Molecularly Targeted Therapy for Childhood Cancer

Molecularly Targeted Therapy for Childhood Cancer w Peter J. Houghton â•‡ â•‡ Robert J. Arceci ● Editors Molecula...

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Molecularly Targeted Therapy for Childhood Cancer

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Peter J. Houghton â•‡ â•‡ Robert J. Arceci ●

Editors

Molecularly Targeted Therapy for Childhood Cancer

Editors Peter J. Houghton Elizabeth M. and Richard M. Ross Chair Director, Center for Childhood Cancer The Research Institute Nationwide Children’s Hospital 700 Children’s Drive Columbus, OH 43205 [email protected]

Robert J. Arceci King Fahd Professor of Pediatric Oncology Professor of Pediatrics Oncology and Cellular and Molecular Medicine Kimmel Comprehensive Cancer Center at Johns Hopkins The Bunting and Blaustein Cancer Research Building 1650 Orleans Street, Suite 207 Baltimore, MD 21231 USA [email protected]

ISBN 978-0-387-69060-5 e-ISBN 978-0-387-69062-9 DOI 10.1007/978-0-387-69062-9 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2010930693 © Springer Science+Business Media, LLC 2010 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Acknowledgments

We would like to especially thank all the authors and investigators who contributed to the content of this book along with the publisher for believing in the importance of directly addressing important issues concerning children with cancer.

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Contents

Part Iâ•… Hematologic Malignancies The Emerging Era of Targeted Therapy in Childhood Acute Lymphoblastic Leukemia..................................................................... William L. Carroll and Rob Pieters

3

Molecular Targeted Therapies in T-CellAcute Lymphoblastic Leukemia................................................................................ Alejandro Gutierrez and A. Thomas Look

19

Molecularly Targeted Therapy for Infant ALL............................................ Patrick A. Brown and Carolyn A. Felix

31

Targeted Therapeutic Approaches for AML................................................. Robert J. Arceci and Donald Small

59

Acute Promyelocytic Leukaemia.................................................................... Andrea Biondi, Anna Maria Testi, and Brenda E.S. Gibson

83

Down Syndrome and Acute Myeloid Leukemia: An Unique Genetic Sensitivity to Chemotherapy......................................... 109 Jeffrey W. Taub, Yubin Ge, and Yaddanapudi Ravindranath Targeting RAS Signaling Pathways in Juvenile Myelomonocytic Leukemia (JMML)........................................................................................... 123 Jennifer O’Hara Lauchle and Benjamin S. Braun Chronic Myeloid Leukemia: Pathophysiology and Therapeutics............... 139 Seth J. Corey and Jorge Cortes Molecularly Targeted Therapies in Pediatric Myelodysplastic Syndromes............................................................................ 155 Lia Gore vii

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Contents

New Therapeutic Frontiers for Childhood Non-Hodgkin Lymphoma............................................................................... 177 Megan S. Lim and Mitchell S. Cairo Molecular Targeting of Post-transplant Lymphoproliferative Disorders........................................................................................................... 215 Michael Wang and Thomas G. Gross Part IIâ•… Solid Tumors Molecularly Targeted Therapies for Astrocytomas...................................... 231 Ian F. Pollack Targeted Therapy in Medulloblastoma in Molecularly Targeted Therapy for Childhood Cancer...................................................... 267 Yoon-Jae Cho and Scott L. Pomeroy Future Treatments of Ependymoma.............................................................. 291 Richard J. Gilbertson Development of Targeted Therapies for Rhabdoid Tumors Based on the Functions of INI1/hSNF5 Tumor Suppressor........................ 305 Ganjam V. Kalpana and Melissa E. Smith Development of Targeted Therapies for Neurofibromatosis Type 1 (NF1) Related Tumors......................................................................... 331 Brigitte Widemann Molecular Therapy for Neuroblastoma......................................................... 351 Yaël P. Mossé and John M. Maris Ewing’s Sarcoma Family of Tumors: Molecular Targets Need Arrows..................................................................................................... 373 Jeffrey A. Toretsky and Aykut Üren Molecular Targeted Therapy for Wilms’ Tumor.......................................... 401 James I. Geller and Jeffrey S. Dome Molecular Therapy for Rhabdomyosarcoma................................................ 425 Raushan T. Kurmasheva, Hajime Hosoi, Ken Kikuchi, and Peter J. Houghton Molecularly Targeted Therapy for Osteosarcoma: Where Do We Go from Here?......................................................................... 459 Rosanna Ricafort and Richard Gorlick

Contents

ix

Nonrhabdomyosarcoma Soft Tissue Sarcoma in Children: Developing New Treatments Based on a Better Understanding of Disease Biology.................................................................. 499 Stephen X. Skapek Index.................................................................................................................. 521

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Introduction to Targeted Therapy Text Robert J. Arceci and Peter Houghton

The dream of “magic bullets” to treat patients with cancer, as defined by Paul Ehrlich in the early 1900s during his Harben Lectures, promised great hope for molecules that would specifically react with and eradicate tumors without harming the host. His lecture was entitled, “Experimental Researches on Specific Therapy. On Immunity with Special Reference to the Relationship between Distribution and Action of Antigens.”1 Many decades have come and gone since those concepts were first presented along with many lives prematurely lost to cancer. One might ask then “Where did we go wrong?” As if trying to push Ehrlich’s ideas more rapidly forward, Hollywood and Warner Brothers Studios premiered in 1940 the movie “Dr. Ehrlich’s Magic Bullets,” starring Emanuel Goldenberg, aka Edward G. Robinson. During that same period, under the auspices of the Department of Defense, Drs. Louis Goodman and Alfred Gilman were enlisted to investigate autopsy results of soldiers who had died following mustard gas exposures, leading to one of the earliest insights into the selective ablation of the hematopoietic elements and subsequently to early clinical trials in patients with primarily lymphoid malignancies.2 During the first half of the twentieth century, the Indian investigator, Dr. Yellapragada Subbarao, was successful in synthesizing a spectrum of antimetabolites, leading in turn to the landmark study by Farber etÂ€ al., “Temporary Remissions in Acute Leukemia in Children Produced by Folic Acid Antagonist, 4-aminopteroyl-glutamic acid (Aminopterin), in 1948.3 In 1956, Li, Hertz, and Spencer reported the effect of methotrexate on gestational related choriocarcinoma leading eventually to a curative therapy and demonstrating that indeed a small molecule could cure a widely metastatic cancer.4 These were of course all examples of targeted cancer therapy. Following the death of her husband, Albert Lasker, from cancer in the 1950s, Mrs. Mary Lasker and her Citizens Committee for the Conquest of Cancer, effectively promoted the conviction that the federal government had the ability and resources to develop a national crusade to cure cancer. A second United States National Cancer Act (The first was in 1937 and established the National Cancer institute.) was signed by President Richard Nixon in 1971. There was a tremendous amount of excitement during those decades for the day when Dr. Ehrlich’s magic bullets would cure all cancers. Comprehensive Cancer Centers arose, small molecule drug screening began on a wide scale leading to the development of new therapies, xi

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training in oncologic specialties expanded, the maturation of the clinical cooperative groups for adults and children occurred, the need for multidisciplinary treatment teams and supportive care became apparent, and the initiation of combination chemotherapy, adjuvant and neoadjuvant approaches to treatment along with sanctuary site prophylaxis were developed. Over the course of those decades, many cancer patients of all ages benefited. For children with cancer, cancer evolved from a disease that was nearly always fatal to a disease in which about 75% of patients can be cured. Yet, a large percentage of patients still could not expect to be cured. And for those who were cured, the adverse consequences of treatment were too often severe. Thus, as combinations of more intense doses and delivery schedules were tested, the very real plateau of balancing cure and toxicity became an increasingly difficult challenge. Had Dr. Ehrlich’s dream come to an end? Fortunately, throughout those decades, basic laboratory and translational investigators were pushing forward with novel tools and approaches to extend our knowledge of the molecular basis of different cancers and the hosts they afflict. This basic understanding of genetics and epigenetics has formed the substrate of a resurgence of hope in the form of the next generation of targeted therapies. The new therapeutic agents are based on the distinct genetic and epigenetic signatures that translate into unique protein expression patterns that can be exploited for the benefit of patients. From the dawn of this resurgence, stemming from the demonstration of targeting BCR-ABL with imatinib,5 a plethora of novel tumor specific or selective approaches are being applied. Targeting these tumor specific pathways is based on the development of small molecule signal transduction modulators to monoclonal antibodies to antitumor vaccines to nanoparticles. In addition, a growing recognition of the confluence of stem cell biology to the behavior of tumor initiating stem cells is provided for the first time in history the correct cellular targets. The treatment of children with cancer has been at center stage addressing the basic causes of cancer as well as developing and exploiting novel molecularly targeted therapies. This volume attempts to bring together in one place some of the successes, but mostly the hope of eradicating the morbidity and mortality of both hematopoietic malignancies and solid tumors in pediatric patients. The chapters, written by expert laboratory and clinical investigators, provide updated information on current and future approaches to targeted therapy in pediatric oncology. Further, there is a strong belief that the development of less toxic and more effective combinations of therapeutic approaches will also be important in improving the outcome of the 85% of children with cancer who live in developing countries and too often cannot be successfully treated. While the pace of progress has continued to accelerate, for too many patients the pace remains too slow. As Mario Andretti once said, “If things seem under control, you’re not going fast enough.” It is with that sense of urgency that the current state of targeted therapy should be catapulted to the next level based on an increasingly detailed molecular description of both tumors and hosts as we enter an era of increasing genetically directed therapies. There still remains an immense need for

Introduction to Targeted Therapy Text

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more financial support, more collaboration, more discovery, more innovation, and more translation, but there has never been, nor will there ever be a lack of passion and hard work. Given the opportunities, those dedicated in eradicating this ancient enemy of the young and old will most assuredly succeed and bring into reality the magic bullets of Paul Ehrlich.

References Witkop B. Paul Ehrlich and His magic Bullets, Revisited. Proceedings of the American Philosophical Society. Vol. 143. Philadelphia, PA: American Philosophical Society; 1999; 540–557. Goodman LS, Wintrobe MM, Dameshek W, Goodman MJ, Gilman A, McLennan MT. Nitrogen mustard therapy. Use of methyl-bis(beta-chloroethyl)amine hydrochloride and tris(beta-chloroethyl)amine hydrochloride for Hodgkin’s disease, lymphosarcoma, leukemia and certain allied and miscellaneous disorders. JAMA. 1946; 251:2255–2261. Farber S, Diamond LK, Mercer RD, Sylvester RF Jr, Wolff JA. Temporary Remissions in acute leukemia in children produced by folic acid antagnonist, 4-aminopteroyl-glutamic acid (aminopterin). NEJM. 1948; 238:787–793. Li M, Hertz R, Spencer DB. Effect of methotrexate therapy upon choriocarcinoma and chorioadenoma. Proc Soc Exp Biol Med. 1956; 93:361–366. Druker BJ, Talpaz M, Resta DJ, etÂ€al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med. 2001; 344:1031–1037.

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Contributors

Robert J. Arceci King Fahd Professor of Pediatric Oncology, Professor of Pediatrics, Oncology and Cellular and Molecular Medicine, Kimmel Comprehensive Cancer Center at Johns Hopkins, The Bunting & Blaustein Cancer Research Building, 1650 Orleans Street, Suite 207, Baltimore, MD 21231, USA [email protected] Andrea Biondi Centro M. Tettamanti, Clinica Pediatrica Università di Milano-Bicocca, Ospedale San Gerardo, Via Pergolesi, 33, 20052 Monza, Italy [email protected] Patrick A. Brown Sidney Kimmel CCC at Johns Hopkins, Bunting-Blaustein Cancer Research Building, 1650 Orleans Street, CRB I-2M49, Baltimore, MD 21231, USA [email protected] Mitchell S. Cairo Division of Pediatric Blood and Marrow Transplantation, New York-Presbyterian Morgan Stanley Children’s Hospital, Columbia University, 3959 Broadway, CHN 10-03, New York, NY 10032, USA [email protected] William L. Carroll New York University Cancer Institute, 522 First Avenue, Smilow 1201, New York, NY 10016, USA [email protected] Seth J. Corey Departments of Pediatrics and Cell & Molecular Biology, Children’s Memorial Hospital and the Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL [email protected]

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Contributors

Jeffrey S. Dome Division of Oncology, Center for Cancer and Blood Disorders, Children’s National Medical Center, 111 Michigan Avenue NW, Washington, DC 20010, USA [email protected] Carolyn A. Felix Division of Oncology, Department of Pediatrics, The Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Colket Translational Research Building, Room 4006, 3501 Civic Center Blvd, Philadelphia, PA [email protected] Lia Gore The University of Colorado Cancer Center and The Center for Cancer and Blood Disorders, The Children’s Hospital, University of Colorado Denver, Pediatrics Mail Stop 8302, P.O. BoxÂ€6511, Aurora, CO 80045, USA [email protected] Richard Gorlick Department of Pediatrics, Pediatric Hematology/Oncology, The Children’s Hospital at Montefiore and the Albert Einstein College of Medicine, 3415 Bainbridge Avenue, Rosenthal 3rd Floor, Bronx, NY 10467, USA [email protected] Richard J. Gilbertson Neurobiology and Brain Tumor Program, St. Jude Children’s Research Hospital, Memphis, TN, USA [email protected] Thomas G. Gross Division of Hematology/Oncology/BMT, The Ohio State University College of Medicine, 700 Children’s Drive, Columbus, OH 43205, USA [email protected] Peter J. Houghton Department of Director, Center for Childhood Cancer, The Research Institute Nationwide Children’s Hospital, 700 Children’s Drive, Columbus, OH 43205 [email protected] Ganjam V. Kalpana Department of Molecular Genetics and Albert Einstein College Cancer Center, Albert Einstein College of Medicine of Yeshiva University, 1300, Morris Park Ave., Ullman 821, Bronx, NY 10461, USA [email protected]

Contributors

xvii

Jennifer O’Hara Lauchle University of California, Helen Diller Family Cancer Research Building (Optional), 1450 3rd Street, Room 264 (Lauchle) and 265 (Braun), San Francisco, CA 94158, USA [email protected] A. Thomas Look Department of Pediatric Oncology, Dana-Farber Cancer Institute and Children’s Hospital Boston, 44 Binney Street, Boston, MA 02115, USA and Harvard Medical School, Boston, MA, USA [email protected] Yaël P. Mossé Division of Oncology, Department of Pediatrics, Children’s Hospital of Philadelphia, University of Pennsylvania, 3615 Civic Center Blvd., ARC 907C, Philadelphia, PA, USA [email protected] Rob Pieters Department of Pediatric Oncology and Hematology, Erasmus MC, Sophia Children’s Hospital, Room Sp2456, Dr Molewaterplein 60, 3015 GJ Rotterdam, The Netherlands [email protected] Ian F. Pollack Department of Neurosurgery, Children’s Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA, USA [email protected] Scott L. Pomeroy Department of Neurology, Children’s Hospital Boston, 300 Longwood Avenue, Enders 270 , 02115 Boston, MA, USA [email protected] Yaddanapudi Ravindranath Children’s Hospital of Michigan, 3901 Beaubien Boulevard, Detroit, MI 48201, USA [email protected] Donald Small Sidney Kimmel CCC at Johns Hopkins, Bunting-Blaustein Cancer Research Building, 1650 Orleans Street, CRB I-252, Baltimore, MD 21231, USA [email protected]

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Contributors

Stephen X. Skapek Pediatric Hematology/Oncology, The University of Chicago Comer Children’s Hospital, 900 E. 57th Street, Chicago, IL 60637, USA [email protected] Jeffrey W. Taub Division of Hematology/Oncology, Children’s Hospital of Michigan, 3901 Beaubien Boulevard, Detroit, MI 48201, USA and Department of Pediatrics, Wayne State University School of Medicine, Detroit, MI 48201, USA and Developmental and Therapeutics Program, Barbara Ann Karmanos Cancer Institute, 4100 John R Street, Detroit, MI 48201, USA e-mail: [email protected] Jeffrey A. Toretsky Departments of Oncology and Pediatrics, Lombardi Comprehensive Cancer Center, Georgetown University, 3970 Reservoir Rd. N.W., Washington, DC 20057-1469, USA [email protected] Brigitte Widemann Pharmacology and Experimental Therapeutics Section, National Cancer Institute, Pediatric Oncology Branch, 10 Center Drive, 10-CRC, Room 1-5750, MSC 1101, Bethesda, MD 20892, USA [email protected]

Part I

Hematologic Malignancies

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The Emerging Era of Targeted Therapy in Childhood Acute Lymphoblastic Leukemia William L. Carroll and Rob Pieters

One of the most fundamental goals of modern cancer research is to develop more effective therapies that specifically target the cancer cell while sparing normal cells from the collateral damage that is common to conventional therapies. The cornerstone of current cancer treatment depends on drugs associated with a very narrow therapeutic index in that the effective dose and the toxic dose frequently overlap. While progress in pediatric oncology, specifically, improved cure rates for the most common childhood malignancy, acute lymphoblastic leukemia (ALL), has outpaced improvements in other cancer subtypes, treatment for ALL still relies on conventional cytotoxic agents thereby exposing children to considerable short- and long-term side effects. Optimally, targeted therapy would converge on a specific lesion or pathway in the cancer cell, not shared by normal cells, that is essential to the maintenance of the population of leukemia cells. In order to be most effective, the agent must have efficient access to the biological target to maximize tumor kill. Fortunately, in the past few years, a spectrum of agents have been, or are currently being developed, that target cancer cells with increasing specificity. With the initial development of monoclonal antibodies to specific leukocyte antigens, it became clear that leukemia cells display a unique profile of surface proteins shared by a minority of normal cells. These antigens have proved to be useful targets for therapeutic antibodies. Likewise, breakthroughs in methods to analyze gross chromosome structure and the subsequent introduction of recombinant DNA technology led to the recognition that all human cancers were the result of disrupted biological pathways due to somatic, and sometimes germline, alterations in DNA copy number (e.g., amplifications, deletions) and/or structure (translocations, mutations, etc.). Thus, there are many examples of where altered biological pathways that drive cancer initiation and maintenance can be traced back to these sentinal genetic lesions.

W.L. Carrollâ•›(*) New York University Cancer Institute, 522 First Avenue, Smilow 1201, New York, NY 10016, USA e-mail: [email protected]

P.J. Houghton and R.J. Arceci (eds.), Molecularly Targeted Therapy for Childhood Cancer, DOI 10.1007/978-0-387-69062-9_1, © Springer Science+Business Media, LLC 2010

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W.L. Carroll and R. Pieters

The identification of the specific genes and pathways involved in transformation and their subsequent validation in various experimental models now provide the first bona fide molecular targets. These studies have also determined that the discovery of a somatically mutated gene in a cancer cell does not necessarily indicate that it will prove to be a useful target, and subsequent analysis to distinguish “driver” from “passenger” mutations will prove key to the prioritization potential targets (Frohling etÂ€ al. 2007). This complex process is now even more daunting with the massive amount of data coming from more recent advances in high throughput sequencing, proteomic approaches, epigenetic profiling, and analysis of non-coding RNAs. While many approaches to targeted therapy are being considered, this overview will focus on those targets and their corresponding targeted agents that are currently in clinical trials that best illustrate the challenges and opportunities for this, the next generation of clinical trials in ALL.

The BCR/ABL Tyrosine Kinase and Ph+ ALL The t(9;22)(q34;q11) or Philadelphia chromosome was the first nonrandom chromosomal abnormality detected in cancer and is identified in 2% of childhood ALL samples and in approximately 20% of adult cases (Moorman etÂ€al. 2007; Schultz etÂ€al. 2007). The translocation results in the well known BCR-ABL protein fusion that is a constitutively active tyrosine kinase (Chan etÂ€al. 1987; Clark etÂ€al. 1988). Historically, the presence of the Ph+ genotype correlates with an extremely poor outcome and has been used in risk stratification (Secker-Walker etÂ€al. 1997; Schultz etÂ€al. 2007). While age, WBC, and early response may select for patients with a better outcome, stem cell transplantation (SCT) strategies have been usually considered for this subgroup of patients (Ribeiro etÂ€ al. 1997; Schrappe etÂ€ al. 1998). However, even if stem cell transplantation is performed after successful induction, many patients will still succumb to the disease. In the largest analysis to date in children, the 5Â€ year disease-free survival is 25±4% with chemotherapy alone compared to 65±8% when SCT is performed (Arico etÂ€al. 2000). The discovery of the biological function of the fusion protein as well as recognition that BCR-ABL was essential for tumor maintenance led to efforts to screen for inhibitors. The ATP binding pocket proved to be an attractive target for selective inhibition. Imatinib mesylate was the first such compound to enter clinical trials. Considerable preclinical data showed that it bound to the inactive confirmation of the kinase domain, blocked kinase activity, and inhibited proliferation of leukemia cells harboring the t(9;22). The results from early trials in chronic myelogenous leukemia led to considerable enthusiasm for integration into treatment strategies for Ph+ ALL (Druker etÂ€al. 1996, 2001, 2006). In phase I studies performed in children and adults, the drug was well tolerated and the majority of patients showed a response including a complete response rate of approximately 20% (Ottmann etÂ€al. 2002). However, these responses were short-lived with most patients showing

The Emerging Era of Targeted Therapy in Childhood Acute Lymphoblastic Leukemia

5

disease progression within weeks of their initial response. Studies incorporating imatinib into a backbone of chemotherapy soon followed these initial observations. A number of protocols using a combination of imatinib and chemotherapy in adults showed that this approach improved the induction rate, led to a lower disease burden, increased the number of patients proceeding to SCT, and most importantly, showed an impact on EFS and overall survival. For example, a study by Thomas and colleagues from MD Anderson Cancer Center investigated the efficacy of imatinib given concurrently with the hyper-CVAD regimen (Thomas etÂ€al. 2004). The CR rate on this study was 93% and the 3Â€year relapse free and overall survival rate was 62% and 55%, respectively, far better than historical controls. The Children’s Oncology Group COG initiated a study for Ph+ ALL (AALL0031) in 2002. Children and adolescents 1 to 21Â€years of age with Ph+ ALL were eligible after initial induction using standard chemotherapy. Imatinib was introduced in a stepwise fashion over five cohorts of patients to assess potential toxicity when used with aggressive chemotherapy. Patients with an HLA identical sibling proceeded to stem cell transplantation after two consolidation courses. Other patients received three additional intensification and re-induction blocks followed by 12 8-week cycles of maintenance chemotherapy for a total protocol duration of just over 2Â€ years. Patients in cohort one received 42Â€ days of imatinib before maintenance whereas patients in cohort 5 received 280Â€days. All groups received 336Â€days of imatinib in maintenance and patients who received SCT had 6Â€months of imatinib post transplant. Imatinib was well tolerated in the context of aggressive chemotherapy on AALL0031. There was modest ALT elevation noted in maintenance that led to a shortening of the duration of imatinib from 21 to 14Â€days for each month of maintenance. In addition, there was an increase in grade III and IV neutropenia during re-induction block 2 and a lower total WBC in consolidation. The 3Â€year EFS of patients on cohort 5 who received continuous imatinib was 80.5±11.2%, well above the 35.0±4.4% EFS reported on previous cooperative group studies. Twenty one patients received matched sibling transplants, and there was no difference in outcome among those receiving chemotherapy versus SCT. Furthermore, end induction minimal residual disease was not prognostic in those patients exposed to continuous imatinib indicating that targeted therapy can overcome the adverse prognostic significance of slow early response to chemotherapy. These striking results, while early, are extremely encouraging and represent an example of the promise of targeted therapy. Has been well documented that imatinib resistance is most commonly due to the development of BCR-ABL mutants (usually in the P loop or ATP-binding site) that alter affinity for imatinib (Branford etÂ€al. 2003; O’Hare etÂ€al. 2007). Newer generation tyrosine kinase inhibitors such as dasatinib and nilotinib, among others, inhibit such mutants, show more potent suppression of BCR-ABL kinase activity, bind to both the inactive and active conformation of BCR-ABL (e.g., dasatinib), and inhibit important collateral pathways such as those involving Src that are known to play a role in Ph+ ALL (Shah etÂ€al. 2004; Weisberg etÂ€al. 2005; Talpaz etÂ€al. 2006). Preliminary data from an ongoing European phase I/II study with dasatinib show promising activity in Â�children with

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W.L. Carroll and R. Pieters

Ph+ ALL who are refractory or intolerant to imatinib (Zwaan etÂ€ al. 2008). Therefore, dasatinib is being used in the current COG study (AALL0622) and is integrated earlier in treatment during induction to maximize early tumor kill.

The FLT-3 Pathway in MLL Rearranged Infant ALL Infants under 1Â€year of age constitute an especially high risk subgroup of patients with ALL. The primary biological basis for this association is that leukemic blasts from such children harbor an 11q23 rearrangement involving the MLL gene, usually t(4;11), t(11;19) or t(9:11) (Silverman 2007). The 6Â€ year event free survival (EFS) for these infants ranges from 22% to 43% and aggressive attempts to improve outcome using augmented chemotherapy including stem cell rescue have largely failed (Pui etÂ€ al. 2002; Hilden etÂ€ al. 2006; Pieters etÂ€ al. 2007). Thus, novel approaches are clearly warranted. The FMS-like tyrosine kinase-3 (FLT-3) is a class III receptor tyrosine kinase, a family that includes the c-KIT and platelet derived growth factor (PDGF) receptors (Agnes etÂ€al. 1994) (Fig.Â€1). FLT-3 is expressed in almost all cases of AML and B-lineage ALL, and up to a third of T ALL (Carow etÂ€al. 1996; Rosnet etÂ€al. 1996).

FLT3L-Binding

Extracellular Domain

Transmembrane Domain Juxtamembrane Domain Kinase Domain

Internal Tandem Duplications Activation Loop Mutations P

P

Fig.Â€1â•… The FLT-3 Receptor. The FLT-3 receptor contains an extracellular domain consisting of five immunoglobulin like domains. The juxtamembrane portion is a target for tandem duplications. Two kinase domains are linked through a tyrosine kinase insert. The kinase domain is mutated in hematological malignancies and most mutations are single point substitutions with insertions and deletions occuring less frequently. Binding of FLT-3 ligand leads to receptor dimerization and phophorylation of the kinase domain that triggers the activation of downstream pathways. (Revised based on reference (Stirewalt and Radich 2003))

The Emerging Era of Targeted Therapy in Childhood Acute Lymphoblastic Leukemia

7

Constitutive activation of FLT-3 is observed through somatic mutation and/or a through co-expression of the FLT-3 ligand as part of an autocrine loop (Brasel etÂ€al. 1995; Drexler 1996; Nakao etÂ€al. 1996). Activating mutations of FLT-3, either in the juxtamembrane region or kinase domain, are seen in 22% of childhood AML samples but are rare in childhood ALL cases (Schnittger etÂ€al. 2002; Thiede etÂ€al. 2002). Juxtamembrane mutations are characterized by internal tandem duplications (FLT/ITD) and insertion/deletion mutations. Importantly, FLT/ITD constitutes an adverse prognostic variable in AML (Meshinchi etÂ€al. 2001). High level mRNA and protein expression of FLT-3 is observed in infant and childhood ALL with rearrangements of MLL and cases associated with hyperdiploidy – two subtypes with distinctly different outcomes (Armstrong etÂ€ al. 2002; Yeoh etÂ€al. 2002). FLT-3 mutations are absent in most subtypes of childhood ALL except for MLL-rearranged and hyperdiploid cases where up to 18 and 25% of samples, respectively, show either kinase mutations (MLL and hyperdiploid) and/ or juxtamembrane mutations (hyperdiploid) (Armstrong etÂ€al. 2004; Taketani etÂ€al. 2004). Other studies showed a much lower incidence of FLT-3 mutations in these subtypes and that the level of expression may be related to outcome in MLL rearranged ALL (Stam etÂ€ al. 2007a, b). Finally, several studies have demonstrated constitutive phosphorylation of FLT-3 in MLL rearranged infant ALL and hyperdiploid cases even in the absence of mutation suggesting an autocrine loop (Brown etÂ€al. 2005; Stam etÂ€al. 2005). Given the importance of FLT-3 in childhood leukemia, there has been great interest in exploring the therapeutic potential of FLT-3 inhibitors. A number of such agents have been developed including CEP-701 (lestaurtinib), SU112248 (sunitinib malate), and PKC412 (midostaurin) among others (Stirewalt and Radich 2003). Lestuartinib is of particular interest because it is currently being evaluated in two Children’s Oncology Group (COG) trials for MLL rearranged infant ALL, as well as, relapsed FLT-3 mutant AML. Preclinical work has demonstrated the therapeutic potential of this agent (Brown etÂ€ al. 2005, 2006). Phase II trials in adults with refractory/relapsed AML demonstrate tolerability and clinical responses, especially in patients with FLT-3 mutant AML (Smith etÂ€al. 2004). A phase I trial in children with heavily pretreated neuroblastoma (lestuartinib also inhibits the Trk neurotropin receptors) has also demonstrated the feasibility of administration. It is noteworthy that preclincal studies with combination chemotherapy show that synergism is achieved when chemotherapy is delivered first followed by FLT-3 inhibition with lestuartinib while the reverse sequence is antagonistic (Brown etÂ€ al. 2006). This important observation underscores the need for careful preclinical evaluation when combining conventional chemotherapy with biologically targeted agents. COG AALL0631 will test whether FLT-3 inhibition with lestuartinib administered on a backbone of chemotherapy will improve outcome for children with MLLrearranged ALL. The trial has a limited institution safety phase where two cohorts of children (99%) plasma protein bound, such that total drug levels measured by standard pharmacokinetic assays give little information regarding drug activity. Furthermore, direct assessment of FLT3 inhibition in leukemic blasts in patients is particularly unfeasible in clinical trials in which the chemotherapy administered beforehand results in clearance of blasts from the blood. Therefore, a plasma inhibitory activity (PIA) was developed as a surrogate for direct assessment of inÂ€ vivo FLT3 inhibition (Levis etÂ€al. 2006). Plasma is collected from patients prior to and at various trough time points after CEP-701 is administered. Aliquots of TF-1/ITD cells (TF-1 human leukemia cell line engineered to express a FLT3/ITD mutation) are exposed to the plasma, then lysed and subjected to FLT3 IP and P-FLT3 Western blotting. The percent inhibition of P-FLT3 compared to cells exposed to the patient’s pretreatment plasma (untreated control) is calculated to give an estimate of FLT3 inhibitory activity present in the patient’s plasma (PIA) at each time point. Validation of this approach has been done by comparing results in cases where FLT3 inhibition could simultaneously be measured directly in leukemic blasts and in plasma for the same patient at the same time point. The PIA is considered adequate if it is >80% at trough time points, corresponding to the degree of prolonged FLT3 inhibition required for cytotoxicity. The adult AML clinical trials with lestaurtinib have successfully correlated clinical responses with FLT3 PIA levels (Smith etÂ€al. 2004; Levis etÂ€al. 2005). PIA analysis is being performed in the ongoing COG clinical trials of lestaurtinib in childhood AML and infant ALL, and excellent levels of inhibitory activity at tolerable dose levels have been observed in some patients, suggesting that an adequate PIA can be achieved in infants and children also. Lestaurtinib is now being tested in the COG for children with relapsed/refractory FLT3-mutant AML. Lestaurtinib is given in sequential combination with reinduction chemotherapy (HiDAC and idarubicin), where the sequence of exposure (chemotherapy followed by FLT3 inhibitor) is based upon preclinical studies demonstrating maximal synergy between FLT3 inhibition with this sequence, compared to only additive effects with simultaneous exposure and antagonism with FLT3 inhibitor followed by chemotherapy (Levis etÂ€al. 2004; Brown etÂ€al. 2006a). The antagonism seen with the sequence of FLT3 inhibition followed by chemotherapy is due to the cell cycle arrest from FLT3 inhibitors, which protects the cells from the cytotoxic effects of chemotherapeutic agents that are cell-cycle dependent. Plans to incorporate FLT3 inhibitors into COG phase III clinical trials for patients with deÂ€novo FLT3/ITD positive AML are in progress.

