to show a meaningful survival benefit of biochemotherapy over chemotherapeutic regimens (Atkins 1997; Eton et  al. 2002; Ives et  al. 2007; Rosenberg et al. 1999). Thus, there is an urgent need to discover new ways to treat these patients with novel drugs. On the basis of all preliminary data, IL-24 therapy is likely to be more efficacious than IL-2 and IFNa and much less toxic (Fisher et al. 2003; Inoue et al. 2006; Ramesh et al. 2004b). This has proven to be the case. Ad-mda-7/IL-24 has been tested in a Phase 1 Clinical Trial in patients with advanced solid tumors.

4.2 Phase I/II intratumoral Ad-mda-7/IL-24 Gene Transfer in Patients with Advanced Solid Tumors This phase I/II clinical trial was in patients with advanced carcinoma for which intralesional injection and multiple biopsies were performed (Cunningham et  al. 2005; Tong et al. 2005). The study enrolled 28 heavily pretreated patients with a total of 15 different tumor types, including melanoma, lymphocytic lymphoma, and carcinomas of the breast, colon, head and neck, adrenal gland, renal cell, and lip. Patients were divided into eight cohorts of increasing IL-24 doses (up to 2 × 1012 viral particles (vp) per injection). Ad-mda-7/IL-24 (also called INGN-241) was administered intratumorally, and the tumors (excised at pre-established times during treatment) evaluated for vector-specific DNA and RNA, transgenic IL-24 expression, and biological effects. Successful gene transfer was demonstrated in 100% of the patients evaluated, with a parallel distribution of vector DNA, vector RNA, IL-24 protein expression, and apoptosis induction observed in all tumors that decreased with distance away from the injection site. The safety analysis reported 15 mild-to-moderate adverse events in patients completing at least one cycle of treatment (n = 22) (Cunningham et al. 2005; Tong et  al. 2005). Toxicity attributable to Ad-mda-7/IL-24 injections was self-limiting and resolved within 48 h; the most common adverse events reported were injection site pain and fever. Skin erythema, which resolved within 96 h post-injection, was reported in the cohort treated at the maximum dose. One grade 3 serious adverse event (fatigue) was reported in a patient who was subsequently removed from the study. Importantly, no maximum tolerated dose (MTD) was achieved (Cunningham et al. 2005). In the first phase of the monotherapy protocol, the injected lesions were excised 24–96  h post-treatment; thus, no conclusions could be drawn about the clinical activity of the treatment. However, minor changes in the morphology of the injected lesions from the higher-dose cohorts were observed. In a repeat-dose protocol, patients in the highest dose cohort received injections twice weekly for 3 weeks and

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had incision or core biopsies taken 30 days after the last Ad-mda-7/IL-24 injection. A clinically significant response to Ad-mda-7/IL-24 (partial regression) was observed in 40% of the patients in this cohort (n = 5) (Cunningham et al. 2005). The most dramatic clinical response occurred in a patient with a 20 × 20 mm lesion at baseline; by the 6th injection, a clear decrease in the size of the lesion was seen that was associated with erythema around the lesion. The erythema resolved and regression continued over the next 2 weeks until there was no clinical evidence of disease at that site. A second course of injections was then started on a second lesion (baseline measurement 18 × 23 mm), and an 84% reduction in size was seen by the fifth injection. After the sixth injection, the lesion was excised. An additional melanoma lesion exhibited a partial response (33% decrease as measured by RECIST). This patient was still alive more than 600  days after initiation of the Ad-mda-7/IL-24 treatment. Molecular markers of vector expression (i.e., beta-catenin redistribution/decrease, TUNEL, and in melanoma, iNOS down-regulation) were positive for all tumor types examined (Tong et al. 2005). The immunohistochemical analysis of IL-24 and TUNEL assay in tumors resected 24–96  h after injection demonstrated substantial IL-24 immunostaining (range, 20–90% positive cells) closest to the injection site and a significant correlation between apoptosis (as measured by TUNEL) and expression of IL-24 (p P5¢ thiophosphoramidate (GRN163) targeting telomerase RNA in human multiple myeloma cells. Cancer Res, 2003;63(19):6187–94. Andrew, T, Aviv, A, Falchi, M, et  al. Mapping genetic loci that determine leukocyte telomere length in a large sample of unselected, female sibling pairs. Am J Hum Genet, 2006;78:480–6. Armanios, MY, Chen, JJ, Cogan, JD, et al. Telomerase mutations in families with idiopathic pulmonary fibrosis. N Engl J Med, 2007;356:1370–2. Aviv, A. Telomeres and human aging: facts and fibs. Sci Aging Knowledge Environ, 2004;51:43–5.

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Aviv, A, Valdes, A, Gardner, JP, Swaminathan, R, Kimura, M, Spector, TD. Menopause modifies the association of leukocyte telomere length with insulin resistance and inflammation. J Clin Endocrinol Metab, 2006;91:635–40. Benetos, A, Okuda, K, Lajemi, M, et al. Telomere length as an indicator of biological aging: the gender effect and relation with pulse pressure and pulse wave velocity. Hypertension, 2001;37:381–5. Benetos, A, Gardner, JP, Zureik, M, et al. Short telomeres are associated with increased carotid atherosclerosis in hypertensive subjects. Hypertension, 2004;43:182–5. Bernhardt, SL, Gjertsen, MK, Trachsel, S, et al. Telomerase peptide vaccination of patients with non-resectable pancreatic cancer: a dose escalating phase I/II study. Br J Cancer, 2006 Dec 4;95(11):1474–82. Bischoff, C, Petersen, HC, Graakjaer, J, et al. No association between telomere length and survival among the elderly and oldest old. Epidemiology, 2006;17:190–4. Blackburn EH. Telomerases. Annu Rev Biochem, 1992;61:113–29. Bodnar, AG, Ouellete, M, Frolkis, M, et al. Extension of lifespan by introduction of telomerase in normal human cells. Science, 1998;279:349–52. Bolonaki, I, Kotsakis, A, Papadimitraki, E, Vaccination of patients with advanced non-small-cell lung cancer with an optimized cryptic human telomerase reverse transcriptase peptide. J Clin Oncol, 2007 Jul 1;25(19):2727–34. Brouilette, S, Singh, RK, Thompson, JR, Goodall, AH, Samani, NJ. White cell telomere length and risk of premature myocardial infarction. Arterioscler Throm Vasc Biol, 2003;23:842–6. 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. Campisi, J. Senescent cells, tumor suppression, and organismal aging: good citizen, bad neighbors. Cell, 2005;120:513–22. Campisi, J, d’Adda di Fagagna, F. Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol, 2007;8:729–40. Carpenter, EL, Vonderheide, RH. Telomerase-based immunotherapy of cancer. Expert Opin Biol Ther, 2006;10:1031–9. Cawthon RM, Smith KR, O’Brien E, Sivatchenko A, Kerber RA. Association between telomere length in blood and mortality in people aged 60 years or older. Lancet, 2003;361:393–5. Cristofari, G, Lingner, J. The telomerase ribonucleoprotein particle. In: de Lange, T, Lundblad, V, and Blackburn, E, (Eds), Telomeres. Second Edition, Cold Spring Harbor Laboratory Press, NY, 2006; pp. 21–48. Danet-Desnoyers, GH, Luongo, JL, Bonnet, DA, Domchek, SM, Vonderheide, RH. Telomerase vaccination has no detectable effect on SCID-repopulating and colony-forming activities in the bone marrow of cancer patients. Exp Hematol, 2005;33:1275–80. de Lange, T. Mammalian Telomeres. In: de Lange, T, Lundblad, V, and Blackburn, E, (Eds), Telomeres. Second edition, Cold Spring Harbor Laboratory Press, NY, 2006; pp. 387–431. de Lange, T, Shiue, L, Meyers, RM, et al. Structure and variability of human chromosome ends. Mol Cell Biol, 1990:10:518–27. Demissie, S, Levy, D, Benjamin, EJ, et al. Insulin resistance, oxidative stress, hypertension, and leukocyte telomere length in men from the Framingham Heart Study. Aging Cell, 2006;5:325–30. Dikmen, ZG, Gellert, GC, Jackson, S, et al. In vivo inhibition of lung cancer by GRN163L – a novel human telomerase inhibitor. Cancer Res, 2005;65:7866–73. Dikmen, ZG, Wright, WE, Shay, JW, Gryaznov, SM. Telomerase targeted oligonucleotide thiophosphoramidates in T24-luc bladder cancer cells. J Cell Biochem, 2008;104:444–52. 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. Domchek, SM, Recio, A, Mick, R, et al. Telomerase-specific T-cell immunity in breast cancer: impact of vaccination on immunosurveillance. Cancer Res, 2007;67:10546–55.

