Chronic Myelogenous Leukemia - A Medical Dictionary, Bibliography, and Annotated Research Guide to Internet References

CHRONIC MYELOGENOUS LEUKEMIA A M EDICAL D ICTIONARY , B IBLIOGRAPHY , AND A NNOTATED R ESEARCH G UIDE TO I NTERNET R E ...

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CHRONIC

MYELOGENOUS LEUKEMIA A M EDICAL D ICTIONARY , B IBLIOGRAPHY , AND A NNOTATED R ESEARCH G UIDE TO I NTERNET R E FERENCES

J AMES N. P ARKER , M.D. AND P HILIP M. P ARKER , P H .D., E DITORS

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ICON Health Publications ICON Group International, Inc. 4370 La Jolla Village Drive, 4th Floor San Diego, CA 92122 USA Copyright 2004 by ICON Group International, Inc. Copyright 2004 by ICON Group International, Inc. All rights reserved. This book is protected by copyright. No part of it may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without written permission from the publisher. Printed in the United States of America. Last digit indicates print number: 10 9 8 7 6 4 5 3 2 1

Publisher, Health Care: Philip Parker, Ph.D. Editor(s): James Parker, M.D., Philip Parker, Ph.D. Publisher's note: The ideas, procedures, and suggestions contained in this book are not intended for the diagnosis or treatment of a health problem. As new medical or scientific information becomes available from academic and clinical research, recommended treatments and drug therapies may undergo changes. The authors, editors, and publisher have attempted to make the information in this book up to date and accurate in accord with accepted standards at the time of publication. The authors, editors, and publisher are not responsible for errors or omissions or for consequences from application of the book, and make no warranty, expressed or implied, in regard to the contents of this book. Any practice described in this book should be applied by the reader in accordance with professional standards of care used in regard to the unique circumstances that may apply in each situation. The reader is advised to always check product information (package inserts) for changes and new information regarding dosage and contraindications before prescribing any drug or pharmacological product. Caution is especially urged when using new or infrequently ordered drugs, herbal remedies, vitamins and supplements, alternative therapies, complementary therapies and medicines, and integrative medical treatments. Cataloging-in-Publication Data Parker, James N., 1961Parker, Philip M., 1960Chronic Myelogenous Leukemia: A Medical Dictionary, Bibliography, and Annotated Research Guide to Internet References / James N. Parker and Philip M. Parker, editors p. cm. Includes bibliographical references, glossary, and index. ISBN: 0-497-00251-5 1. Chronic Myelogenous Leukemia-Popular works. I. Title.

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Disclaimer This publication is not intended to be used for the diagnosis or treatment of a health problem. It is sold with the understanding that the publisher, editors, and authors are not engaging in the rendering of medical, psychological, financial, legal, or other professional services. References to any entity, product, service, or source of information that may be contained in this publication should not be considered an endorsement, either direct or implied, by the publisher, editors, or authors. ICON Group International, Inc., the editors, and the authors are not responsible for the content of any Web pages or publications referenced in this publication.

Copyright Notice If a physician wishes to copy limited passages from this book for patient use, this right is automatically granted without written permission from ICON Group International, Inc. (ICON Group). However, all of ICON Group publications have copyrights. With exception to the above, copying our publications in whole or in part, for whatever reason, is a violation of copyright laws and can lead to penalties and fines. Should you want to copy tables, graphs, or other materials, please contact us to request permission (E-mail: [email protected]). ICON Group often grants permission for very limited reproduction of our publications for internal use, press releases, and academic research. Such reproduction requires confirmed permission from ICON Group International, Inc. The disclaimer above must accompany all reproductions, in whole or in part, of this book.

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Acknowledgements The collective knowledge generated from academic and applied research summarized in various references has been critical in the creation of this book which is best viewed as a comprehensive compilation and collection of information prepared by various official agencies which produce publications on chronic myelogenous leukemia. Books in this series draw from various agencies and institutions associated with the United States Department of Health and Human Services, and in particular, the Office of the Secretary of Health and Human Services (OS), the Administration for Children and Families (ACF), the Administration on Aging (AOA), the Agency for Healthcare Research and Quality (AHRQ), the Agency for Toxic Substances and Disease Registry (ATSDR), the Centers for Disease Control and Prevention (CDC), the Food and Drug Administration (FDA), the Healthcare Financing Administration (HCFA), the Health Resources and Services Administration (HRSA), the Indian Health Service (IHS), the institutions of the National Institutes of Health (NIH), the Program Support Center (PSC), and the Substance Abuse and Mental Health Services Administration (SAMHSA). In addition to these sources, information gathered from the National Library of Medicine, the United States Patent Office, the European Union, and their related organizations has been invaluable in the creation of this book. Some of the work represented was financially supported by the Research and Development Committee at INSEAD. This support is gratefully acknowledged. Finally, special thanks are owed to Tiffany Freeman for her excellent editorial support.

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About the Editors James N. Parker, M.D. Dr. James N. Parker received his Bachelor of Science degree in Psychobiology from the University of California, Riverside and his M.D. from the University of California, San Diego. In addition to authoring numerous research publications, he has lectured at various academic institutions. Dr. Parker is the medical editor for health books by ICON Health Publications. Philip M. Parker, Ph.D. Philip M. Parker is the Eli Lilly Chair Professor of Innovation, Business and Society at INSEAD (Fontainebleau, France and Singapore). Dr. Parker has also been Professor at the University of California, San Diego and has taught courses at Harvard University, the Hong Kong University of Science and Technology, the Massachusetts Institute of Technology, Stanford University, and UCLA. Dr. Parker is the associate editor for ICON Health Publications.

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About ICON Health Publications To discover more about ICON Health Publications, simply check with your preferred online booksellers, including Barnes&Noble.com and Amazon.com which currently carry all of our titles. Or, feel free to contact us directly for bulk purchases or institutional discounts: ICON Group International, Inc. 4370 La Jolla Village Drive, Fourth Floor San Diego, CA 92122 USA Fax: 858-546-4341 Web site: www.icongrouponline.com/health

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Table of Contents FORWARD .......................................................................................................................................... 1 CHAPTER 1. STUDIES ON CHRONIC MYELOGENOUS LEUKEMIA ..................................................... 3 Overview........................................................................................................................................ 3 The Combined Health Information Database................................................................................. 3 Federally Funded Research on Chronic Myelogenous Leukemia ................................................... 4 E-Journals: PubMed Central ....................................................................................................... 59 The National Library of Medicine: PubMed ................................................................................ 62 CHAPTER 2. NUTRITION AND CHRONIC MYELOGENOUS LEUKEMIA.......................................... 111 Overview.................................................................................................................................... 111 Finding Nutrition Studies on Chronic Myelogenous Leukemia................................................ 111 Federal Resources on Nutrition ................................................................................................. 114 Additional Web Resources ......................................................................................................... 114 CHAPTER 3. ALTERNATIVE MEDICINE AND CHRONIC MYELOGENOUS LEUKEMIA ................... 117 Overview.................................................................................................................................... 117 National Center for Complementary and Alternative Medicine................................................ 117 Additional Web Resources ......................................................................................................... 128 General References ..................................................................................................................... 130 CHAPTER 4. DISSERTATIONS ON CHRONIC MYELOGENOUS LEUKEMIA ..................................... 131 Overview.................................................................................................................................... 131 Dissertations on Chronic Myelogenous Leukemia..................................................................... 131 Keeping Current ........................................................................................................................ 131 CHAPTER 5. PATENTS ON CHRONIC MYELOGENOUS LEUKEMIA ................................................ 133 Overview.................................................................................................................................... 133 Patents on Chronic Myelogenous Leukemia .............................................................................. 133 Patent Applications on Chronic Myelogenous Leukemia .......................................................... 141 Keeping Current ........................................................................................................................ 154 CHAPTER 6. PERIODICALS AND NEWS ON CHRONIC MYELOGENOUS LEUKEMIA ...................... 157 Overview.................................................................................................................................... 157 News Services and Press Releases.............................................................................................. 157 Academic Periodicals covering Chronic Myelogenous Leukemia .............................................. 159 CHAPTER 7. RESEARCHING MEDICATIONS .................................................................................. 161 Overview.................................................................................................................................... 161 U.S. Pharmacopeia..................................................................................................................... 161 Commercial Databases ............................................................................................................... 162 Researching Orphan Drugs ....................................................................................................... 163 APPENDIX A. PHYSICIAN RESOURCES .......................................................................................... 167 Overview.................................................................................................................................... 167 NIH Guidelines.......................................................................................................................... 167 NIH Databases........................................................................................................................... 169 Other Commercial Databases..................................................................................................... 171 APPENDIX B. PATIENT RESOURCES ............................................................................................... 173 Overview.................................................................................................................................... 173 Patient Guideline Sources.......................................................................................................... 173 Finding Associations.................................................................................................................. 174 APPENDIX C. FINDING MEDICAL LIBRARIES ................................................................................ 177 Overview.................................................................................................................................... 177 Preparation................................................................................................................................. 177 Finding a Local Medical Library................................................................................................ 177 Medical Libraries in the U.S. and Canada ................................................................................. 177 ONLINE GLOSSARIES................................................................................................................ 183

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Online Dictionary Directories ................................................................................................... 185 CHRONIC MYELOGENOUS LEUKEMIA DICTIONARY................................................... 187 INDEX .............................................................................................................................................. 243

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FORWARD In March 2001, the National Institutes of Health issued the following warning: "The number of Web sites offering health-related resources grows every day. Many sites provide valuable information, while others may have information that is unreliable or misleading."1 Furthermore, because of the rapid increase in Internet-based information, many hours can be wasted searching, selecting, and printing. Since only the smallest fraction of information dealing with chronic myelogenous leukemia is indexed in search engines, such as www.google.com or others, a non-systematic approach to Internet research can be not only time consuming, but also incomplete. This book was created for medical professionals, students, and members of the general public who want to know as much as possible about chronic myelogenous leukemia, using the most advanced research tools available and spending the least amount of time doing so. In addition to offering a structured and comprehensive bibliography, the pages that follow will tell you where and how to find reliable information covering virtually all topics related to chronic myelogenous leukemia, from the essentials to the most advanced areas of research. Public, academic, government, and peer-reviewed research studies are emphasized. Various abstracts are reproduced to give you some of the latest official information available to date on chronic myelogenous leukemia. Abundant guidance is given on how to obtain free-of-charge primary research results via the Internet. While this book focuses on the field of medicine, when some sources provide access to non-medical information relating to chronic myelogenous leukemia, these are noted in the text. E-book and electronic versions of this book are fully interactive with each of the Internet sites mentioned (clicking on a hyperlink automatically opens your browser to the site indicated). If you are using the hard copy version of this book, you can access a cited Web site by typing the provided Web address directly into your Internet browser. You may find it useful to refer to synonyms or related terms when accessing these Internet databases. NOTE: At the time of publication, the Web addresses were functional. However, some links may fail due to URL address changes, which is a common occurrence on the Internet. For readers unfamiliar with the Internet, detailed instructions are offered on how to access electronic resources. For readers unfamiliar with medical terminology, a comprehensive glossary is provided. For readers without access to Internet resources, a directory of medical libraries, that have or can locate references cited here, is given. We hope these resources will prove useful to the widest possible audience seeking information on chronic myelogenous leukemia. The Editors

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From the NIH, National Cancer Institute (NCI): http://www.cancer.gov/cancerinfo/ten-things-to-know.