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P.A. Brown and C.A. Felix Key

Enroll

Induction

A

Post-induction Arm A (SR chemo)

B

Post-induction Arm B (IR/HR chemo)

C

Post-induction Arm C (IR/HR chemo + lestaurtinib)

MLL-G

MLL-R

A

Randomize

B

C

Fig.Â€2â•… Design of COG study AALL0631. MLL-G, germline (wt) MLL; MLL-R, rearranged MLL

Lestaurtinib is also being tested in the COG for infants with newly diagnosed MLL-rearranged ALL. Lestaurtinib is being added in a randomized fashion to the multicourse chemotherapy regimen used in the previous clinical trial for infant ALL (COG P9407). The design of this study (Fig.Â€2) takes into account the preclinical data regarding combinations of lestaurtinib and chemotherapy, as lestaurtinib will be given immediately following the exposure to standard cytotoxic chemotherapy in an effort to maximize potential synergy, and will not be given for at least 24Â€hours prior to chemotherapy to avoid potential for antagonism.

Targeting Anti-apoptotic BCL-2 Family Members Deregulated apoptosis due to imbalanced expression of BCL-2 family proteins is a general avenue to chemotherapy resistance because the cytotoxicity of most anticancer chemotherapy occurs through this pathway. The focus on silencing antiapoptotic BCL2 family members in MLL leukemia in infants grew primarily from protein and gene expression level data on abundant expression of BCL-2 and other pro-survival BCL-2 family members in MLL leukemia in infants (Robinson etÂ€al. 2008; Zhang etÂ€ al. 2008a) coupled with preclinical and clinical adult forerunner data in different cancer cell types suggesting the utility of apoptosis pathway modulation, with selection of compounds to silence anti-apoptotic BCL-2 family members that were already in the clinical phases of development and showed minimal toxicities in adults.

Molecularly Targeted Therapy for Infant ALL

39

BCL-2 family members fall into three classes: multidomain anti-apoptotic proteins homologous in all four BCL-2 homology (BH) domains; multidomain pro-apoptotic proteins homologous in BH1, BH2, and BH3 domains; and BH3only proteins, which all are pro-apoptotic. BCL-2 family protein interactions occur via docking of the BH3 domain with a deep binding groove on the surface of the binding partner (Green 2005; Reed 2002, 2003). Pro-apoptotic multidomain BAX and BAK exist as inactive monomers, but they homo-oligomerize upon BH3-only BIM or BID activation, leading to mitochondrial outer membrane permeabilization and release of apoptotic cofactors cytochrome c and Smac/DIABLO to the cytosol (Danial and Korsmeyer 2004; Nguyen etÂ€al. 2007). Cytochrome c is then bound by Apaf-1, which forms Apaf-1 oligomers and recruits pro-caspase 9. Caspase-9 activation activates effector caspases that execute apoptosis (Danial and Korsmeyer 2004). Many targeted strategies, including antisense therapies, siRNAs, peptides, and small molecule inhibitors have emerged for silencing anti-apoptotic BCL-2 family proteins at the transcript and the protein levels (Reed 2003; Gewirtz etÂ€al. 1998; Letai etÂ€al. 2002; Garber 2005). Since quantitative real-time PCR and Western blot analysis demonstrated that BCL-2 mRNA and protein are abundant and suggested that BCL-2 provides a cell survival mechanism in cases of pediatric/infant ALL and AML with the t(4;11) or other MLL translocations, (Robinson etÂ€al. 2008) the first foray to modulate apoptosis in infant leukemias involved preclinical inÂ€ vitro studies on Genasense™ (Oblimersen, G3139; Genta, Inc.), an 18-mer phosphorothioate antisense oligodeoxynucleotide (ODN), which forms a DNA–RNA hybrid with the first six codons of the BCL-2 mRNA (Nicholson 2000). Among the forerunner adult studies on this agent was an encouraging Phase I trial of Genasense™ with chemotherapy for refractory/relapsed adult acute leukemia, in which 45% of patients had disease response and BCL-2 mRNA decreased in 75% of cases; (Marcucci etÂ€al. 2003) in addition, the toxicities (low-grade fever, transient liver function abnormalities, fatigue, thrombocytopenia) were limited (Marcucci etÂ€al. 2003). Its clinical activity in refractory and relapsed adult B-cell malignancies (O’Brien etÂ€al. 2005; Waters etÂ€al. 2000) also was encouraging. Abundant BCL-2 expression was detected in the cell lines RS4:11, MV4-11, and SEM-K2 with t(4;11), similar to primary MLL leukemia cases, enabling drug activity and mechanistic assays on the cell line models (Robinson etÂ€ al. 2008). Genasense™ abrogated BCL-2 mRNA expression in RS4:11 cells and significantly reduced the protein, though to recognize the changes these molecular analyses were performed at high concentrations (Robinson etÂ€ al. 2008). However, studies of cytotoxicity and effects on apoptosis and cell cycle suggested that lower concentrations were sufficient to change the apoptosis threshold (Robinson etÂ€al. 2008). In MTT assays of cytotoxicity, the most sensitive of the three cell lines to single-agent Genasense™ was RS4:11, and SEM-K2 was the least sensitive (Robinson etÂ€al. 2008). Applying pharmacostatistical response surface modeling of drug interactions, Genasense™ at low concentrations was also found to sensitize RS4:11 and MV4-11 cells to select anti-leukemia cytotoxic drugs (ADR, VP16, ARAC, 6-TG in RS4:11; VP16, 6-TG in MV4-11), indicating synergistic

40

P.A. Brown and C.A. Felix

interactions (Robinson etÂ€al. 2008). In further flow cytometric mechanistic studies, combining Genasense™ with doxorubicin in RS4:11 cells increased active caspase-3 and TUNEL staining compared to doxorubicin alone, consistent with greater apoptosis (Robinson etÂ€ al. 2008). In addition, Genasense™ increased S-phase progression, (Robinson etÂ€al. 2008) which is consistent with an effect on the BCL-2 target transcript since the anti-apoptotic effects of BCL-2 are linked to retardation of the G1/S transition. (Deng etÂ€al. 2003) This evaluation of the effects of the pro-apoptotic BCL-2 antisense compound Genasense™ designed to decrease BCL-2 protein levels via selective BCL-2 mRNA degradation as a prototypic strategy to modulate cell death, indicated that abundant BCL-2 affords a molecular target in leukemias with t(4;11). Although the cardinal anti-apoptotic regulator in the intrinsic cell death pathway, BCL-2, has a general role in chemotherapy resistance (Danial and Korsmeyer 2004), the formation of homo- and heterotypic dimers by many different BCL-2 family proteins with opposing anti- or pro- apoptotic actions together determines the apoptosis threshold and regulates the intrinsic (mitochondrial) cell death pathway. Therefore, an increasing number of small molecule inhibitors also have emerged to restore balance in this pathway. Obatoclax (GX15-070; GeminX, Inc.) is one that antagonizes a broad spectrum of anti-apoptotic BCL-2 family proteins (BCL-2, BCL-XL, A1, BCL-w, MCL-1) (Nguyen etÂ€al. 2007). Removal of the mitochondrial block by obatoclax also may enhance TRAIL-mediated extrinsic apoptosis (Huang etÂ€ al. 2009). Thus, there has been significant impetus for preclinical and clinical obatoclax development to potentiate chemotherapy by activating apoptosis mechanisms in malignancies with inherent or acquired chemotherapy resistance. Obatoclax represents the first molecularly targeted agent for silencing anti-apoptotic BCL-2 family members that will be tested in children. The recently approved COG trial ADVL0816, a Phase I study of obatoclax in combination with vincristine/ doxorubicin/dexrazoxane in children with relapsed/refractory solid tumors or leukemia, incorporates a separate stratum for patients with MLL disease on the basis of preclinical activity data elaborated on below. The pan-anti-apoptotic BCL-2 family small molecule inhibitor obatoclax has the ability to bind to the BH3 binding pocket of the anti-apoptotic BCL-2 family members and block their anti-apoptotic function (Garber 2005). A synthetic derivative of the bacterial prodiginines family of red-pigmented tripyrrolic compounds developed on the basis of structure activity relationships; (Williamson etÂ€al. 2007) obatoclax has been reported to cause apoptosis inÂ€vitro in a wide range of human cancer cells derived from adult leukemias, lymphomas, and solid tumors, (Campas etÂ€al. 2006; Galan etÂ€al. 2005; Perez-Galan etÂ€al. 2007; Li etÂ€al. 2007; Li etÂ€al. 2008b; Martinez-Paniagua etÂ€al. 2007; Trudel etÂ€al. 2007a; Witters etÂ€al. 2007) and single agent responses of several human solid tumor xenografts occurred. In solid tumor models, obatoclax showed synergy with cisplatin in nonsmall cell lung cancer cell lines (Li etÂ€al. 2008b). Synergy with cytosine arabinosine was reported in AML cell lines (Konopleva etÂ€ al. 2008). In mantle cell lymphoma lines, obatoclax induced apoptosis and enhanced the cytotoxicity of vincristine, (Bebb etÂ€al. 2006) proteasome inhibitors and doxorubicin (Yazbeck etÂ€ al. 2006). In addition to inducing


41

synergistic cytotoxicity when combined with cisplatin or doxorubicin (Martinez-Paniagua etÂ€ al. 2007; Hernandez-Ilizaliturri etÂ€ al. 2006a), obatoclax increased rituximabmediated antibody dependent cellular cytotoxicity and complement mediated cytotoxicity in non-Hodgkins lymphoma cell lines (Hernandez-Ilizaliturri etÂ€al. 2006b). Obatoclax also exhibited synergy in combination with dexamethasone in a dexamethasone sensitive multiple myeloma cell line and additive activity in several other multiple myeloma lines (Trudel etÂ€al. 2007a). Therefore, obatoclax rapidly advanced into adult Phase I and II solid tumor and leukemia clinical trials (Borthakur etÂ€ al. 2006; Goy etÂ€ al. 2007a; O’Brien etÂ€ al. 2009; Schimmer etÂ€al. 2007a; Verstovsek etÂ€al. 2007). The salient observations of adult Phase I trials evaluating various infusion durations and schedules have been that obatoclax is well tolerated with minimal toxicities, and that its primary toxicities, which affect the central nervous system (i.e., somnolence, euphoria, confusional state, ataxia), are reversible, infusion-related, and self-limited once the infusion stops. Importantly, modest obatoclax activity was suggested against various relapsed/refractory adult leukemias and solid tumors with minimal toxicity (O’Brien etÂ€ al. 2009; Goy etÂ€ al. 2007b). Remarkably, single agent obatoclax resulted in a complete cytogenetic remission, resolution of cytopenias, and transfusion independence in a patient with secondary AML with the t(9;11) translocation (Schimmer etÂ€al. 2007b). Thus, the attractive toxicity profile and the activity in MLL disease in an adult, made this agent especially suitable for preclinical pediatric studies, especially studies on MLL rearranged leukemia in infants as a potential pediatric cancer where obatoclax might have eventual utility in the clinic and substantial impact. In addition, HOXA9 repression of pro-apoptotic BIM (BCL2L11) was shown in a cell line with t(9;11), (Stubbs etÂ€al. 2007) and silencing of the MLL-AF4 fusion oncoprotein in cell lines with t(4;11) using siRNAs not only decreased BCL-XL protein, but also increased activated caspase 3 and increased apoptosis (Thomas etÂ€al. 2005). The rationale to test obatoclax also extended more generally to pediatric cancers and leukemia. In neuroblastoma, the inherent cancer cell survival mechanism derives from anti-apoptotic MCL-1 (Goldsmith etÂ€al. 2006). In contrast, BCL-XL is believed to be the direct transcriptional target of the PAX3-FKHR translocation, the primary molecular aberration in alveolar rhabdomyosarcoma (Xia etÂ€al. 2002). In childhood ALL, pro-apoptotic BIM expression predicted early response to treatment in the high-risk patients, (Bhojwani etÂ€ al. 2008) and decreased expression of the extrinsic apoptosis effector caspase CASP8 predicted MRD during and following induction (Flotho etÂ€al. 2007). Studies of apoptosis genes in relation to inÂ€vitro drug responses in childhood ALL uncovered the importance of MCL-1 in prednisolone sensitivity and of the new BCL-2 family members BCL2L13 and HRK in L-asparaginase resistance (Holleman etÂ€ al. 2006). Importantly, preclinical inÂ€ vitro studies suggested that obatoclax restored the glucocorticoid response in steroid resistant childhood precursor B-cell and T-cell ALL (Bonapace etÂ€ al. 2007). That not all of the emerging BH3 mimetics have high MCL-1 affinity, (van Delft etÂ€ al. 2006) also made obatoclax an attractive agent to advance in pediatrics.

42


The principal pediatric preclinical studies leading to the ADVL0816 obatoclax Phase I trial in relapsed/refractory solid tumors and leukemia were performed on MLL rearranged leukemia in infants. Quantitative real-time PCR suggested that not only anti- but also several pro-apoptotic BCL-2 family mRNAs, apoptosis execution, and various mitochondrial maintenance mRNAs were abundant in MLL rearranged infant ALL (Zhang etÂ€al. 2008a). Since basal expression levels of cell death and cell survival factors are relevant to finding novel therapeutic targets, these studies formed the basis to evaluate the potential of obatoclax to silence the activity of anti-apoptotic BCL-2 family protein targets in primary MLL leukemia cases (Zhang etÂ€al. 2008b). MTT assays of MLL rearranged infant ALL and bilineal acute leukemias showed variable single-agent sensitivities, many within clinically achievable range (Zhang etÂ€ al. 2008b). In a primary ALL with the MLL-AF4 rearrangement, pharmacostatistical response surface modeling indicated synergy between obatoclax and many of the common antileukemia cytotoxic chemotherapeutic drugs (cytosine arabinoside, doxorubicin, etoposide, dexamethasone, L-asparaginase, vincristine) (Zhang etÂ€ al. 2008b). Interestingly, apoptosis and the alternative cell death pathway of autophagy, in which cellular components are degraded through the lysosomal machinery, are partially overlapping in downstream cell death mediators (Bonapace etÂ€ al. 2007; Klionsky etÂ€ al. 2008; Maiuri etÂ€ al. 2007). Accumulating evidence from standard studies of autophagy (Klionsky etÂ€al. 2008) and autophagy factors is suggesting that the obatoclax cell death mechanism in MLL leukemia involves the autophagy cell death pathway (Zhang etÂ€al. 2008b). Biomarkers in the autophagy pathway will be further studied as the Phase I clinical trial of obatoclax ADVL0816 advances. Beyond the demonstration of preclinical inÂ€vitro activity, obatoclax also is attractive to advance for leukemia in infants because pharmacometric modeling strategies and a pharmacokinetic study carried out in a diseased NOD-scid-IL-2Rgnull (NOG) immunodeficient xenograft model that recapitulates the hyperleukocytosis and extramedullary involvement of MLL leukemias, (Zhang etÂ€al. 2008c) have demonstrated that obatoclax has excellent biodistribution to sites of involvement by MLL leukemia in infants, as indicated by tissue:plasma concentration ratios, including excellent CNS penetration (Zhang etÂ€al. 2008c, 2007). The ADVL0816 Phase I clinical study of obatoclax summarized in Fig.Â€3 was approved by Clinical Trials Evaluation Program (CTEP) and opened for enrollment in COG Phase I Consortium Institutions in 2009. After an initial single agent dose, obatoclax will be administered in combination with vincristine, doxorubicin, and dexrazoxane. Patients will be enrolled in three separate strata. Patients with relapsed/refractory solid tumors (Stratum 1) will be enrolled in a dose escalation trial with a starting obatoclax dose of approximately 70% of the adult MTD. As a result of the striking preclinical obatoclax activity, patients with relapsed/refractory MLL-rearranged leukemia (Stratum 2) will be eligible for enrollment concurrently with the dose finding phase in solid tumor stratum. Once the dose finding phase has been completed, a patient cohort with relapsed/refractory non-MLL leukemia will be enrolled in Stratum 3.


43

Fig.Â€3â•… COG study ADVL0816 treatment schema

The foregoing studies have built a foundation for overcoming drug resistance from abnormal cell death regulation in leukemia in infants where current treatments offer little hope due to the inherent drug resistance and unique vulnerabilities of infants to toxicities from intensive treatments. However, that MLL leukemias exhibit variable preclinical obatoclax sensitivities, (Zhang etÂ€ al. 2008b) although promising, also underscores that no one agent targeting this pathway is expected to be active in all cases. The small molecule inhibitor ABT737, which targets anti-apoptotic BCL-2, BCL-XL, and BCL-w, (van Delft etÂ€al. 2006) is another candidate to evaluate for cell death pathway modulation in leukemia in infants. ABT-737 disrupts BCL-2 family protein interactions by targeting the BH3 binding pocket of select anti-apoptotic BCL-2 family members (van Delft etÂ€ al. 2006). Inhibition of interactions of anti-apoptotic BCL-2 family proteins with proapoptotic BCL-2 family proteins by ABT-737 and activity of ABT-737 alone and in combination with various cytotoxic drugs has been demonstrated in several preclinical studies of leukemia (Kang etÂ€ al. 2007; Trudel etÂ€ al. 2007b; Chauhan etÂ€al. 2007; Konopleva etÂ€al. 2006; Kline etÂ€al. 2007; Kojima etÂ€al. 2006) and ABT263, which has identical pharmacologic properties, is advancing to adult clinic trials. This compound demonstrates mechanism-based single agent activity in lymphomas (Oltersdorf etÂ€al. 2005). Of particular interest, inÂ€vitro activity of ABT737 was recently studied in seven different ALL cell lines [T-cell ALL: COG-LL-317, CEM, MOLT-3, MOLT-4; pre-B ALL: COG-LL-317, Nalm-6, RS4:11], (Kang etÂ€ al. 2007) among which the RS4:11 cell line was established from a case of adult ALL with t(4;11); however, no cases of infant leukemia have been tested. Strongly synergistic or synergistic cytotoxicity as defined by combination indices £0.3 or £0.7, respectively, of ABT-737 combined with cytotoxics, especially L-asparaginase or vincristine, was shown in all of the cell lines, even when there was no single agent sensitivity; the strongest synergy was with Lasparaginase. Further study of the pro-Â�apoptotic effects of the ABT-737/L- asparaginase combination in RS4:11 and COG-LL-317 cells showed evidence of

44


mitochondrial apoptosis pathway Â�activation including an increase in the truncated form of BID, tBID, increased hypophosphorylated BAD, an increase in the activated form of BAX, BAX(s), an increase in the cleaved form of CASP8, which cleaves BID to tBID, and cleavage of CASP9 and CASP3. The ABT-737/L- asparaginase combination in COG-LL-317 cells resulted in increased Annexin V/FITC staining and increased JC-1 probe staining, and Western blot analysis showed increased mitochondrial cytochrome c release. Importantly, ABT-737 did not increase apoptosis in normal PBMCs. ABT-737 showed synergistic cytotoxicity when combined with vincristine, L- asparaginase and dexamethasone in 5 of the 7 cell lines tested, including RS4:11. ABT-737 combined with vincristine, L- asparaginase, and dexamethasone delayed the progression of established xenografts in two NOD/SCID models of relapsed primary ALL and increased EFS compared to chemotherapy alone or ABT-737 alone in one of the two xenografts. As described in detail above, infant acute leukemia exhibits deregulated FLT3 receptor tyrosine kinase signaling as a separate resistance mechanism (Armstrong etÂ€ al. 2003, 2004b; Brown etÂ€ al. 2006b; Taketani etÂ€ al. 2004b; Brown and Small 2004b; Brown etÂ€al. 2005b; Stam etÂ€al. 2005; Stubbs and Armstrong 2007) in addition to the imbalanced expression of cell death regulatory factors (Zhang etÂ€ al. 2008a). Therefore, it may be of substantial interest that Abbott Pharmaceuticals has developed small molecule inhibitors to both pathways. ABT-869 is an ATPcompetitive small molecule inhibitor with specificity for the VEGF and PDGF families of receptor tyrosine kinases (RTKs), thereby targeting multiple resistance mechanisms (Rodila etÂ€al. 2006). There is rationale to test ABT-869 (Carlson etÂ€al. 2005; Dai etÂ€al. 2007; Shankar etÂ€al. 2007; Albert etÂ€al. 2006) alone and combined with the pro-apoptotic agent ABT-737 in leukemias in this population, since FLT3 is a member of the PDGF family of RTKs (Dai etÂ€al. 2007). The activity of ABT-869 is dependent on mutant kinases (Albert etÂ€al. 2006). In the AML cell lines MV4-11 and Molm13 with MLL translocations and FLT3ITD mutations, ABT-869 already has exhibited inÂ€vitro activity in cell proliferation/ viability assays, and downregulation of FLT3, STAT5, and ERK phosphorylation and the expression of the STAT5 target gene Pim-1 (Shankar etÂ€al. 2007). ABT-869 prevented the formation of subcutaneous MV4-11 xenografts, caused the regression of established subcutaneous MV4-11 xenografts, slowed the progression of subcutaneous Molm13 xenografts and prolonged the survival of mice with marrow engraftment of MV4-11 cells (Shankar etÂ€al. 2007). Even though FLT3 tyrosine inhibitors may effectively decrease FLT3 phosphorylation, persistent phosphorylation of downstream signaling proteins (e.g., AKT, MAPK), activating mutations in parallel signaling pathways (e.g., RAS) or overexpression of other RTKs may be avenues to resistance, as has been shown in cell lines with MLL translocations (Piloto etÂ€al. 2007). Importantly, however, the ABT737 pro-apoptotic drug can neutralize resistance to FLT3 tyrosine kinase inhibition (Kohl etÂ€ al. 2007). Studies of ABT-737 and ABT-869 together in leukemia in infants would enable more detailed characterization of additional pathways to deregulated cell death mechanisms and RTK signaling and how these pathways can be targeted.


45

Targeting MLL Fusion Transcripts The plethora of MLL partner genes and heterogeneity in genomic breakpoint locations, which increases transcript heterogeneity even further, would make targeting the fusion transcripts difficult. Nonetheless, it has been possible to silence several of the more common MLL fusion transcripts experimentally using nucleic acid therapeutics (Gewirtz etÂ€al. 1998). Antisense oligodeoxynucleotides have resulted in successful downregulation of translation of MLL fusion proteins inÂ€vitro in cell lines (Kawagoe etÂ€al. 2001; Niitsu etÂ€al. 2001a; Akao etÂ€al. 1998). The MLL-AF9 downregulation in the THP-1 cell line resulted in reduced HOXA7 and HOXA10 gene expression and increased apoptosis (Kawagoe etÂ€ al. 2001). MLL-CBP downregulation using antisense rendered the AML cell line SN-1 sensitive to all-trans retinoic acid or 1a,25-dihydroxyvitamin D3 induced differentiation (Niitsu etÂ€al. 2001a). Importantly, pro-apoptotic effects of targeting the fusion transcript with an antisense oligodeoxynucleotide also were observed in the KOCL33 cell line derived from a case of infant ALL with the MLL-ENL translocation (Akao etÂ€al. 1998). More recently, fusion transcript specific siRNAs were used to deplete the respective fusion oncoproteins resulting from two different MLL-AF4 genomic rearrangements in the SEM-K2 and RS4:11 cell lines (Thomas etÂ€ al. 2005). The inÂ€ vitro effects of the respective siRNAs included decreased colony formation on methylcellulose, decreased proliferation in MTT assays, an increase in G0/G1 phase cells and a decrease in cells in S phase, increased activated caspase 3 and decreased antiapoptotic BCL-XL protein, decreased HOXA7, HOXA9, and MEIS1 gene expression, and decreased expression of the stem cell marker CD133 (Thomas etÂ€ al. 2005). Furthermore, injection of SEM-K2 cells that had been pretreated MLL-AF4 specific siRNA into SCID mice completely suppressed engraftment in a xenotransplantation assay (Thomas etÂ€al. 2005).

PFWT Targeting of MLL Partner Protein Interactions The MLL partner proteins AF4, ENL, AF9, and AF10 interact in a multiprotein complex, which is involved in the regulation of transcriptional elongation and recruitment of the histone H3K79 methyltransferase Dot1 to elongating RNA Pol II (Bitoun etÂ€al. 2007). The PFWT peptide mimetic of the AF9 binding domain of AF4 with a conjugated Penetratin transduction sequence at the amino terminus is in preclinical development for disruption of the AF4-AF9 interaction (Srinivasan etÂ€ al. 2004). The AF-9 interaction domain of AF4, which binds at the AF9 carboxyl terminus, is highly conserved in all LAF4 family proteins (AF4, LAF4, AF5, FMR2) and is retained in the MLL fusion oncoproteins (Srinivasan etÂ€al. 2004; Erfurth etÂ€al. 2004). PFWT disruption of AF4-AF9 complexes was validated in GST pull-down assays and in NIH 3T3 cells, and PFWT decreased cell proliferation of RS4:11 cells, KP-L-RY (MLL-AF5) cells and, to a lesser degree, THP-1 cells with the MLL-AF9

46


rearrangement (Srinivasan etÂ€ al. 2004). In the MV4-11 and Molm13 (MLL-AF9) myeloid cell lines, PFWT rapidly induces ultrastructural changes of necrosis; abrogation of PFWT cytotoxicity by the serine protease inhibitor TLCK implicated serine proteases in the cell death mechanism (Palermo etÂ€al. 2008). In MV4-11 cells, promising synergistic interactions were suggested between PFWT and cytosine arabinoside, etoposide, the HSP-90 inhibitor 17-allylamino17-demethoxygeldanamycin (17-AAG), or the FLT3 inhibitor 3,4-dimethoxybenzoylamide, in Molm13 cells, between PFWT and cytosine arabinoside or etoposide and, in KOPN-8 (MLL-ENL) cells, though only at half the IC50 concentration, between PFWT and cytosine arabinoside (Bennett etÂ€ al. 2009). Further study of MV4-11 cells showed that, with exception of 17-AAG, the penetratin sequence did not itself augment cytotoxicity (Bennett etÂ€al. 2009). Interestingly, in MV4-11 cells in which synergy was sequence-dependent (cytosine arabinoside or etoposide before PFWT), PFWT alone caused an increase in cells in G1 and a decrease in cells in S phase (Bennett etÂ€al. 2009). Treatment with etoposide and PFWT resulted in EM features of both apoptosis and necrosis and an increase activated caspase 3, indicating that the two cell death mechanisms are not mutually exclusive (Bennett etÂ€al. 2009).