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Chapter 14

Gene Therapy for Sarcoma Keila E. Torres and Raphael E. Pollock

Abstract  Soft tissue sarcomas are a group of potentially devastating malignancies. Despite multidisciplinary treatment combining surgery, radiation therapy, and chemotherapy, the overall prognosis for sarcoma patients remains poor. Very few chemotherapeutic agents with meaningful efficacy: toxicity ratios are available, and the development of novel therapeutics will be critical if this currently unacceptable prognosis is to be improved. Much exciting progress in genetic profiling of sarcomas and molecular identification of constituent oncogenes and protein products has recently occurred, which will ultimately enable the development of sarcoma targeted therapies. In this chapter, we discuss the major advances in cytogenetics and the importance of genetic and molecular signatures in sarcoma diagnosis and treatment. In an attempt to create better treatments for cancers several strategies have evolved to administer therapeutic genes that inactivate oncogenes, restore tumor-suppressor genes, and enhance the native immune response. We explore some promising initial data regarding novel gene delivery systems, such as isolated limb perfusion which may be a useful promising technique in delivering growth-suppressing constructs into soft tissue sarcomas. Keywords  Soft tissue sarcoma • Cytogenetics • Expression signatures • Gene therapy

1 Introduction Soft tissue sarcoma comprises a heterogeneous group of rare malignancies consisting of more than 50 distinct histological subtypes that putatively share a common mesenchymal origin and account for approximately 1% of all adult malignancies. R.E. Pollock (*) Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Unit 444, Houston, TX, 77030, USA e-mail: [email protected] J.A. Roth (ed.), Gene-Based Therapies for Cancer, Current Cancer Research, DOI 10.1007/978-1-4419-6102-0_14, © Springer Science+Business Media, LLC 2010

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The American Cancer Society estimates that approximately 10,660 new cases of soft tissue sarcoma will be diagnosed in 2009 and that 3,820 patients will die from the disease (Jemal et  al. 2009). Effective treatment of sarcoma usually requires surgical excision with the addition of radiotherapy and/or chemotherapy. Although conventional therapeutic treatments can effectively reduce the overall tumor mass, they often fail to achieve a curative outcome. Very few chemotherapeutic agents with meaningful efficacy: toxicity ratios are available, and the development of novel therapeutics will be critical if this currently unacceptable prognosis is to be improved. However, progress in this regard has been impeded by the relative rarity of the disease, rendering it a challenge to assemble the requisite investigative resources to support dedicated sarcoma research teams. This problem is compounded by the remarkable histological and cytogenetic heterogeneity underlying the variable clinical behaviors of this tumor cluster. In turn, these issues negatively impact on the ability to generate the precise diagnoses needed for subtype-specific tumor staging, treatment planning, clinical trials accrual, and prognostic inferences needed in contemporary oncology practice. Against this backdrop, over the past several decades significant progress has been achieved due to the development of improved pathological diagnostic criteria, including the advent of molecular diagnostics, enhanced imaging techniques, including metabolic-based dynamic examinations, new surgical applications such as microvascular tissue transfer underlying advances in limb preservation, and the development of a somewhat broader chemotherapy armamentarium. These enhancements have been integrated into multidisciplinary treatment and research programs at the leading sarcoma centers world-wide, thereby providing a critical scaffold upon which alternative therapeutic initiatives such as sarcoma immunotherapy and gene therapy strategies can be actively investigated (Meyers et al. 2008; Mori et al. 2006; Witlox et al. 2007).

2 Cytogenetics of Soft Tissue Sarcoma One of the most significant advances in sarcoma diagnosis has been the development and introduction of molecular probes to detect consistent cytogenetic and molecular abnormalities (Table 1). The identification of numerous simple as well as complex karyotypic abnormalities in patients bearing different sarcoma subtypes suggests that these processes may be important in sarcomagenesis while also presenting prognostic insight (Ludwig 2008). From the molecular point of view, sarcomas can be divided into two major groups based on their genetic alterations. The former group consists of sarcomas showing specific, recurrent genetic alterations, and relatively simple karyotypes, such as Ewing sarcoma. The second group represents sarcomas with variable gene alterations and very complex karyotypes such as leiomyosarcomas (Yang et  al. 2009). Until recent years, gastrointestinal stromal tumors (GISTs) were not clearly delineated from leiomyosarcoma. Leiomyosarcoma only rarely expresses c-kit

14  Gene Therapy for Sarcoma Table 1  Cytogenetic and molecular abnormalities in sarcomas Tumor type Cytogenetic Bone sarcomas Ewing’s sarcoma/PNET t(11;22)(q24;q12) t(21;22)(q12;q22) t(7;22)(p22;q12) Soft tissue sarcomas Malignant fibrous histiocytoma 1q11, 3p12, 11p11, and 19p13 Myxoid/round cell t(12;16)(q13;p11) Liposarcoma t(12;22)(q13;q11±12) Synovial sarcoma t(x,18)(p11;q11) Rhabdomyosarcoma t(2;13)(q35±37;q14) Alveolar t(1;13)(p36;q14) Embryonal trisomy 2q Neuroblastoma del (1p) Desmoplastic small round cell t(11;22)(p13;q12) Myxoid chondrosarcoma t(9;22)(q31;q12) Clear cell sarcoma t(12;22)(q13±14;q12±13) EWS-ATF1 Dermatofibrosarcoma t(17,22)(q22;q13)

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Molecular EWS-FLI1 EWS-ERG EWS-ETV1

CHOP-TLS CHOP-EWS SYT-SSX PAX3-FKHR PAX7-FKHR

EWS-WT1 EWS-TEC PDGFB-COL1A1

when compared to GISTs, in which the majority of cases are positive for c-kit protein as measured by immunohistochemical approaches (Meza-Zepeda et al. 2006; Trent et al. 2007). Soft tissue leiomyosarcomas show multiple gene alterations in tandem with these very complex karyotypes; these include numerous gains and losses of genetic material and/or function. Analysis of about 100 leiomyosarcomas revealed that most of the karyotypes were complex, and there were no consistent recurrent aberrations found at the chromosomal level (Wang et al. 2001). The most frequent losses were detected in 10q and 13q, regions where the tumor suppressor genes PTEN and Rb reside. Interestingly, high level amplifications in 17p were often found in small tumors. These amplifications were not common in the very large tumors, suggesting that the small and large tumors could represent distinct types of leiomyosarcoma. Furthermore, this finding may imply that large leiomyosarcomas do not necessarily progress from the small tumor type or at least do not derive from the same clones. Gains in 1q, 5p, 6q, and 8q were detected in large tumors (El-Rifai et  al. 1998). The oncogenes MYC (located on 8q24) and MYB (located on 6q22) are the most likely involved candidate oncogenes. Analysis of the relative chromosome copy number in 14 cases of leiomyosarcoma revealed frequent gains in 5p15, 8q24, 15q25-26, 17p, and Xp (Otano-Joos et al. 2000). Gain of 17p11-12 was also found in high-grade osteosarcoma as well as in gliomas (van Dartel et al. 2003). The putative oncogenes on 17p are not well characterized. One of the genes located in 17p region is COPS3 (17p11-12). COPS3 has been shown to target p53 protein for proteosome-mediated degradation in osteosarcoma (Henriksen et al. 2003). Interestingly, murine double minute protein 2 (MDM2) amplification or p53 mutation was not found in osteosarcomas that contained COPS3 amplification. Amplification of the COPS3 gene was proposed to target p53 protein for proteosome-mediated degradation in osteosarcoma in a

