Epigenetic Therapy of Cancer

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2 Michael Lübbert Peter A. Jones Editors Epigenetic Therapy of Cancer Preclinical Models and Treatment Approaches 123

3 Epigenetic Therapy of Cancer

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5 Michael Lübbert Peter A. Jones Editors Epigenetic Therapy of Cancer Preclinical Models and Treatment Approaches

6 Editors Michael Lübbert Department of Hematology/Oncology University dical Center Freiburg University of Freiburg Freiburg Germany Peter A. Jones Department of Biochemistry and Molecular Biology Norris Comprehensive Cancer Center University of Southern California Los Angeles, CA USA ISBN ISBN (ebook) DOI / Springer Heidelberg New York Dordrecht London Library of Congress Control Number: Springer-Verlag Berlin Heidelberg 2014 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher's location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Springer is part of Springer Science+Business dia (

7 Preface The view that cancer is primarily a genetic disease has been dramatically challenged by the recent discoveries demonstrating that malignancies are marked by multiple epigenetic abnormalities; the resultant gene silencing may be more ubiquitous than gene inactivation by genetic events. This paradigm change was accompanied by enormous advances in high-throughput sequencing of primary human tumors, leading to the discovery of driver mutations in a plethora of genes encoding chromatin-modifying enzymes. Finally, the last few years also saw a striking expansion in the clinical development of drugs inhibiting several of these enzymes. Whereas in the past, a crucial role for DNA hypermethylation in cancer and thus its pharmacologic reversal as a therapeutic principle were not at all generally accepted, finally two DNA hypomethylating agents are now in routine use in the treatment of several myeloid malignancies, and more in clinical development. Also several histone deacetylase (HDAC) inhibitors have found a place in the standard treatment of two types of T-cell lymphoma, providing robust proof of principle for in vivo inhibition of histone-modifying enzymes as a valid therapeutic approach. In order to systematically review these parallel and interlocking developments which drive fruitful cross-talk between basic epigenetic research, drug development and clinical application in hematology/oncology, we put together the present series of articles authored by leading experts in the field of cancer research and epigenetics. The initial chapters review, from different perspectives, the central role of epigenetic regulation in coordinate normal cell growth, differentiation and the control of stemness, and delineate the consequences of its disturbances for cellular transformation. Thus in the first chapter, Manuel Rodríguez-Paredes and Manel Esteller provide a comprehensive view of different chromatin modifications, the enzymes setting the histone and DNA modification marks and their regulation during these processes. In Chap. 2, Irina Savelyeva and Frank Rosenbauer give in-depth descriptions of the murine models employed for in vivo studies of the effects of altered levels of DNA methylation or histone modifications, particularly as they relate to the induction and propagation of malignancies. In Chap. 3, Chaurasia, Berenzon and Hoffman take a similar view, but from the perspective of the normal hematopoietic cell differentiation model system. Specifically, they also provide their results of successful expansion of the hematopoietic progenitor cell pool through DNA methyltransferase (DNMT) and HDAC inhibitors. Focusing on one particular hematopoietic cell lineage the erythropoietic system Saunthararajah, Lavelle and v