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CHAPTER 1. STUDIES ON CHRONIC MYELOGENOUS LEUKEMIA Overview In this chapter, we will show you how to locate peer-reviewed references and studies on chronic myelogenous leukemia.

The Combined Health Information Database The Combined Health Information Database summarizes studies across numerous federal agencies. To limit your investigation to research studies and chronic myelogenous leukemia, you will need to use the advanced search options. First, go to http://chid.nih.gov/index.html. From there, select the “Detailed Search” option (or go directly to that page with the following hyperlink: http://chid.nih.gov/detail/detail.html). The trick in extracting studies is found in the drop boxes at the bottom of the search page where “You may refine your search by.” Select the dates and language you prefer, and the format option “Journal Article.” At the top of the search form, select the number of records you would like to see (we recommend 100) and check the box to display “whole records.” We recommend that you type “chronic myelogenous leukemia” (or synonyms) into the “For these words:” box. Consider using the option “anywhere in record” to make your search as broad as possible. If you want to limit the search to only a particular field, such as the title of the journal, then select this option in the “Search in these fields” drop box. The following is what you can expect from this type of search: •

Low-energy Laser Therapy in Oral Mucositis Source: Journal of Oral Laser Applications. 1(2): 97-101. Autumn 2001. Contact: Available from Quintessence Publishing Co., Inc. 551 Kimberly Drive, Carol Stream, Ill. 60188. (630) 682-3223. Website: www.quintessenz.de. Email: [email protected]. Summary: The use of high dose chemotherapy as part of the preparative regimen for stem cell transplantation is associated with mucosal damage. Laser irradiation of the oral mucosa may help to decrease the severity of mucositis. This article reports on a pilot trial that was conducted to evaluate the usefulness of low energy laser therapy in

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the control of pain associated with oral mucositis after stem cell transplantation or high dose chemotherapy. Eleven patients with the diagnosis of chronic myelogenous leukemia (n = 4), non-Hodgkin's lymphoma (n = 3), acute myelocytic leukemia (n = 1), and other malignancies (n = 3) were submitted to high dose chemotherapy for myeloablation. Seven patients received autologous stem cell transplantation, two patients received stem cells from allogeneic sources, and two were given high dose chemotherapy only. The oral cavity of all patients was examined by an oral medicine specialist during the pretransplant and prechemotherapy work up. The patients received irradiation with the mucolaser daily until posttransplant day 5. Mucositis was clinically evaluated according the WHO (World Health Organization) scale, and pain was measured by a visual analog scale (VAS). The laser treatment was well tolerated by the patients. Two patients had mucositis grade I or II, 8 patients had grade III to IV, and 1 patient had none. None of the patients had the maximal pain score. Six patients had grade 0 to 3, and 5 patients had grade 5 to 8 by VAS. The majority of patients associated the daily application of laser with prompt pain relief. The authors conclude that the use of low energy laser therapy may play a role in the control of pain associated with oral mucositis. 3 figures. 2 tables. 23 references.

Federally Funded Research on Chronic Myelogenous Leukemia The U.S. Government supports a variety of research studies relating to chronic myelogenous leukemia. These studies are tracked by the Office of Extramural Research at the National Institutes of Health.2 CRISP (Computerized Retrieval of Information on Scientific Projects) is a searchable database of federally funded biomedical research projects conducted at universities, hospitals, and other institutions. Search the CRISP Web site at http://crisp.cit.nih.gov/crisp/crisp_query.generate_screen. You will have the option to perform targeted searches by various criteria, including geography, date, and topics related to chronic myelogenous leukemia. For most of the studies, the agencies reporting into CRISP provide summaries or abstracts. As opposed to clinical trial research using patients, many federally funded studies use animals or simulated models to explore chronic myelogenous leukemia. The following is typical of the type of information found when searching the CRISP database for chronic myelogenous leukemia: •

Project Title: A NOVEL AUTOTRANSPLANT PROTOCOL FOR CML Principal Investigator & Institution: De Lima, Marcos; Medicine; University of Texas Md Anderson Can Ctr Cancer Center Houston, Tx 77030 Timing: Fiscal Year 2004; Project Start 08-JUN-2004; Project End 31-MAY-2006 Summary: (provided by applicant): Imatinib mesylate has shown remarkable promise in the treatment of chronic myeloid leukemia (CML); however, in some patients it is clearly insufficient as a single agent to produce long-term disease-free or overall survival. AIIogeneic hematopoietic cell transplant-based therapies remain an established curative approach, but complications such as graft-versus-host disease and the need for

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Healthcare projects are funded by the National Institutes of Health (NIH), Substance Abuse and Mental Health Services (SAMHSA), Health Resources and Services Administration (HRSA), Food and Drug Administration (FDA), Centers for Disease Control and Prevention (CDCP), Agency for Healthcare Research and Quality (AHRQ), and Office of Assistant Secretary of Health (OASH).

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a suitable donor limit its application. The use of autografts to support high-dose chemotherapy was being explored extensively prior to the introduction of imatinib and promising results with both in vitro and in vivo-purged autologous cells were reported. In the interim, our understanding of the abnormal biology of CML stem cells has advanced significantly and methods for quantitating different types of transplantable normal and leukemic stem cells and for expanding normal cells with rapid, short-term reconstituting potential have been devised. These advances provide an attractive basis for designing an improved strategy for purging CML stem cells from patients' autografts for use with myeloablative chemotherapy in patients without other therapeutic options. Here we will evaluate three purging methods to selectively eliminate CML stem cells: ex vivo pharmacologic treatment with Gleevec and mafosfamide; a novel culture-purging method that exploits the known intrinsically determined, highly defective self-renewal property of CML stem cells under conditions that are predicted to simultaneously amplify residual normal short-term repopulating cells typically present in many CML patients; and a combination of these. We plan to proceed as rapidly as possible to a clinical test of these approaches, in which we will compare the results obtained when patients are randomly assigned to one of the three purging methods. Key to the success of this project is the commitment of two leading groups with complementary expertise in the biology of normal and CML stem cells and the development and clinical evaluation of cultured autografts. The proposed work will thus build on an exciting history in these areas in an attempt to address an emerging need for innovative and broadly applicable approaches to the treatment of CML prompted by a growing recognition of the limitations of imatinib mesylate as a single therapeutic agent. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: ABNORMALITIES/SIGNAL TRANSDUCTION/HEMATOPOIETIC DISEASE Principal Investigator & Institution: D'andrea, Alan D.; Professor; Dana-Farber Cancer Institute 44 Binney St Boston, Ma 02115 Timing: Fiscal Year 2002; Project Start 23-AUG-1996; Project End 31-JUL-2007 Summary: There is no text on file for this abstract. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: ACTION OF INTERFERON IN CHRONIC MYELOGENOUS LEUKEMIA Principal Investigator & Institution: Platanias, Leonidas C.; Professor of Medicine; Robert H. Lurie Comprehensive Cancer Center; Northwestern University Office of Sponsored Research Chicago, Il 60611 Timing: Fiscal Year 2002; Project Start 01-SEP-2002; Project End 31-AUG-2006 Summary: (provided by applicant): Interferon alpha (IFNa) has significant clinical activity in the treatment of chronic myelogenous leukemia (CML), but the mechanisms by which it exhibits its antileukemic effects remain unknown. We have identified a novel signaling cascade activated by the Type I IFN receptor, involving the small GTPase Rac1 and the p38 Map kinase. This pathway acts independently of the Statpathway, but in cooperation with it, to regulate transcriptional regulation of IFNasensitive genes. Our data demonstrate that this signaling cascade is activated in primary granulocytes from CML patients and that pharmacological blockade of its activation reverses the growth inhibitory effects of IFNa on primary leukemia bone marrow

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progenitors. This proposal is a systematic approach to identify the signaling mechanisms by which IFNa exhibits its antileukemic effects. Specific aim A is to determine the mechanisms of regulation of activation of the p38 pathway by the Type I IFN receptor in BCR-ABL expressing cells and to identify downstream effector mechanisms. Studies will be performed to determine the roles of Jak kinases and the vav proto-oncogene product on the activation of the Rac1/p38 pathway in BCR-ABL expressing cells and to define the role of p38-dependent nuclear histone serine phosphorylation in the induction of IFNa-responses in CML cells. Specific aim B is to determine the biological consequences of activation of p38 in CML. It will involve studies to determine whether Rac1 and p38 are essential for the generation of the growth inhibitory effects of IFNa on primary leukemic progenitors and whether defective activation of this pathway correlates with IFNa-resistance. It will also examine the hypothesis that IFNa downregulates BCR-ABL protein expression via a p38dependent mechanism. Specific aim C includes studies to identify the mechanisms by which the BCR-ABL-tyrosine kinase antagonizes IFNa-dependent gene transcription and determine whether the BCR-ABL specific inhibitor, STI571, augments the growth inhibitory effects of IFNa via regulatory effects on the Rac1/p38 and Jak/Stat pathways. Altogether, these studies should provide important information on the mechanisms by which signals are transduced by the Type 1 IFN receptor in CML cells and advance our knowledge on the mechanisms of development of IFNa resistance. Identifying such mechanisms will facilitate the development of novel therapeutic approaches to overcome IFNa-resistance and the design of new pharmacologic agents for the treatment of CML. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: ACTIVATION & PROLIFERATION OF HEMATOPOIETIC STEM CELLS Principal Investigator & Institution: Lansdorp, Peter M.; Professor of Medicine; British Columbia Cancer Research Centre 601 W 10Th Ave Vancouver, Bc Timing: Fiscal Year 2002; Project Start 01-AUG-1990; Project End 31-MAY-2007 Summary: (provided by applicant): Hematopoietic stern cells (HSC), unlike e.g. embryonic stem cells, have a finite potential to divide. Limitations in the replication potential of HSC appear to be important in hematological disorders such as aplastic anemia and chronic myeloid leukemia. Such limitations could furthermore hamper the development of novel therapeutic strategies, including ex vivo stem cell expansion and gene therapy. Based on these considerations, studies that may help define and extend the replicative potential of HSC are important and a general interest. Previous studies with purified human "candidate" HSC funded by this grant have shown that the functional properties of HSC change dramatically during ontogeny and that the loss in HSC proliferative potential with age correlates with measurable shortening of telomeres. Here we propose to further examine the role of telomerase and telomeres in hematopoiesis. Specifically, we want to test the hypothesis that the replication history of HSC can be traced by studies of their telomere length. In order to test this hypothesis, we will examine the telomere length in subsets of purified HSC and their cultured progeny relative to defined populations of more mature cells using refined flow cytometry techniques developed in our laboratory. These techniques will also be used to further study the age related decline in telomere length in nucleated blood cells from normal individuals, patients with various hematological disorders and pedigrees of genotyped normal baboons. We will furthermore attempt to manipulate the telomere length in HSC using gene transfer and protein transduction strategies and study the