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manner similar to MDM2 (Henriksen et al. 2003). These findings suggest the possibility that osteosarcomas and leiomyosarcomas may share a common mechanism for inactivation of the p53 pathway. Mutations in the p53 tumor-suppressor gene have been identified as the most common genetic alterations in soft tissue sarcoma (Das et al. 2007; Latres et al. 1994). These gene mutations are more often observed in metastases than in primary tumors and in high-grade versus low-grade sarcomas. Patients with p53 mutations have a markedly decreased overall survival relative to those containing wildtype p53 genes. Therefore, these mutations are thought to have a considerable negative impact on both overall as well as sarcoma-specific survival (Pollock et al. 1998; Schneider-Stock et  al. 1999; Taubert et  al. 1996). Our investigations of autologous human primary and metastatic sarcoma have demonstrated that clonal expansion of p53-mutated cells in soft tissue sarcoma confers distinct metastatic advantages (Pollock et al. 1996). Furthermore, we have also demonstrated that p53 alterations in soft tissue sarcoma contribute to metastasis-promoting behaviors, including the loss of cell cycle control (Pollock et al. 1998), enhanced angiogenesis (Zhang et al. 2000), invasiveness (Liu et al. 2006), and chemoresistance (Zhan et al. 2001, 2005). Many sarcoma subtypes harbor characteristic chromosomal translocations or nonrandom mutations that can aid in their identification. The majority of the genes located at the chromosomal breakpoints that determine specific sarcoma-associated translocations result in fusion genes encoding aberrant transcription factors and/or transcriptional regulators that alter gene expression. For instance, the fusion of the collagen type I alpha 1 (COL1A1) gene with the platelet-derived growth factor beta-chain (PDGFB) gene (COL1-PDGFB) has been shown to result in overexpression of the growth factor PDGFB (Simon et al. 1997). This growth factor activates platelet derived growth factor receptor beta and platelet-derived growth factor receptor alpha, ultimately leading to unregulated cell growth. A novel treatment is to inactivate the growth factor or receptor at the cellular level. This possibility was confirmed by the remarkable clinical response of patients with dermatofibrosarcoma protuberans who were treated with imatinib, a PDGFR inhibitor (Maki et al. 2002; Rubin et al. 2002). It has been found that mutations or deletions in genes encoding tyrosine kinases can result in constitutive activation leading to unregulated cell growth. For example, more than 90% of gastrointestinal tumors (GISTs) are associated with mutations of the KIT and/or PDGFRA gene (Yang et  al. 2008). Mutations of these genes are believed to be critical in GIST tumorigenesis (Gunawan et  al. 2002; Lasota and Miettinen 2008; Wozniak et  al. 2007). Specific mutations of KIT and PDGFRA gene have been found to correlate with the specific cell morphology, histologic phenotype, metastasis, and prognosis (Lasota and Miettinen 2006; Penzel et  al. 2005; Singer et al. 2002). Furthermore, specific mutations in KIT and PDGFR have been found to lead to differential drug sensitivity (Chen et al. 2004a). Cytogenetic aberrations associated with these tumors thus far described include the loss of 1p, 13q, 14q, or 15q, the loss of heterozygosity of 22q, numeric chromosomal imbalances, and nuclear/mitochondrial microsatellite instability (Chen et al. 2004b; Kose

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et al. 2006; Yamashita et al. 2006). Molecular genetic aberrations include the loss of heterozygosity of p16 (INK4A) and p14 (ARF), methylation of p15(INK4B), homozygous loss of the Hox11L1 gene, and amplification of C-MYC, MDM2, EGFR1, and CCND1 (Gunawan et  al. 2002; Lasota and Miettinen 2006, 2008; Wozniak et al. 2007) . Mutation of the KIT gene results in constitutive activation of this tyrosine kinase (Rubin et  al. 2001) which can result in ligand-independent tyrosine kinase activity, receptor autophosphorylation, and induction of other downstream kinases (e.g., phosphatitidylinositol 3-kinase and mitogen-activated kinases) (Hirota et al. 1998; Lux et al. 2000; Sarlomo-Rikala et al. 1998), ultimately eventuating in unregulated GIST proliferation. Characteristic chromosomal translocations involving the EWSR1 gene have been identified in clear cell sarcomas (Antonescu et al. 2006; Coindre et al. 2006; Hisaoka et  al. 2008; Panagopoulos et  al. 2002). The most common translocation partner is the activating transcription factor-1 (ATF1) gene on chromosome 12q13, of which four chimeric types have been described (Coindre et  al. 2006; Hisaoka et  al. 2008; Panagopoulos et  al. 2002). A less common translocation partner is cyclic AMP responsive-binding protein (CREB1) located on chromosome 2q34 (Hisaoka et al. 2008). ATF1 and CREB1 (cyclic AMP responsive-binding protein) both encode basic leucine zipper transcription factors that are involved in cAMP and Cap2-induced transcriptional activation (Persengiev and Green 2003). Both EWSR1-ATF1 and EWSR1-CREB1 chimeric proteins are believed to play a critical role in clear cell sarcoma oncogenesis (Antonescu et  al. 2006; Panagopoulos et al. 2002). Analysis of clear cell sarcoma tumors from patients indicate that the EWSR1-ATF1 chimeric transcript type 1 is the most common chimeric transcript found, with many cases carrying multiple chimeric transcripts (Wang et al. 2009b). Furthermore, our group has found that EWSR1-CREB1 is not exclusive to gastrointestinal tract tumors, indicating the need of incorporating this variant in routine molecular testing of soft tissue clear cell sarcoma specimens if RT–PCR for EWSR1-ATF1 is negative (Wang et  al. 2009b). The potential prognostic significance of the different chimeric types remains to be defined in future studies. Chromosomal translocations involving EWSR1 gene have also been found in extraskeletal myxoid chondrosarcomas. The majority of extraskeletal myxoid chondrosarcomas harbor a balanced translocation t(9;22)(q22;q12) that fuses EWSR1 with NR4A3. We were able to identify the rearrangement of the EWSR1 locus in 14 (93%) of 16 cases of extraskeletal myxoid chondrosarcomas using fluorescence in situ hybridization (FISH; Wang et  al. 2008). In this study, the vast majority of extraskeletal myxoid chondrosarcomas were associated with a rearrangement at the EWSR1 locus (22q12). Most sporadic desmoids have been associated with activating mutations in exon 3 of the gene that encodes the cell adhesion cofactor and nuclear signaling factor, b-catenin (CTNNB; Miyoshi et al. 1998; Tejpar et al. 1999). Analysis of a large single cohort of sporadic desmoids demonstrated mutations in CTNNB1 in 85% of cases (Lazar et  al. 2008). A threefold increased risk of recurrence in desmoids was strongly associated with a specific CTNNB1 mutation in codon 45 (45F). This study suggests that genotyping of CTNNB1 exon 3 could be

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useful as a diagnostic test in equivocal situations such as in differentiating postsurgical scar from desmoid recurrence. In addition, such genotyping can also provide important prognostic insight regarding the risk of recurrence, and may therefore have bearing on selection of adjuvant therapeutic approaches. Conversely, desmoids arising in the setting of familial adenomatous polyposis (FAP) display germline inactivating mutations in adenosis polyposis coli (APC) tumor suppressor gene. Similar mutations in APC gene have been identified in a small subset of sporadic desmoid tumors (Alman et  al. 1997; Giarola et  al. 1998). Both APC and beta-catenin proteins are part of the Wnt signaling pathway. Activation of the Wnt/beta-catenin pathway is a hallmark of these tumors, and inhibition of this pathway may have clinical utility, as this appears to be the critical molecular event underlying desmoid tumor biology (Kotiligam et al. 2008). These chromosomal aberrations may occur alone or in combination with other genetic events and are believed to underlie malignant transformation. If a chromosomal alteration such as a nonrandom mutation can be connected to the development of a specific, aberrantly expressed protein, a potentially targetable molecular axis may be so identified. There is a growing awareness that more sensitive prognostication and therapeutic decision-making algorithms will need to incorporate relevant cytogenetic and molecular determinants. Furthermore, a new paradigm of classification, integrating the standard clinical and pathological criteria with molecular aberrations, may permit personalized prognosis and treatment.