Targeting Glycogen Synthase Kinase 3 The recent basic research discovery demonstrating that the serine/threonine kinase GSK3 is a required, cell type specific MLL leukemia maintenance factor, suggested GSK3 as a potential therapeutic target. The role of GSK3 in MLL leukemia is paradoxical because GSK3 is involved in tumor suppression in other cancer types, and also because GSK3 normally phosphorylates and inactivates b -catenin, MYCN, and JUN, and inhibits pathways involved in self renewal and proliferation, whereas GSK3 inhibition increases stem cell pluripotency and HSC repopulation (Wang etÂ€al. 2008). Targeting GSK3 using small molecule inhibitors (GSK3-IX, SB216763, alsterpaullone) in MLL-AF4 (SEMK2, RS4:11, MV4-11) and MLL-AF5 (KP-L-RY) cell lines inhibited proliferation and increased b-catenin expression, decreased G1–S phase cell cycle progression, and increased sub-G0-G1 DNA consistent with cell death. Culture of murine primary myeloid progenitors expressing transduced MLL-ENL, MLL-LAF4, MLL-AF6, or MLL-GAS7 in the presence of SB216763, decreased clonogenic potential and proliferation; myeloid differentiation and decreased c-Kit expression were suggested with prolonged exposure (Wang etÂ€al. 2008). Transduction of murine Gsk3b−/− cells with MLL fusion genes, or shRNAmediated Gsk3b knockdown in murine myeloid progenitors transduced with MLL fusion genes, increased sensitivity to pharmacological GSK3 inhibition, even though Gsk3b was not required for transformation, whereas Gsk3a depletion alone


47

did not have these effects. Still cooperativity was suggested since the sensitivity to pharmacological inhibition, decreased proliferation and clonogenicity, and increased myeloid differentiation were more profound in a Gsk3b−/− Gsk3aKD MLL fusion gene model in which both Gsk3 isoforms were depleted, and Gsk3b−/− Gsk3aKD MLL-ENL transduced cells did not induce leukemia in mice (Wang etÂ€al. 2008). Additionally, the treatment of MLL-AF4 leukemia bearing mice with the GSK3 inhibitor lithium carbonate prolonged their survival (Wang etÂ€al. 2008). With GSK3 inhibition, the CDKI protein p27Kip1 significantly increased in the MLL-AF5 leukemia cell line KP-L-RY, and in MLL-ENL transduced murine myeloid progenitors, indicating that the effects of GSK3 in MLL leukemias involve p27Kip1 suppression (Wang etÂ€al. 2008). None of these effects were observed in non-MLL leukemia cell lines (Wang etÂ€al. 2008).

HSP90 as a Potential Therapeutic Target The heat shock protein HSP90 is an ATP-dependent chaperone protein that functions in a multi chaperone protein complex with roles in the regulation of conformational maturation and refolding of client proteins, (Wiech etÂ€al. 1992; Stebbins etÂ€al. 1997; Terasawa etÂ€al. 2005) tertiary structure of the proteasome, (Imai etÂ€al. 2003) and steroid receptor functions and interactions (Terasawa etÂ€ al. 2005). Of particular interest to MLL leukemia, FLT3 is another HSP90 client protein (Yao etÂ€ al. 2003). Interestingly, in cancer cells HSP90 stabilizes PI3K (Belova etÂ€ al. 2008; Fujita etÂ€al. 2002) and AKT (Zhang etÂ€al. 2005) and has an extracellular role in invasion and metastasis via regulation of MMP2 protein maturation (Picard 2004; Eustace and Jay 2004; Eustace etÂ€al. 2004). HSP90 is of interest as a potential therapeutic target in infant leukemia. Proteomics-based 2D-DIGE analysis of whole cell proteins identified HSP-90 as a differentially expressed protein in the MLL-AF4 cell lines RS4:11 and MV4-11 compared to normal CD34+ cells (Yocum etÂ€al. 2006). In studies testing HSP90 inhibition, MLL leukemia cell lines exhibited differential 17-AAG sensitivity depending on FLT3 status; RS4:11 cells in which FLT3 is wild type were less sensitive than MV4-11 or Molm13 with the FLT3 ITD (Yao etÂ€al. 2003). In all three of these cell lines, HSP90 inhibition was associated with decreased FLT3, RAF, and AKT expression (Yao etÂ€ al. 2003). In MV4-11 cells, not only did quantitative proteomics indicate that 17-AAG effectively depleted the HSP90a target, but also 17-AAG resulted in decreased viability and proliferation and increased apoptosis; a decrease in nucleoside diphosphate kinase nm23 suggested nm23 as a novel biomarker of HSP90 inhibition (Yocum etÂ€ al. 2006). More recently, when synergy between 17-AAG and etoposide was tested, greater synergy was observed in Molm13 and MV4;11 with the FLT3 ITD than in RS4:11 cells with wild-type FLT3 (Yao etÂ€al. 2007). Synergistic effects of combining

48


17-AAG with the FLT3 inhibitor GTP14564 in cell lines with FLT3-ITD (MV411) or amplified wild-type FLT3 (SEM-K2) have also been suggested, with reductions in phospho-FLT3 and phospho-STAT5, G0–G1 arrest and increased apoptosis (Yao etÂ€al. 2005).

Epigenetic Strategies In cell lines with the MLL-AF9 translocation (THP-1, Mono-Mac-6, and Molm13), histone deacetylase inhibition with valproic acid has been associated with decreased cell viability, cell cycle arrest in G1, increased apoptosis and induction of p21, and the G cyclin CG2, which is involved in G1 cell cycle arrest (Tonelli etÂ€al. 2006). In the SN-1 AML cell line with the MLL-CBP translocation, various HDAC inhibitors (sodium butyrate, trichostatin A, polyloxyl bytyrate) exhibited synergy with all-trans retinoic acid in inducing differentiation as measured by NBT-reduction (Niitsu etÂ€ al. 2001a). In the SN-1 cell line, and in the KOCL33, KOCL51, and KOCL44 cell lines, all with the MLL-ENL translocation, the demethylating agent 5-aza-2¢-deoxycytidine resulted in p16/INK4A upregulation and sensitivity to differentiation therapy with all-trans retinoic acid and 1a,25-dihydroxyvitamin D3 (Niitsu etÂ€al. 2001b). Taken together these studies may suggest that combinations of epigenetic therapies with differentiation therapies may have utility in MLL disease.

mTOR Inhibition The serine/threonine kinase mTOR (mammalian Target of Rapamycin) is an integrator of several signal transduction pathways, and is involved in the regulation of cell cycle, apoptosis and angiogenesis. The mTOR pathway is aberrantly activated in many tumor types, and the inhibition of mTOR signaling (using sirolimus or one of its analogs, such as temsirolimus or everolimus) has shown antitumor activity in several model systems as well as early phase clinical trials. In fact, temsirolimus was recently FDA-approved for the treatment of advanced renal cell carcinoma (Hudes etÂ€al. 2007). There is some preclinical evidence that inhibition of the serine/ threonine kinase mTOR (mammalian Target of Rapamycin) may be a useful therapeutic strategy in infant leukemia. Sirolimus (and the related mTOR inhibitor temsirolimus) inhibit the growth of precursor B cell ALL cell lines inÂ€vitro and are also active in ALL transgenic and xenograft murine models (Brown etÂ€ al. 2003). In further studies, mTOR inhibitors showed synergistic cytotoxicity with methotrexate against human ALL xenografts (Teachey etÂ€al. 2008). Moreover, sirolimus has been shown to overcome glucocorticoid resistance in ALL cells via anti-apoptotic MCL1 downregulation (Wei etÂ€al. 2006). Although none of these studies were performed on MLL-rearranged leukemias specifically, several groups have suggested that the


49

glucocorticoid resistance in MLL-rearranged infant ALL may be related to the poor prognosis (Pieters etÂ€al. 1998; Palle etÂ€al. 2005). Since mTOR inhibition can reverse glucocorticoid resistance, these studies may provide rationale to consider clinical mTOR inhibition in infant ALL.

Targeting CD33 Cell Surface Antigen Gemtuzumab ozogamicin is a humanized anti-CD33 antibody linked to the antitumor antibiotic calicheamicin. Not only is CD33 positivity observed in a preponderance of the subtypes of pediatric AML including cases with MLL translocations but also this agent may have a role in MLL rearranged ALL because of the frequent coexpression of myeloid cell surface markers (Zwaan etÂ€al. 2003). The results of clinical testing of gemtuzumab ozogamicin for remission induction in conjunction with intensive post induction therapy for relapsed/refractory CD33+ AML have been encouraging (Zwaan etÂ€al. 2003; Arceci etÂ€al. 2005).

Conclusions Several examples of molecularly targeted agents at various stages of development for infant leukemia have been discussed. As the molecular pathobiology of infant leukemia continues to unravel, the characterization of MLL fusion oncoproteins and factors that interact with MLL fusion oncoproteins in multiprotein complexes or cooperate with them in downstream or parallel signaling pathways have led to identification of a number of promising targets and experiments on potentially efficacious molecularly targeted agents that might benefit this patient population. The agents are at various stages of testing along the drug development pipeline from basic to preclinical inÂ€vitro and inÂ€vivo testing, and more recently to testing in clinical trials. In 2010, most still are at the basic and preclinical stages of development. Identification of efficacious agents with a low toxicity profile holds promise to change the outcome of this subset of pediatric cancer that has heretofore been so refractory to more conventional treatments because the mechanisms of action of targeting specific signaling cascades are very different from the more general mechanisms of action of conventional chemotherapy, which result in such profound toxicities in infants in particular. Appropriate choices of molecularly targeted treatments for infant leukemia in the clinic are also expected to be reliant on molecular testing for the relevant target aberrations because of the heterogeneity in MLL partner proteins and the cooperating events in disease pathogenesis within this patient subset. Furthermore, molecular characterization will be critically important to understand mechanisms of response and resistance. As well exemplified by FLT3 tyrosine kinase inhibition, (Piloto etÂ€al. 2007) the future therapeutic opportunities and challenges lie not only in identifying

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primary mutations and coexisting mutations, but also exploiting acquired resistance mechanisms as avenues to new treatment in instances of treatment failure. Moreover, it has also been suggested that the eradication of the leukemia stem cell will likely prove essential to cure MLL disease (Somervaille and Cleary 2006). Acknowledgmentâ•… The work is supported by Leukemia & Lymphoma Society SCOR 7372-07.

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Srinivasan RS, Nesbit JB, Marrero L, Erfurth F, LaRussa VF, Hemenway CS. The synthetic peptide PFWT disrupts AF4-AF9 protein complexes and induces apoptosis in t(4;11) leukemia cells. Leukemia. 2004;18:1364–1372. Erfurth F, Hemenway CS, de Erkenez AC, Domer PH. MLL fusion partners AF4 and AF9 interact at subnuclear foci. Leukemia. 2004;18:92–102. Palermo CM, Bennett CA, Winters AC, Hemenway CS. The AF4-mimetic peptide, PFWT, induces necrotic cell death in MV4-11 leukemia cells. Leuk Res. 2008;32:633–642. Bennett CA, Winters AC, Barretto NN, Hemenway CS. Molecular targeting of MLL-rearranged leukemia cell lines with the synthetic peptide PFWT synergistically enhances the cytotoxic effect of established chemotherapeutic agents. Leuk Res. 2009;33:937–947. Wang Z, Smith KS, Murphy M, Piloto O, Somervaille TC, Cleary ML. Glycogen synthase kinase 3 in MLL leukaemia maintenance and targeted therapy. Nature. 2008;455:1205–1209. Wiech H, Buchner J, Zimmermann R, Jakob U. Hsp90 chaperones protein folding inÂ€vitro. Nature. 1992;358:169–170. Stebbins CE, Russo AA, Schneider C, Rosen N, Hartl FU, Pavletich NP. Crystal structure of an Hsp90-geldanamycin complex: targeting of a protein chaperone by an antitumor agent. Cell. 1997;89:239–250. Terasawa K, Minami M, Minami Y. Constantly updated knowledge of Hsp90. J Biochem. 2005;137:443–447. Imai J, Maruya M, Yashiroda H, Yahara I, Tanaka K. The molecular chaperone Hsp90 plays a role in the assembly and maintenance of the 26S proteasome. EMBO J. 2003;22:3557–3567. Yao Q, Nishiuchi R, Li Q, Kumar AR, Hudson WA, Kersey JH. FLT3 expressing leukemias are selectively sensitive to inhibitors of the molecular chaperone heat shock protein 90 through destabilization of signal transduction-associated kinases. Clin Cancer Res. 2003;9:4483–4493. Belova L, Brickley DR, Ky B, Sharma SK, Conzen SD. Hsp90 regulates the phosphorylation and activity of serum- and glucocorticoid-regulated kinase-1. J Biol Chem. 2008;283:18821–18831. Fujita N, Sato S, Ishida A, Tsuruo T. Involvement of Hsp90 in signaling and stability of 3-phosphoinositide-dependent kinase-1. J Biol Chem. 2002;277:10346–10353. Zhang R, Luo D, Miao R, etÂ€al. Hsp90-Akt phosphorylates ASK1 and inhibits ASK1-mediated apoptosis. Oncogene. 2005;24:3954–3963. Picard D. Hsp90 invades the outside. Nat Cell Biol. 2004;6:479–480. Eustace BK, Jay DG. Extracellular roles for the molecular chaperone, hsp90. Cell Cycle. 2004;3:1098–1100. Eustace BK, Sakurai T, Stewart JK, etÂ€al. Functional proteomic screens reveal an essential extracellular role for hsp90 alpha in cancer cell invasiveness. Nat Cell Biol. 2004;6:507–514. Yocum AK, Busch CM, Felix CA, Blair IA. Proteomics-based strategy to identify biomarkers and pharmacological targets in leukemias with t(4;11) translocations. J Proteome Res. 2006;5:2743–2753. Yao Q, Weigel B, Kersey J. Synergism between etoposide and 17-AAG in leukemia cells: critical roles for Hsp90, FLT3, topoisomerase II, Chk1, and Rad51. Clin Cancer Res. 2007;13:1591–1600. Yao Q, Nishiuchi R, Kitamura T, Kersey JH. Human leukemias with mutated FLT3 kinase are synergistically sensitive to FLT3 and Hsp90 inhibitors: the key role of the STAT5 signal transduction pathway. Leukemia. 2005;19:1605–1612. Tonelli R, Sartini R, Fronza R, etÂ€ al. G1 cell-cycle arrest and apoptosis by histone deacetylase inhibition in MLL-AF9 acute myeloid leukemia cells is p21 dependent and MLL-AF9 independent. Leukemia. 2006;20:1307–1310. Niitsu N, Hayashi Y, Sugita K, Honma Y. Sensitization by 5-aza-2¢-deoxycytidine of leukaemia cells with MLL abnormalities to induction of differentiation by all-trans retinoic acid and 1alpha,25-dihydroxyvitamin D3. Br J Haematol. 2001;112:315–326. Hudes G, Carducci M, Tomczak P, etÂ€al. Temsirolimus, interferon alfa, or both for advanced renalcell carcinoma. N Engl J Med. 2007;356:2271–2281. Brown VI, Fang J, Alcorn K, etÂ€al. Rapamycin is active against B-precursor leukemia inÂ€vitro and inÂ€vivo, an effect that is modulated by IL-7-mediated signaling. Proc Natl Acad Sci U S A. 2003;100:15113–15118. Teachey DT, Sheen C, Hall J, etÂ€al. mTOR inhibitors are synergistic with methotrexate: an effective combination to treat acute lymphoblastic leukemia. Blood. 2008;112:2020–2023.

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Targeted Therapeutic Approaches for AML Robert J. Arceci and Donald Small

Introduction Acute myeloid leukemia comprises about 20% of the acute leukemias in children, but it is responsible for more than half of leukemic deaths due to leukemia. Compared to the tremendous success in the treatment of acute lymphocytic leukemia in the last three decades, resulting in more than 80% cure rate, improvements in AML therapy have been more limited with only about half of patients with AML being cured. Risk-adapted therapy has been the cornerstone of ALL therapy. One of the reasons for the success of this approach in ALL is that standard ALL induction and consolidation have been able to be intensified without causing significant morbidity and mortality. In contrast, the leukemic stem cell in most AML subtypes is inherently more drug resistant requiring significantly intensified courses of near myeloablative combinations of chemotherapeutic agents. This has resulted in a plateau in survival at approximately 50% along with significant morbidity and mortality. Current AML therapy is based on the use of multi-agent combinations of Â�noncross-resistant chemotherapeutic agents, dose intensification, risk-adapted use of allogeneic HSCT as well as aggressive, pre-emptive use of supportive care interventions. Such therapeutic approaches are also relatively nonselective and associatedÂ� with significant treatment-related toxicities. Advances in the molecular basis for AML have provided improvements in subtype classification, a better understanding of risk stratification, and the introduction of molecularly targeted therapies. This increased the understanding of the molecular basis of AML in combination with a growing list of host genetic risk factors provides for the possibility of truly individualized therapy.

R.J. Arceci (*) The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Pediatric Oncology, Johns Hopkins University, Baltimore, MD 21231, USA e-mail: [email protected] P.J. Houghton and R.J. Arceci (eds.), Molecularly Targeted Therapy for Childhood Cancer, DOI 10.1007/978-0-387-69062-9_4, © Springer Science+Business Media, LLC 2010

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R.J. Arceci and D. Small

Mechanisms of Leukemogenesis: Implications for Targeted Therapy Development The explosion of new discoveries relating to our understanding of the cellular and molecular basis of AML in adults and children has significant implications for the development of less toxic and more effective therapies but also is associated with challenges in terms of validating potential therapeutic targets both in preclinical models and in clinical trials. The genetic changes that lead to AML can be both inherited as well as somatically acquired. Many of these molecular changes have been largely derived from abnormal chromosomal rearrangements that in turn have led to the identification of gene translocations and resulting fusion products that play roles in the development of AML. The genes targeted by these abnormal chromosome changes often include transcription factors that are important in regulating myeloid differentiation as well as partner genes that may relocate the DNA binding or regulatory region of the transcription factor to altered sites within the cell or along DNA. For example, although the cytogenetic abnormalities observed in AML are quite heterogeneous, they may result in the altered targeting of the same molecular pathways. For example, all the t(8;21), t(3;21), t(16;16), t(16;21), and inv(16) chromosomal changes observed in AML alter the function of core binding factor complexes that regulate chromatin structure and function. Another example includes translocations that involve PML and RAR alpha in APL. Such genetic abnormalities result in a change in the normal function of these core binding factors leading to transcriptional repression of target genes regulating proliferation, cell survival, and differentiation. Furthermore, the convergence of these altered differentiation regulatory pathways has important implications for therapeutic targeting. For example, instead of developing therapeutic inhibitors directed toward each fusion product, it may be possible to target the critical and shared transcriptional pathways that contribute to this step in AML development (Ichikawa etÂ€al. 2006). However, preclinical murine models designed to test the contribution of important translocations in AML have determined that the presence of only the translocation fusion product is usually insufficient for leukemic transformation. This observation led to a search for cooperating genetic pathways, which, in turn, led to the recognition of the importance of activating mutations of tyrosine kinase receptors such as FLT3 and c-KIT as regulators of leukemia cell survival and proliferation. The presence of activating mutations of specific tyrosine kinase receptors has also been shown to contribute to chemotherapeutic resistance through their activation of downstream resistance pathways as well as increased proliferation and survival of leukemic blasts. Thus, the pathways affected by these mutated receptors and signal transduction molecules also represent additional therapeutic targets.

Targeted Therapeutic Approaches for AML

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Refining the Definition and Relevance of Targeted Therapy The considerable overlap in the phenotypes of AML and normal hematopoietic precursor cells has presented significant challenges in terms of distinguishing them for targeted therapies. The development of fluorescence activated cell sorting in conjunction with monoclonal antibodies directed to lineage specific markers has provided an approach to isolate primitive self-repopulating leukemic stem cells apart from normal progenitors. The AML, self-repopulating primitive cells are quite rare within the population of leukemic blasts, with estimates of their Â�frequency being in the 0.2 to 200 per 106 mononuclear cells range. These data have led to the conclusion that in many instances, the subtype of AML observed clinically depends on the types of initiating and subsequent molecular events that occur in an early stem cell population of hematopoietic precursors rather than from transformation occurring in a later stage, lineage-determined, precursor cell (Jordan 2007; Barabe etÂ€al. 2007; Hope etÂ€al. 2004). The complex hierarchy of the self-initiating AML stem cell creates important challenges for targeted therapy development. Thus, for a pathway to represent a potentially curative target and not simply a cytoreductive one, the altered pathway should have a specific, or at least a highly selective, effect on the leukemia selfinitiating stem cell. Thus, a potentially curative target should (1) be expressed in the relevant cell populationÂ�, (2) survival of the relevant cell type should be dependent on the expression of the target, and (3) modulation of the targeted pathways should result in the demise or permanent inactivity of the relevant cell type. As noted above, the relevant cell type in AML should include the self-replicating leukemic stem cell. Clinical trials to assess the relevance of agents directed toward enumerating and eradicating this Â�self-repopulating AML in order to test the clinical relevance of these concepts are needed.

Development of Targeted Therapies for AML Although many therapies are particularly effective at cytoreducing leukemic cell burden in patients with AML, very few have been selectively developed to target the self-repopulating leukemic stem cell population. In order to make this type of targeting a reality, a significant amount of investigation needs to be done into what distinguishes AML stem cells from their normal counterparts. Little information has thus far been reported on this issue, although some reports are starting to reveal potentially important differences between leukemic and the normal stem cell counterparts (Andersson etÂ€al. 2005a,b; Ross etÂ€al. 2004; Oshima etÂ€al. 2003). In addition, while most cytoreductive agents produce rapid antileukemic responses, agents targeted at the relatively rare but self-repopulating leukemic stem cell population would be expected to result in slow clinical responses. Most clinical trials do not Â�usually provide the opportunity to observe such slow responses, especially in

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Cancer Self-Renewing Stem Cell

Non-Self-Replicating Cancer Cell

Replication, Proliferation and Differentiation

Lineage Specific Antigens Drug Transporter Inhibitors Cytokine Receptors Signal Transduction Pathways Chromatin/Transcription

Replication or Dormancy Immunotherapy

Proliferation and Differentiation Microenvironment

Fig.Â€1â•… Schematic of possible cellular and molecular targets in AML (adapted from Arceci and Cripe (2002))

patients with acute leukemia. Thus, if a targeted agent is not an effective cytoreductive agent, it may still provide important antileukemic effects, particularly when used in Â�vcombination with more traditional cytoreductive chemotherapies. Quantitation of the self-repopulating AML stem cells in such combination trials may be an approach to evaluate the effect of such stem-cell directed treatments. Several pathways and targets are being currently tested in pediatric AML that are directed alone or in combination toward reversing the abnormalities in leukemia cell survival/drug resistance, proliferation, and differentiation. Promising new approaches to more selective treatments include inhibition of proliferation and survival pathways such as FLT3-ITD and c-KIT receptors and their downstream targets, such as RAS, as well as transcriptional or chromatin-based strategies and immunotargeted therapies with monoclonal antibodies and vaccines (Fig.Â€1).

Specific Examples of Targeting AML Targeting Pathways that Alter Leukemia Cell Proliferation and Survival Tyrosine Kinases Laboratory studies demonstrating the importance of cytokines (survival and differentiation factors) and their receptors along with the induced downstream Â�signaling


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pathways that direct normal hematopoiesis were fundamental in elucidating the role of these same pathways in the etiology and pathophysiology of AML. Several of these cytokine receptors, such as FLT3, c-KIT, c-FMS, PDGF-R, and VEGF-R, have been shown to be expressed at high levels or have activating mutations that lead to increased proliferative, survival, and drug resistance in AML (Kiyoi etÂ€al. 1997, 1998; Meshinchi etÂ€al. 2001, 2003a; Yamamoto etÂ€al. 2001). These changes in leukemic cell behavior have furthermore been shown in several instances to changes in clinical outcome of patients. For example, pediatric and adult patients with FLT3/ITD mutations, particularly in the group of patients with normal karyotypes, have a poor outcome. Such observations have provided the rationale to target these mutant receptors and their downstream signaling pathways. Mutations effecting c-KIT occur more frequently in the subset of AML characterized by alterations in core binding factors and have been reported to portend a poor outcome in an otherwise favorable group of patients (Schnittger etÂ€al. 2006, 2007; Shimada etÂ€al. 2006). In both the examples of FLT3/ITD and c-KIT, the mutant receptors result in distinct changes in signal transduction compared to activation of the respective wild type receptors, leading to cytokine independent survival, increased proliferation, and greater resistance to chemotherapeutic drugs (Tse etÂ€ al. 2000; Ning etÂ€ al. 2001a,b,c). Another important observation regarding FLT3/ITD is that at least some cases of AML appear to have the mutation in a leukemia repopulating subset of cells as well as their progeny, thus in part addressing the issues raised above concerning the targeting of the leukemia initiating stem cell as well as the right molecular target (Levis etÂ€al. 2005a; Pollard etÂ€ al. 2006). However, in some patients with FLT3/ITD positive AML, relapsed samples lacked the FLT3/ITD, suggesting that in these patients, FLT3/ITD is more likely to be a secondary mutational event rather than arising in a leukemia selfrepopulating cell; an alternative explanation would be that more than one leukemia initiating cell was initially present and differentially selected for during treatment (Kottaridis etÂ€al. 2002; Cloos etÂ€al. 2006). Several inhibitors of FLT3/ITD, such as CEP-701 (Cephalon) (Brown etÂ€al. 2006; Levis etÂ€al. 2002, 2004; Smith etÂ€al. 2004), MLN518 (Millenium) (DeAngelo etÂ€al. 2006) and PKC412 (Novartis) (Cools etÂ€ al. 2003; Stone etÂ€ al. 2005), have been developed and tested in preclinical models and early clinical trials. One of these inhibitors, CEP-701 (lestaurtinib), showed excellent activity in a preclinical model (Levis etÂ€al. 2002) followed by clinical trials as a single agent (Smith etÂ€al. 2004; Levis etÂ€ al. 2006). In these clinical trials, decreases in peripheral blood leukemic blasts were observed but no significant marrow responses leading to complete remissions. Such results have also been observed with other inhibitors of FLT3 (DeAngelo etÂ€al. 2006; Stone etÂ€al. 2005; Knapper etÂ€al. 2006). However, an important aspect of the single agent clinical trials with CEP-701 was that methods were developed and validated which showed that sufficiently high plasma levels of the inhibitor could be obtained to inhibit the activated, mutant receptor (Levis etÂ€al. 2006). Additional preclinical work demonstrated increased efficacy of combining CEP701 with cytosine arabinoside (Levis etÂ€al. 2004) which subsequently led to encouraging early results in a randomized phase II trial in adults with relapsed AML expressing FLT3/ITD receptors (Levis etÂ€al. 2005b). In the preliminary data reported

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from this trial, 76% (13 of 17) of patients showed plasma levels of drug that inhibited FLT3 activity by at least 85%. Further, true clinical bone marrow responses were observed primarily in the group of patients whose AML was sensitive and who had sufficient, inhibitory plasma levels of CEP-701 (Levis etÂ€al. 2005b). COG AAML06P1 in combination with chemotherapy (idarubicin and cytosine arabinoside) for pediatric patients with relapsed AML harboring FLT3/ITD mutations. In addition, the increased expression of wild type FLT3 in AML, particularly in infant leukemia, has led to a COG clinical trial combining CEP-701 with chemotherapy in this very high risk leukemia in infants (Brown etÂ€al. 2006; Armstrong etÂ€al. 2002). There are plans to test the addition of FLT3 inhibitors into the COG phase III trial for patients with newly diagnosed AML expressing the FLT3/ITD mutant receptor. The c-KIT tyrosine kinase receptor has also been associated with poor outcome in the subtype of otherwise good prognostic core binding factor AML (Schnittger etÂ€al. 2006; Shimada etÂ€al. 2006) although a recent report from COG was not able to confirm a worse prognosis associated with c-KIT mutations (Poland et al. 2009). Mutations of c-KIT involve single base pair changes leading to amino acid changes in the kinase domain and resulting in an auto-activating receptor. Less common in AML, some c-KIT mutations involve the cytoplasmic, juxtamembrane portion of the receptor; such mutations have different kinase inhibitor characteristics than the kinase domain mutations (Roberts etÂ€al. 2007). c-KIT mutations are observed in only about 3 to 5% of AML, but in 20 to 30% of AML characterized by changes in core binding factors (Schnittger etÂ€al. 2006; Shimada etÂ€al. 2006; Meshinchi etÂ€al. 2003b). Thus, kinase inhibitors with activity against such c-KIT mutations would be expected to be potentially useful in this subset of AML. Imatinib, first developed for the inhibition of the BCR-ABL fusion kinase and the treatment of CML, also has significant activity against c-KIT as well as the platelet derived growth factor receptor (PDGFR) (Heinrich etÂ€al. 2000; Fernandez etÂ€al. 2007). In vitro results have demonstrated that imatinib selectively inhibits wild type and mutant c-KIT activity as well as its activation of downstream signaling molecules, such as MAPK and AKT (Heinrich etÂ€al. 2000). An initial phase II study of imatinib in patients with refractory or recurrent AML showed disappointing results with no significant clinical responses (Kindler etÂ€al. 2004). However, a subsequent clinical trial using imatinib in 21 patients with refractory AML expressing c-KIT demonstrated 5 out of 21 patients with hematologic responses, two of which were complete hematologic remissions in patients who had relatively low blast counts in the bone marrow or in peripheral blood (Ito etÂ€al. 2005; Jentsch-Ullrich etÂ€al. 2004; Pompetti etÂ€al. 2007). Imatinib and low dose cytosine arabinoside has been tested in older adults in a phase II trial based on inÂ€vitro data that showed synergistic cytotoxicity with the combination compared to either agent used alone (Heidel etÂ€al. 2007). However, the clinical response rate was only 11% and did not appear significantly better than AraC alone based on comparison with historical controls (Heidel etÂ€al. 2007). There are several potential problems with such studies. First is that imatinib may not be a particularly potent or specific inhibitor of mutant c-KIT receptors; second, the inhibition of the c-KIT receptor