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functional properties of cells with extended telomeres in vitro and in vivo. The specific aims are:1) To study the telomere length in purified HSC and nucleated blood cells from normal individuals and patients with various hematological disorders before and after therapy.2) To study the telomere length in nucleated blood cells from baboons in relation to their age and genotype.3) To study the effect of artificial telomere elongation and telomerase inhibition on the proliferation, differentiation and replicative potential of purified "candidate" HSC in vitro and in vivo.Taken together, these studies will provide crucial baseline information on the role of telomeres in the biology of HSC. Such information is relevant for a basic understanding of hematopoiesis as well as applications of stem cells in and outside hematology. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: ACTIVATION OF SIGNAL TRANSDUCTION PATHWAYS IN STABLE PHASE CHRONIC MYELOID Principal Investigator & Institution: Griffin, James D.; Professor; Dana-Farber Cancer Institute 44 Binney St Boston, Ma 02115 Timing: Fiscal Year 2002; Project Start 01-AUG-2002; Project End 31-JUL-2007 Summary: (provided by applicant): Chronic myeloid leukemias are caused by activated tyrosine kinase oncogenes, most often by BCR/ABL, or the related oncogenes TEL/ABL, TEL/JAK2, or TEL/PDGFR. The goal of this project is to understand in detail the signal transduction pathways activated by BCR/ABL and related oncogenes that are relevant for transformation of hematopoietic cells. Using BCR/ABL as the best-studied example, this kinase is believed to function by phosphorylating itself and adjacent cell proteins, and by phosphorylating other proteins that are brought in by adapter molecules. This results in activation of a variety of signaling pathways that ultimately block apoptosis, deregulate cell cycle control, alter adhesion and homing, and cause genetic instability. A particular focus of this project period will be phosphotidylinositol signaling, which we and others have shown is required for transformation, probably because of prominent effects on apoptosis and cell cycle deregulation. We would like to understand how PI3K is activated and determine the downstream targets particularly those related to viability signaling. Also, in preliminary studies we have shown that SHIP, an inositol 5-phophatase, is downregulated by BCR/ABL. SHIP activity would be expected to modulate the lipids that accumulated downstream of PI3K. This is of interest since a SHIP knock out mouse develops a myeloproliferative syndrome, suggesting that there may be certain PI3K products that are more important for hematopoiesis than others. Finally, efforts will be focused on understanding the differences in signaling by the 3 known forms of BCR/ABL, encoding p190, p210, or p230; and understanding the differences between BCR/ABL and v-ABL. In particular, pathways initiated because of phosphorylation of Y177 of BCR seem to be of particular interest, as this single tyrosine residue is needed to generate a myeloproliferative disorder in mice. Overall, identification of critical signaling intermediates will be useful for many reasons, but particularly to identify potential targets for drug development, especially for drugs that would be synergistic with STI571. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: ALLOIMMUNITY TO CML IN A NOVEL MURINE MODEL Principal Investigator & Institution: Shlomchik, Warren D.; Assistant Professor; Internal Medicine; Yale University 47 College Street, Suite 203 New Haven, Ct 065208047 Timing: Fiscal Year 2002; Project Start 01-SEP-1998; Project End 31-AUG-2003

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Summary: (Applicant's Description): This proposal outlines a 5 year plan for the candidate to develop the necessary skills and experience to become an independent investigator studying the alloimmunology of bone marrow transplantation. Dr. Shlomchik is board certified in Internal Medicine and Hematology, and is board eligible in Oncology. He has been in the sponsor's lab for 3 years studying Graft-vs.Host Disease (GVHD), T cell tolerance post alloBMT, and methods of gene transfer into human T cells. Chronic myelogenous leukemia (CML) is uniquely sensitive to T cell alloimmune therapies, yet the scientific basis for CML's remarkable susceptibility is unknown. Understanding the details of this anti-CML response, including the nature of antigen presentation, critical effectors, and factors that render CML cells vulnerable to immune attack, may facilitate the development of strategies to enhance alloimmune responses against other less immunogenic neoplasms. The identification of factors that render CML sensitive have been impeded by the absence of: 1) tools needed to isolate individual components of alloimmune responses; and 2) a relevant murine CML model. The first obstacle can now be approached using mice deficient in genes critical for antigen presentation and T cell effector function. For example, the applicants have exploited mice lacking the beta-2-microblobulin gene to demonstrate that radioresistant host cells are critical for initiating GVHD responses. The second problem has been addressed by a new CML murine model wherein bone marrow infected ex vivo with retrovirus expressing the bcr-abl fusion cDNA is transferred into irradiated syngeneic recipients. They will combine these approaches to determine: 1) the identity of anti-CML effectors; 2) the identity of antigen presenting cells in anti-CML responses, and 3) the roles of molecules accessory for target cell vulnerability such as Fas and TNF-R by establishing CML in mice genetically deficient in them. Dr. Shlomchik's sponsor, Dr. Emerson, is chief of the hematology and oncology section. He has a Ph.D. in immunology and was scientific director of the alloBMT program at the University of Michigan. He has launched a new alloBMT program at Penn and is committed to Dr. Shlomchik's development into an independent clinician scientist. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: ANTILEUKEMIA ACTIVITY OF PERILLYL ALCOHOL Principal Investigator & Institution: Clark, Steven S.; Associate Professor; Human Oncology; University of Wisconsin Madison 750 University Ave Madison, Wi 53706 Timing: Fiscal Year 2002; Project Start 01-JAN-2000; Project End 31-DEC-2003 Summary: The Bcr/Abl oncogene encodes a tyrosine kinase that is expressed in leukemias that carry the Philadelphia chromosome translocation (Ph+). The kinase interacts with different cell signaling pathways to cause factor-independent growth, resistance to apoptosis and oncogenic transformation. These pleiotropic activities of Bcr/Abl affect the pathogenesis of Ph+ leukemias by inhibiting the normal rate of cell death and by enabling Ph+ cells to resist conventional chemotherapy that induces apoptosis in other leukemias. A central hypothesis of this proposal is that inhibition of signaling pathways downstream of the Bcr/Abl kinase should render leukemia cells dependent on growth factors and sensitive to apoptosis. Perillyl alcohol (POH) belongs to a new family of chemotherapy agents and has shown excellent therapeutic rations in rodent carcinoma models. The range of potential anti-tumor activities of POH overlaps with some signaling pathways that are affected by the Bcr/Abl kinase. Thus, POH is a logical compound to test for antileukemia activity in Bcr/Abl-induced leukemia. Preliminary experiments demonstrated that in Bcr/Abl-transformed cells, POH rapidly induced G1 arrest and apoptosis. In contrast, Bcr/Abl- transformed cells were resistant to apoptosis induced by different conventional chemotherapy agents. This anti-leukemia

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activity of POH closely correlated with inhibition of the Raf-ERK signaling pathway downstream of Bcl/Abl. On the other hand, POH treatment did not affect other Bcr/Abl signals that are responsible for maintaining expression of c- Myc. Normally, expression of c-Myc is cell cycle regulated, however, when c-Myc expression is enforced during G1 arrest, cells undergo apoptosis. Therefore, POH may uncouple a Bcr/Abl signaling pathway through Raf that is necessary for maintaining cell growth, while not affecting other Bcr/Abl signals that induce constitutive c-Myc expression. This combination may lead to apoptosis in leukemia cells. This model will be evaluated further by examining 1) the role of the Bcr/Abl oncogene in sensitizing cells to POH, 2) how POH affects signaling through Raf, and 3) whether POH induces Myc-dependent apoptosis in leukemia cells. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: ARSENIC BASED THERAPY OF BCR ABL POSITIVE LEUKEMIAS Principal Investigator & Institution: Bhalla, Kapil N.; Professor of Medicine; Moffitt Cancer Center; University of South Florida 4202 E Fowler Ave Tampa, Fl 33620 Timing: Fiscal Year 2002; Project Start 15-JUN-2001; Project End 31-MAY-2004 Summary: The leukemic clone in virtually all of the patients with chronic myeloid leukemia with blast crisis (CML-BC) and approximately one-third of the adults with acute lymphoblastic leukemia (ALL) expresses the bcr-abl fusion gene encoded p210 and p185 Bcr-Abl tyrosine kinase (TK), respectively. Although the chemotherapeutic regimens or bone marrow stem cell transplantation employed against acute leukemias can also produce complete remissions in Bcr-Abl positive ALL and CML-BC, these remissions are not durable and the overall clinical outcome remains dismal. This creates a strong rationale to test novel strategies in this clinical setting. Arsenic Trioxide (As2O3 or AT) is clinically active against relapsed or refractory acute promyelocytic leukemia (APL), where it downregulates the levels of the fusion protein PML-RARdelta causing partial differentiation and apoptosis of APL cells. Recently, we have demonstrated that clinically achievable levels of AT can also reduce p210 or p185 Bcr-Abl fusion protein levels and induce apoptosis of CML-BC cells. Based on these findings, we propose to conduct a Phase II clinical-pharmacologic trial of AT (NCI/CTEP sponsored) as the induction therapy for relapsed and refractory; Bcr-Abl positive adult ALL and CML-BC. In vitro studies on the patient derived leukemic blasts are proposed to determine the molecular correlates of the clinical response and apoptosis induced by AT. We have also shown that STI571, a relatively specific inhibitor of Bcr-Abl TK activity, induces differentiation and apoptosis of Bcr-Abl positive leukemic cells. Therefore, we also propose to investigate the in vitro apoptotic and differentiation effects of a combination of AT and STI571 in Bcr-Abl positive leukemic blasts. The specific aims of this proposal are: AIM 1: To determine the clinical efficacy, i.e., the rate and duration of clinical and hematologic response and overall survival, secondary to treatment with daily intravenous AT in adult patients with Philadelphia chromosome (bcr-abl fusion gene) positive relapsed or refractory ALL or CML-BC. AIM 2: To determine the pharmacokinetic parameters of AT, i.e., AUC and Css and correlate these with the clinical and cytogenetic response in patients with Bcr-Abl positive ALL or CMLBC. AIM 3: To correlate the clinical response to AT with the decline in the bcr-abl mRNA levels determined by real-time RT-PCR. AIM 4: To correlate the clinical and in vivo molecular response to AT with AT-induced in vitro downregulation of Bcr-Abl and Akt protein levels, histone hyperacetylation as well as differentiation and apoptosis, utilizing the pre-treatment samples of leukemic blasts. AIM 5: To determine the in vitro differentiation and apoptotic effects of STI-571 alone and in combination with AT in the