3 Expression Signatures Much exciting progress in genetic profiling of sarcomas and molecular identification of constituent oncogenes and protein products has recently occurred, which will ultimately enable the development of sarcoma targeted therapies. Sarcoma expression profiles can be divided into two groups. One group consists of those that have consistent collections of upregulated and downregulated genes, thereby allowing specimen clustering such as with GIST (Nielsen et  al. 2002), synovial sarcoma (Nagayama et  al. 2002), and extraskeletal myxoid chondroma (Subramanian et al. 2005). The second group includes those that do not have genes that up- or downregulated, and therefore cannot be consistently grouped together by gene expression profiling such as pleomorphic sarcomas (Nielsen et al. 2002; Segal et al. 2003). Comparison of genes expressed in tumors and their precursor lesions can reveal important information about tumor progression. Malignant peripheral nerve sheath tumors, when compared with Schwann cells, exhibit a significant loss of expression of many genes related to Schwannian differentiation (SOX10, PMP22, NGFR, S100B) and concurrent upregulation of a smaller set of genes, many of which are markers of neural crest and mesenchymal stem cells (SOX9, TWIST1) (Miller et al. 2006). Using a large tissue microarray, our laboratory has shown that Ki67, vascular endothelial growth factor, p53, and pMEK are all overexpressed in malignant

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peripheral nerve sheath tumors (MPNST) compared to benign neurofibromas (Zou et al. 2009). In a multivariable analysis incorporating both molecular factors and traditional staging criteria, only tumor size and nuclear p53 expression were found to be independent prognosticators of MPNST outcome (Zou et  al. 2009). It has been found that neurofibromatosis-associated and sporadic cases of malignant peripheral nerve sheath tumor are remarkably similar on a molecular level (Holtkamp et al. 2004; Watson et al. 2004). Similar studies examining at malignant progression of chondrosarcoma have shown that this process correlates with an increased expression of genes that facilitate anaerobic metabolism, as well as a decreased production of matrix (Rozeman et al. 2005). This type of study can be used as a diagnostic tool by incorporating highly expressed genes for a particular tumor type, seeking to identify biomarkers that can be tested by immunohistochemistry. Microarray technology cannot only assist in the diagnosis of patients with sarcoma, but can also facilitate prognostication of patient outcomes. For example, analysis of gene expression profiles of Ewing sarcoma patients has led to the identification of a select set of genes that correlate with tumor resistance to chemotherapy in these individuals (Scotlandi et al. 2009). Molecular signatures allow the classification of sarcoma patients into high- and low-risk groups based on their clinical outcome. This classification has a practical value at diagnosis for selecting patients with sarcoma who are unresponsive to current treatments. Approaches such as these may enable definition of sarcoma molecular targets suitable for novel personalized therapy while also helping refine sarcoma staging algorithms. Microarrays have been a critical tool to identify new and relevant soft tissue sarcoma targetable loci. Nielsen et al. 2002 reported expression profiles for GIST involving Kit as a discriminator gene. Microarray approaches have been used to reveal involvement of the retinoic acid pathway in monophasic synovial sarcomas (Nielsen et al. 2003). It has been demonstrated that epidermal growth factor receptor (EGFR) is expressed in monophasic synovial sarcoma while erb-B2 is expressed in epithelial components of the biphasic synovial sarcoma variant (Allander et  al. 2002; Nielsen et al. 2002). The finding was further elaborated in in vitro studies demonstrating that EGFR blockade increased apoptosis, a p53-independent G(0)-G(1) cell cycle arrest, and decreased cyclin D1 expression (Ren et al. 2008). Additionally, in vivo studies using an EGFR tyrosine kinase inhibitor, Iressa plus doxorubicin markedly decreased the cell growth of HT1080 human fibrosarcoma cells in nude mice (Ren et al. 2008). These studies demonstrated that EGFR blockade combined with conventional chemotherapy results in antitumor activity in vitro and in vivo, suggesting the possibility that combining these synergistic treatments will improve sarcoma treatment. It has been demonstrated that ras protein has an active role in tumorigenesis. The ras protein is overexpressed in neurofibromatosis syndrome 1 (NF1), which confers a predisposition to the development of MPNST, a problem which affects as many as 10% of NF1 patients (Woods et al. 2002). It has been shown that inhibition of ras protein trafficking to the plasma cell membrane can be achieved using farnesylation inhibitors (Scappaticci and Marina 2001). Treatment with farnesylation

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inhibitors induces accumulation of cytoplasmic nonfarnesylated H-ras that is able to bind Raf and form inactive cytoplasmic ras/Raf complexes. The use of such targeted inhibitors may be therapeutically relevant in sarcomas overexpressing ras protein. Gene expression in leiomyosarcoma has been evaluated using microarray analysis arrays containing approximately 12,000 known genes and 48,000 expressed sequence tags (Skubitz and Skubitz 2003). Cyclin-dependent kinase (CDK) inhibitor 2A, diaphanous 3, doublecortin, calpain 6, interleukin-17B, and proteolipid 1 were overexpressed in uterine leiomyosarcoma compared with normal myometrium. In contrast several genes were found to be underexpressed in uterine leiomyosarcoma, including alcohol dehydrogenase 1A-polypeptide, alcohol dehydrogenase 1B polypeptide, insulin-like growth factor 1, c-jun, c-fos, and TU3A. Ragazzini et  al. (2004) analyzed whether the genetic amplification of CDK4, MDM2, GLI, and SAS genes of the 12q13-15 region in a group of soft tissue sarcomas correlated with overexpression of the protein products. This group observed that CDK4, MDM2, GLI and SAS were frequently altered and/or highly expressed in leiomyosarcomas and rhabdomyosarcomas, indicating that genes located at 12q13-15 may be important for tumorigenesis of these neoplasms. It was also found that most cases with CDK4, MDM2, or GLI gene alteration also demonstrated simultaneous high level of protein product expression. Conversely, another group found no MDM2 and CDK4 amplification in an additional leiomyosarcoma tissue collection so analyzed (Shimada et al. 2006). Therefore the role of MDM2 and CDK4 in leiomyosarcoma remains controversial and a subject for future elucidation. Another example of how overexpression of a specific protein can modulate tumor to chemoresistance is observed in cells overexpressing the Rad51 protein. Rad51 is believed to have a central role in homologous recombination. Excessive or uncontrolled homologous recombination poses a threat to genome integrity by inducing chromosome fusion, aberrant karyotype formation, and augmenting resistance to DNA damage-induced apoptosis (Raderschall et al. 2002a). Rad51 overexpression is sufficient to promote DNA strand exchange. High levels of Rad51 are associated with elevated rates of DNA recombination as well as enhanced resistance to DNA-damaging chemotherapies and/or ionizing radiation in several experimental tumor systems (Raderschall et  al. 2002b; Vispe et  al. 1998; Xu et al. 2005). Antisense strategies have been successfully used to attenuate Rad51mediated radioresistance in in  vitro and in  vivo (Ohnishi et  al. 1998; Taki et  al. 1996). We have shown that Rad51 protein is overexpressed in primary, recurrent, and metastatic human soft tissue sarcoma specimens of various histologic subtypes (Hannay et al. 2007b). In addition, inhibiting Rad51 expression using anti-Rad51 siRNA markedly increases chemosensitivity to low-dose doxorubicin. Interestingly, reintroduction of wtp53 into a human mutated p53 STS cell line (SKLMS-1 leiomyosarcoma) resulted in decreased Rad51 mRNA and protein expression. Examination of the Rad51 gene promoter suggested a putative activator protein 2 (AP2) binding site. AP2 is known to directly interact with p53. To confirm that AP2 binding to the Rad51 promoter leads to transcriptional repression, we transiently