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kinase activity and/or downstream signaling pathways were not assessed in leukemic blasts from patients, thus raising the possibility that the kinase was not sufficiently inhibited. Although c-KIT remains a potentially important therapeutic target in a subset of patients with AML, more selective and potent inhibitors need to develop along with synergistic combinations conventional chemotherapy or other targeting agents. Although their name suggests the unlikely possibility that the IGF-I and VEGF receptors would play a role in AML, there are data to the contrary. IGF-IR expression has been demonstrated on AML cells although activating mutations have not been reported (Hizuka etÂ€al. 1987). In addition, IGF-I has been shown to contribute along with other cytokines to the survival and proliferation of AML cells as well as their response to AraC (Abe etÂ€al. 2006; Frostad and Bruserud 1999). In vitro studies have further shown that blockade of the IGF-IR was able to induce apoptosis in AraC resistant AML cells (Abe etÂ€al. 2006). As IGF-IR inhibitors, whether small molecules or monoclonal antibodies, are further studied in the laboratory and in clinical trials (Cosaceanu etÂ€al. 2007; Jerome etÂ€al. 2006), a potential role of these approaches alone or in combination with other targeted therapies, may prove worthwhile in AML. Several lines of evidence have established a role for VEGF and its receptor in hematopoiesis and in AML. Laboratory studies have demonstrated that a high percentage of AML cells express VEGF as well as VEGFR (Perl and Carroll 2007). Furthermore, the secretion of VEGF by AML blasts can stimulate bone marrow stromal fibroblasts to secrete hematopoietic cytokines such as G-CSF and GM-CSF, which, in turn, may contribute to further myeloid progenitor expansion (Aguayo 2004; Aguayo etÂ€al. 1999, 2003; Hussong etÂ€al. 2000; Kessler etÂ€al. 2007; Schuch etÂ€ al. 2002). In addition, increased bone marrow angiogenesis, observed in both MDS and AML, may be in part a result of VEGF secretion. Thus, VEGF may play both an autocrine and paracrine role in MDS and AML. Leukemic cell proliferation, survival, and resistance to chemotherapy have also been directly linked to signaling through VEGF-C and its activation of VEGFR-3 (Dias etÂ€ al. 2002; Liersch etÂ€ al. 2007). Inhibitory monoclonal antibodies that bind and inactivate VEGF (e.g., Avastin or Bevacizumab) (Karp etÂ€al. 2004), or small molecule inhibitors of VEGFR (e.g., SU5416 or AZD2171) (Fiedler etÂ€al. 2003; Maris etÂ€al. 2008), have been developed for therapeutic anti-angiogenic treatment approaches. Karp etÂ€al. (2004) tested Bevacizumab post chemotherapy treatment in adults with AML and showed clearance of blasts and decreased bone marrow microvasculature. Bevacizumab is also being tested in adults with newly diagnosed AML in combination with idarubicin and AraC. Initial clinical studies with SU5416 reported a complete remission in a patient with AML, although subsequent studies have not shown significant response rates to this agent when used alone (Perl and Carroll 2007; Fiedler etÂ€al. 2003). It is possible that the selective preference of SU5416 may be too narrow or mechanisms of resistance are activated to prevent significant leukemic cell killing. Current attempts to combine such agents with chemotherapy or to use tyrosine kinase inhibitors with a broader spectrum of targets are being developed (Perl and Carroll 2007).

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The above examples demonstrate several key principles for targeted therapy in AML using TK inhibitors. First, unlike in CML, such approaches using single agents are unlikely to be highly effective in AML. However, these TK inhibitors appear to share the characteristic of sensitizing AML cells to conventional chemotherapeutic drugs, without necessarily resulting in significant overlapping toxicities. Nevertheless, because some of the activated TKs signal through common, downstream pathways, investigators are attempting to determine whether inhibition of these shared, convergence points can be therapeutically exploited (Fig.Â€2). However, similar to imatinib, resistance to TK inhibitors has been demonstrated, although the pathways leading to resistance may be different (Piloto etÂ€al. 2007).

Inhibition of Signaling Pathways Downstream of TK Receptors Not long after its initial identification as an important dominant oncogene in malignant transformation, RAS mutations were documented in about 25% of AML/MDS samples (Farr etÂ€al. 1991). Further, in the absence of RAS mutations, RAS activation through its conversion from GDP to GTP and farnesylated tethering to the inner cell membrane is known to play a critical role in the development of AML as well as the myeloproliferative syndrome, juvenile myelomonocytic leukemia (JMML) (Loh etÂ€al. 2004). While specific inhibitors of GTP activation of RAS have not yet been successfully reported, farnesyl transferase inhibitors (FTI) have been tested in patients with AML (Karp 2001, 2005; Emanuel etÂ€al. 2000; Gotlib 2005). Phase I studies in adults with AML reported significant clinical responses that did not appear to be dose dependent (Karp 2001). A subsequent multi-institutional phase II study reported a 5% complete remission rate using tipifarnib as a single agent in adult patients with relapsed and/or refractory disease (Harousseau etÂ€ al. 2007). Tipifarnib as a single agent in patients aged 65 or older with high risk AML showed a response rate of 10 to 20% (Lancet etÂ€al. 2007). Based on such results, a randomized phase III trial comparing tipifarnib to supportive care in patients aged 70 years or more with AML is being conducted in Europe. A pediatric randomized trial of tipifarnib versus no drug in the postallogeneic HSCT setting by the COG in patients with relapsed AML. While tipifarnib has shown a low, but real level of activity in AML as a single agent, future use and testing will likely be in combination with other targeted agents and/or conventional chemotherapy. Of note, however, is that the mechanism of action of such FTI in AML and whether they are mediating their effect through the RAS pathway remain unclear. Downstream of RAS are the RAF, MEK, ERK, and STAT pathways, all of which are being considered as potential therapeutic targets in AML (Fig.Â€2). For example, small molecule inhibitors of the MAPK pathway, such as PD98059 and PD184352, show significant and selective induction of cell cycle arrest and apoptosis in AML cells (Milella etÂ€al. 2001, 2002, 2003). MAPK inhibition can also result in increased sensitization to chemotherapy of AML cells, particularly from AML expressing the

Fig.Â€2â•… Schematic of FLT3 signaling pathways as an example potential downstream molecular targets as a result of activating mutations of tyrosine kinases. From Doepfner etÂ€al. (2007)

Targeted Therapeutic Approaches for AML 67

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stem cell marker, CD34 (James etÂ€ al. 2003). RAF-1 kinase inhibitors, such as sorafenib, are also being clinically developed. Of note, sorafenib also has significant activity against c-KIT (Tong etÂ€al. 2006). The mTOR (mammalian Target Of Rapamycin) serine/threonine kinase plays a primary role as a regulator of cell cycle progression, proliferation, apoptosis, and angiogenesis in both normal and neoplastic cells. mTOR functions as a type of Â�utility player of signal transduction and in that it is a central regulator of a variety of signaling pathways originating from TK receptor activation, including FLT3, c-KIT, PDGFR, c-FMS, and VEGF, as well as through RAS, various phosphatases, such as PTEN and SHP-1/2 (Weisberg etÂ€al. 2008). Coactivation of the PI3K/AKT pathway is also usually observed with mTOR activation (Tamburini etÂ€al. 2008; Xu etÂ€al. 2003, 2005). In each of these examples, a role of mTOR has been demonstrated in the proliferation, survival, and drug resistant phenotype of approximately 70% of AML cases examined (Xu etÂ€al. 2003; Recher etÂ€al. 2005; Min etÂ€al. 2003). Several highly selective inhibitors of mTOR, including rapamycin, CCI-779, RAD001, AP23573, and MK-8669 have been developed. A clinical trial in adults with refractory/relapsed or secondary AML reported that four of nine patients had partial responses to rapamycin when used as a single agent (Recher etÂ€al. 2005). A phase I/II study using RAD001 (everolimus) as a single agent in adults with refractory and/or relapsed hematologic malignancies showed that a 10Â€mg daily dose was well tolerated and two of five patients with MDS had a major and minor platelet responses. No patients with AML were included on this trial (Yee etÂ€ al. 2006). Because of its central role and activation in a majority of patients with AML, mTOR inhibition as a therapeutic approach, is being planned for clinical trial testing in pediatric patients with relapsed/ refractory AML. Combining mTOR inhibitors with conventional chemotherapy, particularly ARAC containing regimens, or with other signal transduction inhibitors will likely be the best approach for definitive clinical testing.

Drug Resistance Mechanisms and Leukemic Cell Survival As discussed above, in addition to conveying a proliferative advantage, mutant or highly expressed tyrosine kinase receptors and their downstream signaling pathways can also lead to increased drug resistance on the part of AML blasts. Other molecular pathways may also lead to deÂ€novo or acquired drug resistance as well. The identification of the drug efflux transporter P-glycoprotein (Pgp 170 or MDR1 P-glycoprotein) as an important mechanism of multidrug resistance in cancer cells provided a potential therapeutic target. To this end, a variety of competitive inhibitors, including cyclosporine A and its nonimmunosuppressive D isomer, PSC833, as well as a number of other small molecules, were developed to block the drug efflux capacity of this ATP-dependent transporter. Inhibition of the MDR1 transporter results in decreased efflux of multiple chemotherapeutic, natural product drugs, such as anthracyclines and vinca alkaloids, leading to their increased intracellular concentration and subsequent cell death. The observations that many tumor types that are resistant to


69

such chemotherapeutic agents also express high levels of the MDR1 P-glycoprotein and could be resensitized to chemotherapy in the presence of an MDR1 inhibitor provided the experimental foundation to clinically test this approach. Of note, the expression of MDR1 P-glycoprotein was found to be high in adult AML and, furthermore, showed even higher expression at the time of relapse (Baer etÂ€al. 2002; Leith etÂ€al. 1995, 1999; Willman 1996). Such high levels of MDR1 P-glycoprotein were correlated with poor outcome (Willman 1997). Expression in childhood AML was reported to be considerably lower (Sievers etÂ€al. 1995). After the completion of phase I/II trials with MDR1 P-glycoprotein inhibitors, it became clear that significant dose adjustments of the chemotherapeutic drugs needed to be made to correct for decreased drug elimination secondary to inhibition of cellular efflux in normal cells of the liver, kidney, and intestine (List etÂ€al. 1993; Dahl etÂ€ al. 2000). Most randomized phase III trials in adults with AML tested the impact on outcome of inclusion of an MDR1 inhibitor, e.g., PSC833, in combination with chemotherapy, did not show an improvement compared to chemotherapy along (Greenberg etÂ€al. 2004). One exception was a randomized comparison of cyclosporine A and no cyclosporine in combination with daunorubicin and ARAC during consolidation for adults with poor-risk AML (List etÂ€al. 2001). Although the rate of complete remission was not different in the two groups of patients (39 vs. 33%, pâ•›=â•›0.14), relapse free survival and overall survival were significantly improved in the group receiving cyclosporine (RFS 34 vs. 9% at 2 years, pâ•›=â•›0.031 and OS 22 vs. 12%, pâ•›=â•›0.46, respectively). Currently, the Eastern Cooperative Oncology group is testing the nonpharmacologically active inhibitor, zosuquidar and the Southwest Oncology Group is testing cyclosporine A in randomized, phase III trials. Phase I and II studies in pediatric patients with relapsed/refractory AML established the dose of cyclosporine A and chemotherapy when used in combination (Dahl etÂ€al. 2000). Based on these results, the POG 9421 study randomized patients to receive in consolidation cyclosporine or no cyclosporine in combination with mitoxantrone and ARAC during the consolidation phase of treatment (Becton etÂ€al. 2006). In addition, this study randomized high dose to standard dose ARAC during induction therapy. The results showed that the best outcome was associated receiving high dose ARAC during induction and cyclosporine plus mitoxantrone and ARAC during consolidation. However, no statistically significant differences in outcome were observed for either the high dose ARAC or the addition of cyclosporine when considered as independent variables (Becton etÂ€al. 2006). The lack of a definitive benefit for MDR1 P-glycoprotein inhibition may be related to the emergence of other mechanisms of resistance in the presence of MDR1 inhibition, including the possible role of one or more of the dozens of other transport pumps expressed in cells, increased expression of the MDR1 P-glycoprotein as well as ineffective blockade and increased toxicity. While further MDR transporter inhibition trials in pediatrics are unlikely in the near future, one exception might be in conjunction with anti-CD33-calicheamicin immunotargeted therapy because a known mechanism of resistance to calicheamicin is MDR1 P-glycoprotein efflux (Arceci etÂ€al. 2005; Linenberger etÂ€al. 2001).

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A second pathway to chemotherapy-induced resistance is the upregulation of bcl-2. Extensive work on the role of bcl-2 in drug resistance has established an important role in AML. The ability of inhibitors of bcl-2 to sensitize AML cells to chemotherapeutic agents has led to the development of clinical trials using bcl-2 inhibitors (e.g., antisense oligonucleotide oblimersen or Genasense) (Marcucci etÂ€ al. 2003, 2005). While this approach is being tested in adults with AML, no immediate clinical trials are being planned in pediatric AML (Moore etÂ€al. 2006). An exciting new approach of therapeutically targeting protein degradation mechanisms in cancer cells as a means to sensitize them to chemotherapy has arisen out of experimental studies demonstrating the extreme dependency of tumor cells on such pathways (Zavrski etÂ€al. 2007). In part because of the increased metabolic rate, the increased concentration of reactive oxygen species and the expression of multiple, mutated or aberrant proteins, malignant cells heavily depend on proteasome degradation. By blocking this system, proteasome inhibitors have been shown to increase the sensitivity of tumor cells to chemotherapeutic agents as well as induce apoptosis. Thus, this “proteasome addiction” of tumor cells, similar to their “kinase addiction” may make them more susceptible to inhibition of these pathways than normal cells. The observations that AML stem cells express high levels of the transcription and survival promoting factor, NF-KB, along with the expression of the NF-KB inhibitor, IKB, provides a potentially important link between survival factors and proteasome inhibition (Guzman etÂ€al. 2001, 2002, 2007). For example, the use of the proteasome inhibitor, bortezomib (PS-341 or Velcade), has been shown to decrease the degradation of IKB and thereby reducing the amount of translocation of NF-KB to the nucleus, resulting in increased apoptosis of AML self-repopulating or stem cells (Guzman etÂ€al. 2001, 2002). Further, the apoptotic effects of an anthracycline, for example, idarubicin, were greatly enhanced in AML stem cells in the presence of proteasome inhibition (Guzman etÂ€al. 2001, 2002). The induction of the proapoptotic factors, BIM and Bax, have also been observed to be increased in AML cells exposed to idarubicin and proteasome inhibition (Pigneux etÂ€al. 2007). Based on such data, the COG is planning to conduct a clinical trial with bortezemib in combination with chemotherapy in children with relapsed and/or refractory AML. This type of approach should help resolve the issue of the ability to selectively target AML stem cells in a clinical setting.

Epigenetic and Chromatin Remodeling-Directed Targets The modifications of DNA that help regulate its expression, whether cytosine methylation, histone type and/or modification or changes in other DNA-associated proteins, are considered epigenetic events (Holliday 2006). Epigenetic modifications of the genome have been causally linked to altered gene expression, often involving gene silencing of tumor suppressor genes and the development of various types of cancer. Several of the chromosomal translocations that characterize


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various subtypes of AML are also known to generate fusion proteins of gene products that profoundly influence DNA and/or chromatin structure and function. For instance, several of these fusion proteins, including the t(15;17)/PML/RAR alpha in APL, those involving core binding factors such as AML1/ETO resulting from t(8;21), or those involving the MLL gene product, have been demonstrated to recruit repression-associated chromatin remodeling protein complexes to specific genomic regions (Wood etÂ€al. 2005; Nie etÂ€al. 2003). In a significant percentage of AML, silencing of expression coincides with promoter hypermethylation of the tumor suppressor gene, p15INK4b or the mismatch repair gene, hMLH1, is observed (Preisler etÂ€al. 2001; Herman etÂ€al. 1997; Seedhouse etÂ€al. 2003). Other chromatin marks of the repression of gene expression are decreased histone H3 acetylation as well as trimethylation of lysine 27 of histone H3 (Bernstein etÂ€al. 2007; Ting etÂ€al. 2006). Rearrangements of the MLL gene along with its fusion to other partner genes results in both activation and repression of a subset of genes involved in AML, particularly the upregulation of HOX gene expression (Nie etÂ€ al. 2003; So etÂ€ al. 2003, 2004). Although the epigenetic changes that characterize different subtypes of AML are complex, many of them may converge on several critical changes, similar to the convergence of some downstream signaling pathways following activation of different TK receptors. Thus, strategies to reprogram the aberrant epigenetic changes observed in cancer and, in particular, MDS and AML are being pursued. Small molecule inhibitors or modulators of both DNA methylation and histone deacetylation have been developed and are being tested alone and in combination with the intent of inducing the reactivation of expression of genes which may promote leukemia cell maturation, senescence, or apoptosis (Ghoshal and Bai 2007; Santini etÂ€al. 2007). Studies on MDS in adults led to the approval by the US Food and Drug Administration for the DNA methylation inhibitor 5¢-azacytidine (Garcia-Manero etÂ€ al. 2007; Gattermann etÂ€ al. 2007; Plimack etÂ€ al. 2007; Kaminskas etÂ€ al. 2005; Silverman 2001). However, inÂ€ vitro and subsequently inÂ€vivo data have demonstrated synergy in terms of the induction of gene expression with the use of combinations of inhibitors of DNA methylation and HDACs (Griffiths and Gore 2008; Qin etÂ€al. 2007). Clinical trials testing the DNA methyltransferase inhibitors, 5¢-azacytidine or the deoxy-form, decitabine, alone and in combination with HDAC inhibitors, such as suberoylanilide hydroxamic acid (SAHA, Vorinostat or Zolinza), the benzamide derivative, MS275, depsipeptide, and valproic acid, are being intensely pursued in adults with MDS and high risk AML (Griffiths and Gore 2008; Gore etÂ€al. 2006). Some of the results from these trials have demonstrated clinical responses that are comparable to conventional chemotherapy in older adults with high risk MDS/AML (Garcia-Manero etÂ€ al. 2006; Issa etÂ€ al. 2004; Soriano etÂ€ al. 2007; Kantarjian etÂ€ al. 2007). Trials using DNA methyltransferase and HDAC inhibitors alone and in combination are either ongoing or being planned in pediatric patients. Several challenges exist for this type of therapeutic targeting. The first is how specific or global the effects of the inhibitors should be in terms of effecting epigenetic change. An unwanted result would be if DNA demethylation, for example,

72


induced genes that would prevent apoptosis or induce drug resistance; there is some precedent for this occurring (Li etÂ€al. 1999). Another challenge is how best to use epigenetically directed therapies in combination with each other, with other targeting agents or with conventional chemotherapy. For example, DNA replication is required for the effects of DNA methyltransferase inhibitors to be most effective. HDAC inhibitors have been known to cause cell cycle arrest. Thus, the sequencing of agents with these characteristics will be a significant issue to solve. A third challenge is to determine how such epigenetic therapies will impact the AML stem cell population. Increasing evidence suggests that the cancer stem cells share many epigenetic marks with embryonic stem cells. How such complex genomic signatures can be therapeutically manipulated remains an immense challenge.

Targeted Immunotherapy and Immunostimulatory Therapy Immense potential of the discovery of how to generate antigen specific, monoclonal antibodies from B cell/myeloma hybridomas by Kohler and Milstein has only started to be realized in the last decade. An outcome of such work in target AML has been thus far most using monoclonal antibodies directed against the differentiation antigen, CD33, a sialic acid-dependent cell adhesion molecule that is differentially expressed during myeloid differentiation. Gemtuzumab ozogamicin (GO; Mylotarg™) is a humanized, IgG4 subtype, monoclonal antibody conjugated to calicheamicin and directed against CD33; GO has demonstrated significant activity in AML and was the first toxin/monoclonal antibody fusion approved by the US FDA (Larson etÂ€al. 2005). Initial clinical trials in adults and children with relapsed or primary refractory disease AML showed response rates of approximately 30 to 35% when GO is used as a single agent (Arceci etÂ€ al. 2005; Larson etÂ€ al. 2005). Of particular interest, the response in children with primary refractory disease was the same as that for patients with relapsed disease who had achieved a prior remission, suggesting that some conventional resistance mechanisms can be circumvented with this agent (Arceci etÂ€al. 2005). The maximal tolerated dose (MTD) for children was determined to be 6Â€mg/m2 on the pediatric phase I study. The main toxicity was myelosuppression. However, there was a 24% (7 of 29 patients) overall incidence of veno-occlusive disease (VOD) with 6 of 13 (40%) developing VOD during a subsequent HSCT. VOD was most frequently observed in patients undergoing allogeneic SCT in less than 3.5 months from receiving GO. A COG phase I trial using GO in combination with either mitoxantrone plus high dose ARAC (regimen A) or with Capizzi II chemotherapy (regimen B) showed a maximum tolerated dose of GO to be 3 and 2Â€ mg/m2, respectively (Aplenc etÂ€ al. 2008). Significant toxicities included those typically observed in patients with AML. DLTs for the Capizzi II included grade III transaminitis, pancreatitis and hyperbilirubinemia requiring a dose de-escalation to 2Â€mg/m2. The starting dose of 3Â€mg/m2 of GO in combination with mitoxantrone plus high dose ARAC was well tolerated. Responses included 52 and 40% CR


73

or CRp for regimens A and B, respectively (Aplenc etÂ€ al. 2008). The MRC has performed a randomized trial of GO plus induction chemotherapy with ARAC, daunomycin, and etoposide (ADE) as well as with the second intensification course of mitoxantrone plus ARAC (Kell etÂ€al. 2003). Results from this trial have been reported in abstract form and an advantage of EFS and OS for good risk patients who received GO (Burnett etÂ€al. 2006). The current COG trial is test in a randomized fashion whether the addition of GO to induction and intensification courses of treatment results in an improved outcome for pediatric patients with newly diagnosed AML. In addition to the potential role of GO in improving the outcome of patients with AML, several additional questions remain. The question of why many patients are resistant to GO therapy has been partially addressed by the findings that calicheamicin is a substrate for the MDR1 P-glycoprotein drug transporter (Arceci etÂ€al. 2005; Linenberger etÂ€al. 2001). Thus, AML cells that express high levels of this transporter are more likely to be resistant to GO. These observations have led to the consideration of a clinical trial in pediatric patients with relapsed or refractory AML that combines GO with an inhibitor of the MDR1 transporter. A second question is whether GO will augment killing of AML repopulating stem cells. There is now definitive evidence of expression of CD33 on the surface of some AML stem cells, although the expression does not appear to be uniform across all AML (Pollard etÂ€al. 2006; Taussig etÂ€al. 2005). Phase III randomized trials will help answer the question of the utility of adding GO to conventional chemotherapy, but trials developed to look more specifically at the effects of such combination on AML stem cells should also be performed. The potential for targeting AML with monoclonal antibodies remains an important avenue for future research. Defining antigens that are more selectively or specifically expressed on AML stem cells remains an important challenge. CLL-1 has been reported to be such a specific AML stem cell antigen although its utility as a therapeutic target will need to be definitively tested in clinical trials (van Rhenen etÂ€al. 2007). The hope that the inherent ability of the immune system to recognized foreign antigens and cells could be directed toward antitumor therapy has been a challenge for many decades. Several preclinical models for vaccine-mediated treatment of AML have shown excellent antileukemic effects, including cures (Arceci 1998; DunussiJoannopoulos etÂ€al. 1996, 1997, 1998). Most of these preclinical models have involved upon the transduction of costimulatory receptors into AML cells or the use of cytokines to induce immune costimulation (Cheuk and Guinn 2008; Chan etÂ€al. 2005). Clinical trials in adults with AML have involved similar approaches but remain in very early stages of development (Houtenbos etÂ€al. 2006). Another approach has been to expose patients with potentially immunogenic antigens that are preferentially expressed on AML blasts (Greiner etÂ€ al. 2004, 2005). For example, WT1 peptides are being tested in an attempt to augment anti-AML responses (Greiner etÂ€al. 2005, 2006; Gaiger etÂ€al. 2000). Attempts to stimulate anti-AML immune responses in pediatric patients have been tested in the randomized CCG-2961 trial, which used a limited course of IL-2 in the postremission period (Lange etÂ€al. 2008). No significant difference in out-

74


come was observed between the patients who received IL-2 and those who did not (Lange etÂ€al. 2008). This result could certainly have been due to the relatively short course or dosing of IL-2. Based in part on the empiric evidence that significant graft versus host disease is correlated with a lower relapse rate for patients with AML undergoing allogeneic HSCT, the use of donor lymphocytes and attempts to modulate graft versus tumor responses with cytokines and or early withdrawal of immunosuppressive drugs have been tested but with limited success. Another possibility, supported by preclinical models, is that this approach simply may not be sufficiently robust to elicit significant antitumor responses. Attempts to augment anti-AML immune responses in the allogeneic HSCT setting are also being tested in a COG trial attempting to exploit HLA mismatching and blockade of natural killer cell inhibition.

Future Challenges A major hope for improving the outcome of patients with AML is that more molecularly targeted therapies that will increase antileukemic activity without adding significant toxicity will be developed. Some of these agents, such as antibody-directed therapies and tyrosine kinase inhibitors, are currently in phase II and III clinical trials. The results of such trials should determine whether these approaches will be integrated into future regimens, establishing a new standard backbone for AML therapy. Although the thought of eliminating the need to use currently available cytotoxic chemotherapeutic agents with their short- and long-term associated adverse sequelae may seem improbable at this time, this should be our goal. Subtypes of AML with distinct genomic, gene expression or proteomic signatures or epigenetic patterns as well as with mutations of genes encoding critical molecules affecting signaling pathways should be prime targets for this type of therapy. In addition, essential to future strategies will be the important consideration of targeting leukemic self-repopulating progenitors in clinical trials. There is also a significant need for more predictive inÂ€vitro and animal models for the many subtypes of AML. Such truly predictive inÂ€ vitro or inÂ€ vivo models would help greatly in the prioritization of new agents. However, at the current time and in the conceivable future, the definitive proof that a drug will improve outcomes in patients is to demonstrate this in the context of a well-designed and statistically powered clinical trial. The increasing emphasis on subgroup analysis, prognostic factor stratification and patient characteristics should lead to more individualized therapeutic approaches. The historical approach of using one or two hammers to hit a heterogeneous group of nails will be challenged by the biologically based delineation of increasingly smaller subgroups of patients for testing targeted therapies. Thus, the accrual to phase III trials will also continue to be a challenge with diminishing numbers of patients characterized by specific subgroups. The use of smaller, albeit less statistically robust, trials, such as randomized phase II or trials comparing an experimental treatment with a comparable historical


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control group, may provide approaches to allow for both the testing of novel agents and decision-making concerning how to incorporate them into future regimens. We should also not forget that about 85% of children with cancer reside in Â�developing countries. And although significant obstacles may need to be overcome, it is becoming increasingly clear that approaches for effectively performing international trials need to be established. In addition, the predicted reduction in toxicity to be realized with molecularly targeted therapies is likely to be of significant benefit to patients in developing parts of the world where the complications associated with intensive chemotherapy regimens are often difficult to manage.

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Acute Promyelocytic Leukaemia Andrea Biondi, Anna Maria Testi, and Brenda E.S. Gibson

Introduction Since the time of its first description (Degos 2003), Acute Promyelocytic Leukaemia (APL) has drawn the attention of clinicians and scientists for its clinical and biological features (Degos 2003). APL is notable for distinctive clinical and biological characteristics, including its particular morphological features, the presence of potentially devastating hemorrhagic syndrome that is related to disseminated intravascular coagulopathy and abnormal fibrinolysis, and finally, the sensitivity to anthracyclinecontaining chemotherapy (Bernard etÂ€ al. 1973). The availability of differentation therapy with All-Trans Retinoic Acid (ATRA), and more recently, the discovery of the beneficial effect of arsenic trioxide (ATO), together with the molecular characterization of the t(15;17) specific translocation (Grignani etÂ€al. 1994) have produced a remarkable improvement in patient outcome in the last decade. Most patients with APL are now cured (Sanz 2006). APL is a paradigm of a malignant disease that can be treated by cell modulation, using agents that act specifically on oncogenic, molecular events. This chapter reviews its impact in children APL.