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pre-treatment samples of Bcr-Abl positive leukemic blasts. These in vitro and in vivo studies are designed to evaluate AT-based novel strategies against Bcr-Abl positive human leukemias. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: ASSAYING TYROSINE KINASE ACTIVITY: A NEW PARADIGM Principal Investigator & Institution: Sims, Christopher E.; Physiology and Biophysics; University of California Irvine Irvine, Ca 926977600 Timing: Fiscal Year 2004; Project Start 16-APR-2004; Project End 31-MAR-2005 Summary: (provided by applicant): New approaches are needed in the molecular analyses of the growth-promoting signal transduction pathways involved in the development and maintenance of cancer. The Laser Micropipet System (LMS), a new technology for the biochemical assay of kinases, holds the promise of directly assaying the activities of protein tyrosine kinases in primary patient cells. The LMS was developed for the assay of serine/threonine kinases in individual cells grown in tissue culture. This R21/R33 application proposes to expand the applications of this instrumentation to directly determine the activity of oncogenic tyrosine kinases in primary cells from patients. Assay of Bcr-Abl in chronic myelogenous leukemia (CML) will demonstrate the power of this tool for research, clinical, and pharmacologic applications. While this assay will be of value in the study of the biology and resistance mechanisms in CML, the studies will lay the groundwork for a new paradigm in the study of molecular mechanisms in cancer signal transduction and will have general applicability. The R21 phase of this proposal is designed to establish the feasibility of assaying Bcr-Abl activity and to demonstrate the necessary assay conditions for measurements in cells derived from a patient with CML. The specific aims are to: 1) identify and characterize a reporter of intracellular Bcr-Ab1 kinase activity, 2) define and optimize conditions for measuring Bcr-Ab1 activity in human cells, 3) compare reporter phosphorylation in cells expressing Bcr-Ab1 vs. cells lacking this kinase, and 4) demonstrate the feasibility of using primary patient cells in kinase assays. The goal of the R33 phase is to demonstrate the potential for the LMS-based kinase assay in applications for the study of the biology of pharmacologic resistance in CML, including from primary patient cells. The R33 specific aims are to: 1) develop a membranepermeant Bcr-Ab1 kinase reporter, 2) demonstrate the ability to detect imatinib resistance in CML cell lines, 3) determine Bcr-Ab1 activity in primary cells from patients with CML and their response to pharmacologic inhibition, and 4) determine if the assay can detect the emergence of imatinib resistance in CML patients and can differentiate the mechanisms of drug resistance. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: ATTACKING ANTHRAX ACTION BY BLOCKING RECEPTOR SIGNALING Principal Investigator & Institution: Chan, Joanne; Research Associate & Instructor; Dana-Farber Cancer Institute 44 Binney St Boston, Ma 02115 Timing: Fiscal Year 2003; Project Start 30-SEP-2003; Project End 31-AUG-2005 Summary: (provided by applicant): Application for R21, NOT-AI-02-023, Biodefense and Emerging Infectious Diseases Research Anthrax toxin, isolated from the bacterium, Bacillus anthracis, enters human cells and disrupts cellular function. The toxin consists of a receptor binding component, protective antigen (PA) which can associate with the enzymatic components, edema factor (EF) and/or lethal factor (LF) to form the anthrax

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toxin. The toxin binds a cell surface receptor which mediates the internalization of the toxin complex. Recently, work from the laboratories of John Young and John Collier identified the receptor for the anthrax toxin, named the anthrax toxin receptor (ATR; Bradley et al., 2001). Since the ability of the anthrax toxin to cause harm is dependent upon receptor binding, targeting the anthrax receptor might provide additional therapies that could be useful even after suspected exposure to anthrax spores. In many cases, signaling downstream of cell surface receptors involves the activation of protein and lipid kinases. Successful use of the small molecule kinase inhibitor Gleevec in chronic myelogenous leukemia has shown that targeting kinases might provide an efficient means to treat various diseases. This goal of this project is to study the ATR signaling pathway in order to identify key effector molecules as targets for inhibition. The role of ATR in mediating toxin internalization will be dissected using biochemical and immunofluorescence methods in mammalian cells. The zebrafish will be used as an animal model to study ATR function in vivo and to assess its potential as an animal model for testing drugs aimed at blocking toxin action. The applicant has 3 specific aims which will be undertaken in collaboration with the Young and Collier labs. Aim 1. To investigate the role of the cytoplasmic domain of the anthrax receptor for potential therapeutic intervention. Aim 2. To determine the physiological role of the anthrax receptor by functional interference during normal embryonic development in the zebrafish. Aim 3. To examine anthrax toxicity in the zebrafish for use as an additional inexpensive model system for drug or vaccine testing. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: BCR/ABL SIGNALING IN HUMAN LEUKEMIAS Principal Investigator & Institution: Pendergast, Ann M.; Associate Professor; Pharmacology and Cancer Biology; Duke University Durham, Nc 27710 Timing: Fiscal Year 2002; Project Start 15-JUN-1994; Project End 31-JAN-2004 Summary: The long-term objective of this research is to identify the intracellular signaling pathways that are critical for oncogenic transformation by altered forms of the ABL tyrosine kinase. Oncogenic forms of ABL are linked to the development of human, murine and feline leukemias. Activation of ABL may occur as a consequence of chromosomal translocations. The chimeric BCR-ABL oncogene is produced by a reciprocal chromosomal translocation that fuses varying amounts of the BCR gene on chromosome 22 with sequences upstream of the second exon of the c-ABL gene on chromosome 9. Three different BCR-ABL proteins may be produced: P210 which is the causative agent of greater than 95 percent of chronic myelogenous leukemia (CML). P185 which is associated with a subset of acute lymphocytic leukemias (ALL), and P230 which is associated with chronic neutrophilic leukemia (CNL), a rare myeloproliferative disorder characterized by a mild hematologic phenotype. The P185 and P210 forms of BCR-ABL have been proposed to transform cells through their ability to enhance cell proliferation, block apoptosis, alter cell adhesion and increase cell motility. Multiple proteins have been identified as downstream targets of BCR-ABL. However, only a small subset of these proteins have been shown to play critical roles in the biological activities associated with BCR-ABL expression. The specific aims of this proposal are: 1) to test the hypothesis that the ubiquitin-dependent degradation of specific cellular proteins by the oncogenic BCR-ABL tyrosine kinases constitutes a novel mechanism for the functional inactivation of growth inhibitors/tumor suppressors, and 2) to identify intracellular signaling pathways that are differentially regulated by the oncogenic forms of BCR-ABL (such as P210) and by the weakly leukemogenic P230 protein that is associated with an indolent or benign clinical disease. Comparative analysis of the BCR-

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ABL proteins may allow the identification of critical molecular components required for malignant transformation by BCR-ABL. Furthermore, our finding that oncogenic tyrosine kinases trigger the destruction of specific target proteins via the ubiquitin proteasome machinery provides a potentially important pathway for the elimination of growth inhibitors/tumor suppressors during tumor progression. Definition of this pathway may allow for the development of therapeutic reagents for the treatment of leukemias and other cancers that are caused by the activation of nonreceptor tyrosine kinases. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: BCR-ABL KINASE ASSAYS FOR STI571 SENSITIVITY OF RESPONSE Principal Investigator & Institution: Kron, Stephen J.; Associate Professor; Molecular Genetics & Cell Biol; University of Chicago 5801 S Ellis Ave Chicago, Il 60637 Timing: Fiscal Year 2003; Project Start 17-JUL-2003; Project End 31-DEC-2004 Summary: (provided by applicant): This phased innovation award proposal is focused on developing a robust approach to quantitative assay of specific protein tyrosine kinase activities from cancer cells. Our model is the oncogenic BCR-ABL fusion protein, the gene product of the t(9;22) Philadelphia chromosome (Ph1) translocation observed in the vast majority of Chronic Myelogenous Leukemia (CML) and in up to 30% of adults with Acute Lymphoblastic Leukemia and in other hematological neoplastic diseases. The activation of Abl kinase by fusion to BCR that is inferred to underly the malignant transformation of Phl positive CML is effectively opposed by the orally administered tyrosine kinase inhibitor (TKI) Imatinib Mesylate (IM, STI-571, Gleevec). The activity of IM as an Abl kinase inhibitor in vitro is thought to be the critical determinant of its efficacy in vivo. Nonetheless, a clinically useful assay for IM inhibition of BCR-ABL kinase activity in circulating CML leukemia cells is lacking. We propose to develop a protein/peptide chip-based assay for BCR-ABL that can detect the degree of inhibition by IM to evaluate dosing and drug resistance. Insofar as other activated tyrosine kinases may be critical mediators of malignancy in both leukemias and solid tumors, developing such an assay would be a powerful tool in evaluating other TKI drug candidates targeting these kinases for their efficacy in vivo. Thus, this project is directed at two major discovery objectives and three development objectives. First, in the initial project year, we intend to use our established methods for anti-phosphotyrosine antibodybased detection of purified Abl kinase activity on a peptide chip to 1) Recapitulate our Abl kinase assay with undiluted whole cell extracts from cell lines expressing BCR-ABL and 2) Use this assay to measure the inhibition of BCR-ABL by IM both in extracts and intact cells. During the development phase, we intend to use both BCR-ABL expressing cancer cell lines and circulating leukemic cells from treated patients as samples to 1) Optimize the BCR-ABL substrate and reaction conditions to enhance sensitivity and specificity of phosphorylation, 2) Examine alternative detection methods for BCR-ABL activity based on phosphospecific antibodies and thiophosphate targeted chemistry, and 3) Evaluate different chemistries for immobilizing BCR-ABL substrates on a surface and geometries for detection of phosphorylation. By these aims we intend to develop a highly versatile kinase assay system which can be applied to monitoring of patient response to IM and as a tool for discovery and testing of new TKI cancer drugs. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: BCR-ABL RNA LEVELS TO MONITOR STI571 LEUKEMIA THERAPY Principal Investigator & Institution: Press, Richard D.; Pathology; Oregon Health & Science University Portland, or 972393098

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Timing: Fiscal Year 2003; Project Start 14-FEB-2003; Project End 31-JAN-2005 Summary: (provided by applicant): The recent discovery, validation, and accelerated FDA approval of the tyrosine kinase inhibitor STI571 (Gleevec) for the treatment of chronic myeloid leukemia (CML), the first such rationally-designed, small molecule cancer therapy, confirms the validity of the molecular targeting approach to cancer drugs. Aberrant CML cell growth is caused by the constitutive expression of the bcr-abl kinase, a chimeric fusion protein resulting from a leukemia-specific chromosomal translocation [t(9;22)]. STI571's targeted inhibition of this kinase activity specifically represses the leukemic clone without the typical toxicity of conventional nonspecific cancer drugs. Although >95% of chronic phase CML patients undergo a complete hematologic response to STI571, only a minority experience a complete cytogenetic response, the best current surrogate marker of long-term survival. After 18 months of daily STI571 therapy, approximately 28% of chronic phase patients lose their complete hematologic response. STI571 also induces an initial hematologic response in approximately 50-80% of CML patients with accelerated phase or blast crisis CML, but again these responses are typically only transient. This heterogeneity of responses suggests the pressing need for better laboratory methods to monitor and predict STI571 treatment efficacy to identify patients likely to have disease progression who may benefit from additional (more toxic) therapies. The considerable institutional resources in expertise and STI571-treated patient volume that have been built by co-investigator and STI571 co-developer Brian Druker represent a unique opportunity to develop and test novel laboratory methods to molecularly monitor STI571's treatment efficacy. This proposal's specific aims are then to: 1) validate a novel, quantitative, ultra-sensitive bcrabl RT-PCR assay for quantifying minimal residual disease in STI571-treated patients, 2) assess whether serial bcr-abl RNA levels in STI571-treated patients can be used to predict and/or monitor clinical disease progression, and 3) establish a storage bank of clinical samples from STI571-treated patients to enable future studies of STI571 drug resistance mechanisms. As STI571 is the first of perhaps many soon-to-come molecularly targeted cancer therapies, the successful achievement of these aims will likely serve as the model by which the efficacy of other novel therapies may be similarly monitored. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: BCR-ABL SUBSTRATES AND INHIBITORS Principal Investigator & Institution: Clarkson, Bayard D.; Chief; Sloan-Kettering Institute for Cancer Res New York, Ny 100216007 Timing: Fiscal Year 2003; Project Start 10-JUL-2003; Project End 30-JUN-2008 Summary: It is the broad, long-term goal of this project to understand, on a molecular level, how the p210- bcr-abl protein tyrosine kinase activity ultimately causes a myeloid expansion in chronic phase Chronic Myelogenous Leukemia (CML), and to search for selective therapies efficacious for the treatment of CML. In order to accomplish this, the following Aims are proposed: (1) Expression of Bcr-Abl and its substrates in human primary hematopoietic cells will be determined during hematopoietic differentiation and maturation. CD34+ normal and CML progenitor cells and subpopulations will be stimulated with single and multiple cytokines that induce proliferation and maturation. Expression of Bcr-Abl and its substrates will be assessed and effects of specific inhibitors. (2) The role of SHIP-1 and SHIP-2 in Ber-Abl signaling will be examined. SHIP proteins will be constitutively targeted to the plasma membrane and the effects on Bcr-Abl mediated signaling will be determined in Rat1p210 cells. (3) The role of Dok-1 in c-kit signaling will be determined. The abilities of wt and mutant forms of Dok proteins to interfere with c-kit signaling will be analyzed. The kinase(s) that phosphorylates Dok