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transfected SKLMS1 cells with a specific AP2 siRNA, leading to decreased AP2 expression in these cells and simultaneously increased Rad51 expression. The increase in Rad51 after AP2 inhibition in SKLMS1 cells harboring mutated p53 suggests that AP2 can also repress the Rad51 promoter in SKLMS1 cells in a wild type (wt) p53-independent manner. Taken together, these data suggest that p53induced Rad51 transcriptional repression is mediated by the binding of AP2 to the Rad51 promoter. Integration of gene expression profiles and cytogenetic profiles will provide additional information regarding the molecular basis of sarcoma development. While selective inhibition of signaling pathways holds great promise as a new strategy applicable to cancer therapy, it is important to bear in mind that there may be redundancy in signaling pathways or function such that abrogation of one might be obviated by the tumor shifting to an alternative mechanism, leading to therapeutic resistance. Consequently, it is more likely that the combinations of signal transduction inhibitors will lead to maximal and perhaps even synergistic constraints on tumor growth.

4 Gene Therapy Gene therapy offers an attractive approach to control specific genes and short nucleic acid sequences. The strategy is to transfer genetic material or nucleic acid constructs, such as plasmid DNA, viral RNA and DNA vectors, ribozymes, antisense molecules, decoy oligodeoxynucleotides, small interfering RNA (siRNA), and deoxyribozymes (DNAzymes) into cells in an attempt to achieve a therapeutic effect. The transfer of these gene therapy constructs can be achieved via several approaches, including viral-mediated vectors and nonviral-mediated vehicles using liposome delivery or DNA protein complexes. There have been some promising initial experiences utilizing these tools in the treatment of sarcoma.

5 Nonviral Vectors In 1996, Maelandsmo et al. (1996) constructed hammerhead ribozymes to target the calcium protein placental homolog (Cap1) gene, which has a putative role in enhancing the development of human cancer metastasis. This group demonstrated marked reduction of Cap1 transcript levels in vitro utilizing human osteosarcoma cell lines. While they were not able to demonstrate any in  vivo antiproliferation effects, they did observe a decrease in the metastatic burden in xenograft recipients treated with ribozyme targeted to Cap1. Some studies have explored interfering RNA (siRNA) for the treatment of osteosarcoma. For instance, expression of specific Ape1 siRNA was shown to inhibit tumor growth rate by as much as 62% in human osteosarcoma xenografts when

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combined with endostatin, an antiangiogenic agent (Wang et al. 2007). Ape1 was thought to enhance tumor sensitivity to antiangiogenic therapy in this preclinical experimental context. Additionally, in  vitro studies have demonstrated that the treatment with antivascular endothelial growth factor (anti-VEGF) siRNA reduces microvascular growth resulting in apoptosis of osteosarcoma tumors (Mei et al. 2008). The use of siRNA has the theoretical advantage of being resistant to nuclease degradation, thereby potentially allowing slightly longer therapeutic effects (Li et  al. 2006). Arrayed against this possible positive treatment consideration is the reality that naked RNA is usually unable to penetrate cellular lipid membranes and reach the cytoplasm of target cells. Moreover, the short half-life of siRNA and its rapid rate of excretion rate may contribute to the lack of durable effects if used as single application treatment (Dave and Pomerantz 2003). Wang et  al. (2009a) addressed this issue utilizing chitosan nanoparticle-mediated delivery of a short hairpin RNA (shRNA) expressing a vector to inhibit TGFB1 expression in the RD human rhabdomyosarcoma cell line. Knockdown of TGFB1 by shRNA resulted in a decrease in RD cell growth in vitro and a decrease of tumorigenicity in nude mice (Wang et al. 2009a). These results support chitosan nanoparticle-mediated delivery as a potentially valuable gene therapy vehicle. DNAzymes are highly sequence-specific RNases-independent nucleotides which bind and cleave RNA (Breaker and Joyce 1994; Papachristou et al. 2003), and are also a potential tool for cancer therapy. Immunohistochemical studies have indicated increased levels of activated c-Jun and c-Fos in osteosarcoma (Papachristou et al. 2003). Dass et al. (2008a) demonstrated the use of chitosan nanoparticles as delivery system for DNAzyme designed against the c-Jun transcript, resulting in knockdown of c-Jun, thereby inhibiting the osteosarcoma cell growth. The same group was also able to demonstrate that c-Jun knockdown sensitizes osteosarcoma cells to doxorubicin (Dass et al. 2008b).

6 Viral Vectors There have been significant improvements in gene therapy technology involving the development of third-generation lentiviral vectors, adenoviral vectors, and encapsulated methods of delivering naked DNA (Dubensky et al. 2000; Vigna and Naldini 2000). In particular, adenoviral-mediated gene therapy using the tumor suppressor gene p53 has demonstrated promise in preclinical sarcoma models (Milas et al. 2000). Over the years, several strategies targeted in cancer have evolved to administer therapeutic genes that inactivate oncogenes, restore tumor-suppressor genes and enhance the native immune response. Numerous investigations have focused on p53 gene therapy, aiming to restore the important tumor-suppressor functions of this molecule. Thirty to fifty percent of adult soft-tissue sarcomas contain genetic mutations in the p53 gene (Das et  al. 2007; Latres et  al. 1994; Schneider-Stock et  al. 1999). In vitro studies using SKLMS-1 cells, a human-derived leiomyosarcoma cell

