Advances in Immunology Associate Editors K

VOLUME ONE HUNDRED AND SEVENTEEN

ADVANCES IN IMMUNOLOGY ASSOCIATE EDITORS K. Frank Austen Harvard Medical School, Boston, Massachusetts, USA Tasuku Honjo Kyoto University, Kyoto, Japan Fritz Melchers University of Basel, Basel, Switzerland Jonathan W. Uhr University of Texas, Dallas, Texas, USA Emil R. Unanue Washington University, St. Louis, Missouri, USA VOLUME ONE HUNDRED AND SEVENTEEN

ADVANCES IN IMMUNOLOGY

Edited by FREDERICK W. ALT Howard Hughes Medical Institute, Boston, Massachusetts, USA

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier Academic Press is an imprint of Elsevier 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA 225 Wyman Street, Waltham, MA 02451, USA The Boulevard, Langford Lane, Kidlington, Oxford, OX5 1GB, UK 32 Jamestown Road, London, NW1 7BY, UK Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands

First edition 2013

No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher

Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made ISBN: 978-0-12-410524-9 ISSN: 0065-2776

For information on all Academic Press publications visit our website at store.elsevier.com

Printed and bound in USA 13 14 15 16 11 10 9 8 7 6 5 4 3 2 1 CONTENTS

Contributors vii

1. Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression 1 Panagiotis Ntziachristos, Jasper Mullenders, Thomas Trimarchi, and Iannis Aifantis

1. Introduction 2 2. Aberrant DNA Methylation in Leukemia 6 3. Disruption of Histone-Modifying Complexes Polycomb and MLL in Leukemia 14 4. Other Epigenetic Writers, Erasers, and Readers 19 5. Novel Aspects and Technologies in Epigenetics: Implications for Leukemia 25 Acknowledgments 27 References 28

2. Translocations in Normal B Cells and Cancers: Insights from New Technical Approaches 39 Roberto Chiarle 1. Mechanistic Elements that Generate Chromosomal Translocations 40 2. Novel High-Throughput Methods to Study Chromosomal Translocations 50 3. New Findings on Translocation Formation Obtained by HTGTS and TC-Seq 52 4. Landscape of Translocations in Cancers 57 5. Perspectives 63 Acknowledgments 64 References 64

3. The Intestinal Microbiota in Chronic Liver Disease 73 Jorge Henao-Mejia, Eran Elinav, Christoph A. Thaiss, and Richard A. Flavell

1. Introduction 74 2. Role of the Intestinal Microbiota on Chronic Liver Diseases 75 3. Role of the Interactions Between the Innate Immune System and the Intestinal Microbiota on Chronic Liver Diseases 81 4. Probiotics and their Potential Role in Liver Disease Therapy 89

v vi Contents

5. Conclusions 90 References 91

4. Intracellular Pathogen Detection by RIG-I-Like Receptors 99 Evelyn Dixit and Jonathan C. Kagan 1. General Principles of the Antiviral Innate Immune Response 99 2. RLRs are RNA Sensors 101 3. RIG-I Activation and Receptor Proximal Signal Propagation 109 4. Regulatory Mechanisms of RIG-I Signaling 113 5. Conclusions and Future Directions 117 Acknowledgments 118 References 118

Index 127 Contents of Recent Volumes 133 CONTRIBUTORS

Iannis Aifantis Howard Hughes Medical Institute; Department of Pathology, New York University School of Medicine; NYU Cancer Institute, New York University School of Medicine, and Helen and Martin S. Kimmel Stem Cell Center, New York University School of Medicine, New York, USA Roberto Chiarle Department of Pathology, Children’s Hospital Boston and Harvard Medical School, Boston, Massachusetts, USA, and Department of Molecular Biotechnology and Health Sciences, University of Torino, Italy Evelyn Dixit Harvard Medical School and Division of Gastroenterology, Boston Children’s Hospital, Boston, Massachusetts, USA Eran Elinav Immunology Department, Weizmann Institute of Science, Rehovot, Israel Richard A. Flavell Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, and Howard Hughes Medical Institute, Chevy Chase, Maryland, USA Jorge Henao-Mejia Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA Jonathan C. Kagan Harvard Medical School and Division of Gastroenterology, Boston Children’s Hospital, Boston, Massachusetts, USA Jasper Mullenders Howard Hughes Medical Institute; Department of Pathology, New York University School of Medicine; NYU Cancer Institute, New York University School of Medicine, and Helen and Martin S. Kimmel Stem Cell Center, New York University School of Medicine, New York, USA Panagiotis Ntziachristos Howard Hughes Medical Institute; Department of Pathology, New York University School of Medicine; NYU Cancer Institute, New York University School of Medicine, and Helen and Martin S. Kimmel Stem Cell Center, New York University School of Medicine, New York, USA Christoph A. Thaiss Immunology Department, Weizmann Institute of Science, Rehovot, Israel Thomas Trimarchi Howard Hughes Medical Institute; Department of Pathology, New York University School of Medicine; NYU Cancer Institute, New York University School of Medicine, and Helen and Martin S. Kimmel Stem Cell Center, New York University School of Medicine, New York, USA

vii Intentionally left as blank CHAPTER ONE

Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression

Panagiotis Ntziachristos*,†,‡,},1, Jasper Mullenders*,†,‡,},1, Thomas Trimarchi*,†,‡,}, Iannis Aifantis*,†,‡,},2 *Howard Hughes Medical Institute, New York, USA †Department of Pathology, New York University School of Medicine, New York, USA ‡NYU Cancer Institute, New York University School of Medicine, New York, USA }Helen and Martin S. Kimmel Stem Cell Center, New York University School of Medicine, New York, USA 1These authors contributed equally to this work 2Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2 1.1 Leukemia as a heterogeneous and multifactorial disease 2 1.2 Epigenetic factors and their possible roles in leukemia 4 2. Aberrant DNA Methylation in Leukemia 6 2.1 The role of DNA methylation in hematopoietic malignancies 6 2.2 The role of DNMT3A in leukemia 7 2.3 The biology of TET proteins and their perturbations in leukemia 10 2.4 IDH1 and IDH2 oncometabolic proteins 12 3. Disruption of Histone-Modifying Complexes Polycomb and MLL in Leukemia 14 3.1 PRC2 in hematological neoplasms 14 3.2 Role of PRC1 in leukemia 17 3.3 MLL function 17 4. Other Epigenetic Writers, Erasers, and Readers 19 4.1 Arginine methyltransferases 19 4.2 Lysine demethylases (KDMs) 21 4.3 Histone demethylases inhibitors (KDMi) 22 4.4 Histone acetyl transferases 22 4.5 Histone deacetylases 23 4.6 Bromodomain-containing proteins 24 4.7 Plant homeodomain-containing proteins 24 4.8 Chromatin remodeling complexes 25 5. Novel Aspects and Technologies in Epigenetics: Implications for Leukemia 25 5.1 Combinatorial epigenetic marks 25 5.2 Novel aspects of regulation and epigenetic factors in cancer 26 Acknowledgments 27 References 28

Abstract Over the past decade, it has become clear that both genetics and epigenetics play pivotal roles in cancer onset and progression. The importance of epigenetic regulation in proper maintenance of cellular state is highlighted by the frequent mutation of chromatin modulating factors across cancer subtypes. Identification of these mutations has created an interest in designing drugs that target enzymes involved in DNA methylation and posttranslational modification of histones. In this review, we discuss recurrent genetic alterations to epigenetic modulators in both myeloid and lymphoid leukemias. Furthermore, we review how these perturbations contribute to leukemogenesis and impact disease outcome and treatment efficacy. Finally, we discuss how the recent advances in our understanding of chromatin biology may impact treatment of leukemia.

1. INTRODUCTION 1.1. Leukemia as a heterogeneous and multifactorial disease Hematopoietic malignancies are a broad category of diseases (Gilliland, 2001). Leukemia is one of the most aggressive among them and is characterized as the abnormal proliferation of immature cells of the hematopoietic system. Different types of leukemias can arise from lymphocytes (lymphocytic leukemia), myeloid cells (myeloid leukemia), erythrocytes (erythro- cytic leukemia), and others in the bone marrow, lymph nodes, or spleen. Regardless of the cell type of origin, leukemia generally proceeds in either a chronic or an acute manner. Chronic disease consists of a long incubation period, whereas acute leukemia is associated with an abrupt accumulation of immature blood cells in the peripheral blood, bone marrow, and secondary lymphoid organs. Certain disorders are marked by both a chronic and acute phases, which are categorized based on several factors. Among the most common forms of leukemia are two chronic variants, chronic myeloid leukemia (CML) and chronic lymphoblastic leukemia (CLL), and two acute variants, acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). We briefly review these disease types below.

1.1.1 Chronic myeloid leukemia CML is a unique case of leukemia that is characterized by the presence of the Philadelphia chromosome. This reciprocal translocation between chromosomes 9 and 22 leads to the formation of a chimeric protein consisting of the breakpoint cluster region (BCR) gene with the abelson kinase (ABL1) gene. Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression 3

The resulting Bcr-Abl oncogene is characterized by constitutive tyrosine kinase activity leading to activation of downstream targets (Bartram et al., 1983; Druker, 2008). The current standard of care for CML is the small-molecule kinase inhibitor Imatinib, a very specific inhibitor of the Bcr-Abl fusion protein. Treatment with Imatinib results in a 5-year progression free survival rate of approximately 89% (Druker et al., 2006). Resistance to Imatinib occurs in certain cases usually through mutations in the Imatinib binding site in Bcr-Abl (Deininger, Goldman, & Melo, 2000; Holtz, Forman, & Bhatia, 2005). It is also worth noting that Imatinib does not erad- icate the disease, as it apparently does not target the CML leukemia- initiating cells (LICs).

1.1.2 Acute myeloid leukemia AML is the most common acute leukemia and its incidence increases with age (Daver & Cortes, 2012). AML can either occur de novo or be preceded by a premalignant state. Several preleukemic conditions exist (Byrd et al., 2002) which have the potential to progress to AML. Myelodysplastic syndromes (MDS) or -myeloproliferative neoplasms (MPN) are characterized by a block in differentiation leading to accumulation of myeloid progenitor cells. Included in MDS and MPN are refractory anemia (RA), chronic myelomonocytic leukemia (CMML), polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis (MF). Around one-third of the MDS cases progresses and gives rise to AML. AML is a heterogeneous disease that can be classified in as many as seven subtypes (de Jonge, Huls, & de Bont, 2011). These subtypes are characterized by a variety of cytogenetic and cell surface markers. Unlike CML, there is no unifying way of treating AML patients. In general, AML is treated with an array of chemotherapeutic drugs; in some cases, chemotherapy is followed by bone marrow transplantation. Overall, AML can be very hard to treat, resulting in a relatively high mortality, which is reported to account approximately to 10,000 deaths per year in the United States.

1.1.3 Acute lymphoblastic leukemia ALL is an acute disorder of either B-lymphocytes (B-ALL) or T-lymphocytes (T-ALL). ALL is the most common form of cancer in children (Pui & Evans, 2006). The genetics of ALL are quite complex and are comprised of a variety of chromosome fusions. Similar to AML, these chromosome fusions can be used to distinguish different subtypes of disease, which are associated with distinct clinical features and outcome. 4 Panagiotis Ntziachristos et al.

In T-ALL, the most common genetic event is the activation of the Notch pathway. Mutations leading to enhanced Notch signaling are present in more than 50% of patients (Aifantis, Raetz, & Buonamici, 2008). This can be explained by the fact that in thymic development, signaling through the Notch receptor promotes cell cycle progression and proliferation and Notch1 therefore acts as a proto-oncogene in this setting. Treatment of ALL especially in children has become very effective, leading to cure rates as high as 80% (Chessells et al., 2003; Rivera et al., 2005). This is mainly achieved by the use of advanced chemotherapy regimen. Although this is an outstanding clinical achievement, novel less toxic treatments should still be pursued.

1.1.4 Chronic lymphocytic leukemia Chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia (Cramer & Hallek, 2012). CLL is a disease of the B-cell lymphocytes that is characterized by a very slow progression. The incidence of CLL increases with aging. Progression of the disease can be at such a low rate that treatment is sometimes postponed till later stage. As in the leukemias described above, chromosomal aberrations and gene mutations (including mutations in the NOTCH pathway) are common in CLL. And, again, these genetic variants also determine disease outcome.

1.2. Epigenetic factors and their possible roles in leukemia The focus of this review is the regulation and deregulation of epigenetic processes in different types of leukemia. The term epigenetics was coined by C.H. Waddington in the 1940s and is a fusion of words “genetics” and “epigenesis.” The major meaning of epigenesis at that time was that the embryo gradually changes into the adult organism in contrast to the preva- iling idea of that era that the adult is preformed at the embryo stage. A more modern definition of epigenetics has been proposed as “a change in the state of expression of a gene that does not involve a mutation, but that is nevertheless inherited in the absence of the signal (or event) that initiated the change” (Ptashne, 2007). The term is used for phenomena such as genomic imprinting, paramutation, polycomb complex-mediated gene silencing, and position effect variegation. Model organisms have proven to be incredible tools to obtain insight in epigenetic phenomena. For instance, Drosophila development allows the study of stem cells that are responsible for the formation of adult structures in the fly. Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression 5

Epigenetic phenomena must fit at least one of the following three criteria (Bonasio, Tu, & Reinberg, 2010): (i) a mechanism for propagation, the signal must be propagated through DNA replication/cell division; (ii) the signal must be transmitted to the progeny; and (iii) the signal should affect gene expression. Among, the modifications that fit these criteria are histone modifications, histone variants, DNA methylation, relative nucleosomal position, and occupancy and larger chromatin domains (Margueron & Reinberg, 2010). To date, DNA methylation is the only epigenetic mark that fulfills all three criteria. Many different mechanisms have been proposed that would explain the propagation and transmission of histone marks, or the histone variants; however, these processes are not completely understood currently and require further investigation (Bonasio et al., 2010). Recently, it has become clear that disruption of epigenetic processes contributes to leukemic transformation. Traditionally, mutations in leukemia were thought to involve two discreet classes of genes. One class contains genes whose mutation can give a proliferation or survival advantage to the cell and is not specific to the hematopoietic system. This would include components of RAS-MAPK signaling, PI3-kinase/AKT signaling, and others. A second class of genes mutated in leukemia consists of regulators of hematopoiesis which do not necessarily give a growth or survival advantage but result in differentiation defects (Gilliland, 2001; Shih, Abdel-Wahab, Patel, & Levine, 2012). Although epigenetic regulators often do not belong to either of these two classes of genes, they are nonetheless frequently mutated in leukemia. This is exemplified by the translocations that are commonly found in leukemia and affect mixed lineage leukemia (MLL), polycomb repressive complex 2 (PRC2), or the ten-eleven translocation (TET ) family. It has been proposed that deregulation of epigenetic factors can provide a tumor cell the plasticity needed to adapt to different situations. Similarly, it is thought that perturbation of epigenetic regulators prior to full transformation may be a priming event that allows a more permissive environment for leukemogenesis upon acquisition of additional mutations (Feinberg, 2007). Apart from the enzymes that catalyze the histone or DNA modifications (epigenetic writers), there are proteins that specifically bind modified histone residues (readers), as well as enzymes that remove covalent modifications (erasers). There are enzymes containing the appropriate domains for both reading and writing of the marks. Mutations that alter enzymatic function can be found in all these types of chromatin-interacting proteins. In this review, we will discuss the major perturbations to epigenetic processes found in leukemia. For purposes of clarity, we will divide this review 6 Panagiotis Ntziachristos et al. into three sections comprising the following: (1) DNA methylation; (2) polycomb and MLL complexes and their roles in physiology and disease; and (3) other epigenetic modulators, with an emphasis on the ones that are mutated in leukemia. Moreover, novel therapeutic options will be mentioned throughout the review, such as inhibitors of epigenetic modulators and their combinations with current therapies. Finally, emerging technologies and biological paradigms and how the potential for novel-targeted therapies will be discussed. Of course, the field is enormous and this review cannot cover every aspect of epigenetic regulation in leukemia; we thus apologize to our colleagues for any potential omissions.

2. ABERRANT DNA METHYLATION IN LEUKEMIA 2.1. The role of DNA methylation in hematopoietic malignancies DNA methylation is the most common epigenetic modification. Methyla- tion of CpG islands in the promoters of genes is generally associated with reduced expression from that locus. CpG islands can be at least 200 bases in size with a GC content of at least 50%. CpG dinucleotides are quite rare in mammalian genomes ( 1%) (Esteller, 2008), despite which about 60% of human promoters contain CpG islands. Although the majority of CpG islands are unmethylated, a small percentage ( 6%) becomes methylated in a tissue-specific manner during early development or in differentiated tis- sues (Straussman et al., 2009). Besides CpG island methylation in the promoter, DNA methylation of the gene body is common. This is mainly seen in ubiquitously expressed genes and is positively correlated with gene expression (Hellman & Chess, 2007). It has been proposed that gene body DNA methylation might increase elongation efficiency and prevent spurious initiation of transcription (Zilberman, Gehring, Tran, Ballinger, & Henikoff, 2007). Aberrant methylation patterns are considered to be one of the characteristics of the cancer epigenome (Laird & Jaenisch, 1996). In general, global DNA hypomethylation is observed which can lead to chromosomal instability (Eden, Gaudet, Waghmare, & Jaenisch, 2003; Gaudet et al., 2003; Holm et al., 2005; Nishigaki et al., 2005). This general hypomethylation can lead to aberrant activation of oncogenes such as cyclin D2 and maspin (Oshimo et al., 2003). On the other hand, hypermethylation of the promoters of tumor-suppressor genes such as retinoblastoma 1, CDKN2A (also known as cyclin-dependent kinase inhibitor p16), the von Hippel–Lindau Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression 7 tumor suppressor, and MutL protein homologue 1 can lead to aberrant silencing (Esteller, 2007; Herman & Baylin, 2003; Jones & Baylin, 2002). Therefore, it is not surprising that DNA methyltransferase (DNMT) enzymes, which catalyze the addition of a methyl group to CpG dinucleotides, play key roles in development and disease. DNMT1 is considered to be the maintenance methyltransferase and can act on unmethylated DNA. DNMT3A and DNMT3B are the de novo DNMTs, whereas DNMT3-like lacks catalytic activity but acts as cofactor for DNMT3A/B and interacts and colocalizes with them in the nucleus. Only recently, it became clear that mutations in DNMT3A are common in AML (Ley et al., 2010; Yan et al., 2011). Moreover, there has been an advent of specific DNMT inhibitors that are used against MDS with very encouraging results (Dawson & Kouzarides, 2012).

2.2. The role of DNMT3A in leukemia 2.2.1 DNMT3A mutations in hematopoietic malignancies Mutations in DNMT3A (Fig. 1.1) were reported in approximately 20% of cases of AML of various subtypes (Ley et al., 2010). Identical mutation per- centages were found in the AML-M5 subtype that is classified as acute monocytic leukemia (Yan et al., 2011). In addition, it was reported that DNMT3A is mutated in other hematopoietic malignancies albeit at a lower frequency (Thol et al., 2011; Walter et al., 2011). DNMT3A mutations seem not to be restricted to leukemias from the myeloid lineage, as recently mutations have also been found in T-cell lymphoma and T-ALL (Couronne, Bastard, & Bernard, 2012; Simon et al., 2012). In pediatric AML, however, mutations in DNMT3A have not been found, despite the sequencing and analysis of a cohort consisting of 180 patients (Ho et al., 2011).

2.2.2 Functional consequence of DNMT3A mutations So far, mutations identified in DNMT3A are found to be exclusively heterozygous. Specifically, a very clear hotspot can be identified for DNMT3A mutations, as around 50% of the mutations occur in residue R882 (Ley et al., 2010). In vitro experiments showed that AML-linked mutations in DNMT3A lead to a severe loss of enzymatic activity. How- ever, as DNMT3A is one of two de novo DNMTs in the human genome, it is unclear what the molecular consequence of DNMT3A mutations is. One study compared the DNA methylation status of DNMT3A mutant versus wild-type AML samples. This revealed that, as expected, some OH OH A O O O IDH1 or IDH2 Mutant OH O IDH1 or IDH2 HO O O

OH OH OH Isocitrate Oxoglutarate O

OH O OH

2-Hydroxyglutarate

NH NH NH 2 2 OH 2 CH3 N DNMTs N TETs N

N O N O N O H H H ++ Oxoglutarate Succinate

B (i) CpG CpG RNAP2 CpG

DNMT DNMT DNMT (ii) X CpG CpG RNAP2 CpG

(iii) TET2 TET2 TET2 ? CpG CpG Unmodified CpG island CpG RNAP2 CpG

CpG Methylated CpG island Hydroxy methylated CpG CpG island Figure 1.1 The role of DNA methylation in leukemia. (A) Wild-type IDH converts isocitrate to oxoglutarate. Mutations in IDH1 and 2 as found in myeloid leukemias change the activity of the enzyme. Mutant IDH converts oxoglutarate to 2-hydroxyglutarate. Oxoglutarate is a cofactor to dioxygenases like the TET proteins. TET proteins convert 5-methylcytosine to 5-hydroxymethylcytosine. This potentially leads to a demethylation of the DNA, which will permit transcription from a previously silent locus. (B) Overview of the effect of the different enzymes that regulate DNA methylation. When CpG islands are unmethylated, transcription can occur from that locus. (i) DNMT enzymes methylate CpG islands in the promoter, this leads to repression of transcription from this locus. (ii) TET proteins can oxidize the methylcytosine to 5-hydroxymethylcytosine. (iii) The outcome of this reaction is not yet fully understood, but it is suggested that this leads to demethylation permits transcription. Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression 9

CpG islands in promoters indeed become hypomethylated. In AML samples with mutant DNMT3A, a lower level of methylation of CpG islands in the HoxA-gene cluster was detected. In addition, there was a clear correlation with increased expression of the HoxA genes, which leads to a less differentiated phenotype (Yan et al., 2011).

2.2.3 Mouse models of DNMT3 function Initially, no apparent hematopoietic stem cell (HSC) differentiation defect was observed in mice mutant for either DNMT3A or DNMT3B (Tadokoro, Ema, Okano, Li, & Nakauchi, 2007). It was found that cells deficient for DNMT3A or DNMT3B could still give rise to a variety of pro- genitors. In addition, it was shown that HSCs depleted for both DNMT3A and DNMT3B could not reconstitute hematopoiesis of a recipient animal. After the identification of the DNMT3A mutations in AML, Challen et al. (2012) further examined the DNMT3A knockout phenotype. In this case, it was found that loss of DNMT3A led to decreased differentiation of mouse HSCs. This phenotype could be correlated with higher expression of genes that are involved in maintaining multipotency of HSCs. Strikingly, when comparing methylation patterns in wild type and DNMT3A mutant cells, no significant changes were found in overall DNA methylation. However, further analysis of specific loci revealed that some genes were hypo- while others were hypermethylated in DNMT3A knockout animals. Genes that were found to be hypomethylated and consequently higher expressed include the well-known HSC homeostasis genes RUNX1 and GATA3.

2.2.4 Is mutant DNMT3A a prognostic marker in myeloid leukemia? The genetics of AML are very complex, but, nevertheless, it has been reported that the mutation status of DNMT3A by itself is a significant prognostic marker for disease outcome in AML (Ley et al., 2010; Marcucci et al., 2012; Ribeiro et al., 2012). Common genetic lesions co-occurring with mutant DNMT3A are mutations in NPM1 and FLT3. Especially, the combination of an FLT3- ITD mutation combined with mutant DNMT3A seems to be associated with unfavorable outcome in this disease (Patel et al., 2012). Moreover, one report showed that patients with DNMT3A mutations could benefit from higher than normal dose of chemotherapy (Daunorubicin) (Patel et al., 2012).

2.2.5 DNMT inhibitors Currently, two DNMT inhibitors, vidaza (5-azacytidine) and decitabine (5-aza-2-deoxycytidine), are approved for the treatment of cancer patients. Both vidaza and decitabine are analogues of the nucleotide cytosine. 10 Panagiotis Ntziachristos et al.

The relatively low side effects make these DNMT inhibitors the drug of choice for the treatment of MDS. However, in the more advanced AML, the use of decitabine is debated and is reported to have very little effect. Nevertheless, combination of inhibitors for histone deacetylases (HDACs) with DNMTi has given even more very promising outputs (Gore, 2011). Another preliminary study used a small cohort of patients in which it seems that DNMT3A mutant AMLs are more sensitive to the DNA methylation inhibitor Decitabine (Metzeler et al., 2012). The mechanism behind these responses is currently unknown.

2.3. The biology of TET proteins and their perturbations in leukemia 2.3.1 TET proteins In a search for proteins that are homologous to the J-binding proteins from the parasite leishmania, the only homologous proteins identified in the human genome were the TET proteins (Tahiliani et al., 2009). J-binding proteins were known for their capacity to bind a modified DNA base, base J that is unique for the parasite. Base J is a glycosylated derivative of the base thymidine. This suggested a role for the TET proteins in modifying DNA directly. Indeed, further studies showed that in TET proteins resides the catalytic activity to modify 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) (Tahiliani et al., 2009). Soon after this finding, Delhommeau et al. (2009) reported frequent mutations of TET2 in AML again suggesting a key role for DNA methylation in leukemogenesis. Some of these mutations were later verified to be true loss-of-function variants (Ko et al., 2010); however, the role of 5-hmC in tumor development remains to be fully appraised. A variety of technical issues have hampered the study of TET2 and 5-hmC in leukemia. The lack of a TET2 antibody, for instance, has made it difficult to study its genomic occupancy. However, recent technical advances have made genome-wide 5-hmC profiling possible with base-pair resolution (Booth et al., 2012; Yu et al., 2012).

2.3.2 Mutational status of TET proteins in leukemia First identified as a gene (TET1) in a chromosomal translocation in AML, it took some time to appreciate the importance of the TET proteins in leukemia. The initial report described a fusion between chromosomes Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression 11

10 and 11 in AML that lead to a chimeric protein consisting of the MLL amino(N)-terminus and the carboxyl(C)-terminus of TET1 (Lorsbach et al., 2003). In addition, despite the fact that fusions and deletions in chromosome 4 were detected in a number of AML patients (Viguie et al., 2005), it was only later understood that a single gene was present in this locus. The gene product showed a high level of sequence conser- vation with the previously identified TET1 protein and was therefore named TET2. Further analysis of the human genome identified one more protein homologous to both TET1 and TET2, that is, TET3 (Delhommeau et al., 2009). Fusions of TET1 and deletions of the TET2 locus indicated an important role of the TET proteins in hematopoietic malignancies. And, indeed, sequencing efforts confirmed that TET2 (Fig. 1.1) is a commonly mutated gene in myeloid leukemia and premalignant stages of leukemia. So far, TET2 mutations have been found in AML, CMML, MDS, and other myeloid malignancies. The largest studies suggest that TET2 mutations can be identified in 2–10% of PV and ET patients, and in 10–20% of patients with primary MF or post-PV/ET MF (Abdel-Wahab et al., 2010, 2009; Cimmino, Abdel-Wahab, Levine, & Aifantis, 2011; Tefferi et al., 2009). In addition, studies of paired MPN and AML samples from individual patients demonstrated that TET2 mutations are commonly acquired during transformation to AML from a chronic myeloid neoplasm (Abdel-Wahab et al., 2010). Sur- prisingly, mutational analysis of both TET1 and TET3 has not been as fruitful. Mutations in TET1 and TET3 have been reported in patients with CLL, but the overall incidence of these mutations is currently unknown (Quesada et al., 2012). One of the few studies that carefully investigated the status of all the three TET family members in myeloid malignancies found only TET2 and not TET1 nor TET3 mutated (Abdel-Wahab et al., 2009). Recently, TET2 has been found to be also mutated in lymphoid neoplasms (Couronne et al., 2012; Quivoron et al., 2011). Finally, recent studies point to a role for TET proteins in solid tumors, as sporadic mutations have been identified in brain (Parsons et al., 2011) and prostate cancers (Grasso et al., 2012).

2.3.3 Consequence of TET2 mutations in AML The majority of our knowledge of the TET proteins comes from studies performed in nonhematopoietic cells. For instance, one study that sheds light on how TETs could be involved in active DNA methylation was performed in the brain (Guo, Su, Zhong, Ming, & Song, 2011). The proposed mechanism 12 Panagiotis Ntziachristos et al. involves activation-induced (cytidine) deaminase (AID) and APOBEC proteins, which promote the conversion of 5-hmC into an unmodified cytosine and thereby lead to active demethylation of the DNA. If this mechanism were proven to be universal, the consequence of loss of TET2 function would be increased DNA methylation. This does pose some sort of a conundrum as we have discussed earlier that loss-of-function mutations in DNMT3A are common in AML. So far, however, it is not clear what happens on the level of DNA methylation in TET2 mutant cells. There is conflicting evidence in the literature reporting that TET2 loss could lead to either increased or decreased DNA methylation (Figueroa et al., 2010; Ko et al., 2010).

2.3.4 TET2 mouse models Several research groups, including ours, have modeled the loss of TET2 in the hematopoietic system (Li et al., 2011; Moran-Crusio et al., 2011; Quivoron et al., 2011). In all cases, the loss of TET2 leads to a decrease in 5-hmC levels as expected. Deletion of TET2 in the HSC compartment causes an increase in self-renewal capacity. During the maturation of the TET2 knockout animals, an increase in the frequency of both myeloid and lymphoid cells can be observed. This premalignant state develops into a myeloproliferative neoplasm as the mice become older; this results in splenomegaly and either an MDS or a CMML-like disease.

2.4. IDH1 and IDH2 oncometabolic proteins 2.4.1 IDH1 and IDH2 mutations in leukemia The isocitrate dehydrogenase (IDH) enzymes are NADP-dependent molecules that normally function as homodimers to catalyze the oxidative decar- boxylation of isocitrate to alpha-ketoglutarate (a-KG) with the concomitant production of NADPH. Mutations in IDH1 and IDH2 are important for our discussion for two reasons. First, the mutations occur in a hotspot resulting in the alteration of the enzymatic activity of the enzyme. Second, inhibition of TET2 seems to be part of the mechanism by which mutations in IDH1 and IDH2 cause leukemia (Figueroa et al., 2010). The first indica- tion that the IDH enzymes were involved in carcinogenesis came from a study in gliomas (Yan et al., 2009). Strikingly, it was found that nearly all mutations occur in a couple of residues in either IDH1 (R132) or IDH2 (R140 or R172) (Fig. 1.1). Not much later mutations in IDH1 and IDH2 were detected in hematopoietic malignancies. Especially, myeloid malignancies are reported to have mutations in either IDH1 or Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression 13

IDH2 at a rate of around 9% (Green & Beer, 2010; Ward et al., 2010). In particular, mutations have been reported in MDS and MPN (Kosmider et al., 2010).

2.4.2 Consequence of IDH mutations As mentioned above, mutations in the IDH1 and IDH2 proteins occur in the protein catalytic site. Wild-type IDH enzymes can convert isocitrate to a-KG. IDH1 is predominantly cytoplasmic, while IDH2 can be found in the cell mitochondria. So far, only heterozygous IDH mutations have been found, leaving one allele intact. One very exciting finding was the fact that mutations in IDH proteins do not abrogate its enzymatic function but change the outcome of its reaction toward isocitrate. Mutant IDH proteins have a much higher output of 2-hydroxyglutarate (2-HG) at the expense of a-KG (Ward et al., 2010). 2-HG is an oncometabolite that can be used as a marker to distinguish wild-type IDH from mutant IDH cancers. At the molecular level, this also somehow explains how mutations in an enzyme, so critical for cellular homeostasis, can be tolerated. Mutations in IDH1 and IDH2 seem to have similar effects on the enzyme function. It is therefore not surprising that mutations in IDH1 and IDH2 are mutually exclusive. Recent studies showed a role for oncometabolites, such as 2-HG, in the function of epigenetic modulators (Teperino, Schoonjans, & Auwerx, 2010). Under normal conditions, a-KG is produced in the trichloroacetic acid (TCA) cycle from isocitrate and is a cofactor for dioxygenases. Among these dioxygenases are the Jumonji-domain-containing histone demethylases, as well as the Tet family of hydroxymethylases. As we have discussed earlier, mutations in IDH1 and IDH2 lead to the increased production of 2-HG, leading to reduced catalytic activity of certain dioxygenase enzymes. In an elegant study, this hypothesis was proven (Figueroa et al., 2010). First, it was shown that DNA isolated from IDH mutant AMLs is more hypermethylated. Sec- ond, it was shown that mutations in IDH proteins inhibit the conversion of 5-mC into 5-hmC by TET proteins. This observation is supported by the fact that TET2 and IDH mutations are mutually exclusive in AML (Figueroa et al., 2010). Other enzymes that are affected by IDH mutations are the histone demethylases, especially the H3K9me3 demethylase KDM4C (Lu & Thompson, 2012; Lu et al., 2012; Turcan et al., 2012). Along these lines, it is possible that mutations of other enzymes in the TCA cycle can cause similar effects. Examples of these are loss-of-function mutations in the enzymes succinate dehydrogenase and fumarate hydratase (Kaelin, 2011). 14 Panagiotis Ntziachristos et al.

2.4.3 Animal models for IDH gene function Very recently, the IDH1 mutation commonly found in AML (R132H) was modeled in a mouse (Figueroa et al., 2010). This mutation was created in a conditional fashion in one of the endogenous IDH1 alleles. This murine model showed that mutant IDH1 is indeed sufficient to disrupt the hematopoietic system homeostasis. IDH1 mutant mice show extra medullary hematopoiesis and a loss of cells from the bone marrow. This phenotype is associated with an increase in methylation of both DNA and histones.

3. DISRUPTION OF HISTONE-MODIFYING COMPLEXES POLYCOMB AND MLL IN LEUKEMIA Posttranslational modification of N-terminal histone tails is a more recently appreciated mechanism of regulation of gene expression patterns in development and disease. Since Jenuwein and Allis (2001) first proposed the “histone code” hypothesis in 2001, there has been an explosion in research aimed at cataloging all posttranslational modifications added to the histones and their distribution across genomes as well as association with particular transcriptional states (Ernst et al., 2011; Tan, Luo, et al., 2011). Additionally, many groups have focused on understanding how enzymes that catalyze or remove these modifications and other proteins with the ability to “read” histone marks are involved in global regulation of chromatin states (Shih et al., 2012; Zhou, Goren, & Bernstein, 2011). The importance of such enzymes in disease development is highlighted by frequent mutation of many key histone modifiers in human cancer (Abdel-Wahab et al., 2012; Dawson, Kouzarides, & Huntly, 2012; Ntziachristos et al., 2012; Patel et al., 2012; Shih et al., 2012; van Haaften et al., 2009; Zhang, Ding, et al., 2012), including both solid tumors and hematological neoplasms. Deregulation of mechanisms regulating histone modification seems to have a particularly important role in leukemic transformation as genetic lesions targeting such proteins are often considered driver mutations, with potent oncogenic activity. Here, we will focus on two histone-modifying complexes, the PRC, including both PRC2 and PRC1, and MLL complexes, which are frequently perturbed in human leukemia of several different blood lineages.

3.1. PRC2 in hematological neoplasms PRC2 is a large multimeric enzymatic complex that includes the set- domain-containing methyltransferase EZH2. Other key components include chromodomain-containing protein EED, SUZ12, and histone Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression 15

A Polycomb repressive complex 2 B EZH2

EZH2 CDKN2A/B NH SANT SANTCXC SET SUZ12 2 EED Set HOXA JARID2 RBBP4/7

Nonsense/InDels T-ALL Y641 Missense Myeloid disorders DLBCL

KDM6A KDM6B MLL-fusion COMPASS-like methyltransferase CDcomplex complex MLL protein RBBP5 AF10 TIP60 Breakpoint ASH2L ENL HOXA AT CxxC PHD TA SET MLL MEIS1 MLL NH2 Hooks WDR5 AF9 DOT1L CBX8 Set RNAP2 MLL-fusion proteins

AT Hooks CxxC AF-9

AT CxxC AF-4 H3K4me3 H3K79me2 H3K27me3 Hooks

AT Hooks CxxC ENL Figure 1.2 Genetic perturbations impacting EZH2 and MLL proteins. (A) EZH2, the catalytic subunit of PRC2, represses gene activity by methylation of H3 on lysine 27. (B) Representative distribution of EZH2 mutations reported in T-ALL, myeloid disorders (MDS, MPN, CMML, AML), and DLBCL. (C) The wild-type MLL protein is the catalytic subunit of mammalian COMPASS-like complexes which enhances gene activity through methylation of H3 on lysine 4. MLL-fusion proteins frequently associate with members of DotCom to regulate methylation of H3 on lysine 79. (D) MLL-fusion proteins typically do not involve the Set methyltransferase domain but rather the N-terminal AT hooks and CxxC domain. Frequent MLL-fusion partners include AF-9, AF-4, and ENL. chaperone RBBP4/7 (Margueron & Reinberg, 2011)(Fig. 1.2A). As its name suggests the main function of this complex is to silence gene expression at specific loci through catalysis of trimethylation of lysine 27 of histone 3 (H3K27me3). The presence of this mark not only enhances the activity of PRC2 itself but is also read by the polycomb repressive complex 1 (PRC1), leading to monoubiquitylation of histone 2A lysine 119 and subsequent chromatin compaction (Simon & Kingston, 2009). Gene silencing by PRC2 is critical for establishing proper lineage commitment during development by inactivating genes required for alternative cell fates. With a critical role in nearly every developmental system, it is not surprising that deregulation of PRC2 function contributes to tumorigenesis (Bracken & Helin, 2009; Margueron & Reinberg, 2011; Sauvageau & Sauvageau, 2010; Sawarkar & Paro, 2010). Although components of PRC2 are heavily mutated in many types of cancer, the consequences of such mutations in leukemia are especially intriguing with reports of both oncogenic and tumor-suppressor function 16 Panagiotis Ntziachristos et al. of the complex in neoplasms derived from different lineages (Ernst et al., 2010; Morin et al., 2010)(Fig. 1.2B). Originally suggested to be a loss-of-function mutation, recurrent mutations in EZH2 residue Y641 have now been shown to enhance PRC2 activity by cooperating with complexes containing the wild-type EZH2 protein leading to more efficient catalysis and hypertrimethylation of lysine 27 of histone 3 (H3K27me3) in follicular and diffuse large B-cell lymphoma (DLBCL) (McCabe et al., 2012; Sneeringer et al., 2010; Yap et al., 2011). Conversely, in T-cell acute lymphoblastic leukemia (T-ALL), loss-of-function mutations to several PRC2 subunits including EZH2, SUZ12, and EED have been reported to result in a more aggressive phenotype compared to wild-type tumors (Ntziachristos et al., 2012; Simon et al., 2012), suggesting a tumor- suppressor role for the complex in this context. Unlike DLBCL, T-ALL mutations targeting PRC2 components consist mainly of nonsense mutations upstream of the catalytic domain of EZH2 and larger deletions of the locus, suggesting a true loss-of-function outcome. Further highlighting the duality of PRC2 function in hematological tumors, it has been suggested that within different subtypes of myeloid disease both an oncogenic and a tumor-suppressor function for this complex exist. Ernst et al. (2010) have shown loss-of-function EZH2 mutations in MDS and MPN with poorer overall survival in patients with mutant alleles. However, in mouse models of MLL-AF9 positive AML, it seems that PRC2 is required for efficient transformation, suggesting a role for the complex in contributing to aberrant self-renewal of LICs (Neff et al., 2012; Shi et al., 2012). These results suggest that proper maintenance of the H3K27me3 modification is critical for normal cell homeostasis. Although the results discussed above are compelling, our understanding of the mechanism through which deregulated H3K27me3 might lead to leukemic transformation is very poorly understood. As a tumor suppressor, we might imagine an antagonistic relationship between PRC2 and oncogenic transcription factor networks. In diseases driven by transcriptional activators, we support a model where genes targeted for activation by the oncogenic factor might in turn be occupied for silencing by PRC2. Thus, loss of PRC2 function may create a more permissive environment for the activity of oncogenic transcription factors. As an oncogene, there is evidence that PRC2 can act to directly repress key tumor-suppressor genes such as the CDKN1A, CDKN1B (Velichutina et al., 2010), or CDKN2A/CDKN2B (Chen et al., 2009) loci providing a mechanism of epigenetic silencing in lieu of genetic inactivation. Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression 17

Recently, mutations of the protein ASXL1, that is part of the polycomb- repressive deubiquitylase complex, were identified in human malignancies (Abdel-Wahab et al., 2012; Shih et al., 2012). This complex catalyzes the deubiquitination of H2AK119, the modification left by PRC1, suggesting a possible antagonistic relationship. Surprisingly, inactivation of ASXL1 has been shown to have a potent effect on PRC2 function leading to global decreases in H3K27me3 although the mechanism by which this occurs is unclear. However, loss of ASXL1 and PRC2 function at the HOXA cluster was shown to correlate with increased HOXA9 expression which is known to contribute to myeloid transformation (Abdel-Wahab et al., 2012).