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during c-kit stimulation will be identified as well as the site(s) of tyrosine phosphorylation. (4) Binding partners for SHIP proteins in normal and Bcr-Abl expressing hematopoietic cells will be isolated and identified. (5) Analogues of PD173955, a potent inhibitor of Bcr-Abl kinase, will be analyzed in vitro and in vivo. In collaboration with Drs. William Bornmann and John Kuriyan, we will design and synthesize PD173955 analogues and test their activity in hematopoietic cells in vitro and in animal models of CML. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: BIOLOGY AND TRANSPLANTATION OF THE HUMAN STEM CELL Principal Investigator & Institution: Mcglave, Philip B.; Professor and Director; Medicine; University of Minnesota Twin Cities 200 Oak Street Se Minneapolis, Mn 554552070 Timing: Fiscal Year 2002; Project Start 25-AUG-1995; Project End 31-MAY-2005 Summary: The them of this competitive renewal is unchanged: Understanding the biology of human hematopoietic stem cell (HSC) and their progeny will lead to improved stem cell transplantation therapy for a variety of methal malignant and nonmalignant diseases. In Project 1, Dr. Catherine Verfaillie will perform in vitro and xenogeneic transplant experiments to determine how apoptosis, differentiation and proliferation affect symmetrical self-renewal of HSC. She will use this information to develop clinically relevant methods for HSC expansion and gene transfer applicable to clinical trials proposed in Projects 2 and 3. In Project 2, Dr. John Wagner will investigate engraftment of ex vivo expanded umbilical cord blood (UCB) HSC in a xenogeneic transplant model. He will conduct human transplantation trials using a single UCB graft containing a subpopulation of ex vivo expanded and genetically marked UCB. Alternatively, transplants will be performed with two HLA-identical UCB grafts-one expanded and genetically marked UCB. Alternatively, transplants will be performed with two HLA-identical UCB grants-one expanded ex vivo and one unmanipulated These studies will determine if ex vivo expansion of HSC has been achieved and what effects ex vivo expansion have on homing and engraft of HSC to the marrow. In Project 3, Dr. Daniel Weisdorf will examine how mobilization regimen, cell source (marrow or blood) and phenotype (CD34+ or CD34-) influence HSC capacity for expansion, gene transfer and transplantation. These studies will be performed in vitro, in mouse and sheep xenogeneic transplant models and in clinical trials of autologous transplant therapy for lymphoma. Human clinical transplant trials performed in PROJECTS 2 and 3 will also validate the use of surrogate in vitro and in vivo HSC assays. In Project 4, Dr. Jeffrey Miller will investigate the contribution of BCR/ABL-mediated defective natural killer based immune surveillance and therapy in chronic myelogenous leukemia (CML). He will then test our immunotherapy with normal, allogeneic NK cells following autologous HSC transplant therapy for CM;. Finally, our group has demonstrate that marrow stromal cells can be derived in vitro from a mesodermal progenitor cell (MPC), and that in Hurler syndrome MPC- derived stromal cells contain abnormal heparan sulfate (HS) which inhibits normal HSC growth. In Project 5, Dr. Charles Peters and his co- investigators will explore the role of abnormal HS in Hurler stroma on defective hematopoietic support. He will also perform clinical trials in which normal, allogeneic MPC are transplanted in combination with hematopoietic cells (MPC/HCT) in order to "cross/correct" Hurler stromal defects and improve HSC engraftment and clinical outcome. These projects are supported by administrative and biostatistical cores, as well as cores to provide cell collection and processing NOD/SCID and other mouse assays and production of vectors suitable of clinical gene transfer.

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Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CAAX PROCESSING ENZYMES AS ANTICANCER TARGETS Principal Investigator & Institution: Bergo, Martin O.; J. David Gladstone Institutes Box 419100, 365 Vermont St San Francisco, Ca 94103 Timing: Fiscal Year 2004; Project Start 01-APR-2004; Project End 31-MAR-2006 Summary: (provided by applicant): Many intracellular signaling proteins (e.g., the Ras and Rho proteins) and several nuclear lamins terminate with a carboxyl-terminal CAAX motif. CAAX proteins undergo three sequential posttranslational modifications. First, the cysteine (i.e., the C of the CAAX motif) is farnesylated or geranylgeranylated by a pair of cytosolic enzymes--farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I). Second, the last three amino acids (i.e., the -AAX) are cleaved off by Ras and a-factor converting enzyme (Rcel), an integral membrane protease of the endoplasmic reticulum (ER). Third, the newly exposed carboxyl-terminal isoprenylcysteine is methylated by another ER protein, isoprenylcysteine carboxyl methyltransferase (Icmt). These posttranslational modifications render the C-terminus of CAAX proteins more hydrophobic, enhancing the attachment of the proteins to membrane surfaces and facilitating certain protein-protein interactions. Activating Ras mutations have been detected in 30% of all human cancers, and are common in leukemia and myeloproliferative diseases. Inhibitors of FTase have been used to treat cancers that harbor mutationally activated Ras proteins. Unfortunately, K-Ras and N-Ras--the Ras isoforms most often implicated in human cancers--are readily geranylgeranylated by GGTase I in the setting FTase inhibition. This alternate isoprenylation pathway has focused attention on other enzymes in the pathway, such as GGTase I, Rcel, and Icmt. Surprisingly, there are no data on the impact of inhibiting these other enzymes on the development of cancer in mice. In this project, this void will be addressed. In preliminary studies, mice harboring both a Cre-inducible latent oncogenic Kras2 allele (KrasLsL) and the inducible Mx1-Cre transgene have been generated. Induction of Cre in those mice activates the latent Ras allele and results in a full-fledged, lethal, myeloproliferative disease that is reminiscent of chronic myelogenous leukemia or juvenile myelomonocytic leukemia in humans. Recently, conditional "floxed" alleles for the posttranslational processing enzymes (FTase, GGTase I, Rcel, and Icmt) have been generated. Thus, it is now possible to breed mice in which Cre expression can be used to simultaneously activate the latent oncogenic K-Ras allele and inactivate the CAAX processing enzymes. Using these mice, we will define the impact of defective CAAX processing on the development, progression, and lethality of Ras-induced myeloproliferative disease. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: CELL SURVIVAL PATHWAYS AND INHIBITORS IN LEUKEMIA Principal Investigator & Institution: Eastman, Alan R.; Professor and Associate Director; Pharmacology and Toxicology; Dartmouth College 11 Rope Ferry Rd. #6210 Hanover, Nh 03755 Timing: Fiscal Year 2002; Project Start 30-JAN-2002; Project End 30-NOV-2003 Summary: (provided by applicant): Tumors characteristically exhibit mutations that enhance cell proliferation and survival. Two well-recognized cell survival pathways are RAF to MEK to ERK and PI3-kinase to Akt. Many inhibitors of these pathways are now in clinical trials or at earlier stages of development. However, early results suggest these inhibitors are more likely to suppress growth than kill the tumor cells. Our recent

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observations have demonstrated that such inhibitors may be more valuable when used in combination with more traditional anticancer agents. Specifically, it has been shown that a MEK inhibitor can dramatically enhance the rate of apoptosis induced by vinblastine in myeloid leukemia ML-1 cells and HL6O cells. However, U937 cells are insensitive to the MEK inhibitor but are sensitized to vinblastine by an inhibitor of PI3kinase. These observations have led to the hypothesis that different leukemias preferentially use different survival signaling pathways, and that by defining which pathway a specific leukemia uses, effective drug combinations can be individualized for that patient. The goal of this project is to study freshly-isolated human leukemia cells and define the frequency with which they are sensitized to chemotherapy by inhibitors of these two cell survival pathways. The specific aims are to assay leukemia cells for phosphorylation of ERK and Akt as indicators of the signaling pathways used, and to combine inhibitors of these signaling pathways with vinca alkaloids ex vivo to determine the rate of induction of apoptosis. Additional experiments will determine whether normal leukocyte progenitors, which do not have an oncogene-enhanced cell survival pathway, are resistant to these drug combinations thereby suggesting such a therapy may be selective for the tumor. Finally, activation of Jun N-terminal kinase (JNK) will be assayed in leukemia patients receiving vincristine therapy, to confirm that this pathway is activated at drug concentrations tolerated by patients. Activation of the JNK pathway is necessary for the enhanced apoptosis induced by inhibitors of the Erk and Akt pathways. Successful completion of these aims will identify which leukemia patients might benefit from administration of inhibitors of these signaling pathways, and facilitate the design of clinical trials to test their efficacy in combination with other anticancer agents. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: COMMUNITY CLINICAL ONCOLOGY PROGRAM Principal Investigator & Institution: Locker, Gershon Y.; Chief; Evanston Northwestern Healthcare Evanston, Il 60201 Timing: Fiscal Year 2002; Project Start 01-SEP-1983; Project End 31-MAY-2003 Summary: (Applicant's Description) Evanston Hospital Corporation which has been renamed as Evanston Northwestern Health care (ENH), has been a CCOP since 1093 and has participated in studies of the Eastern Cooperative Oncology Group (ECOG), the National Surgical Adjuvant Breast and Bowel Program (NSABP), and propose to accrue patients to the Gynecology Oncology Group (GOG). It accrued 259 patients with 272 credits to therapeutic trials between June of 1992 to May of 1997. ENH also contributed non-COP patients to NIH-sponsored studies on brain tumors. ENH investigators have chaired ECOG protocols in genitourinary, breast, and hematologic malignancies. They have also chaired steering committees and served in leadership roles in these groups. Currently, Dr. Ann Thor is on the Executive and directs the ECOG Pathology Coordination Office. Dr. David Calls chains the Health Behavior and Practices Committee and the Outcomes Subcommittee. The CCOP has participated in approved cancer control projects in the NSABP-sponsored breast cancer prevention trial with tamoxifen, the Prostate Cancer Prevention Trial, and other cancer control studies. During the 5 years, 279.5 cancer control credits were awarded. ENH investigators have been active in several cancer control projects outside the CCOP pertaining to epidemiology, diagnosis, "diagnostic marker" and dietary manipulation. These include a NCI funded study of low- fat diet in post-menopausal breast cancer, and the Women's Health Initiative, treatment of post-mastectomy arm lymphedema. The CCOP has been reorganized to increase accrual by: recruitment of new investigators, adding Swedish