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line with a codon 245 p53 point mutation, indicated that stable transfectants expressing wild-type (wt) p53 exhibit reduced tumor growth and clonogenicity, and decreased tumorigenicity in severe combined immunodeficient (SCID) mice (Milas et  al. 2000; Pollock et al. 1998). These results encouraged the examination of p53 gene restoration in sarcomas by treating mice bearing tumors induced by subcutaneous infection of the SKLMS-1 cells with an adenoviral-mediated wt p53 gene delivery system. We were able to demonstrate that wt p53 sensitizes soft tissue sarcoma cells to doxorubicin by downregulating multidrug resistance-1 protein expression (Zhan et  al. 2001). The use of this strategy in conjunction with systemic chemotherapy could conceivably result in synergistic promotion of apoptosis in sarcoma. To test the above in vitro and in vivo findings, our group has developed an isolated limb perfusion (ILP) model in the sarcoma-bearing nude rat (Hannay et al. 2007a). This regional approach allows for the delivery of high gradients of biochemotherapy directly to the tumor in a concentrated intensity that is not feasible using standard systemic drug delivery systems. The feasibility of regional ILP delivery of potentially therapeutic adenovirus-based gene therapy against human soft tissue sarcoma was tested in a soft tissue sarcoma-xenograft nude rat hind limb model, modeling a traditional chemotherapy technique in widespread practice. An incompetent adenovirus bearing FLAG-tagged wild-type p53 and a fiber-modified, replication selective oncolytic adenovirus was administered into human leiomyosarcoma xenografts by ILP, utilizing escalating doses of the constructs. After 72 h of treatment, expression of FLAGp53 was confirmed by reverse transcriptionpolymerase chain reaction. Diffuse upregulation of p21CIP1/WAF1 was observed in ILP-treated tumors. These experiments demonstrated that the reintroduction of functional wild-type p53 into soft tissue sarcoma cells harboring a p53 mutation is possible when delivered via ILP. ILP delivery of therapeutic viruses theoretically has a number of inherent advantages over systemic or intratumoral delivery; these include minimization of systemic exposure to the therapeutic agent so delivered, attainment of higher regional concentrations of the therapeutic agent, and treatment of in-transit disease. This novel gene delivery system may be useful in delivering growth-suppressing constructs to cells bearing p53 mutations, and suggest the possibility of combining p53 gene therapy with current multimodality treatment options to improve sarcoma patient outcomes. Cell cycle regulatory proteins are an additional potential novel target for gene therapy. The CDK inhibitor p27 has been shown to be downregulated in myxoid and round-cell liposarcomas (Oliveira et al. 2000). This low expression of p27 correlated with decreased metastasis-free and overall survival. Theoretically, if the expression of this factor could be increased, an aggressive sarcoma phenotype could potentially be altered. Rexin-G, a target gene therapy vector bearing a cytocidal dominant negative cyclin G1 construct, is currently being tested simultaneously in three phase I/II clinical trials for chemotherapy-resistant metastatic sarcoma, pancreatic cancer, and breast cancer (Chawla et al. 2009). It is also being tested in one phase II study for chemotherapy resistant metastatic osteosarcoma (Chawla et al. 2009), preliminary studies suggesting that Rexin-G is well tolerated and may have activity in controlling tumor growth.

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Transcription factors of the E2F family are essential for cell cycle transition from the G1 to the S phase. While all the E2F transcription factors can induce proliferation and differentiation, only E2F-1 is known to effectively induce apoptosis in a broad variety of cancer cells (Phillips and Vousden 2001). Vorburger et  al. (2005) investigated the efficacy of E2F-1 gene therapy in leiomyosarcoma in vitro and in vivo. A replication-deficient adenovirus carrying the E2F-1 gene (Ad5E2F) was used to induce E2F-1 overexpression in the p53-mutated leiomyosarcoma cell line SKLMS-1. E2F-1 overexpression led to cell apoptosis following infection with Ad5E2F. In vivo experiments revealed that the treatment of SKLMS-1 tumorbearing BALB/c mice with intratumoral injections of Ad5E2F viral particles resulted in significant inhibition of tumor growth compared with control animals; complete disappearance of all tumors was observed in two of seven Ad5E2F-treated mice. Furthermore, these investigations demonstrate that adenovirus-mediated overexpression of E2F-1 resulted in upregulation of the protein kinase PKR, a kinase known to regulate several transcription factors involved in cell growth, differentiation, proliferation, and induction of apoptosis. Several challenges must be confronted when using viral vectors for gene transfer. Viruses are not endogenous to the human body; their introduction can induce the immune system to recognize viral capsid proteins, resulting in adverse immunological side effects (Hartman et al. 2008). Additionally, viral genetic material will not always incorporate in the host genome as desired resulting in oncogenesis, as has been previously observed in several well-known patient episodes (Hacein-BeyAbina et al. 2003; Kaiser 2007; Tack et al. 2006). Despite these adverse events, viral vectors remain attractive due to their high transfection rate and rapid transcription of material that has been inserted into the viral genome. However, there are additional concerns that will need to be addressed prior to the clinical utilization of viral vectors in sarcoma treatment. These include viral proinflammatory effects, technical difficulties inherent in attempting to incorporate foreign DNA into viral genomes, sporadic wild-type mutations of viral constructs, and the potential for oncogenesis.

7 Summary The use of gene therapy for the treatment of sarcomas will be greatly accelerated by an enhanced understanding of the biology of these tumors. Insight into the expression of the antiapoptotic factors and cell cycle regulatory proteins will enable the design of gene therapy strategies to replace or antagonize these factors in sarcoma cells. Selective delivery of genes to sarcoma cells with long-term gene expression remains a great challenge and a frontier for future scientific research. It is also necessary to define and verify more appropriate clinical parameter to assess efficacy in sarcoma treatment. Tumor shrinkage in the form of partial or complete response is the standard measure used to define chemoefficacy. However, since gene therapies may convert cancer into a chronic process without total eradication

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of disease, tumor stabilization, and freedom from progression maybe more meaningful parameters of clinical impact. Discovery of gene expression and cytogenetic profiles may provide additional information regarding the molecular basis of sarcomagenesis while presenting even more specific targets for therapy. More sensitive prognostication and therapeutic decision-making algorithms will require the incorporation of such relevant cytogenetic and molecular determinants. New paradigms of classification, integrating standard clinical and pathological criteria with molecular aberrations, will also facilitate personalized prognosis and even treatment. Moreover, the utilization of novel gene delivery systems such as ILP may be useful in delivering growth-suppressing constructs into soft tissue sarcomas. Hopefully, the use of these novel approaches coupled with current multimodality treatment options may improve the therapeutic options for sarcoma patients, thereby enhancing their overall prognostic outlook.

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Index

A Adenoviruses (Ads) based gene therapy, 194, 261 Delta-24-RGD, 23–24 description, 21–22 E1A products, 22 E2F-1 overexpression, 262 oncolytic virus, 242–243 ONYX-015, 22–23 retargeting adapter-based, 145 Ad5 vectors, 144–145 cancer gene therapy, 143 delivery, adjunct technologies, 149 description, 141–142 gene expression, cellular control, 146–148 genetic modifications, 146 immunotherapy, 143 life cycle and genomic organization, 142–143 virotherapy, 144 Ad-mda-7/IL-24 metastatic melanoma dacarbazine (DTIC), 188 IL-2 based regimens, 188–189 phase II intratumoral injection clinical results, 191 cutaneous lesions, 190 peripheral immune system, 192–193 pro-apoptotic and anti-proliferative effects, 191–192 phase I/II trial gene transfer intralesional injection and multiple biopsies, 189 monotherapy protocol, 189–190 TUNEL assay, 190 Alternative lengthening of telomeres (ALT), 81

Androgen suppression therapy (AST), 34 Antiangiogenesis therapy, lentiviral vectors, 168–169 B Brain tumors ICP47, 209 malignant gliomas causes, 17–18 clinical trials testing, 18, 19 viral vectors, 18 oncolytic viruses adeno, 21–24 HSV-1, 24–25 measles, 26 NDV, 26–27 reovirus, 25–26 replication-deficient viral vectors Ad-p53, 18, 20 HSVtk/GCV gene therapy, 20–21 tk-deficient HSV1, 206 C Cancer gene therapy vectors Ad-based, 143 HSV1 vectors, 207 lentiviruses (See Lentiviruses) Carcinogenesis, 168 Cell-based vaccines allogenic tumor vaccine, 43 GVAX cells, 43–44 immune infiltrates, 43 prednisone, 44 Chemotherapeutic drugs dacarbazine, 188 immune activation, hTERT promoter-driven, 85