3.2. Role of PRC1 in leukemia Like PRC2, PRC1 has also been suggested to play a role both in maintenance of HSCs and transformation in vivo. However, unlike PRC2, there are very few reports of mutations in PRC1 complex members in cancer. Specifically, there are studies showing that PRC1 component BMI1 is required for normal HSC function and similarly for maintenance of leukemic stem cell function in MLL-rearranged leukemia (Oguro et al., 2012; Park et al., 2003). It has been proposed in both settings that BMI1 is essential for maintaining PRC1-mediated suppression of the CDKN2A/CDKN2B locus, thus allowing cells to evade cellular senescence. Nevertheless, mutations in BMI1 have not been described so far.

3.2.1 Histone methyltransferase inhibitors Chaetocin, deazaneplanocin (DZNep), and BIX-01294 are the best characterized histone methyltransferase inhibitors. All these inhibitors have so far only been tested in the preclinical environment. However, at this stage, results are promising; for example, chaetocin has anticancer properties against multiple myeloma (MM) cells (Greiner, Bonaldi, Eskeland, Roemer, & Imhof, 2005; Isham et al., 2007). Combination of the PRC2 (EZH2) inhibitor DZNep and a HDAC inhibitor (HDACi) (Panobinostat) has been shown to kill AML cells in vitro (Fiskus et al., 2009).

3.3. MLL function The MLL gene is the human homologue of Drosophila melanogaster trithorax. Trithorax was initially described as a regulator of homeotic gene expression in flies. Now, it has become clear that MLL is a key component of mammalian COMPASS-like complexes, which play critical roles in both embryonic 18 Panagiotis Ntziachristos et al. development and hematopoiesis. COMPASS complexes contain hSET1A and B, MLL1, MLL2, MLL3, or MLL4 as the catalytic subunit and have a critical role in activating transcription by catalyzing mono-, di-, and trimethylation on lysine 4 of histone 3 (H3K3me1, H3K4me2, H3K4me3). WDR5, RBBP5, and ASH2L are important core subunits that modulate the action of the methyltransferase (Dou et al., 2006). What determines the catalytic specificity of the complex regarding the component constitution is still unknown as deletion of any of the four catalytic subunits leads to minimal effects in the H3K4me3 levels possibly because of redun- dancy. However, deletion of the core subunits brings about global loss of H3K4me3 (Lubitz, Glaser, Schaft, Stewart, & Anastassiadis, 2007; Wang, Lin, et al., 2009). In this regard, loss of MLL2 in mouse embryonic stem cells (ESCs) leads to skewed differentiation, but evidence for a connection to H3K4 methylation is weak (Lubitz et al., 2007). MLL- deficient ESCs are defective in hematopoiesis (Ernst et al., 2004), but we do not know if this holds true for MLL3, MLL4, or SET1. Some studies support the role of the recently characterized DPY-30 protein as a critical regulator of MLL function (Jiang et al., 2011), although further investigation is required.

3.3.1 MLL fusions in leukemia Leukemias harboring 11q23 translocations involving MLL have characteristic clinical and biological outcomes (Bernt & Armstrong, 2011). MLL- rearranged leukemias include lymphoid, myeloid, and mixed-phenotype acute leukemias phenotypes. They are found in >70% of infants with ALL and in 35–50% of infants with AML. Children with MLL-rearranged B-ALL exhibit an overall survival of 50% versus an overall survival of >80% for children that do not harbor the translocation. MLL rearrangements with more than 60 translocation partners have been documented. These translocation partners share no single unifying feature or functional association. The resulting MLL-fusion proteins contain the amino-terminal domain of MLL and the carboxy-terminal domain of the translocation partners. As the fusion proteins no longer contain the MLL SET domain, the oncogenic action of this chimeric protein is independent of the H3K4me3 mark. The majority of the MLL-fusion partners are part of nuclear proteins (Fig. 1.2C). Members of the so-called super elongation complex (SEC) (AF1, AF9, ENL, ELL, and AF4) are frequent fusion partners. MLL can also be fused to components of the Dot1-containing complex (DotCom) (Mohan, Lin, Guest, & Shilatifard, 2010; Smith, Lin, & Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression 19

Shilatifard, 2011) such as ENL (Tkachuk, Kohler, & Cleary, 1992) and AF-9 and AF-4 (Gu et al., 1992)(Fig. 1.2D). DOT1L is the catalytic component of DotCom, which facilitates di- and trimethylation of lysine 79 on histone 3 (H3K79me2, H3K79me3) (Fig. 1.2C). This histone mark is associated with actively transcribed genes and is essential for transformation by MLL-AF9 (Bernt et al., 2011; Daigle et al., 2011). Interestingly, cross talk between the MLL-AF9 fusion protein and polycomb protein CBX8 was recently revealed in leukemia. The essential role of CBX8 in MLL-AF9-driven leukemia shows that the relationship between trithorax and polycomb group proteins is not yet fully understood (Tan, Jones, et al., 2011).

4. OTHER EPIGENETIC WRITERS, ERASERS, AND READERS Apart from enzymes that directly add or remove epigenetic marks (writers/erasers), there are proteins that can “read” these marks. These readers can recruit other proteins that can propagate the signal and subsequent repress or activate target genes or bear themselves catalytic activity. Here, we discuss genetic and posttranslational perturbation of writers, erasers, and readers and drugs that are used against these proteins in preclinical and clinical settings in leukemia studies (Fig. 1.3A).

4.1. Arginine methyltransferases The role of arginine methyltransferases and demethylases in tumorigenesis is poorly understood and is briefly discussed here. One methyltransferase, PRMT5, is of particular interest as this protein has been implicated in myeloproliferative neoplasms (Wysocka, Allis, & Coonrod, 2006; Zhang & Abdel-Wahab, 2012). It was shown that PRMT5 is aberrantly phosphorylated by mutant JAK2 (V617F, with increased activity) leading to decreased methylation of histones H2A and H4 and alterations in gene expression (Liu et al., 2011). Importantly, a specific inhibitor of the mutant JAK2 (Ruxolitinib) is used against MF. Inversely, the action of CCND1/CDK4 can lead to increased PRMT5 enzymatic activity in mouse lymphomas (Aggarwal et al., 2010). A putative role for PRMTs in cancers is further suggested by the fact that expression levels of both PRMT1 and 6 have been found to be elevated in different types of cancer (Yoshimatsu et al., 2011). 20 Panagiotis Ntziachristos et al.

A KMTi HATi DNMTi vidaza PRC2 DNMT decitabine HATs Set

CpG

Sirtuins HDACs

KDM HDACi PRMT5 JAK2 vorinostat SIRTi romidepsin (V617F) KDMi

JAKi Ruxolitinib

B Monoclonal antibodies

EGFR

Cell membrane

Lysine methylation Inhibitors of signal BCR-ABL transduction Lysine acetylation Nuclear envelope

Arginine methylation Epigenetic inhibitors Alkylating agents

Cisplatin phosphorylation DNMT

CpG CpG CpG

Figure 1.3 Major epigenetic modifiers that are genetically affected in leukemia, their associated marks and the corresponding inhibitors. (A) Major epigenetic modifiers with the corresponding inhibitors (marked with the letter i). Inhibitors that are being used for the treatment of hematopoietic malignancies are shown in red. HDAC (vorinostat and romidepsin) and DNMT inhibitors vidaza (5-azacytidine) and decitabine (5-aza-2- deoxycytidine) are currently used against MDS and CTCL correspondingly. Ruxolitinib is a JAK2 inhibitor used against myelofibrosis. HAT inhibitors, such as curcumin, have Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression 21

4.2. Lysine demethylases (KDMs) Lysine demethylases are very important for homeostasis and cancer. There are two major families of lysine demethylases. One consists of the amine oxi- dases and the second of the dioxygenases. Amine oxidation by the LSD family of flavin adenine dinucleotide-dependent demethylases (Shi et al., 2004) represents a type of active demethylation reaction. KDM1A (LSD1), the first reported histone demethylase (Shi et al., 2004), catalyzes demethylation of H3K4me1 and H3K4me2 and can also demethylate H3K9me1 and H3K9me2. KDM1A has also been shown to catalyze the demethylation of nonhistone targets, such as p53 (Huang et al., 2007) as well as DNMT1 and E2F1 (Wang, Hevi, et al., 2009; Xie et al., 2011). A second type of demethylation reaction is hydroxylation by JmjC-domain-containing proteins (Kooistra & Helin, 2012; Tsukada et al., 2006; Yamane et al., 2006). This broad family contains proteins, which catalyze demethylation of different histone and nonhistone substrates. Jumonji (Jarid2), the founding member of this family, lacks catalytic activity but plays important roles in pluripotency and development by modulating the PRC2 complex activity. JMJD3 or KDM6B, an H3K27me3 demethylase, has been reported to facilitate transcriptional initiation and elongation (Chen et al., 2012). UTX (KDM6A) and JMJD3 interact with the chromatin remodeling complex SWI/SNF (Miller, Mohn, & Weinmann, 2010), as well as MLL complexes, showing the diversity of interactions and actions of the group. The role of lysine demethylases in tumorigenesis has been exemplified by KDM1A and KDM2B (FBXL10) (Harris et al., 2012; He, Nguyen, & Zhang, 2011; Schenk et al., 2012). In addition, mutations in the lysine demethylase UTX, which can remove the H3K27me3 mark, have been found in human cancers (van Haaften et al., 2009). A recent study focusing specifically on ALL reported a low frequency of UTX mutations (Mar et al., 2012). Most of these mutations were found in clinically defined high-risk patients suggesting possible future therapeutic or prognostic relevance (Mar et al., 2012). been used in clinical trials against leukemia and other hematopoietic malignancies. Other inhibitors used in the lab include histone (lysine), methyltransferase (KMTi) and demethylase (KDMi) inhibitors, and sirtuins inhibitors (SIRTi). (B) Recently, different combinations of different epigenetic inhibitors, as well as combinations of epigenetic inhibitors with drugs inhibiting signaling transduction pathways, or chemotherapy (such as alkylating agents) are being used in clinical trials. 22 Panagiotis Ntziachristos et al.

4.3. Histone demethylases inhibitors (KDMi) The inhibition of histone demethylases as anticancer treatment has great potential. However, so far, the use of these inhibitors has been restricted to preclinical studies. For example, recently, it was shown that tranylcypromine (TCP, a LSD1 inhibitor) has activity against myeloid leukemia cell lines that are driven by the MLL-AF9 oncogene (Harris et al., 2012). So far, it has been reported that only acute promyelocytic leukemia (a subtype of AML) is sensitive to all-trans retinoic acid (ATRA) treatment. However more recently, in vitro combinatorial use of ATRA and TCP yielded promising results for other types of AML as well (Schenk et al., 2012). This further underlines the importance of combinatorial use of drugs in treatment of leukemia (Fig. 1.3B). Moreover, it is very encouraging that the advent of new technologies including high-throughput screens and advanced crystallographic techniques are paving the way to specific drugs, which target structurally similar molecules that fulfill different functions in the cell. A recent example is the generation of the first specific inhibitor for the H3K27me3 demethylases (Kruidenier et al., 2012), which can allow selective pharmacological intervention across the Jumonji family.

4.4. Histone acetyl transferases The family of histone acetyl transferases (HATs) consists of epigenetic modifiers that include CREB-binding protein (CBP), GCN5, and CLOCK. Mutations that inactivate the action of CBP were recently identified in ALL (Mullighan et al., 2011) and in B-cell lymphoma (Pasqualucci et al., 2011). The MOZ (monocytic leukemia zinc-finger protein) and MORF (MOZ-related factor) HATs are important for different developmental programs and have been implicated in leukemogenesis and other tumorigenic processes. In AML, the MOZ gene on chromosome 8p11 is fused to the CBP gene on 16p13, producing a transcript encoding the fusion protein MOZ-CBP (Borrow et al., 1996). Interestingly, the MORF gene has been identified (Champagne et al., 1999) fused to CBP in AML or MDS (Kojima et al., 2003; Yang & Ullah, 2007).

4.4.1 HAT inhibitors Three HAT inhibitors (HATi) have been described to date. Curcumin (Shehzad, Wahid, & Lee, 2010) is broad acting inhibitor that also targets p300/CBP. Garcinol (Balasubramanyam et al., 2004) and anacardic acid Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression 23

(Sun, Jiang, Chen, & Price, 2006) are both p300 and KAT2B inhibitors. These three inhibitors are currently in preclinical development.

4.5. Histone deacetylases HDACis typically play a repressive role in transcription as they remove the activating acetylation marks from gene control elements. However, HDACs have also been detected at the promoters of transcribed genes (Wang, Zang, et al., 2009). There are four classes of HDACs and three of them depend on the substrate-Zn chelation in their active site. Sirtuins (Type III HDACs) are the exception to this rule as they depend on NADþ for their action. No genetic perturbations affecting HDACs have been described in leukemia to date. However, differential expression of the HDACs has been associated with other types of cancer. In the absence of retinoic acid, RARa plays a suppressive role in transcription through the recruitment of corepressors such as NcoR, SMRT, Sin3a, and HDACs. The PML-RARa fusion protein is a stronger repressor than endogenous RARa (Uribesalgo & Di Croce, 2011), thereby warranting the use of HDACis in this scenario.

4.5.1 HDAC inhibitors HDACis can be chemically classified as short-chain fatty acids, hydroxamic acids, cyclic peptides, and benzamide derivatives (Masetti, Serravalle, Biagi, & Pession, 2011). HDAC inhibition can lead to different outcomes, such as cell cycle arrest, differentiation, or apoptosis. The most widely used class of HDACis is the hydroxamic acids, which include trichostatin A and vorinostat (SAHA). SAHA has been approved for the treatment of several hematological malignancies, including cutaneous T-cell lymphoma (CTCL). Another hydroxamic acid, Panobinostat, is currently being subjected to trials in CML, refractory CTCL, and MMs (Wolf et al., 2012). Belinostat is another investigational HDACi and has demonstrated encouraging results in peripheral T-cell lymphoma (Copeland, Buglio, & Younes, 2010). In addition, romidepsin is a cyclic peptide (FK228) approved for CTCL. Benzamide derivatives (MGCD-0103) are a separate class of investigational drugs, in clinical development for the treatment of hematological malignancies and solid tumors. Sirtuin inhibitors have not been comprehensively studied to date. Cambinol, a sirtuin inhibitor that is structurally unrelated to other HDACi, has been shown to lead to apoptosis in BCL6-expressing Burkitt’s lymphoma cells through inhibition of SIRT1 and SIRT2 (Heltweg et al., 2006). Overall, this is a very big family of inhibitors, having two members FDA approved for CTCL treatment. 24 Panagiotis Ntziachristos et al.

4.6. Bromodomain-containing proteins Another family of histone readers is the bromodomain (BRD)-containing protein family. The BRD recognizes acetylated residues and comprises a highly conserved, four-helix, left-twisted bundle with a characteristic hydrophobic cleft between two conserved loops. The BRD is present in the bromodomain and extra-terminal (BET) proteins as well as in members of the chromatin remodeling complexes (Snf2), the MLL complex and members of the SEC (Belkina & Denis, 2012). Recently, BET domain-containing proteins have been found to play a key role in the development of MM (Delmore et al., 2011) mainly through the induction of the c-Myc gene. Another example is MLL-fusion proteins containing components of the SEC, including PAFc and pTEFb that contain BET proteins (Dawson et al., 2011). These MLL-fusion proteins can activate transcription of potent oncogenes, such as BCL2, MYC, and CDK6.

4.6.1 BRD inhibitors Recently, James Bradner and his group modified a thienodiazepine molecule so that it inhibited the binding of BRD4 to the acetylated residues of histone H4 (Filippakopoulos et al., 2010). This so-called JQ1 inhibitor abruptly inhibits MYC expression and the MYC-associated transcriptional signatures in MM. In MLL-fusion leukemias (Dawson et al., 2011; Delmore et al., 2011; Filippakopoulos et al., 2010), inhibition of the BET proteins with a specific inhibitor (GSK1210151A (I-BET151)) lead to displacement of BRD3/4 and components of the SEC from chromatin improving the survival in mouse models of MLL-rearranged leukemia (Dawson et al., 2011). While BET proteins are involved in broad cellular processes, these two examples show that their inhibition may actually be feasible as a potential cancer therapy.

4.7. Plant homeodomain-containing proteins The plant homeodomain (PHD) recognizes the various methylation states of lysine 4 residue on histone 3 (H3K4). In addition, affinity of the PHD for H3K9me3 has also been documented in the case of JARID1C (Iwase et al., 2007). JARID1C is a histone demethylase for H3K4me3, which suggests cross talk between different histone marks. Translocation of PHD-containing proteins is highly prevalent in hematopoietic malignancies (Chi, Allis, & Wang, 2010). Specifically, the PHD of Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression 25

PHF23 and KDM5A that recognizes H3K4me3/2 has been found to be fused to the Nucleoporin 98 (NUP98) gene. NUP98 is a nuclear pore complex component. The NUP98 chimeric protein leads to aberrant transcriptional activation. The resulting fusion protein inhibits the removal of H3K4me3 and the repressive action of EZH2 complex (Wang, Song, et al., 2009).

4.8. Chromatin remodeling complexes Intriguingly, no mutations in chromatin remodeling complexes, such as the BRG family, have been identified to date in hematopoietic malignancies (Wilson & Roberts, 2011). This probably suggests that this family has key roles in cellular physiology and mutations, even heterozygote ones, could affect key cellular processes.

5. NOVEL ASPECTS AND TECHNOLOGIES IN EPIGENETICS: IMPLICATIONS FOR LEUKEMIA 5.1. Combinatorial epigenetic marks Recent progress suggests that the histone marks do not act alone but in highly concerted combinations. The first example came from studies on ESCs, where the so-called bivalent domains (Bernstein et al., 2006) consist of the activating mark H3K4me3 and the repressive mark H3K27me3. Genes that display these marks are poised for activation or repression and their levels of transcription are fine-tuned by the relative levels of the two marks or by other stimuli. During differentiation, these genes are either up- or downregulated leading to the subsequent removal of the respective mark. Another example of combinatorial histone marks can be found on active genes. These genes can display the simultaneous presence of both H3K4me3 and the elongating mark H3K36me3 (Guenther, Levine, Boyer, Jaenisch, & Young, 2007). There are several other paradigms of cross talk between epigenetic marks (Zhou et al., 2011). In addition, these marks can occur both on histone tails and the DNA itself. For example, H3K4me3 is typically associated with low levels of DNA methylation (Meissner et al., 2008; Weber et al., 2007). It is not surprising that cancer cells have aberrations in their combinatorial histone marks. For example, Fraga et al. (2005) have reported that loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Strikingly, this loss of histone acetylation leads to 26 Panagiotis Ntziachristos et al. a following loss of DNA methylation. This implies that physiological and cancer histone marks must be interpreted in a combinatorial mode. One of the challenges has been to define how combinations of epigenetic marks reflect different chromatin states. One method to establish this is by studying the genome-wide localization of epigenetic marks. This has been done, for example, in human cancer cell lines (Ernst et al., 2011), D. melanogaster (Filion et al., 2010), and mammals (Ram et al., 2011). A recent study generated by Bernstein and colleagues described the binding of multiple chromatin modulators and transcription factors in a myeloid cell line and in ESCs. Analysis of the results revealed six classes of chromatin modules. These chromatin modules are characterized by different combinations of chromatin regulators. This study showed that although chromatin regulators might reside at different loci in the genome of different cell types, they do act on these loci in similar fashion (Ram et al., 2011). The findings described above provide us with the tools to understand how mutations of epigenetic regulators in cancer could affect combinatorial chromatin modules. For instance, there is the fact that a lot of these factors interact with each other (such as EZH2 and DNMT (Vire et al., 2006), UTX and MLL (Issaeva et al., 2007; Lee et al., 2007)). Different models have been used to describe the functional outcome of various epigenetic states (Ernst & Kellis, 2010; Hon, Hawkins, & Ren, 2009).

5.2. Novel aspects of regulation and epigenetic factors in cancer Recently, a novel class of RNAs, termed long noncoding RNAs (lncRNAs) was discovered. Strikingly, specific lncRNAs, such as HOTAIR, have also been found to promote cancer metastasis. The effect of HOTAIR has been reported to be through interaction with the PRC2 complex (Gupta et al., 2010). Another study by the same group showed that HOTAIR could actually interact with both PRC2 and LSD1 complexes bridging by this way H3K27 methylation with H3K4me3 demethylation leading to gene repression (Tsai et al., 2010). Another lncRNA, HOTTIP, has been found to mediate activation of the distal HOXA genes through recruitment of and MLL-containing methyltransferase complex (Wang et al., 2011). Moreover, a number of studies displayed the importance of chromosomal interactions and the integrity of the nuclear architecture in cancer. For example, Roix, McQueen, Munson, Parada, and Misteli (2003) demonstrated that there is a correlation between the spatial proximity of two loci in normal cell development and the likelihood of translocation during Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression 27 carcinogenesis. Specifically, in physiological circumstances, the MYC gene resides in close proximity to the IGH and IGL loci. These are the exact translocation partners for Myc as found in leukemia (Chiarle et al., 2011; Klein et al., 2011). Lieberman-Aiden et al. (2009) capitalized on techniques such as HiC to map the landscape of inter- and intrachromosomal associations in myeloid leukemia and lymphoblastoid cell lines. This paved the way for new studies that assayed the differences in interchromosomal associations, the translocation landscape, and the transcriptome between normal and cancer cells. A comprehensive study by Zhang, McCord, et al. (2012) evaluated the genome-wide correlation between translocations and chromosomal interactions. This study provided further evidence for the fact that spatial proximity is positively correlated with the potential to generate chromosomal translocations. In addition, a recent study by Hakim et al. (2012) correlated the action of AID, an enzyme that causes breaks to DNA, to the presence of translocations. This study showed that stimuli, such as DNA damage, can also affect the frequency of translocations. The importance of nuclear architecture in the process of tumorigenicity is further underscored by the fact that lamin, a protein important for the maintenance of nuclear architecture, is also strongly associated with epigenetic regulation. Moreover, DNA methylation studies in prostate cancer showed that lamin-associated areas exhibit local hypermethylation (Berman et al., 2012). A recent study showed the extent of associations resulting from RNA polymerase activity in cancer cell lines, and the association between the respective loci and various disease states (Li et al., 2012). Taken together, it has become clear that in order to understand cancer we will have to look at the full picture. This includes mutations, epigenetic changes, transcriptional changes, and possibly larger order chromatin interactions. Nowadays, there is no reliable epigenetic marker that can be used as a prognostic or diagnostic marker for leukemia. DNA methylation, particularly of CpG islands of DNA repair enzymes, has been shown a potential to be a useful prognostic marker in some types of cancer (Van Neste et al., 2012), but there is a long way to go before this becomes an established practice. Overview of the cancer’s full properties will allow us to better estimate its potential. Ultimately, this leads to a better prognosis estimate and potentially it will allow for prediction of treatment outcome.

ACKNOWLEDGMENTS We thank the members of the Aifantis’ laboratory for critical reading of the chapter and useful comments on the work. I. A. is a Howard Hughes Medical Institute (HHMI) Early Career 28 Panagiotis Ntziachristos et al.

Scientist and is also supported by the National Institutes of Health (RO1CA133379, RO1CA105129, R21CA141399, RO1CA149655, and RO1GM088847), the Leukemia & Lymphoma Society, the V Foundation, the American Cancer Society (RSG0806801), the Irma T. Hirschl Trust, and the Dana Foundation. P. N. is supported by a fellowship from Lady Tata Foundation for Leukemia. J. M. is financially supported by the Netherlands Organisation for Scientific Research (NWO Rubicon) and by the Dutch Cancer Society (KWF Fellowship Buit 2012-5358). T. T. is supported by the NIH (training grant T32 CA009161).

REFERENCES Abdel-Wahab, O., Adli, M., Lafave, L. M., Gao, J., Hricik, T., Shih, A. H., et al. (2012). ASXL1 mutations promote myeloid transformation through loss of PRC2-mediated gene repression. Cancer Cell, 22, 180–193. Abdel-Wahab, O., Manshouri, T., Patel, J., Harris, K., Yao, J., Hedvat, C., et al. (2010). Genetic analysis of transforming events that convert chronic myeloproliferative neoplasms to leukemias. Cancer Research, 70, 447–452. Abdel-Wahab, O., Mullally, A., Hedvat, C., Garcia-Manero, G., Patel, J., Wadleigh, M., et al. (2009). Genetic characterization of TET1, TET2, and TET3 alterations in myeloid malignancies. Blood, 114, 144–147. Aggarwal, P., Vaites, L. P., Kim, J. K., Mellert, H., Gurung, B., Nakagawa, H., et al. (2010). Nuclear cyclin D1/CDK4 kinase regulates CUL4 expression and triggers neoplastic growth via activation of the PRMT5 methyltransferase. Cancer Cell, 18, 329–340. Aifantis, I., Raetz, E., & Buonamici, S. (2008). Molecular pathogenesis of T-cell leukaemia and lymphoma. Nature Reviews. Immunology, 8, 380–390. Balasubramanyam, K., Altaf, M., Varier, R. A., Swaminathan, V., Ravindran, A., Sadhale, P. P., et al. (2004). Polyisoprenylated benzophenone, garcinol, a natural histone acetyltransferase inhibitor, represses chromatin transcription and alters global gene expression. The Journal of Biological Chemistry, 279, 33716–33726. Bartram, C. R., de Klein, A., Hagemeijer, A., van Agthoven, T., Geurts van Kessel, A., Bootsma, D., et al. (1983). Translocation of c-ab1 oncogene correlates with the presence of a Philadelphia chromosome in chronic myelocytic leukaemia. Nature, 306, 277–280. Belkina, A. C., & Denis, G. V. (2012). BET domain co-regulators in obesity, inflammation and cancer. Nature Reviews. Cancer, 12, 465–477. Berman, B. P., Weisenberger, D. J., Aman, J. F., Hinoue, T., Ramjan, Z., Liu, Y., et al. (2012). Regions of focal DNA hypermethylation and long-range hypomethylation in colorectal cancer coincide with nuclear lamina-associated domains. Nature Genetics, 44, 40–46. Bernstein, B. E., Mikkelsen, T. S., Xie, X., Kamal, M., Huebert, D. J., Cuff, J., et al. (2006). A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell, 125, 315–326. Bernt, K. M., & Armstrong, S. A. (2011). Targeting epigenetic programs in MLL-rearranged leukemias. Hematology/the Education Program of the American Society of Hematology, 2011, 354–360. Bernt, K. M., Zhu, N., Sinha, A. U., Vempati, S., Faber, J., Krivtsov, A. V., et al. (2011). MLL-rearranged leukemia is dependent on aberrant H3K79 methylation by DOT1L. Cancer Cell, 20, 66–78. Bonasio, R., Tu, S., & Reinberg, D. (2010). Molecular signals of epigenetic states. Science, 330, 612–616. Booth, M. J., Branco, M. R., Ficz, G., Oxley, D., Krueger, F., Reik, W., et al. (2012). Quan- titative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science, 336, 934–937. Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression 29

Borrow, J., Stanton, V. P., Jr., Andresen, J. M., Becher, R., Behm, F. G., Chaganti, R. S., et al. (1996). The translocation t(8;16)(p11;p13) of acute myeloid leukaemia fuses a putative acetyltransferase to the CREB-binding protein. Nature Genetics, 14, 33–41. Bracken, A. P., & Helin, K. (2009). Polycomb group proteins: Navigators of lineage pathways led astray in cancer. Nature Reviews. Cancer, 9, 773–784. Byrd, J. C., Mrozek, K., Dodge, R. K., Carroll, A. J., Edwards, C. G., Arthur, D. C., et al. (2002). Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: Results from Cancer and Leukemia Group B (CALGB 8461). Blood, 100, 4325–4336. Challen, G. A., Sun, D., Jeong, M., Luo, M., Jelinek, J., Berg, J. S., et al. (2012). Dnmt3a is essential for hematopoietic stem cell differentiation. Nature Genetics, 44, 23–31. Champagne, N., Bertos, N. R., Pelletier, N., Wang, A. H., Vezmar, M., Yang, Y., et al. (1999). Identification of a human histone acetyltransferase related to monocytic leukemia zinc finger protein. The Journal of Biological Chemistry, 274, 28528–28536. Chen, H., Gu, X., Su, I. H., Bottino, R., Contreras, J. L., Tarakhovsky, A., et al. (2009). Polycomb protein Ezh2 regulates pancreatic beta-cell Ink4a/Arf expression and regen- eration in diabetes mellitus. Genes & Development, 23, 975–985. Chen, S., Ma, J., Wu, F., Xiong, L. J., Ma, H., Xu, W., et al. (2012). The histone H3 Lys 27 demethylase JMJD3 regulates gene expression by impacting transcriptional elongation. Genes & Development, 26, 1364–1375. Chessells, J. M., Veys, P., Kempski, H., Henley, P., Leiper, A., Webb, D., et al. (2003). Long-term follow-up of relapsed childhood acute lymphoblastic leukaemia. British Jour- nal of Haematology, 123, 396–405. Chi, P., Allis, C. D., & Wang, G. G. (2010). Covalent histone modifications— Miswritten, misinterpreted and mis-erased in human cancers. Nature Reviews. Cancer, 10, 457–469. Chiarle, R., Zhang, Y., Frock, R. L., Lewis, S. M., Molinie, B., Ho, Y. J., et al. (2011). Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells. Cell, 147, 107–119. Cimmino, L., Abdel-Wahab, O., Levine, R. L., & Aifantis, I. (2011). TET family proteins and their role in stem cell differentiation and transformation. Cell Stem Cell, 9, 193–204. Copeland, A., Buglio, D., & Younes, A. (2010). Histone deacetylase inhibitors in lymphoma. Current Opinion in Oncology, 22, 431–436. Couronne, L., Bastard, C., & Bernard, O. A. (2012). TET2 and DNMT3A mutations in human T-cell lymphoma. The New England Journal of Medicine, 366, 95–96. Cramer, P., & Hallek, M. (2012). Hematological cancer in 2011: New therapeutic targets and treatment strategies. Nature Reviews. Clinical Oncology, 9, 72–74. Daigle, S. R., Olhava, E. J., Therkelsen, C. A., Majer, C. R., Sneeringer, C. J., Song, J., et al. (2011). Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor. Cancer Cell, 20, 53–65. Daver, N., & Cortes, J. (2012). Molecular targeted therapy in acute myeloid leukemia. Hema- tology, 17(Suppl. 1), S59–S62. Dawson, M. A., & Kouzarides, T. (2012). Cancer epigenetics: From mechanism to therapy. Cell, 150, 12–27. Dawson, M. A., Kouzarides, T., & Huntly, B. J. (2012). Targeting epigenetic readers in cancer. The New England Journal of Medicine, 367, 647–657. Dawson, M. A., Prinjha, R. K., Dittmann, A., Giotopoulos, G., Bantscheff, M., Chan, W. I., et al. (2011). Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia. Nature, 478, 529–533. de Jonge, H. J., Huls, G., & de Bont, E. S. (2011). Gene expression profiling in acute myeloid leukaemia. The Netherlands Journal of Medicine, 69, 167–176. 30 Panagiotis Ntziachristos et al.

Deininger, M. W., Goldman, J. M., & Melo, J. V. (2000). The molecular biology of chronic myeloid leukemia. Blood, 96, 3343–3356. Delhommeau, F., Dupont, S., Della Valle, V., James, C., Trannoy, S., Masse, A., et al. (2009). Mutation in TET2 in myeloid cancers. The New England Journal of Medicine, 360, 2289–2301. Delmore, J. E., Issa, G. C., Lemieux, M. E., Rahl, P. B., Shi, J., Jacobs, H. M., et al. (2011). BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell, 146, 904–917. Dou, Y., Milne, T. A., Ruthenburg, A. J., Lee, S., Lee, J. W., Verdine, G. L., et al. (2006). Regulation of MLL1 H3K4 methyltransferase activity by its core components. Nature Structural and Molecular Biology, 13, 713–719. Druker, B. J. (2008). Translation of the Philadelphia chromosome into therapy for CML. Blood, 112, 4808–4817. Druker, B. J., Guilhot, F., O’Brien, S. G., Gathmann, I., Kantarjian, H., Gattermann, N., et al. (2006). Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. The New England Journal of Medicine, 355, 2408–2417. Eden, A., Gaudet, F., Waghmare, A., & Jaenisch, R. (2003). Chromosomal instability and tumors promoted by DNA hypomethylation. Science, 300, 455. Ernst, T., Chase, A. J., Score, J., Hidalgo-Curtis, C. E., Bryant, C., Jones, A. V., et al. (2010). Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders. Nature Genetics, 42, 722–726. Ernst, P., Fisher, J. K., Avery, W., Wade, S., Foy, D., & Korsmeyer, S. J. (2004). Definitive hematopoiesis requires the mixed-lineage leukemia gene. Developmental Cell, 6, 437–443. Ernst, J., & Kellis, M. (2010). Discovery and characterization of chromatin states for systematic annotation of the human genome. Nature Biotechnology, 28, 817–825. Ernst, J., Kheradpour, P., Mikkelsen, T. S., Shoresh, N., Ward, L. D., Epstein, C. B., et al. (2011). Mapping and analysis of chromatin state dynamics in nine human cell types. Nature, 473, 43–49. Esteller, M. (2007). Cancer epigenomics: DNA methylomes and histone-modification maps. Nature Reviews. Genetics, 8, 286–298. Esteller, M. (2008). Epigenetics in cancer. The New England Journal of Medicine, 358, 1148–1159. Feinberg, A. P. (2007). An epigenetic approach to cancer etiology. Cancer Journal, 13, 70–74. Figueroa, M. E., Abdel-Wahab, O., Lu, C., Ward, P. S., Patel, J., Shih, A., et al. (2010). Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell, 18, 553–567. Filion, G. J., van Bemmel, J. G., Braunschweig, U., Talhout, W., Kind, J., Ward, L. D., et al. (2010). Systematic protein location mapping reveals five principal chromatin types in Drosophila cells. Cell, 143, 212–224. Filippakopoulos, P., Qi, J., Picaud, S., Shen, Y., Smith, W. B., Fedorov, O., et al. (2010). Selective inhibition of BET bromodomains. Nature, 468, 1067–1073. Fiskus, W., Wang, Y., Sreekumar, A., Buckley, K. M., Shi, H., Jillella, A., et al. (2009). Combined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A and the histone deacetylase inhibitor panobinostat against human AML cells. Blood, 114, 2733–2743. Fraga, M. F., Ballestar, E., Villar-Garea, A., Boix-Chornet, M., Espada, J., Schotta, G., et al. (2005). Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nature Genetics, 37, 391–400. Gaudet, F., Hodgson, J. G., Eden, A., Jackson-Grusby, L., Dausman, J., Gray, J. W., et al. (2003). Induction of tumors in mice by genomic hypomethylation. Science, 300, 489–492. Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression 31

Gilliland, D. G. (2001). Hematologic malignancies. Current Opinion in Hematology, 8, 189–191. Gore, S. D. (2011). New ways to use DNA methyltransferase inhibitors for the treatment of myelodysplastic syndrome. Hematology/The Education Program of the American Society of Hematology, 2011, 550–555. Grasso, C. S., Wu, Y. M., Robinson, D. R., Cao, X., Dhanasekaran, S. M., Khan, A. P., et al. (2012). The mutational landscape of lethal castration-resistant prostate cancer. Nature, 487, 239–243. Green, A., & Beer, P. (2010). Somatic mutations of IDH1 and IDH2 in the leukemic transformation of myeloproliferative neoplasms. The New England Journal of Medicine, 362, 369–370. Greiner, D., Bonaldi, T., Eskeland, R., Roemer, E., & Imhof, A. (2005). Identification of a specific inhibitor of the histone methyltransferase SU(VAR)3-9. Nature Chemical Biology, 1, 143–145. Gu, Y., Nakamura, T., Alder, H., Prasad, R., Canaani, O., Cimino, G., et al. (1992). The t(4;11) chromosome translocation of human acute leukemias fuses the ALL-1 gene, related to Drosophila trithorax, to the AF-4 gene. Cell, 71, 701–708. Guenther, M. G., Levine, S. S., Boyer, L. A., Jaenisch, R., & Young, R. A. (2007). A chromatin landmark and transcription initiation at most promoters in human cells. Cell, 130, 77–88. Guo, J. U., Su, Y., Zhong, C., Ming, G. L., & Song, H. (2011). Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Cell, 145, 423–434. Gupta, R. A., Shah, N., Wang, K. C., Kim, J., Horlings, H. M., Wong, D. J., et al. (2010). Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature, 464, 1071–1076. Hakim, O., Resch, W., Yamane, A., Klein, I., Kieffer-Kwon, K. R., Jankovic, M., et al. (2012). DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes. Nature, 484, 69–74. Harris, W. J., Huang, X., Lynch, J. T., Spencer, G. J., Hitchin, J. R., Li, Y., et al. (2012). The histone demethylase KDM1A sustains the oncogenic potential of MLL-AF9 leukemia stem cells. Cancer Cell, 21, 473–487. He, J., Nguyen, A. T., & Zhang, Y. (2011). KDM2b/JHDM1b, an H3K36me2-specific demethylase, is required for initiation and maintenance of acute myeloid leukemia. Blood, 117, 3869–3880. Hellman, A., & Chess, A. (2007). Gene body-specific methylation on the active X chromosome. Science, 315, 1141–1143. Heltweg, B., Gatbonton, T., Schuler, A. D., Posakony, J., Li, H., Goehle, S., et al. (2006). Antitumor activity of a small-molecule inhibitor of human silent information regulator 2 enzymes. Cancer Research, 66, 4368–4377. Herman, J. G., & Baylin, S. B. (2003). Gene silencing in cancer in association with promoter hypermethylation. The New England Journal of Medicine, 349, 2042–2054. Ho, P. A., Kutny, M. A., Alonzo, T. A., Gerbing, R. B., Joaquin, J., Raimondi, S. C., et al. (2011). Leukemic mutations in the methylation-associated genes DNMT3A and IDH2 are rare events in pediatric AML: A report from the Children’s Oncology Group. Pediatric Blood & Cancer, 57, 204–209. Holm, T. M., Jackson-Grusby, L., Brambrink, T., Yamada, Y., Rideout, W. M., 3rd., & Jaenisch, R. (2005). Global loss of imprinting leads to widespread tumorigenesis in adult mice. Cancer Cell, 8, 275–285. Holtz, M. S., Forman, S. J., & Bhatia, R. (2005). Nonproliferating CML CD34 progen- itors are resistant to apoptosis induced by a wide range of proapoptotic stimuli.þLeukemia: Official Journal of the Leukemia Society of America, Leukemia Research Fund, UK, 19, 1034–1041. 32 Panagiotis Ntziachristos et al.