8 vi Preface DeSimone in Chap. 4 review the concert of epigenetic regulatory mechanisms governing one of the classical cell differentiation models of tissue- and developmentspecific gene expression, i.e. globin gene regulation during the different stages of erythropoiesis. They elegantly demonstrate that (despite the non-malignant features of hemoglobinopathies) the study of dysregulated globin genes continues to deepen our understanding of epigenetic aberrations as therapeutic targets in the hematopoietic system, aside from the therapeutic implications of epigenetics for these very frequent genetic disorders. Next, the focus is on the pathophysiological role of epigenetic abnormalities in hematopoietic cancers, as exemplified by myeloid neoplasias, with particular focus on acute myeloid leukemia (AML). Claus, Schmutz, Greve and Bullinger in Chap. 5 provide a timely look to the role of gene-specific and global DNA methylation abnormalities in these malignant disorders, expanding also on the new methodological possibilities provided by high-throughput technology. In Chap. 6, Duque-Afonso, Lübbert and Cleary describe several distinct AML syndromes, each bearing a striking genotype/phenotype relationship defined by chromosomal translocations generating fusion genes, which in turn act as epigenetic modifiers. These fusion genes, resulting in the AML1/ETO and MLL-rearranged oncofusion proteins, not only have a causal role in mediating the abnormal epigenotype of the resultant leukemias, but their pharmacologic antagonization has increasingly important therapeutic implications. Chapters 7 and 8 provide a comprehensive overview on clinical applications of DNA hypomethylating agents (primarily azanucleoside drugs) to treat hematologic malignancies, as well as on the far from resolved in vivo mechanism of action of these drugs. Specifically, Daskalakis, Joeckel, Lübbert and Kuendgen review published and ongoing clinical trials of hypomethylating agents being tested in myelodysplastic syndromes (MDS), AML and other hematopoietic malignancies. Their chapter is complemented by that of Griffiths, Momparler and Karpf on the pharmacodynamic responses to these drugs. Here the often debated similarities and differences of the two clinically approved DNA hypomethylating drugs are in focus. Not only the introduction of DNMT inhibitors has significantly expanded our armamentarium to treat hematopoietic cancers, also cancer-associated histone modifications can be reversed by inhibition of the enzymes mediating them. Chapters 9 and 10 are devoted to drug development and clinical applications for inhibitor drugs of histone methyltransferases (as comprehensively reviewed by Richard Chesworth and colleagues), and of HDACs (with a timely update on clinical trials with HDAC inhibitors provided by Kristina Keller and Manfred Jung). The final three chapters summarize our present knowledge of epigenetic disturbances in different solid tumor types, specifically in lung cancer, colorectal and ovarian cancer, and other frequent malignancies. Again, a firm link is provided between the description of these tissue-specific or ubiquitous abnormalities conceptually as druggable as those found in hematopoietic cancers to the present status of clinical trial development. In Chap. 11, David Schrump reviews the abnormal patterns of DNA methylation observed in primary lung cancer cells, with a particular focus on the cancer/testis antigens (CTAs); he then outlines the remarkable potential of epigenetic drug treatment to enhance lung cancer cell

9 Preface vii immunogenicity. In Chap. 12, Sarah Derks and Manon van Engeland detail the role of altered DNA methylation patterns in colorectal cancer, particularly as regards their predictive and prognostic impact. In the final chapter, Robert Brown and colleagues discuss the priming concept of combination treatment in ovarian cancer based on sequential exposure to DNMT inhibitors followed by platinum-based chemotherapy. They also provide a comprehensive and up-to-date overview on clinical trials with DNMT and HDAC inhibitors (alone and in combination with cytotoxic agents, differentiation-inducing or immunostimulating molecules etc.), in a broad array of solid tumors. The remarkable degree of activity in this area demonstrates how far the concept of epigenetic therapy has already entered the clinic, but the authors also discuss current challenges when comparing the results to those obtained in hematologic malignancies. Being able to provide this broad overview over a field that has been active for decades but only entered center-stage over the last 5 10 years, we wish to thank all the authors for their dedicated and timely contributions, and gratefully acknowledge the editorial assistance provided by Juliane Steinmann, with support from Gregor Klaus, and the valuable input by Rainer Claus. Freiburg, Germany Los Angeles, CA, USA Michael Lübbert Peter A. Jones