Demographic Features APL represents approximately 4 to 8% of paediatric AML (Kaspers and Creutzig 2005). The median age at presentation is probably similar to that of other AML subtypes (7 to 9Â€years), but APL has rarely been reported in the first year of life. A remarkable epidemiologic feature of APL is its high incidence in certain ethnic groups (Ribeiro and Rego 2006). Douer described the increased incidence of APL

A. Biondiâ•›(*) Centro M. Tettamanti, Clinica Pediatrica Università Milano-Bicocca, Ospedale San Gerardo,Via Pergolesi, 33, 20052 Monza, Italy e-mail: [email protected]


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among patients of Latin American descent (Douer 2003). Several series from hospitalbased registries in Italy (Biondi etÂ€al. 1994), Spain (Tomas and Fernandez-Ranada 1996), Mexico (Ruiz-Arguelles 1997), Perù (Otero etÂ€ al. 1996), and Nicaragua (Malta Corea etÂ€al. 1993) have noted a higher-than expected frequency of APL. It is still unknown whether such differences may suggest a genetic predisposition to APL and/or exposure to distinct environmental factor(s).

Pathogenesis of APL Acute promyelocytic leukaemia (APL) is characterized by rearrangements of the retinoic acid receptor-a(RARA) gene on chromosome 17q21 reviewed by Melnick and Licht (1999). To date, six different partner genes have been identified, with the vast majority of cases having the majority of the RAR gene fused to the PML gene on chromosome 15q21 reviewed by Grignani etÂ€al. (1994). Determination of the underlying molecular lesion is critical for appropriate management of APL, with the presence of an underlying PML-RARA fusion gene predicting a favorable response to molecularly targeted therapies in the form of ATRA and ATO. Two rare subtypes of retinoid-sensitive APL have been reported in the paediatric population, that is, the NPM1-RARA and NuMA-RARA fusions resulting from t(5;17)(q34-35;q21) and t(11;17)(q13;q21), respectively (Redner etÂ€al. 1996; Wells etÂ€al. 1997; Grimwade etÂ€al. 2000). The response of these rare subtypes of APL to ATO has not been established in patients. PLZF-RARA fusion, associated with t(11;17)(q23;q21) has only been reported in adults with the disease but is clinically important since it is resistant to ATRA and ATO reviewed by Mistry etÂ€al. (2003). The STAT5b-RARA fusion resulting from an interstitial deletion on chromosome 17 is also considered to be retinoid-resistant (Arnould etÂ€al. 1999). Fusion of the PRKAR1A to RARA was described as a variant APL. (Catalano etÂ€al. 2007). The presence of the PML-RARA fusion gene is responsible for the peculiar response to ATRA and ATO. RARA (there are three homologous RAR proteins, called a, b and g) is a ligand-dependent transcriptional activator that binds through its zinc (Zn) finger domain, as heterodimer with members of the RXR family of nuclear receptors (Grignani etÂ€al. 1994), to specific DNA sequences (called retinoic acid responsive elements, RARE) found in the promoters of retinoic acid-responsive genes (Umesono etÂ€al. 1991). This process is accomplished in concert with a coactivator complex that includes proteins such as p300 and pCAF. As shown schematically in Fig.1, in the absence of retinoic acid, a co-repressor complex is recruited comprising either Nuclear Receptor Co-Repressor (N-CoR) or Silencing Mediator of Retinoid and Thyroid Receptors (SMRT), which binds to another protein called Sin3 and then to HDAC1 (Umesono etÂ€ al. 1991; Alland etÂ€ al. 1997; Laherty etÂ€al. 1997; Heinzel etÂ€al. 1997; Redner etÂ€al. 1999). HDAC1 is a histone deacetylase protein, which epigenetically alters histones to keep DNA in an untranscribable form. The expression of retinoic-acid responsive genes is essential for

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Differentiation RAR (wild-type) PML-RARa

Cofactors

in3

N-CoR

mS

RAR

N-CoR

3

in mS

HDAC

HDAC

RAR

PML

Myelocyte

All-transretinoic acid RAR

X Transcription arrest

APL blast cell

PML

PML (dispersed)

Metamyelocyte PML-RARα degradation

PML in PODs

Granulocyte

Fig.Â€1â•… A schematic representation of the effects of PML-RAR and ATRA in the blast cells of acute promyelocytic leukaemia. DNA-bound PML-RARA interacts with N-CoR (or SMRT) and recruits the m-Sin3-HD complex, decreasing hystone acetylation and producing repressive chromatin organisation and transcriptional repression. Retinoic acid interaction with PML/RAR leads to rapid degradation of this fusion protein and assembly of the wild-type PML protein into normal nuclear structures called PODs (PML oncogenic domains). Moreover, it induces dissociation of the N-CoR-mSin3-HD complex, recruitment of coactivators with acetyltransferase activity, increased levels of hystone acetylation, chromatin remodelling, and transcriptional activation

normal myeloid development (Tsai and Collins 1993). In APL, the presence of the PML-RARA fusion gene generates a chimeric protein that is capable of DNA binding, but it recruits the N- CoR/Sin3/HDAC1 co-repressor complex and prevents transcription of RAR target genes (Collins 1998) and myeloid cell differentiation (Tsai and Collins 1993) under normal conditions. Additionally, the PML-RARA forms a heterodimer with the wild type PML protein and disrupts the PML nuclear body/PML oncogenic domain (Koken etÂ€al. 1994). ATRA functions by binding to the RAR and causing degradation of the PML-RAR protein through both the ubiquitin-proteosome and caspase systems, thus allowing for the terminal differentiation of leukemic promyelocytes (Nervi etÂ€ al. 1998; Zhu etÂ€ al. 1999; Liu etÂ€ al. 2000). This binding also results in N-CoR being dissociated from the PML-RAR fusion protein, and subsequently, the nuclear co-activator complex is recruited to reverse histone deacetylase-mediated repression Fig.Â€1. Finally, PML nuclear bodies are restored to their normal structure.

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Diagnosis of APL Compared with diagnosis of other AML subtypes, the identification of APL conveys unique therapeutic and prognostic implications. In fact, (1) this leukaemia is a medical emergency, and up to 10% of early hemorrhagic deaths are currently recorded even in patients receiving modern state-of-the-art treatments; (2) the optimised front-line approach (ATRA plus chemotherapy) is different from that used in other AMLs and is effective also in controlling the life-threatening coagulopathy. Figureâ•›2 summarises the different tools available for the proper diagnosis of APL. According to FAB classification, the term M3-AML was assigned to hypergranular promyelocytic leukaemia characterised by blast cells with heavy azurophilic granules, bundles of Auer rods, and a reniform or bilobed nucleus (Fig.Â€2 panel A). Although the vast majority of M3 cases fit the description of hypergranular or classical M3, a cytological hypogranular or microgranular variant form, M3v, has been identified (Fig.Â€2 panel B). It is commonly associated with hyperleukocytosis, accounts for 15 to 20% of APL cases, and shares the same t(15;17). Albeit less frequently observed, M3v occurs even in paediatric patients (Rovelli etÂ€al. 1992). As compared with control cases with the classical t(15;17) translocation, in the cases with PLZF/RARA rearrangements, the majority of blasts had (1) a regular nucleus, and, (2) an abundant cytoplasm with either coarse granules or, less frequently, with fine or no granules and Chediak-like granules are rarely detectable in such cases Sainty etÂ€al. (2000). The presence of a regular nuclear outline is a key feature of t(11;17)-associated APL. The few cases so far described of APL expressing NPM/RARA (Redner etÂ€al. 1996) could be classified as M3 and presented with hyperleukocytosis the remaining case had an M3v morphology. As for morphology and molecular genetics, the immunophenotype of acute promyelocytic leukaemia is also very distinctive. Both in adults and children, APL blasts show a typical surface marker expression characterised by positivity for CD33, CD13, CD9, absence of HLA-DR and rare expression of CD10, CD7, and CD11b (Guglielmi etÂ€al. 1998). The t(15;17) is the diagnostic hallmark of APL and initially had been considered to be present in all patients with this condition reviewed by Grimwade etÂ€al. (2000). Conventional cytogenetics or Fluorescence in situ hybridization (FISH) enable the identification of both derivatives chromosome 15q and 17q (Fig.Â€ 2, panel C). In addition to the classic form of the t(15;17) translocation, the existence of cryptyic translocations or microinsertion have been reported (Grimwade etÂ€al. 2000). The study of the PML distribution pattern in leukemic cells provides a rapid, specific, low-cost, and relatively simple diagnostic approach (Dyck etÂ€ al. 1995; Falini etÂ€al. 1997). Different from the wild-type (speckled) staining, which corresponds to the localization of PML into 5 to 20 discrete nuclear particles (so-called “nuclear bodies”), APL cells show a characteristic and easily distinguishable nuclear PML positivity known as “microspeckled,” resulting from the disruption of the nuclear bodies and redistribution of the protein into greater than 50 small

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Fig.Â€2â•… Diagnosis of acute promyelocytic leukaemia. Representative morphology of M3-hypergranular (Panel a) and M3v (Panel b). Bone marrow smears were stained with May-Grumwald-Giemsa. ×1,000-fold magnification. Panel c: FISH analysis of an APL case. The result shown has been obtained by using the Vysis probe set, which is designed to detect only the PML-RARA fusion gene. It comprises a mixture of directly labelled probes: a PML probe, which begins in intron 7 and extends toward the centromere for 180Â€kb, and a RARA probe, which begins in intron 4 and extends toward the telomere for 400Â€kb, Panel d: Immunolabeling APL blast with thePG-M3MoAb shows a microgranular distribution of the PML/RARa protein within the nucleus. Panel e: Results of the RT-PCR analysis of the PML-RARA fusion gene. Ethidium bromide-stained agarose gel showing bcr1+ and bcr3+ nested PCR products according to the procedures in van Dongen et al. (1999). Pt.pos: positive samples, Pt:neg: negative samples; Ctrl neg: negative controls, MK: markers

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granules/per cell. Either immunocytochemistry or immunofluorescence have been successfully used as detection systems. The use of Reverse-Transcription PCR (RT-PCR) for the detection of the PMLRARA and RARA/PML fusion genes is the only technique that defines the PML breakpoint type and that allows the definition of a correct strategy for subsequent MRD monitoring. The advantage of routinely using this assay at diagnosis to better address treatment (Miller etÂ€ al. 1992; Biondi etÂ€ al. 1992) has been subsequently validated in prospective multicenter reviewed by Grimwade (2002). According to most investigators, high-quality RNA and efficient RT are the crucial determinants for successful RT-PCR of PML-RARA (van Dongen etÂ€ al. 1999). As shown in Fig.Â€2, panel E, a proper set of primers has been identified that allow the detection of all the different PML-RARA junctions, generated by the existence of different PML breakpoint regions in the PML locus and the presence of alternative PML splicing of PML transcripts. Moreover, the alternative usage of two alternative usage of RARA sites generates extra PML-RARA transcripts of different size (van Dongen etÂ€al. 1999).

Treatment of APL Despite areas of controversy in the management of APL, there is reasonable consensus in both adults and children for the approach of induction therapy with simultaneous ATRA and anthracycline, two or three blocks of anthracycline-based consolidation with simultaneous ATRA, with or without cytarabine or nonintercalating agents, and 1 or 2Â€ years of maintenance therapy with intermittent ATRA, with or without 6-mercaptopurine and methotrexate. Consolidation may be risk adapted and usually based on the presenting white cell count (WCC). Many frontline protocols incorporate serial molecular monitoring during treatment and at three month intervals for 2Â€years following completion of therapy to detect persistent or recurrent molecular disease, which would be an indication for further treatment. There is no role for hematopoietic stem cell transplantation (HSCT) in frontline therapy, except for the small number of patients with persistent minimal residual disease (MRD) at the end of consolidation who may proceed to either allogenic or autologous transplantation based on molecular status after further treatment. While the incorporation of ATO in frontline studies appears encouraging, there are limited data regarding children and none based on a randomized comparison of ATO-based therapy against ATRA and anthracycline based therapy. Pediatric APL trials, which include ATRA with anthracycline-based chemotherapy, report a 5Â€year overall survival (OS) of 87 to 90%, disease-free survival (DFS) of 78 to 82% and event-free survival (EFS) of 71 to 77% (Mann etÂ€al. 2001; de Botton etÂ€ al. 2004; Ortega etÂ€ al. 2005; Testi etÂ€ al. 2005) (Table 1). However, ATRA and anthracycline-based chemotherapy is only appropriate for children confirmed positive for a fusion gene associated with ATRA sensitivity and should not be used for children with APL who lack these fusion genes, and who should receive standard intensive AML type therapy with anthracyclines and cytarabine.


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TableÂ€1â•… Four studies of children with newly diagnosed APL treated with combinations of ATRA and anthracycline-based chemotherapya Study results German-Austrian- European Trial Swiss BFM APL 93 GIMEMA PETHEMA No. of children 22 31 124 66 Study period 1994–2000 1993–1998 1993–2000 1996–2004 Treatment: ATRA dose (mg/m2/d) 25(15) 45(7) 45 25 25 Anthracycline dose DNR 180 or IDR DNR 495 IDR 80â•›+â•›MTZ IDR 80/100â•›+â•›MTZ (mg/m2) 36â•›+â•›ADR 120 50 50 ATRA toxicity (%): APL DS 14 13 7.5 19.5 Definite 2 4.5 Indeterminate 6 15 Pseudotumour cerebri 5 16 9 6 Headache 27 39 13 30 Results: CR Rate % 95 97 96 92 Deaths in CR 0 0 0 0 NA NA 3 3 Molecular persistence at the end of consolidation 5-year EFS, % 76 71 76 77 5-year DFS, % NA 78 82 5-year OS, % 87 90 89 87 GIMEMA Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto group; PETHEMA Programa de Estudio y Tratamiento de las Hemopatias Malignas Group; NA not available; DNR daunorubicin; IDR idarubicin; ADR adriamycin; MTZ mitoxantrone; CR complete remission; APL DS APL differentiation syndrome; EFS event-free survival; OS overall survival; DFS disease free survival a Adapted from Ortega etÂ€al. (2005)

Induction Therapy The simultaneous use of ATRA and anthracycline monotherapy is considered optimal induction chemotherapy (Fenaux etÂ€al. 1999; Sanz etÂ€al. 2003). Primary resistance to ATRA and idarubicin is extremely rare and there may be no advantage for the addition of cytarabine or other chemotherapeutic agents to ATRA and anthracyclines in induction. A study, which did not include ATRA, found no advantage in CR or EFS for dual therapy with idarubicin and cytarabine compared to monotherapy with idarubicin (Avvisati etÂ€al. 2002), although a larger anthracycline dose may have conferred an advantage in the monotherapy arm. However, an increase in the relapse rate, but no difference in CR, was reported when cytarabine was omitted from a schedule employing daunorubicin as the anthracycline (Ades etÂ€al. 2006). Daunorubicin and idarubicin have not been randomly compared in APL although idarubicin is often favored because of its relatively long half-life and CNS penetration.

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In two separate trials employing simultaneous anthracycline (idarubicin) and ATRA as induction therapy, the CR and EFS for children were reported at 92 and 96% and 77 and 76%, respectively (Ortega etÂ€al. 2005; Testi etÂ€al. 2005). While ATRA followed sequentially by chemotherapy is superior in terms of CR and RR to single agent ATRA or chemotherapy alone (Ortega etÂ€al. 2005; Fenaux etÂ€ al. 2000; Tallman etÂ€ al. 1997; Kanamaru etÂ€ al. 1995), the simultaneous use of chemotherapy in combination with ATRA is superior to the sequential use (de Botton etÂ€ al. 2004; Fenaux etÂ€ al. 1999; Burnett etÂ€ al. 1999; Mandelli etÂ€ al. 1997; Tallman etÂ€al. 2002). The European APL 93 study (Fenaux etÂ€al. 1999) randomized patients to simultaneous induction treatment (ATRA, with chemotherapy added on day 3) or sequential treatment (ATRA followed by chemotherapy after CR to a maximum of 90 days) in patients with a WCCâ•›â•›5â•›×â•›109/L. Fifty-five percent of patients randomized to sequential treatment had chemotherapy added before CR achievement because of a rising WCC. The CR rate was similar for simultaneous and sequential treatment at 94 and 95% (pâ•›=â•›0.79) respectively, but the estimated RR at 2Â€ years was in favor of simultaneous treatment (pâ•›=â•›0.04). This implies that the advantage of simultaneous over sequential ATRA is due to a reduction in relapse risk rather than a significant increase in CR or reduction in early deaths. The number of children less than 18Â€years of age and treated on this trial was too small (31; 5%) to compare simultaneous with sequential treatment, but overall, there was no difference in outcome between adults and children, except for a significantly better survival in children after adjustment for WCC (Ortega etÂ€al. 2005). Within the pediatric age group, there are limited data on the advantage of ATRA and anthracycline monotherapy over ATRA and polychemotherapy and on simultaneous over sequential ATRA with strategies extrapolated from adult studies. AML- BFM 93 reported a CR rate of 95% with a single induction death (4.5%) in a cohort of 22 children who received 3 days of ATRA followed by simultaneous multi-agent chemotherapy, compared to a CR rate of 64% with seven induction deaths (32%) in a historical cohort of 22 children who were treated with similar chemotherapy alone (Mann etÂ€al. 2001) and no ATRA (pâ•›10â•›×â•›109/L) and in those with M3 variant. The severe coagulopathy initiated or exacerbated by chemotherapy, can result in both hemorrhagic and thrombotic complications. While ATRA significantly reduces the incidence of intracranial hemorrhage, this complication remains a cause of early death in 3 to 4% of children despite increased awareness, preemptive therapy, and the use of ATRA (Mann etÂ€al. 2001; de Botton etÂ€al. 2004; Ortega etÂ€al. 2005; Testi etÂ€al. 2005). The risk of early hemorrhagic death, often prior to, or in the first week of treatment, classifies APL as a medical emergency, and ATRA, which improves the coagulopathy and may reduce the risk period, should be started immediately on morphological suspicion (Sanz etÂ€ al. 1999; Falanga 2003; Huang etÂ€al. 1988; Chomienne etÂ€al. 1990; Tallman etÂ€al. 2004a) and not delayed until the diagnosis is molecularly confirmed. The rapid initiation of combined ATRA and anthracycline-based chemotherapy is particularly important in patients with a high WCC, who should not undergo leucopheresis as this can exacerbate the coagulopathy (Tallman etÂ€al. 2004b). Coagulation parameters remain abnormal (low fibrinogen, elevated d-dimers) until hypocellularity of the bone marrow is achieved; a median of about 9 days. The coagulation screen and platelet count should be checked twice daily during induction therapy. Fibrinogen levels should be maintained above 1.5 to 2Â€g/L with fresh frozen plasma (FFP) or cryoprecipitate and the platelet count above 50â•›×â•›109/L until the coagulopathy resolves (Sanz etÂ€al. 1999; Chomienne etÂ€al. 1990; Tallman etÂ€al. 2004a, b; Milligan etÂ€al. 2006). There is no proven benefit for the use of heparin, Tranexamic acid, or antifibrinolytic drugs and these drugs should not be used routinely. Indeed, anti-fibrinolytic agents, when combined with ATRA, may increase the risk of thrombosis. Anti-fibrinolytic agents and NOVOSEVEN may be considered in life-threatening hemorrhage unresponsive to platelets, FFP, and cryoprecipitate. The APL differentation syndrome (APL DS) is characterized by fluid retention and capillary leak and is associated with a high mortality if not promptly treated (Sanz etÂ€al. 2005a; Vahdat etÂ€al. 1994; Frankel etÂ€al. 1992; De Botton etÂ€al. 1998). It is thought to be related to surface adhesion molecule modulation and cytokine release following differentiation of APL cells and can occur at any time from day 1 to day 35 after the start of induction therapy with a mean of day 7 (Frankel etÂ€al. 1992). Patients with a high WCC (>10â•›×â•›109/L) or a rising WCC are at greatest risk and should receive simultaneous chemotherapy and ATRA, which is reported to reduce the incidente (Frankel etÂ€al. 1992; De Botton etÂ€al. 1998). Early recognition and prompt treatment with dexamethasone reduces the associated mortalità (Milligan etÂ€al. 2006; De Botton etÂ€al. 1998). At the first sign or symptom suggesting APL differentation syndrome, dexamethasone should be promptly initiated at a dose of 5Â€mg/m2â•›bd (max single dose 10Â€mg) iv, until disappearance of symptoms and signs, and for a minimum of 3 days. ATRA should be stopped if symptoms are severe or unresponsive to steroid (Milligan etÂ€ al. 2006; de Botton etÂ€ al. 2003). ATRA can be cautiously reintroduced because the occurrence of APL differentiation syndrome during induction, linked to the differentiation of APL blasts, is not a contraindication to its subsequent use. Although the prophylactic use of steroids

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with induction therapy is practised, its benefit is uncertain (Firkin etÂ€ al. 1999; Wiley and Firkin 1995). APL differentiation syndrome appears to occur with the same incidence in children as in adults. In adults receiving combined ATRA and chemotherapy and the early use of dexamethasone, the incidence is reported at 15%, but with a low associated mortality of 1.2% (De Botton etÂ€ al. 1998). The incidence in similarly treated children of definite APL differentiation syndrome is reported at 3 to 5% (de Botton etÂ€al. 2004; Ortega etÂ€al. 2005; Testi etÂ€al. 2005) with an additional 5 to 15% having indeterminate APL differentiation syndrome and a mortality rate of less than 1% (Mann etÂ€ al. 2001; de Botton etÂ€ al. 2004; Ortega etÂ€al. 2005; Testi etÂ€al. 2005). Children have a higher incidence of ATRA-associated headache and pseudotumor cerebri than adults (Mahmoud etÂ€ al. 1993). Symptoms are reversible with discontinuation of the drug, which may be tolerated when reinstituted at a lower dose. The incidence of headaches may be dose-dependent and age-dependent (â•›10â•›×â•›109/L predicting a significantly increased risk of induction death and relapse risk reviewed by Grimwade (2002). Relapse risk among patients treated with ATRA and anthracycline-based chemotherapy with WBCâ•›90%) and that combined ATO and ATRA is superior to ATO or ATRA alone (Niu etÂ€al. 1999; Soignet etÂ€al. 2001; Shen etÂ€al. 2004). ATRA and ATO may act in part synergistically in triggering down regulation of telomerase causing telomerase shortening and subsequent cell death (Tarkanyi etÂ€al. 2005). A small single center pediatric study using monotherapy with ATO reported a haematological and molecular remission rate of 91% with a RFS and OS of 81 and 91%, respectively at 30Â€months (George etÂ€al. 2004). The addition of ATO to consolidation following remission induction with ATRA and chemotherapy has been reported to significantly improve the EFS and OS in adults with newly


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diagnosed APL (Powell 2007). While ATO appears to be excellent at inducing remission, its long-term benefit and comparability with ATRA-based chemotherapy is less clearly defined in frontline therapy, especially in children, where there are many unanswered questions. ATO may have a role in both induction and consolidation, combined with either ATRA or chemotherapy and used with the aim of minimizing or eliminating chemotherapy. ATO is usually well tolerated, although its use is associated with serious adverse events including hyperleukocytosis, the APL differentiation syndrome, and prolongation of QT interval. Although severe neuropathy has been rarely observed, mild peripheral neuropathy, which usually resolves after ATO discontinuation, has been reported in approximately 40% of patients reviewed by Sanz etÂ€al. (2005b). Little is known of late ATO cardio and neurological long-term toxicity. There is a need to define toxicity in children and to assess the role and best strategy for the use of ATO within a pediatric study.

What to Do When Treatment Fails? Failure to treatment currently constitutes an infrequent event in APL, involving only one-fourth of patients, owing to the high efficacy of frontline therapy. Failure is represented by disease recurrence at hematological and/or molecular level (relapse rate 20 to 25%) and by the persistence of molecular disease at recovery from 2 to 3 consolidation cycles (about 3 to 5%). Relapse occurs more frequently in those patients with a WCC countâ•›>â•›10â•›×â•›109/L at presentation (Tallman etÂ€al. 2002; Sanz etÂ€al. 2004; Tallman 2007). Approximately 3 to 5% of APL patients develop extramedullary (EM) sites of relapse, predominating in the central nervous system (CNS) and the skin, followed by other sites (testes, sites of vascular access, external ear, and auditory canal). It has been suggested that EM relapse also may be associated with initial adverse prognostic features such as high WCC count, and bcr3 PML-RARA isoform, but the risk factors for this kind of relapse have not been precisely defined (Tallman 2007; de Botton etÂ€al. 2006; Specchia etÂ€al. 2001; Breccia etÂ€al. 2003). Nonetheless, the adequate management of relapsed APL patients is still unclear, and few studies have analyzed the outcome of these patients in a prospective fashion. In particular, very limited information is available in paediatric age, with only few sporadic cases, mainly included in adult series, described in literature. In any case, the prognosis following hematological relapse is usually dismal; on the contrary, institution of therapy at the first evidence of molecular disease results in more favorable outcome, reinforcing the rationale of PCR-triggered therapy in APL (Lo Coco etÂ€al. 2003). Reinduction with ATRA alone in patients did not provide long-term benefit, since second complete remission (CR) achieved (approximately 50%) in this manner, lacked durability and required consolidation by further intensive chemotherapy (Castagnola etÂ€al. 1998). Better results have been achieved in relapsed APL with salvage therapy consisting of ATRA and chemotherapy for induction, generally containing high-dose cytarabine, followed by further chemotherapy and/or hematopoietic stem cell transplant (HSCT) (CR rate 87%; disease-free-survival 50 to 60

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(99, 100). In these studies, the choice of transplant modality was mainly based on PCR status achieved with chemotherapy: autologous HSCT was the preferred option in patients without detectable MRD, while allogeneic HSCT was chosen for patients failing to achieve a second molecular remission. However, the outcome of patients treated in molecular relapse compared favorably to those who received the treatment for hematological relapse, with longer survival and lower relapse risk (Specchia etÂ€ al. 2001). Additionally, molecular response to salvage therapy predicted longer leukaemia-free survival (LFS), independently by the therapeutic consolidation choice (Castagnola etÂ€al. 1998; Esteve etÂ€al. 2007). Currently, given the high antileukemic efficacy observed with ATO in APL patients relapsing after ATRA-containing regimen, this agent is regarded as the best option in this context. ATO has emerged as the single most active agent in patients with relapsed APL (Tallman 2007; Lo Coco etÂ€al. 2007; Ghavamzadeh etÂ€al. 2006). In the several published trials with relatively small numbers of patients, the CR rate ranges from approximately 80 to 90%, accompanied by molecular remission after two cycles in the majority of cases. ATO is administered at the dosage of 0.15Â€mg/ kg/day by 3Â€hours intravenous infusion, until CR achievement, and for a maximum of 50 days. Combining ATO with other agents may also be useful: in a report from China, the combination of ATO and anthracycline resulted in a superior outcome compared to ATO alone. However, the addition of ATRA with ATO, providing high CR rate in newly diagnosed patients, does not appear to be beneficial in the setting of relapse disease. In the randomised French study, ATO alone was compared to ATO plus ATRA in 20 patients with relapsed APL. Haematological and molecular response (8/10 patients in each treatment group), time necessary to reach CR (42 days in the two groups) and outcome (2-year overall survival 59% in the two groups) were comparable in both treatment groups (Tallman 2007; Lo-Coco etÂ€al. 2004b, 2007; Ghavamzadeh etÂ€al. 2006; Raffoux etÂ€al. 2003). Also in these experiences, best results are achieved in patients treated in molecular relapse; in these cases molecular and durable remission can be obtained after ATO monotherapy with less risk of ATO-related toxicity. The quality of CR obtained after ATO salvage treatment remains an open question. However, the best consolidation strategy after ATO-induced second remission is unknown. Several options are available, including repeated cycles of ATO (0.15Â€ mg/kg/day, ×5 days ×5Â€ weeks; 1 to 2 courses), combination with standard chemotherapy, HSCT, and recently, antiCD33. Gemtuzumab ozogamicin combines an anti-CD33 antibody with calicheamicin a cytotoxic agent with similarities to anthracyclines, and therefore, this targeted therapy might be expected to have activity in APL. CD33 is expressed in virtually 100% of APL cases with a highly homogenous expression pattern and calicheamicin belongs to the anthracycline family. While it has been used mainly in relapsed disease, it has shown efficacy in adult frontline studies (Lo-Coco etÂ€al. 2004b; Takeshita etÂ€al. 2005). CR rates comparable to ATRA and anthracycline have been reported for ATRA and Gemtuzumab (Takeshita etÂ€al. 2005). Nonetheless, the precise role of this agent in the management of relapsed APL remains unsettled. In a limited series, molecular response was obtained in 9/11 (91%) patients tested by RT-PCR after two doses and in all 13 (100%) patients tested after the third dose. GO resulted in mild


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myelosuppression and little extrahematologic toxicity, even in heavily pretreated patients (Zheng etÂ€al. 2007). Furthermore, it is presumable that combining GO with simultaneous ATRA or other agents, such as ATO, may result in improved outcome also in more advanced APL (Takeshita etÂ€al. 2005; Zheng etÂ€al. 2007). The selection of one of the above-mentioned options for consolidation therapy, as well as the modality of HSCT, should take into account various variables that may influence the outcome. Recent data published from the EBMT Group indicate that HSCT has continued to be part of treatment strategy for patients with second CR (CR2) (Sanz etÂ€al. 2007). These results show that a high proportion of patients in CR2 achieve a long-term overall survival (OS) after auto- and allo-HSCT, and both procedures present valid therapeutic options in this setting. The choice of one or other procedures will depend on the molecular status, age, the availability of an HLA identical donor and the time from diagnosis to transplant. For the 195 autografted patients in CR2, the 5-year cumulative incidence of LFS was 51% and the transplant-related mortality (TRM) 16%; for the 137 patients allografted in CR2, the 5-year LFS and TRM were 59% and 24%, respectively. The use of peripheral blood stem cells was associated with decreased TRM as compared to bone marrow (12 and 31%). In the near future the results of all consolidation procedures in similar series of CR2 patients need to be compared for better understanding how to improve the prognosis in this group of patients.