Hon, G. C., Hawkins, R. D., & Ren, B. (2009). Predictive chromatin signatures in the mammalian genome. Human Molecular Genetics, 18, R195–R201. Huang, J., Sengupta, R., Espejo, A. B., Lee, M. G., Dorsey, J. A., Richter, M., et al. (2007). p53 is regulated by the lysine demethylase LSD1. Nature, 449, 105–108. Isham, C. R., Tibodeau, J. D., Jin, W., Xu, R., Timm, M. M., & Bible, K. C. (2007). Chaetocin: A promising new antimyeloma agent with in vitro and in vivo activity mediated via imposition of oxidative stress. Blood, 109, 2579–2588. Issaeva, I., Zonis, Y., Rozovskaia, T., Orlovsky, K., Croce, C. M., Nakamura, T., et al. (2007). Knockdown of ALR (MLL2) reveals ALR target genes and leads to alterations in cell adhesion and growth. Molecular and Cellular Biology, 27, 1889–1903. Iwase, S., Lan, F., Bayliss, P., de la Torre-Ubieta, L., Huarte, M., Qi, H. H., et al. (2007). The X-linked mental retardation gene SMCX/JARID1C defines a family of histone H3 lysine 4 demethylases. Cell, 128, 1077–1088. Jenuwein, T., & Allis, C. D. (2001). Translating the histone code. Science, 293, 1074–1080. Jiang, H., Shukla, A., Wang, X., Chen, W. Y., Bernstein, B. E., & Roeder, R. G. (2011). Role for Dpy-30 in ES cell-fate specification by regulation of H3K4 methylation within bivalent domains. Cell, 144, 513–525. Jones, P. A., & Baylin, S. B. (2002). The fundamental role of epigenetic events in cancer. Nature Reviews. Genetics, 3, 415–428. Kaelin, W. G., Jr. (2011). Cancer and altered metabolism: Potential importance of hypoxia- inducible factor and 2-oxoglutarate-dependent dioxygenases. Cold Spring Harbor Sympo- sia on Quantitative Biology, 76, 335–345. Klein, I. A., Resch, W., Jankovic, M., Oliveira, T., Yamane, A., Nakahashi, H., et al. (2011). Translocation-capture sequencing reveals the extent and nature of chromosomal rearrangements in B lymphocytes. Cell, 147, 95–106. Ko, M., Huang, Y., Jankowska, A. M., Pape, U. J., Tahiliani, M., Bandukwala, H. S., et al. (2010). Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2. Nature, 468, 839–843. Kojima, K., Kaneda, K., Yoshida, C., Dansako, H., Fujii, N., Yano, T., et al. (2003). A novel fusion variant of the MORF and CBP genes detected in therapy-related myelodysplastic syndrome with t(10;16)(q22;p13). British Journal of Haematology, 120, 271–273. Kooistra, S. M., & Helin, K. (2012). Molecular mechanisms and potential functions of histone demethylases. Nature Reviews. Molecular Cell Biology, 13, 297–311. Kosmider, O., Gelsi-Boyer, V., Slama, L., Dreyfus, F., Beyne-Rauzy, O., Quesnel, B., et al. (2010). Mutations of IDH1 and IDH2 genes in early and accelerated phases of myelodysplastic syndromes and MDS/myeloproliferative neoplasms. Leukemia: Official Journal of the Leukemia Society of America, Leukemia Research Fund, UK, 24, 1094–1096. Kruidenier, L., Chung, C. W., Cheng, Z., Liddle, J., Che, K., Joberty, G., et al. (2012). A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Nature, 488, 404–408. Laird, P. W., & Jaenisch, R. (1996). The role of DNA methylation in cancer genetic and epigenetics. Annual Review of Genetics, 30, 441–464. Lee, M. G., Villa, R., Trojer, P., Norman, J., Yan, K. P., Reinberg, D., et al. (2007). Demethylation of H3K27 regulates polycomb recruitment and H2A ubiquitination. Science, 318, 447–450. Ley, T. J., Ding, L., Walter, M. J., McLellan, M. D., Lamprecht, T., Larson, D. E., et al. (2010). DNMT3A mutations in acute myeloid leukemia. The New England Journal of Medicine, 363, 2424–2433. Li, Z., Cai, X., Cai, C. L., Wang, J., Zhang, W., Petersen, B. E., et al. (2011). Deletion of Tet2 in mice leads to dysregulated hematopoietic stem cells and subsequent development of myeloid malignancies. Blood, 118, 4509–4518. Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression 33

Li, G., Ruan, X., Auerbach, R. K., Sandhu, K. S., Zheng, M., Wang, P., et al. (2012). Exten- sive promoter-centered chromatin interactions provide a topological basis for transcription regulation. Cell, 148, 84–98. Lieberman-Aiden, E., van Berkum, N. L., Williams, L., Imakaev, M., Ragoczy, T., Telling, A., et al. (2009). Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science, 326, 289–293. Liu, F., Zhao, X., Perna, F., Wang, L., Koppikar, P., Abdel-Wahab, O., et al. (2011). JAK2V617F-mediated phosphorylation of PRMT5 downregulates its methyltransferase activity and promotes myeloproliferation. Cancer Cell, 19, 283–294. Lorsbach, R. B., Moore, J., Mathew, S., Raimondi, S. C., Mukatira, S. T., & Downing, J. R. (2003). TET1, a member of a novel protein family, is fused to MLL in acute myeloid leukemia containing the t(10;11)(q22;q23). Leukemia: Official Journal of the Leukemia Soci- ety of America, Leukemia Research Fund, UK, 17, 637–641. Lu, C., & Thompson, C. B. (2012). Metabolic regulation of epigenetics. Cell Metabolism, 16, 9–17. Lu, C., Ward, P. S., Kapoor, G. S., Rohle, D., Turcan, S., Abdel-Wahab, O., et al. (2012). IDH mutation impairs histone demethylation and results in a block to cell differentiation. Nature, 483, 474–478. Lubitz, S., Glaser, S., Schaft, J., Stewart, A. F., & Anastassiadis, K. (2007). Increased apoptosis and skewed differentiation in mouse embryonic stem cells lacking the histone methyltransferase Mll2. Molecular Biology of the Cell, 18, 2356–2366. Mar, B. G., Bullinger, L., Basu, E., Schlis, K., Silverman, L. B., Dohner, K., et al. (2012). Sequencing histone-modifying enzymes identifies UTX mutations in acute lymphoblastic leukemia. Leukemia: Official Journal of the Leukemia Society of America, Leukemia Research Fund, UK, 26, 1881–1883. Marcucci, G., Metzeler, K. H., Schwind, S., Becker, H., Maharry, K., Mrozek, K., et al. (2012). Age-related prognostic impact of different types of DNMT3A mutations in adults with primary cytogenetically normal acute myeloid leukemia. Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology, 30, 742–750. Margueron, R., & Reinberg, D. (2010). Chromatin structure and the inheritance of epigenetic information. Nature Reviews. Genetics, 11, 285–296. Margueron, R., & Reinberg, D. (2011). The Polycomb complex PRC2 and its mark in life. Nature, 469, 343–349. Masetti, R., Serravalle, S., Biagi, C., & Pession, A. (2011). The role of HDACs inhibitors in childhood and adolescence acute leukemias. Journal of Biomedicine and Biotechnology, 2011, 148046. McCabe, M. T., Graves, A. P., Ganji, G., Diaz, E., Halsey, W. S., Jiang, Y., et al. (2012). Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27). In: Proceedings of the National Academy of Sciences of the United States of America, 109, 2989–2994. Meissner, A., Mikkelsen, T. S., Gu, H., Wernig, M., Hanna, J., Sivachenko, A., et al. (2008). Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature, 454, 766–770. Metzeler, K. H., Walker, A., Geyer, S., Garzon, R., Klisovic, R. B., Bloomfield, C. D., et al. (2012). DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia. Leukemia: Official Journal of the Leukemia Society of America, Leu- kemia Research Fund, UK, 26, 1106–1107. Miller, S. A., Mohn, S. E., & Weinmann, A. S. (2010). Jmjd3 and UTX play a demethylase- independent role in chromatin remodeling to regulate T-box family member-dependent gene expression. Molecular Cell, 40, 594–605. Mohan, M., Lin, C., Guest, E., & Shilatifard, A. (2010). Licensed to elongate: A molecular mechanism for MLL-based leukaemogenesis. Nature Reviews. Cancer, 10, 721–728. 34 Panagiotis Ntziachristos et al.

Moran-Crusio, K., Reavie, L., Shih, A., Abdel-Wahab, O., Ndiaye-Lobry, D., Lobry, C., et al. (2011). Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation. Cancer Cell, 20, 11–24. Morin, R. D., Johnson, N. A., Severson, T. M., Mungall, A. J., An, J., Goya, R., et al. (2010). Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin. Nature Genetics, 42, 181–185. Mullighan, C. G., Zhang, J., Kasper, L. H., Lerach, S., Payne-Turner, D., Phillips, L. A., et al. (2011). CREBBP mutations in relapsed acute lymphoblastic leukaemia. Nature, 471, 235–239. Neff, T., Sinha, A. U., Kluk, M. J., Zhu, N., Khattab, M. H., Stein, L., et al. (2012). Pol- ycomb repressive complex 2 is required for MLL-AF9 leukemia. In: Proceedings of the National Academy of Sciences of the United States of America, 109, 5028–5033. Nishigaki, M., Aoyagi, K., Danjoh, I., Fukaya, M., Yanagihara, K., Sakamoto, H., et al. (2005). Discovery of aberrant expression of R-RAS by cancer-linked DNA hypomethylation in gastric cancer using microarrays. Cancer Research, 65, 2115–2124. Ntziachristos, P., Tsirigos, A., Van Vlierberghe, P., Nedjic, J., Trimarchi, T., Flaherty, M. S., et al. (2012). Genetic inactivation of the polycomb repressive complex 2 in T cell acute lymphoblastic leukemia. Nature Medicine, 18, 298–301. Oguro, H., Yuan, J., Tanaka, S., Miyagi, S., Mochizuki-Kashio, M., Ichikawa, H., et al. (2012). Lethal myelofibrosis induced by Bmi1-deficient hematopoietic cells unveils a tumor suppressor function of the polycomb group genes. The Journal of Experimental Med- icine, 209, 445–454. Oshimo, Y., Nakayama, H., Ito, R., Kitadai, Y., Yoshida, K., Chayama, K., et al. (2003). Promoter methylation of cyclin D2 gene in gastric carcinoma. International Journal of Oncology, 23, 1663–1670. Park, I. K., Qian, D., Kiel, M., Becker, M. W., Pihalja, M., Weissman, I. L., et al. (2003). Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature, 423, 302–305. Parsons, D. W., Li, M., Zhang, X., Jones, S., Leary, R. J., Lin, J. C., et al. (2011). The genetic landscape of the childhood cancer medulloblastoma. Science, 331, 435–439. Pasqualucci, L., Dominguez-Sola, D., Chiarenza, A., Fabbri, G., Grunn, A., Trifonov, V., et al. (2011). Inactivating mutations of acetyltransferase genes in B-cell lymphoma. Nature, 471, 189–195. Patel, J. P., Gonen, M., Figueroa, M. E., Fernandez, H., Sun, Z., Racevskis, J., et al. (2012). Prognostic relevance of integrated genetic profiling in acute myeloid leukemia. The New England Journal of Medicine, 366, 1079–1089. Ptashne, M. (2007). On the use of the word ‘epigenetic’. Current Biology, 17, R233–R236. Pui, C. H., & Evans, W. E. (2006). Treatment of acute lymphoblastic leukemia. The New England Journal of Medicine, 354, 166–178. Quesada, V., Conde, L., Villamor, N., Ordonez, G. R., Jares, P., Bassaganyas, L., et al. (2012). Exome sequencing identifies recurrent mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia. Nature Genetics, 44, 47–52. Quivoron, C., Couronne, L., Della Valle, V., Lopez, C. K., Plo, I., Wagner-Ballon, O., et al. (2011). TET2 inactivation results in pleiotropic hematopoietic abnormalities in mouse and is a recurrent event during human lymphomagenesis. Cancer Cell, 20, 25–38. Ram, O., Goren, A., Amit, I., Shoresh, N., Yosef, N., Ernst, J., et al. (2011). Combinatorial patterning of chromatin regulators uncovered by genome-wide location analysis in human cells. Cell, 147, 1628–1639. Ribeiro, A. F., Pratcorona, M., Erpelinck-Verschueren, C., Rockova, V., Sanders, M., Abbas, S., et al. (2012). Mutant DNMT3A: A marker of poor prognosis in acute myeloid leukemia. Blood, 119, 5824–5831. Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression 35

Rivera, G. K., Zhou, Y., Hancock, M. L., Gajjar, A., Rubnitz, J., Ribeiro, R. C., et al. (2005). Bone marrow recurrence after initial intensive treatment for childhood acute lymphoblastic leukemia. Cancer, 103, 368–376. Roix, J. J., McQueen, P. G., Munson, P. J., Parada, L. A., & Misteli, T. (2003). Spatial proximity of translocation-prone gene loci in human lymphomas. Nature Genetics, 34, 287–291. Sauvageau, M., & Sauvageau, G. (2010). Polycomb group proteins: Multi-faceted regulators of somatic stem cells and cancer. Cell Stem Cell, 7, 299–313. Sawarkar, R., & Paro, R. (2010). Interpretation of developmental signaling at chromatin: The Polycomb perspective. Developmental Cell, 19, 651–661. Schenk, T., Chen, W. C., Gollner, S., Howell, L., Jin, L., Hebestreit, K., et al. (2012). Inhibition of the LSD1 (KDM1A) demethylase reactivates the all-trans-retinoic acid differentiation pathway in acute myeloid leukemia. Nature Medicine, 18, 605–611. Shehzad, A., Wahid, F., & Lee, Y. S. (2010). Curcumin in cancer chemoprevention: Molec- ular targets, pharmacokinetics, bioavailability, and clinical trials. Archiv der Pharmazie, 343, 489–499. Shi, Y., Lan, F., Matson, C., Mulligan, P., Whetstine, J. R., Cole, P. A., et al. (2004). Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell, 119, 941–953. Shi, J., Wang, E., Zuber, J., Rappaport, A., Taylor, M., Johns, C., et al. (2012). The Polycomb complex PRC2 supports aberrant self-renewal in a mouse model of MLL- AF9;Nras(G12D) acute myeloid leukemia. Oncogene, 32, 930–938. Shih, A. H., Abdel-Wahab, O., Patel, J. P., & Levine, R. L. (2012). The role of mutations in epigenetic regulators in myeloid malignancies. Nature Reviews. Cancer, 12, 599–612. Simon, C., Chagraoui, J., Krosl, J., Gendron, P., Wilhelm, B., Lemieux, S., et al. (2012). A key role for EZH2 and associated genes in mouse and human adult T-cell acute leukemia. Genes & Development, 26, 651–656. Simon, J. A., & Kingston, R. E. (2009). Mechanisms of polycomb gene silencing: Knowns and unknowns. Nature Reviews. Molecular Cell Biology, 10, 697–708. Smith, E., Lin, C., & Shilatifard, A. (2011). The super elongation complex (SEC) and MLL in development and disease. Genes & Development, 25, 661–672. Sneeringer, C. J., Scott, M. P., Kuntz, K. W., Knutson, S. K., Pollock, R. M., Richon, V. M., et al. (2010). Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas. In: Proceedings of the National Academy of Sciences of the United States of America, 107, 20980–20985. Straussman, R., Nejman, D., Roberts, D., Steinfeld, I., Blum, B., Benvenisty, N., et al. (2009). Developmental programming of CpG island methylation profiles in the human genome. Nature Structural and Molecular Biology, 16, 564–571. Sun, Y., Jiang, X., Chen, S., & Price, B. D. (2006). Inhibition of histone acetyltransferase activity by anacardic acid sensitizes tumor cells to ionizing radiation. FEBS Letters, 580, 4353–4356. Tadokoro, Y., Ema, H., Okano, M., Li, E., & Nakauchi, H. (2007). De novo DNA methyltransferase is essential for self-renewal, but not for differentiation, in hematopoietic stem cells. The Journal of Experimental Medicine, 204, 715–722. Tahiliani, M., Koh, K. P., Shen, Y., Pastor, W. A., Bandukwala, H., Brudno, Y., et al. (2009). Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science, 324, 930–935. Tan, J., Jones, M., Koseki, H., Nakayama, M., Muntean, A. G., Maillard, I., et al. (2011). CBX8, a polycomb group protein, is essential for MLL-AF9-induced leukemogenesis. Cancer Cell, 20, 563–575. 36 Panagiotis Ntziachristos et al.

Tan, M., Luo, H., Lee, S., Jin, F., Yang, J. S., Montellier, E., et al. (2011). Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification. Cell, 146, 1016–1028. Tefferi, A., Pardanani, A., Lim, K. H., Abdel-Wahab, O., Lasho, T. L., Patel, J., et al. (2009). TET2 mutations and their clinical correlates in polycythemia vera, essential thrombocythemia and myelofibrosis. Leukemia: Official Journal of the Leukemia Society of America, Leukemia Research Fund, UK, 23, 905–911. Teperino, R., Schoonjans, K., & Auwerx, J. (2010). Histone methyl transferases and demethylases; can they link metabolism and transcription? Cell Metabolism, 12, 321–327. Thol, F., Winschel, C., Ludeking, A., Yun, H., Friesen, I., Damm, F., et al. (2011). Rare occurrence of DNMT3A mutations in myelodysplastic syndromes. Haematologica, 96, 1870–1873. Tkachuk, D. C., Kohler, S., & Cleary, M. L. (1992). Involvement of a homolog of Drosoph- ila trithorax by 11q23 chromosomal translocations in acute leukemias. Cell, 71, 691–700. Tsai, M. C., Manor, O., Wan, Y., Mosammaparast, N., Wang, J. K., Lan, F., et al. (2010). Long noncoding RNA as modular scaffold of histone modification complexes. Science, 329, 689–693. Tsukada, Y., Fang, J., Erdjument-Bromage, H., Warren, M. E., Borchers, C. H., Tempst, P., et al. (2006). Histone demethylation by a family of JmjC domain-containing proteins. Nature, 439, 811–816. Turcan, S., Rohle, D., Goenka, A., Walsh, L. A., Fang, F., Yilmaz, E., et al. (2012). IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Nature, 483, 479–483. Uribesalgo, I., & Di Croce, L. (2011). Dynamics of epigenetic modifications in leukemia. Briefings in Functional Genomics, 10, 18–29. van Haaften, G., Dalgliesh, G. L., Davies, H., Chen, L., Bignell, G., Greenman, C., et al. (2009). Somatic mutations of the histone H3K27 demethylase gene UTX in human cancer. Nature Genetics, 41, 521–523. Van Neste, L., Herman, J. G., Otto, G., Bigley, J. W., Epstein, J. I., & Van Criekinge, W. (2012). The epigenetic promise for prostate cancer diagnosis. The Prostate, 72, 1248–1261. Velichutina, I., Shaknovich, R., Geng, H., Johnson, N. A., Gascoyne, R. D., Melnick, A. M., et al. (2010). EZH2-mediated epigenetic silencing in germinal center B cells contributes to proliferation and lymphomagenesis. Blood, 116, 5247–5255. Viguie, F., Aboura, A., Bouscary, D., Ramond, S., Delmer, A., Tachdjian, G., et al. (2005). Common 4q24 deletion in four cases of hematopoietic malignancy: Early stem cell involvement? Leukemia: Official Journal of the Leukemia Society of America, Leukemia Research Fund, UK, 19, 1411–1415. Vire, E., Brenner, C., Deplus, R., Blanchon, L., Fraga, M., Didelot, C., et al. (2006). The Polycomb group protein EZH2 directly controls DNA methylation. Nature, 439, 871–874. Walter, M. J., Ding, L., Shen, D., Shao, J., Grillot, M., McLellan, M., et al. (2011). Recurrent DNMT3A mutations in patients with myelodysplastic syndromes. Leukemia: Official Jour- nal of the Leukemia Society of America, Leukemia Research Fund, UK, 25, 1153–1158. Wang, J., Hevi, S., Kurash, J. K., Lei, H., Gay, F., Bajko, J., et al. (2009). The lysine demethylase LSD1 (KDM1) is required for maintenance of global DNA methylation. Nature Genetics, 41, 125–129. Wang, P., Lin, C., Smith, E. R., Guo, H., Sanderson, B. W., Wu, M., et al. (2009). Global analysis of H3K4 methylation defines MLL family member targets and points to a role for MLL1-mediated H3K4 methylation in the regulation of transcriptional initiation by RNA polymerase II. Molecular and Cellular Biology, 29, 6074–6085. Mechanisms of Epigenetic Regulation of Leukemia Onset and Progression 37

Wang, G. G., Song, J., Wang, Z., Dormann, H. L., Casadio, F., Li, H., et al. (2009). Haematopoietic malignancies caused by dysregulation of a chromatin-binding PHD finger. Nature, 459, 847–851. Wang, K. C., Yang, Y. W., Liu, B., Sanyal, A., Corces-Zimmerman, R., Chen, Y., et al. (2011). A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature, 472, 120–124. Wang, Z., Zang, C., Cui, K., Schones, D. E., Barski, A., Peng, W., et al. (2009). Genome- wide mapping of HATs and HDACs reveals distinct functions in active and inactive genes. Cell, 138, 1019–1031. Ward, P. S., Patel, J., Wise, D. R., Abdel-Wahab, O., Bennett, B. D., Coller, H. A., et al. (2010). The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer Cell, 17, 225–234. Weber, M., Hellmann, I., Stadler, M. B., Ramos, L., Paabo, S., Rebhan, M., et al. (2007). Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome. Nature Genetics, 39, 457–466. Wilson, B. G., & Roberts, C. W. (2011). SWI/SNF nucleosome remodellers and cancer. Nature Reviews. Cancer, 11, 481–492. Wolf, J. L., Siegel, D., Goldschmidt, H., Hazell, K., Bourquelot, P. M., Bengoudifa, B. R., et al. (2012). Phase II trial of the pan-deacetylase inhibitor panobinostat as a single agent in advanced relapsed/refractory multiple myeloma. Leukemia & Lymphoma, 53, 1820–1823. Wysocka, J., Allis, C. D., & Coonrod, S. (2006). Histone arginine methylation and its dynamic regulation. Frontiers in Bioscience, 11, 344–355. Xie, Q., Bai, Y., Wu, J., Sun, Y., Wang, Y., Zhang, Y., et al. (2011). Methylation-mediated regulation of E2F1 in DNA damage-induced cell death. Journal of Receptor and Signal Transduction Research, 31, 139–146. Yamane, K., Toumazou, C., Tsukada, Y., Erdjument-Bromage, H., Tempst, P., Wong, J., et al. (2006). JHDM2A, a JmjC-containing H3K9 demethylase, facilitates transcription activation by androgen receptor. Cell, 125, 483–495. Yan, H., Parsons, D. W., Jin, G., McLendon, R., Rasheed, B. A., Yuan, W., et al. (2009). IDH1 and IDH2 mutations in gliomas. The New England Journal of Medicine, 360, 765–773. Yan, X. J., Xu, J., Gu, Z. H., Pan, C. M., Lu, G., Shen, Y., et al. (2011). Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute monocytic leukemia. Nature Genetics, 43, 309–315. Yang, X. J., & Ullah, M. (2007). MOZ and MORF, two large MYSTic HATs in normal and cancer stem cells. Oncogene, 26, 5408–5419. Yap, D. B., Chu, J., Berg, T., Schapira, M., Cheng, S. W., Moradian, A., et al. (2011). Somatic mutations at EZH2 Y641 act dominantly through a mechanism of selectively altered PRC2 catalytic activity, to increase H3K27 trimethylation. Blood, 117, 2451–2459. Yoshimatsu, M., Toyokawa, G., Hayami, S., Unoki, M., Tsunoda, T., Field, H. I., et al. (2011). Dysregulation of PRMT1 and PRMT6, Type I arginine methyltransferases, is involved in various types of human cancers. International Journal of Cancer, 128, 562–573. Yu, M., Hon, G. C., Szulwach, K. E., Song, C. X., Zhang, L., Kim, A., et al. (2012). Base- resolution analysis of 5-hydroxymethylcytosine in the Mammalian genome. Cell, 149, 1368–1380. Zhang, S. J., & Abdel-Wahab, O. (2012). Disordered epigenetic regulation in the pathophysiology of myeloproliferative neoplasms. Current Hematologic Malignancy Reports, 7, 34–42. 38 Panagiotis Ntziachristos et al.

Zhang, J., Ding, L., Holmfeldt, L., Wu, G., Heatley, S. L., Payne-Turner, D., et al. (2012). The genetic basis of early T-cell precursor acute lymphoblastic leukaemia. Nature, 481, 157–163. Zhang, Y., McCord, R. P., Ho, Y. J., Lajoie, B. R., Hildebrand, D. G., Simon, A. C., et al. (2012). Spatial organization of the mouse genome and its role in recurrent chromosomal translocations. Cell, 148, 908–921. Zhou, V. W., Goren, A., & Bernstein, B. E. (2011). Charting histone modifications and the functional organization of mammalian genomes. Nature Reviews. Genetics, 12, 7–18. Zilberman, D., Gehring, M., Tran, R. K., Ballinger, T., & Henikoff, S. (2007). Genome- wide analysis of Arabidopsis thaliana DNA methylation uncovers an interdependence between methylation and transcription. Nature Genetics, 39, 61–69. CHAPTER TWO

Translocations in Normal B Cells and Cancers: Insights from New Technical Approaches

Roberto Chiarle*,†,1 *Department of Pathology, Children’s Hospital Boston and Harvard Medical School, Boston, Massachusetts, USA †Department of Molecular Biotechnology and Health Sciences, University of Torino, Italy 1Corresponding author: e-mail address: [email protected]

Contents 1. Mechanistic Elements that Generate Chromosomal Translocations 40 1.1 DNA DSB formation 40 1.2 DNA-repair mechanisms involved in translocations 45 1.3 Chromosome territories and gene proximity in translocations 47 1.4 Spatial organization of the genome: Implications for translocations 48 2. Novel High-Throughput Methods to Study Chromosomal Translocations 50 2.1 High-throughput genomic translocation sequencing 51 2.2 Translocation-capture sequencing 52 3. New Findings on Translocation Formation Obtained by HTGTS and TC-Seq 52 3.1 RAG1/2 translocation hotspots in pro-B lymphocytes 53 3.2 AID hotspots in activated B lymphocytes 53 3.3 Gene density, transcription, and translocations 54 3.4 Role of nuclear positioning and chromosomal structure in translocations 55 4. Landscape of Translocations in Cancers 57 4.1 Distribution of chromosomal translocations in cancers 57 4.2 Chromothripsis in cancer genomes 58 4.3 Repetitive patterns and heterogeneity of translocations involving oncogenes 62 5. Perspectives 63 Acknowledgments 64 References 64

Abstract Chromosomal translocations are recurrent genetic events that define many types of cancers. Since their first description several decades ago as defining elements in cancer cells, our understanding of the mechanisms that determine their formation as well as their implications for cancer progression and therapy has remarkably progressed. Chro- mosomal translocations originate from double-strand breaks (DSBs) that are brought into proximity in the nuclear space and joined inappropriately by DNA-repair pathways.

The frequency and pattern of translocations are influenced by perturbations of any of these events. DSB formation is heavily determined by physiologic processes, such as the activity of RAG1/2 and AID enzymes during B-cell development or maturation, or by pathologic factors, such as ionizing radiations, ROS, or fragile sites. Cellular processes of mRNA transcription, DNA replication, and repair can influence the chromosomal territories and modify the relative position and proximity of genes inside the nucleus. DNA- repair factors contribute not only to the maintenance of genome integrity but also to translocations in normal and cancer cells. Next-generation sequencing techniques provide an unprecedented and powerful tool to approach the field of chromosomal translocations. Using specific examples, we will explain how genome-wide translocation mapping methods, such as high-throughput genomic translocation sequencing (HTGTS) and translocation-capture sequencing, combined with large-scale methods to determine nuclear proximity of genes or chromosome domains, such as 4C and Hi-C, have changed our view of the factors and the rules governing translocation formation in noncancer cells. Finally, we will review chromosomal rearrangements and newly described findings, such as chromothripsis, in cancer cells based on these novel rules on translocation formation.

1. MECHANISTIC ELEMENTS THAT GENERATE CHROMOSOMAL TRANSLOCATIONS Chromosomal translocations require a series of consecutive events for their formation (Fig. 2.1). The first event is the generation of at least a pair of DNA double-strand breaks (DSBs). Then, two DSBs need to find themselves within close enough proximity for the DNA-repair machinery to join them. When appropriate, the ligation of two DSBs restores DNA integrity, whereas “illegitimate” joining results in a chromosomal rearrangement. Various types of structural chromosomal rearrangements can be generated, including inversions, deletions, and intrachromosomal or interchromosomal translocations. Here, we will quickly review these types of events.

1.1. DNA DSB formation The generation of DSBs in a cell can result from physiologic or pathologic mechanisms. Physiologic mechanisms may be defined as those in which programmed DSBs are introduced within a restricted window during the development and maturation of B and T cells by specific enzymes, including recombination-activating genes (RAG) 1 and 2 and activation-induced cytidine deaminase (AID). In contrast, pathologic DSBs are generated by external agents, intracellular biochemical agents, or failure of the DNA replication machinery. Translocation in Normal and Cancer Cells 41

Pathologic DNA double strand breaks (DSBs) – ROS Physiologic DNA double – Ionizing radiations strand breaks (DSBs) – Stalled replication forks – Common fragile sites and ERFSs – RAG1/2 induced V(D)J recombination – Oncogene-induced replication breaks – AID induced CSR and SHM – Topoisomerases – DNA damage in micronuclei – “Off-target” activity of RAG1/2 – “Off-target” activity of AID

DSB

Legitimate DNA repair Illegitimate DNA repair C-NHEJ (all cell cycle) C-NHEJ HR (S/G2 phases) A-EJ FoSTeS (collapsed replication fork) FoSTeS MMBIR (collapsed replication fork) MMBIR

DNA integrity Translocations–Duplications–Chromothripsis Figure 2.1 Mechanisms of chromosomal translocation formation. Chromosomal translocations are initiated by double-strand breaks (DSBs) formation. DSBs can be programmed by physiologic events, or generated by pathologic processes. Once formed, two DSBs are in large majority correctly repaired to restore chromosome integrity. Less frequently, an erroneous joining of DSBs originates chromosomal translocations.

1.1.1 Physiologic breaks induced by RAG1/2 and AID enzymatic activity 1.1.1.1 RAG-initiated DSBs and translocations The process of V(D)J recombination during B- and T-cell development is initiated by the activity of the RAG1 and RAG2 proteins (reviewed in Jung, Giallourakis, Mostoslavsky, & Alt, 2006). The IgH variable (VH), diversity (D), and joining ( JH) gene segments are assembled into the IgH variable region exons in a process that starts with RAG1 and RAG2 introducing DSBs at their borders. RAG1 and RAG2 form a complex that is absolutely required for V(D)J recombination. The RAG complex recognizes recombination signal sequences (RSSs) that flank V, D, and J segments and contain nonamers and heptamers flanked separately by 12- or 23-bp spacers (according to the so-called 12/23 rule). The RAG complex generates DSBs from a pair of RSS ends in the form of blunt 50-phosphorylated DSBs and 42 Roberto Chiarle hairpin-sealed coding ends (Honjo, Alt, & Neuberger, 2004). The 12/23 rule and additional “beyond 12/23” restrictions (Gostissa, Alt, & Chiarle, 2011) target RAG complex activity to defined segments in the IgH and TCR loci and limit its “off-target” activity in other sites in the genome. The mechanisms by which RAG activity is targeted to the correct loci have been partially elucidated. The binding of RAG1 and RAG2 proteins to target V(D)J sequences in the Ig and T-cell receptor (TCR) genes requires the precise orchestrationofprotein–DNAcomplexestonarrowthepossibilityofoff-target effects (Schatz & Swanson, 2011). RAG proteins bind DNA in a very focal pattern, in particular, within small regions of chromatin in the Igk and TCRa J-gene segments as well as the IgH and TCRb J-gene and J-proximal D-gene segments. These small regions, called recombination centers, involve multiple proteins in addition to RAG, such as the high-mobility-group proteins HMGB1 or HMGB2 (Swanson, 2004), and depend on canonical RSSs for access to RAG binding. RSSs’ accessibility depends on chromatin conformation that, in turn, is controlled by enhancers and active promoters in the region. These modifications depend on enzymes that modify and remodel chromatin structure and promote transcription by allowing for Pol II binding, thus revealing an essential role for chromatin in RAG activity and specificity (Krangel, 2007). In this context, RAG1 binds directly to RSSs via domains that directly interact with the nonamers and heptamers of the RSSs (Swanson, 2004). In contrast, RAG2 has limited capability for DNA binding and instead binds to histone H3 trimethylated at lysine 4 (H3K4me3), a marker of active and poised promoters (Liu, Subrahmanyam, Chakraborty, Sen, & Desiderio, 2007; Matthews et al., 2007). Accordingly, in vivo RAG1 binding sites were found preferentially in regions containing RSSs, whereas bound RAG2 was found within thousands of H3K4me3-enriched sites across the genome (Ji et al., 2010). The mechanisms that regulate RAG DNA binding and cleavage are exquisitely important for restricting the generation of RAG-mediated DSBs to their proper sites. Failure of this regulation, or off-target activity, results in the generation of DSBs that could be improperly repaired and could lead to the formation of chromosomal translocations. Indeed, off-target RAG activity is responsible for low-frequency DSBs that are observed throughout the genome, and the misrepair of DSBs at the Ig and TCR loci causes genomic instability and translocations in lymphoid cells (Lieber, Yu, & Raghavan, 2006; Mills, Ferguson, & Alt, 2003). Aberrant RAG activity has been implicated in the development of human malignancies (Tsai et al., 2008), whereas RAG2 integrity is essential to maintain genomic stability and prevent Translocation in Normal and Cancer Cells 43 complex chromosomal translocations, amplifications, and deletions in the TCR and IgH loci (Deriano et al., 2011). Translocations found in B- or T-cell acute lymphoblastic leukemia (B-ALL or T-ALL, respectively), in mouse models of these diseases (Gladdy et al., 2003; Zha et al., 2010; Zhu et al., 2002) and in humans (Ku¨ppers, 2005), are thought to be initiated by RAG activity. Other examples of RAG-mediated translocations are the recurrent translocations observed in B-cell lymphomas, such as (1) the t(8;14) translocations that involve IgH and c-myc in endemic Burkitt’s lymphoma (BL); (2) the t(11;14) translocations found in mantle cell lymphomas (MCL) involving IgH and the bcl-1 loci; (3) the t(14;18) translocation in follicular lymphoma (FL) that translocates IgH and bcl-2; and (4) the t(1;14) translocation in mucosa-associated lymphoid tissue (MALT) lymphomas that involves IgH and bcl-10 (Ku¨ppers & Dalla-Favera, 2001).

1.1.1.2 AID-initiated DSBs and translocations AID is a B-cell-specific enzyme required for class switch recombination (CSR) as well as somatic hypermutation (SHM). It is mostly expressed by IgM-positive naı¨ve B cells upon antigen stimulation, typically in the germinal center (GC) and to a lesser extent in the extrafollicular areas of secondary lymphoid organs, such as the spleen and lymph nodes. AID-mediated CSR generates DSBs in the IgH CH locus that are frequently involved in translocations, whereas SHM very rarely leads to DSB formation and translocation (Pasqualucci et al., 2001). AID is a single-strand (ss)-specific DNA cytidine deaminase that targets repetitive GC-rich sequences within CSR switch regions and catalyzes dC-to-dU deamination. The presence of GC-rich sequences induces the formation of stable RNA:DNA complexes, resulting in displacement of the nontemplate strand as ssDNA (R-loops). AID targets R-loops in the nontemplate strand, whereas its access to the template strand depends on the RNA exosome, a cellular RNA-processing/ degradation complex (Basu et al., 2011). Transcription of mRNA is necessary for AID activity, as demonstrated by studies showing that AID is directed to DNA sites where RNA polymerase II (Pol II) activity is stalled (as a result of its association with Spt5; Pavri et al., 2010). This relationship between AID, mRNA transcription, and Pol II stalling has likely implications for the mechanisms of AID-initiated translocations. AID expression, nuclear localization, and activity are finely regulated in B cells to limit its genotoxic effects. Indeed, AID expression is transient in B cells, owing to tight transcriptional control of the promoter as well as by 44 Roberto Chiarle miR-155 (Teng et al., 2008), as a mutation in the miR-155 binding site in the AID 30-UTR has been shown to increase cellular AID levels and, consequently, the frequency of IgH-myc translocations (Dorsett et al., 2008). The protein levels of AID are also tightly regulated. After induction, AID is quickly degraded (Basu, Franklin, & Alt, 2009), with the majority of AID being retained in the cytoplasm and only a small amount entering the nucleus, and its enzymatic activity being controlled by protein kinase-A-mediated phosphorylation (Basu et al., 2005). AID off-target activity outside the IgH locus has been implicated in chromosomal translocations not only in B cells but also in nonlymphoid cells. Indeed, signs of AID off-target activity, in the form of SHM, have been found in up to 25% of the expressed genes analyzed in GC B cells (Liu et al., 2008), and AID is required for DSBs generated in the c-myc locus (Robbiani et al., 2008; Wang et al., 2009). In agreement with these findings, ectopic expression of AID in mice induces DSBs and tumor formation in B- and non-B cells (Okazaki, Kotani, & Honjo, 2007; Robbiani et al., 2009). In human tumors, AID is thought to initiate DSBs when translocations involve the IgH switch regions. Typical examples of such translocations are: (1) IgH and c-myc in sporadic BL, t(8;14); (2) IgH and Bcl-3 in chronic lymphocytic leukemia (CLL), t(14;19); (3) IgH and Bcl-6 in diffuse, large B-cell lymphoma (DLBCL), t(3;14); (4) IgH and Pax5 in lymphoplasmacytic (LP) lymphoma, t(9;14); and (5) the t(4;14), t(14;16), and t(6;14) translocations that recur in multiple myeloma (MM) (Ku¨ppers & Dalla-Favera, 2001; Mitelman, Johansson, & Mertens, 2007). In some translocations, such as the t(14;18) found in FL and the t(11;14) in MCL, it is thought that RAG and AID activity might collaborate in the generation of DSBs at the Bcl-2 and Bcl-1 loci (Tsai et al., 2008). In non-B cells, AID expression has been found in gastric (Matsumoto et al., 2007), liver, and colorectal tumors (Marusawa, 2008), and a role has also been suggested in germ cell tumors (Okazaki et al., 2007), breast cancer (Pauklin, Serna´ndez, Bachmann, Ramiro, & Petersen-Mahrt, 2009), and prostate cancer (Lin et al., 2009).

1.1.2 Pathologic induction of DSBs in normal and tumor cells Nonprogrammed pathologic DSBs can originate in G1-arrested or cycling cells by a variety of mechanisms resulting from (1) exposure to physical agents, such as ionizing radiations; or (2) the malfunctioning of cellular biochemical processes, such as the production of reactive oxygen species (ROS) or breaks in fragile sites during impaired replication. Ionizing (g ) radiation À Translocation in Normal and Cancer Cells 45 can directly induce DSBs in DNA in a dose-dependent manner (Lieber, 2010; Tsai & Lieber, 2010). Oxidative stress generates ROS that can react with DNA and induce DSB formation by triggering the induction of two neighboring SSBs (Kryston, Georgiev, Pissis, & Georgakilas, 2011). Fragile sites are regions of DNA that can generate DSBs when DNA synthesis is partially inhibited (Durkin & Glover, 2007). Some fragile sites are common to all cells. In cancer cells, they are implicated in DNA damage associated with replication stress and were shown to be involved in constitutional and cancer rearrangements in vivo (Arlt, Durkin, Ragland, & Glover, 2006). As examples, the FRA6E and FRA6F fragile sites are associated with break points in ALL and acute myelogenous leukemia (AML) (Sinclair, Harrison, Jarosova´, & Foroni, 2005), and some BL show c-myc translocations close to the FRA8C and FRA8D fragile sites (Sinclair et al., 2005). Interest- ingly, oncogene-induced replication stress can induce the collapse of stalling replication forks and DSB formation at fragile sites (Halazonetis, Gorgoulis, & Bartek, 2008). Early replication fragile sites (ERFSs) have been recently described to form during cell cycle progression and DNA replication. ERFSs colocalize with highly expressed gene clusters in B lymphocytes subjected to replication stress (Barlow et al., 2013). DSBs in ERFSs are relevant, since greater than 50% of recurrent amplifications/deletions in human diffuse large B cell lymphoma map to ERFSs (Barlow et al., 2013). Finally, topoisomerases can induce DSBs. For example, in prostate cancer, topoisomerase IIb has been shown to induce DSB formation that results in the hallmark TMPRSS2-ERG translocation (Haffner et al., 2010). Nota- bly, this DSB activity has been observed during topoisomerase inhibition by anticancer drugs (Felix et al., 2006). 1.2. DNA-repair mechanisms involved in translocations In response to DSB generation, cells activate an intrinsic DNA-damage- response (DDR) pathway to resolve DSBs and facilitate DNA repair. Notable molecular players within the DDR include ATM, the RAD50/MRE11/ NBS1 complex, H2AX, and 53BP1 (for a detailed review, see Lieber, 2010; Alt et al., 2013; Gostissa et al., 2011). The two major DDR pathways typically operate depending on the sequence homology of the DSB and division state of the cell. In dividing diploid cells, sequence homology is used by the homologous recombination (HR) pathway or by the single-strand annealing and breakage-induced replication pathways (San Filippo, Sung, & Klein, 2008). In nondividing cells, or when homology is not present, cells utilize a form of direct joining called nonhomologous DNA end joining (NHEJ). 46 Roberto Chiarle

Classical NHEJ (C-NHEJ) relies on a series of factors that participate in a multistep DNA-repair process. First, Ku proteins (Ku70/86) bind to the DSB to generate a Ku–DNA complex. The Ku–DNA complex serves as a docking point for other DNA-repair complexes, such as the nuclease complex formed by Artemis and DNA-PKs, the DNA polymerases Pol l and m, and the ligase complex composed of XLF, XRCC4, and DNA Ligase IV (for a detailed description of these factors, see Lieber, 2010 and Gostissa et al., 2011). The recruitment and activity of these higher-order complexes result in the direct joining of DNA or minimal (2–3 base) microhomology (MH) of the ends. C-NHEJ is considered to be a highly efficient method to rapidly repair DSBs and restore chromosomal and genomic integrity. In particular, it is used to repair physiologic DSBs introduced by RAG1/2 during V(D)J recombination (see above) and most DSBs introduced by AID during CSR (Gostissa et al., 2011). Also, it shows a predisposition toward repairing DSBs on the same chromosome (Ferguson et al., 2000). When one essential core factor (e.g., Ku or Ligase IV) of the C-NHEJ pathway is missing, such as in knock-out mouse models or in human patients with specific genetic defects, cells can still join nonhomologous DNA ends by one or more mechanisms known collectively as the alternative end-joining (A-EJ) pathway. Many factors have been implicated in A-EJ, among them Nbs1, Mre11, CtlP, DNA Lig3, Parp1, and XRCC1, but their roles are still under investigation (Alt et al., 2013; Gostissa et al., 2011; Lieber, 2010). The signature function of A-EJ is to repair DSBs via short sequences of MH present at the ends of DSBs. However, MH is not absolutely required since A-EJ can also generate a substantial fraction of direct joins (Boboila et al., 2010b). A-EJ is considered to be a slower (Han & Yu, 2008)andmoretranslocation-prone pathway than C-NHEJ (Boboila et al., 2010a). The fact that translocations are also observed in normal cells indicates either that the C-NHEJ can sometimes mediate inappropriate DNA-repair responses that result in chromosomal translocation or that A-EJ might work in parallel with the C-NHEJ to repair some DSBs, possibly when the C-NHEJ is overwhelmed. In this context, I-SceI was shown to mediate translocation in WT mouse cells in which C-NHEJ and A-EJ were both intact (Gostissa et al., 2011; Klein et al., 2011; Weinstock, Elliott, & Jasin, 2006), showing a bias toward MH usage, possibly indicating a coexistence of C-NHEJ and A-EJ functions. A similar bias toward MH usage is observed in translocation junctions from cancer cells (Zhang & Rowley, 2006). The role of C-NHEJ in suppressing translocations and maintaining chromosomal integrity is exemplified by knock-out mouse models of C-NHEJ factors. Translocation in Normal and Cancer Cells 47

Bcellsdeficientforvirtuallyanysingle C-NHEJ factor, such as Ku70, Ku86, XRCC4, Lig4, DNA-PKcs, Artemis, or XLF, show increases in DSB formation and translocation (Gostissa et al., 2011). When the p53-dependent G1/S check- / point was missing, as in a p53À À background,micedeficientinKu86,XRCC4, Lig IV or DNA-Pks, or Artemis expression as well as mice deficient in ATM developed pro-B-cell lymphomas that often carried IgH/c-myc translocations with c-myc amplifications. In Artemis-deficient mice, IgH/N-myc translocations were also observed. Medulloblastomas occurred in XLF-deficient mice and, together with lymphomas, were also found in other C-NHEJ-deficient mice (Gostissa et al., 2011). Overall, C-NHEJ maintains genome integrity and suppresses translocation formation, either by promptly repairing DSBs or by suppressing the translocation-prone repair activity of A-EJ.