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11 Contents 1 The Fundamental Role of Epigenetic Regulation in Normal and Disturbed Cell Growth, Differentiation, and Stemness Manuel Rodríguez-Paredes and Manel Esteller 2 Mouse Models to Study DNA thylation in Cancer Research Irina Savelyeva and Frank Rosenbauer 3 Epigenetic Regulation of Normal Hematopoietic Development Pratima Chaurasia, Dmitriy Berenzon, and Ronald Hoffman 4 Epigenetic Regulation of Globin Genes and Disturbances in Hemoglobinopathies Yogen Saunthararajah, Donald Lavelle, and Joseph DeSimone 5 DNA thylation Abnormalities in Hematopoietic Disorders: Biological Significance and thodological Approaches Rainer Claus, Maximilian Schmutz, Gabriele Greve, and Lars Bullinger 6 Epigenetic Modifications diated by the AML1/ETO and MLL Leukemia Fusion Proteins Jesús Duque-Afonso, Michael Lübbert, and Michael L. Cleary 7 Treatment of Hematologic Malignancies with DNA Hypomethylating Agents Michael Daskalakis, Tina E. Joeckel, Michael Lübbert, and A. Kuendgen 8 Pharmacodynamic Responses to DNA thyltransferase Inhibition Elizabeth Griffiths, Richard L. Momparler, and Adam R. Karpf 9 Histone thyltransferases: Opportunities in Cancer Drug Discovery Richard Chesworth, Tim J. Wigle, Kevin W. Kuntz, Jesse J. Smith, and Victoria M. Richon ix

12 x Contents 10 Histone Deacetylase (HDAC) Inhibitors in Recent Clinical Trials for Cancer Therapy Kristina Keller and Manfred Jung 11 Clinical Implications of Epigenetic Alterations in Lung Cancer David S. Schrump 12 Epigenetic Disturbances in Colorectal Cancer Sarah Derks and Manon van Engeland 13 Epigenetic Therapies in Solid Tumours: From Preclinical Models to Clinical Trial Results Robert Brown, Juliane Steinmann, Janet Graham, and Ros Glasspool Index

13 The Fundamental Role of Epigenetic Regulation in Normal and Disturbed Cell Growth, Differentiation, and Stemness 1 Manuel Rodríguez-Paredes and Manel Esteller 1.1 Introduction Originally coined by Conrad Waddington in the 1940s, the term epigenetics currently defines the mitotically and/or meiotically heritable changes in gene expression that take place without changes in the DNA sequence (Berger et al ). More specifically, epigenetics includes a series of cellular mechanisms that, in response to external signals, are capable of modifying chromatin packaging, thus creating transient or permanent, global or local, condensed, and decondensed chromatin states that modulate access of the different enzymatic machineries involved in the main genetic processes (transcription, replication, recombination, and DNA repair). These patterns of more closed or open chromatin (also called heterochromatin and euchromatin, respectively) display special features during stemness. Subsequently, during differentiation, they undergo a series of changes, remain fixed, and become stably transmitted through multiple cycles of cell division. This gives rise to the different gene expression patterns that underlie cell-type identity and lineage specification, as well as adult cell renewal. Epigenetic layers also play a key role in processes like X-chromosome inactivation and genomic imprinting (Portela and Esteller 2010 ). M. Rodríguez-Paredes (*) Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), L Hospitalet, Barcelona, Spain mrodriguezp@idibell.cat M. Esteller (*) Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), L Hospitalet, Barcelona, Spain Department of Physiological Sciences II, School of dicine, University of Barcelona, Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain mesteller@iconcologia.net M. Lübbert, P.A. Jones (eds.), Epigenetic Therapy of Cancer, DOI / _1, Springer-Verlag Berlin Heidelberg