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Covenant Hospital as an affiliate, and GOG as a research base. Efforts are underway to encompass minority enrollment. A 24-bed Clinical Pharmacology Unit sponsored by Searle is operation, with the PI on the advisory committee. We have expanded our education activities through Grand Rounds and lecture series. In the last 4 years, ENH investigators published 63 papers and 10 abstracts pertaining to clinical cancer treatment and control. A research effort in cellular and molecular biology has been developed with the establishment of a program in molecular genetics. Thus, a vertical integration, e.g., from laboratory studies to delivery of care in the local community is being sought. Support is asked for ENH's continued participation in the CCOP. Funding is sought for continued accrual of patients to cancer therapy and cancer control studies of the ECG, NSABP, and GOG. Thus, our participation in cancer control and therapeutic trials will promoter medical advances as well as stimulate better patient care. These in turn will impact favorably on the level of knowledge of staff and physicians within the community. Since 1983, we have successfully participated in the CCOP program, and our record and proposed changes promise continued success in the future. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CORE--CLINICAL RESEARCH SUPPORT COMPONENT Principal Investigator & Institution: Stone, Richard M.; Professor; Dana-Farber Cancer Institute 44 Binney St Boston, Ma 02115 Timing: Fiscal Year 2002; Project Start 01-AUG-2002; Project End 31-MAR-2003 Summary: (provided by applicant): The ultimate objective of the research projects in this program project application is to improve the therapeutic results for patients with myeloid malignancies including acute myeloid leukemia, myelodysplastic syndrome, and chronic myeloid leukemia. To achieve this objective, tumor cells and other relevant clinical samples from patients will be collected, catalogued, and distributed to the relevant projects for analysis of the expected therapeutic targets and other molecules that might be important in prognosis or pathophysiology. Secondly, a clinical infrastructure is required to carry out the clinical trials described in Project 5 and additional clinical studies that will emanate from the developmental approaches outlined in Projects 1, 8, 9 & 10. Clinical Research Core resources are required to carry out these functions which extend beyond direct patient care and the clinical laboratory. Without the clinical research support provided in the Core it would be impossible to coordinate the proper collection of multiple research specimens, the adherence to novel complex therapeutic schedules and timely follow-up of patients enrolled on research studies. Also critical to this success of the project is the collaboration of individuals in the Core with the staff from the Biostatistics Core who will provide a quality control system for specimen tracking, computerized data entry, quality of control data and will assist in the design and analysis of the clinical research protocols. The purpose of the Clinical Research Support Core is to provide the following services that will be utilized by all the clinical research studies: 1. To collect research specimens and coordinate patient follow-up at Dana- Farber Partners Cancer Center and collaborating institutions. 2. To act as liaison with outside physicians, hospitals, and biotechnology companies to coordinate the collection of research specimens and follow-up data. 3. To insure that study parameters are followed, ancillary specimens are collected on time and processed properly, confirm eligibility, and patient registration. 4. To insure the accuracy of submitted data from outside sources. 5. To provide data management for the collection of individual patient information. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: CORE--LEUKEMIA TISSUE BANK Principal Investigator & Institution: Caligiuri, Michael A.; Professor and Director; Ohio State University 1960 Kenny Road Columbus, Oh 43210 Timing: Fiscal Year 2003; Project Start 13-MAY-2003; Project End 31-MAR-2009 Summary: (provided by applicant): The CALGB Leukemia Tissue Bank (LTB) serves as a centralized tissue bank of blood and bone marrow specimens procured from patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL) and other hematologic malignancies enrolled on CALGB treatment protocols. The specific aims of this proposal will be accomplished through CALGB protocol 9665, a protocol for the procurement of relevant tissue specimens. The associated work will be performed in the laboratory of Michael Caligiuri, M.D. at The Ohio State University. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: CTCL STAGING USING GENE EXPRESSION PROFILES Principal Investigator & Institution: Showe, Louise C.; Associate Professor, Director Genomics c; Wistar Institute Philadelphia, Pa 191044268 Timing: Fiscal Year 2002; Project Start 30-SEP-1999; Project End 31-MAR-2004 Summary: The cutaneous T-cell lymphomas (CTCL) including Mycosis fungoides (MF) and Sezary syndrome (SS) are indolent lymphomas that progress in stages, starting with skin lesions, sometimes proceeding through a leukemic phase with circulating tumor cells and eventually spreading to the visceral organs. Treatments for CTCL vary in efficacy even for patients with what appears to be similar level of disease, emphasizing the likely existence of undetectable heterogeneity. These characteristics added to a the availability of a large archive of patient samples make it a good candidate for tumor staging by molecular profiles. SS, the leukemia form of CTCL will be the initial focus of these studies as it provides easy access to large numbers of purified malignant cells. RNA from 10 patients, with diverse patterns of disease presentation and progression, will be analyzed during the first year against arrays of cDNA probes for 20,000 sequence verified Unigene clusters in order to determine the global gene expression patterns of these cells. Samples will be selected from newly diagnosed SS patients and from an archive of viably frozen SS cells including samples collected at progressive stages of disease over a period of greater than 10 years. Since CTCL cells represent Th-2 T-cells, RNA from healthy donor PBL, stimulated to develop a TH-2 phenotype will be used as controls. Genes that are over or under-expressed in patient RNAs, compared to controls, will be candidate tumor markers for a reduced panel of genes that will be used to screen a larger group of patient samples. In the second phase of the study, 100 patients will be selected for gene expression studies with a reduced panel of 1000-2000 genes. These expression profiles will be analyzed, using statistical techniques, to identify groups of genes that behave in a similar fashion in subsets of patients. The results of these analyses will be a putative diagnostic panel of genes whose expression levels describe classes of tumors. The correlation between expression levels and tumor groups will be confirmed using alternative methods for measuring gene expression. Finally, clinical information from patient histories will be compared with tumors clustered by gene expression levels to determine whether important clinical outcomes, e.g., responsiveness to treatment, can be predicted from the specific gene expression patterns. Concurrent with the above studies, samples from patients with MF, the skin-associated early form of CTCL, will be queried with the panel of genes identified as being diagnostic for SS to determine

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whether the same genes are also sufficient to characterize different classes of MF. If novel gene clusters are found, they will be added to the data base of candidate markers. If not, up to 10 MF patients will be analyzed on 20,000-gene filters for genes whose expression pattern distinguishes MF from SS patients. If found, these will be added to the panel of candidate SS genes. Finally, techniques will be developed to assay expression profiles in a clinical setting. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CYTOKINE MEDIATED DIFFERENTIATION THERAPY Principal Investigator & Institution: Smith, B D.; Oncology; Johns Hopkins University 3400 N Charles St Baltimore, Md 21218 Timing: Fiscal Year 2002; Project Start 01-SEP-2000; Project End 31-AUG-2005 Summary: Dr. Smith is a junior faculty member at Johns Hopkins Oncology Center who has spent the past several years developing an understanding of the basic biology of drug resistance in acute leukemia. His observations have clearly pointed to the association between anti-apoptotic signals and drug resistance in both in vitro and in vivo studies. He has also been involved in developing clinical strategies to overcome the drug resistance that results from inhibited apoptosis. His career development plan is to obtain formal training in the theory and methods of clinical investigation leading to a Master of Health Sciences degree, while conducting translational research with mentorship from Richard Jones, M.D., the Director of the Bone Marrow Transplantation Program. Steven Piantadosi, M.D, Ph.D. will serve as co-mentor with expertise in epidemiology, biostatistics, and clinical trial design. It is now clear that signals that block apoptosis are important mechanisms by which cancers are pan-resistant to cytotoxic anticancer agents and result in poor clinical outcomes. Our preliminary data suggest that growth factor-mediated differentiation therapy is one strategy that may circumvent resistance secondary to anti-apoptotic signals. The net effect of growth factors on a tumor cell population is determined by a balance of their pleiotropic effects on cell self-renewal, survival, and differentiation. Preferential enhancement of selfrenewal and/or survival could hasten tumor progression; conversely, selective induction of differentiation would exhaust the neoplastic clone. We have found that growth factors induce selective terminal differentiation of chronic myeloid leukemia (CML) at doses that normal hematopoetic progenitors require for optimal growth. Moreover, agents that induce growth arrest, including interferon and bryostatin-1 enhance growth factor- mediated differentiation of CML and other myeloid malignancies in vitro. Based on pre-clinical work that suggests growth factor- mediated induction of differentiation is an effective anti-tumor strategy in pan-resistant myeloid malignancies, three clinical trials of growth factor-mediated differentiation therapy are proposed: 1) GM-CSF + autologous BMT for CML, 2) interferon + GM- CSF in chronic phase CML and 3) bryostatin-1 + GM-CSF in refractory myeloid leukemias. Throughout the course of our proposed trials, we will have the opportunity to further study the biologic impact of growth factor-mediated differentiation. Our current data suggest that the anti-tumor effects of our therapy results from terminal differentiation, however, the agents we are studying in the clinical trials (e.g., GM-CSF, interferon, bryostatin-1) also have immunomodulatory effects which may contribute to an anti- tumor effect. A major objective of our studies will be to evaluate the relative roles that induction of differentiation and immunomodulation play in the anticipated anti-tumor effects of the growth factors and cytostatic agents in the clinical trials. One intriguing possibility is that the induction of differentiation and immunomodulation could be potentially linked. It is now clear that CML, and even AML, progenitors can be differentiated in vitro into

20


dendritic cells (DC) by cytokines that include GM-CSF; moreover, these leukemic DC can stimulate autologous anti-leukemic T cell responses in vitro. Thus, it is possible that strategies that induce differentiation of myeloid malignancies could lead to eradication of the malignant clone via both induction of terminal differentiation and enhanced immunologic anti-leukemic activity. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: DESIGN OF ABL SH2 AND SH3 INHIBITORS FOR LEUKEMIA Principal Investigator & Institution: Overduin, Michael J.; Associate Professor; Pharmacology; University of Colorado Hlth Sciences Ctr P.O. Box 6508, Grants and Contracts Aurora, Co 800450508 Timing: Fiscal Year 2002; Project Start 19-APR-2001; Project End 31-MAR-2003 Summary: (provided by applicant) This project targets the src homology domains of the Abl oncogene for rational anti-cancer drug design. The team consists of a partnership between my structural biology research group at the University of Colorado and a drug discovery program at OSI Pharmaceuticals. This biotechnology company has developed a series of peptidomimetic compounds as novel anticancer agents targeted at signaling domains responsible for the localization and regulation of kinases and phosphatases. My laboratory is determining the three-dimensional structures of complexes of lead compounds bound to src homology 2 (SH2) and src homology 3 (SH3) domains by nuclear magnetic resonance (NMR) spectroscopy. The specific targets are the SH2 and SH3 domains of Abl, an oncogenic tyrosine kinase for which inhibitors are in phase II clinical trials. The SH2 domain is inhibited by a pyridone derivative that mimics its natural phosphotyrosine ligand, and the SH3 domain is inhibited by spirolactam-based compound that resembles its polyproline ligands. These templates possess affinities and binding modes comparable to physiological ligands, as demonstrated by nuclear magnetic resonance spectroscopy (NMR) and surface plasmon resonance (SPR). Modifications of these lead compounds that enhance selectivity and potency will be suggested based on protein structures, NMR and SPR data, and computational modeling. Synthetic organic chemistry expertise and access to combinatorial libraries is provided by OSIP. This complementary partnership will allow the rational optimization of lead compounds with significant therapeutic potential for cancers including chronic myelogenous leukemia. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: DOK IN CELL GROWTH AND P210 BCR-ABL SIGNALING PATHWAYS Principal Investigator & Institution: Van Aelst, Linda; Associate Professor; SloanKettering Institute for Cancer Res New York, Ny 100216007 Timing: Fiscal Year 2003; Project Start 10-JUL-2003; Project End 30-JUN-2008 Summary: The broad long term goal of this project is to define the molecular mechanism(s) by which p62(dok) exerts its effect(s) on p210bcr-abl-mediated transformation and cell proliferation in general, and to obtain a better understanding of the establishment of the myeloproliferative phenotype of p210 bcr-abl in CML at a molecular level. p62(dok) is a protein identified as being constitutively tyrosine phosphorylated in chronic phase progenitor cells of CML patients and has been found to be a common substrate of many receptor and membrane-associated tyrosine kinases. Several lines of evidence indicate that p62(dok) plays a negative role in growth factorinduced cell proliferation, and in p210bcr-abl mediated transformation. An intriguing