1.3. Chromosome territories and gene proximity in translocations For a chromosomal translocation to be formed, two DSBs must be in close proximity to allow the DNA-repair pathways to join them. It is not clear, however, whether the two loci involved in a translocation should be close to each other before the DSBs are generated or whether DSBs have some mobility inside the nucleus. In classical cytogenetic studies using fluorescence in situ hybridization (FISH), the nuclear distance between c-myc and IgH, Igk, and Igl directly correlated with the respective frequency of translocations found in BL. Fur- thermore, in the interphase nucleus, the IgH locus was found to be proximal to some of its translocation partners found in lymphomas, such as the CCND1, Bcl-2, and Bcl-6 genes (Roix, McQueen, Munson, Parada, & Misteli, 2003). In a mouse model of lymphoma, the IgH locus was found to frequently colocalize with its translocation partners c-myc and Igl (Wang et al., 2009). This colocalization was tissue specific and limited to a relatively small portion of the chromosome, as it was lost with the chromosomal segment located at 15 Mb distance from Igl (Wang et al., 2009). Chromosomal proximity is similarly thought to influence translocation frequency in leukemias, including the ABL and BCR genes in chronic myelogenous leukemia (CML) and the promyelocytic leukemia (PML) and RARA genes in PML (Neves, Ramos, da Silva, Parreira, & Parreira, 1999), and in solid tumors such as prostate cancers, where androgen stimulation was shown to increase proximity in the frequently translocated TMPRSS2 and ERG genomic loci (Lin et al., 2009; Mani et al., 2009). However, the definition of proximity in such studies was probabilistic and quite arbitrary, as 48 Roberto Chiarle it was found only in a fraction of the cells examined and was established based on the limited resolution of confocal microscopes. A more precise determination of the physical contact between loci has been established by modern techniques, such as 4C and Hi-C (see Section 3.4).

1.4. Spatial organization of the genome: Implications for translocations The nucleus is a highly dynamic structure where chromosomes as well as many fundamental cellular processes, including transcription, replication, and DNA repair, are organized and carried out within defined compartments (Fig. 2.2). Insidetheinterphasenucleus,chromosomes,smallergenomicregions,andeven single genes are not randomly distributed but rather interact within highly ordered 3D structures known as chromosome territories. Chromosome territories can be directly visualized by in situ hybridization approaches, which highlight the localization of individual chromosomes in distinct patterns (Bolzer etal.,2005).Thepatternsofchromosometerritorieschangewithdifferentiation and development and are cell-type specific (Cremer et al., 2006; Misteli, 2007). The functions of chromosome territories are still unclear. Human lymphocytes show a strong correlation between the position of chromosomes and their gene density, with gene-rich chromosomes clustered toward the nuclear interior (Boyle et al., 2001). Gene-rich regions correlate with

Chromosome territories Transcription factories Replication factories DNA repair centers

Active A domain Inactive B domain mRNA

mRNA DNA RNA polymerases polymerases PCNA C-NHEJ A-EJ

mRNA mRNA

Short chromosomes Long chromosomes

Figure 2.2 Spatial organization of the genome. The nucleus is a highly dynamic structure. Chromosomes are organized in territories. Short chromosomes interact more frequently with each other than with long chromosomes. Within each chromosome, active A domains interact more frequently with other A domains, whereas inactive B domains are more frequently associated with B domains. Transcription factories are nuclear compartments (calculated in about 10,000 in HeLa cells) where transcription factors are recruited, assembled, and disassembled within few seconds to achieve efficient transcription of clustered genes. Replication factories and DNA repair centers are dis- crete compartments of the nucleus where DNA synthesis and DNA repair are achieved through the recruitment of specific factors (see text for details). Translocation in Normal and Cancer Cells 49 decondensed chromatin status, whereas gene-poor regions are associated with condensed chromatin (Gilbert et al., 2004). Chromosomal territories can also be determined by the size of single chromosomes, with smaller chromosomes generally located toward the center of the nucleus (Bolzer et al., 2005). Indeed, very recent data collected with Hi-C mapping in G1-arrested pro-B cells showed that the longest chromosomes more frequently interact with each other than with smaller chromosomes (Zhang et al., 2012). The transcription of mRNA by Pol II polymerases predominantly occurs in centralized structures called “transcription factories” (Cook, 1999). The concentration of transcription factors into these factories allows for efficient transcription, and cotranscribed genes are recruited together by modifications of chromosome structure and changes in chromatin conformation (Misteli, 2007). In this process of transcription-driven recruitment, single genes can change their positioning with respect to the nucleus. For example, in lymphocytes, activated IgH and CD4 relocalize from the periphery toward the center of the nucleus (Kosak et al., 2002; Kim et al., 2004). Similar principles of nuclear compartmentalization and dynamics also apply to replication and DNA repair. During the S phase, within the so- called replication factories, multiple factors involved in the replication machinery are rapidly assembled and disassembled within minutes to allow for efficient DNA synthesis. DNA repair occurs in “repair centers” where distinct foci of accumulating factors are recruited to ensure efficient repair of DSBs (Bekker-Jensen et al., 2006). Taken together, this evidence shows that the nucleus is a highly dynamic structure in which entire chromosomes, gene clusters, or even single genes can rapidly change their position inside the geometry of the nucleus or with respect to other chromosome or gene loci. Therefore, rather than assuming that the proximity between translocation-prone genes is fixed, their probability of contact appears to be somewhat fluid in a variable fraction of cells and influenced by factors such as cell-cycle phase (predominantly G1 or G1/S/G2 phases). Modern techniques for genome-wide contact analysis, such as 4C and Hi-C, have improved our understanding of chromosomal organization within the nucleus. These techniques capture the physical contact made between gene segments via the formalin-mediated cross-linking of histones that are in physical contact in the nucleus (Lieberman-Aiden et al., 2009; Simonis et al., 2006; Zhao et al., 2006). With 4C, it was found that actively transcribed loci, such as the b-globin locus, preferentially contacted other 50 Roberto Chiarle actively transcribed loci and that active and inactive genes are involved in long-range intra- and interchromosomal contacts (Simonis et al., 2006). Recent studies relied on Hi-C approaches to build spatial proximity maps of the human genome at 1 Mb resolution. These maps revealed that, in both cycling cells and G1-arrested cells, chromatin conformation is consistent with a fractal globule in which intrachromosomal interactions are much more frequent than interchromosomal interactions, and in each chromosome, the probability of contact was directly proportional to genomic distance (Lieberman-Aiden et al., 2009; Zhang et al., 2012). These physical data are entirely consistent with the concept of chromosome territories. Fur- thermore, genomic loci in A domains, characterized by their open chromatin conformation and tendency to correlate with gene-rich areas, showed higher probability of contact with other loci in A domains than with loci in the closed and transcriptionally inactive B domains, and vice versa (Lieberman-Aiden et al., 2009). These concepts—that is, the clustering of genes during transcription, transcription factories, the higher probability of contact with regions of the same chromosome, and the segregation of open and closed chromatin to form two genome-wide compartments— have profound implications for the interpretation of mechanistic factors that regulate translocation (Fig. 2.2). These implications will be analyzed below.

2. NOVEL HIGH-THROUGHPUT METHODS TO STUDY CHROMOSOMAL TRANSLOCATIONS To better study chromosomal translocation formation, the development of powerful detection methods in relatively simple assays in vivo is required. In the past decade, most translocation assays were performed by a direct PCR approach, a rather simple technique in which a series of primers were designed to detect translocations between two known genes, such as c-myc and the IgH locus (Ramiro et al., 2006, 2004; Wang et al., 2009), or multiple pairs of genes (Jankovic et al., 2010). Although direct PCR provides reliable quantitative measurements of translocation frequency between two loci, only defined translocations generated within a few kilobases from where primers are located can be detected, thus limiting its use- fulness when translocation partners are unknown or larger regions of the genome are involved. The introduction of next-generation sequencing techniques has allowed for the development of broader, genome-wide methods to assay for chromosomal translocation formation. Translocation in Normal and Cancer Cells 51

2.1. High-throughput genomic translocation sequencing HTGTS was recently developed to clone translocation junctions from a baited DSB end (Chiarle et al., 2011). This bait was provided by DSBs generated via the homing endonuclease I-SceI that cuts a canonical recognition sequence of 18 bp (Liang, Romanienko, Weaver, Jeggo, & Jasin, 1996) targeted into the IgH or c-myc locus (Chiarle et al., 2011; Klein et al., 2011). Therefore, HTGTS isolates junctions between a chromosomal DSB introduced at a fixed site and other genic or intergenic regions in the whole genome. In this study, primary splenic B cells were isolated and activated in vitro for up to 4 days in conditions that allow for AID expression and CSR induction (Fig. 2.3). This relatively short time of activation

AID-induced DSBs and translocations

Mature B cells IgM+

DNA isolation AID induction Translocations Linker-mediated PCR 4 days stimulation IL-4 + CD40 or LPS I-Scel-mediated DSBs

Proliferation CSR to IgG1+ B cells Next-generation Translocation sequencing maps

A-MuLV Pro-B cells G1 arrest

RAG1/2 induction Translocations DNA isolation STI-571 Linker-mediated PCR

I-Scel-mediated DSBs

RAG1/2-induced DSBs and translocations

Figure 2.3 Strategies to generate translocations maps from normal mature B cells and pro-B cells. Mature B cells are freshly isolated from spleens and activated to induce AID expression and class switch recombination. I-SceI-mediated DSBs are generated in the IgH and c-myc loci either by retrovirus-mediated I-SceI expression or by hormone- mediated induction of I-SceI–glucocorticoid receptor (GR) fusion. After 4 days, cells are collected and translocations junctions are cloned as described in the text. For pro-B cells, A-MuLV transformants are generated by Bcr-Abl transduction. In the presence of Bcl2 overexpression, the inhibition of Bcr-Abl tyrosine kinase activity by STI-571 induces G1 cell-cycle arrest and RAG1/2 induction. DSBs are generated by I-SceI–GR fusion activation and translocations junctions are cloned as for mature B cells. 52 Roberto Chiarle minimized cellular selection and avoided biases related to the biological consequences of translocations on cell growth and survival. To clone translocation junctions, genomic DNA was digested into relatively small fragments by restriction enzymes and ligated to an adapter or, alternatively, to generate circular fragments. Up to three rounds of nested PCRs were performed with adapter- and locus-specific primers, and pooled PCR products were then sequenced by 454 sequencing and aligned to a reference genome after several steps of filtering to eliminate nonspecific products and artifacts. The method proved to be highly specific, with artificial nonspecific junctions being lower than 1% of detected sequences. By this method, almost 150,000 independent junctions from independent mice and libraries were generated. All sequences contained the translocation junction, thus allowing for MH studies (Chiarle et al., 2011).

2.2. Translocation-capture sequencing Translocation-capture sequencing (TC-Seq) was developed in parallel with HTGTS based on similar principles (Klein et al., 2011; Oliveira et al., 2012). Fixed DSBs were generated in activated B cells in the c-myc locus targeted with the I-SceI recognition sequence. Translocations to I-SceI-c-myc were cloned with an adapter-based PCR approach. Genomic DNA was fragmen- ted by sonication, blunted, ligated to double-stranded asymmetric linkers, and cut with I-SceI to eliminate native nontranslocated loci. Next, two rounds of seminested PCR were performed and the fragments assembled into a paired-end Illumina library. By this method, over 160,000 translocations were obtained from WT and AID-deficient B cells (Klein et al., 2011).

3. NEW FINDINGS ON TRANSLOCATION FORMATION OBTAINED BY HTGTS AND TC-Seq HTGTS and TC-Seq have greatly expanded our understanding and interpretation of translocation mechanisms. For example, using these methods, it was discovered that DSBs generated from fixed loci show a much wider genomic distribution than previously expected. Chromosomal translocations were mostly found within the same chromosome where the original DSB was located, in particular, within a relatively narrow (about 1 Mb) region around the DSB site. This intrachromosomal preference for DSB joining could be explained by the documented preference for C-NHEJ to repair DSBs within the same chromosome (Ferguson et al., 2000; Mahowald et al., 2009; Zarrin et al., 2007) and/or by recent data showing Translocation in Normal and Cancer Cells 53 that regions within the same chromosome are much more likely to be in physical contact than regions between two different chromosomes (Lieberman-Aiden et al., 2009; Zhang et al., 2012; see Section 3.4). By HTGTS, the analysis of SNPs in translocation junctions showed that translocations are 6–10 times more likely to occur within the same allele where the DSBs are located (Zhang et al., 2012).

3.1. RAG1/2 translocation hotspots in pro-B lymphocytes New translocation-mapping techniques, such as HTGTS, provide a viable tool to analyze RAG-mediated target and off-target DSB formation. In pro-B cell lines transformed with Abelson murine leukemia virus (A-MuLV), RAG expression is induced by cell-cycle arrest mediated by the inhibition of v-abl activity by the kinase STI571 (Bredemeyer et al., / 2006). In ATMÀ À pro-B cell lines, translocations were enriched in the expected RAG target sites (IgH, Igk, Igl) in TCR loci (TCRa/d, TCRg), which are considered to be inaccessible due to a closed chromatin conformation in pro-B cells. Thus, similar to AID in peripheral B cells, RAG is the major source of DSBs in pro-B cells, where it largely dictates the landscape of translocations (Zhang et al., 2012).

3.2. AID hotspots in activated B lymphocytes HTGTS and TC-Seq translocation maps found a strong enrichment of expected AID targets. A prediction of AID targets for DSBs initiation was obtained by deep sequencing of genomic AID-binding sites. With this approach, AID was found to bind preferentially promoter-proximal regions where stalled polymerases and chromatin-activating marks were enriched (Yamane et al., 2011). In translocation maps, when B cells were stimulated with CD40 or LPS and IL-4, AID generated DSBs in the Sm,Sg1, Sg3, and Se regions, as expected. Chromosomal translocations to S regions were up to 20-fold higher than any other recurrent translocation in B cells, revealing the IgH S region as a major translocation hotspot in B cells and highlighting AID as the most important DSB-generating enzyme in a normal B cell (Chiarle et al., 2011; Klein et al., 2011). A series of known and unknown AID targets were also found to be involved in translocations or deletions. For example, (1) Pim1, Il21r, and Gas5 all translocate with Bcl6 in DLBCL (Nakamura et al., 2008; Ueda et al., 2002; Yoshida et al., 1999); (2) Pax5 and Ddx6 are translocated with IgH in DLBCL and in LP lymphoma (Iida, Rao, Ueda, Chaganti, & Dalla-Favera, 1999; Lu & Yunis, 1992; Yoshida et al., 1999); 54 Roberto Chiarle

(3) c-myc and Pvt1 are repeatedly translocated in human BL and mouse plasmacytoma (Cory, Graham, Webb, Corcoran, & Adams, 1985; Einerson et al., 2006; Ku¨ppers, 2005); (4) Aff3 and Grhpr translocate with Bcl2 and Bcl6, respectively, in FL (Akasaka, Lossos, & Levy, 2003; Impera et al., 2008); (5) Ccnd2 and Bcl2l11 are translocated or deleted (respectively) in MCL (Bea et al., 2009; Gesk et al., 2006); and (6) Birc3 is translocated with Malt1 in MALT lymphoma (Murga Penas et al., 2006) and with mir142 in B-cell prolymphocytic leukemia (Gauwerky, Huebner, Isobe, Nowell, & Croce, 1989).

3.3. Gene density, transcription, and translocations By HTGTS, about 10–20% of translocations were found to be interchromosomal and were widely distributed across all chromosomes (Chiarle et al., 2011; Klein et al., 2011). Strikingly, translocations strongly correlated with gene density and gene activity in each chromosome, clustering at a higher frequency in regions enriched in transcribed genes, and much less so in regions devoid of active genes (Chiarle et al., 2011). Furthermore, translocations were enriched in genes with Pol II and marks of active histones, such as H3K4 trimethylation, H3 acetylation, and H3K36 trimethylation (Klein et al., 2011). In actively transcribed genes, translocations accumulated in the promoter region, within a few kilobases of the transcription starting site, whereas in inactive genes, translocations were evenly dispersed throughout the gene. The clustering of translocations in the promoter region of WT B cells was found not only in AID target genes but also in genes not targeted by AID and in AID-deficient cells (Chiarle et al., 2011). The observed correlation between gene transcription and translocation is very intriguing. Transcription generates genetic instability by multiple mechanisms that are still poorly understood. Transcribed genes likely offer regions of genomic fragility, owing to the single-strand DNA conformation of transcribed gene segments, the collision of transcription machinery with replication forks, and the formation of DNA–RNA hybrids (R-loops) (Aguilera, 2002; Ruiz, Gomez-Gonzalez, & Aguilera, 2011). In B cells, CSR (see Section 1.1.1.2) is a recurrent source of DSBs and is heavily dependent on transcription. Transcription of the switch (S) regions determines the formation of stable DNA structures, in which the C-rich template strand forms R-loop intermediates, whereas the G-rich nontemplate strand generates secondary structures. AID deaminates DNA on the C-rich template strand of actively transcribing S regions, to allow for eventual DSB Translocation in Normal and Cancer Cells 55 generation (Chaudhuri et al., 2003; Nambu et al., 2003). Binding of AID to transcribed regions is mediated by the transcription elongation complex (Besmer, Market, & Papavasiliou, 2006) and is enhanced by transcriptional pausing and stalling of Pol II (Canugovi, Samaranayake, & Bhagwat, 2009). The protein Spt5 is thought to mediate AID binding to stalled transcription sites (Pavri et al., 2010). Therefore, AID might be recruited not only to transcriptionally stalled sites in the immunoglobulin (Ig) loci to induce CSR and SHM but also to non-Ig loci, thus favoring DSBs and translocations in genes such as c-myc, Bcl6, Pim1, and Pax5 that are repeatedly translocated in human lymphomas (Pavri & Nussenzweig, 2011). Indeed, TC-Seq studies showed strong overlapbetweentranslocationsand AID-andSpt5-binding sites (Klein et al., 2011; Yamane et al., 2011). In the absence of AID, translocations were likely to result from DSB formation during physiological processes related to transcription and DNA replication (Branzei & Foiani, 2010). In this context, ERFSscould contribute to explain more than 50%of chromosomal aberrations in human diffuse large B cell lymphomas (Barlow et al., 2013).

3.4. Role of nuclear positioning and chromosomal structure in translocations Physical proximity has always been considered a key determinant in chromosomal translocations in cancer. Early DNA–FISH studies in the interphase nuclei of mouse and human B cells showed that loci frequently involved in lymphoma translocations, such as IgH and c-Myc, or IgH and Bcl2, were often located in close proximity to each other (Parada, McQueen, & Misteli, 2004; Roix et al., 2003; see Section 1.3). Similarly, translocation partners in other hematologic malignancies, such as BCR–ABL1 in chronic myeloid leukemia, RET–CDC6 in thyroid malignancies, TMPRSS2–ERG/ ETV1 in prostate cancer, and PML–RARA in acute PML, were frequently proximal in the cells considered to be tumor precursors (Mitelman et al., 2007). Proximity between loci seems to be dictated, at least in some instances (such as the IgH and c-myc loci), by the recruitment of a common transcription factory (see Section 1.4; Osborne et al., 2007). Pro-B cells undergo V(D)J rearrangements due to the expression of RAG enzymes that recognize specific sequences in the genome (see Section 1.1.1). Application of HTGTS translocation-mapping approaches to pro-B cells revealed high genomic stability in WT cells with / a limited number of cloned translocations. In contrast, in ATMÀ À cells, 56 Roberto Chiarle

RAG-mediated breaks dominated the landscape of translocation (see Section 3.1). Thus, DSB frequency in RAG targets strictly determines the translocation pattern. In contrast, when DSBs were no longer a limiting factor, as in cells treated with ionizing radiation, most of the translocations (between 25% and 40%) were found in the same chromosome where the bait DSBs were located, supporting a strong influence of chromosomal territories on translocation formation (Zhang et al., 2012). Furthermore, translocations on the same chromosome were mostly in cis on the same allele. Strikingly, by combining HTGTS with Hi-C studies, it was found that translocations on the same chromosome, as well as translocations in trans with other chromosomes, strongly correlated with regions with a higher contact probability (Zhang et al., 2012). 4C-seq maps generated from genes that are actively transcribed in mature B cells, such as IgH and c-myc, showed that these genes shared similar genome-wide interactions, despite being on different chromosomes (chr. 12 for IgH and Chr. 15 for c-myc). The highest frequency of contacts was found in cis within the same chromosome 12 and 15, respectively, consistent with the data obtained in pro-B cells (see above; Zhang et al., 2012) and with the concept that chromosomes are organized in defined territories (Cremer & Cremer, 2010; Lieberman-Aiden et al., 2009). In contrast, trans interactions were likely to be driven by transcriptional activity and chromatin conformation, as IgH and c-myc interacted preferentially with loci associated with activating histone acetylation marks, Pol II binding, and active transcription (Hakim et al., 2012). These findings are consistent with the notion that chromosomes are organized into areas of compact and open chromatin, with open chromatin regions having a distinct nuclear organization and being enriched in genes (Gilbert et al., 2004). Genes within areas of active or inactive chromatin have a higher probability of contact than do genes between these areas (Lieberman-Aiden et al., 2009; Simonis et al., 2006). In mature B cells, in the absence of AID, DSBs are likely to be generated by common mechanisms associated with transcription and replication. In this setting, translocation pattern was shown to strictly correlate with the interaction frequencies between loci. In contrast, in the presence of AID, translocations correlated not with contact frequency but with DSB / frequency, as determined by RPA binding in 53BP1À À cells (Hakim et al., 2012). Overall, these new genome-wide correlations between translocations, DSBs, and nuclear proximity indicate that in the presence of a dominant source of DSBs in B cells (i.e., RAG1/2 in pro-B cells and AID in mature Translocation in Normal and Cancer Cells 57

B cells), the translocation landscape is mainly dictated by the frequency of DSBs in any given locus. In contrast, in the absence of such DSBs (such / as in AIDÀ À B cells), the relative nuclear position mainly regulates translocation frequency. Future studies are needed to address these correlations in non-B cells, where a dominant mechanism for DSB formation is likely absent.

4. LANDSCAPE OF TRANSLOCATIONS IN CANCERS The landscape of chromosomal translocation in human cancers varies from tumors containing minimal structural variations to tumors with highly complex genomic rearrangements (CGRs). The recurrent presence of such structural abnormalities in cancers can be interpreted as “driver” events that are selected and enriched during tumor progression, or “passenger” events that originate during the life cycle of a tumor without particular selective forces that fix them in the cancer genome. Typical driver chromosomal translocations are those that define different categories of hematologic malignancies or specific subtypes of solid tumors, such as the BCR-ABL translocation in CML, various translocations in AML, most translocations in human lymphomas (see below), and recurrent translocations in solid cancers, such as EWSR1 fusions in Ewing sarcoma (Toomey, Schiffman, & Lessnick, 2010), ETS fusions in prostate cancer (Rubin, Maher, & Chinnaiyan, 2011), and anaplastic lymphoma kinase (ALK) fusions in lymphoma, lung carcinoma, and other cancers (Chiarle, Voena, Ambrogio, Piva, & Inghirami, 2008). In such tumors, the structural landscape of the tumor genome is dominated by the driver translocation events, with minimal additional structural variations in other chromosomes (Mitelman et al., 2007). Driver translocations were first discovered more than three decades ago with classical cytogenetic techniques because they are highly recurrent in cancers and, therefore, more easily identified. In contrast, our understanding of the more elusive nonrecur- rent passenger chromosomal rearrangements has only recently become clearer, owing to the development of genome-wide analysis tools such as next-generation DNA sequencing, RNA sequencing, SNP-array analyses and, of course, adequate bioinformatics methods.

4.1. Distribution of chromosomal translocations in cancers Many human cancers show complex genomic structural rearrangements. These rearrangements can be divided into different types and can be intrachromosomal or interchromosomal. Intrachromosomal rearrangements are 58 Roberto Chiarle typically observed as deletions, tandem duplications, inversions, or other complex noninverted intrachromosomal rearrangements. Interchromosomal rearrangements are translocations between different chromosomes that account for less than 10% of all the structural variations in cancers with complex genomic structural rearrangements (Pleasance et al., 2010). Next- generation DNA sequencing allows for the improved characterization of such complex cancer genomes. Strikingly, in many cancer types, highly CGRs are confined to one or few chromosomes, where tens or hundreds of chromosomal rearrangements are clustered. Such events have been termed “chromothripsis” (Stephens et al., 2011).

4.2. Chromothripsis in cancer genomes The first evidence of chromothripsis came from whole-genome sequencing of CLL patients. In this series, it was found that chromosomal rearrangements generally clustered within one entire chromosome and more frequently in smaller regions, such as an entire chromosomal arm or even in segments just a few tens of megabases or kilobases in length (Stephens et al., 2011). In these regions of chromothripsis, chromosomal rearrangements were both inverted and noninverted in orientation. Strikingly, there was an equal representation of the four major possible patterns of intrachromosomal rearrangements, that is, deletions, head-to-head and tail-to-tail inversions, and tandem duplications (Stephens et al., 2011). Chromothripsis has been found in a range (3–25%) of other cancer types, such as neuroblastoma (Molenaar et al., 2012); medulloblastoma (Northcott et al., 2012; Rausch et al., 2012); bone cancers (Stephens et al., 2011); MM (Magrangeas, Avet-Loiseau, Munshi, & Minvielle, 2011); and lung, renal, and thyroid cancers (Forment, Kaidi, & Jackson, 2012). Chromothripsis can also be characterized in terms of its limited copy-number state, where the rearranged regions vary between one or two copies, with segments exhibiting a loss of heterozygosity alternating with segments with retained heterozygosity (Kloosterman et al., 2011; Stephens et al., 2011). These extensive chromosomal rearrangements that occur during chromothripsis are thought to originate from a single catastrophic event rather than as a consequence of progressive sequential rearrangements. In the progressive rearrangement model, complex, localized clustering of rearrangements originates during many cell cycles, generating increasing complexity in genomic structure. This is the mechanism typically thought to cause genomic amplifications. As an example, amplifications of the c-myc Translocation in Normal and Cancer Cells 59 locus are thought to derive from the so-called breakage–fusion–bridge cycle, where regional weakness of the DNA structure may lead to repeated cycles of DNA breakage and repair (Gostissa et al., 2011; Zhang et al., 2010). In contrast, during chromothripsis, the entire chromosome or chromosomal regions are shattered into several fragments in a narrow window of time and then joined. The minimal or absent sequence homology between the translocated pieces favors mechanisms of repair based on C-NHEJ or other end-joining pathways (Forment et al., 2012). The fact that tens to hundreds of pieces of chromosomal DNA can be rejoined within the same chromosome suggests that chromothripsis could occur when chromosomes are in a condensed state, such as during mitosis. Alternatively, it might be the result of the organization of chromosomes into territories where DNA repair, mostly C-NHEJ, favors joining within the same chromosome (Gostissa et al., 2011; Zhang et al., 2010). Indeed, mouse models of translocations indicate that, in the presence of a catastrophic event, such as ionizing radiations, rejoining of DSBs and translocations are preferentially clustered in cis within the same chromosomal allele (Zhang et al., 2012). Alternatively, other repair mechanisms could be involved, such as replication fork stalling and template switching (Branzei & Foiani, 2010) or microhomology-mediated break-induced replication (MMBIR) (Liu et al., 2011). The question on how such catastrophic events are generated is still open (Fig. 2.4). The fact that DNA shattering involves only a limited number of chromosomes, or limited segments within chromosomes, suggests that the DNA-damaging event should occur when chromosomes are at a condensed stage, such as mitosis. DSBs could be caused by an environmental stimulus, such as exposure to free radicals or ionizing radiation (Tsai & Lieber, 2010), or by DNA replication stress with premature termination of replication forks and DSB formation at potentially fragile sites (Halazonetis et al., 2008). Recently, it was shown that aberrant and persistent DNA replication within micronuclei can generate DNA damage and chromosome pulverization (Crasta et al., 2012). Additional catastrophic DNA-damaging events could occur during telomere attrition (Sahin & Depinho, 2010) or apoptotic events that would kill a normal but not a cancerous cell (Tubio & Estivill, 2011). Interestingly, germ line and somatic TP53 mutations were associated with chromothripsis both in medulloblastoma and AML (Rausch et al., 2012). This finding indicates that defects in checkpoint pathways designed to repair DNA damage predisposes to chromothripsis, likely by facilitating cell-survival mechanisms that operate during catastrophic 60 Roberto Chiarle

Mitosis Chromosome shattering

Micronucleus pulverization

Radiation MMBIR

Predisposition to cancer due to oncogenes and oncosuppressors’ alterations NHEJ DNA repair MMBIR Chromosome territory P53 deficiency Postmitotic cell with chromothripsis

Mitosis exit

Figure 2.4 Proposed mechanisms of chromothripsis in normal and cancer cells. During mitosis, chromosomes are condensed and focal DNA damage can be induced by radiation, microhomology-mediated break-induced replication (MMBIR), or chromosome lagging, micronuclei formation, and pulverization due to inappropriate chromosome segregation. After chromosome shattering, DNA fragments are joined by NHEJ within chromosomal territories. Repaired chromosomes contain chromosomal rearrangements such as deletions, head-to-head and tail-to-tail inversions, and tandem duplications. With mitosis exit, the chromothriptic chromosome can be reincorporated into the nucleus. Deficiency of DNA-damage checkpoints, such as P53 deficiency, facilitates the survival of cells during the chromothriptic process. As a result, chromothripsis can induce oncogene activations by translocations or duplications as well as loss of oncosuppressor genes by deletions or locus disruptions, thus facilitating cancer progression. events, such as the favoring of low-fidelity repair or bypassing of G2/M cell- cycle checkpoints (Maher & Wilson, 2012). Chromothripsis could have important implications in the evolution of cancers. First, because regions of heterozygosity are conserved inside clusters of rearrangements, chromothripsis is thought to occur early in cancer cell development (Forment et al., 2012; Stephens et al., 2011), suggesting that such rearrangements might themselves influence the progression of cancer cells. Second, a particular cancer cell undergoing chromothripsis could derive some selective advantages. Chromosomal segments generated during massive chromosomal fragmentation might not be reincorporated into the derivative chromosome but instead might form double-minute chromosomes. In this Translocation in Normal and Cancer Cells 61 context, one case of bone cancer chromothripsis was shown to generate one double-minute chromosome approximately 1.1 Mb in length that contained multiple amplified copies of c-myc (Stephens et al., 2011), likely giving a selective advantage to the cell. Alternatively, the catastrophic event could simultaneously disrupt multiple tumor-suppressor genes, as in the case of a chordoma sample in which the cyclin-dependent kinase inhibitor 2A (CDKN2A), the F-box and WD-40 domain containing protein 7 (FBXW7), and the Werner syndrome ATP-dependent helicase (WRN) genes were disrupted as a consequence of one single chromothriptic event (Forment et al., 2012). Interestingly, the pattern of intrachromosomal rearrangements resulting from chromothripsis in cancer cells has been faithfully reproduced in normal B cells in mouse models of induced translocations. In these models, a dominant source of DSBs is generated by the targeting of I-SceI substrate sequences in the IgH locus or in the c-myc locus. Those I-SceI-induced DSB loci are embedded in regions with a high density of spontaneous DSBs, generated in B cells by either AID in the IgH switch regions flanking the I-SceI site, or by AID-dependent and -independent mechanisms in the closely localized c-myc and Pvt1 genes. Indeed, the Pvt1 gene is frequently translocated not only in BL but also in nonlymphoid cancers such as lung cancer and medulloblastoma (Northcott et al., 2012; Pleasance et al., 2010). Therefore, these models show that a high density of synchronous DSBs generated in normal cells, within a chromosomal region of few kilobases, might simulate a chromothriptic event. Remarkably, normal cells react to these localized chromothriptic events by generating a pattern of chromosomal rearrangements highly similar to cancer cells, with deletions, head-to-head and tail-to-tail inversions represented in approximately equal frequency (Chiarle et al., 2011; Klein et al., 2011). Similarly, in human medulloblastoma, an approximately 200-kb region flanking the Pvt1 locus was found to be frequently involved in chromothriptic events, with frequent oncogenic rearrangements occurring with the c-myc locus upstream or the NDRG1 locus downstream of Pvt1 (Northcott et al., 2012). Additionally, events similar to chromothripsis can be observed in some genomic disorders where CGRs are identified (Zhang, Carvalho, & Lupski, 2009). These genomic disorders are thought to originate from germ line rearrangements during gametogenesis or early postzygotic development. In these conditions, multiple copy-number changes can be found, including deletions, duplications, and extensive translocations and inversions. To explain these events, a MMBIR model has been proposed for complex 62 Roberto Chiarle rearrangements, where copy gains or losses involve the generation and repair of DSBs in regions with domains of MH (Hastings, Lupski, Rosenberg, & Ira, 2009). It is speculated that MMBIR and subsequent replication- mediated repair by C-NHEJ could explain both CGRs in genomic disorders as well as chromothripsis in cancer cells (Kloosterman et al., 2012; Liu et al., 2011). Thus, the structural similarities between chromosomal rearrangements observed in normal B cells, CGR genomic disorders, and chromothripsis strongly argue in favor of similar mechanisms for DSB repair operating in normal and cancer cells.

4.3. Repetitive patterns and heterogeneity of translocations involving oncogenes In human patients, some translocations are recurrently found in specific subtypes of tumors. The IgH-c-myc translocations are typically found in BL and DLBCL, IgH-Bcl-2 translocations in B-cell lymphomas (mainly FL, CLL, and DLBCL), IgH-Bcl-1 translocations in MCL and MM, and Bcl-6 translocations in DLBCL. It is thought that these translocations are generated from the joining of one physiologic DSB in the IgH locus with one pathologic DSB in the oncogene as consequence of off-target activity of B-cell- specific genes such as RAG and AID (see Section 1.1). However, some oncogenes have much more heterogeneous patterns of translocations. For example, PAX5 is involved in the t(9:14) translocation with IgH in PL and other more aggressive lymphomas (Poppe et al., 2005), but 2.6% of pediatric B-ALL patients show multiple different translocation partners for PAX5. PAX5-ETV6 translocation was the first to be reported (Cazzaniga et al., 2001), but many others were subsequently discovered, including translocations of PAX5 with the transcription factors ETV6, FOXP1, ZNF521, PML, DACH1, DACH2, the chromatin regulators NCoR1, BRD1, the protein kinases JAK2, HIPK1, and others (Coyaud et al., 2010; Medvedovic, Ebert, Tagoh, & Busslinger, 2011). Similarly, the Bcl-6 oncogene is frequently involved in translocation events with many non-IgH genes in lymphoma, including CIITA, Pim-1, eif4AII, TFRC, RHOH, Ikaros, and up to 20 different partners (Ohno, 2006). The explanation for how such non-IgH translocations in lymphoid cells occur is not totally clear. Some are likely to be off-target AID translocation partners. Pax5, Pim-1, and RHOH frequently show SHM in B cells as a result of AID activity (Liu et al., 2008) and are found as hotspots in translocation- mapping experiments (see Section 2). However, a clear role for AID or RAG has not yet been proved for many other genes, and other mechanisms Translocation in Normal and Cancer Cells 63 for DSB formation could still be responsible for initiating such translocation. Even more compelling, specific cell-type mechanisms likely occur when the same oncogene translocates with specific partners in different tumor types. The ALK oncogene was first discovered as a partner of the Nucleophosmin 1(NPM1)-ALKtranslocationinanaplastic-large-celllymphoma(Morris et al., 1994). In the past 20 years, at least 20 different ALK translocation partners were discovered in lymphoma and recently also in solid tumors, such as myofibroblastic inflammatory tumors and lung, colorectal, and renal carcinoma (Chiarle et al., 2008; Kohno et al., 2012; Lipson et al., 2012; Soda et al., 2007; Takeuchi et al., 2012). For such translocations, the initiating events are very poorly understood. Strikingly, some translocations are highly tumor specific. For example, the NPM-ALK translocation, by far the most frequent translocation in lymphoma, is never encountered in solid tumors. In contrast, the EML4-ALK translocation that is predominant in lung carcinoma is never observed in lymphoma. Similarly, differential tissue-specific patterns are also found in other recurring oncogene translocations, such as RET, which is involved in translocations in thyroid and lung cancers (Lipson et al., 2012; Nikiforov & Nikiforova, 2011). Therefore, tissue-specific differences in translocation patterns do exist. Factors such as tissue-specific DSB formation, transcriptional activity of the implicated genes, or nuclear conformation and chromosome distribution must be investigated to explain the tissue specificity of translocation patterns.

5. PERSPECTIVES Recent breakthroughs in technology have critically advanced our ability to investigate the mechanisms that regulate translocation formation. We are now able to move on from studies focusing on a few specific genes to studies that can generate genome-wide maps of translocations and contacts in both normal and neoplastic cells. From these maps, we can rank the relative specific weight of the different factors required for translocation formation. DSB frequency seems to largely determine the pattern of translocations in normal cells and likely in cancer cells as well, but more studies are needed to prove this. In the absence of a dominant source of DSBs (i.e., in the absence of RAG or AID in B cells), translocations seem to follow the rules dictated by the proximity of chromosomal regions where the DSBs occur. The active transcription characteristics of genes influence their frequency of translocation, but it remains to be determined whether this effect depends on the increased probability of DSBs in transcribed genes or on increased 64 Roberto Chiarle proximity due to clustering in transcription factors, or both. Defects in DNA-repair pathways increase the overall frequency of translocation, but the exact roles of the various NHEJ—and possibly HR—factors in translocations remain to be fully elucidated. Finally, other important questions await answers. What is the role of chromatin conformation in translocations? Is there a tissue specificity in translocations of the same oncogene found in different tumor types? Are the translocation mechanisms identical in normal or cancer cells? The new combination of system-level tools and techniques for mining genomic data will likely allow us to answer most of these questions in the near future.