14 2 M. Rodríguez-Paredes and M. Esteller CRC Gene ON Wrong TSS OFF M M M M M M Ac Ac Ac Ac Ac Ac Ac Promoter and TSS Gene body H3K4me3 H3K36me3 Ac H3/H4ac Ac H2A.Zac M methylcpg Fig. 1.1 Summary of the classical epigenetic mechanisms acting on a typical active gene in normal cells. This figure, depicting a typical transcriptionally active gene in a healthy cell, serves as an introduction to the various classic epigenetic mechanisms. Thus, the promoter and the region around the TSS present different activating covalent histone modifications which, recognized by ATP-dependent chromatin-remodeling complexes (CRCs), lead to a more open configuration of chromatin. Nucleosomes with histone variants like H2A.Z (in brown ), upstream and downstream of the TSS, contribute to this transcriptionally permissive state. In the body of the gene, while other histone marks indicate active transcription, DNA methylation aborts spurious initiations The classic epigenetic mechanisms are DNA methylation, a wide range of covalent histone modifications, and nucleosome positioning (through large ATPdependent chromatin-remodeling complexes and the substitution of canonical histones with different variants) (Fig. 1.1 ). One should also highlight the role of noncoding RNAs (ncrnas) such as micrornas (mirnas), which directly affect, or are affected by, all the former mechanisms. These are commonly included as part of the epigenetic setting phenomenon although, as with histone modifications, it is possible that not all of them may be self-perpetuating or inherited (Riddihough and Zahn 2010 ). Dysregulation of any of these mechanisms, which continuously work in close collaboration, is known to provoke misexpression of critical genes, problems in DNA repair, etc., leading to diseases like cancer (Portela and Esteller 2010 ; Rodriguez-Paredes and Esteller 2011 ). Throughout this chapter, we will summarize the current knowledge about the operation of the main epigenetic mechanisms in normal cells, the way their disruption can promote tumorigenesis, and, finally, their importance in stemness. Along the way, we will also describe how all these mechanisms continuously coordinate in a given cell to yield what is currently called its epigenome, the epigenetic status that, starting from a single mammalian genome, gives rise to the diverse cell types and developmental stages of the organism.

15 1 The Fundamental Role of Epigenetic Regulation Epigenetic chanisms in Normal and Cancer Cells DNA thylation In Normal Cells DNA methylation is historically the most extensively studied epigenetic mechanism, and its main effect is to silence genes and noncoding genomic regions. It normally consists of the covalent addition of a methyl group (-CH 3 ) at the 5 position of the cytosine ring within CpG dinucleotides, but in embryonic stem (ES) cells, about 25 % of the total DNA methylation occurs in a non-cpg context (Lister et al ). This unusual DNA methylation might be important for the origin and maintenance of pluripotency, as it disappears upon differentiation and is restored in induced pluripotent stem cells (Lister et al ; Laurent et al ). The majority of the CpG dinucleotides are concentrated either in the so-called CpG islands or in regions of large repetitive sequences like centromeres and retrotransposon elements (Deaton and Bird 2011 ; Esteller 2008 ). CpG islands are CpG-rich DNA regions spanning, on average, 1,000 base pairs and are normally located near transcription start sites (TSSs) at approximately 70 % of annotated gene promoters (almost all those corresponding to housekeeping genes and some of the tissue-specific and developmental regulator gene promoters) (Larsen et al ; Saxonov et al ; Zhu et al ). Although recent work has uncovered a large number of new CpG islands, called orphan CpG islands because they are located far away from the annotated TSSs, they may be canonical CpG islands associated with either alternative promoters of nearby annotated genes or with ncrna promoters (Illingworth et al ; Maunakea et al ). While the function of DNA methylation at repetitive sequences is to protect chromosomal integrity by preventing chromosome instability and transposition (parasitic sequence elements may represent more than 35 % of our genome), DNA methylation at CpG islands leads to the silencing of the corresponding genes (Esteller 2008 ). Intriguingly, while most CpG islands remain unmethylated during development and in differentiated tissues, only 6 % of the genes become methylated, and thus silenced, in this process. Among them are pluripotency and germ line-specific genes, different tissue-specific genes depending on each cell type, and, finally, genes involved in genomic imprinting and X-chromosome inactivation (Straussman et al ; Suzuki and Bird 2008 ; Mohn et al ). In this regard, it has been reported that extensive DNA methylation changes due to differentiation do occur at CpG island shores, regions of comparatively lower CpG density located near canonical CpG islands (Doi et al ; issner et al ). The fact that most tissue-specific DNA methylation takes place at these CpG island shores is supported by the observation that they account for 70 % of the differentially methylated regions when reprogramming somatic cells (Doi et al ; Ji et al ). The strong influence of DNA methylation upon cell identity is also underlined by the discovery that the efficiency of this reprogramming is greatly improved when methylation levels are artificially decreased (Huangfu et al ).