269

270 Chemotherapeutic drugs (cont.) vs. myeloprotection, lentiviral vector, 169 stable lentiviral transduction, HCT116 colon cancer cells, 168 SV40 Tag expression, 97 Conditionally replication adenoviral vectors (CRads) advantages, 127 human MSC effect, 127–128 Cytotoxic T lymphocytes (CTLs) generation, MM-bearing mice, 100 induction, IFN-g, 85 interleukins, 126 MHC I expression, 99 peptide-pulsed cells, 237 D DCs. See Dendritic cells Delayed type hypersensitivity (DTH), 43 Delta-24-RGD, 23–24 Dendritic cells (DCs) Ad5 vectors, 149 characteristics, 85 description, 12 human, 239 IL, 126 production, 239 skin-derived (sDC), 167 telomerase vaccination, 238 DOTAP: cholesterol (DOTAP:Chol.) FUS1-expressing plasmid vector, 73 mda7/IL-24, 194–196 systemic therapy, 197 DTH. See Delayed type hypersensitivity E Enzyme/prodrug gene therapy replication-competent advantages, 37 efficacy, 40 oncolytic, 44–45 phase 1 trials, 39 PSADT lengthening, 38 PSA responses, 37 salvage therapies, 38–39 replication-defective biopsy results, 35 HSV-1 TK, 34–35 nitroreductase, 36 osteocalcin promoter, 36 suicide gene therapy, 35

Index F Familial adenomatous polyposis (FAP), 256 FUS1 gene DOTAP:Chol. nanoparticles, 195, 196 inactivation, 72 nanoparticle, 73 3p21.3-deficient NSCLC cells, 72, 73 G Gancyclovir (GCV) cytotoxic gancyclovir triphosphate, 166 hrR3, 208 HSVtk’s effect, 99 HSV1 tumor cells, 205 production, 206 tk gene, 210 Gastrointestinal stromal tumors (GISTs) expression profiles, 256, 257 leiomyosarcoma, 252–253 GCV. See Gancyclovir Gene-directed enzyme prodrug therapy (GDEPT), 20 Gene silencing gene replacement and, 168 and RNAi, 52, 168 siRNA-mediated, 10–11 GISTs. See Gastrointestinal stromal tumors GV1001, telomerase immunotherapy Heptovax, 238 pancreatic cancer, 238 phase II studies, 237 H Herpes simplex virus-1 (HSV-1) clinical trials, 212–213 cyclophosphamide, 221–222 extracellular matrix, 222–223 G207 anti-tumor efficacy, rodents, 210 beta galactosidase, 209–210 humans, 210–211 genome, 204–205 HF10 description, 218–219 paclitaxel, 219 skin nodules, 219–220 HSV1716 adverse events, 217 759 base pair deletion, 211 electron microscopy and DNA fragmentation, 214 gliomas, 215, 216

Index head and neck squamous cell carcinoma, 215, 217 intracerebral inoculation, 215 ICP 47 host immunity, 208–209 US11 viral gene, 209 ICP 34.5/RL1 amino acids, 206 transcriptionally targeted viruses, 207 virus replication, 206–207 NV1020/RV7020 antitumor efficacy, 218 description, 217 oncolytic viruses, 24–25 OncoVex gm-csf doses, 220–221 head and neck, 221 Us11gene, 220 properties, 206 ribonucleotide reductase/ICP 6, 208 TK, 205–206 Herpes simplex virus (HSV) HSV-1, 24–25 HSVtk/GCV gene therapy, 20–21 HSV-1. See Herpes simplex virus-1 hTERT promoter-driven oncolytic adenovirus immune activation chemotherapeutic drugs, 85 DCs, 85 structure E1A/E4, 82 E1B gene, 82 telomelysin replication, 82 in vivo antitumor effects, 83–84 I IFN. See Interferons IFN-b. See Interferon-b IL. See Interleukins ILP. See Isolated limb perfusion Immunotherapy vectors, 143 Interferon-b (IFN-b) melanoma apoptosis, 188 MSC as cellular delivery system, 124 Interferons (IFN), 125–126 Interleukin-24 gene therapy, melanoma Ad mda-7/IL-24 metastatic melanoma, 188–189 phase II intratumoral injection, 190–193 phase I/II trial, advanced solid tumors, 189–190

271 cytokine properties low amino acid sequence homology, 184 skin biology, 185 expression, melanocytes and melanoma immunohistochemical analysis, 185 loss, 186 non-viral nanoparticle-based gene delivery systems clinical trials, 195–196 DOTAP, chol mda7/IL-24, 194–195 tumor suppressor properties Ad-mda/IL-24, intratumoral administration, 183 anti-angiogenic activity, 184 human cancer cells, 182 mda-7/IL-24 gene, 181–182 tumor suppressor, re-expression bystander antitumor effect, 186–187 co-administration, 188 induced cell death, 187 viral delivery phase II trial, 193 recombinant adeno-associated virus, 194 Interleukins (IL) IL-24 gene therapy (See Interleukin-24 gene therapy, melanoma) MSC, 126–127 Isolated limb perfusion (ILP) growth-suppressing constructs, soft tissue sarcomas, 263 regional delivery, 261 L Leiomyosarcoma c-kit expression, 252–253 E2F-1 overexpression, 262 gene expression, 258 human-derived, 260–261 Lentiviral immunotherapy host immune response, 166–167 TAA, 167 Lentiviral vectors, lentiviruses See Lentiviruses applications anti-angiogenesis therapy, 168–169 gene transfer efforts, 166 immunotherapy, 166–167 myelo-protection, chemotherapeutics, 169 replacement and silencing, gene, 168 suicide gene therapy, 166 gene transfer clinical trials, 169–170 HIV-1 derived

272 Lentiviral vectors, lentiviruses See Lentiviruses (cont.) description, 159 design and improvement, 160–163 non-HIV-1, 163–164 packaging system, 160 production, 164–165 pseudotyping, 164 Lentiviruses description, 157 genome and structure function, 158 proteins, 157 life cycle DNA flap, 159 gp120, 158–159 reverse transcriptase, 159 M Malignant mesothelioma (MM) description, 95 IFN-b cytokine effect, 101 Malignant pleural mesothelioma (MPM) anti-angiogenesis, 98 apoptosis induction CRI1 gene, 98 downstream inducers, 97 p16INK4A mutations, 97 p53 mutations, 96–97 REIC/Dickkopf-3 (Dkk-3), 97–98 cytokine gene therapy Ad.IFN-b dose escalation, 105 Ad.IFN vector, 105–106 intratumoral cytokine gene delivery, 104 vero cells, 105 gene therapy, target, 96 immuno-gene therapy anti-mesothelioma effects, 101 intraperitoneal b-galactosidase delivery, 100 protein gene-65 delivery, 100 MM, description, 95 suicide-gene therapy dose-related intratumoral HSVtk gene transfer, 103 E1/E4-deleted adenoviral vector, 104 GCV, 99 HSVtk DNA transfer, 99–100 immunogenic killing, 99 tumor-selective oncolytic viral vectors, replication, 101–102 Measles virus characteristics, 26 live-attenuated, 102

Index Mesenchymal stem/stromal cells (MSC) alternative mesenchymal tissues, 129–130 cell vehicles, cancer, 124–125 chemokines and growth factor antagonists, 128 CRads advantages, 127 human MSC effect, 127–128 fibroblasts and stromal precursors, 117–118 IFN, 125–126 IL, 126–127 migratory factors EGFR, 121–122 wound repair, 121 stem cells antitumor proteins, 123 IFNb, 124 lung tumor nodules, 123 NSC, 122 stroma cancer induced stroma, 116 description, 114 desmoplastic reaction, 116 precusor cells, 120 TAF and EMT, 116–117 target, BM-derived cells, 119–120 TFG-b signaling, 115 suicide genes, 128–129 TRAIL, 129 tropism, wounds and tumors, 118–119 tumor cell-centric, 113–114 MM. See Malignant mesothelioma MPM. See Malignant pleural mesothelioma MSC. See Mesenchymal stem/stromal cells N Newcastle disease virus (NDV), 26–27 O Off-target effects non-specific shRNA, 59 siRNA immunologic effect, 58–59 specific shRNA, 58 siRNA, 58 OncoVex gm-csf doses, 220–221 head and neck, 221 prodrug arming, 222 Us11gene, 220