ACKNOWLEDGMENTS The work was supported by grants from the Associazione Italiana per la Ricerca sul cancro (AIRC), from the International Association for Cancer Research (AICR), and Grant FP7 ERC-2009-StG (Proposal No. 242965—“Lunely”). Disclosure Statement. The author is not aware of affiliations, memberships, funding, or financial holdings that might affect the objectivity of this review.

REFERENCES Aguilera, A. (2002). The connection between transcription and genomic instability. The EMBO Journal, 21, 195–201. Akasaka, T., Lossos, I. S., & Levy, R. (2003). BCL6 gene translocation in follicular lymphoma: A harbinger of eventual transformation to diffuse aggressive lymphoma. Blood, 102, 1443–1448. Alt, F. W., Zhang, Y., Meng, F. L., Guo, C., & Schwer, B. (2013). Mechanisms of programmed DNA lesions and genomic instability in the immune system. Cell, 152, 417–429. Arlt, M. F., Durkin, S. G., Ragland, R. L., & Glover, T. W. (2006). Common fragile sites as targets for chromosome rearrangements. DNA Repair, 5, 1126–1135. Barlow, J. H., Faryabi, R. B., Callen, E., Wong, N., Malhowski, A., Chen, H. T., et al. (2013). Identification of early replicating fragile sites that contribute to genome instability. Cell, 152, 620–632. Basu, U., Chaudhuri, J., Alpert, C., Dutt, S., Ranganath, S., Li, G., et al. (2005). The AID antibody diversification enzyme is regulated by protein kinase A phosphorylation. Nature, 438, 508–511. Basu, U., Franklin, A., & Alt, F. W. (2009). Post-translational regulation of activation- induced cytidine deaminase. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences, 364, 667–673. Basu, U., Meng, F. L., Keim, C., Grinstein, V., Pefanis, E., Eccleston, J., et al. (2011). The RNA exosome targets the AID cytidine deaminase to both strands of transcribed duplex DNA substrates. Cell, 144, 353–363. Bea, S., Salaverria, I., Armengol, L., Pinyol, M., Fernandez, V., Hartmann, E. M., et al. (2009). Uniparental disomies, homozygous deletions, amplifications, and target genes in mantle cell lymphoma revealed by integrative high-resolution whole-genome profiling. Blood, 113, 3059–3069. Translocation in Normal and Cancer Cells 65

Bekker-Jensen, S., Lukas, C., Kitagawa, R., Melander, F., Kastan, M. B., Bartek, J., et al. (2006). Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. The Journal of Cell Biology, 173, 195–206. Besmer, E., Market, E., & Papavasiliou, F. N. (2006). The transcription elongation complex directs activation-induced cytidine deaminase-mediated DNA deamination. Molecular and Cellular Biology, 26, 4378–4385. Boboila, C., Jankovic, M., Yan, C. T., Wang, J. H., Wesemann, D. R., Zhang, T., et al. (2010). Alternative end-joining catalyzes robust IgH locus deletions and translocations in the combined absence of ligase 4 and Ku70. In: Proceedings of the National Academy of Sciences of the United States of America, 107, 3034–3039. Boboila, C., Yan, C., Wesemann, D. R., Jankovic, M., Wang, J. H., Manis, J., et al. (2010). Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase 4. The Journal of Experimental Medicine, 207, 417–427. Bolzer, A., Kreth, G., Solovei, I., Koehler, D., Saracoglu, K., Fauth, C., et al. (2005). Three- dimensional maps of all chromosomes in human male fibroblast nuclei and prometaphase rosettes. PLoS Biology, 3, e157. Boyle, S., Gilchrist, S., Bridger, J. M., Mahy, N. L., Ellis, J. A., & Bickmore, W. A. (2001). The spatial organization of human chromosomes within the nuclei of normal and emerin-mutant cells. Human Molecular Genetics, 10, 211–219. Branzei, D., & Foiani, M. (2010). Maintaining genome stability at the replication fork. Nature Reviews. Molecular Cell Biology, 11, 208–219. Bredemeyer, A. L., Sharma, G. G., Huang, C.-Y., Helmink, B. A., Walker, L. M., Khor, K. C., et al. (2006). ATM stabilizes DNA double-strand-break complexes during V(D)J recombination. Nature, 442, 466–470. Canugovi, C., Samaranayake, M., & Bhagwat, A. S. (2009). Transcriptional pausing and stalling causes multiple clustered mutations by human activation-induced deaminase. The FASEB Journal, 23, 34–44. Cazzaniga, G., Daniotti, M., Tosi, S., Giudici, G., Aloisi, A., Pogliani, E., et al. (2001). The paired box domain gene PAX5 is fused to ETV6/TEL in an acute lymphoblastic leukemia case. Cancer Research, 61, 4666–4670. Chaudhuri, J., Tian, M., Khuong, C., Chua, K., Pinaud, E., & Alt, F. W. (2003). Transcription-targeted DNA deamination by the AID antibody diversification enzyme. Nature, 422, 726–730. Chiarle, R., Voena, C., Ambrogio, C., Piva, R., & Inghirami, G. (2008). The anaplastic lymphoma kinase in the pathogenesis of cancer. Nature Reviews. Cancer, 8, 11–23. Chiarle, R., Zhang, Y., Frock, R. L., Lewis, S. M., Molinie, B., Ho, Y. J., et al. (2011). Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells. Cell, 147, 107–119. Cook, P. R. (1999). The organization of replication and transcription. Science, 284, 1790–1795. Cory, S., Graham, M., Webb, E., Corcoran, L., & Adams, J. M. (1985). Variant (6;15) translocations in murine plasmacytomas involve a chromosome 15 locus at least 72 kb from the c-myc oncogene. The EMBO Journal, 4, 675–681. Coyaud, E., Struski, S., Prade, N., Familiades, J., Eichner, R., Quelen, C., et al. (2010). Wide diversity of PAX5 alterations in B-ALL: A Groupe Francophone de Cytogenetique Hematologique study. Blood, 115, 3089–3097. Crasta, K., Ganem, N. J., Dagher, R., Lantermann, A. B., Ivanova, E. V., Pan, Y., et al. (2012). DNA breaks and chromosome pulverization from errors in mitosis. Nature, 482, 53–58. Cremer, T., & Cremer, M. (2010). Chromosome territories. Cold Spring Harbor Perspectives in Biology, 2, a003889. 66 Roberto Chiarle

Cremer, T., Cremer, M., Dietzel, S., Muller, S., Solovei, I., & Fakan, S. (2006). Chromo- some territories—A functional nuclear landscape. Current Opinion in Cell Biology, 18, 307–316. Deriano, L., Chaumeil, J., Coussens, M., Multani, A., Chou, Y., Alekseyenko, A. V., et al. (2011). The RAG2 C terminus suppresses genomic instability and lymphomagenesis. Nature, 471, 119–123. Dorsett, Y., McBride, K. M., Jankovic, M., Gazumyan, A., Thai, T. H., Robbiani, D. F., et al. (2008). MicroRNA-155 suppresses activation-induced cytidine deaminase- mediated Myc-Igh translocation. Immunity, 28, 630–638. Durkin, S. G., & Glover, T. W. (2007). Chromosome fragile sites. Annual Review of Genetics, 41, 169–192. Einerson, R. R., Law, M. E., Blair, H. E., Kurtin, P. J., McClure, R. F., Ketterling, R. P., et al. (2006). Novel FISH probes designed to detect IGK-MYC and IGL-MYC rearrangements in B-cell lineage malignancy identify a new breakpoint cluster region designated BVR2. Leukemia, 20, 1790–1799. Felix, C. A., Kolaris, C. P., & Osheroff, N. (2006). Topoisomerase II and the etiology of chromosomal translocations. DNA Repair (Amst), 5, 1093–1108. Ferguson, D. O., Sekiguchi, J. M., Chang, S., Frank, K. M., Gao, Y., DePinho, R. A., et al. (2000). The nonhomologous end-joining pathway of DNA repair is required for genomic stability and the suppression of translocations. In: Proceedings of the National Academy of Sciences of the United States of America, 97, 6630–6633. Forment, J. V., Kaidi, A., & Jackson, S. P. (2012). Chromothripsis and cancer: Causes and consequences of chromosome shattering. Nature Reviews. Cancer, 12, 663–670. Gauwerky, C. E., Huebner, K., Isobe, M., Nowell, P. C., & Croce, C. M. (1989). Activa- tion of MYC in a masked t(8;17) translocation results in an aggressive B-cell leukemia. In: Proceedings of the National Academy of Sciences of the United States of America, 86, 8867–8871. Gesk, S., Klapper, W., Martin-Subero, J. I., Nagel, I., Harder, L., Fu, K., et al. (2006). A chromosomal translocation in cyclin D1-negative/cyclin D2-positive mantle cell lymphoma fuses the CCND2 gene to the IGK locus. Blood, 108, 1109–1110. Gilbert, N., Boyle, S., Fiegler, H., Woodfine, K., Carter, N. P., & Bickmore, W. A. (2004). Chromatin architecture of the human genome: Gene-rich domains are enriched in open chromatin fibers. Cell, 118, 555–566. Gladdy, R. A., Taylor, M. D., Williams, C. J., Grandal, I., Karaskova, J., Squire, J. A., et al. (2003). The RAG-1/2 endonuclease causes genomic instability and controls CNS complications of lymphoblastic leukemia in p53/Prkdc-deficient mice. Cancer Cell, 3, 37–50. Gostissa, M., Alt, F. W., & Chiarle, R. (2011). Mechanisms that promote and suppress chromosomal translocations in lymphocytes. Annual Review of Immunology, 29, 319–350. Haffner, M. C., Aryee, M. J., Toubaji, A., Esopi, D. M., Albadine, R., Gurel, B., et al. (2010). Androgen-induced TOP2B-mediated double-strand breaks and prostate cancer gene rearrangements. Nature Genetics, 42, 668–675. Hakim, O., Resch, W., Yamane, A., Klein, I., Kieffer-Kwon, K. R., Jankovic, M., et al. (2012). DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes. Nature, 484, 69–74. Halazonetis, T. D., Gorgoulis, V. G., & Bartek, J. (2008). An oncogene-induced DNA damage model for cancer development. Science, 319, 1352–1355. Han, L., & Yu, K. (2008). Altered kinetics of nonhomologous end joining and class switch recombination in ligase IV-deficient B cells. The Journal of Experimental Medicine, 205, 2745–2753. Hastings, P. J., Lupski, J. R., Rosenberg, S. M., & Ira, G. (2009). Mechanisms of change in gene copy number. Nature Reviews. Genetics, 10, 551–564. Translocation in Normal and Cancer Cells 67

Honjo, T., Alt, F. W., & Neuberger, M. (2004). Molecular Biology of B Cells. London, UK: Elsevier Academic Press. Iida, S., Rao, P. H., Ueda, R., Chaganti, R. S., & Dalla-Favera, R. (1999). Chromosomal rearrangement of the PAX-5 locus in lymphoplasmacytic lymphoma with t(9;14)(p13; q32). Leukemia & Lymphoma, 34, 25–33. Impera, L., Albano, F., Lo Cunsolo, C., Funes, S., Iuzzolino, P., Laveder, F., et al. (2008). A novel fusion 50AFF3/30BCL2 originated from a t(2;18)(q11.2;q21.33) translocation in follicular lymphoma. Oncogene, 27, 6187–6190. Jankovic, M., Robbiani, D. F., Dorsett, Y., Eisenreich, T., Xu, Y., Tarakhovsky, A., et al. (2010). Role of the translocation partner in protection against AID-dependent chromosomal translocations. In: Proceedings of the National Academy of Sciences of the United States of America, 107, 187–192. Ji, Y., Resch, W., Corbett, E., Yamane, A., Casellas, R., & Schatz, D. G. (2010). The in vivo pattern of binding of RAG1 and RAG2 to antigen receptor loci. Cell, 141, 419–431. Jung, D., Giallourakis, C., Mostoslavsky, R., & Alt, F. W. (2006). Mechanism and control of V(D)J recombination at the immunoglobulin heavy chain locus. Annual Review of Immu- nology, 24, 541–570. Kim, S. H., McQueen, P. G., Lichtman, M. K., Shevach, E. M., Parada, L. A., & Misteli, T. (2004). Spatial genome organization during T-cell differentiation. Cytogenetic and Genome Research, 105, 292–301. Klein, I. A., Resch, W., Jankovic, M., Oliveira, T., Yamane, A., Nakahashi, H., et al. (2011). Translocation-capture sequencing reveals the extent and nature of chromosomal rearrangements in B lymphocytes. Cell, 147, 95–106. Kloosterman, W. P., Hoogstraat, M., Paling, O., Tavakoli-Yaraki, M., Renkens, I., Vermaat, J. S., et al. (2011). Chromothripsis is a common mechanism driving genomic rearrangements in primary and metastatic colorectal cancer. Genome Biology, 12, R103. Kloosterman, W. P., Tavakoli-Yaraki, M., van Roosmalen, M. J., van Binsbergen, E., Renkens, I., Duran, K., et al. (2012). Constitutional chromothripsis rearrangements involve clustered double-stranded DNA breaks and nonhomologous repair mechanisms. Cell Reports, 1, 648–655. Kohno, T., Ichikawa, H., Totoki, Y., Yasuda, K., Hiramoto, M., Nammo, T., et al. (2012). KIF5B-RET fusions in lung adenocarcinoma. Nature Medicine, 18, 375–377. Kosak, S. T., Skok, J. A., Medina, K. L., Riblet, R., Le Beau, M. M., Fisher, A. G., et al. (2002). Subnuclear compartmentalization of immunoglobulin loci during lymphocyte development. Science, 296, 158–162. Krangel, M. S. (2007). T cell development: Better living through chromatin. Nature Immu- nology, 8, 687–694. Kryston, T. B., Georgiev, A. B., Pissis, P., & Georgakilas, A. G. (2011). Role of oxidative stress and DNA damage in human carcinogenesis. Mutation Research, 711, 193–201. Ku¨ppers, R. (2005). Mechanisms of B-cell lymphoma pathogenesis. Nature Reviews. Cancer, 5, 251–262. Ku¨ppers, R., & Dalla-Favera, R. (2001). Mechanisms of chromosomal translocations in B cell lymphomas. Oncogene, 20, 5580–5594. Liang, F., Romanienko, P. J., Weaver, D. T., Jeggo, P. A., & Jasin, M. (1996). Chromosomal double-strand break repair in Ku80-deficient cells. In: Proceedings of the National Academy of Sciences of the United States of America, 93, 8929–8933. Lieber, M. R. (2010). The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annual Review of Biochemistry, 79, 181–211. Lieber, M. R., Yu, K., & Raghavan, S. C. (2006). Roles of nonhomologous DNA end joining, V(D)J recombination, and class switch recombination in chromosomal translocations. DNA Repair, 5, 1234–1245. 68 Roberto Chiarle

Lieberman-Aiden, E., van Berkum, N. L., Williams, L., Imakaev, M., Ragoczy, T., Telling, A., et al. (2009). Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science, 326, 289–293. Lin, C., Yang, L., Tanasa, B., Hutt, K., Ju, B. G., Ohgi, K., et al. (2009). Nuclear receptor- induced chromosomal proximity and DNA breaks underlie specific translocations in cancer. Cell, 139, 1069–1083. Lipson, D., Capelletti, M., Yelensky, R., Otto, G., Parker, A., Jarosz, M., et al. (2012). Iden- tification of new ALK and RET gene fusions from colorectal and lung cancer biopsies. Nature Medicine, 18, 382–384. Liu, M., Duke, J. L., Richter, D. J., Vinuesa, C. G., Goodnow, C. C., Kleinstein, S. H., et al. (2008). Two levels of protection for the B cell genome during somatic hypermutation. Nature, 451, 841–845. Liu, P., Erez, A., Nagamani, S. C., Dhar, S. U., Kolodziejska, K. E., Dharmadhikari, A. V., et al. (2011). Chromosome catastrophes involve replication mechanisms generating complex genomic rearrangements. Cell, 146, 889–903. Liu, Y., Subrahmanyam, R., Chakraborty, T., Sen, R., & Desiderio, S. (2007). A plant homeodomain in RAG-2 that binds Hypermethylated lysine 4 of histone H3 is necessary for efficient antigen-receptor-gene rearrangement. Immunity, 27, 561–571. Lu, D., & Yunis, J. J. (1992). Cloning, expression and localization of an RNA helicase gene from a human lymphoid cell line with chromosomal breakpoint 11q23.3. Nucleic Acids Research, 20, 1967–1972. Magrangeas, F., Avet-Loiseau, H., Munshi, N. C., & Minvielle, S. (2011). Chromothripsis identifies a rare and aggressive entity among newly diagnosed multiple myeloma patients. Blood, 118, 675–678. Maher, C. A., & Wilson, R. K. (2012). Chromothripsis and human disease: Piecing together the shattering process. Cell, 148, 29–32. Mahowald, G. K., Baron, J. M., Mahowald, M. A., Kulkarni, S., Bredemeyer, A. L., Bassing, C. H., et al. (2009). Aberrantly resolved RAG-mediated DNA breaks in Atm-deficient lymphocytes target chromosomal breakpoints in cis. In: Proceedings of the National Academy of Sciences of the United States of America, 106, 18339–18344. Mani, R. S., Tomlins, S. A., Callahan, K., Ghosh, A., Nyati, M. K., Varambally, S., et al. (2009). Induced chromosomal proximity and gene fusions in prostate cancer. Science, 326, 1230. Marusawa, H. (2008). Aberrant AID expression and human cancer development. The Inter- national Journal of Biochemistry & Cell Biology, 40, 1399–1402. Matsumoto, Y., Marusawa, H., Kinoshita, K., Endo, Y., Kou, T., Morisawa, T., et al. (2007). Helicobacter pylori infection triggers aberrant expression of activation-induced cytidine deaminase in gastric epithelium. Nature Medicine, 13, 470–476. Matthews, A. G., Kuo, A. J., Ramon-Maiques, S., Han, S., Champagne, K. S., Ivanov, D., et al. (2007). RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination. Nature, 450, 1106–1110. Medvedovic, J., Ebert, A., Tagoh, H., & Busslinger, M. (2011). Pax5: A master regulator of B cell development and leukemogenesis. Advances in Immunology, 111, 179–206. Mills, K. D., Ferguson, D. O., & Alt, F. W. (2003). The role of DNA breaks in genomic instability and tumorigenesis. Immunological Reviews, 194, 77–95. Misteli, T. (2007). Beyond the sequence: Cellular organization of genome function. Cell, 128, 787–800. Mitelman, F., Johansson, B., & Mertens, F. (2007). The impact of translocations and gene fusions on cancer causation. Nature Reviews. Cancer, 7, 233–245. Molenaar, J. J., Koster, J., Zwijnenburg, D. A., van Sluis, P., Valentijn, L. J., van der Ploeg, I., et al. (2012). Sequencing of neuroblastoma identifies chromothripsis and defects in neuritogenesis genes. Nature, 483, 589–593. Translocation in Normal and Cancer Cells 69

Morris, S. W., Kirstein, M. N., Valentine, M. B., Dittmer, K. G., Shapiro, D. N., Saltman, D. L., et al. (1994). Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin’s lymphoma. Science, 263, 1281–1284. Murga Penas, E. M., Kawadler, H., Siebert, R., Frank, M., Ye, H., Hinz, K., et al. (2006). A novel fusion of the MALT1 gene and the microtubule-associated protein 4 (MAP4) gene occurs in diffuse large B-cell lymphoma. Genes, Chromosomes & Cancer, 45, 863–873. Nakamura, Y., Takahashi, N., Kakegawa, E., Yoshida, K., Ito, Y., Kayano, H., et al. (2008). The GAS5 (growth arrest-specific transcript 5) gene fuses to BCL6 as a result of t(1;3) (q25;q27) in a patient with B-cell lymphoma. Cancer Genetics and Cytogenetics, 182, 144–149. Nambu, Y., Sugai, M., Gonda, H., Lee, C. G., Katakai, T., Agata, Y., et al. (2003). Transcription-coupled events associating with immunoglobulin switch region chromatin. Science, 302, 2137–2140. Neves, H., Ramos, C., da Silva, M. G., Parreira, A., & Parreira, L. (1999). The nuclear topography of ABL, BCR, PML, and RARalpha genes: Evidence for gene proximity in specific phases of the cell cycle and stages of hematopoietic differentiation. Blood, 93, 1197–1207. Nikiforov, Y. E., & Nikiforova, M. N. (2011). Molecular genetics and diagnosis of thyroid cancer. Nature Reviews. Endocrinology, 7, 569–580. Northcott, P. A., Shih, D. J., Peacock, J., Garzia, L., Morrissy, A. S., Zichner, T., et al. (2012). Subgroup-specific structural variation across 1,000 medulloblastoma genomes. Nature, 488, 49–56. Ohno, H. (2006). Pathogenetic and clinical implications of non-immunoglobulin; BCL6 translocations in B-cell non-Hodgkin’s lymphoma. Journal of Clinical and Experimental Hematopathology, 46, 43–53. Okazaki, I. M., Kotani, A., & Honjo, T. (2007). Role of AID in tumorigenesis. Advances in Immunology, 94, 245–273. Oliveira, T. Y., Resch, W., Jankovic, M., Casellas, R., Nussenzweig, M. C., & Klein, I. A. (2012). Translocation capture sequencing: A method for high throughput mapping of chromosomal rearrangements. Journal of Immunological Methods, 375, 176–181. Osborne, C. S., Chakalova, L., Mitchell, J. A., Horton, A., Wood, A. L., Bolland, D. J., et al. (2007). Myc dynamically and preferentially relocates to a transcription factory occupied by Igh. PLoS Biology, 5, e192. Parada, L. A., McQueen, P. G., & Misteli, T. (2004). Tissue-specific spatial organization of genomes. Genome Biology, 5, R44. Pasqualucci, L., Neumeister, P., Goossens, T., Nanjangud, G., Chaganti, R. S., Ku¨ppers, R., et al. (2001). Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas. Nature, 412, 341–346. Pauklin, S., Serna´ndez, I. V., Bachmann, G., Ramiro, A. R., & Petersen-Mahrt, S. K. (2009). Estrogen directly activates AID transcription and function. The Journal of Experimental Medicine, 206, 99–111. Pavri, R., Gazumyan, A., Jankovic, M., Di Virgilio, M., Klein, I., Ansarah-Sobrinho, C., et al. (2010). Activation-induced cytidine deaminase targets DNA at sites of RNA polymerase II stalling by interaction with Spt5. Cell, 143, 122–133. Pavri, R., & Nussenzweig, M. C. (2011). AID targeting in antibody diversity. Advances in Immunology, 110, 1–26. Pleasance, E. D., Stephens, P. J., O’Meara, S., McBride, D. J., Meynert, A., Jones, D., et al. (2010). A small-cell lung cancer genome with complex signatures of tobacco exposure. Nature, 463, 184–190. Poppe, B., De Paepe, P., Michaux, L., Dastugue, N., Bastard, C., Herens, C., et al. (2005). PAX5/IGH rearrangement is a recurrent finding in a subset of aggressive B-NHL with complex chromosomal rearrangements. Genes, Chromosomes & Cancer, 44, 218–223. 70 Roberto Chiarle

Ramiro, A. R., Jankovic, M., Callen, E., Difilippantonio, S., Chen, H.-T., Mcbride, K. M., et al. (2006). Role of genomic instability and p53 in AID-induced c-myc-Igh translocations. Nature, 440, 105–109. Ramiro, A. R., Jankovic, M., Eisenreich, T., Difilippantonio, S., Chen-Kiang, S., Muramatsu, M., et al. (2004). AID is required for c-myc/IgH chromosome translocations in vivo. Cell, 118, 431–438. Rausch, T., Jones, D. T., Zapatka, M., Stutz, A. M., Zichner, T., Weischenfeldt, J., et al. (2012). Genome sequencing of pediatric medulloblastoma links catastrophic DNA rearrangements with TP53 mutations. Cell, 148, 59–71. Robbiani, D. F., Bothmer, A., Callen, E., Reina-San-Martin, B., Dorsett, Y., Difilippantonio, S., et al. (2008). AID is required for the chromosomal breaks in c-myc that Lead to c-myc/IgH translocations. Cell, 135, 1028–1038. Robbiani, D. F., Bunting, S., Feldhahn, N., Bothmer, A., Camps, J., Deroubaix, S., et al. (2009). AID produces DNA double-strand breaks in non-Ig genes and mature B cell lymphomas with reciprocal chromosome translocations. Molecular Cell, 36, 631–641. Roix, J. J., McQueen, P. G., Munson, P. J., Parada, L. A., & Misteli, T. (2003). Spatial proximity of translocation-prone gene loci in human lymphomas. Nature Genetics, 34, 287–291. Rubin, M. A., Maher, C. A., & Chinnaiyan, A. M. (2011). Common gene rearrangements in prostate cancer. Journal of Clinical Oncology, 29, 3659–3668. Ruiz, J. F., Gomez-Gonzalez, B., & Aguilera, A. (2011). AID induces double-strand breaks at immunoglobulin switch regions and c-MYC causing chromosomal translocations in yeast THO mutants. PLoS Genetics, 7, e1002009. Sahin, E., & Depinho, R. A. (2010). Linking functional decline of telomeres, mitochondria and stem cells during ageing. Nature, 464, 520–528. San Filippo, J., Sung, P., & Klein, H. (2008). Mechanism of eukaryotic homologous recombination. Annual Review of Biochemistry, 77, 229–257. Schatz, D. G., & Swanson, P. C. (2011). V(D)J recombination: Mechanisms of initiation. Annual Review of Genetics, 45, 167–202. Simonis, M., Klous, P., Splinter, E., Moshkin, Y., Willemsen, R., de Wit, E., et al. (2006). Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C). Nature Genetics, 38, 1348–1354. Sinclair, P., Harrison, C. J., Jarosova´, M., & Foroni, L. (2005). Analysis of balanced rearrangements of chromosome 6 in acute leukemia: Clustered breakpoints in q22-q23 and possible involvement of c-MYB in a new recurrent translocation, t(6;7) (q23;q32 through 36). Haematologica, 90, 602–611. Soda, M., Choi, Y. L., Enomoto, M., Takada, S., Yamashita, Y., Ishikawa, S., et al. (2007). Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature, 448, 561–566. Stephens, P. J., Greenman, C. D., Fu, B., Yang, F., Bignell, G. R., Mudie, L. J., et al. (2011). Massive genomic rearrangement acquired in a single catastrophic event during cancer development. Cell, 144, 27–40. Swanson, P. C. (2004). The bounty of RAGs: Recombination signal complexes and reaction outcomes. Immunological Reviews, 200, 90–114. Takeuchi, K., Soda, M., Togashi, Y., Suzuki, R., Sakata, S., Hatano, S., et al. (2012). RET, ROS1 and ALK fusions in lung cancer. Nature Medicine, 18, 378–381. Teng, G., Hakimpour, P., Landgraf, P., Rice, A., Tuschl, T., Casellas, R., et al. (2008). MicroRNA-155 is a negative regulator of activation-induced cytidine deaminase. Immu- nity, 28, 621–629. Toomey, E. C., Schiffman, J. D., & Lessnick, S. L. (2010). Recent advances in the molecular pathogenesis of Ewing’s sarcoma. Oncogene, 29, 4504–4516. Translocation in Normal and Cancer Cells 71

Tsai, A. G., & Lieber, M. R. (2010). Mechanisms of chromosomal rearrangement in the human genome. BMC Genomics, 11(Suppl. 1), S1. Tsai, A. G., Lu, H., Raghavan, S. C., Muschen, M., Hsieh, C.-L., & Lieber, M. R. (2008). Human chromosomal translocations at CpG sites and a theoretical basis for their lineage and stage specificity. Cell, 135, 1130–1142. Tubio, J. M., & Estivill, X. (2011). Cancer: When catastrophe strikes a cell. Nature, 470, 476–477. Ueda, C., Akasaka, T., Kurata, M., Maesako, Y., Nishikori, M., Ichinohasama, R., et al. (2002). The gene for interleukin-21 receptor is the partner of BCL6 in t(3;16)(q27;p11), which is recurrently observed in diffuse large B-cell lymphoma. Oncogene, 21, 368–376. Wang, J. H., Gostissa, M., Yan, C. T., Goff, P., Hickernell, T., Hansen, E., et al. (2009). Mechanisms promoting translocations in editing and switching peripheral B cells. Nature, 460, 231–236. Weinstock, D. M., Elliott, B., & Jasin, M. (2006). A model of oncogenic rearrangements: Differences between chromosomal translocation mechanisms and simple double-strand break repair. Blood, 107, 777–780. Yamane, A., Resch, W., Kuo, N., Kuchen, S., Li, Z., Sun, H. W., et al. (2011). Deep- sequencing identification of the genomic targets of the cytidine deaminase AID and its cofactor RPA in B lymphocytes. Nature Immunology, 12, 62–69. Yoshida, S., Kaneita, Y., Aoki, Y., Seto, M., Mori, S., & Moriyama, M. (1999). Identifica- tion of heterologous translocation partner genes fused to the BCL6 gene in diffuse large B-cell lymphomas: 50-RACE and LA - PCR analyses of biopsy samples. Oncogene, 18, 7994–7999. Zarrin, A. A., Del Vecchio, C., Tseng, E., Gleason, M., Zarin, P., Tian, M., et al. (2007). Antibody class switching mediated by yeast endonuclease-generated DNA breaks. Science, 315, 377–381. Zha, S., Bassing, C. H., Sanda, T., Brush, J. W., Patel, H., Goff, P. H., et al. (2010). ATM- deficient thymic lymphoma is associated with aberrant tcrd rearrangement and gene amplification. The Journal of Experimental Medicine, 207, 1369–1380. Zhang, F., Carvalho, C. M., & Lupski, J. R. (2009). Complex human chromosomal and genomic rearrangements. Trends in Genetics, 25, 298–307. Zhang, Y., Gostissa, M., Hildebrand, D. G., Becker, M. S., Boboila, C., Chiarle, R., et al. (2010). The role of mechanistic factors in promoting chromosomal translocations found in lymphoid and other cancers. Advances in Immunology, 106, 93–133. Zhang, Y., McCord, R. P., Ho, Y. J., Lajoie, B. R., Hildebrand, D. G., Simon, A. C., et al. (2012). Spatial organization of the mouse genome and its role in recurrent chromosomal translocations. Cell, 148, 908–921. Zhang, Y., & Rowley, J. D. (2006). Chromatin structural elements and chromosomal translocations in leukemia. DNA Repair, 5, 1282–1297. Zhao, Z., Tavoosidana, G., Sjolinder, M., Gondor, A., Mariano, P., Wang, S., et al. (2006). Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions. Nature Genetics, 38, 1341–1347. Zhu, C., Mills, K. D., Ferguson, D. O., Lee, C., Manis, J., Fleming, J., et al. (2002). Unrepaired DNA breaks in p53-deficient cells lead to oncogenic gene amplification subsequent to translocations. Cell, 109, 811–821. Intentionally left as blank CHAPTER THREE

The Intestinal Microbiota in Chronic Liver Disease

Jorge Henao-Mejia*,1, Eran Elinav†,1, Christoph A. Thaiss†,1, Richard A. Flavell*,‡,2 *Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA †Immunology Department, Weizmann Institute of Science, Rehovot, Israel ‡Howard Hughes Medical Institute, Chevy Chase, Maryland, USA 1Equal contributors 2Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 74 2. Role of the Intestinal Microbiota on Chronic Liver Diseases 75 2.1 Nonalcoholic fatty liver disease 75 2.2 Cirrhosis and associated comorbidities 78 2.3 Hepatocellular carcinoma 80 2.4 Autoimmune liver disease 80 3. Role of the Interactions Between the Innate Immune System and the Intestinal Microbiota on Chronic Liver Diseases 81 3.1 Toll-like receptors 82 3.2 Inflammasomes 84 3.3 C-type lectins 86 3.4 Dysbiosis associated with innate immune deficiency and its implications for liver disease 87 4. Probiotics and their Potential Role in Liver Disease Therapy 89 5. Conclusions 90 References 91

Abstract Recent evidence indicates that the intestinal microflora plays a critical role in physiological and pathological processes; in particular, it is now considered a key determinant of immune pathologies and metabolic syndrome. Receiving the majority of its blood supply from the portal vein, the liver represents the first line of defense against food antigens, toxins, microbial-derived products, and microorganisms. Moreover, the liver is critically positioned to integrate metabolic outcomes with nutrient intake. To accom- plish this function, the liver is equipped with a broad array of immune networks. It is now evident that, during pathological processes associated with obesity, alcohol-intake, or autoimmunity, the interaction between these immune cell populations and the

intestinal microbiota promotes chronic liver disease progression and therefore they represent a novel therapeutic target. Herein, we highlight recent studies that have shed new light on the relationship between the microbiome, the innate immune system, and chronic liver disease progression.

1. INTRODUCTION The human gastrointestinal tract contains 10–100 trillion bacteria and approximately 500–1500 different bacterial species (Lozupone, Stombaugh, Gordon, Jansson, & Knight, 2012). These microorganisms have critical functions in multiple aspects of human physiology such as regulation of metabolic processes, education of the immune system, and promotion of epithelial cell responses that are essential to maintain mutualism (Maynard, Elson, Hatton, & Weaver, 2012; Tremaroli & Backhed, 2012). The intestinal microflora differs quantitatively and qualitatively among species and individuals. Life style, age, dietary habits, exposure to antibiotics, and host genotype play essential roles in the composition of the intestinal microflora (Claesson et al., 2012; Turnbaugh et al., 2009); moreover, disruption of the delicate balance that represents the ecosystem of bacterial communities of the gastrointestinal tract can lead to severe metabolic and inflammatory pathologies. The close functional relationship between the liver and the gastrointestinal tract (gut–liver axis) is highlighted by multiple important physiological processes that intimately interconnect these organs. The liver, the largest organ in the body, has a dual blood supply. The hepatic artery, which arises from the celiac artery, supplies oxygenated blood to the liver, and the portal vein conducts venous blood from the intestines and the spleen. Approxi- mately 75% of hepatic blood flow is derived from the hepatic portal vein (1000–1200 mL/min), and therefore, the liver is constantly exposed to nutrients, toxins, food-derived antigens, microbial products, and microorganisms derived from the intestinal tract (Miyake & Yamamoto, 2013). This strategic location confers critical metabolic, immunologic, and detoxifying roles to the liver and stresses the crucial role of the intestinal microbiota on hepatic pathophysiology. In thisreview,weexaminetheimpact ofgutmicrobiota onhepatic diseases, focusing on how dysbiosis and immune responses triggered by microbiota- derived products shape the progression of chronic liver pathologies. The Intestinal Microbiota in Chronic Liver Disease 75

2. ROLE OF THE INTESTINAL MICROBIOTA ON CHRONIC LIVER DISEASES 2.1. Nonalcoholic fatty liver disease Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease in Western societies, with a prevalence ranging from 20% to 40% in the general population and up to 75–100% in obese individuals (Ludwig, Viggiano, McGill, & Oh, 1980; Sheth, Gordon, & Chopra, 1997). NAFLD is considered the hepatic manifestation of metabolic syndrome (Marchesini et al., 2003), with many patients developing other comorbidities including insulin resistance, hyperlipidemia, cardiovascular disease, polycystic ovary syndrome, and obstructive sleep apnea (Cerda et al., 2007; Tolman, Fonseca, Dalpiaz, & Tan, 2007). While most patients with NAFLD remain asymptomatic, 20% progress to develop chronic hepatic inflammation (nonalcoholic steatohepatitis, NASH), which in turn can lead to cirrhosis, portal hypertension, hepatocellular carcinoma (HCC), and increased mortality (Caldwell et al., 1999; Propst, Propst, Judmaier, & Vogel, 1995; Shimada et al., 2002). NASH can be classified as primary NASH (associated with obesity, type 2 diabetes (T2DM), and hyperlipemia) and secondary NASH (occurring after pharmacological interventions, parenteral nutrition, jejunoileal bypass surgery, or Wilson’s disease). Despite its high prevalence, factors leading to progression from NAFLD to NASH remain poorly understood and no treatment has proved effective (Charlton, 2008; Hjelkrem, Torres, & Harrison, 2008). A “two-hit” mechanism is proposed to drive NAFLD/NASH pathogenesis (Day & James, 1998). The first hit, hepatic steatosis, is closely associated with lipotoxicity-induced mitochondrial abnormalities that predispose the liver to additional proinflammatory insults (second hits) that promote disease progression. Second hits include increased generation of reactive oxygen species, increased lipid peroxidation, and gut-derived factors. Most likely, the parallel action of these hepatic tissue insults is required for the development of steatohepatitis (Sanyal et al., 2001). In the past decade, a growing body of research functionally links the intestinal microbiota with the development of steatosis (first hit) and with the progression to NASH (second hit). Obesity is considered the most common risk factor for NAFLD in humans (Younossi et al., 2011). Several lines of evidence unequivocally link the intestinal microflora with body weight and body fat composition 76 Jorge Henao-Mejia et al.

Type 2 diabetes • Endotoxemia • Insulitis • Insulin resistance Intestinal microbiota Steatosis

Obesity • Increased calorie extraction • Cleavage of dietary polysaccharides • Dyslipidemia

Decreased choline metabolism Figure 3.1 Effects of the intestinal microbiota on the risk factors that promote NAFLD development. The microbiota can regulate the progression of multiple associated comorbidities that are associated with NAFLD pathogenesis such as choline metabolism, obesity, and diabetes mellitus.