Extramedullary Relapse Because of the rarity of this event in APL, the outcome of these patients remains undetermined and only few studies have been reported in literature. However, since the introduction of ATRA in the treatment of this disease, cases of EM relapse have been increasingly described in the last years (3-year cumulative incidence from 1 to 5 to 12% of the first relapse) (Tallman 2007; de Botton etÂ€al. 2006; Specchia etÂ€al. 2001; Breccia etÂ€ al. 2003). The reason for the potential increase in EM relapse observed with ATRA treatment are unknown. One possibility is that the longer survival of patients may increase the number of patients at risk of relapse. The role of ATRA in mediating increased expression of adhesion molecules has been implicated as possible mechanism in the infiltration of EM sites. EM relapses largely predominate in the CNS and are frequently associated with overt or molecular relapse (about 80% of cases). Some reports found, in patients with EM relapse, a high incidence of presenting features such as microgranular M3 variant, the bcr3 PML-RARalpha isoform, both features being correlated to high WBC counts and younger age. Finally, it has been suggested that occurrence of APL differentation syndrome during induction, might be associated with an increased risk of EM relapse (Tallman 2007; de Botton etÂ€al. 2006; Specchia etÂ€al. 2001; Breccia etÂ€al. 2003; Knipp etÂ€al. 2007). The available data suggest that EM relapse in patients with APL should be regarded as systemic disease. CNS-directed therapy with intrathecal methotrexate and cytarabine and cranial radiotherapy, has to be associated to systemic treatment

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including drugs administered at high doses and HSCT. Heterogeneous protocols have been used for the treatment of EM relapse; second CR is achieved in almost 90% of the patients. Despite intensive systemic therapy followed by autologous or allogeneic HSCT in most cases, patients with EM relapse have a poor outcome and the overall survival is comparable to those patients who relapsed at hematological level only. The new drugs, such as ATO, alone or in combination with other chemotherapeutic agents, were recently employed in the treatment of EM relapse; ATO crosses the blood-brain-barrier but the concentrations in Cerebral Spinal Fluid (CSF) achieved by intravenous infusion, are probably insufficient for the treatment of meningeal leukemia (Knipp etÂ€ al. 2007). Also, the described ATO neurological toxicity might be increased by the previous or subsequent intrathecal and radiotherapy, necessary for the whole treatment of CNS relapse. For these reasons and because of the small number of patients, it is difficult to give more exact indications about the treatment of CNS relapse in APL. In response to the above considerations, many institutions have adopted CNS intrathecal prophylaxis in the frontline protocols (starting from consolidation phase), only for those patients with unfavorable presenting features, at higher risk of CNS relapse.

New Drugs Recent novel therapeutic agents potentially effective in APL are currently under investigation. In particular, there is great promise for the FLT-3 inhibitors in APL. FLT-3 aberrations, in the form of an internal tandem duplication (ITD) or mutation at the activation loop position 835, D835, have been reported in 20 to 45% of APL cases (Arrigoni etÂ€al. 2003; Au etÂ€al. 2004; Sohal etÂ€al. 2003). The FLT-3 ITD has also frequently associated with high WBC, microgranular morphology, and PML breakpoint at bcr3. The FLT-3 inhibitors have been shown to be effective with ATRA in a APL mouse model (Fazi etÂ€al. 2005). These raise the enticing possibility that APL may be more effectively managed in the future using orally bioavailable drugs that molecular target both classes of mutation that contribute to the pathogenesis of APL: FLT-3 inhibitors for FLT-3 ITD and ATRA that targets the PML/ RARalpha fusion. The upfront combination of ATRA and FLT-3 antagonists could be highly effective for the treating of APL patients possessing these adverse factors. On the contrary, FLT-3 aberrations might only exist in subclones or represent secondary changes in relapse, and finally might be lost at relapse. The use of FLT-3 antagonists in APL patients in relapse might not be warranted. These data should be addressed in future large prospective trials. A potential role of histone deacetylase (HDAC) inhibitors has been also suggested in the setting of APL. The addition of HDAC inhibitor to ATRA, may restore the sensitivity to retinoids in ATRA-resistant relapse. The effects of a novel mixed retinoic/butyric hyaluronan ester (HBR) were explored inÂ€vitro and inÂ€ vivo on a retinoic acid (RA)-sensitive human myeloid cell line and on its RA-resistant subclone. The results showed that HBR blocks cell growth in both RA-sensitive and RA-resistant cell lines and induces terminal granulocytic


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differentiation in RA-sensitive cells and apoptosis in RA-resistant cells. The antiproliferative effect observed inÂ€vitro is confirmed by inÂ€vivo significant prolongation of the lifetime expectancy in treated immunodeficient mice. Also, HBR increased the host life spam similarly to maximum tolerated doses of RA alone. HBR, due to its strong affinity for the CD44 membrane receptors could be a promising antineoplastic agent for APL patients regardless of their possible resistance to RA. Other retinoid/HDCA inhibitor associations are currently under investigation with promising results. Finally, a possible role for alemtuzumab, an anti-CD52 monoclonal antibody, has also been suggested in APL. ATRA treatment of a patient’s APL cells inÂ€vitro, induced high level of CD52 expression on the leukemic cells. This antigen may become a new target for antibody therapy in patients with APL.

Conclusions Much of the data on which strategies for the treatment of children with APL are based have been extrapolated from adult trials. Questions which require to be addressed exclusively in children include both the optimal ATRA and cumulative anthracycline dose, the role of lipsomal anthracyclines and cardioprotectants, the optimal risk-adapted therapy (as children often have a higher WCC at presentation compared to adults), optimal maintenance and its duration, the role of GO and ATO in combination with traditional first line therapies, the ideal frequency of molecular monitoring and best preemptive therapy, and finally, the role that novel therapies will play in this disease.

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Down Syndrome and Acute Myeloid Leukemia: An Unique Genetic Sensitivity to Chemotherapy Jeffrey W. Taub, Yubin Ge, and Yaddanapudi Ravindranath

Introduction Following the first description of leukemia in a Down syndrome (DS) child in 1930 (Brewster and Cannon 1930), a national survey in 1957 confirmed that DS individuals had an increased risk of developing leukemia (Krivit and Good 1957). It has been estimated that DS children have a 10 to 20-fold increased risk of developing both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) compared to nonDS children (Taub 2001). A Danish population-based study reported that the cumulative risk of developing leukemia in DS children by the age of 5 was 2.1%, and there was an approximately fourfold higher standardized incidence ratio of AML compared to ALL (Hasle etÂ€al. 2000). The Nordic Society of Pediatric Hematology and Oncology (NOPHO) cooperative group reported that DS children with ALL and AML comprised 2.1% and 14%, respectively, of total childhood leukemia cases (Zeller etÂ€al. 2005). In the Children’s Cancer Group (CCG) 2891 study, 15% of the AML patients had DS, indicating that DS children comprise one of the largest subgroup of AML patients. Acute megakaryocytic leukemia (AMkL; M7) is the most common FrenchAmerican-British (FAB) subtype of DS AML patients, as reported by the Pediatric Oncology Group (POG), CCG, Berlin-Frankfort-Münster (BFM)-AML, NOPHO, Medical Research Council (MRC), Toronto and Japanese Childhood AML

J.W. Taub (*) Division of Hematology/Oncology, Children’s Hospital of Michigan, 3901 Beaubien Boulevard, Detroit, MI 48201, USA and Department of Pediatrics, Wayne State University School of Medicine, Detroit, MI 48201, USA and Developmental and Therapeutics Program, Barbara Ann Karmanos Cancer Institute, 4100 John R Street, Detroit, MI 48201, USA e-mail: [email protected]


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Â�cooperative groups (Zeller etÂ€al. 2005; Ravindranath etÂ€al. 1992, 1996; Gamis etÂ€al. 2003; Creutzig etÂ€ al. 2005; Al-Ahmari etÂ€ al. 2006; Kudo etÂ€ al. 2005; Rao etÂ€ al. 2006) (TableÂ€1). In contrast, AMkL is estimated to represent approximately 10% of pediatric AML cases and 1 to 2% of adult AML cases. The reported frequencies of the AMkL phenotype in DS AML cases have ranged from 39% to 100% and the discrepancy among the reported frequencies is likely due to variations in identifyingÂ� megakaryoblasts by either morphology or expression of platelet-associated membrane antigens (glycoprotein IIb/IIIa) using CD41/61 antibodies. Zipursky has estimated that DS children have a 500-fold increased risk of developing AMkL compared to nonDS children (Zipursky etÂ€al. 1994). Prior to the early 1990s, the prognosis for DS AML patients was considered to be extremely poor. In 1992, 12 DS AML patients treated on the POG 8498 study, had a 100% event-free survival (EFS) rate compared to 28% for nonDS AML patients treated with the identical therapies (Ravindranath etÂ€ al. 1992). Subsequent studies from multiple pediatric oncology cooperative groups have confirmed this observation (TableÂ€1). The improved survival rates highlighted by studies from these groups coincided with the utilization of high-dose cytosine arabinoside (ara-C)-based therapy for DS AML patients (Ravindranath etÂ€ al. 1992, 1996; Lange etÂ€ al. 1998; Gamis etÂ€ al. 2003). The high EFS rates of DS AML patients (particularly patients with AMkL) contrasts with the extremely poor complete remission (CR) rates and EFS rates for nonDS pediatric and adult patients with AMkL (Athale etÂ€ al. 2001; Barnard etÂ€ al. 2007; Reinhardt etÂ€ al. 2005; Ravindranath etÂ€ al. 2005; Lie etÂ€ al. 2005; Ruiz-Arguelles etÂ€ al. 1992; Duchayne etÂ€al. 2003; Tallman etÂ€al. 2000; Oki etÂ€al. 2006; Pagano etÂ€al. 2002) (TableÂ€2). The EFS rates of nonDS AMkL patients are also markedly lower compared

TableÂ€1â•… Treatment outcome of Down syndrome children with AML/MDS Induction failure/ Rx relapse deaths Protocol N M6/7 EFS References POG 8498 12 58% 100% 0% 0% Ravindranath etÂ€al. (1992) POG 8821 34 47% 68% 15% 18% Ravindranath etÂ€al. (1996) POG 9421 62 91% 80% 11% 11% CCG-2891 161 55% 77% 13% 4% Gamis etÂ€al. (2003) NOPHO 38 66% 83% 11% 0% Zeller etÂ€al. (2005) BFM AML98 58 97% 89% 6% 6% Creutzig etÂ€al. (2005) Japan-AML72 90% 83% 9% 9% Kudo etÂ€al. (2005) Down MRC AML10/12 46 39% 74% 3% 27% Rao etÂ€al. (2006) Toronto (low 18 100% 72% 28% 0% Al-Ahmari etÂ€al. dose ara-C) (2006) Toronto (standard 16 100% 75% 25% 0% Al-Ahmari etÂ€al. dose ara-C) (2006)

Down Syndrome and Acute Myeloid Leukemia: An Unique Genetic Sensitivity TableÂ€2â•… Treatment results of non-Down syndrome AMkL cases Group N CR rate EFS St. Jude 35 (12.7%) 60.5% 14% De novo 20% Secondary CCG 2891 52 (5.9%) 66% 22.5% BFM 89 (7.1%) 75% 34%

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References Athale etÂ€al. (2001) Barnard etÂ€al. (2007) Reinhardt etÂ€al. (2005) Ravindranath etÂ€al. (2005) Lie etÂ€al. (2005) Ruiz-Argüelles etÂ€al. (1992) Duchayne etÂ€al. (2003)

POG 8821

32 (6.5%)

Not reported

25%

NOPHO-AML93 Mexico

16 (6.6 %) 10 children 35 adults 23 children

Not reported 90% 74% 77%

35% 30% (OS) 9% (OS) 26%

23 adult 20 (1.2%) 37 (2%)

33% 50% 43%

0% 10% 0%

Tallman etÂ€al. (2000) Oki etÂ€al. (2006)

24 (0.6%)

50%

10% (OS)

Pagano etÂ€al. (2002)

Groupe Francais De Cytogenetique Hematologique ECOG (adult) MD Anderson (adult) Gimema (adult)

to current EFS rates for nonDS pediatric AML patients, overall. This indicates that the AMkL group is one of the highest risk AML subgroups, despite the use of intensive chemotherapy protocols.

Chemotherapy Sensitivity and Down Syndrome What is the basis for the significantly higher EFS rates of DS AML patients compared to other subtypes of AML? In the 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) inÂ€vitro drug sensitivity assays, DS megakaryoblasts (nâ•›=â•›22) were 4.5-fold more sensitive to ara-C (median IC50: 77.5Â€ nm) and 12-fold more sensitive to daunorubicin (median IC50: 5.8Â€nm) compared to a large sample (nâ•›=â•›362) of nonDS AML blasts (median IC50’s: 350.9Â€nm and 71.2Â€nm, respectively) (Taub and Ge 2005). Further, DS megakaryoblasts generated significantly higher levels (4.4-fold) of the active intracellular metabolite, ara-CTP, following inÂ€vitro incubations with 3H-ara-C, indicating that the metabolism of ara-C in DS cells is altered (Taub etÂ€al. 1999). Increased inÂ€vitro sensitivities of DS AML blasts to ara-C and anthracyclines have been confirmed by other groups (Frost etÂ€al. 2000; Zwaan etÂ€al. 2002). These studies suggest that chromosome 21-localized genes, which may be overexpressed in trisomy 21 containing cells, contribute to enhanced drug sensitivities of DS AML blasts. Do the increased chemotherapy sensitivities of DS megakaryoblasts translate into increased treatment related toxicity of DS children with AML therapy? For the CCG-2891 AML study (standard timed induction), there were no significant Â�differences in Grade III/IV toxicity at any site between DS and nonDS patients

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treated with the high-dose ara-C (Capizzi II) cycle (Gamis etÂ€ al. 2003). NonDS patients experienced significantly greater hepatic toxicity (7.4% vs. 2%; Pâ•›=â•›0.036) compared to DS patients. In contrast, DS patients experienced greater mucositis (9.6% vs. 3.7%; Pâ•›=â•›0.023) and skin toxicity (3.7% vs. 0.4%; 0.024) compared to nonDS patients. However, other studies have reported a high frequency of treatment-related toxicity and mortality of DS patients. The intensive-timed DCTER regimen used in the CCG 2861/2891 AML protocol was associated with a 32% mortality rate for DS patients (Lange etÂ€ al. 1998). Similarly, the MRC AML 10/12 studies reported a 27% treatment related mortality rate for DS patients, which used no modification in chemotherapy dosing for DS compared to nonDS AML patients (Rao etÂ€ al. 2006). Infectious complications have also had a negative impact on the outcome of DS AML patients in the BFM studies (Lehrnbecher etÂ€ al. 2004). A study from Argentina reported seven treatment-related deaths among 11 DS AML patients (Zubizarreta etÂ€al. 1998). Studies from the POG have suggested that DS children have an increased risk of anthracycline-induced cardiac toxicity. A multivariate analysis of 6,493 children treated on POG protocols from 1974 to 1990 with anthracycline chemotherapy found that DS children had a relative risk of 3.4 to develop cardiac toxicity (Krischer etÂ€al. 1997). On the POG 9421 AML study (total cumulative anthracycline dose: daunorubicin: 135Â€ mg/m2; mitoxantrone: 80Â€ mg/m2), symptomatic Â�cardiomyopathy developed in 10 (17.5%) DS patients during or soon after completion of treatment, with three patients dying of congestive heart failure (O’Brien etÂ€al. 2006). The treatment outcome of DS ALL patients have been distinctly different Â�compared to DS AML patients. Prior studies have reported inferior survival rates of DS ALL patients compared to nonDS ALL patients (Whitlock 2006) and Â�frequently DS patients have experienced treatment-related toxicity, particularly with methotrexate (Peeters and Poon 1987). In vitro drug sensitivity studies have also demonstrated that DS lymphoblasts do not have the same patterns of increased inÂ€vitro chemotherapy sensitivities as in DS AML cases (Frost etÂ€al. 2000; Zwaan etÂ€ al. 2002). This suggests that DS ALL and DS AML cells differ biologically, Â�possibly reflecting the lack of GATA1 mutations (discussed below) in DS ALL and potential differences in expression of chromosome 21-localized genes.

The Role of Chromosome 21-Localized Genes and Chemotherapy Sensitivity in Down Syndrome AMkL Several chromosome 21-localized genes may be involved in the increased Â�sensitivity of DS megakaryoblasts to chemotherapy agents. The transsulfuration pathway enzyme, cystathionine-ß-synthase (CBS; localized to chromosome 21q22.3), Â�catalyzes the condensation of serine and homocysteine to form Â�cystathionine, an intermediate step in the synthesis of cysteine. Increased CBS

Down Syndrome and Acute Myeloid Leukemia: An Unique Genetic Sensitivity

113

activity is associated with significantly lower homocysteine, methionine, and S-adenosylmethionine (AdoMet) levels in DS individuals, which may have downstream effects on reduced folate metabolism. Increased CBS activity may also indirectly impact metabolism of nucleotides (including ara-C) and their antileukemic activity, analogus to the established synergism of sequential methotrexate and ara-C therapy (Newman etÂ€al. 1990). We previously hypothesized that increased CBS expression in DS leukemia cells leads to decreased allosteric regulation of 5,10-methylenetetrahydrofolate reductase (MTHFR) by AdoMet, which diverts increased levels of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. The lower 5,10-methylenetetrahydrofolate pools could result in decreased dTMP and dTTP, which would relieve inhibition of deoxycytidylate deaminase (DCD) and result in lower dCTP pools. The decreased dCTP pools, in turn, would result in increased deoxycytidine kinase (dCK) catalytic activity and greater activation of ara-C to the active intracellular metabolite, araCTP, and decreased competition with ara-CTP for incorporation into DNA (Taub etÂ€al. 1999, 1996) (Fig.Â€1). Portions of this hypothesis have been validated experimentally in both clinical leukemia samples and leukemia cell line models. (1) The CBS gene was expressed at significantly higher levels in DS leukemia cells (median 12-fold higher) Â�compared to nonDS leukemia cells and correlated with both inÂ€ vitro ara-CTP Â�generation and ara-C sensitivities (Taub etÂ€al. 1999). Interestingly, CBS transcripts were not detected in DS and nonDS ALL blasts. (2) Transfection of the CBS-null CCRF-CEM leukemia cell line with the CBS coding cDNA resulted in significantly increased inÂ€vitro and inÂ€vivo ara-C sensitivities compared to wild-type CEM cells (Taub etÂ€al. 2000). (3) In AMkL cell lines with gene expression patterns similar to clinical AML samples, high CBS transcripts in DS CMK cells were accompanied by tenfold greater ara-C sensitivities and 2.4-fold higher levels of ara-CTP Â�generation compared to the CBS-null, nonDS AMkL cell line, CMS (Ge etÂ€al. 2003). A relationship may also exist between the presence of the CBS 844ins68 polymorphism and inÂ€vitro ara-C sensitivities of DS and nonDS AML blast cells (Ge etÂ€al. 2002). Copper/zinc superoxide dismutase (SOD1, gene localized to 21q22) catalyzes the reaction from superoxide anions to hydrogen peroxide, resulting in the generation of hydroxyl free radicals. Imbalances of oxygen radicals secondary to increased SOD activity are known to be involved in the pathophysiology of DS, reflecting increased generation of oxygen free radicals and increased susceptibility of cells to undergo apoptosis (Busciglio and Yankner 1995; de Haann etÂ€ al. 1997). The increased susceptibility of DS cells to undergo apoptosis via oxygen radicals, may contribute to enhanced chemotherapy sensitivity, particularly following daunorubicin therapy, which mediates its cytotoxicity via the generation of oxygen radicals (Chien etÂ€al. 2004). Consistent with this notion, SOD transcripts were significantly higher (median fourfold) in DS megakaryoblasts compared to nonDS AML cells with a trend toward a correlation with inÂ€vitro daunorubicin sensitivities (Taub etÂ€al. 1999). Altered oxygen radical metabolism in DS may also contribute to the increased risk of anthracycline-induced cardiotoxicity.

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a

Down syndrome Megakaryoblast

B O N E

GATA1s (40-kDa)

ARA-U

E C

J

CDA

A

ARA-C

I

F

ARA-C dCTP

H

B

D

BST2

M A R R O W S T R O M A

ARA-CTP G

Fig.Â€1â•… The Role of GATA1 in cytidine deaminase expression and cytarabine metabolism in AML cells. After facilitated intracellular transport via the nucleoside transporter, HENT1 (a), cytarabine (ara-C) undergoes three sequential phosphorylation steps to form the active metabolite ara-CTP. The first phosphorylation step is rate limiting and is catalyzed by deoxycytidine kinase (dCK) (b), which is negatively regulated by dCTP. Cytidine deaminase (CDA) deaminates ara-C to the inactive metabolite, uridine arabinoside (ara-U) (c). Ara-CTP competes with dCTP for incorporation into DNA and inhibits DNA polymerase (d). Down syndrome (DS) acute megakaryocytic leukemia (AMkL) blasts are characterized by the presence of somatic mutations in exon 2 of the X-linked transcription factor gene, GATA1, resulting in the sythesis of a truncated GATA1 protein, GATA1s (40-kDa), which has reduced transactivation activity compared to the wild-type 50-kDa GATA1 protein (e). The CDA gene consists of a CDAsf (short-form) intronic promoter that acts as an enhancer to the CDAlf (long-form) promoter, which may enhance the transcription of the CDA gene. Therefore, lower CDA expression in DS megakaryoblasts compared to nonDS AMkL, may be due to the synthesis of the GATA1s protein, secondary to GATA1 mutations (f). Decreased CDA in DS megakaryoblasts results in greater ara-C phosphorylation and higher ara-CTP levels compared to nonDS megakaryoblast cells (g). Increased CBS expression in DS AMkL blasts results in altered intracellular reduced folate pools and decreased dCTP pools. This leads to: (1) reduced feedback inhibition of dCK resulting in greater phosphorylation of ara-C to form ara-CTP and (2) greater ara-CTP incorporation into DNA due to reduced competition from the lower dCTP pools (h). Bone marrow stromal cell antigen 2 (BST2), is a cell surface membrane protein expressed in normal tissues and malignant cells and supports the stromal cell-dependent growth of other cells. The transcription of BST2 gene is regulated by GATA1. Stimulation of BST2 promoter activity by the GATA1s protein is substantially reduced compared to GATA1, resulting in lower BST2 expression in DS megakaryoblasts. This results in reduced leukemia cell protection from ara-C-induced cytotoxicity modulated by bone marrow stromal cells (i). Other potential genes regulated by GATA1, may modulate the cytotoxicity of ara-C and other chemotherapy drugs via apoptosis pathways (e.g., HSP70, bcl-2) (j). In nonDS megakaryoblasts, higher CDA expression results in greater ara-C deamination to ara-U (k), decreased generation of ara-CTP and reduced ara-C cytotoxicity. Increased BST2 expression leads to greater leukemic blast protection from ara-C-induced apoptosis in the presence of bone marrow stromal cells (l)


b

115

Non-Down Syndrome Megakaryoblast

B O N E

GATA1 (50-kDa) K

E

ARA-U

L C

ARA-C

A

F

J

CDA

BST2

M A R R O W

ARA-C B

dCTP

H

D

S T R O M A

ARA-CTP G

Fig.Â€1â•… (continued)

Relationship Between GATA1 and Chemotherapy Drug Sensitivities A seminal discovery in understanding the biology of AML in DS children was initially reported in 2002, (Wechsler etÂ€ al. 2002) and subsequently confirmed by other groups (Hitzler etÂ€al. 2003; Rainis etÂ€al. 2003; Ahmed etÂ€al. 2004). Acquired somatic mutations of the transcription factor gene, GATA1 (localized to Xp11.23), have been detected with nearly 100% penetrance in DS AMkL and transient myeloproliferative disorder (TMD) cases, while mutations have not been detected in nonDS AML and nonAMkL DS leukemia cases. Hence, GATA1 mutations appear to be synonymous with the DS AMkL phenotype and are not detected in remission bone marrows. GATA1 encodes a zinc finger transcription factor that binds to the WGATAR motif and is essential for normal erythroid and megakaryocytic differentiation. Reported sequence alterations in the region encoding the N-terminal activation domain of GATA1 include insertion, deletion, missense, nonsense, and splice site mutations at the exon 2/intron boundary (Muntean etÂ€al. 2006). The net effect of GATA1 mutations is the introduction of stop codons and synthesis of a shorter GATA1 (designated GATA1s) protein (40-kDa), initiated from a downstream initiation site and distinguishable from the wild-type GATA1 protein (50-kDa) (Wechsler etÂ€al. 2002). Both GATA1s and the wild-type GATA1 proteins show similar DNA binding abilities and interact with FOG1, though the GATA1s protein exhibits altered transactivation capacity due to the loss of the N-terminal activation domain.

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Thus, mutations at the GATA1 locus in DS are believed to result in the accumulationÂ� of poorly differentiated megakaryocytic precursors and represent initiating or early genetic hits in a multistep process of leukemogenesis. The uniform detection of somatic mutations in the GATA1 gene in DS AMkL cases suggested the possibility that the mutant GATA1 protein in DS AMkL may contribute to the high EFS rates. A relationship between GATA1 and AML outcome has been suggested by a Japanese study in which nonDS AML patients with lower GATA1 expression had the highest complete remission rates (Shimamoto etÂ€al. 1995). It is conceivable that GATA1 may regulate the expression of several differentially expressed genes (based on the localization of GATA1 binding sites in their promoters), leading to altered activity of chemotherapy drugs. One likely target is cytidine deaminase (CDA; gene localized to chromosome 1p), which deaminates ara-C to the inactive metabolite, uridine arabinoside (ara-U). CDA transcripts were a median 5.1-fold lower in DS megakaryoblasts compared to nonDS AML blast cells (Ge etÂ€al. 2005). The CDA transcript is transcribed from a CDA “long form” promoter (CDAlf) while a “short form” promoter (CDAsf) acts as an enhancer for the CDAlf promoter (Ge etÂ€ al. 2004). Thus, mutations of the GATA1 gene in DS AMkL blasts that generate a functionally altered GATA1 protein, CDA enhancer activity and decreased overall CDA expression, decreased net conversion of ara-C to ara-U (Fig.Â€1). To assess the relationship between ara-C and GATA1, the DS AMkL cell line, CMK, was stably transfected with the full-length GATA1 cDNA (Ge etÂ€al. 2005). The CMK transfectants expressing the full-length GATA1 protein were approximately 8 to 17-fold less sensitive to ara-C compared to wild-type and mock-transfected CMK cells. The GATA1-transfected cells showed slightly decreased daunorubicin (approximately 2-fold) sensitivity compared with the wild-type and mock-Â�transfected CMK cells, though the relationship between GATA1 and genes involved in daunorubicin metabolism/activity is unknown.