Index P PDGFR. See Platelet-derived growth factor receptor Personalized cancer therapy delivery strategies, 59–60 RNAi shRNA, 55–57 siRNA, 53–55 signal transduction networks, 52–53 SiRNA vs. shRNA Dicer/Drosha expression, RNAi effector suitability, 57 off-target effects, 58–59 p53 gene adenoviral-mediated gene therapy, 260 cisplatin, 69 gene replacement adenovirus, 66, 67 biomarkers, 67 lung cancer, 65–66 neck cancer, 66 radiation therapy, 69–71 retrovirus vector, 66 therapeutic benefit, 65 mutations, 254 pathway regulation, 65 restoration, sarcomas, 261 PKR. See Protein kinase R Platelet-derived growth factor receptor (PDGFR) differential drug sensitivity, 254 RTKs, 24 Prostate cancer, gene therapy enzyme/prodrug replication-competent adenoviruses, 36–40 replication-defective adenoviruses, 34–36 replication-competent, oncolytic adenoviruses CG7870, 45 CV706, intraprostatic injection, 44–45 vaccine-based cell, 43–44 Poxvirus, 41–43 Protein kinase R (PKR) as anti-viral protective mechanism, 24 cellular, 207 lung cancer cells, 182 mediated translation shutoff, 207 R Radiation therapy, p53 gene replacement adenovirus p53 expressing vector, 70–71 Ad-p53 injection, 70, 71 biopsies, 70

273 NPC, 71 NSCLC, 72–73 Receptor tyrosine kinases (RTKs), 24 Reovirus, 25–26 Replication competent lentiviruses (RCL), 160 Retroviral vector-producing cells (RVPCs), 21 RNA interference (RNAi) biodistribution and target modulation, 10–11 clinical trials, 11–12 delivery strategies, clinical translation, 59–60 gene regulatory therapy description, 2 development, 3 siRNA, 2–3 genetic dysregulation, 2 off-target effects classification, 8–9 non-specific, 9–10 search engines, siRNA design and validation, 9 sequences, siRNA, 9 processing steps characterization, 3 Dicer and Drosha levels, 3, 4 shRNA (See Short hairpin RNA (shRNA)) siRNA chemical modifications, 5, 6 delivery systems, 5, 7 local delivery, 4 minicells, 8 naked, 5 RISC, 53–54 and shRNA, 8 vs. shRNA, 55–59 synthetic, 55 RNA-interfering silencing complex (RISC), 53–54 RTKs. See Receptor tyrosine kinases RVPCs. See Retroviral vector-producing cells S Sarcoma, gene therapy constructs, 259 expression signatures leiomyosarcoma, 258 malignant peripheral nerve sheath tumor, 256–257 microarray technology, 257 profile divisions, 256 Rad51 protein, 258–259 ras protein, 257–258 SKLMS1 cells, 259

274 Sarcoma, gene therapy (cont.) non-viral vectors Cap1 gene, 259 immunohistochemical studies, 260 siRNA use, 259–260 soft tissue, cytogenetics chromosomal aberrations, 256 EWSR1 and CREB1, 255 leiomyosarcoma, 252–253 molecular abnormalities, 253 molecular probes, 252 17p11-12 gain, 253–254 p53 mutations, 254 sporadic desmoids, 255–256 unregulated cell growth, 254–255 telomelysin, 83 viral vectors E2F family, 262 ILP model, 261 p53 gene, 260 Rexin-G, 261 SCLC. See Small-cell lung cancer Short hairpin RNA (shRNA) bi-functional cleavage-dependant and-independent expression, 56–57 expression unit design, 56 dicer-mediated endonucleolytic cleavage, 55 vs. SiRNA comparative efficacy, 57 Dicer/Drosha expression, 57 off-target effects, 58–59 synthesis, 55 Small-cell lung cancer (SCLC), 71–72 Small interfering RNA (siRNA) chemical modifications, 5, 6 chemosensitivity, 258 clinical trials, 11–12 delivery systems, 5, 7 local delivery, 4 minicells, 8 naked, 5 nuclease degradation, resistance, 260 off-target effects classification, 8–9 non-specific, 9–10 search engines, design and validation, 9 sequences, siRNA, 9 osteosarcoma, 259–260 RISC assembly, 53–54 cleavage independent, 54–55 component, 53

Index and shRNA, 8 vs. shRNA comparative efficacy, 57 Dicer/Drosha expression, 57 off-target effects, 58–59 Suicide-gene therapy adenoviral vector, 242 dose-related intratumoral HSVtk gene transfer, 103 E1/E4-deleted adenoviral vector, 104 GCV, 99 HSVtk DNA transfer, 99–100 immunogenic killing, 99 lentiviral vectors, 160 MSC, 128–129 syngeneic murine MM models, 102 T TAA. See Tumor-associated antigens TAF. See Tumor-associated fibroblasts Targeted oncolytic adenovirus Ad5CMV-p53, 80 cytotoxic efficacy, 80–81 description, 79–80 oncolytic vectors, 80 telomerase-specific cancer therapeutics, hTERT promoter-driven, 81–85 clinical application, 88 ex vivo imaging, GFP fluorescence, 86–87 hTERT promoter-driven GFPexpressing, 86 in vivo imaging, GFP fluorescence, 87 transcriptional cancer, telomerase activity ALT, 81 enzyme, 81 Telomerase activity, transcriptional cancer targeting, 81 Ad-hTR-NTR, adenoviral suicide gene therapy vector, 242 anti-telomerase cancer therapy activity, 236 normal somatic cells, 235 associated gene therapy, 242 clinical application, 88 components, human, 233 early cancer detection, 234–235 GFP fluorescence ex vivo imaging, human circulating tumor cells, 86–87 lymph node micrometastasis, in vivo imaging, 87

Index GRN163L, oligonucleotide enzyme inhibitor apoptotic cell death, 241 clinical trial, 241–242 sequence, 239, 241 template region, 239 hTERT promoter-driven GFP-expressing, 86 immune activation, 85 preclinical studies, 82–84 structure, 81–82 immunotherapy dendritic cells, 238–239 GV1001, 237–238 Heptovax, 238 HLA molecules, 236–237 low affinity epitopes, 237 nondendritic cell-based cancer vaccine, 239 specific oncolytic virus adenoviruses, 242 hTERTp-TRAD gene therapy, 243 telomeres (See Telomeres) Telomeres bypass, 233 human, 231–232 length, 232 senescence, 232, 233 shortened, 232–233 Thymidine kinase (TK) description, 205–206 HSVtk/GCV gene therapy, 20 tk-deficient virions, 206 TRAIL. See Tumor necrosis factor related apoptosis inducing ligand Transductional targeting adapter-based, 145 Ad genome, 147 cryptic transcription, 147 DNA regulatory sequences, 147 genetic modifications, 146

275 Translational targeting, 148 Tumor-associated antigens (TAA), 143, 167 Tumor-associated fibroblasts (TAF), 116 Tumor necrosis factor related apoptosis inducing ligand (TRAIL) antitumor effects, 122 MSC, 129 Tumor-specific replication competent Adenoviral (hTERTp-TRAD) gene therapy, 243 Tumor suppressor gene therapy gene replacement chemotherapy, 69 DNA damaging agents, 68–69 p53, radiation therapy, 69–71 lung cancers, 64 malignant growth, 63 metastases cationic immunoliposome system, 73–74 FUS1 gene, 72 nanoscale synthetic particles, 72 NSCLC, 72–73 p53 protein expression, 71 3p21.3 region genes, 73 SCLC, 71–72 p53 gene product, 64–65 gene replacement, 65–68 pathway regulation, 65 protein, 64 V Vaccine-based gene therapy, Poxvirus advantages, 41 recombinant, 42 vaccinations, 41–42 Vesicular stomatitis virus G glycoprotein (VSVG), 164