(Fig. 3.1). In animal studies, germ-free mice have a lower body fat content than conventionally raised mice; moreover, the inoculation of germ-free mice with microbiota from wild-type mice results in a significant increase in body fat accumulation (Turnbaugh et al., 2006). The phyla Bacteroidetes and Firmicutes represent a large proportion of the intestinal microbiota composition in mice and humans; however, their relative abundance profoundly affects the body composition of individuals (Ley et al., 2005; Ley, Turnbaugh, Klein, & Gordon, 2006). Genetically obese mice (ob/ob) have a significant increase in the Bacteroidetes to Firmicutes ratio when compared with lean littermate controls, but perhaps more importantly, germ-free mice colonized with microbiota from genetically obese mice gained weight faster and harvest calories more efficiently than mice colonized with intestinal microflora from lean mice (Turnbaugh et al., 2006). These findings indicate that the composition of the microbiota directly influences calorie extraction, body fat composition, and body weight. In humans, several lines of evidence now correlate the composition of the intestinal microbiota with multiple metabolic and inflammatory parameters as well as dietary habits (Claesson et al., 2012; Ley et al., 2006; Muegge et al., 2011). Similar to mice, obese individuals have increased levels of Bacteroidetes and the reduction of this phylum in the intestinal microflora is significantly associated with weight loss either by The Intestinal Microbiota in Chronic Liver Disease 77 fat- or carbohydrate-restricted diets, suggesting that Bacteroidetes may be responsive to calorie intake (Ley et al., 2006). Metagenome-wide association studies have recently demonstrated that T2DM patients are characterized by gut microbial dysbiosis, a decrease in the abundance of butyrate-producing bacteria and an increase in various opportunistic bacterial pathogens. More- over, these gut microbial markers can be useful for classifying T2DM, indicating that specific conformations of the intestinal microbiota play critical roles in the pathogenesis of T2DM and associated disorders (Qin et al., 2012). Calorie intake of Western society diets is a key determinant of metabolic syndrome. Long-term dietary habits have a profound effect on the human gut microbiota and therefore on potential deleterious metabolic outcomes. It has been proposed that the human gut microbiota should be divided into three compositions (enterotypes), yet this notion is still debated and merits further validation. Each suggested enterotype is dominated by a different genus—Bacteroides, Prevotella, or Ruminococcus—(Arumugam et al., 2011). Interestingly, enterotypes dominated by Bacteroides are associated with diet rich in protein and animal fat (Western diet), while Prevotella- dominated enterotypes are associated with the consumption of a diet rich in carbohydrates/fiber (De Filippo et al., 2010; Wu et al., 2011), suggesting that the gut microbiota is shaped by the different diets to maximize energy extraction. Taken together, these studies show that the composition of the microbiota is a critical player in the metabolic status of the host and its dis- turbance is associated with metabolic abnormalities that are associated with the “first hit” (steatosis) during NAFLD pathogenesis. Although it is now clear that the intestinal microflora plays critical roles in body fat accumulation and weight gain, the role of gut-derived factors on NAFLD progression has just begun to be elucidated. Progression from steatosis to steatohepatitis is mainly an inflammatory process that likely reflects the concerted deleterious effects of multiple noxious stimuli. Several lines of evidence now suggest that intestinal bacterial communities might play an important part in this process. Jejunoileal bypass, small intestinal diverticulosis, total parenteral nutrition, and intestinal failure are associated with NASH progression (Carter & Karpen, 2007; Corrodi, 1984; Nazim, Stamp, & Hodgson, 1989; Quigley, Marsh, Shaffer, & Markin, 1993; Vanderhoof, Tuma, Antonson, & Sorrell, 1982); interestingly, small intestinal bacterial outgrowth (SIBO) as a consequence of low intestinal motility has been proposed as a key determinant factor for NAFLD progression in these conditions in humans (Carter & Karpen, 2007; Pappo et al., 1992; Quigley et al., 1993). In concordance with this, antibiotic treatment or 78 Jorge Henao-Mejia et al. surgical removal of the bypassed section of the intestine reverses SIBO and steatohepatitis. Similarly, rats fed under total parenteral nutrition are characterized by severe liver injury secondary to bowel hypomotility, which leads to the expansion of Gram-negative bacterial populations and increased hepatotoxic mediators such as endotoxin or tumor necrosis factor (Pappo et al., 1992). The role of the intestinal microbiota in the more highly prevalent primary NASH is less clear. The prevalence of SIBO is significantly increased in obese individuals as compared with healthy lean subjects (Sabate et al., 2008), but its role in NAFLD progression has largely been overlooked. Nev- ertheless, a recent study conducted by Miele et al. (2009) evaluated intestinal permeability, SIBO, and NAFLD disease stage. Interestingly, patients with NAFLD were reported to have significantly increased gut permeability and SIBO when compared with healthy individuals, suggesting that overgrowth of the intestinal bacterial flora gut could lead to bacterial translocation, portal endotoxemia, and ultimately hepatic injury (Miele et al., 2009). In concordance with this possibility, multiple studies have found high levels of SIBO prevalence in different cohorts of NASH patients (Sajjad et al., 2005; Wigg et al., 2001); moreover, we recently demonstrated that inflammasome- mediated dysbiosis characterized by an expansion of the Prevotellaceae and Porphyromonadaceae families as well as the TM7 taxa promotes NAFLD progression in different mouse models (Henao-Mejia, Elinav, Jin, et al., 2012). Collectively, these studies indicate that different compositions of the bacterial communities of the intestines might regulate NAFLD progression in humans and therefore represent a novel therapeutic target. Characterization of the bacterial communities at different stages of NAFLD and the exact role of metabolites derived from the bacterial microflora in disease progression should shed some light on the precise role of the microbiome in liver disease in the context of metabolic syndrome.

2.2. Cirrhosis and associated comorbidities Cirrhosis is the final clinical–histopathological stage of a wide array of liver diseases. The intestinal microbiota is a common denominator of the major complications of liver cirrhosis, including spontaneous bacterial peritonitis, hepatic encephalopathy (HE), and esophageal variceal bleeding (Basile & Jones, 1997; Campillo et al., 1999; Guarner & Soriano, 1997; Husova et al., 2005; Thalheimer, Triantos, Samonakis, Patch, & Burroughs, 2005). The process of liver fibrogenesis promotes dysbiosis and intestinal The Intestinal Microbiota in Chronic Liver Disease 79 barrier dysfunction through multiple pathological processes. Cirrhotic patients have decreased blood flow through the portal vein and intestinal vascular congestion, which results in increased gut permeability (Bauer et al., 2001; Gunnarsdottir et al., 2003). Moreover, impaired liver function promotes changes in bacterial communities in the gut through decreased bile acid production and defective intestinal motility that leads to SIBO (Sung, Shaffer, & Costerton, 1993). Thus, it is now well recognized that impaired fluid/liver physiology and innate immunity in combination with dysbiosis are key pathological processes that promote bacterial translocation to the peritoneum. HE is a broad term that encompasses a constellation of neuropsychiatric abnormalities observed in patients with liver dysfunction (Bajaj, 2010). Overt HE is diagnosed in up to 45% of patients with cirrhosis, while minimal HE is observed in 60–80% of the patients (Bajaj, 2010). In healthy individuals, the liver protects the brain from ammonia by converting it to urea, which is then excreted by the kidneys. In the context of severe liver dysfunction, ammonia becomes the critical driver of HE pathogenesis and the intestinal microbiota is by far its predominant source (Williams, 2007). In particular, Urease-producing bacteria such as Klebsiella and Proteus species seem to play a critical role in increased ammonia production and HE development (Basile & Jones, 1997). In concordance with the concept of HE being a bacterial-driven disease, treatment with nonabsorbable antibiotics such as Neomycin and Rifaximinis is associated with a significant decrease in the risk of breakthrough episodes of HE, relapses, or hospital- ization due to this neuropsychiatric complication (Bajaj et al., 2011; Bass et al., 2010; Sidhu et al., 2011). Recently, the role of specific bacterial families in cirrhosis has begun to be addressed. Two studies have performed nonculture-based methods to determine the composition of the microbiota in patients with cirrhosis and HE. Both studies found a higher concentration of Streptococcaceae and a negative correlation between cirrhosis and the abundance of Lachnospiraceae (Bajaj et al., 2012; Chen et al., 2011). Interestingly, Bajaj et al. (2012) found that in addition to changes in the intestinal microbiota between healthy and cirrhotic individuals, there was a significant increase in the abundance of different bacterial families (Enterobacteriaceae, Alcaligenaceae, and Streptococcaceae)in patients with confounded HE. Moreover, a positive correlation between cognitive dysfunction and the presence of Alcaligenaceae and Porphyromonadaceae was observed by standardized cognitive testing (Bajaj et al., 2012). The investigation of the gut microbiome in cirrhosis and its correlation to severe clinical 80 Jorge Henao-Mejia et al. complications is still in its early stages, but identification of bacterial species that specifically drives disease progression will greatly improve our understanding of the pathogenesis of these complex human diseases.

2.3. Hepatocellular carcinoma HCC is one of the most frequent human cancers worldwide. Approximately 80–90% of HCCs are preceded by chronic liver disease, hepatic fibrosis, and cirrhosis (Nordenstedt, White, & El-Serag, 2010). Therefore, it has been speculated that microbial-derived products are essential determinants of HCC progression. Indeed, recent studies performed using a mouse model of HCC showed that hepatocarcinogenesis in chronically injured livers depended on the intestinal microbiota and Toll-like receptor 4 (TLR4) activation in non-bone-marrow-derived resident liver cells. Importantly, TLR4 and the gut microbiota are not required for HCC initiation but for HCC progression as intestinal sterilization restricted late stages of hepatocarcinogenesis (Dapito et al., 2012). The role of the microbiome on human HCC is an unexplored area that warrants further investigation in the following years.

2.4. Autoimmune liver disease Primary sclerosing cholangitis (PSC) is a chronic liver disease characterized by inflammation and eventual obstruction of biliary ducts (Levy & Lindor, 2006). Although the pathogenesis of PSC remains undetermined, intestinal microbiota is considered to be a major factor in its etiology. The role of intestinal bacterial communities in ulcerative colitis (UC) pathogenesis is well characterized. Interestingly, approximately 75% of patients with PSC have UC and nearly 3% of patients with UC have PSC as a concomitant comorbidity (Bambha et al., 2003; Bergquist et al., 2008; Hashimoto et al., 1993; Joo et al., 2009; O’Toole et al., 2012; Sano et al., 2011; Ye et al., 2011). Moreover, PSC is more frequent in UC patients with total colonic involvement suggesting a strong positive correlation between intestinal inflammation and PSC development (Joo et al., 2009; O’Toole et al., 2012). Several lines of evidence point to the microbiota as a common denominator driving liver and intestinal inflammation in this condition. In the bile of PSC patients, Candida and enteric bacteria such as Escherichia coli are frequently detected (Rudolph et al., 2009). End-stage PSC liver shows The Intestinal Microbiota in Chronic Liver Disease 81 significantly increased expression and activation of critical genes involved in innate immune pathways (Miyake & Yamamoto, 2013). Finally, serum atypical perinuclear antineutrophil cytoplasmic antibodies (pANCA) are frequently found in patients with PSC (Mulder et al., 1993; Terjung et al., 1998). Recently, the autoantigen of this atypical pANCA has been reported to be b-tubulin, but perhaps more importantly, pANCA cross- reacts with FtsZ, a bacterial cytoskeletal protein present in all intestinal bacteria (Terjung et al., 2010). Thus, identifying the specific bacterial species that trigger PCS is a clinically relevant problem that deserves further investigation. Primary biliary cirrhosis (PBC) affects approximately 40 per 100,000 peo- ple in the United States. PBC is an autoimmune liver disorder characterized by immune cell activation and directed damage of cholangiocytes, which results in cholestasis that ultimately leads to hepatic fibrogenesis and liver failure in 26% of patients within 10 years of diagnosis (Washington, 2007). The presence in the serum of antimitochondrial antibodies (AMAs) is the hallmark of PBC. AMAs are detected in approximately 95% of PBC patients and their cross-reaction with bacterial components is proposed as a critical event for the early pathogenesis of PBC (Bogdanos et al., 2004; Hopf et al., 1989). AMAs have been reported to react with proteins of E. coli isolated from PBC patients (Bogdanos et al., 2004; Hopf et al., 1989). Moreover, IgG3 antibodies in approximately 50% of PBC patients cross-react with b-galactosidase of Lacto- bacillus delbrueckii, and in 25% of PBC patients, the serum reacts specifically with proteins of Novosphingobium aromaticivorans from stool specimens (Bogdanos et al., 2005; Selmi et al., 2003). Given this association, further study is warranted to determine if modulation of gut microbiota might aid in the treatment of this catastrophic disease.

3. ROLE OF THE INTERACTIONS BETWEEN THE INNATE IMMUNE SYSTEM AND THE INTESTINAL MICROBIOTA ON CHRONIC LIVER DISEASES The complex interplay between the host and its indigenous microflora is mediated by a large array of pattern-recognition receptors (PRRs) of the innate immune system (Carvalho, Aitken, Vijay-Kumar, & Gewirtz, 2012). Originally mainly appreciated for their role in recognizing invading pathogenic microbes and for the initiation of adaptive immune responses, these receptors and their downstream signaling cascades are increasingly regarded 82 Jorge Henao-Mejia et al. as pivotal for the recognition of the commensal microbiota. This microbial recognition plays an important role under homeostatic conditions, and dysfunction in innate signaling in the intestine has been associated with aberrant development of the intestinal immune system, failure in maintenance of intestinal epithelial homeostasis and barrier function, and exacerbated intestinal injury (Michelsen & Arditi, 2007). Importantly, this innate sensing function also serves to locally contain the microbiota and to exclude intestinal microorganisms from the systemic circulation (Slack et al., 2009). The innate receptors expressed in the gastrointestinal tract represent the first line of defense against invasion of microorganisms. However, in cases of increased microbial translocation through the gastrointestinal barrier, the liver as first line of defense requires the expression of innate PRRs in order to set in place a secondary surveillance system of microbial products potentially draining from the gastrointestinal tract. Indeed, intrahepatic expression of innate immune receptors has been described for Kupffer cells (Visvanathan et al., 2007), liver sinusoidal endothelial cells (Hosel et al., 2012), hepatic stellate cells (Wang et al., 2009), biliary epithelial cells (Yokoyama et al., 2006), and hepatocytes (Wang et al., 2005). Consequently, the liver has to master a delicate balance between its ability to induce systemic tolerance toward innocuous food particles and occasional translocation of commensal microbial products and its role in promoting inflammation when a persistent microbial stimulus caused by intestinal breech is indicative of systemic microbial spread. In the following sections, we will discuss how hepatic PRR signaling mediates host–microbial interactions in this vital organ, and how aberrations in PRR expression and signaling contribute to the molecular etiology of liver disease.

3.1. Toll-like receptors TLRs were the first class of PRRs discovered. They recognize a wide range of microbial ligands, ranging from bacterial and fungal cell wall components to nucleic acid (Kawai & Akira, 2010). TLRs are expressed in a wide variety of liver cells and have long been recognized to be involved in the pathogenesis of liver diseases. In particular, Kupffer cells express high levels of TLR2, TLR3, and TLR4, and respond to LPS stimulation with the production of TNF-a, IL-6, and IFN-g. Moreover, the expression of TLRs has been found on hepatocytes, biliary epithelial cells, hepatic stellate cells, and liver sinusoidal endothelial cells (Miyake & Yamamoto, 2013)(Fig. 3.2). The TLR4–MyD88–NF-kB signaling axis has been found to play a critical role in various pathophysiological settings in the liver, including cirrhosis, The Intestinal Microbiota in Chronic Liver Disease 83

Steatosis Hepatocyte: TLR2-4 Kupffer cell: TLR2-4

Stellate cell: TLR1-9

TNF IL-6 Inflammatory response in the liver LSEC: TLR2

Flux of PRR ligands

Enterocytes: NLRP6

Intestinal dysbiosis Figure 3.2 Multiple layers of pattern-recognition receptor involvement in the pathogenesis of liver disease. Functional expression of the NLRP6 inflammasome in the intestine is necessary to avoid dysbiosis. Chronic intestinal inflammation is associated with increased translocation of microbes across the gastrointestinal tract and influx of microbial products into the liver. There, TLR expression on a variety of cell types mediates an aberrant respond to the increased microbial load, initiating an exaggerated inflammatory response that can lead to hepatitis. fibrosis, viral hepatitis, HCC, and fatty liver disease. For instance, in mice on a high-fat diet, TLR4 deficiency ameliorates hepatic steatosis (Li et al., 2011). In addition, signaling through TRIF downstream of TLR4 in Kupffer cells has been shown to promote alcoholic liver disease (Gao et al., 2011). Further, hepatic TLR4 expression is increased in animal models of NASH (Thuy et al., 2008), PSC (Mueller et al., 2011), and PBC (Wang et al., 2005). These animal studies have been supported by genetic data from humans. A polymorphism in the gene encoding TLR4, which attenuates the signaling downstream of the receptor in response to LPS stimulation, has been associated with a decreased risk to develop cirrhosis (Figueroa et al., 2012; Huang et al., 2007). Another TLR which has been repeatedly associated with enhanced severity of inflammatory liver disease is TLR9, which signals through IRF-7 to induce the expression of type I interferons (IFNs). Interestingly, type I IFNs 84 Jorge Henao-Mejia et al. were recently described to protect from TLR9-associated liver damage, and this effect was mediated by the endogenous IL-1 receptor antagonist (Petrasek, Dolganiuc, Csak, Kurt-Jones, & Szabo, 2011). The same authors also found a protective role for type I IFNs in a TLR4-driven model of alcoholic liver disease (Petrasek, Dolganiuc, Csak, Nath, et al., 2011). The involvement of TLRs in a multitude of liver pathologies clearly implied a role for increased microbial translocation across the gastrointestinal tract and hepatic recognition of microbial products (Fig. 3.2), but direct evidence for this notion has been lacking until recently. First insight came from a study by Seki et al. who showed an involvement of the microbiota in the development of hepatic fibrosis. Antibiotic treatment, as well as TLR4- or MyD88-deficiency, reduced fibrosis after bile duct ligation. TLR4 expression on hepatic stellate cells led to enhanced TGF-b signaling and recruitment of Kupffer cells to the fibrotic liver (Seki et al., 2007). As detailed below, we recently described that, under conditions of intestinal inflammation, the influx of microbial products into the liver promotes the development and progression of NAFLD in a TLR4- and TLR9-dependent manner (Henao-Mejia, Elinav, Jin, et al., 2012). In concordance with these results, Lin et al. recently used the concanavalin A (ConA) model of fulminant liver injury to demonstrate that the intestinal microbiota is critically involved in TLR4-mediated hepatitis. Treatment of mice with broad-spectrum antibiotics as well as TLR4 deficiency greatly ameliorated liver damage, as evidenced by reduced release of aminotrans- ferases into the blood, dampened production of proinflammatory cytokines, and decreased hepatic cell death (Lin et al., 2012). In contrast, administration of purified LPS potentiated liver pathology in the ConA model. Adoptive transfer experiments using TLR4-deficient or sufficient splenocytes revealed that immune cells contribute to disease progression through TLR4 expression.

3.2. Inflammasomes Inflammasomes are a group of cytosolic multiprotein complexes, classically consisting of an upstream sensor protein of the NOD-like receptor (NLR) family, the adaptor protein ASC, and the downstream effector caspase-1 (Henao-Mejia, Elinav, Strowig, & Flavell, 2012). To date, the NLR proteins NLRP1, NLRP2, NLRP3, NLRP6, NLRP7, NLRC4, and the HIN-200 family member AIM2 have been reported to initiate the formation of an inflammasome. Upon stimulation with a diverse set of microbial The Intestinal Microbiota in Chronic Liver Disease 85 or damage-associated molecular patterns, inflammasome assembly leads to the autocatalytic cleavage of caspase-1 and processing of pro-IL-1b and pro-IL-18 into their mature and bioactive forms (Strowig, Henao-Mejia, Elinav, & Flavell, 2012). Inflammasome activity is thought to require two sequential stimuli. The first stimulus drives transcription of the proforms of IL-1b and IL-18, while the second stimulus is required for the formation of the inflammasome complex (Latz, 2010). Inflammasomes fulfill a dual role, recognizing both endogenous damage-associated substances such as ATP or crystal particles and initiating immune responses in reaction to pathogen-associated molecular patterns during bacterial, viral, fungal, and parasitic infections (Elinav, Strowig, Henao-Mejia, & Flavell, 2011). In addition, the inflammasomes are critically involved in the complex interplay between the intestinal immune system and the gut microbiota, which will be covered in more detail below. Recently, inflammasomes were identified to play a role in the pathogenesis of liver disease. Inflammasome components are expressed by various cell types in the liver. Kupffer cells and sinusoidal endothelial cells express high level of NLRP1, NLRP3, and AIM2, and hepatocytes upregulate NLRP3 expression in an LPS-dependent manner (Boaru, Borkham-Kamphorst, Tihaa, Haas, & Weiskirchen, 2012). Imaeda et al. (2009) initially demonstrated an involvement of the NLRP3 inflammasome in the development of acetaminophen-induced hepatotoxicity and showed reduced mortality in acetaminophen-treated mice lacking any component of the NLRP3 inflammasome, although others could not find a role for NLRP3 in acetaminophen-mediated liver failure (Williams, Farhood, & Jaeschke, 2010). Watanabe et al. (2009) revealed expression of inflammasome components in hepatic stellate cells and demonstrated an involvement of the inflammasome in a mouse model of liver fibrosis using carbon tetrachloride or thioacetamide. Similarly, knockdown of NLRP3 ameliorated liver inflammation and protected ischemia–reperfusion injury in mice by preventing excessive production of inflammatory cytokines and NF-kB activity (Zhu et al., 2011). These early studies mainly focused on the role of the inflammasome in the response against tissue damage in sterile injury-mediated models of liver disease. Subsequent reports, however, have also demonstrated an involvement of the inflammasome in liver pathology caused by microbial components or live microorganisms, such as in a model of Propionibacterium acnes-induced sensitization to LPS-induced liver injury (Tsutsui, Imamura, Fujimoto, & Nakanishi, 2010) and in Schistosoma mansoni infection (Ritter et al., 2010). 86 Jorge Henao-Mejia et al.

In these studies, a cooperative behavior of TLR signaling and inflammasome activation was noticed to be a driving force in the development of overt liver inflammation, suggesting concerted recognition events of microbial- and damage-associated molecules. Interestingly, Csak et al. (2011) recently showed an involvement of the NLRP3 inflammasome in the development and progression of NASH. Upon induction of a mouse model of NASH, expression of inflammasome components was upregulated in the liver and inflammasome activation occurred in isolated hepatocytes. Mechanistically, palmitic acid, a saturated fatty acid, was found to activate the inflammasome and sensitized hepatocytes to IL-1b secretion in response to LPS. The results from this study indicated that both microbial and nonmicrobial PRR ligands act in concert to induce pathogenic inflammasome responses in the liver. A later study confirmed NLRP3 activation in the liver and showed that LPS stimulation alone is sufficient to drive hepatic production of inflammatory cytokines downstream of NLRP3 inflammasome activation (Ganz, Csak, Nath, & Szabo, 2011).

3.3. C-type lectins C-type lectin (CTL) receptors and their downstream adaptor molecules are mediating recognition of glycosylated ligands on microorganisms (Sancho & Reis e Sousa, 2012). Dectin-1 and 2 are two CTLs involved in the immune response against fungal pathogens. The recognition of fungal-associated molecular patterns elicits a downstream cascade through the signaling molecules caspase recruitment domain-containing protein 9 (CARD9) and Syk (Kerrigan & Brown, 2011). A recent study found hepatic mRNA expression in humans of many factors involved in CTL signaling, including Dectin-1, Syk, and CARD9 (Lech et al., 2012). Interestingly, CARD9, which is known as a susceptibility locus in inflammatory bowel disease (IBD), has recently been associated with PSC, along with Rel and IL-2, two other IBD risk loci (Janse et al., 2011). Rel is a member of the NF-kB family of transcription factors, CARD9 induces NF-kB signaling, and IL-2 is an NF-kB target gene, potentially combining all three susceptibility loci into one pathway. The involvement of three members of a fungal recognition pathway in PSC implies a functional role of innate immune recognition of fungal microorganisms in the pathogenesis of this disease. CARD9 is essential for the control of fungal infection, and CARD9-deficient mice show high rates of The Intestinal Microbiota in Chronic Liver Disease 87 early mortality after infection with Candida albicans (Gross et al., 2006). As mentioned above, Candida is detected in the bile fluid of 1 in every 10 PSC patients. In most cases, the detection of fungi in the bile negatively influences the prognosis on disease severity (Rudolph et al., 2009). Functional studies are needed in the future to delineate the mechanisms and the importance of host–fungal interactions in the pathophysiology of liver disease. Intriguingly, the recently suggested link between alterations in commensal fungal sensing and susceptibility to IBD may potentially provide a mechanistic explanation for the substantial susceptibility for PSC among chronic IBD patients (Iliev et al., 2012). Taken together, the involvement of PRRs of the innate immune system in the pathogenesis of inflammatory liver disease has so far been interpreted in the context of local responses to endogenous signal of damage. While PRR-mediated recognition of damage-associated molecular patterns certainly plays a critical role in disease development and progression, recent evidence indicates that one should also consider microbial ligands as drivers in hepatic inflammatory disorders.

3.4. Dysbiosis associated with innate immune deficiency and its implications for liver disease The cases described above are examples of a liver-intrinsic role of microbial recognition and its association with disease pathogenesis. Recent studies, however, point to a new role of extrahepatic innate immune-microbial cross talk in the initiation and progression of liver disease. First evidence came from a report demonstrating that mice lacking TLR5, the receptor recognizing bacterial flagellin, develop features of metabolic syndrome as a consequence of altered microbial composition in the gut (Vijay-Kumar et al., 2010). Although a recent study has argued that familial transmission, rather than genetic deficiency, might be the dominant driver of dysbiosis in mice (Ubeda et al., 2012), the intriguing notion that defective host–microbiome interactions in the intestine might have consequences that are not limited to regulating inflammation in the gastrointestinal tract, but rather affect systemic metabolism and liver disease, has prompted further investigation. We recently found that the intestinal tracts of mice deficient in the inflammasome components NLRP3, NLRP6, ASC, and Caspase-1, as well as mice lacking the downstream effector cytokine IL-18, harbor an aberrant microbial community which is characterized by the overrepresentation of anaerobic bacterial species of the Prevotellaceae family and the candidate phylum TM7 (Elinav, Strowig, Kau, et al., 2011). This indicates that 88 Jorge Henao-Mejia et al. inflammasomeactivityintheintestineisrequiredforthemaintenanceofastable microflora composition, partially through the secretion of IL-18. The altered microbiota found in inflammasome-deficient mice was transferable to wild- type mice upon cohousing in the same cage, demonstrating a dominant population effect, and was reversible upon antibiotic treatment. The biogeograph- ical niche enabling the outgrowth of Prevotellaceae seemed to be the area close to the colonic epithelial layer and the colonic crypts, an area which is normally less densely colonized with microbes due to mechanisms involving antimicrobial peptide production and mucus secretion. This altered intestinal flora leads to mild chronic inflammation and greatly predisposes to experimental colitis. Mechanistically, the colitogenic bacteria present in inflammasome-deficient mice leads to enhanced epithelial production of the chemokine CCL5, which in turn recruits proinflammatory immune cell populations to the intestinal lamina propria (Elinav, Strowig, Kau, et al., 2011). Most importantly, however, we found that the inflammatory processes regulated by the colitogenic flora were not limited to the regulation of local immune responses. When inflammasome-deficient mice were fed a methionine/choline-deficient diet, a model commonly used to induce NAFLD, they featured a dramatic outgrowth of bacterial species of the Porphyromonadaceae family and enhanced translocation of microbial products, in particular, TLR4 and TLR9 ligands, to the portal circulation (Henao- Mejia, Elinav, Jin, et al., 2012). Again, this increased microbial translocation across the gastrointestinal tract was dependent on dysbiosis-induced CCL5 production and intestinal inflammation. In the liver, the increased stimulation of TLR4 and TLR9 led to augmented production of TNF-a via MyD88/TRIF signaling, which initiated an inflammatory process leading to the development of NASH. The altered microbiota alone, when transferred from inflammasome- deficient or IL-18-deficient mice to wild-type recipients, was able to enhance susceptibility to NASH in a CCL5-, TLR4-, TLR9-, MyD88/TRIF-, and TNF-dependent manner, demonstratingthatdysbiosis,rather than genetic deficiency, was responsible for increased disease susceptibility and that metabolic disease might feature infectious, that is, transmissible microbial, components. Correspondingly, antibiotic treatment of inflammasome-deficient mice fed an MCD diet not only ameliorated NASH severity but also inhibited transmission of the phenotype to wild-type recipients. Moreover, the abnormal microflora also influenced other manifestations of metabolic syndrome in other mouse models of disease. Genetically obese leptin receptor-deficient mice gained markedly more weight when cohoused with inflammasome-deficient mice and so did ASC-deficient The Intestinal Microbiota in Chronic Liver Disease 89 mice and cohoused wild-type mice fed a high-fat diet. Antibiotic treatment reversed not only weight gain but also fasting plasma insulin amounts and glucose intolerance to normal levels, showing the strong influence of the microbial component on systemic metabolic parameters (Henao-Mejia, Elinav, Jin, et al., 2012). These results demonstrated that homeostatic extrahepatic expression of PRRs is necessary to prevent the development of dysbiosis in the gastrointestinal tract, which in turn predisposes to liver disease via the tight anatom- ical connection between both organ systems (Fig. 3.2). They also provide an example where multistage host–microbial interactions via different kinds of PRRs and their downstream signaling are involved in disease progression, both at distal (in this case, inflammasomes) and proximal sites (in this case, TLRs). The changes induced by the colitogenic microflora affect inflammatory processes locally (induction of CCL5 and leukocyte recruitment to the intestine), at the most proximal sites draining the intestine (inflammatory cytokine production in the liver), and even beyond (multiorgan regulation of weight gain and insulin sensitivity).

4. PROBIOTICS AND THEIR POTENTIAL ROLE IN LIVER DISEASE THERAPY The recognition of the importance of dysbiosis in the development and progression of liver disease opens new avenues for the development of therapeutic approaches. Similar to diseases in which a contribution of dysbiosis has long been appreciated, such as IBD, therapeutic intervention with the aim of adjusting the composition of the intestinal microflora might prove a valuable tool in the treatment of liver diseases. Probiotics and prebiotics are actively exploited for their therapeutic effects in IBD (DuPont & DuPont, 2011). Probiotics are live microorganisms given as dietary supplements to modify their relative representation in the intestinal ecosystem, while prebiotics are nondigestible dietary substances which promote the growth of one or more types of microorganisms in the gut, with the aim of increasing their relative abundance in the intestinal microflora. The benefits of pro- and prebiotics include direct effects, such as the increased release of metabolic products, and indirect effects, in particular, through microbe–microbe interactions and changes in population dynamics of intestinal microbial communities. Interestingly, the initial studies have demonstrated beneficial effects of probiotic interventions in liver disease. In a rat model of liver damage 90 Jorge Henao-Mejia et al. provoked by ischemia–reperfusion, intestinal dysbiosis was observed, including the outgrowth of Enterobacteriaceae and a decrease in Bacteroides spp., Lactobacillus spp., and Bifidobacter spp. These changes could be reversed by dietary supplementation with Lactobacillus paracasei, which remarkably led to reduced liver inflammation, as evidenced by ameliorated production of the proinflammatory cytokines IL-1b, IL-6, and TNF-a (Nardone et al., 2010). Similarly, after chemical liver injury, probiotic therapy with Lactobacillus spp. reduced hepatic inflammation by supporting intestinal barrier function and reducing microbial translocation across the gastrointestinal tract (Osman, Adawi, Ahrne, Jeppsson, & Molin, 2007). Interestingly, in our inflammasome dysbiosis mouse model, representation of Lactobacillus was significantly reduced (Elinav, Strowig, Kau, et al., 2011), pointing toward potential involvement of this commensal family in prevention of local mucosal inflammation and the related tendency toward systemic metabolic complications. Indeed, probiotic interventions were shown to influence hepatic metabolism, as was demonstrated in a rat model of high-cholesterol diet, in which Lactobacillus spp. supplementation in the food reduced the levels of cholesterol and triglycerides in the liver (Xie et al., 2011). Further, HE in cirrhotic patients was ameliorated by probiotic therapy leading to decreased representation of E. coli and reduced blood ammonia levels (Liu et al., 2004). Future studies are clearly needed to understand the mechanisms by which dietary manipulation of the intestinal ecosystem exerts its effects on liver metabolism. Gnotobiotic mice represent an excellent tool to study the contribution of individual microorganisms and their metabolic pathways to liver function.

5. CONCLUSIONS The mesenteric lymph node is the “first pass” organ for nutrients and microbial substances entering the lymph fluid in the intestinal lamina propria. As such, it serves as a key site for tolerance induction to food particles but at the same time acts as a firewall to prevent systemic spread of microorganisms. Similarly, the liver is exposed to all substances leaving the gastrointestinal tract via the portal blood circulation and faces similar challenges balancing tolerance to innocuous particles draining from the intestine and barrier function to potentially harmful microbial substances. In contrast to the mesenteric lymph node, the liver is the body’s prime metabolic organ, and any aberrations from the homeostatic state of host–microbial interactions in the liver may affect its metabolic functions. The Intestinal Microbiota in Chronic Liver Disease 91

We are convinced that the realization that both intrahepatic and extrahepatic host–microbial interactions, and in particular, innate immune system–microflora interactions, drastically influence systemic physiologic and pathophysiologic processes will guide future efforts to exploit this new insight in preclinical and clinical settings.

REFERENCES Arumugam, M., Raes, J., Pelletier, E., Le Paslier, D., Yamada, T., Mende, D. R., et al. (2011). Enterotypes of the human gut microbiome. Nature, 473, 174–180. Bajaj, J. S. (2010). Review article: The modern management of hepatic encephalopathy. Alimentary Pharmacology & Therapeutics, 31, 537–547. Bajaj, J. S., Heuman, D. M., Wade, J. B., Gibson, D. P., Saeian, K., Wegelin, J. A., et al. (2011). Rifaximin improves driving simulator performance in a randomized trial of patients with minimal hepatic encephalopathy. Gastroenterology, 140, 478–487 e471. Bajaj, J. S., Ridlon, J. M., Hylemon, P. B., Thacker, L. R., Heuman, D. M., Smith, S., et al. (2012). Linkage of gut microbiome with cognition in hepatic encephalopathy. American Journal of Physiology. Gastrointestinal and Liver Physiology, 302, G168–G175. Bambha, K., Kim, W. R., Talwalkar, J., Torgerson, H., Benson, J. T., Therneau, T. M., et al. (2003). Incidence, clinical spectrum, and outcomes of primary sclerosing cholangitis in a United States community. Gastroenterology, 125, 1364–1369. Basile, A. S., & Jones, E. A. (1997). Ammonia and GABA-ergic neurotransmission: Interre- lated factors in the pathogenesis of hepatic encephalopathy. Hepatology, 25, 1303–1305. Bass, N. M., Mullen, K. D., Sanyal, A., Poordad, F., Neff, G., Leevy, C. B., et al. (2010). Rifaximin treatment in hepatic encephalopathy. The New England Journal of Medicine, 362, 1071–1081. Bauer, T. M., Steinbruckner, B., Brinkmann, F. E., Ditzen, A. K., Schwacha, H., Aponte, J. J., et al. (2001). Small intestinal bacterial overgrowth in patients with cirrhosis: Prevalence and relation with spontaneous bacterial peritonitis. The American Journal of Gastroenterology, 96, 2962–2967. Bergquist, A., Montgomery, S. M., Bahmanyar, S., Olsson, R., Danielsson, A., Lindgren, S., et al. (2008). Increased risk of primary sclerosing cholangitis and ulcerative colitis in first- degree relatives of patients with primary sclerosing cholangitis. Clinical Gastroenterology and Hepatology, 6, 939–943. Boaru, S. G., Borkham-Kamphorst, E., Tihaa, L., Haas, U., & Weiskirchen, R. (2012). Expression analysis of inflammasomes in experimental models of inflammatory and fibrotic liver disease. Journal of Inflammation (London), 9, 49. Bogdanos, D. P., Baum, H., Grasso, A., Okamoto, M., Butler, P., Ma, Y., et al. (2004). Microbial mimics are major targets of crossreactivity with human pyruvate dehydrogenase in primary biliary cirrhosis. Journal of Hepatology, 40, 31–39. Bogdanos, D. P., Baum, H., Okamoto, M., Montalto, P., Sharma, U. C., Rigopoulou, E. I., et al. (2005). Primary biliary cirrhosis is characterized by IgG3 antibodies cross-reactive with the major mitochondrial autoepitope and its Lactobacillus mimic. Hepatology, 42, 458–465. Caldwell, S. H., Oelsner, D. H., Iezzoni, J. C., Hespenheide, E. E., Battle, E. H., & Driscoll, C. J. (1999). Cryptogenic cirrhosis: Clinical characterization and risk factors for underlying disease. Hepatology, 29, 664–669. Campillo, B., Pernet, P., Bories, P. N., Richardet, J. P., Devanlay, M., & Aussel, C. (1999). Intestinal permeability in liver cirrhosis: Relationship with severe septic complications. European Journal of Gastroenterology & Hepatology, 11, 755–759. 92 Jorge Henao-Mejia et al.

Carter, B. A., & Karpen, S. J. (2007). Intestinal failure-associated liver disease: Management and treatment strategies past, present, and future. Seminars in Liver Disease, 27, 251–258. Carvalho, F. A., Aitken, J. D., Vijay-Kumar, M., & Gewirtz, A. T. (2012). Toll-like receptor-gut microbiota interactions: Perturb at your own risk!. Annual Review of Phys- iology, 74, 177–198. Cerda, C., Perez-Ayuso, R. M., Riquelme, A., Soza, A., Villaseca, P., Sir-Petermann, T., et al. (2007). Nonalcoholic fatty liver disease in women with polycystic ovary syndrome. Journal of Hepatology, 47, 412–417. Charlton, M. (2008). Cirrhosis and liver failure in nonalcoholic fatty liver disease: Molehill or mountain? Hepatology, 47, 1431–1433. Chen, Y., Yang, F., Lu, H., Wang, B., Lei, D., Wang, Y., et al. (2011). Characterization of fecal microbial communities in patients with liver cirrhosis. Hepatology, 54, 562–572. Claesson, M. J., Jeffery, I. B., Conde, S., Power, S. E., O’Connor, E. M., Cusack, S., et al. (2012). Gut microbiota composition correlates with diet and health in the elderly. Nature, 488, 178–184. Corrodi, P. (1984). Jejunoileal bypass: Change in the flora of the small intestine and its clinical impact. Reviews of Infectious Diseases, 6(Suppl. 1), S80–S84. Csak, T., Ganz, M., Pespisa, J., Kodys, K., Dolganiuc, A., & Szabo, G. (2011). Fatty acid and endotoxin activate inflammasomes in mouse hepatocytes that release danger signals to stimulate immune cells. Hepatology, 54, 133–144. Dapito, D. H., Mencin, A., Gwak, G. Y., Pradere, J. P., Jang, M. K., Mederacke, I., et al. (2012). Promotion of hepatocellular carcinoma by the intestinal microbiota and TLR4. Cancer Cell, 21, 504–516. Day, C. P., & James, O. F. (1998). Steatohepatitis: A tale of two “hits”? Gastroenterology, 114, 842–845. De Filippo, C., Cavalieri, D., Di Paola, M., Ramazzotti, M., Poullet, J. B., Massart, S., et al. (2010). Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. In: Proceedings of the National Academy of Sciences of the United States of America, 107, 14691–14696. DuPont, A. W., & DuPont, H. L. (2011). The intestinal microbiota and chronic disorders of the gut. Nature Reviews. Gastroenterology & Hepatology, 8, 523–531. Elinav, E., Strowig, T., Henao-Mejia, J., & Flavell, R. A. (2011). Regulation of the antimicrobial response by NLR proteins. Immunity, 34, 665–679. Elinav, E., Strowig, T., Kau, A. L., Henao-Mejia, J., Thaiss, C. A., Booth, C. J., et al. (2011). NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis. Cell, 145, 745–757. Figueroa, L., Xiong, Y., Song, C., Piao, W., Vogel, S. N., & Medvedev, A. E. (2012). The Asp299Gly polymorphism alters TLR4 signaling by interfering with recruitment of MyD88 and TRIF. The Journal of Immunology, 188, 4506–4515. Ganz, M., Csak, T., Nath, B., & Szabo, G. (2011). Lipopolysaccharide induces and activates the Nalp3 inflammasome in the liver. World Journal of Gastroenterology, 17, 4772–4778. Gao, B., Seki, E., Brenner, D. A., Friedman, S., Cohen, J. I., Nagy, L., et al. (2011). Innate immunity in alcoholic liver disease. American Journal of Physiology. Gastrointestinal and Liver Physiology, 300, G516–G525. Gross, O., Gewies, A., Finger, K., Schafer, M., Sparwasser, T., Peschel, C., et al. (2006). Card9 controls a non-TLR signalling pathway for innate anti-fungal immunity. Nature, 442, 651–656. Guarner, C., & Soriano, G. (1997). Spontaneous bacterial peritonitis. Seminars in Liver Dis- ease, 17, 203–217. Gunnarsdottir, S. A., Sadik, R., Shev, S., Simren, M., Sjovall, H., Stotzer, P. O., et al. (2003). Small intestinal motility disturbances and bacterial overgrowth in patients with liver cirrhosis and portal hypertension. The American Journal of Gastroenterology, 98, 1362–1370. The Intestinal Microbiota in Chronic Liver Disease 93

Hashimoto, E., Ideta, M., Taniai, M., Watanabe, U., Okuda, H., Nagasako, K., et al. (1993). Prevalence of primary sclerosing cholangitis and other liver diseases in Japanese patients with chronic ulcerative colitis. Journal of Gastroenterology and Hepatology, 8, 146–149. Henao-Mejia, J., Elinav, E., Jin, C., Hao, L., Mehal, W. Z., Strowig, T., et al. (2012). Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity. Nature, 482, 179–185. Henao-Mejia, J., Elinav, E., Strowig, T., & Flavell, R. A. (2012). Inflammasomes: Far beyond inflammation. Nature Immunology, 13, 321–324. Hjelkrem, M. C., Torres, D. M., & Harrison, S. A. (2008). Nonalcoholic fatty liver disease. Minerva Medica, 99, 583–593. Hopf, U., Moller, B., Stemerowicz, R., Lobeck, H., Rodloff, A., Freudenberg, M., et al. (1989). Relation between Escherichia coli R(rough)-forms in gut, lipid A in liver, and primary biliary cirrhosis. The Lancet, 2, 1419–1422. Hosel, M., Broxtermann, M., Janicki, H., Esser, K., Arzberger, S., Hartmann, P., et al. (2012). Toll-like receptor 2-mediated innate immune response in human non- parenchymal liver cells toward adeno-associated viral vectors. Hepatology, 55, 287–297. Huang, H., Shiffman, M. L., Friedman, S., Venkatesh, R., Bzowej, N., Abar, O. T., et al. (2007). A 7 gene signature identifies the risk of developing cirrhosis in patients with chronic hepatitis C. Hepatology, 46, 297–306. Husova, L., Lata, J., Husa, P., Senkyrik, M., Jurankova, J., & Dite, P. (2005). Bacterial infection and acute bleeding from upper gastrointestinal tract in patients with liver cirrhosis. Hepato-Gastroenterology, 52, 1488–1490. Iliev, I. D., Funari, V. A., Taylor, K. D., Nguyen, Q., Reyes, C. N., Strom, S. P., et al. (2012). Interactions between commensal fungi and the C-type lectin receptor Dectin-1 influence colitis. Science, 336, 1314–1317. Imaeda, A. B., Watanabe, A., Sohail, M. A., Mahmood, S., Mohamadnejad, M., Sutterwala, F. S., et al. (2009). Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome. The Journal of Clinical Investigation, 119, 305–314. Janse, M., Lamberts, L. E., Franke, L., Raychaudhuri, S., Ellinghaus, E., Muri Boberg, K., et al. (2011). Three ulcerative colitis susceptibility loci are associated with primary sclerosing cholangitis and indicate a role for IL2, REL, and CARD9. Hepatology, 53, 1977–1985. Joo, M., Abreu-e-Lima, P., Farraye, F., Smith, T., Swaroop, P., Gardner, L., et al. (2009). Pathologic features of ulcerative colitis in patients with primary sclerosing cholangitis: A case-control study. The American Journal of Surgical Pathology, 33, 854–862. Kawai, T., & Akira, S. (2010). The role of pattern-recognition receptors in innate immunity: Update on Toll-like receptors. Nature Immunology, 11, 373–384. Kerrigan, A. M., & Brown, G. D. (2011). Syk-coupled C-type lectins in immunity. Trends in Immunology, 32, 151–156. Latz, E. (2010). The inflammasomes: Mechanisms of activation and function. Current Opinion in Immunology, 22, 28–33. Lech, M., Susanti, H. E., Rommele, C., Grobmayr, R., Gunthner, R., & Anders, H. J. (2012). Quantitative expression of C-type lectin receptors in humans and mice. Interna- tional Journal of Molecular Sciences, 13, 10113–10131. Levy, C., & Lindor, K. D. (2006). Primary sclerosing cholangitis: Epidemiology, natural his- tory, and prognosis. Seminars in Liver Disease, 26, 22–30. Ley, R. E., Backhed, F., Turnbaugh, P., Lozupone, C. A., Knight, R. D., & Gordon, J. I. (2005). Obesity alters gut microbial ecology. In: Proceedings of the National Academy of Sciences of the United States of America, 102, 11070–11075. Ley, R. E., Turnbaugh, P. J., Klein, S., & Gordon, J. I. (2006). Microbial ecology: Human gut microbes associated with obesity. Nature, 444, 1022–1023. 94 Jorge Henao-Mejia et al.