Differential Gene Expression Studies and Down Syndrome AMkL Two studies have used microarrays to identify differentially expressed genes between DS and nonDS AMkL cases, (Ge etÂ€al. 2006; Bourquin etÂ€al. 2006), which may account for the biological differences between the two groups of AMkL and identify genes linked to the increased chemotherapy responses of DS AMkL cases and/or chemotherapy resistance of nonDS AMkL cases. In the study by Ge etÂ€al. (2006), 551 differentially expressed genes (105 overexpressed in the DS group) with a minimum twofold change were identified between DS and nonDS AMkL cases. Among the genes overexpressed in the nonDS AMkL group was bone Â�marrow stromal cell antigen 2 (BST2; localized to 19p13.2), a surface membrane protein expressed in normal tissues, malignant cells (e.g., nonDS AMkL cell line, Mo7, multiple myeloma cells). BST2 has been reported to support the stromal


117

Â� cell-dependent growth of the DW34 cell line (Ishikawa etÂ€al. 1995; Ohtomo etÂ€al. 1999). Coincubation of leukemia cells with bone marrow stromal cells protects the former from chemotherapy-induced apoptosis, while megakaryocytic differentiation of progenitor cells is inhibited by direct contact with bone marrow stromal cells (Garrido etÂ€al. 2001; Konopleva etÂ€al. 2002; Zweegman etÂ€al. 1999). The BST2 promoter region contains numerous putative cis elements including GATA1. Using gel shift and chromatin immunoprecipitation (ChIP) assays, BST2 was confirmed to be a GATA1 target gene with transcript levels significantly higher in nonDS AMkL blasts compared to DS megakaryoblasts (Ge etÂ€ al. 2006). Interestingly, stable transfection of the BST2 cDNA into the DS AMkL cell line, CMK, resulted in reduced ara-C-induced apoptosis of the CMK transfectant cells in the presence of bone marrow stromal cells compared to a mock transfectant (Ge etÂ€ al. 2006). These results suggest that bone marrow stromal cells protect leukemia cells through cell interaction mediated by cell surface antigens (e.g., BST2) and receptors. In addition to bone marrow stromal cells, bone marrow derived mesenchymal cells have also been found to protect leukemia cells from asparaginase cytotoxicity (Iwamoto etÂ€al. 2007). Additional genes overexpressed in the nonDS AMkL group including bcl-2 and HSP70, (Ge etÂ€al. 2006) have known antiapoptosis activity, suggesting a potential common mechanism of resistance to chemotherapy drugs in nonDS megakaryoblasts. Interestingly, the chromosome 21-localized gene, AML1 (also known as RUNX1) is underexpressed in the DS AMkL group compared to the nonDS AMkL group despite the gene dosage effect of trisomy 21 (Bourquin etÂ€al. 2006). Knockdown expression of AML1 in a cell line model was associated with increased ara-C sensitivities (Edwards et al. 2009). Savasan etÂ€al. (2006) used CD 36 expression by flow cytometry as a marker for megakaryocytic blast maturation in DS and nonDS AMkL blasts. Interestingly, in a small sample of nonDS megakaryoblasts expressing CD36 at similar levels as DS AMkL blasts (high CD 36 expression is universal in DS AMkL), exhibited the same level of inÂ€vitro sensitivities as DS megakaryoblasts and the corresponding patients had markedly superior outcome compared to nonDS AMkL patients with low CD36 expression (Savasan etÂ€al. 2006).

Future Challenges The optimal therapy for DS AML patients has not been clearly defined, balancing curative therapy against the potential risk of significant morbidity and treatmentrelated mortality. The current COG AAML041 clinical trial “Treatment of Down Syndrome Children with AML and MDS Under the Age of 4Â€Years” is designed to determine whether intensification of ara-C therapy during the second cycle of induction therapy, a reduction of the total cumulative anthracycline dose by 25% and a reduction in the number of intrathecal chemotherapy treatments, can maintain the high EFS rates of DS patients. Correlative pharmacologic and genetic studies may also

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identify DS patients who could be treated with further reduced intensive therapies, such as low dose ara-C containing regimens, as pioneered by Zipursky (Al-Ahmari etÂ€ al. 2006) and/or identify “high risk” DS AML patients. In an analysis of DS patients treated on the POG 9421 and CCG-2891 (standard timing arm) AML studies, 11% (24/218) of the patients relapsed with an overall survival of only 12%, despite intensive salvage therapies including bone marrow transplantation (Loew etÂ€al. 2004). In addition, DS AML patients >4Â€years of age (which comprise 5% of total DS AML cases), have EFS rates of 4Â€years of age. It is conceivable that these cases may represent true deÂ€novo AMLs rather than the classic DS AMkL phenotype associated with GATA1 mutations.

Conclusion Can the mechanisms of enhanced chemotherapy sensitivities in DS AMkL be translated to the treatment of nonDS children with AMkL, and possibly AML overall? From the studies to date, it appears that the high cure rates of DS AML patients, and in particular, patients with the AMkL phenotype, are multifactorial based upon the trisomy 21 phenotype (overexpression of the chromosome 21 localized genes, CBS and potentially SOD1) and the unique defining genetic abnormality in DS megakaryoblasts (mutations in the GATA1 gene leading to the synthesis of the 40-kDa GATA1s protein). Using gene targeting methods to knock down GATA1 expression in nonDS AMkL may be one potential strategy, though difficulties in targeting genes inÂ€ vivo exist. Alternative approaches include identifying downstream GATA1 target genes (e.g., genes in the apoptotic pathways), which mediate chemotherapy sensitivity/resistance. Ultimately, a universal mechanism linking the generation of GATA1 mutations, leukemogenesis, and enhanced chemotherapy sensitivities in DS AMkL may be identified. Acknowledgmentsâ•… Supported by grants RO1 CA92308 and CA120772 from the National Cancer Institute, the Leukemia and Lymphoma Society, The Elana Fund, The Ring Screw Textron Chair in Pediatric Cancer Research and The Georgie Ginopolis Chair for Pediatric Cancer and Hematology.

References Ahmed, M., Sternberg, A., Hall, G., Thomas, A., Smith, O., O’Marcaigh, A., Wynn, R., Stevens, R., Addison, M., King, D., Stewart, B., Gibson, B., Roberts, I., Vyas, P. 2004. Natural history of GATA1 mutations in Down Syndrome. Blood 103:2480–2489.


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Al-Ahmari, A., Shah, N., Sung, L., Zipursky, A., Hitzler, J. 2006. Long-term results of an ultra low-dose cytarabine-based regimen for the treatment of acute megakaryoblastic leukaemia in children with Down syndrome. Br J Haematol 133:646–648. Athale, U.H., Razzouk, B.I., Raimondi, S.C., Tong, X., Behm, F.G., Head, D.R., Srivastava, D.K., Rubnitz, J.E., Bowman, L., Pui, C.H., Ribeiro, R.C. 2001. Biology and outcome of childhood acute megakaryoblastic leukemia: a single institution’s experience. Blood 97:3727–3732. Barnard, D.R., Alonzo, T.A., Gerbing, R.B., Lange, B., Woods, W.G. 2007. Comparison of childhood myelodysplastic syndrome, AML FAB M6 or M7, CCG 2891: report from the Children’s Oncology Group. Pediatr Blood Cancer 49:17–22. Bourquin, J.P., Subramanian, A., Langebrake, C., Reinhardt, D., Bernard, O., Ballerini, P., Baruchel, A., Cave, H., Dastugue, N., Hasle, H., Kaspers, G.L., Lessard, M., Michaux, L., Vyas, P., van Wering, E., Zwaan, C.M., Golub, T.R., Orkin, S.H. 2006. Identification of distinct molecular phenotypes in acute megakaryoblastic leukemia by gene expression profiling. Proc Natl Acad Sci USA 103:3339–3344. Brewster, H.F., Cannon, H.E. 1930. Acute lymphatic leukemia: report of a case in an eleventh month mongolian idiot. New Orleans Med Surg J 82:872–873. Busciglio, J., Yankner, B.A. 1995. Apoptosis and increased generation of reactive oxygen species in Down’s syndrome neurons inÂ€vitro. Nature 378:776–779. Chien, M.T., Buck, S., Johnson, R.M., Stout, M., Ravindranath, Y. 2004. Increased generation of reactive oxygen species correlates with cytotoxicity in Acute Myeloid Leukemia (AML) of Down Syndrome and is augmented by cytotoxic agents affecting the mitochondrial electron transport chain. Pediatr Res 55: (abstract 1681). Creutzig, U., Reinhardt, D., Diekamp, S., Dworzak, M., Stary, J., Zimmermann, M. 2005. AML patients with Down syndrome have a high cure rate with AML-BFM therapy with reduced dose intensity. Leukemia 19:1355–1360. de Haann, J.B., Wolvetang, E.J., Cristiano, F., Iannello, R., Bladier, C., Kelner, M.J., Kola, I. 1997. Reactive oxygen species and their contribution to pathology in Down syndrome. Adv Pharmacol 38:379–402. Duchayne, E., Fenneteau, O., Pages, M.P., Sainty, D., Arnoulet, C., Dastugue, N., Garand, R., Flandrin, G., Groupe Francais d’Hematologie Cellulaire, Groupe Francais de Cytogenetique Hematologique. 2003. Acute megakaryoblastic leukaemia: a national clinical and biological study of 53 adult and childhood cases by the Groupe Francais d’Hematologie Cellulaire (GFHC). Leuk Lymphoma 44:49–58. Edwards, H., Xie, C., LaFiura, K. M., Dombkowski, A., Buck, S., Boerner, J., Taub, J. W., Matherly, L. H., Ge, Y. 2009. RUNX1 regulates phosphoinositide 3-kinase/AKT pathway: role in chemotherapy sensitivity in acute megakaryocytic leukemia. Blood 114:2744–2752. Frost, B.M., Gustafsson, G., Larsson, R., Nygren, P., Lonnerholm, G. 2000. Cellular cytotoxic drug sensitivity in children with acute leukemia and Down’s syndrome: an explanation to differences in clinical outcome? Leukemia 14:943–944. Gamis, A.S., Woods, W.G., Alonzo, T.A., Buxton, A., Lange, B., Barnard, D.R., Gold, S., Smith, F.O. Children’s Cancer Group Study 2891. 2003. Increased age at diagnosis has a significantly negative effect on outcome in children with Down syndrome and acute myeloid leukemia: a report from the Children’s Cancer Group Study 2891. J Clin Oncol 21:3415–3422. Garrido, S.M., Appelbaum, F.R., Willman, C.L., Banker, D.E. 2001. Acute myeloid leukemia cells are protected from spontaneous and drug-induced apoptosis by direct contact with a human bone marrow stromal cell line (HS-5). Exp Hematol 29:448–457. Ge, Y., Jensen, T., James, S.J., Becton, D.L., Massey, G.V., Weinstein, H.J., Ravindranath, Y., Matherly, L.H., Taub, J.W. 2002. High frequency of the 844Â€ins 68 cystathionine-b-synthase gene variant in Down Syndrome children with acute myeloid leukemia. Leukemia 16:2339–2341. Ge, Y., Jensen, T.L., Matherly, L.H., Taub, J.W. 2003. Transcriptional regulation of the cystathionine-ß-synthase gene in Down syndrome and non-Down syndrome megakaryocytic leukemia cell lines. Blood 101:1551–1557.

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Targeting RAS Signaling Pathways in Juvenile Myelomonocytic Leukemia (JMML) Jennifer O’Hara Lauchle and Benjamin S. Braun

Juvenile myelomonocytic leukemia (JMML) is an aggressive, clonal Â�myeloproliferative disorder (MPD) of childhood characterized by the overproduction of myelomonocytic cells that infiltrate the spleen, lung, and gastrointestinal tract (Arico etÂ€al. 1997; Emanuel etÂ€al. 1996). Children frequently present with anemia, thrombocytopenia, splenomegaly, and failure to thrive. The median age of diagnosis is 2Â€years of age. Without definitive treatment, the median survival of JMML patients is less than 1Â€year. Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative therapy with a probability of event-free survival at 5Â€ years of 50% (Liu etÂ€ al. 2004). The main cause of treatment failure continues to be leukemia relapse with death due to organ infiltration, infection, or transformation to acute myeloid leukemia. Stem cell transplantation is also associated with significant acute and chronic morbidity in young children. Therefore, new approaches to therapy are needed for children with newly diagnosed and relapsed JMML. Hematopoietic progenitors isolated from the peripheral blood and bone marrow of JMML patients are hypersensitive to multiple cytokines, particularly granulocytemacrophage colony-stimulating factor (GM-CSF) (Emanuel etÂ€ al. 1991). This is demonstrated by increased growth of granulocyte-macrophage colony forming units (CFU-GM) at low doses of GM-CSF compared to progenitor cells from healthy children. The molecular genetics of JMML implicates hyperactive RAS as an essential initiating event, as 85% of patients harbor mutations in either NRAS, KRAS, NF1, PTPN11 or CBL (reviewed in Lauchle etÂ€al. 2006; Loh et al. 2009). Due to deregulation of the RAS pathway in many malignancies, a tremendous effort has been expended developing novel inhibitors targeted at RAS signaling for cancer therapies. We will summarize recent experience with preclinical and clinical evaluation of targeted therapeutics for JMML and discuss opportunities to study agents currently in Â�development. With molecular insights into pathogenesis, new assays to evaluate molecular response and preclinical testing systems in murine

J. O’Hara Lauchle (*) University of California, Helen Diller Family Cancer Research Building (Optional), 1450 3rd Street, Room 264 (Lauchle) and 265 (Braun), San Francisco, CA 94158, USA e-mail: [email protected]


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models and human samples, JMML researchers are uniquely poised to evaluate novel mechanism-based therapeutics for RAS pathway malignancies.

GM-CSF and Deregulated RAS Signaling in Pathogenesis of JMML RAS Signaling is a critical determinant of proliferative cell output in response to extracellular stimuli including GM-CSF and other hematopoietic growth factors. Upon ligand binding, the GM-CSF receptor activates a receptor associated tyrosine kinase, JAK2. JAK2 phosphorylates the b-chain of the GM-CSF receptor and other substrates including Signal Transducer and Activator of Transcription (STAT) proteins. The phosphorylated GM-CSF receptor also recruits adaptor and signaling molecules such as SHP-2 and results in activation of RAS signaling pathways. RAS Proteins are molecular switches that cycle between an inactive GDP-bound state and an active GTP-bound state after receptor activation. Guanine nucleotide exchange factors (GEFs), such as son of sevenless homolog (SOS1), increase the proportion of RAS bound to GTP by disassociating guanine nucleotides from RAS and favoring passive binding to GTP, which is abundant in the cytosol. GTP bound RAS interacts with effector molecules including phosphatidylinositol 3-kinase (PI3K), RAF, and Ral-GDS to activate downstream kinase cascades. Fig.Â€1 shows a simplified schematic of RAS pathway effector cascades, although both cell context specific interactions between effectors and higher order network regulation are likely to be relevant. RAS signaling is terminated by hydrolysis of bound GTP to GDP by a slow intrinsic GTPase, which is markedly accelerated by the binding of GTPase activating proteins (GAPs), including p120GAP and neurofibromin. RAS Proteins are encoded by the closely related KRAS, NRAS, and HRAS genes, which undergo somatic point mutations in approximately one-third of human cancers. NRAS and KRAS mutations have been documented in 25% of JMML cases, though HRAS mutations are rare (Flotho etÂ€al. 1999; Miles etÂ€al. 1996). These mutations introduce amino acid substitutions that lead to accumulation of RAS in the GTP-bound conformation due to defective intrinsic GTPase activity and insensitivity to GAPs. Children with neurofibromatosis type 1 (NF1) are predisposed to developing several distinct malignancies, especially JMML, brain tumors, and malignant peripheral nerve sheath tumors. The NF1 gene encodes neurofibromin, which is a GAP that negatively regulates RAS signaling by accelerating the conversion of RAS-GTP to RAS-GDP (reviewed in refs. Cichowski and Jacks 2001; Donovan etÂ€al. 2002). Genetically, NF1 functions as a classic tumor suppressor gene. Patients with NF1 demonstrate germline heterozygosity for NF1 and develop malignancies that have undergone biallelic inactivation of NF1 (Schubbert etÂ€al. 2005; Shannon etÂ€al. 1994). In JMML, homozygous inactivation of NF1 has been shown to result in reduction in neurofibromin-specific GAP activity and elevated levels of RAS-GTP (Bollag etÂ€al. 1996). Recent studies have demonstrated that there is acquired isodisomy of the mutant NF1 allele in these patients, a commonly observed phenomenon in human cancer (Flotho etÂ€al. 2007; Michaelson etÂ€al. 2005).

Targeting RAS Signaling Pathways in Juvenile Myelomonocytic Leukemia (JMML)

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GF

JAK2 XL019 lestaurtinib INCB018424

P

SHP-2

P

Shc

Gab2 Grb2 SOS

RAS GDP

GAP NF1

RAS GTP

tipifarnib salirasib

CBL

IC87114 GDC0941 PX866 XL147 BGT226

PI3K

sorafenib

Raf

RalGDS

PDK

PD0325901 AZD6244 XL518

MEK

JNKK

ERK

JNK

NVP-BEZ235 XL765 Akt rapamycin everolimus temsirolimus

mTOR

Fig.Â€1â•… RAS Pathway signaling and inhibitors in clinical development. A simplified schematic of the RAS signal transduction network is depicted. RAS cycles between active (GDP-bound) and inactive (GTP-bound) states upon activation of growth factor (GF) receptors, and in turn stimulates multiple cytoplasmic signal transduction cascades (see text). JMML-associated mutations in KRAS, NRAS, NF1, CBL, or PTPN11 (SHP-2) activate this system. Selected agents in clinical development that might be used to treat JMML are shown with their most relevant targets. Note that, in some cases, “off-target” proteins not shown in this figure are inhibited to an equal or greater degree. As reviewed in the text, initial efforts were focused on inhibition of RAS. Current efforts are more focused on targeting downstream RAS effectors, singly or in combination. An alternative approach is to inhibit upstream biochemical events, such as JAK2 activation

Germline PTPN11 mutations are present in 40 to 50% cases of Noonan syndrome, which shares clinical features with NF1 (Sun etÂ€al. 2003). Similar to NF1, the occurrence of JMML and transient myeloproliferative disorders in children with Noonan syndrome led investigators to screen nonsyndromic patients with deÂ€novo JMML for somatic mutations in PTPN11. Somatic mutations in PTPN11 are found in 35% of nonsyndromic JMML cases (Locatelli etÂ€ al. 2005; Tartaglia etÂ€ al. 2001). PTPN11 encodes the nonreceptor protein tyrosine phosphatase (PTP) SHP-2. SHP-2 contains two src homology 2 (SH2) domains and a PTP domain. In its basal state, the PTP activity is blocked by an interaction with the N-SH2 domain. Ligand binding disrupts this auto-inhibitory interaction resulting in PTP activation and enhanced RAS/ERK pathway activation. Most PTPN11 mutations in JMML alter residues in the interface between the N-SH2 and PTP domains thereby preventing basal auto-inhibition (Keilhack etÂ€al. 2005). Leukemia associated alleles encode SHP-2 variants, such as D61Y and E76K, that tend to have higher PTP activity than those found in germline mutations found in NS (Nakamura etÂ€al. 2005; Rotblat etÂ€al. 2008).

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More recently, mutations in CBL have been found in approximately 10% of JMML cases (Loh et al. 2009). The Cbl protein has multiple roles in signal transduction. A ubiquitin ligase function targets various growth factor receptors for degradation to terminate signaling, whereas other domains promote signal transduction by formation of signaling complexes. Leukemia associated point mutations appear to disrupt ubiquitin transfer activity but leave adapter functions intact, leading to a net activation of signal transduction by aberrant CBL proteins (Sanada et al. 2009). Complete loss of Cbl ubiquitin ligase activity may be important, because the normal allele is usually lost (specifically, by acquired uniparental isodisomy). In one child with JMML, the mutant allele was found to be heterozygous in buccal DNA, suggesting possible germline inheritance. In this scenario, patients that inherit a heterozygous point mutation would be at risk for developing JMML upon acquired somatic homozygosity, in a manner similar to patients heterozygous for NF1. Mutations in PTPN11, RAS and NF1 are rarely found in the same patient, supporting the hypothesis that hyperactive RAS signaling is essential to the pathogenesis of JMML. The molecular events in 25% of JMML cases are unknown, yet they are likely to also dysregulate the RAS pathway. Analysis of JMML samples has not yet uncovered mutations in GM-CSF, SOS1, SHC1, GRB2, GAB1, BRAF, MEK1, or MEK2 (de Vries etÂ€al. 2007a; Kratz etÂ€al. 2007). In addition, FLT3 and JAK2 mutations, relatively common in other myeloid leukemias and myeloproliferative disorders, are rarely detected in JMML (de Vries etÂ€al. 2007b; Winter-Vann and Casey 2005). In addition to providing insights into pathogenesis, detection of mutations in NF1, RAS, PTPN11, or CBL will be incorporated into revised diagnostic criteria for JMML, and may have prognostic implications. For therapeutic studies, mutation detection provides a way to evaluate molecular response to treatment. Specific quantitative assays for JMML-associated mutant alleles have been developed and used to detect relapse following stem cell transplant with high sensitivity (Archambeault etÂ€al. 2008). Importantly, this assay could yield objective response criteria for therapies given prior to transplantation or in the context of minimal residual disease. Because the morphology of neoplastic cells in JMML is nearly normal, quantitative assessment of the mutant clone by DNA analysis provides the only realistic way to measure disease burden in these settings. More recently, a population of cells with increased STAT5 phosphorylation in response to GM-CSF was identified in diagnostic JMML samples with sensitivity of 91% and a specificity of 95% (Kotecha etÂ€al. 2008). Presence of this population correlated with clinical disease status in several patients for whom diagnostic, remission, and relapse samples were evaluated (Kotecha etÂ€al. 2008). Application of this assay to evaluate therapeutic response and disease status may be especially important in cases for which PCR assays are not available such as those patients without a known mutation or with a diagnosis of NF1. Beyond its potential use as a diagnostic tool, intracellular phosphoprotein analysis of JMML samples may provide opportunities to identify new disease targets, facilitate preclinical drug testing, and evaluate clinical responses. For example, inÂ€ vitro exposure of JMML samples to a JAK2 inhibitor abrogated both the phospho-STAT5 and phospho-ERK response to GM-CSF (Kotecha etÂ€al. 2008). This work follows prior efforts to develop assays using patient samples for drug Â�development but will


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need to be linked to leukemia growth inÂ€ vitro, and most importantly, clinical responses. The GM-CSF hypersensitive myeloid progenitor colony growth that aids in diagnosis has also been applied as an inÂ€vitro assay to screen new therapeutic compounds (Emanuel etÂ€ al. 2000; Loh etÂ€ al. 2004). Inhibition of spontaneous versus GM-CSF stimulated colonies may be assessed, and therapeutic thresholds can be defined when colony growth from normal bone marrow progenitors is evaluated concurrently. Engraftment of JMML samples into NOD/SCID mice provides another method to test new agents on patient-derived samples (Mohi etÂ€al. 2005).

Mouse Models Genetically engineered mouse models have substantial potential to address several important issues in drug development. Clinical research in patients is limited by high cost, long duration, and the need to protect the safety of subjects. The rarity of JMML is also a major barrier, as even cooperative groups can expect to enroll fewer than 50 patients each year. Hopefully, mouse models that recapitulate the genetic, biochemical, and cell biologic features of JMML will have useful positive and negative predictive value for screening candidate therapeutics. Studies in these animal models can be performed at far lower cost, complexity, and time in comparison to conventional human trials. Several strategies have been used to model JMML in the mouse. The first used gene targeting to disrupt Nf1, and heterozygous mice indeed developed a JMML-like disease associated with somatic loss of the wild type allele (Bollag etÂ€al. 1996; Jacks etÂ€al. 1994). This occurred only in old mice, however, so homozygous loss of Nf1 was accomplished by breeding Nf1−/− mice. Because of late-term embryonic lethality, hematopoietic cells were harvested from Nf1−/− fetal livers and transplanted into irradiated recipients (Largaespada etÂ€al. 1996). This method reliably produced mice with MPD but also required significant effort and introduced potentially confounding effects of radiation. To create a more tractable and physiologic model, a conditional Nf1flox allele was constructed by placing loxP recombination sites around exons 31 and 32 (Zhu etÂ€al. 2001). When the Cre recombinase protein is expressed, it performs a splicing event between the loxP sites that removes the intervening DNA. This results in the deletion of these two exons and inactivation of Nf1. Importantly, this lesion is similar to mutations found in NF1 patients, who frequently have deletion, nonsense, or frameshift mutations in this region. The inducible Mx1-Cre transgene allows Nf1 disruption to be induced in hematopoietic stem cells after birth by treating mice with interferon or substances that elicit interferon production such as polyinosinic-polycytidilic acid (pIpC). Nf1flox/flox, Mx1-Cre mice that are treated with pIpC at 3–5Â€days of age uniformly develop a monocytic MPD that closely resembles JMML (Le etÂ€al. 2004). This represents a major technical advance over the earlier systems, and the conditional system is much more amenable to preclinical studies. Similar conditional alleles have also been used to model effects of dominant oncogenes such as Kras and Ptpn11 (Braun etÂ€al. 2004; Chan etÂ€al. 2004, 2009). In this case, an oncogenic mutation is introduced in the germ line, but it is initially

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silenced by a repressor cassette (“LoxP-Stop-LoxP” or LSL). The LSL is then excised by Cre in juvenile mice. In contrast to the indolent MPD seen in Nf1flox/flox and LSL-Ptpn11D61Y mice, which survive for 6–12Â€months, the LSL-KrasG12D model is characterized by aggressive disease leading to death in less than 3Â€months. The LSL-KrasG12D model is also the most readily transplanted into naïve recipients. Interestingly, despite broad phenotypic similarities among all these models, there remain distinctions between them with respect to erythroid maturation, progenitor distribution, signaling biochemistry, and stem cell function. It is yet unknown as to which of these features will reflect genotype-specific findings in JMML patients and which will be unique to mice. Nonetheless, it is a testament to the efficiency of modern genetic engineering that three of the 5 known JMML oncogenes have now been modeled using conditional targeted alleles in mice. Such systems have significant advantages, most notably (a) ease of use, (b) preservation of normal cis-acting regulatory control of the targeted locus, including normal copy number, and (c) the ability to study the effects of somatic mutation in animals that have undergone normal development. In principle, these mouse cancer models will allow researchers to conduct well-controlled experiments aimed at both discovering fundamental mechanisms of leukemogenesis and evaluating potential therapies prior to human testing. However, it is prudent to note that no inÂ€vitro or inÂ€vivo model system has yet been used to identify a clinically important therapy for JMML.

Targeted Therapeutics Considering its role in many cancers, inhibition of activated RAS is a logical therapeutic strategy. However, there are several obstacles to this approach. The importance of RAS signaling in diverse physiologic processes in normal cells raises the concern of systemic toxicities from any therapies in which normal RAS proteins or effectors are inhibited. In addition, oncogenic mutations in RAS lead to decreased GTPase activity, and developing agents that help restore rather than inhibit enzyme function has not been achieved to date. Competitive GTP inhibitors would need to confront the very high affinity of RAS for GTP, as well as the high intracellular concentration of GTP. With this in mind, researchers turned their focus to inhibiting posttranslational RAS processing at the C-terminal CAAX sequence. The multistep process of CAAX protein modification renders proteins hydrophobic at their C-termini, allowing for membrane association. The rate-limiting step in normal and mutant RAS protein modification is prenylation, which covalently attaches an isoprenoid lipid at the CAAX motif at the carboxyl terminus. Based on the terminal amino acid (X) of the CAAX motif, proteins are prenlyated by farnesyltransferase (FTase), which transfers a 15 carbon farnesyl isoprenoid, or geranylgeranyltransferase (GGtase), which in turn, transfers a 20 carbon geranylgeranyl isoprenoid. As prenylation of H-RAS, N-RAS, and K-RAS is catalyzed by FTase, farnesyltransferase inhibitors (FTIs) were developed to prevent RAS localization to the plasma membrane and inhibit RAS signaling. Colony formation of JMML cells was reduced by 25 to 50% at Â�concentrations of the FTI L739,749 that only modestly reduced colony growth


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of control hematopoietic progenitors (Emanuel etÂ€al. 2000). Treatment of Nf1 deficient murine models of JMML with a similar FTI, L744,832, did not inhibit Nf1−/− hematopoietic cell growth or splenic infiltration with myeloid progenitors (Mahgoub etÂ€ al. 1999). FTI treatment was associated with a decrease in farnesylation of H-RAS but not N-RAS (Mahgoub etÂ€al. 1999). Response rate and acute toxicities of a nonpeptidic CAAX-competitive selective inhibitor of FTase tipifarnib ( R115777 ) was studied in a Children’s Oncology Group (COG) Phase II treatment window in newly diagnosed JMML (Castleberry etÂ€ al. 2005). The majority of JMML patients treated had partial clinical responses defined by decreased white blood count and organomegaly prior to transplantation; however, event-free survival was not impacted by FTI treatment. Similar to other clinical trials with tipifarnib, clinical response did not correlate with the degree of inhibition of FTase activity as measured by farnesylation of the heat shock molecule, HDJ2 (Castleberry etÂ€al. 2005). Residual FTase activity at clinical doses, alternative prenylation of N-RAS and K-RAS by GGTase, as well as a lack of correlation between RAS mutation status and response in other tumor models underscore that the authentic target(s) of FTIs are unknown. The cellular effects of FTIs may require inhibition of any of the many cellular proteins that require farnesylation such as the Rho family GTPases. As N-RAS and K-RAS have been shown to be prenylated by GGTase during FTase inhibition, GGTase inhibitors have been developed and their clinical applications are under investigation (Park etÂ€al. 2008; Side etÂ€al. 1997). Prenylation is one step in a larger metabolic pathway that targets RAS to plasma membranes in both physiologic signaling and transformation. After prenyl transfer by FTase or GGTase, cleavage of the AAX residues occurs by RAS converting enzyme 1 (Rce1). This is followed by methylation catalyzed by isoprenylcystine carboxyl methyltransferase (ICMT). These steps increase efficiency of membrane association of the RAS proteins. A few small molecule inhibitors of Rce1 protease and ICMT have recently been described and remain interesting for preclinical evaluation (reviewed in Wilhelm etÂ€al. 2004). Analysis of Rce1 and Icmt null cells suggests that farnesylated RAS and other farnesylated proteins are mislocalized to a greater degree than geranylated proteins (Mahgoub etÂ€ al. 1999). Disruption of Icmt or Rce1 results in embryonic lethality, suggesting an important physiologic role for this pathway and potential for systemic toxicities from its inhibition (Bergo etÂ€al. 2001; Kim etÂ€al. 1999). In the Kras MPD model, conditional deletion of Icmt in the malignant populations reduced severity of the disease but did not correct anemia or prolong survival (Wahlstrom etÂ€ al. 2007). Paradoxically, conditional deletion of Rce1 caused acceleration of MPD, perhaps due to effects of Rce1 on CAAX-containing proteins that inhibit RAS signaling (Tartaglia etÂ€al. 2003). Overall, investigation of agents targeting RAS processing has revealed complex and poorly predicted effects on growth of normal and malignant cells. These problems can be attributed both to ineffective targeting of mutant RAS, in the case of FTIs that spare GGTase modification of N-RAS and K-RAS, and to a large class of “off-target” substrates other than RAS with both positive and negative effects on cell growth. This experience underscores the need to investigate the therapeutic targets, mechanism, and selectivity of these drugs if we are to understand which diseases and patients might benefit from their clinical application.