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possibility is that p62(dok) could influence the establishment or duration of the chronic phase of CML. In light of this, the characterization of the mechanism(s) by which p62(dok) exerts its effect on growth factor and p210bcr-abl-mediated signaling is essential, as is comprehensive knowledge of bcr-abl-induced signaling pathways, and aberrations therein. To this end, we propose the following specific aims: 1) To map and compare the sites of p62(dok) tyrosine phosphorylated by p210 bcr-abl with those phoshorylated upon PDGF stimulation, 2) To define mechanisms by which p62(dok) [suppresses PDGF-triggered proliferation and p210bcr-abl-nduced transformation, 3) To identify and characterize p62(dok)-interacting signaling components which mediate the negative effect of p62(dok) on PDGF-triggered proliferation and p210bcr-abl-induced transformation, 4) To define signal transduction pathways affected by p210 bcr-abl by microarray analysis and clustering resulting data, and 5) To characterize the target genes involved in the p210 bcr-abl signal transduction pathways identified. This project offers biochemical, molecular and cellular approaches to the study of p62(dok) with respect to its involvement in CML and to the identification of bcr-abl target genes. The health-relatedness of this project is that defining the role of p62(dok) in p210 bcr-abl signaling, and the identification of novel target genes of bcr-abl, will contribute to a better understanding of the establishment of human CML. Furthermore the identification of novel target genes of p210 bcr-abl signaling may provide new potential targets for therapeutic intervention. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: DOK PROTEINS IN ONTOGENESIS AND LEUKEMOGENESIS Principal Investigator & Institution: Pandolfi, Pier Paolo; Professor; Sloan-Kettering Institute for Cancer Res New York, Ny 100216007 Timing: Fiscal Year 2003; Project Start 10-JUL-2003; Project End 30-JUN-2008 Summary: Chronic myelogenous leukemia (CML) is characterized by the presence of the chimeric p210bcr-abl protein which shows elevated protein tyrosine kinase activity relative to the normal c-abl tyrosine kinase. Although many of the p210bcr-abl substrates have now been identified, the relevance of these phosphorylation events in the pathogenesis of CML and the normal function of these p210bcr-abl substrates and are still poorly understood. The focus of this proposal is to elucidate, in vivo in the mouse, the functions of three p210bcr-abl phosphorylation targets, Dok1, 2 and 3, and to determine how these relate to the pathogenesis of CML by a direct genetic approach with the following Specific Aims: 1) To define, in single knock out mice and null cells, the role of Dok1, 2, 3 in ontogenesis and hemopoiesis. We have disrupted the Dok 1, 2, 3 genes and mice lacking their functions (-/-, null) have been generated. Ontogenesis and hemopoiesis will be studied in these mutants. 2) To define in double or triple knock-out mutants the role of Dok1, 2, 3 in ontogenesis and hemopoiesis. We will intercross the various Dok-/- mice among them in order to generate double or triple knock-out mutants. We will define the developmental role of these genes and their role in hemopoiesis by characterizing the embryonic and adult phenotype resulting from their concomitant inactivation. 3) To establish the role of Dok proteins in leukemia and cancer promotion/progression. We will examine spontaneous or physically/chemically induced tumorigenesis as well as leukemogenesis by p210bcr-abl in the various K.O. mutants and in double and triple Dok-/- mutants. We will cross Dok1-/- mutants with mice lacking Nf1 or Pten gone products to test whether Dok1 cooperates with these proteins in tumor suppression. 4) To identify genes critical for Dok1, 2 and 3 function and for CML pathogenesis. We will utilize purified cell populations from our single,

22


double and triple K.O. mutants to identify, on a comparative basis, target genes relevant for their function. We will test whether these genes are also deregulated in CML blasts. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: FLAVOPIRIDOL AND IMATINIB IN BCR/ABL+ LEUKEMIA Principal Investigator & Institution: Grant, Steven; Professor of Medicine, Biochemistry, And; Internal Medicine; Virginia Commonwealth University Richmond, Va 232980568 Timing: Fiscal Year 2003; Project Start 11-SEP-2003; Project End 31-AUG-2005 Summary: (provided by applicant): Recent evidence suggests that neoplastic cells are particularly susceptible to a strategy involving simultaneous interruption of survivalassociated signal transduction and cell cycle regulatory pathways. Consistent with this notion, we have observed in preclinical studies that the cyclin-dependent kinase inhibitor flavopiridol (NSC 649890) interacts synergistically with the Bcr/Abl kinase inhibitor imatinib (STI571; Gleevec) to induce mitochondrial injury, caspase activation and apoptosis in Bcr/Abl+ human leukemia cells, including those highly resistant to imatinib. These events are associated with multiple perturbations in survival signaling and cell cycle-related pathways, including down-regulation of Mcl-1 and Bcl-xL, reduced expression of cyclin D1, activation of JNK, and inactivation of CREB and Stat5. Based upon these findings, a multi-institutional Phase I trial has been developed in which patients with progressive CML (chronic and accelerated phase) or CML-BC or Philadelphia chromosome+ AML or ALL will be treated with escalating doses of daily imatinib in conjunction with flavopiridol administered as a 1-hr infusion weekly x 3 q month. The goals of this Phase I trial are to define the MTD for these agents, characterize dose-limiting toxicities, and gain preliminary information regarding activity of the regimen. Correlative laboratory studies will test the hypothesis that in vivo administration of imatinib in conjunction with flavopiridol will induce perturbations in apoptotic regulatory proteins in peripheral blood Bcr/Abl+ cells (e.g., diminished expression of Mcl-1, Bcl-xL, and cyclin D1, inactivation of Stat5 and CREB, activation of JNK) similar to those observed in Bcr/Abl+ cell lines exposed to these agents in vitro. Other studies will investigate a) effects of the imatinib/flavopiridol regimen on Stat5 phosphorylation of Bcr/Abl+ peripheral blood cells by flow cytometry; b) the pharmacokinetics of imatinib and flavopiridol when administered together; and c) the presence of Bcr/Abl mutations as well as increased Bcr/Abl expression/activity in cells from imatinib-resistant patients, and their possible relationship to imatinib/flavopiridol pharmacodynamics. Information derived from this trial will provide a foundation for a successor Phase II trial and correlative laboratory studies which will address issues of regimen activity and imatinib/flavopiridol molecular interactions more definitively. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: GENE EXPRESSION PROFILE OF PROGRESSION & RESPONSE IN CML Principal Investigator & Institution: Radich, Jerry P.; Full Member; Fred Hutchinson Cancer Research Center Box 19024, 1100 Fairview Ave N Seattle, Wa 98109 Timing: Fiscal Year 2002; Project Start 30-SEP-1999; Project End 31-MAR-2004 Summary: Chronic myeloid leukemia (CML) is a hematopoetic stem cell disease with distinct biological and clinical features, presenting as a relatively clinically benign state ( chronic phase ), which invariably evolves to an incurable aggressive disease ( blast crisis ). Treatment can range from low intensity chemotherapy to the curative yet potentially lethal therapy of bone marrow transplantation (BMT). Unfortunately little is known