Li, L., Chen, L., Hu, L., Liu, Y., Sun, H. Y., Tang, J., et al. (2011). Nuclear factor high- mobility group box1 mediating the activation of Toll-like receptor 4 signaling in hepatocytes in the early stage of nonalcoholic fatty liver disease in mice. Hepatology, 54, 1620–1630. Lin, Y., Yu, L. X., Yan, H. X., Yang, W., Tang, L., Zhang, H. L., et al. (2012). Gut-derived lipopolysaccharide promotes T-cell-mediated hepatitis in mice through Toll-like receptor 4. Cancer Prevention Research (Philadelphia, PA), 5, 1090–1102. Liu, Q., Duan, Z. P., Ha, D. K., Bengmark, S., Kurtovic, J., & Riordan, S. M. (2004). Syn- biotic modulation of gut flora: Effect on minimal hepatic encephalopathy in patients with cirrhosis. Hepatology, 39, 1441–1449. Lozupone, C. A., Stombaugh, J. I., Gordon, J. I., Jansson, J. K., & Knight, R. (2012). Diver- sity, stability and resilience of the human gut microbiota. Nature, 489, 220–230. Ludwig, J., Viggiano, T. R., McGill, D. B., & Oh, B. J. (1980). Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed disease. Mayo Clinic Proceedings, 55, 434–438. Marchesini, G., Bugianesi, E., Forlani, G., Cerrelli, F., Lenzi, M., Manini, R., et al. (2003). Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome. Hepatology, 37, 917–923. Maynard, C. L., Elson, C. O., Hatton, R. D., & Weaver, C. T. (2012). Reciprocal interactions of the intestinal microbiota and immune system. Nature, 489, 231–241. Michelsen, K. S., & Arditi, M. (2007). Toll-like receptors and innate immunity in gut homeostasis and pathology. Current Opinion in Hematology, 14, 48–54. Miele, L., Valenza, V., La Torre, G., Montalto, M., Cammarota, G., Ricci, R., et al. (2009). Increased intestinal permeability and tight junction alterations in nonalcoholic fatty liver disease. Hepatology, 49, 1877–1887. Miyake, Y., & Yamamoto, K. (2013). Role of gut microbiota in liver diseases. Hepatology Research, 43(2), 139–146. Muegge, B. D., Kuczynski, J., Knights, D., Clemente, J. C., Gonzalez, A., Fontana, L., et al. (2011). Diet drives convergence in gut microbiome functions across mammalian phylog- eny and within humans. Science, 332, 970–974. Mueller, T., Beutler, C., Pico, A. H., Shibolet, O., Pratt, D. S., Pascher, A., et al. (2011). Enhanced innate immune responsiveness and intolerance to intestinal endotoxins in human biliary epithelial cells contributes to chronic cholangitis. Liver International, 31, 1574–1588. Mulder, A. H., Horst, G., Haagsma, E. B., Limburg, P. C., Kleibeuker, J. H., & Kallenberg, C. G. (1993). Prevalence and characterization of neutrophil cytoplasmic antibodies in autoimmune liver diseases. Hepatology, 17, 411–417. Nardone, G., Compare, D., Liguori, E., Di Mauro, V., Rocco, A., Barone, M., et al. (2010). Protective effects of Lactobacillus paracasei F19 in a rat model of oxidative and metabolic hepatic injury. American Journal of Physiology. Gastrointestinal and Liver Physiology, 299, G669–G676. Nazim, M., Stamp, G., & Hodgson, H. J. (1989). Non-alcoholic steatohepatitis associated with small intestinal diverticulosis and bacterial overgrowth. Hepato-Gastroenterology, 36, 349–351. Nordenstedt, H., White, D. L., & El-Serag, H. B. (2010). The changing pattern of epidemiology in hepatocellular carcinoma. Digestive and Liver Disease, 42(Suppl. 3), S206–S214. O’Toole, A., Alakkari, A., Keegan, D., Doherty, G., Mulcahy, H., & O’Donoghue, D. (2012). Primary sclerosing cholangitis and disease distribution in inflammatory bowel disease. Clinical Gastroenterology and Hepatology, 10, 439–441. Osman, N., Adawi, D., Ahrne, S., Jeppsson, B., & Molin, G. (2007). Endotoxin- and D- galactosamine-induced liver injury improved by the administration of Lactobacillus, Bifidobacterium and blueberry. Digestive and Liver Disease, 39, 849–856. The Intestinal Microbiota in Chronic Liver Disease 95

Pappo, I., Bercovier, H., Berry, E. M., Haviv, Y., Gallily, R., & Freund, H. R. (1992). Poly- myxin B reduces total parenteral nutrition-associated hepatic steatosis by its antibacterial activity and by blocking deleterious effects of lipopolysaccharide. Journal of Parenteral and Enteral Nutrition, 16, 529–532. Petrasek, J., Dolganiuc, A., Csak, T., Kurt-Jones, E. A., & Szabo, G. (2011). Type I interferons protect from Toll-like receptor 9-associated liver injury and regulate IL-1 receptor antagonist in mice. Gastroenterology, 140, 697–708 e694. Petrasek, J., Dolganiuc, A., Csak, T., Nath, B., Hritz, I., Kodys, K., et al. (2011). Interferon regulatory factor 3 and type I interferons are protective in alcoholic liver injury in mice by way of crosstalk of parenchymal and myeloid cells. Hepatology, 53, 649–660. Propst, A., Propst, T., Judmaier, G., & Vogel, W. (1995). Prognosis in nonalcoholic steatohepatitis. Gastroenterology, 108, 1607. Qin, J., Li, Y., Cai, Z., Li, S., Zhu, J., Zhang, F., et al. (2012). A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature, 490, 55–60. Quigley, E. M., Marsh, M. N., Shaffer, J. L., & Markin, R. S. (1993). Hepatobiliary complications of total parenteral nutrition. Gastroenterology, 104, 286–301. Ritter, M., Gross, O., Kays, S., Ruland, J., Nimmerjahn, F., Saijo, S., et al. (2010). Schistosoma mansoni triggers Dectin-2, which activates the Nlrp3 inflammasome and alters adaptive immune responses. In: Proceedings of the National Academy of Sciences of the United States of America, 107, 20459–20464. Rudolph, G., Gotthardt, D., Kloters-Plachky, P., Kulaksiz, H., Rost, D., & Stiehl, A. (2009). Influence of dominant bile duct stenoses and biliary infections on outcome in primary sclerosing cholangitis. Journal of Hepatology, 51, 149–155. Sabate, J. M., Jouet, P., Harnois, F., Mechler, C., Msika, S., Grossin, M., et al. (2008). High prevalence of small intestinal bacterial overgrowth in patients with morbid obesity: A contributor to severe hepatic steatosis. Obesity Surgery, 18, 371–377. Sajjad, A., Mottershead, M., Syn, W. K., Jones, R., Smith, S., & Nwokolo, C. U. (2005). Ciprofloxacin suppresses bacterial overgrowth, increases fasting insulin but does not correct low acylated ghrelin concentration in non-alcoholic steatohepatitis. Alimentary Phar- macology & Therapeutics, 22, 291–299. Sancho, D., & Reis e Sousa, C. (2012). Signaling by myeloid C-type lectin receptors in immunity and homeostasis. Annual Review of Immunology, 30, 491–529. Sano, H., Nakazawa, T., Ando, T., Hayashi, K., Naitoh, I., Okumura, F., et al. (2011). Clinical characteristics of inflammatory bowel disease associated with primary sclerosing cholangitis. Journal of Hepato-Biliary-Pancreatic Sciences, 18, 154–161. Sanyal, A. J., Campbell-Sargent, C., Mirshahi, F., Rizzo, W. B., Contos, M. J., Sterling, R. K., et al. (2001). Nonalcoholic steatohepatitis: Association of insulin resistance and mitochondrial abnormalities. Gastroenterology, 120, 1183–1192. Seki, E., De Minicis, S., Osterreicher, C. H., Kluwe, J., Osawa, Y., Brenner, D. A., et al. (2007). TLR4 enhances TGF-beta signaling and hepatic fibrosis. Nature Medicine, 13, 1324–1332. Selmi, C., Balkwill, D. L., Invernizzi, P., Ansari, A. A., Coppel, R. L., Podda, M., et al. (2003). Patients with primary biliary cirrhosis react against a ubiquitous xenobiotic- metabolizing bacterium. Hepatology, 38, 1250–1257. Sheth, S. G., Gordon, F. D., & Chopra, S. (1997). Nonalcoholic steatohepatitis. Annals of Internal Medicine, 126, 137–145. Shimada, M., Hashimoto, E., Taniai, M., Hasegawa, K., Okuda, H., Hayashi, N., et al. (2002). Hepatocellular carcinoma in patients with non-alcoholic steatohepatitis. Journal of Hepatology, 37, 154–160. Sidhu, S. S., Goyal, O., Mishra, B. P., Sood, A., Chhina, R. S., & Soni, R. K. (2011). Rifaximin improves psychometric performance and health-related quality of life in patients with minimal hepatic encephalopathy (the RIME Trial). The American Journal of Gastroenterology, 106, 307–316. 96 Jorge Henao-Mejia et al.

Slack, E., Hapfelmeier, S., Stecher, B., Velykoredko, Y., Stoel, M., Lawson, M. A., et al. (2009). Innate and adaptive immunity cooperate flexibly to maintain host-microbiota mutualism. Science, 325, 617–620. Strowig, T., Henao-Mejia, J., Elinav, E., & Flavell, R. (2012). Inflammasomes in health and disease. Nature, 481, 278–286. Sung, J. Y., Shaffer, E. A., & Costerton, J. W. (1993). Antibacterial activity of bile salts against common biliary pathogens. Effects of hydrophobicity of the molecule and in the presence of phospholipids. Digestive Diseases and Sciences, 38, 2104–2112. Terjung, B., Herzog, V., Worman, H. J., Gestmann, I., Bauer, C., Sauerbruch, T., et al. (1998). Atypical antineutrophil cytoplasmic antibodies with perinuclear fluorescence in chronic inflammatory bowel diseases and hepatobiliary disorders colocalize with nuclear lamina proteins. Hepatology, 28, 332–340. Terjung, B., Sohne, J., Lechtenberg, B., Gottwein, J., Muennich, M., Herzog, V., et al. (2010). p-ANCAs in autoimmune liver disorders recognise human beta-tubulin isotype 5 and cross-react with microbial protein FtsZ. Gut, 59, 808–816. Thalheimer, U., Triantos, C. K., Samonakis, D. N., Patch, D., & Burroughs, A. K. (2005). Infection, coagulation, and variceal bleeding in cirrhosis. Gut, 54, 556–563. Thuy, S., Ladurner, R., Volynets, V., Wagner, S., Strahl, S., Konigsrainer, A., et al. (2008). Nonalcoholic fatty liver disease in humans is associated with increased plasma endotoxin and plasminogen activator inhibitor 1 concentrations and with fructose intake. The Jour- nal of Nutrition, 138, 1452–1455. Tolman, K. G., Fonseca, V., Dalpiaz, A., & Tan, M. H. (2007). Spectrum of liver disease in type 2 diabetes and management of patients with diabetes and liver disease. Diabetes Care, 30, 734–743. Tremaroli, V., & Backhed, F. (2012). Functional interactions between the gut microbiota and host metabolism. Nature, 489, 242–249. Tsutsui, H., Imamura, M., Fujimoto, J., & Nakanishi, K. (2010). The TLR4/TRIF- mediated activation of NLRP3 inflammasome underlies endotoxin-induced liver injury in mice. Gastroenterology Research and Practice, 2010, 641865. Turnbaugh, P. J., Hamady, M., Yatsunenko, T., Cantarel, B. L., Duncan, A., Ley, R. E., et al. (2009). A core gut microbiome in obese and lean twins. Nature, 457, 480–484. Turnbaugh, P. J., Ley, R. E., Mahowald, M. A., Magrini, V., Mardis, E. R., & Gordon, J. I. (2006). An obesity-associated gut microbiome with increased capacity for energy harvest. Nature, 444, 1027–1031. Ubeda, C., Lipuma, L., Gobourne, A., Viale, A., Leiner, I., Equinda, M., et al. (2012). Familial transmission rather than defective innate immunity shapes the distinct intestinal microbiota of TLR-deficient mice. The Journal of Experimental Medicine, 209, 1445–1456. Vanderhoof, J. A., Tuma, D. J., Antonson, D. L., & Sorrell, M. F. (1982). Effect of antibiotics in the prevention of jejunoileal bypass-induced liver dysfunction. Digestion, 23, 9–15. Vijay-Kumar, M., Aitken, J. D., Carvalho, F. A., Cullender, T. C., Mwangi, S., Srinivasan, S., et al. (2010). Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor 5. Science, 328, 228–231. Visvanathan, K., Skinner, N. A., Thompson, A. J., Riordan, S. M., Sozzi, V., Edwards, R., et al. (2007). Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein. Hepatology, 45, 102–110. Wang, A. P., Migita, K., Ito, M., Takii, Y., Daikoku, M., Yokoyama, T., et al. (2005). Hepatic expression of toll-like receptor 4 in primary biliary cirrhosis. Journal of Autoim- munity, 25, 85–91. Wang, B., Trippler, M., Pei, R., Lu, M., Broering, R., Gerken, G., et al. (2009). Toll-like receptor activated human and murine hepatic stellate cells are potent regulators of hepatitis C virus replication. Journal of Hepatology, 51, 1037–1045. The Intestinal Microbiota in Chronic Liver Disease 97

Washington, M. K. (2007). Autoimmune liver disease: Overlap and outliers. Modern Pathol- ogy, 20(Suppl. 1), S15–S30. Watanabe, A., Sohail, M. A., Gomes, D. A., Hashmi, A., Nagata, J., Sutterwala, F. S., et al. (2009). Inflammasome-mediated regulation of hepatic stellate cells. American Journal of Physiology. Gastrointestinal and Liver Physiology, 296, G1248–G1257. Wigg, A. J., Roberts-Thomson, I. C., Dymock, R. B., McCarthy, P. J., Grose, R. H., & Cummins, A. G. (2001). The role of small intestinal bacterial overgrowth, intestinal permeability, endotoxaemia, and tumour necrosis factor alpha in the pathogenesis of nonalcoholic steatohepatitis. Gut, 48, 206–211. Williams, R. (2007). Review article: Bacterial flora and pathogenesis in hepatic encephalopathy. Alimentary Pharmacology & Therapeutics, 25(Suppl. 1), 17–22. Williams, C. D., Farhood, A., & Jaeschke, H. (2010). Role of caspase-1 and interleukin- 1beta in acetaminophen-induced hepatic inflammation and liver injury. Toxicology and Applied Pharmacology, 247, 169–178. Wu, G. D., Chen, J., Hoffmann, C., Bittinger, K., Chen, Y. Y., Keilbaugh, S. A., et al. (2011). Linking long-term dietary patterns with gut microbial enterotypes. Science, 334, 105–108. Xie, N., Cui, Y., Yin, Y. N., Zhao, X., Yang, J. W., Wang, Z. G., et al. (2011). Effects of two Lactobacillus strains on lipid metabolism and intestinal microflora in rats fed a high- cholesterol diet. BMC Complementary and Alternative Medicine, 11, 53. Ye, B. D., Yang, S. K., Boo, S. J., Cho, Y. K., Yang, D. H., Yoon, S. M., et al. (2011). Clinical characteristics of ulcerative colitis associated with primary sclerosing cholangitis in Korea. Inflammatory Bowel Diseases, 17, 1901–1906. Yokoyama, T., Komori, A., Nakamura, M., Takii, Y., Kamihira, T., Shimoda, S., et al. (2006). Human intrahepatic biliary epithelial cells function in innate immunity by producing IL-6 and IL-8 via the TLR4-NF-kappaB and -MAPK signaling pathways. Liver International, 26, 467–476. Younossi, Z. M., Stepanova, M., Afendy, M., Fang, Y., Younossi, Y., Mir, H., et al. (2011). Changes in the prevalence of the most common causes of chronic liver diseases in the United States from 1988 to 2008. Clinical Gastroenterology and Hepatology, 9, 524–530, e521; quiz e560. Zhu, P., Duan, L., Chen, J., Xiong, A., Xu, Q., Zhang, H., et al. (2011). Gene silencing of NALP3 protects against liver ischemia-reperfusion injury in mice. Human Gene Therapy, 22, 853–864. Intentionally left as blank CHAPTER FOUR

Intracellular Pathogen Detection by RIG-I-Like Receptors

Evelyn Dixit, Jonathan C. Kagan1 Harvard Medical School and Division of Gastroenterology, Boston Children’s Hospital, Boston, Massachusetts, USA 1Corresponding author: e-mail address: [email protected]

Contents 1. General Principles of the Antiviral Innate Immune Response 99 2. RLRs are RNA Sensors 101 2.1 Common and distinct features of RLRs and their signaling capabilities 101 2.2 Structural characteristics of synthetic RLR ligands 104 2.3 Viruses 106 2.4 Bacteria 108 3. RIG-I Activation and Receptor Proximal Signal Propagation 109 4. Regulatory Mechanisms of RIG-I Signaling 113 4.1 Regulators of RLR signaling 113 4.2 Regulation of RLR signal transduction by subcellular compartmentalization 115 5. Conclusions and Future Directions 117 Acknowledgments 118 References 118

Abstract The RIG-I-like receptors (RLRs) RIG-I, MDA5, and LGP2 trigger innate immune responses againstviralinfectionsthatservetolimitvirusreplicationandtostimulateadaptiveimmunity. RLRsare cytosolic sensors for virus-derivedRNA and thusresponsible for intracellular immune surveillance against infection. RLR signaling requires the adapter protein MAVS to induce type I interferon, interferon-stimulated genes, and proinflammatory cytokines. This review focuses on the molecular and cell biological requirements for RLR signal transduction.

1. GENERAL PRINCIPLES OF THE ANTIVIRAL INNATE IMMUNE RESPONSE Viruses are obligate intracellular parasites and thus depend strictly on the biosynthetic machinery of the host in order to replicate and spread. As a result, the virus-driven exploitation of the host cell’s metabolic pathways and

Advances in Immunology, Volume 117 # 2013 Elsevier Inc. 99 ISSN 0065-2776 All rights reserved. http://dx.doi.org/10.1016/B978-0-12-410524-9.00004-9 100 Evelyn Dixit and Jonathan C. Kagan reprogramming of cellular processes often lead to cell death. The struggle for survival between virus and host is ancient and as a consequence both have evolved multiple strategies to antagonize each other. While mammalian hosts developed sophisticated mechanisms of antiviral immunity, viruses acquired strategies to evade the immune response. Therefore, it is critical for the host to mount an effective innate and adaptive immune response immediately upon infection in order to successfully combat the pathogen. The innate immune response constitutes the earliest phase of the host’s defense against pathogens and will stimulate and modulate the later onset adaptive response (Palm & Medzhitov, 2009). It operates through a set of germ line-encoded pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) of viruses, bacteria, fungi, and protozoa. PAMPs are conserved within broad classes of pathogens. They are typically products of biosynthetic pathways that are essential for the survival of the pathogen and thus lack the potential for immune evasion through genetic variability (Medzhitov, 2007). Owing to the panel of PAMPs that is recognized by PRRs, the innate immune system achieves an impressively complete coverage of pathogens despite the genetically limited number of available receptors. Engagement of antiviral PRRs by their cognate PAMPs activates signaling pathways that lead to the production of defense factors such as proinflammatory cytokines, type I interferons (IFN-a and IFN-b), or interferon-stimulated genes (ISGs). ISGs induced by IFN secretion or cell-autonomously upon viral infection collectively establish an antiviral state that limits viral replication and prevents further spread of the infection (Katze, He, & Gale, 2002). Detection of viruses poses a particular challenge to the host as they lack features in line with the postulated characteristics of PAMPs, that is, invariant structures required for survival. With few exceptions, viral proteins are highly variable without being functionally compromised by mutation. Moreover, viruses are obligate parasites relying on the host metabolism for their replication. The evolutionary solution to this problem is to recognize viral nucleic acids, either virus genomes or replication intermediates. Undoubtedly, nucleic acid is not a PAMP that is unique to viruses and thus virus detection comes at the cost of the potential for autoimmunity (Barton & Kagan, 2009). Nucleic acid detection is accomplished by a growing list of PRRs, namely, the cytosolic RIG-I-like receptors (RLRs) RIG-I and MDA5 (Yoneyama et al., 2005, 2004); the endosomal Toll-like receptors TLR3, TLR7/8, TLR9, and TLR13 (Kawai & Akira, 2010); the Ifi16/ cGAS/STING axis (Ishikawa, Ma, & Barber, 2009; Sun, Wu, Du, Chen, & RIG-I-Like Receptor Signaling 101

Chen, 2012; Unterholzner et al., 2010; Wu et al., 2012); and the AIM2 inflammasome (Burckstummer et al., 2009; Fernandes-Alnemri, Yu, Datta, Wu, & Alnemri, 2009; Hornung et al., 2009; Roberts et al., 2009). This review will focus on virus-induced signaling by RLRs; nucleic acid sensing by other receptor families is reviewed elsewhere (Barbalat, Ewald, Mouchess, & Barton, 2011).

2. RLRs ARE RNA SENSORS 2.1. Common and distinct features of RLRs and their signaling capabilities RLRs detect RNA derived from RNA viruses and in some instances DNA viruses. In terms of specificity and signaling output, RLRs are most similar to TLR3, as both detect viral RNA and induce ISGs, type I IFN, and proinflammatory cytokines (Alexopoulou, Holt, Medzhitov, & Flavell, 2001; Matsumoto et al., 2003; Schulz et al., 2005). However, there is a fundamental conceptual difference in nucleic acid detection between TLRs and RLRs. The nucleic acid-specific endosomal TLRs TLR3, TLR7/8, and TLR9 recognize extracellular nucleic acids having reached the endosomes through endocytosis (Takeda & Akira, 2005), whereas RLRs are cytosolic receptors required for detection of intracellular viral RNA from actively replicating viruses (Kawai & Akira, 2006). As such, RLRs represent an indis- pensable means for determining if a given cell is infected or not. In line with this key role in antiviral immunity, RLR signaling operates in most cell types. In contrast, TLR expression is restricted to specialized immune cells such as macrophages and dendritic cells. Even though RLRs are expressed in plasmacytoid dendritic cells, TLRs but not RLRs are required for IFN-a production in this cell type (Kato et al., 2005). Three highly related proteins constitute the family of RLRs: the founding member RIG-I, MDA5, and LGP2. They are characterized by a central ATPase containing DExD/H box helicase domain. RIG-I and MDA5 contain N-terminal tandem CARD domains that mediate downstream signaling, whereas LGP2 lacks a CARD (Yoneyama et al., 2005, 2004). RIG-I and LGP2 also harbor a repressor domain (RD) in their C-terminal regulatory domains (CTDs) (Fig. 4.1). Due to the presence of the RD in RIG-I, its overexpression in the absence of an activating ligand does not result in signaling, whereas MDA5 overexpression is sufficient to activate the pathway. In accordance with their domain architecture, RLRs lacking the CARDs have a dominant negative phenotype. RIG-I devoid of 102 Evelyn Dixit and Jonathan C. Kagan

RIG-I CARD CARD ATPase/helicase RD/CTD 1 87 92 172 251 735 925

MDA5 CARD CARD ATPase/helicase RD-like 7 97 110 190 316 882 1025

LGP2 ATPase/helicase RD/CTD 11 476 678

MAVS CARD Pro TM 10 77 103 153 514 534 540 Figure 4.1 Domain architecture of RLRs and MAVS. Domain boundaries are indicated for human RIG-I, MDA5, LGP2, and MAVS proteins according to www.uniprot.org. Note that MDA5 harbors an RD-like domain in the C-terminus that does not participate in autoregulation. the CTD or the N-terminal fragment comprising solely the CARDs signal constitutively (Cui et al., 2008; Saito et al., 2007; Takahasi et al., 2008). All RLRs are present at low levels in resting cells, but their expression is strongly induced by type I IFN creating a feed forward loop for a robust antiviral response (Kang et al., 2004; Yoneyama et al., 2005, 2004). Despite different ligand specificities for viral RNA, both RIG-I and MDA5 rely on the same signaling cascade to trigger the expression of type I IFNs, ISGs, and proinflammatory cytokines (Yoneyama et al., 2005). The adapter protein MAVS (also known as IPS-1, VISA, and Cardif ) (Kawai et al., 2005; Meylan et al., 2005; Seth, Sun, Ea, & Chen, 2005; Xu et al., 2005) acts immediately downstream of the receptors and represents a node from which RLR signaling branches in several directions in order to promote the activation of NF-kB through the canonical IKKs, IKK-a, IKK-b, and IKK-g, of ATF2/c-jun through MAPK activation and most importantly of members of the interferon regulatory factor (IRF) family of transcription factors (Kawai et al., 2005; Meylan et al., 2005; Mikkelsen et al., 2009; Poeck et al., 2010; Seth et al., 2005; Xu et al., 2005). IRF3 and IRF7 are the essential transcription factors for IFN-b gene transcription, as activation of NF-kB and ATF-2/c-Jun alone is not sufficient for IFN-b induction. Interestingly, in dendritic cells, IRF5 can also function to promote IFN-b expression (Lazear et al., 2013). They reside in the cytosol in their latent forms until viral infection activates the non- canonical IKKs, TBK1 and IKK-i. Phosphorylation of IRF3 and IRF7 by these kinases causes hetero- or homodimerization and nuclear translocation. IRF3 and/or IRF7, NF-kB, and ATF-2/c-Jun together with RIG-I-Like Receptor Signaling 103 the transcriptional coactivator CBP/p300 and the architectural protein HMG I(Y) assemble in an enhanceosome to direct IFN-b transcription (Hiscott, 2007; Honda, Takaoka, & Taniguchi, 2006)(Fig. 4.2). Once IFN-b is secreted, it binds to the IFN-a/b receptor (IFNAR) in an autocrine and paracrine manner resulting in JAK-STAT signaling and thus expression of several hundred ISGs by the ISGF3 transcription factor, which consists of STAT1, STAT2, and IRF9 (Platanias, 2005). However, despite their namesake, ISGs may also be induced independent of a preceding secretion of type I IFN (Collins, Noyce, & Mossman, 2004; Mossman et al., 2001).

Influenza virus WNV EMCV NDV Dengue virus Theiler’s virus SeV Reovirus VSV HCV JEV PPP Cytoplasm TRIM25 Riplet RIG-I MDA-5 RNF125 PKC-a/b NLRX1 MAVS

IKK-i IKK-a MAPKs ? TBK1 IKK-b IKK-g

IRF3/7 NF-kB ATF2/c-Jun

Type I IFN Cytokines ISGs Nucleus

Figure 4.2 RLR signaling on a glance. The repertoire of viruses detected by RIG-I and MDA5, respectively, reflects their different ligand specificities. Both receptors use common signaling components to activate three sets of transcription factors required for expression of type I IFN, proinflammatory cytokines, and ISGs. 104 Evelyn Dixit and Jonathan C. Kagan

Many ISGs function as direct antiviral effectors, acting to prevent viral genome replication, viral particle assembly, or virion release from infected cells. Others encode components of signaling pathways such as receptors for pathogen recognition or transcription factors resulting in a stronger IFN response and thereby creating a positive feedback loop. The role of LGP2 in antiviral immunity is less clear. LGP2 lacks a CARD domain (Fig. 4.1). Devoid of a signaling domain, LGP2 was proposed to be a negative regulator of RLR signaling. Overexpression of LGP2 does not activate IFN-b induction. On the contrary, reduced IRF3 activation was observed when LGP2 overexpressing cells were infected with Newcastle disease virus (NDV) (Rothenfusser et al., 2005; Yoneyama et al., 2005). In vivo experiments with different lines of LGP2-deficient mice strongly contradict the previous data generated by in vitro studies and implicate LGP2 as a positive regulator (Satoh et al., 2010; Venkataraman et al., 2007). In the absence of LGP2, both RIG-I and particularly MDA5-dependent responses to RNA virus infection are impaired, whereas responses to synthetic ligands of these RLRs are unaffected (Satoh et al., 2010). Presumably, LGP2 facilitates binding of viral RNA—potentially in complex with protein—to its cognate receptor, whereas the affinity of RIG-I and MDA5 is sufficiently strong to bind to “naked” synthetic agonists. Structural analysis of the binding interface of RNA with the CTD of RIG-I supports this model, as it predicts weaker affinity of MDA5 than RIG-I to its ligand (Takahasi et al., 2009). In addition to confirming the role of LGP2 as a positive, yet nonessential regulator of RLR signaling, a recent report implicates LGP2 as a cell-intrinsic regulator of virus-specific CD8þ T cell survival and effector functions. CD8þ T cells are crucial for controlling West Nile virus (WNV) pathology in the brain. LGP2-deficient mice displayed higher viral burden and significantly lower WNV-specific CD8þ T cells in the brain leading to increased mortality as compared to wild-type animals (Suthar et al., 2012). Nonethe- less, further clarification is required to determine the role of LGP2 in RLR signaling.

2.2. Structural characteristics of synthetic RLR ligands The two best characterized RLRs, RIG-I and MDA5, recognize structurally distinct RNA species that have reached the cytosol by infection or by means of transfection. Being cytosolic receptors, RIG-I and MDA5 do not respond to extracellular nucleic acid. RIG-I-Like Receptor Signaling 105

The RIG-I ligand comprises an RNA molecule with two features: (i) a 50-triphosphate (Hornung et al., 2006; Pichlmair et al., 2006) and (ii) base pairing at the 50-end due to secondary RNA structures such as hairpin or panhandle conformations (Schlee et al., 2009; Schmidt et al., 2009). Studies aimed at the characterization of molecular features of the RIG-I ligand largely rely on in vitro transcripts. In vitro-transcribed RNA by all known RNA poly- merasesleavesatriphosphateatthe50 endofthetranscript(pppRNA)(Schlee& Hartmann, 2010). Transfection of pppRNA into monocytes resulted in robust IFN-a secretion, whereas RNA lacking a triphosphate did not (Hornung et al., 2006).Similarly,highlyimmunogenicRNAextractedfrominfluenza-infected cells was rendered nonstimulatory after phosphatase treatment (Pichlmair et al., 2006). However, a 50-triphosphate alone is not sufficient to mark a single- stranded (ss) RNA molecule as nonself and to render it immunogenic. In support of this notion, synthetic 50-triphosphate-ssRNA did not activate RIG-I signaling. In contrast, when the same ssRNA molecule was generated by in vitro transcription, it was stimulatory. Reverse cloning and sequencing of the latter RNA species revealed the presence of sequences generated by self- coding intramolecular 30-extension leading to blunt-ended RNA with complementary 50- and 30-ends. Thus, aberrant in vitro transcription products are responsible for the immunostimulatory properties of such preparations. The minimal length of the 50-base paired region was found to be 19 bp. Further- more, a 30-overhang of 2 nt reduced the stimulatory activity by 70%, while no 50-overhang was tolerated (Schlee et al., 2009). Alternative to 50-base pairing, sequence composition may contribute to the stimulatory potential of pppRNA. Hepatitis C virus (HCV) genomic ssRNA is characterized by polyuridine motifs with interspersed C nucleotides (referred to as poly-U/ UCmotifs) anda 50-triphosphate.Deletionof thepoly-U/UCmotifabrogated the stimulatory activity of HCV genomic RNA (Saito, Owen, Jiang, Marcotrigiano, & Gale, 2008; Uzri & Gehrke, 2009). Thus, both panhandle structures and poly-U/UC may serve as a secondary PAMP for pppRNA. However, short synthetic double-stranded (ds) RNA without a 50- triphosphate was reported to activate RIG-I as well (Kato et al., 2008; Takahasi et al., 2008).Notably, theantiviral protein RNaseL can cleavessRNA of virus or host origin and thereby generate short (200 nt) ligands devoid of a 50-triphosphate for RIG-I and MDA5 (Malathi, Dong, Gale, & Silverman, 2007). The structural features responsible for the immunogenicity of RNaseL-generated ligands have not been identified. The molecular nature of the MDA5 ligand remains poorly characterized. The stereotypic MDA5 agonist is polyI:C (Gitlin et al., 2006; 106 Evelyn Dixit and Jonathan C. Kagan

Kato et al., 2006), a synthetic RNA molecule lacking 50-triphosphates that is generated by the annealing of poly-inosine strands to poly-cytidine strands of various lengths. Thus, polyI:C contains an ill-defined mix of ramified ds and ssRNA. Size fractionation of polyI:C revealed that MDA5 responds to high-molecular-weight (HMW) polyI:C, whereas polyI:C shorter than 1000 nucleotides acts as a RIG-I agonist (Kato et al., 2008). Size fractionation of total RNA isolated from encephalomyocarditis virus (EMCV)- infected cells yielded a prominent dsRNA fraction of 11 kb and an even larger-molecular-weight RNA aggregate with variable ss and dsRNA content. Of note only the RNA aggregate, but not the dsRNA, stimulated MDA5 activity. Furthermore, this fraction required its intact secondary and tertiary structure to remain fully active (Pichlmair et al., 2009). Thus, MDA5 preferentially binds to HMW dsRNA that presumably adopts a web-like conformation much like the synthetic RNA analog polyI:C.

2.3. Viruses The structural features of viral RNA that are displayed by a given virus depend on its replication cycle. As a consequence, the different ligand specificities of RIG-I and MDA5 are reflected by the largely non- overlapping pattern of virus susceptibility of mice deficient in either of the two RLRs. RIG-I is required for innate responses to many ssRNA viruses. The best-studied examples among these are the negative-stranded viruses of the orthomyxoviridae, for example, influenza A and B virus, paramyxoviridae, for example, NDV, Sendai virus (SeV), respiratory syncytial virus, and measles virus, and rhabdoviridae, for example, vesicular stomatitis virus (VSV) and rabies virus (Hornung et al., 2006; Kato et al., 2006; Loo et al., 2008; Plumet et al., 2007). Moreover, detection of positive-stranded flaviviruses including HCV and Japanese encephalitis virus is RIG-I dependent (Kato et al., 2006; Saito et al., 2007; Sumpter et al., 2005). In addition, recognition of cytoplasmic DNA can also feed into the RIG-I pathway. RIG-I does not detect DNA directly but can do so after RNA polymerase III-mediated transcription of AT-rich DNA. IFN induction in response to infection with DNA viruses such as adenovirus, herpes simplex virus-1, and Epstein–Barr virus relies on this pathway (Ablasser et al., 2009; Chiu, Macmillan, & Chen, 2009; Samanta, Iwakiri, Kanda, Imaizumi, & Takada, 2006). MDA5 is required for protection against picornaviruses such as EMCV, Theiler’s virus, mengovirus, murine norovirus, and murine hepatitis virus (Gitlin RIG-I-Like Receptor Signaling 107 et al., 2006; Kato et al., 2006; McCartney et al., 2008; Roth-Cross, Bender, & Weiss, 2008). Similar to RIG-I, MDA5 has also been implicated in DNA virus detection. Vaccinia virus, a dsDNA virus of the poxvirus family, activates MDA5 via a yet-to-be-characterized mechanism (Pichlmair et al., 2009). Some viruses such as WNV, Dengue virus, reovirus, and lymphocytic choriomeningitis virus (Fredericksen, Keller, Fornek, Katze, & Gale, 2008; Loo et al., 2008; Zhou et al., 2010)triggerboth RIG-I- and MDA5-dependent innate immune responses. RLR dependence of the aforementioned viruses was determined by infection of different RLR-deficient cell types or mice with purified virions and is summarized in Table 4.1.