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Direct targeting of RAS by strategies that do not inhibit prenylation is also being evaluated. Farnesylthiosalicylic acid (FTS, Salirasib) mimics the carboxy-terminal farnesyl cysteine carboxymethyl ester of RAS and competes with RAS-GTP for binding sites on cellular membrane proteins, galectin 1 (Gal-1) and galectin 3 (Gal-3) (Ashery etÂ€al. 2006; Rinehart etÂ€al. 2004). It is postulated that disruption of galectin binding displaces all isoforms of RAS-GTP from the plasma membrane interfering with effector interactions and increasing the rate of inactivation to GDP (Ashery etÂ€al. 2006; Rinehart etÂ€al. 2004). FTS was recently shown to inhibit growth of NF1 mutant MPNST cell lines in a dose-dependent fashion that correlated with basal RAS-GTP levels (Barkan etÂ€al. 2006). FTS treatment shortened duration of RAS-GTP induction following serum stimulation and was associated with lower levels of phosphorylated ERK, AKT, and Ral-GTP (Barkan etÂ€al. 2006). Phase I and II trials of Salirasib in pancreatic and nonsmall cell lung cancer are currently enrolling patients and will be important in evaluating the pharmacodynamics and toxicity of this compound in malignancies associated with activated RAS signaling. Finally, N-RAS needs to be both palmitoylated and prenylated for stable membrane association, and inihibition of the palmitoylation step has been proposed as a therapy for N-RAS mutant leukemias (Cuiffo and Ren 2010). However, the K-RAS 4B isoform is not palmitoylated and therefore such a strategy would not apply to cases with K-RAS mutations. As targeting mutated RAS, SHP-2, and neurofibromin is difficult, inhibiting effectors of RAS signaling has emerged as an alternative therapeutic strategy. Nonmutated effector proteins would be inhibited to the same degree in neoplastic and normal cells, and thus, a therapeutic response would arise only if cancer cells respond differently to pathway inhibition. Cellular adaptation to oncogenic signals can alter the dependence on specific signals and determine the outcome of pathway inhibition, a process referred to as “oncogene addiction” (Hingorani and Tuveson 2003; Watanabe etÂ€ al. 1996). One potential advantage of this strategy is that inhibiting a critical cellular signal that is altered by diverse molecular mechanisms has broader clinical applicability. Given the complexity of normal and malignant signaling networks, it is hard to predict a priori whether inhibition of one or more signaling proteins will be selectively toxic to cancer cells. The two RAS effector pathways most highly associated with cancer are the Raf/MEK/ERK and the PI3K/AKT/mTOR networks, though their individual contributions to malignant phenotypes are not clear. A growing number of compounds targeting various components of these systems are now available for preclinical and clinical investigations. Studying cellular responses and signaling in the presence of specific inhibitors will inform the pathobiology of cancer and hopefully yield therapeutic discoveries.

RAF/MEK/ERK RAS-GTP associates with and activates the Raf kinases (A-Raf, B-Raf, and c-Raf /Raf-1). Raf phosphorylates and activates mitogen-activated protein kinase kinase (MEK1 and MEK2). MEK, in turn, activates ERK, which phosphorylates diverse


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nuclear and cytosolic substrates. RAF gene mutations that encode constitutively active proteins are found in several cancers, and therefore, it is reasonable to hypothesize that inhibiting the Raf/MEK/ERK cascade has therapeutic potential in cancers associated with elevated RAS-GTP. Degradation of Raf-1 with a DNA enzyme resulted in a reduction of spontaneous colony formation and GM-CSF hypersensitivity in JMML samples without effect on colony formation in normal bone marrow cells (Iversen etÂ€ al. 2002). Reduced levels of GM-CSF mRNA and protein were observed in a panel of treated JMML samples (Iversen etÂ€al. 2002). Immunodeficient mice transplanted with primary JMML cells and treated for 4Â€weeks with an Raf-1 degrading enzyme experienced a greater than 50% reduction in JMML bone marrow cell mass compared to control mice (Iversen etÂ€al. 2002). Several Raf inhibitors have been developed that vary in their specificity for Raf isoforms and mutant (V600E) B-Raf commonly found in melanoma (reviewed in Caraglia etÂ€al. 2006). To date, the largest clinical experience with Raf inhibitors is with Sorafenib (BAY 43-9006), which is approved by the FDA for treatment of advanced renal cell carcinoma and hepatocellular carcinoma. Sorafenib is a novel bi-aryl urea that has been shown to inhibit Raf-1 (c-Raf) as well as B-Raf, and reduces tumor proliferation in several human tumor xenograft models (Weinstein 2002). In preclinical testing, antitumor activity was noted to correlate with a reduction in MEK and ERK phosphorylation in the presence of Sorafenib in diverse tumor cell lines and murine models (Weinstein 2002). However, Sorafenib is also active against a variety of other kinases, including VEGFR-2 and 3, PDGFR-b, FGFR-1, FLT3 and c-KIT (Weinstein 2002). RAS and BRAF mutation status failed to correlate with growth inhibition in xenograft models suggesting the importance of inhibiting angiogenesis in these responses. Early phase testing is currently being conducted in pediatric solid tumor patients. A future study of this FDA-approved multikinase inhibitor in relapsed JMML is feasible, yet the preclinical activity in JMML cells is unknown. The broad kinase inhibition of Sorafenib may have more disadvantages than benefits in hematopoietic malignancies, as inhibition of angiogenesis may cause toxicities and is unlikely to have therapeutic benefit. These potential “off-target” toxicities may be avoided with more specific inhibitors of Raf or MEK. Although not frequently mutated in cancers, MEK activation is implicated in tumor development based on the frequency of RAS and RAF mutations in tumors. MEK is an attractive target because the only known substrate is ERK. Highly selective inhibition of MEK can be achieved with non competitive inhibitors that bind a novel region and lock the kinase in a catalytically inactive state (Niihori etÂ€ al. 2005). The addition of U0126, a MEK inhibitor, to myeloid colony assays selectively inhibited the growth of Ptpn11E76K expressing cells when compared with wild-type cells (Miyauchi etÂ€ al. 1994). In contrast, the MEK inhibitor CI-1040 inhibited myeloid colony growth in Nf1 mutant bone marrow only at high concentrations that equivalently inhibited myeloid colonies in wild-type bone marrow (Lauchle etÂ€al. 2007). Treatment with CI-1040 twice a day at the maximal tolerated dose (MTD) is known to inhibit phosphorylation of ERK for 6 to 8Â€ hours

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(Peterson etÂ€al. 2006). Four weeks of treatment with this regimen failed to decrease WBC or Â�splenomegaly in the Nf1flox/flox MPD model (Lauchle etÂ€al. 2007). There may be a role for MEK inhibition for patients with refractory JMML, especially in those patients that transform to acute myeloid leukemia. This is suggested by the inhibition of Nf1 mutant acute myeloid leukemic colony growth at low doses of CI-1040 and prolonged survival of mice transplanted with Nf1 mutant AMLs when treated with CI-1040 (Lauchle etÂ€al. 2007). Recently developed MEK inhibitors (PD0325901 and AZD 6244) that have more favorable pharmacodynamic profiles are currently in clinical trials, including a Phase II trial of AZD 6244 in adults with acute myeloid leukemia (Adjei etÂ€al. 2008).

PI3K/AKT/mTOR The PI3K/AKT/mTOR pathway is activated in human cancers by diverse genetic events including RAS mutations, activating PI3K mutations, and inactivation of PTEN, a negative regulator of PI3K. The largest clinical experience is with PI3K/ AKT/mTOR pathway inhibitors that target the more distal component of the pathway, mTOR. Rapamycin and its analogs, temsirolimus (CCI-779) and everolimus (RAD001), bind to the FK506 binding protein, FKBP-12, which then binds to and inhibits mTOR. Initially discovered in 1975, rapamycin is FDA approved as an immunosuppressant and is frequently used after HSCT to prevent graft versus host disease. In JMML, antileukemic activity of low dose rapamycin (1 to 10Â€nM) has been observed in Ptpn11E76K mutant transformed cells and in JMML patient samples (Le etÂ€al. 2004; Miyauchi etÂ€al. 1994). In Ptpn11 mutant cells, the addition of a MEK inhibitor to rapamycin resulted in a synergistic decrease in colony formation (Miyauchi etÂ€al. 1994). Inhibition of mTOR by rapamycin results in AKT activation in some cell systems. This, combined with the potential of other effectors downstream of PI3K/ AKT activation to contribute to the malignant phenotype, has increased interest in chemical inhibitors of more proximal pathway components. The first generation PI3K inhibitors, wortmannin and LY294002, both target the p110 catalytic subunit of PI3K, yet they also inhibit other kinases. They are associated with broad cellular toxicities that limit therapeutic evaluation of PI3K pathway inhibition. Recently, development of more specific inhibitors of PI3K (PX-866, GDC-0941, IC87114, XL147, BGT226) has revitalized the study of the PI3K pathway in malignancies, and several agents are under clinical investigation. In addition, several dual inhibitors of PI3K and mTOR (NVP-BEZ235, XL765 and PI-103) are under active preclinical and clinical study. Antileukemic activity in AML has recently been reported for PI-103, a dual p110 and mTOR inhibitor, which was not induced either by selective PI3K inhibition with a p110 inhibitor, IC87714, or by mTOR inhibition with everolimus (Pardanani 2008). In AML samples, these authors noted both AKT activation during mTOR inhibition as well as mTOR activation during PI3K inhibition, supporting a rationale for combined inhibition of pathway components


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(Pardanani 2008). AKT inhibitors with diverse mechanisms including ATP Â�competition, prevention of membrane localization, and allosteric inhibition provide another approach to inhibit this complex pathway.

PTPN11/SHP-2 As SHP-2 is the most commonly mutated protein in sporadic JMML and promotes signal transduction through RAS pathway, inhibiting the nonreceptor protein tyrosine phosphatase (PTP) is a rational therapeutic strategy for JMML. By screening the NCI diversity set chemical library, NSC-87877 was identified as a specific inhibitor of SHP-2 and SHP-1 (Chen etÂ€ al. 2006). Site directed mutagenesis and modeling suggested that NSC-87877 inhibits SHP-2 PTP activity by binding to the catalytic cleft (Chen etÂ€al. 2006). NCS-87877 may preferentially inhibit activated SHP-2, as 50% inhibition of PTP activity in fetal liver cells transduced with fulllength wild-type SHP-2 occurred at a NSC-87877 dose 4.5-fold higher than the dose required to achieve the same inhibition of the E76K mutant SHP-2 or the wildtype PTP domain lacking the autoinhibitory N-SH2 domain (Chen etÂ€ al. 2006). Cells exposed to inhibitor had lower RAS-GTP and phospho-ERK levels following cytokine stimulation (Chen etÂ€al. 2006). The cytotoxic effects of NSC-87877 on a human breast cancer cell line were not significantly different than those seen with a MEK inhibitor or a PI3K inhibitor (LY294002) alone; however, greater cell death was noted with the combination of NSC-878777 and LY294002 (Chen etÂ€al. 2006). A second compound, phenylhydrazonopyrazolone (PHSP1) was identified in a screen modeling binding interactions with the SHP-2 catalytic site (Hellmuth etÂ€al. 2008). PHSP1 decreased growth factor stimulated SHP-2 PTPase activity, phosphorylation of ERK, and SHP-2 dependent morphologic changes in epithelial cells (Hellmuth etÂ€al. 2008). PHSP1 inhibited growth and colony formation in a panel of human tumors in a SHP-2 dependent manner, with high expression of mutant SHP-2 predicting strong inhibition (Hellmuth etÂ€al. 2008). A reasonable next step is to study these compounds in clinical and murine JMML samples to evaluate therapeutic potential of SHP-2 inhibitors as they are currently unavailable for clinical administration.

JAK2-STAT5 Cellular responses to GM-CSF are primarily regulated by the JAK2 kinase (Wahlstrom etÂ€al. 2008). With the recent discovery of STAT5 phosphorylation in response to low dose GM-CSF in JMML cells and a prominent role of activating JAK2 mutations in myeloproliferative disorders, it is logical to evaluate the functional consequences of inhibiting JAK signaling (Kotecha etÂ€al. 2008). Based on the gain of function mutation JAK2 V617F, identified in the majority of patients with

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polycythemia vera, essential thrombocythemia, and primary myelofibrosis, a number of inhibitors (XL019, INCB018424, and TG101348) that inhibit wild-type and mutant JAK2 are in various stages of preclinical and clinical trial testing (Ohren etÂ€al. 2004). In addition, several agents designed to inhibit Aurora kinases (VX680, and AT9283) or FLT3 (CEP-701, lestaurtinib) have significant JAK2 kinase inhibition and Phase I and II clinical trials in MPDs are being conducted (Hexner etÂ€al. 2008; Ohren etÂ€ al. 2004). Therefore, these agents may be among the first signal transduction inhibitors used in clinical trials for JMML. In a preclinical setting, the orally available JAK inhibitor (XL019) abrogated the phosphorylation of both STAT5 and ERK in response to GM-CSF (Kotecha etÂ€al. 2008). Further correlating an inhibitor’s effect on RAS signaling proteins and colony formation may be used to evaluate the role of JAK/STAT5 signaling in JMML disease pathogenesis.

Conclusion Therapy with selective small molecule inhibitors has revolutionized cancer treatment for several hematopoietic malignancies, most notably CML. Advances in the molecular understanding of JMML have arrived concurrently with development of many inhibitors directed at RAS signaling. This provides an unprecedented opportunity to evaluate the impact of targeted inhibitors alone and in combination in JMML treatment. At a minimum, the drugs reviewed in this chapter will serve as chemical probes to explore the critical biochemical alterations in JMML cells and inform future drug development. Ultimately, the successful identification of a new therapy for JMML will require collaborative research to generate and analyze preclinical data necessary to prioritize the growing number of drugs available for clinical trial. Trial design must give careful consideration to incorporation of novel agents and biologic correlates into current clinical trial design to evaluate each target thoroughly. The options for JMML therapy are expanding rapidly and hold promise for improved clinical outcomes.

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Chronic Myeloid Leukemia: Pathophysiology and Therapeutics Seth J. Corey and Jorge Cortes

Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative disorder Â�characterized by a translocation between the long arms of chromosomes 9 and 22. Often referred to as the Philadelphia (Ph) chromosome, this genetic rearrangement results in an oncogenic tyrosine kinase, Bcr-Abl. The disease may smolder for years before terminating in a blast crisis. Once curable, mostly with bone marrow transplantation and only seldom with interferon alpha-based therapy, its management has been radically changed with the introduction of imatinib mesylate, an orally available Abl kinase inhibitor, in 1998. More common in adults, only an estimated 50 pediatric cases occur annually in North America. The treatment of CML will remain in flux as second-generation Bcr-Abl kinase inhibitors are studied clinically. Because of its rarity in pediatrics, clinical insights must come from Â�experience with adult CML. CML serves as the paradigm of how molecular understanding of a cancer’s pathophysiology can produce a revolutionary form of Â�targeted therapy, providing hope for those pediatric cancers with a well-defined genetic lesion.

Pathophysiology of Bcr-Abl in CML More than 95% of all adult and pediatric CML possess the Ph chromosome (designated as Ph+ or Ph1) (Melo and Barnes 2007; Sherbenou and Druker 2007). This results from a reciprocal and balanced translocation in hematopoietic stem cells, which may be more precisely described t(9;22)(q34;q11.2) (Nowell and Hungerford 1960; Rowley 1973). Consequently, the Abl gene on chromosome 9 fuses to the Bcr

S.J. Corey (*) Departments of Pediatrics and Cell & Molecular Biology, Children’s Memorial Hospital and the Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL e-mail: [email protected]


139

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S.J. Corey and J. Cortes

gene on chromosome 22 (Ben-Neriah etÂ€al. 1986; Shtivelman etÂ€al. 1985). The fusion mRNA transcript encodes a protein, Bcr-Abl, with constitutive tyrosine kinase activity. Bcr stands for breakpoint cluster region, because there are variable breakpoints at which the translocation arises. The two most common breakpoints produce transcripts designated as e13a2 and e14a2 (also known as b2a2 and b3a2) typically found in CML. Mapping specific sites of chromosomal translocations aids in the design of quantitative PCR, which is commercially available, to monitor disease activity. The fusion of the partial genes for Bcr and Abl produce transcripts encoding either a 210 or approximately 190Â€kDa protein (Shtivelman etÂ€al. 1985). The larger (p210 Bcr-Abl) is found predominantly in CML, while the smaller form (p190 Bcr-Abl) occurs most frequently in Ph+ acute lymphoblastic leukemia (ALL) (Chan etÂ€al. 1987). Discussion of therapeutically targeting the p190 Bcr-Abl in ALL is discussed in Chap. 20. Bcr itself is a serine/threonine kinase and contains regulatory domains that affect the activities of RAS-related proteins Rac and Cdc42 (Diekmann etÂ€al. 1991; Maru and Witte 1991). Named after a viral transforming gene (v-Abl, Abelson murine leukemia virus) that Herb Abelson discovered as pediatric hematology fellow (Abelson and Rabstein 1970; Wang etÂ€al. 1984), cellular Abl (c-Abl) is a tyrosine kinase (Groffen etÂ€ al. 1983). Weakly active in its proto-oncogenic form, Abl becomes a much more potent intracellular tyrosine kinase that is constitutively activated upon fusion with Bcr. (Fig.Â€1). Bcr-Abl kinase drives cellular proliferation and inhibits apoptosis of myeloid progenitor and precursor cells. Ultimately, differentiation to neutrophils is impaired and blasts accumulate, leading to an acute leukemia-like presentation known as blast phase (BP). As the disease evolves, additional chromosomal abnormalities and genetic mutations may arise. Corresponding to the progression are three clinically defined phases: chronic, accelerated, and blast (discussed below). Murine retroviral transplantation experiments and the clinical success of imatinib confirm that Bcr-Abl is necessary and sufficient for CML. Bone marrow mononuclear cells harvested from healthy mice can be infected with retrovirus containing the Bcr-Abl gene (Daley etÂ€al. 1990). These modified cells, now expressing Bcr-Abl kinase, can be transplanted into irradiated, genetically related mice. Within several weeks, the transplant engrafts and a fatal myeloproliferative disease develops. At the same time that the mouse model for CML was being developed, organic chemists were synthesizing an array of tyrosine kinase inhibitors (Lydon and Druker 2004). Tyrosine kinases may be found in one of about a dozen families of receptors, such as those for epidermal growth factor, insulin, and Â�platelet-derived growth factor. At least eight families of non-receptor (or cytosolic) tyrosine kinases exist: Csk, Src, Btk/Itk, Syk/Zap-70, Fes, FAK/Pyk2, Jak, and Abl/Arg. Structural similarities among the several hundred receptor and cytosolic tyrosine kinases exist, which result in only relative specificity for most inhibitors. One lead compound (CGP57148B, later renamed STI571, and now known as imatinib mesylate) was discovered by scientists at Ciba-Geigy and showed potency against Abl and PDGF Receptors (Buchdunger etÂ€ al. 1996). Administration of that Abl tyrosine kinase inhibitor, eventually known as imatinib mesylate Â�(commercially known as

Chronic Myeloid Leukemia: Pathophysiology and Therapeutics

141

Fig.Â€1â•… Structure and signaling function of Bcr-Abl. Upper panel, structure of Bcr and Abl. Arrows point to sites of where the two proteins fuse forming either a p190 or, more commonly, p210 fusion. For Bcr, portions of the N-terminal Ser/Thr kinase is preserved. For Abl, most of the domains, except for SH3, are retained. Lower panel, the Bcr-Abl fusion kinase recruits multiple signaling pathways. A few of the more important ones are shown here. When phosphorylated, Grb2 binds to Tyr177. This recruits two pathways, Sos-RAS-Raf-MAP Kinase and Gab2-PI 3¢kinase-Akt. Additional signaling come through activation of the Src kinase Lyn and the transcription factor STAT5. Mitochondrial associated apoptosis is deregulated through effects on Bad and Bcl-XL by Akt and STAT5, respectively. Altogether, these signaling pathways affect proliferation, survival, altered adhesiveness, and loss of differentiation

Gleevec™ or Glivec™), to these retrovirally transduced cell lines (Druker etÂ€ al. 1996) or mice (le Coutre etÂ€ al. 1999) or patients (Druker etÂ€ al. 2001) prevented development of the fatal disease. Additional genetic lesions contribute to or drive the disease through its stages. However, Bcr-Abl remains a critical target in disease progression (Shah etÂ€al. 2007). The structure and signaling function of Bcr-Abl has been thoroughly investigated, and still, new insights into how this fusion tyrosine kinase causes CML arise (Melo and Barnes 2007). Bcr-Abl tyrosine kinase’s activity is absolutely required for leukemogenesis. Increased genomic instability arising from the abnormal kinase likely contributes to disease progression with accumulation of additional genetic mutations. The constitutive tyrosine kinase activity of Bcr-Abl Â�phosphorylates a number of substrates, which activate a variety of signaling pathways (Fig.Â€ 1). Abl’s kinase activity autophosphorylates tyrosine 177 of Bcr-Abl, which serves as

142


a docking site for the SH2 domain of Grb2 (Pendergast etÂ€al. 1993). In turn, Grb2 recruits SOS, a GTP exchange factor for RAS. RAS activates both PI 3¢kinase and Raf (and subsequently MAP Kinases). Grb2 also binds another scaffolding protein, Gab2, which also activates PI 3¢kinase (Sattler etÂ€al. 2002). PI 3¢kinase phosphorylates phosphatidylinositol at the 3¢ position of inositol. This leads to several 3¢OH-phosphoinositides. One major effect is to activate the serine/threonine kinase Akt (Skorski etÂ€al. 1997). Akt phosphorylates a variety of substrates involved in the regulation of metabolism and apoptosis control. One physiologically important substrate is Bad, which when phosphorylated, can no longer promote mitochondrial-associated apoptosis. Through less understood mechanisms, Bcr-Abl activates Src family kinases, such as Lyn and Hck (Danhauser-Riedl etÂ€al. 1996). These may serve to recruit additional or salvage growth-promoting pathways. Bcr-Abl also leads to activation of the transcription factor STAT5 (Carlesso etÂ€al. 1996; Ilaria and Van Etten 1996). STAT5 promotes the expression of the anti-apoptotic factor, Bcl-XL. Another group of Bcr-Abl substrates are cytoskeletal proteins, such as CrkL, paxillin, talin, and focal adhesion kinase, which affect cellular adhesiveness (Salgia etÂ€al. 1997). As a result of fusion of Bcr with Abl, normal cellular functions of c-Abl (and Bcr) are lost. Since c-Abl binds to DNA and is involved in mediating genotoxic repair, genetic instability results. Altogether, these signaling events promote cell cycle division, survival, altered adhesiveness, and genetic instability (Skorski 2002). Ultimately, a loss of differentiation occurs (Schuster etÂ€al. 2003).

Clinical Phases Pediatric CML is a rare disease with an annual incidence of 1 case per million children (the incidence in adults is approximately ten times greater) (Linet etÂ€ al. 1999). The clinical characteristics do not differ from those seen in the adult population, with the exception of age at presentation, and perhaps, white blood cell count (Millot etÂ€al. 2005). There is a slight preponderance of boys affected (ratio of 1.5:1) with a median age at diagnosis of 12.5 years. A third of the children will be diagnosed within the first decade of life. Almost all children with CML (approximately 95%) are diagnosed during the chronic phase (CP). Common presenting symptoms are: weakness, abdominal discomfort or fullness, weight loss, bleeding, fever, and bone pain. More than 2/3 of the children will have a palpable spleen on examination; most will not have a palpable liver or enlarged lymph nodes. Hematologic findings consist of elevated white blood cell count, with almost two-thirds having a count greater than 100â•›×â•›109/L. Two-thirds will also have an anemia (hemoglobin less than 12Â€g/dL) and thrombocytosis (greater than 450â•›×â•›109/L). The CP of CML presents as a myeloproliferative disease, with an increasing number of normal appearing and functioning granulocytes and their precursors (metamyelocytes and promyelocytes). Blasts count remains below 15% in both the peripheral circulation and the bone marrow. On examination, the spleen becomes increasingly enlarged. The CP typically lasts between 3 and 5 years. Before the disease terminates in a BP, which may be either myeloid (predominantly) or

Chronic Myeloid Leukemia: Pathophysiology and Therapeutics

143

Â� lymphoid, there is frequently an intermediary accelerated phase (AP). Almost all of the children never go through AP, or it goes unrecognized. There are several criteria the different stages of CML (Cortes etÂ€al. 2006). As defined by the World Health Organization, criteria for AP include any of the following: 10 to 19% blasts in either the periphery or bone marrow, greater than 20% basophils in blood or marrow, platelet count either less than 100â•›×â•›109â•›L or greater than 1,000â•›×â•›109â•›L, new cytogenetic abnormalities, or increasing splenomegaly or white blood count unresponsive to therapy. However, these criteria have never been used prospectively, and several characteristics do not appear to remain as predictive of outcome in patients treated with tyrosine kinase inhibitors. The duration of AP is short, lasting 1 to 2 years, and usually terminates with a BP. Though there may not be an antecedent AP, BP occurs when there are at least 30% blasts (20% according to the WHO classification) in either the periphery or bone marrow or evidence of a chloroma or large clusters of blasts in the bone marrow. Blasts most frequently display myeloid markers, but a few will be lymphoid, and some undifferentiated.

Targeted Therapies Imatinib is a small molecule tyrosine kinase inhibitor, a 2-phenylaminopyrimidine compound originally identified in a drug screen for activity against the PDGF Receptor (Lydon and Druker 2004; Buchdunger etÂ€al. 1996). Pre-clinical evaluation of the compound (known then as CGP57148B and later as STI571) demonstrated its exquisite sensitivity against Bcr-Abl-transfected cell lines, cells derived from patients with CML, and mouse retroviral transduction models (Druker etÂ€al. 2001). Active also against stem cell factor receptor (c-Kit) and Abl, imatinib was tested first in patients with interferon-resistant CML, and subsequently, in patients with gastrointestinal stromal cell tumor (GIST) where activating mutations of c-Kit are commonly found (Demetri etÂ€al. 2002). Of note, imatinib interferes with the ATP binding domain of the inactive form of Bcr-Abl. For both diseases, responses were swift and dramatic. Complete cytogenetic remission can now be achieved in more than 80% of patients with CML in CP. The initial phase I trial of imatinib in patients with CP CML resistant to interferon-a was conducted in 1998 (Druker etÂ€al. 2001). Though a maximally tolerated dose was not estAblished, even at doses up to 1,000Â€ mg daily, an oral dose of 400Â€mg daily for adults was selected for phase II trials. Responses in CML may be defined as hematologic, cytogenetic, or molecular (TableÂ€1). A phase III International Randomized Study of Interferon and STI571 (IRIS) cross-over study compared imatinib 400Â€mg/day with combination of interferon-a and cytarabine in untreated CML patients in CP (O’Brien etÂ€al. 2003). As reported in 2003, complete hematologic response was seen in 96% of patients treated with imatinib (76% with Â�complete cytogenetic responses). With 5 years of additional follow-up, projected rates of complete hematologic responses were 98% and complete cytogenetic responses were 87%. Responses are mostly durable, with 5-year event-free survival rates of 83%, with 93% alive and free from transformation to accelerated or BP.

144 TableÂ€1â•… Response criteria Hematologic â•… Complete

â•… Partial

Cytogenetic â•… Complete â•… Major â•… Minor Molecular â•… Complete


Complete normalization of peripheral blood counts Leukocyte count

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