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about the molecular events that trigger the evolution of chronic phase to blast crisis. Thus, tailoring therapy to individual patient s risk is impossible. This proposal aims to identify changes in gene expression that occur in the evolution of the chronic phase to blase crisis, as well as discovering gene expression patterns that are associated with good outcomes to conventional interferon-based therapy. Specifically, we will: 1) optimize and validate the expression array technology, then 2) use mRNA expression arrays to identify genes involved in the progression of chronic phase to blast phase CML; and 3) identify genes associated with good or poor outcome following conventional interferon-based therapy. These studies will allow us to begin to study the biology of CML transformation, and understand at a genetic level why some patients respond to conventional therapy, while other patients are refractory to therapy, and quickly transform to highly aggressive disease. The identification of low v high risk patients will allow theraphy to be appropriately tailored to each individual s disease. In addition, the application of large-scale expression analysis in this model system will be ideal to iron out unforeseen technical problems, and thus the experience gained may be very valuable in future investigations other more complex tumor systems. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: GRAFT REJECTION Principal Investigator & Institution: Mcniece, Ian K.; Professor; Oncology; Johns Hopkins University 3400 N Charles St Baltimore, Md 21218 Timing: Fiscal Year 2003; Project Start 08-APR-2003; Project End 31-MAR-2005 Summary: (provided by applicant): Non-myeloablative transplants are being used increasingly for a number of diseases, including Non Hodgkins lymphoma, Hodgkin's disease, myeloma, acute leukemia, chronic lymphoytic leukemia (CLL) and chronic myelogenous leukemia (CML). A reduction in toxicity compared to fully myeloabative allogeneic stem cell transplantation is stimulating the use of this strategy particularly for older patients. Donor engraftment is achieved in the majority of patients, however, a significant number of patients, particularly those with myelodisplastic syndrome (MDS) experience secondary loss of donor engraftment. This can be fatal for some patients due to the prolonged pancytopenia that follows. There are several possibilities for the loss of donor engraftment; 1) donor graft rejection by recipient T cells, 2) late donor graft failure due to insufficient stem cells in the graft, or 3) dominance of recipient stem cells due to competitive repopulation. In allogeneic transplants to support high dose chemotherapy, graft failure/graft rejection occurs early after transplant within the first month, however, for patients that achieve early donor engraftment secondary graft failure/graft rejection is rare and only occurs in less than 5% of patients [1,2]. In contrast, nearly a 100% of patients receiving mini-allogeneic transplants achieve donor engraftment within 2 months of transplant. Graft failure/graft rejection occurs at 2 to 4 months post transplant in approximately 15% of mini-allogeneic recipients. We hypothesize that loss of donor grafts in non-myeloablative stem cell transplant (NST) recipients can occur due to rejection of the donor cells by recipient T cells and/or low numbers of donor stem cells in the graft resulting in secondary graft failure. The aim of this proposal is to develop methods to evaluate graft rejection and determine the mechanisms responsible for late donor graft failure in recipients of NST. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: HEMATOPOIETIC TRANSFORMATION BY TYROSINE KINASE FUSIONS Principal Investigator & Institution: Gilliland, D Gary.; Associate Professor; Dana-Farber Cancer Institute 44 Binney St Boston, Ma 02115 Timing: Fiscal Year 2002; Project Start 01-AUG-2002; Project End 31-JUL-2007 Summary: (provided by applicant): The overall hypothesis of this proposal is that a detailed characterization of the signal transduction properties of constitutive activated tyrosine kinases associated with human leukemias will identify the critical targets that are required for transformation of hematopoietic cells. These targets can then be exploited for the development of novel therapeutic approaches to leukemia. During the previous funding period, we accomplished each of the goals that were set forward. In particular, we cloned and characterized the transforming properties of a spectrum of tyrosine kinase fusions associated with recurring chromosomal translocations in human leukemias, using both cell culture systems and murine models of leukemia. These include, in addition to the TEL/PDGFbetaR and TEL/ABL fusions, the HIP1/PDGFbetaR, H4/PDGFbetaR, TEL/JAK2 and TEL/TRKC fusion proteins. The availability of a spectrum of constitutively activated tyrosine kinases provides a unique opportunity to identify critically important pathways of transformation through comparative analysis of the signal transduction pathways and target genes activated by these fusion proteins, with validation in animal models. In Specific Aim 1, we will address the hypothesis that characterization of the signal transduction pathways and target genes activated by TEL/PDGFbetaR, TEL/JAK2, TEL/TRKC, and TEL/ABL, and BCR/ABL, will identify critical pathways and targets that are required for transformation. We will utilize standard approaches to analysis of signal transduction, as well as expression arrays. In Specific Aim 2, we will test the hypothesis that the putative critical effectors of transformation identified in Specific Aim 1 can be evaluated and validated in our model systems. We will utilize a combination of approaches that focus on inhibition of transformation in cell culture systems, as well as murine bone marrow transplant models of leukemia using hematopoietic progenitors from donor mice are that are genetically deficient in one or more of the putative targets. In Specific Aim 3, we will test the hypothesis that acute leukemia phenotypes are a collaboration between constitutively activated tyrosine kinases and transcription factor fusion genes such as AML1/ETO, AML1/EVI1 and NUP98/HOXA9. We hypothesize that coexpression of tyrosine kinase fusions and transcription factor fusions will cause an acute leukemia phenotype in murine models, and that these leukemias will retain sensitivity to specific tyrosine kinase inhibitors. This proposal will provide insights into signal transduction and target genes critical for transformation of hematopoietic cells, as well as novel therapies for leukemia. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: IMMUNOTHERAPY AFTER TRANSPLANT TO PREVENT RELAPSE

AUTOLOGOUS

STEM

CELL

Principal Investigator & Institution: Miller, Jeffrey S.; Professor; University of Minnesota Twin Cities 200 Oak Street Se Minneapolis, Mn 554552070 Timing: Fiscal Year 2002; Project Start 01-DEC-2001; Project End 30-NOV-2002 Summary: This study proposes a series of clinical and related preclinical investigations to minimize peritransplant tumor burden and to reduce the chances of posttransplantation recurrence of certain lymphomas and of other cancers. Autologous IL-2 activated natural killer cells have been shown to fill a broad spectrum of tumor targets.

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Thus, autologous lymphocytes may have anti-tumor activity which may be most effective in a minimal residual disease setting indeced by autologous transplantation. In this study, we will test the safety and tolerance of subcutaneous, low dose IL-2 +/- GMCSF on an outpatient basis in subjects who have had an autologous transplant for lymphoma, breast cancer, chronic myelogenous leukemia, or multiple myeloma. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: INDUCTION AND MANIPULATION OF HUMAN CML IN NOD/SCID Principal Investigator & Institution: Conrad, Patricia D.; Children's Research Institute 700 Children's Dr Columbus, Oh 432052664 Timing: Fiscal Year 2002; Project Start 20-SEP-1999; Project End 31-AUG-2003 Summary: This proposal describes an intensive, five-year training program designed to allow the principal investigator to successfully bridge the gap between clinical and basic science training. The ultimate goal of the performance of this training will be to achieve scientific independence in the fields of normal and malignant hematopoiesis. Dr. Conrad is board certified in pediatrics and is currently a third year pediatric hematology/oncology fellow at the Children's Hospital of Philadelphia. She has been working for the last 18 months in the laboratory of Stephen G. Emerson, M.D., Ph.D. During this time, Dr. Conrad has intensively pursued laboratory investigation and is ready for the next phase of training, progressively independent work. The candidate's research has focused on 1) investigating functional and biological properties of umbilical cord blood (UCB) hematopoietic stem cells (HSCs) and 2) establishing the NOD/SCID assay using cord blood as the source for stem cells. UCB stem cells are not identical to their bone marrow or peripheral blood counterparts, and seem to act as a more primitive population. Immunodeficient mice, including NOD/SCID, have become the preferred in vivo assay for HSCs. Chronic myelogenous leukemia (CML) is believed to be a myeloproliferative disorder of stem-cell origin. Therefore, experiments designed to investigate fundamental questions relating to pathogenesis of CML will be optimized utilizing a system of UCB HSCs and NOD/SCID mice. Dr. Conrad's sponsor, Dr. Emerson, is division chief of the hematology/oncology section at the University of Pennsylvania. He holds a Ph.D. in cell biology and immunology and was scientific director of the alloBMT program at the University of Michigan. He has significantly contributed to and remains committed to Dr. Conrad's growth into a physician scientist. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: LEUKEMIA ANTIGENS FOLLOWING DONOR LEUKOCYTE INFUSION Principal Investigator & Institution: Wu, Catherine J.; Dana-Farber Cancer Institute 44 Binney St Boston, Ma 02115 Timing: Fiscal Year 2002; Project Start 01-SEP-2000; Project End 31-AUG-2005 Summary: The infusion of donor lymphocytes (DLI) induces remission in approximately 75 percent of patients with relapsed chronic myelogenous leukemia (CML) after allogeneic bone marrow transplantation. The efficacy of DLI reveals that immune responses against leukemia cells can be mounted in the absence of complicating chemotherapy or radiation treatment, and is thus a unique model for studying tumor immunity. How DLI mediates this anti-leukemic effect is unclear. T cells are required for this response, but their antigenic targets are unknown. Recent reports have demonstrated the presence of autoantibodies reactive to various tumors and have

26


utilized autologous sera to identify novel tumor-associated antigens, which were later shown to be recognized by T cells as well (for review, see Old et al., 1998). Our preliminary results reveal that an analogous approach may succeed in identifying CMLassociated antigens in patients who have responded to DLI. In our trial which used CD4+-DLI to treat relapsed CML, many patients were observed to develop marked elevations in their peripheral B cell number and frequent lymphocytosis and plasmacytosis on bone marrow biopsies, suggesting the presence of a potent B cell response. Immunoscreening of a CML cDNA expression library with high titer post-DLI sera identified eight gene products with antibody reactivity to patient sera collected after but not before DLI. Herein, we propose to characterize the humoral and cellular response to these DLI-associated targets. The CML cDNA expression library will be further screened with other patients' post-DLI sera in order to expand the panel of DLIassociated antigens. Our questions will focus on determining whether the identified antigens are shared among DLI-treated patients (in effort to understand whether leukemia-specific targets or host-donor differences explain the basis of anti-tumor immunity), on delineating the time course of antibody response; and on ascertaining whether the presence of antibody reactivity correlates with a clinical response to DLI, or to the development of graft-versus-host disease (GVHD). To gain insight into whether the identified genes play a role in leukemogenesis, the genes will be fully sequenced, and differential expression between normal and malignant cells will be examined by Northern analysis. Lastly, because T cells are critical to DLI, the gene products will be tested for their ability to induce an antigen- specific T cell response in CML patients' peripheral blood leukocytes in culture. The experiments described in this proposal will lead to a deeper understanding of the mechanisms underlying the antitleukemic effect of DLI and provide a basis for improved diagnosis and treatment of CML and potentially other hematological malignancies. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: LEUKEMOGENESIS BY ABL AND OTHER ONCOGENES Principal Investigator & Institution: Baltimore, David; Institute Professor; None; California Institute of Technology Mail Code 201-15 Pasadena, Ca 91125 Timing: Fiscal Year 2002; Project Start 15-DEC-1989; Project End 31-DEC-2004 Summary: This proposal has 5 parts representing 5 on-going efforts by different groups of junior investigators in the laboratory. They are: 1. Conditional Abl knockout. Having knocked out both the Abl and Arg genes and found that one of the two genes is needed for development beyond embryonic day 10, we plan to construct a conditional knockout of Abl on an Arg-deficient background so that the roles of the genes in later stages of mouse development and function can be examined. 2. Abl in C. elegans. Abl function has been investigated in mice and Drosophila but the putative functions of the gene are so variable that examination of another organism is warranted. We have chosen C. elegans because its entire genome is known and it is possible to examine it genetically at very high resolution. 3. NF-kappaB control of transcription. While there is much fragmentary knowledge of the genes controlled by NF-kappaB, a global analysis of gene expression in cells with defined genetic lesions in the NF-kappaB-related proteins will give more precise knowledge of which genes are controlled and by which subunits. 4. NF-kappaB activation by TANK and TBK1. There are numerous pathways of NFkappaB activation but none are known in precise detail. We have found a new kinase, TBK1, that interacts with TANK and appears to act along with TRAF proteins and may phosphorylate them. A deeper knowledge of the details of the biochemistry of these events could help to understand the precise mechanisms of NFkappaB activation. 5.

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ATR kinase. This very large kinase has been implicated in checkpoint control of the cell cycle in mammalian cells. We have knocked out the gene for this protein and the mice die very early in gestation, in a manner similar to mice lacking the BRCA genes. We plan to construct a conditional allele of this gene to examine in more detail its function and, in particular, its relation to p53, BRCA1 and 2, and Chk1. These studies are all aimed at a deeper understanding of both the processes of oncogenic transformation and the normal development and function of mammals. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: MDS NORDIAN GAMMACELL-40 EXACTOR WHOLE ANIMAL IRRADIATOR Principal Investigator & Institution: Monroe, John G.; Professor; Pathology and Lab Medicine; University of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104 Timing: Fiscal Year 2002; Project Start 01-APR-2002; Project End 31-MAR-2003 Summary: Funds are requested for a MDS Nordian Inc. Gammacell-40 exactor selfcontained low dose rate research animal (rodent) irradiator to support the ongoing NIHfunded individual-initiated research programs in a new state of the art animal facility located within a recently erected research building (BRBII) on the campus of the University of Pennsylvania School of Medicine. Twelve core laboratories comprise the list of the major users of the requested instrument. Each have laboratories in the BRBII and will house their mice and perform ongoing experiments requiring whole animal irradiation and extended maintenance of irradiated animals in ongoing experiments in the animals in the basement of this building. These core laboratories will constitute the majority (>75%) of the usage of the irradiator. In addition, there is a group of over 25 additional users that will utilize this instrument on an infrequent (

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