Table 4.1 RIG-I and MDA5 detect different sets of viruses

Viruses detected by RIG-I Orthomyxoviridae Influenza A virus Kato et al. (2006) ( ) ssRNA, NS À Influenza B virus Loo et al. (2008) Paramyxoviridae Sendai virus Kato et al. (2006) ( ) ssRNA, NS À Newcastle disease virus Kato et al. (2006) Respiratory syncytial Loo et al. (2008) virus Measles virus Plumet et al. (2007) Rhabdoviridae ( ) Vesicular stomatitis Kato et al. (2006) ssRNA, NS À virus Rabies virus Hornung et al. (2006) Flaviviridae ( ) Hepatitis C virus Saito et al. (2007) and Sumpter et al. ssRNA NS þ (2005) Japanese encephalitis Kato et al. (2006) virus dsDNA-viruses Epstein–Barr virus Ablasser et al. (2009), Chiu et al. (2009), and Samanta et al. (2006) Herpes simplex virus-1 Chiu et al. (2009) Adenovirus Chiu et al. (2009) Continued 108 Evelyn Dixit and Jonathan C. Kagan

Table 4.1 RIG-I and MDA5 detect different sets of viruses—cont'd

Viruses detected by MDA5 Picornaviridae ( ) Encephalomyocarditis Gitlin et al. (2006) and Kato et al. ssRNA, NS þ virus (2006) Theiler’s virus Kato et al. (2006) Mengovirus Kato et al. (2006) Caliciviridae ( ) Murine norovirus-1 McCartney et al. (2008) ssRNA, NS þ Coronaviridae ( ) Murine hepatitis virus Roth-Cross et al. (2008) ssRNA NS þ Viruses detected by RIG-I and MDA5 Flaviviridae ( ) Dengue virus Loo et al. (2008) ssRNA, NS þ West Nile virus Fredericksen et al. (2008) and Loo et al. (2008) Reoviridae Reovirus Loo et al. (2008) dsRNA S Arenaviridae ( ) Lymphocytic Zhou et al. (2010) ssRNA, S À choriomeningitis virus

RLR dependence to various viruses is listed according to virus families. The respective genome type is indicated as single-stranded (ss) or double-stranded (ds) RNA or DNA with negative ( ) or positive ( ) genome orientation featuring segmentation (S) or nonsegmentation (NS). À þ

2.4. Bacteria Various bacteria including Francisella tularensis, Mycobacteria tuberculosis, Brucella abortis, group B streptococcus (GBS), Listeria monocytogenes, and Legionella pneumophila have been shown to induce type I IFN in a TLR-independent manner (Charrel-Dennis et al., 2008; Henry, Brotcke, Weiss, Thompson, & Monack, 2007; O’Riordan, Yi, Gonzales, Lee, & Portnoy, 2002; Opitz et al., 2006; Roux et al., 2007; Stanley, Johndrow, Manzanillo, & Cox, 2007; Stetson & Medzhitov, 2006). While it is well appreciated that viral replication is inhibited by type I IFN, the role of IFN in bacterial infections is less clear; for example, IFN has a protective effect during GBS infection (Mancuso et al., 2007), whereas it is disadvantageous during Listeria infection (Auerbuch, Brockstedt, Meyer-Morse, O’Riordan, & Portnoy, 2004; Carrero, Calderon, & Unanue, 2004; O’Connell et al., 2004). Even less clear is which bacterial ligands and host receptors trigger IFN secretion. RIG-I-Like Receptor Signaling 109

The intracellular gram-negative bacterium L. pneumophila infects macrophages and causes Legionnaires’ disease. IFN-b induction in lung epithelial cells and macrophages depends on MAVS (Monroe, McWhirter, & Vance, 2009; Opitz et al., 2006). However, the signaling events upstream of MAVS activation are a matter of debate. Chiu et al. propose that AT-rich DNA reaches the host cytosol and is transcribed into an RNA ligand for RIG-I in an RNA polymerase III-dependent manner (Chiu et al., 2009). In contrast, Monroe et al. argue that the IFN response to Legionella genomic DNA does not require MAVS in mouse macrophages as MAVS-deficient and wild-type macrophages display comparable levels of IFN. Instead, their data support a model where Legionella RNA is directly detected by both RIG-I and MDA5 as macrophages deficient in either receptor display a partial phenotype (Monroe et al., 2009). Shigella flexneri, the causative agent of bacillary dysentery, infects macrophages of the colonic epithelium and rapidly induces cell death by pyroptosis. Escaping bacteria invade colonic epithelial cells where they replicate in the cytosol. Type II IFN-g is critical for inhibiting S. flexneri cytosolic growth. It is at this stage that IFN-g exerts its antimicrobial effect through RIG-I signaling in nonmyeloid cells. Both RIG-I- and MAVS- deficient mouse embryonic fibroblasts (MEFs) failed to restrict IFN-g- dependent S. flexneri replication. Inhibition of RNA polymerase III also reduced the antimicrobial effect of IFN-g suggesting that RIG-I signaling is triggered by RNA polymerase III-generated RNA mediates. Interest- ingly, type I IFN induction is not required for this effect as IFNAR-deficient MEFs that are completely unresponsive to type I IFNs do not impair IFN-g- mediated growth inhibition of S. flexneri. In contrast, in primary macrophages, RIG-I signaling is dispensable for IFN-g-mediated growth arrest ( Jehl, Nogueira, Zhang, & Starnbach, 2012). These findings underscore the importance of the interplay of distinct innate immunity pathways in order to successfully combat pathogens.

3. RIG-I ACTIVATION AND RECEPTOR PROXIMAL SIGNAL PROPAGATION RLR activation is a multistage process that requires a well-coordinated interplay of receptor, ligand, and several accessory proteins. In contrast to RIG-I, the specific requirements for efficient MDA5 activation are unclear, but it stands to reason that both proinflammatory RLRs follow a similar mechanism. As exemplified by RIG-I, our current understanding of this 110 Evelyn Dixit and Jonathan C. Kagan process involves the following sequence of events: (1) In resting cells, RIG-I adopts a closed conformation resulting in an autoinhibited (nonsignaling) state. (2) pppRNA binding to RIG-I induces conformational changes that lead to dimerization and exposure of CARDs in the open conformation. (3) Dephosphorylation of RIG-I and TRIM25-dependent ubiquitination events fully activate the signaling capability of RIG-I. (4) RIG-I associates with MAVS in a CARD-dependent manner. (5). MAVS accumulates in signaling aggregates by a prion-like mechanism. In the absence of infection, RIG-I is kept in an autoinhibited state by intramolecular interactions between the CARDs and the helicase domain, which sterically hinders RNA binding to the helicase domain and prevents the CARDs from signaling (Kowalinski et al., 2011; Saito et al., 2007). Accordingly, the N-terminus of RIG-I comprising the two CARDs has a constitutively active phenotype when overexpressed (Yoneyama et al., 2004). Furthermore, phosphorylation of threonine 170 (and serine 8 in pri- mate orthologs) by PKC-a and PKC-b suppresses RIG-I activity at steady state (Gack, Nistal-Villan, Inn, Garcia-Sastre, & Jung, 2010; Maharaj, Wies, Stoll, & Gack, 2012; Nistal-Villan et al., 2010). Only upon ligand binding does the closed conformation open up to facilitate downstream signaling by the CARDs. Biochemical studies have identified the CTD as the sensor for pppRNA. Receptor–ligand interactions were examined by measuring ATPase activity of purified deletion mutants of RIG-I lacking the CARDs (DCARD), the CTD (DCTD), or both (helicase) in response to treatment with a panel of RNA ligands derived from the rabies virus leader (RVL) sequence, that is, pppRNA (pppRVL), nonphosphorylated ssRNA (ssRVL), as well as dsRNA (dsRVL). ssRVL did not activate ATPase activity in any of the RIG-I variants. pppRVL strongly stimulated ATPase activity of wild-type RIG-I. Deletion of the CARDs did not interfere with pppRVL-stimulated ATPase activity. Neither the helicase domain alone nor RIG-I lacking the CTD displayed ATPase activity in response to pppRVL. dsRNA weakly stimulated wild-type RIG-I and the isolated helicase domain. Of note, dsRNA activated DCARD more efficiently than pppRVL achieving ATPase activity levels comparable to wild- type RIG-I in complex with pppRVL. These findings suggest that the CARDs inhibit dsRNA binding in an inactive conformation, while CTD promotes pppRVL binding in an active conformation. Further binding studies clearly demonstrated that the pppRNA binding site resides within the CTD. X-ray crystallography of the CTD revealed two features that are required for pppRNA binding: (1) A zinc coordination site comprising RIG-I-Like Receptor Signaling 111 four highly conserved cytidine residues (C810, C813, C864, C869). These cytidines are conserved in a paralogous and orthologous manner within the family of RLRs. (2) A conserved groove with a positively charged patch at the center of which an RIG-I invariant lysine is located (K888) (Cui et al., 2008). Crystallographic structures of RIG-I give detailed insight into the conformational changes triggered by ligand binding and required for signal initiation. The structural data suggest a model where in the autorepressed state the CTD is devoid of intramolecular interactions and thus can freely engage in pppRNA binding. This initial event increases the local RNA concentration and leads to cooperative binding of RNA and ATP to the helicase domain resulting in dramatic rearrangements within the helicase domain that are orchestrated by the pincher domain that connects the helicase domain with the CTD. The helicase domain and the CTD completely surround the RNA clasping onto the helix by numerous intermolecular interactions. This channel covers 9–10 bp along the RNA. Longer RNA molecules allow the binding of two RIG-I monomers simultaneously. However, this apparent dimerization is devoid of a protein–protein interface but much rather reflects an RNA-guided oligomerization (Kowalinski et al., 2011; Luo et al., 2011). In line with the structural data of RNA-bound RIG-I, full-length RIG-I but not the DCTD mutant or MDA5 eluted as dimers after gel filtration when incubated with pppRNA (Cui et al., 2008). Downstream signaling by ligand-activated RIG-I is achieved by the N-terminal tandem CARDs. Deletion of the CARDs results in a dominant negative phenotype of RIG-I (Yoneyama et al., 2004). Huh7.5 cells, a sub- population of the hepatocyte cell line Huh7 that is characterized by a threonine to isoleucine mutation at position 55 (T55I) in the first CARD of RIG-I, fail to respond to HCV infection. As a consequence, the absence of a functional antiviral response creates conditions permissive for HCV replication in Huh7.5 (Sumpter et al., 2005). The T55I mutant interferes with the binding of the TRIM25 E3 ubiquitin ligase that is required for activation of RIG-I signaling. Gack et al. demonstrated that TRIM25 binds to the first CARD domain via its SPRY domain. Prerequisite for TRIM25 binding is dephosphorylation of RIG-I at T170 (and S8 in primates) by an unidentified phosphatase. The phosphomimetic mutation T170E abrogated binding of TRIM25 to RIG-I and interfered with downstream signaling events and antiviral activity of RIG-I (Gack et al., 2010). TRIM25 transfers K63-linked ubiquitin moieties to the lysine 172 residue (K172) within the second CARD using its RING domain. Oligomerization of RIG-I with the adapter 112 Evelyn Dixit and Jonathan C. Kagan protein MAVS critically depends on this modification. Accordingly, TRIM25-deficient MEFs do not secrete IFN-b after SeV infection. The absence of antiviral defenses is reflected by markedly higher viral titers upon VSV infection (Gack et al., 2007). Although TRIM25 does not attach ubiquitin moieties to MDA5, polyubiquitin binding by MDA5 is required for its signaling functions ( Jiang et al., 2012). The requirement for ubiquitination of RIG-I for initiation of downstream signaling was challenged by a study using a cell-free system to identify the minimal components for RIG-I signal transduction. The RIG-I pathway was reconstituted in a mixture containing affinity-purified RIG-I, crude mitochondria and peroxisomes (containing the adapter MAVS), cytosolic extracts (containing TBK1), in vitro-synthesized transcription factor IRF3, and ATP. RIG-I activation was quantified by measuring dimerization of IRF3, a read- out for its activation. With this in vitro assay in place, the authors recapitulated key aspects of RIG-I signaling and revealed new regulatory mechanisms. IRF3 activation required MAVS and TRIM25 as depletion of these proteins by RNAi interfered with IRF3 dimerization. RIG-I needed to be isolated from virus-infected cells, be activated by RNA ligand in vitro, or be present as an N-terminal CARD fragment for IRF3 activation to occur. The ubiquitination machinery responsible for RIG-I activation was shown to be comprising E1, the E2 Ubc5 and Ubc13, and the E3 TRIM25, as the mitochondrial fraction of virus-infected cells depleted from Ubc5 (isoform b and c) and Ubc13 no longerelicited IRF3dimerization. Inline withthe notionthatUbc13isspecific for synthesis of lysine 63 (K63)-linked ubiquitin and previous findings on the importance of K63-linked polyubiquitin for RIG-I activation, ubiquitin proteins with a sole lysine residue at position 63 were capable to activate the pathway in the cell-free in vitro system (Zeng et al., 2010). Thus, a requirement for both TRIM25 and K63-linked ubiquitin for IFN-b induction by RIG-I were confirmed in this experimental setup. The major discrepancy between the studies by Gack et al. and Zeng et al. is the attachment of polyubiquitin. While in the former study covalent linkage to the K172 residue of RIG-I was proposed, the latter study suggested that unanchored polyubiquitin chains serve as essential cofactors for RIG-I activation. Two major lines of evidence support this proposition: (1) RIG-I CARDs isolated from E. coli that lack an ubiquitination system-activated IRF3 when ubiquitin polymers were added to the cell-free system. (2) Endogenous polyubiquitin was coprecipitated with RIG-I CARDs from mammalian cells and subsequently recovered from the complex by selective heat denaturation. This preparation promoted IRF3 dimerization, but lost RIG-I-Like Receptor Signaling 113 its activity when treated with the deubiquitination enzyme IsoT. Even though the K172 residue is not required as an acceptor for ubiquitination in this situation, its relevance for RIG-I signaling remains undisputed as it is critical for the binding affinity to polyubiquitin (Zeng et al., 2010). Both RIG-I and MDA5 signaling depends on the adapter protein MAVS to link receptor activity to the downstream kinases TBK1 and IKK-i (Fig. 4.2). MAVS is a 540 aa protein comprising an N-terminal CARD domain, a central proline-rich region (Pro), and a C-terminal transmembrane domain (Seth et al., 2005)(Fig. 4.1). While the transmembrane domain targets the adapter to its proper subcellular locations (mitochondria, peroxisomes, and mitochondria-associated membranes (MAM); see Section 4.2), the CARD domain is required for signaling (Dixit et al., 2010; Horner, Liu, Park, Briley, & Gale, 2011; Seth et al., 2005). When MAVS was initially characterized as an RLR signaling adapter, the authors noted that viral infection results in the formation of detergent-resistant aggregates (Seth et al., 2005). Recent studies by the same group defined these aggregates as highly organized, self- propagating prion-like fibrils. Using the cell-free system for in vitro reconstitution of RLR signaling as described earlier, complexes of MAVS larger than the 26S proteasome were detected 9 h after SeV infection which coincided with IRF3 dimerization. These complexes displayed several features characteristic for prions: (1) The MAVS CARD is necessary and sufficient for formation of fiber-like structures as determined by electron microscopy. (2) These fibrils are resistant to protease K treatment and detergent solubilization. (3) Protease-resistant fibrils convert MAVS on mitochondria that were extracted from uninfected cells into functional aggregates leading to IRF3 activation. Interestingly, however, these MAVS aggregated did not stain with Congo Red, a dye that typically stains “classic” prion structures (chen prion paper). Conversely, mitochondria depleted of MAVS by RNAi prior to extraction did not result in IRF3 dimerization. Importantly, MAVS aggregates form within minutes upon activation of RLR signaling in the cell-fee reconstitution assay indicating that prion-like MAVS fibrils are a bona fide determinant of the MAVS activation status (Hou et al., 2011).

4. REGULATORY MECHANISMS OF RIG-I SIGNALING 4.1. Regulators of RLR signaling Several proteins regulate RLR signaling along the pathway in order to tailor the response. Various E3 ubiquitin ligases regulate RIG-I activity. TRIM25 as discussed in Section 3 and Riplet (also known as RNF135 or REUL) 114 Evelyn Dixit and Jonathan C. Kagan positively regulate RIG-I activity through K63-linked ubiquitination at its N- or C-terminus, respectively (Gack et al., 2007; Gao et al., 2009; Oshiumi, Matsumoto, Hatakeyama, & Seya, 2009; Oshiumi et al., 2010). In contrast, RNF125 mediates K48-linked ubiquitination that targets RIG-I for degradation and thus acts as a negative regulator (Arimoto et al., 2007). Recently, ZAPS was identified as a cofactor for RIG-I signaling. ZAPS is a member of the poly (ADP-ribose) polymerase (PARP) family but lacks the PARP-like domain present in ZAPS due to alternative splicing. ZAPS was shown to directly associate with RIG-I in a ligand-dependent manner and to amplify downstream signaling events such as activation of the transcription factors IRF3 and NF-kB and induction of type I IFN. As a result, ZAPS inhibited viral replication after infection with RIG-I-dependent viruses such as influenza virus or NDV (Hayakawa et al., 2011). While a continuously growing number of accessory proteins that modify RIG-I signaling activity emerges, the interplay between these proteins, the order in which they act upon RIG-I, and their relative signif- icance for signaling output remain elusive until further systematic studies are done to address these questions. NLRX1 (also known as Nod9) was proposed to control RLR signal transduction at the level of MAVS; however, its role is a matter of debate. NLRX1 was reported to reside at the outer mitochondrial membrane from where it physically disrupts the virus-induced RLR–MAVS interaction (Moore et al., 2008)(Fig. 4.2). Alternatively, NLRX1 was found to be localized within the mitochondrial matrix which deems impossible the proposed function as a direct interactor of MAVS to modulate its activity. Rather, it was shown that NLRX1 promotes the generation of reactive oxygen species (ROS) (Arnoult et al., 2009; Tattoli et al., 2008). Interestingly, several lines of evidence implicate ROS as modulators of RLR signaling. Cells deficient in autophagy accumulate dysfunctional mitochondria which entails increased ROS levels and display enhanced RLR signaling. Treatment with antioxidant reverses the effect (Tal et al., 2009). Conversely, mitochondrial uncoupling—a process by which ROS generation is decreased—reduced RLR signaling (Koshiba, Yasukawa, Yanagi, & Kawabata, 2011). Addi- tional research is required to delineate the mechanism by which ROS regulate RLR-dependent antiviral responses. STING (also known as MITA, MPYS, or ERIS) (Ishikawa & Barber, 2008; Jin et al., 2011; Sun et al., 2009; Zhong et al., 2008) was originally identified as a regulator of RIG-I signaling owing to its ability to directly bind to RIG-I, MAVS, and TBK1 and to its knockout phenotype. RIG-I-Like Receptor Signaling 115

Overexpression of the constitutively active fragment of RIG-I failed to induce IFN in STING-deficient MEFs. Moreover, VSV infection of STING-deficient mice resulted in significantly poorer survival rates and lower type I IFN serum levels relative to control littermates. It is of note that the response to transfected polyI:C remained unchanged in the absence of STING (Ishikawa et al., 2009). While STING was shown to play an undisputed role in the IFN response to cytosolic DNA from viruses or synthetic agonists, its implication in RLR signaling may not be essential.

4.2. Regulation of RLR signal transduction by subcellular compartmentalization All three receptorsof the RLR family are cytosolic proteins, and they have not been found to be associated with any subcellular structure at steady state. However, several signaling components downstream of the receptors are membrane proteins whose functional domains project into the cytosol from the surface of the respective organelles. More importantly, proper localization of these proteins is a prerequisite for their biological activity. The best characterized example is the adapter protein MAVS. MAVS resides on the outer mitochondrial membrane (Seth et al., 2005), peroxisomes (Dixit et al., 2010) and MAMs (Horner et al., 2011), a specialized subdomain of the ER that connects mitochondria and peroxisomes (Hayashi, Rizzuto, Hajnoczky, & Su, 2009; Vance, 1990). Both peroxisomal and mitochondrial MAVS signal to induce ISG expression in MEFs. While mitochondrial MAVS induces type I IFN and as a consequence ISG expression in response to reovirus and influenza virus infection, peroxisomal MAVS directly induces ISG expression which creates a transient yet functional antiviral state. The lack of type I IFN induction by peroxisomal MAVS was also observed in macrophages. Unlike MEFs, macrophages upregulate not only expression of ISGs but also proinflammatory cytokines after reovirus infection (Dixit et al., 2010). A different study confirms the localization of MAVS on mitochondria and peroxisomes, and adds MAMs to the list of subcellular pools of MAVS. Moreover, the authors propose the MAM as an innate immune synapse for antiviral responses that coordinates MAVS-dependent signaling from mitochondria and peroxisomes (Horner et al., 2011). HCV-infected Huh7 hepatocytes are unable to induce IFN expression due to MAVS cleavage by the viral protease NS3/4A (Loo et al., 2006; Meylan et al., 2005). Others and we have shown that cytosolic MAVS is unable to signal (Dixit et al., 2010; Seth et al., 2005). Given that NS3/4A cleaves MAM-localized MAVS, but not mitochondrial MAVS, the authors conclude that—at least for HCV 116 Evelyn Dixit and Jonathan C. Kagan infections—mitochondrial MAVS is dispensable for RIG-I signaling. This notion is further supported by the finding that RIG-I is recruited specifically to MAM-resident MAVS upon HCV infection (Horner et al., 2011). In fact, a ternary complex consisting of active open-conformation RIG-I, TRIM25, and the chaperone 14-3-3e is redistributed to MAMs upon infection (Liu et al., 2012). MFN2 tethers the ER to mitochondria and thus maintains the MAM mitochondrial contacts (de Brito & Scorrano, 2008). Depletion of MFN2 by RNAi destabilizes the antiviral synapse, which shifts MAVS to peroxisomes and thereby increases RIG- I-mediated signaling in response to SeV, VSV, and HCV (at early time points before MAVS cleavage by NS3/4A) infection (Horner et al., 2011). It would be interesting to test the effect of MFN2 on the organelle-specific outcome of RLR signaling using cells with organelle-restricted MAVS expression. In addition to MFN2, MFN1 has been implicated in regulation of RIG-I signaling as well. Activation of RLRs by infection with SeV, NDV, influenza virus, VSV, Sindbis virus, or EMCV and by transfection with pppRNA resulted in redistribution of mitochondrial MAVS. While some mitochondria accumulate MAVS, others become devoid of it during a process that depends on MFN1. RIG-I is evenly distributed throughout the cytosol in uninfected cells but is concentrated in foci upon infection. However, no colocalization between RIG-I and MAVS was observed. On the contrary, RIG-I colocalized with viral nucleocapsid. As a consequence, type I IFN induction after NDV infection was completely abolished in MFN1-deficient MEFs. These findings led the authors to propose a model where RIG-I is recruited to virus factories to maximize the chances of receptor–ligand interaction. Mitochondria serve as vehicles that position MAVS. Some mitochondria enrich MAVS through repeated fission and fusion events and surround the foci of active viral replication in order to enable IFN induction (Onoguchi et al., 2010). While this model outlines how mitochondrial signaling is optimized to perpetuate IFN induction for the duration of infection and to establish a sustained antiviral immune response, it leaves two important questions unanswered. First, what are the kinetics of this process? The earliest time point presented in the study is 9 h postinfection. Second, what triggers mitochondrial remodeling and accumulation of MAVS? Regardless of whether activation of RLR signaling or a different stimulus initiates the rearrangement, this model does not explain RNA detection at the very first instance of virus encounter. Much rather it demands additional and disparate means of RLR signaling that RIG-I-Like Receptor Signaling 117 ensure an immediate antiviral response until MAVS-enriched mitochondria are recruited to the periphery of virus factories.

5. CONCLUSIONS AND FUTURE DIRECTIONS RLR signaling is a crucial pathway for detection of intracellular viruses and mounting protective antiviral defenses. Since the identification of RIG-I and its related proteins MDA5 and LGP2, tremendous progress has been made in terms of the core components of this pathway and the regulatory mechanisms. Still, many open questions remain on the pathogen as well as the host side. What are the biological ligands that arise during a given viral infection? Viral genomes, viral transcripts, or replication intermediates are likely candidates. Do these naturally occurring ligands match the postulated structural features that were identified in vitro? Baum, Sachidanandam, and Garcia-Sastre (2010) sought to characterize such ligands by immunopre- cipitation of endogenous RIG-I/RNA complexes from SeV and influenza virus-infected cells and subsequent deep sequencing. Copy-back defective interfering particles were identified as the natural ligand of both SeV and influenza virus. RIG-I also bound to (preferentially short segments of) genomic RNA of influenza virus. This study confirms the requirement for both a 50 triphosphate and a panhandle structure for RIG-I activation during SeV and influenza virus infection (Baum et al., 2010). How accessible are these ligands during infection? In the light of coevolution of virus and host, it stands to reason that viral PAMPs are spatially segregated from the respective PRRs. Is RLR-mediated virus detection merely possible by accidental escape of PAMPs or are mechanisms in place that actively sample sites of viral replication? Regarding the host factors required for an effective antiviral response, our understanding of the spatiotemporal control of this pathway is very limited. Despite the designation of RLRs as cytosolic receptors, the signal transduction cascade initiated upon ligand engagement is certainly not cytosolic, but strictly dependent on proper subcellular localization of many components of this pathway. The adaptor protein MAVS resides on and signals distinctively from peroxisomes, MAM, and mitochondria (Dixit et al., 2010; Horner et al., 2011; Seth et al., 2005). The negative regulator NLRX1 is also localized on mitochondria (Moore et al., 2008). In the course of infection, mitochondria are rearranged to surround sites of viral replication in an MFN1-dependent manner. Failure to do so severely abrogates an antiviral response (Onoguchi et al., 2010). What is the benefit for the host of 118 Evelyn Dixit and Jonathan C. Kagan such an elaborate subcellular arrangement of a signal transduction pathway? Perhaps, recruitment of molecules concentrated on an organelle might be faster and more energy efficient than recruiting every single molecule independently. Considering the different responses mediated by peroxisomal and mitochondrial MAVS, distribution of this pathway on two organelles might facilitate targeting of factors specifically required for each of the responses. A similar situation can be found with TLR4, the receptor for the prototypical PAMP lipopolysaccharide. Perhaps, a positive regulator of direct ISG induction is only targeted to peroxisomes or an inhibitorof such a signaling pathway is located on mitochondria. The TLR4 pathway exemplifies how the spatial distribution of signaling components governs the signaling output. While plasma membrane-bound TLR4 induces cytokine expression in an MyD88-dependent manner (Medzhitov, Preston-Hurlburt, & Janeway, 1997; Medzhitov et al., 1998), endocytosis of TLR4 induces type I IFN induction in a TRIF-dependent manner (Kagan et al.,2008;Yamamoto et al., 2002). For TLR4 signaling, TRAF3 was proposed to be limited in its mobility. The inability of TRAF3 to be recruited to TLR4 at the plasma membrane neces- sitates TLR4 to be endocytosed. It is at the endosome that the TRAM–TRIF adaptor pair is recruited to engage TRAF 3 and to enable type I IFN signaling (Kagan et al., 2008). Similarly, an essential factor for direct ISG induction may be available exclusively at peroxisomes. Experimental evidence for the organelle-specific presence of regulators of RLR signaling comes from NLRX1. Overexpression of NLRX1 inhibits signaling mediated by mitochondrial MAVS, but not by peroxisomal MAVS (Dixit et al., 2010). The spatial regulation may also be indicative of RLR signaling being a multistage process, wherein in an initial wave a nascent infection is sensed, and in a later phase the process is optimized for a robust response during infection and finally is turned off. In order to address this possibility, kinetic studies rather than late end points after infection would be helpful.

ACKNOWLEDGMENTS E. D. is supported by the Erwin Schro¨dinger Fellowship ( J3295-B22) of the Austrian Science Fund (FWF). The National Institutes of Health grants AI093589 and P30 DK34854 support the work performed in the laboratory of J. K. Dr. J. K. holds an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund.

REFERENCES Ablasser, A., Bauernfeind, F., Hartmann, G., Latz, E., Fitzgerald, K. A., & Hornung, V. (2009). RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate. Nature Immunology, 10, 1065. RIG-I-Like Receptor Signaling 119

Alexopoulou, L., Holt, A. C., Medzhitov, R., & Flavell, R. A. (2001). Recognition of double- stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature, 413, 732. Arimoto, K., Takahashi, H., Hishiki, T., Konishi, H., Fujita, T., & Shimotohno, K. (2007). Negative regulation of the RIG-I signaling by the ubiquitin ligase RNF125. In: Proceed- ings of the National Academy of Sciences of the United States of America, 104, 7500. Arnoult, D., Soares, F., Tattoli, I., Castanier, C., Philpott, D. J., & Girardin, S. E. (2009). An N-terminal addressing sequence targets NLRX1 to the mitochondrial matrix. Journal of Cell Science, 122, 3161. Auerbuch, V., Brockstedt, D. G., Meyer-Morse, N., O’Riordan, M., & Portnoy, D. A. (2004). Mice lacking the type I interferon receptor are resistant to Listeria monocytogenes. The Journal of Experimental Medicine, 200, 527. Barbalat, R., Ewald, S. E., Mouchess, M. L., & Barton, G. M. (2011). Nucleic acid recognition by the innate immune system. Annual Review of Immunology, 29, 185. Barton, G. M., & Kagan, J. C. (2009). A cell biological view of Toll-like receptor function: Regulation through compartmentalization. Nature Reviews. Immunology, 9, 535. Baum, A., Sachidanandam, R., & Garcia-Sastre, A. (2010). Preference of RIG-I for short viral RNA molecules in infected cells revealed by next-generation sequencing. In: Proceedings of the National Academy of Sciences of the United States of America, 107, 16303. Burckstummer, T., Baumann, C., Bluml, S., Dixit, E., Durnberger, G., Jahn, H., et al. (2009). An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome. Nature Immunology, 10, 266. Carrero, J. A., Calderon, B., & Unanue, E. R. (2004). Type I interferon sensitizes lymphocytes to apoptosis and reduces resistance to Listeria infection. The Journal of Experimental Medicine, 200, 535. Charrel-Dennis, M., Latz, E., Halmen, K. A., Trieu-Cuot, P., Fitzgerald, K. A., Kasper, D. L., et al. (2008). TLR-independent type I interferon induction in response to an extracellular bacterial pathogen via intracellular recognition of its DNA. Cell Host & Microbe, 4, 543. Chiu, Y. H., Macmillan, J. B., & Chen, Z. J. (2009). RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway. Cell, 138, 576. Collins, S. E., Noyce, R. S., & Mossman, K. L. (2004). Innate cellular response to virus particle entry requires IRF3 but not virus replication. Journal of Virology, 78, 1706. Cui, S., Eisenacher, K., Kirchhofer, A., Brzozka, K., Lammens, A., Lammens, K., et al. (2008). The C-terminal regulatory domain is the RNA 50-triphosphate sensor of RIG-I. Molecular Cell, 29, 169. de Brito, O. M., & Scorrano, L. (2008). Mitofusin 2 tethers endoplasmic reticulum to mitochondria. Nature, 456, 605. Dixit, E., Boulant, S., Zhang, Y., Lee, A. S., Odendall, C., Shum, B., et al. (2010). Perox- isomes are signaling platforms for antiviral innate immunity. Cell, 141, 668. Fernandes-Alnemri, T., Yu, J. W., Datta, P., Wu, J., & Alnemri, E. S. (2009). AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature, 458, 509. Fredericksen, B. L., Keller, B. C., Fornek, J., Katze, M. G., & Gale, M., Jr. (2008). Estab- lishment and maintenance of the innate antiviral response to West Nile Virus involves both RIG-I and MDA5 signaling through IPS-1. Journal of Virology, 82, 609. Gack, M. U., Nistal-Villan, E., Inn, K. S., Garcia-Sastre, A., & Jung, J. U. (2010). Phos- phorylation-mediated negative regulation of RIG-I antiviral activity. Journal of Virology, 84, 3220. Gack, M. U., Shin, Y. C., Joo, C. H., Urano, T., Liang, C., Sun, L., et al. (2007). TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature, 446, 916. Gao, D., Yang, Y. K., Wang, R. P., Zhou, X., Diao, F. C., Li, M. D., et al. (2009). REUL is a novel E3 ubiquitin ligase and stimulator of retinoic-acid-inducible gene-I. PLoS One, 4, e5760. 120 Evelyn Dixit and Jonathan C. Kagan

Gitlin, L., Barchet, W., Gilfillan, S., Cella, M., Beutler, B., Flavell, R. A., et al. (2006). Essen- tial role of mda-5 in type I IFN responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus. In: Proceedings of the National Academy of Sciences of the United States of America, 103, 8459. Hayakawa, S., Shiratori, S., Yamato, H., Kameyama, T., Kitatsuji, C., Kashigi, F., et al. (2011). ZAPS is a potent stimulator of signaling mediated by the RNA helicase RIG- I during antiviral responses. Nature Immunology, 12, 37. Hayashi, T., Rizzuto, R., Hajnoczky, G., & Su, T. P. (2009). MAM: More than just a house- keeper. Trends in Cell Biology, 19, 81. Henry, T., Brotcke, A., Weiss, D. S., Thompson, L. J., & Monack, D. M. (2007). Type I interferon signaling is required for activation of the inflammasome during Francisella infection. The Journal of Experimental Medicine, 204, 987. Hiscott, J. (2007). Convergence of the NF-kappaB and IRF pathways in the regulation of the innate antiviral response. Cytokine & Growth Factor Reviews, 18, 483. Honda, K., Takaoka, A., & Taniguchi, T. (2006). Type I interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors. Immunity, 25, 349. Horner, S. M., Liu, H. M., Park, H. S., Briley, J., & Gale, M., Jr. (2011). Mitochondrial- associated endoplasmic reticulum membranes (MAM) form innate immune synapses and are targeted by hepatitis C virus. In: Proceedings of the National Academy of Sciences of the United States of America, 108, 14590. Hornung, V., Ablasser, A., Charrel-Dennis, M., Bauernfeind, F., Horvath, G., Caffrey, D. R., et al. (2009). AIM2 recognizes cytosolic dsDNA and forms a caspase- 1-activating inflammasome with ASC. Nature, 458, 514. Hornung, V., Ellegast, J., Kim, S., Brzozka, K., Jung, A., Kato, H., et al. (2006). 50-Triphosphate RNA is the ligand for RIG-I. Science, 314, 994. Hou, F., Sun, L., Zheng, H., Skaug, B., Jiang, Q. X., & Chen, Z. J. (2011). MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response. Cell, 146, 448. Ishikawa, H., & Barber, G. N. (2008). STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature, 455, 674. Ishikawa, H., Ma, Z., & Barber, G. N. (2009). STING regulates intracellular DNA- mediated, type I interferon-dependent innate immunity. Nature, 461, 788. Jehl, S. P., Nogueira, C. V., Zhang, X., & Starnbach, M. N. (2012). IFN gamma inhibits the cytosolic replication of Shigella flexneri via the cytoplasmic RNA sensor RIG-I. PLoS Pathogens, 8, e1002809. Jiang, X., Kinch, L. N., Brautigam, C. A., Chen, X., Du, F., Grishin, N. V., et al. (2012). Ubiquitin-induced oligomerization of the RNA sensors RIG-I and MDA5 activates antiviral innate immune response. Immunity, 36, 959. Jin, L., Hill, K. K., Filak, H., Mogan, J., Knowles, H., Zhang, B., et al. (2011). MPYS is required for IFN response factor 3 activation and type I IFN production in the response of cultured phagocytes to bacterial second messengers cyclic-di-AMP and cyclic-di- GMP. Journal of Immunology, 187, 2595. Kagan, J. C., Su, T., Horng, T., Chow, A., Akira, S., & Medzhitov, R. (2008). TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon-beta. Nature Immu- nology, 9, 361. Kang, D. C., Gopalkrishnan, R. V., Lin, L., Randolph, A., Valerie, K., Pestka, S., et al. (2004). Expression analysis and genomic characterization of human melanoma differentiation associated gene-5, mda-5: A novel type I interferon-responsive apoptosis- inducing gene. Oncogene, 23, 1789. Kato, H., Sato, S., Yoneyama, M., Yamamoto, M., Uematsu, S., Matsui, K., et al. (2005). Cell type-specific involvement of RIG-I in antiviral response. Immunity, 23, 19. RIG-I-Like Receptor Signaling 121

Kato, H., Takeuchi, O., Mikamo-Satoh, E., Hirai, R., Kawai, T., Matsushita, K., et al. (2008). Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5. The Journal of Experimental Medicine, 205, 1601. Kato, H., Takeuchi, O., Sato, S., Yoneyama, M., Yamamoto, M., Matsui, K., et al. (2006). Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature, 441, 101. Katze, M. G., He, Y., & Gale, M., Jr. (2002). Viruses and interferon: A fight for supremacy. Nature Reviews. Immunology, 2, 675. Kawai, T., & Akira, S. (2006). Innate immune recognition of viral infection. Nature Immu- nology, 7, 131. Kawai, T., & Akira, S. (2010). The role of pattern-recognition receptors in innate immunity: Update on toll-like receptors. Nature Immunology, 11, 373. Kawai, T., Takahashi, K., Sato, S., Coban, C., Kumar, H., Kato, H., et al. (2005). IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nature Immunology, 6, 981. Koshiba, T., Yasukawa, K., Yanagi, Y., & Kawabata, S. (2011). Mitochondrial membrane potential is required for MAVS-mediated antiviral signaling. Science Signaling, 4, ra7. Kowalinski, E., Lunardi, T., McCarthy, A. A., Louber, J., Brunel, J., Grigorov, B., et al. (2011). Structural basis for the activation of innate immune pattern-recognition receptor RIG-I by viral RNA. Cell, 147, 423. Lazear, H. M., Lancaster, A., Wilkins, C., Suthar, M. S., Huang, A., Vick, S. C., et al. (2013). IRF-3, IRF-5, and IRF-7 coordinately regulate the type I IFN response in myeloid dendritic cells downstream of MAVS signaling. PLoS Pathogens, 9, e1000607. Liu, H. M., Loo, Y. M., Horner, S. M., Zornetzer, G. A., Katze, M. G., & Gale, M., Jr. (2012). The mitochondrial targeting chaperone 14-3-3epsilon regulates a RIG-I trans- locon that mediates membrane association and innate antiviral immunity. Cell Host & Microbe, 11, 528. Loo, Y. M., Fornek, J., Crochet, N., Bajwa, G., Perwitasari, O., Martinez-Sobrido, L., et al. (2008). Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity. Journal of Virology, 82, 335. Loo, Y. M., Owen, D. M., Li, K., Erickson, A. K., Johnson, C. L., Fish, P. M., et al. (2006). Viral and therapeutic control of IFN-beta promoter stimulator 1 during hepatitis C virus infection. In: Proceedings of the National Academy of Sciences of the United States of America, 103, 6001. Luo, D., Ding, S. C., Vela, A., Kohlway, A., Lindenbach, B. D., & Pyle, A. M. (2011). Struc- tural insights into RNA recognition by RIG-I. Cell, 147, 409. Maharaj, N. P., Wies, E., Stoll, A., & Gack, M. U. (2012). Conventional protein kinase C-alpha (PKC-alpha) and PKC-beta negatively regulate RIG-I antiviral signal transduction. Journal of Virology, 86, 1358. Malathi, K., Dong, B., Gale, M., Jr., & Silverman, R. H. (2007). Small self-RNA generated by RNase L amplifies antiviral innate immunity. Nature, 448, 816. Mancuso, G., Midiri, A., Biondo, C., Beninati, C., Zummo, S., Galbo, R., et al. (2007). Type I IFN signaling is crucial for host resistance against different species of pathogenic bacteria. Journal of Immunology, 178, 3126. Matsumoto, M., Funami, K., Tanabe, M., Oshiumi, H., Shingai, M., Seto, Y., et al. (2003). Subcellular localization of Toll-like receptor 3 in human dendritic cells. Journal of Immu- nology, 171, 3154. McCartney, S. A., Thackray, L. B., Gitlin, L., Gilfillan, S., Virgin, H. W., & Colonna, M. (2008). MDA-5 recognition of a murine norovirus. PLoS Pathogens, 4, e1000108. Medzhitov, R. (2007). Recognition of microorganisms and activation of the immune response. Nature, 449, 819. 122 Evelyn Dixit and Jonathan C. Kagan

Medzhitov, R., Preston-Hurlburt, P., & Janeway, C. A., Jr. (1997). A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature, 388, 394. Medzhitov, R., Preston-Hurlburt, P., Kopp, E., Stadlen, A., Chen, C., Ghosh, S., et al. (1998). MyD88 is an adaptor protein in the hToll/IL-1 receptor family signaling pathways. Molecular Cell, 2, 253. Meylan, E., Curran, J., Hofmann, K., Moradpour, D., Binder, M., Bartenschlager, R., et al. (2005). Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature, 437, 1167. Mikkelsen, S. S., Jensen, S. B., Chiliveru, S., Melchjorsen, J., Julkunen, I., Gaestel, M., et al. (2009). RIG-I-mediated activation of p38 MAPK is essential for viral induction of interferon and activation of dendritic cells: Dependence on TRAF2 and TAK1. Journal of Bio- logical Chemistry, 284, 10774. Monroe, K. M., McWhirter, S. M., & Vance, R. E. (2009). Identification of host cytosolic sensors and bacterial factors regulating the type I interferon response to Legionella pneumophila. PLoS Pathogens, 5, e1000665. Moore, C. B., Bergstralh, D. T., Duncan, J. A., Lei, Y., Morrison, T. E., Zimmermann, A. G., et al. (2008). NLRX1 is a regulator of mitochondrial antiviral immunity. Nature, 451, 573. Mossman, K. L., Macgregor, P. F., Rozmus, J. J., Goryachev, A. B., Edwards, A. M., & Smiley, J. R. (2001). Herpes simplex virus triggers and then disarms a host antiviral response. Journal of Virology, 75, 750. Nistal-Villan, E., Gack, M. U., Martinez-Delgado, G., Maharaj, N. P., Inn, K. S., Yang, H., et al. (2010). Negative role of RIG-I serine 8 phosphorylation in the regulation of interferon-beta production. Journal of Biological Chemistry, 285, 20252. O’Connell, R. M., Saha, S. K., Vaidya, S. A., Bruhn, K. W., Miranda, G. A., Zarnegar, B., et al. (2004). Type I interferon production enhances susceptibility to Listeria monocytogenes infection. The Journal of Experimental Medicine, 200, 437. O’Riordan, M., Yi, C. H., Gonzales, R., Lee, K. D., & Portnoy, D. A. (2002). Innate recognition of bacteria by a macrophage cytosolic surveillance pathway. In: Proceedings of the National Academy of Sciences of the United States of America, 99, 13861. Onoguchi, K., Onomoto, K., Takamatsu, S., Jogi, M., Takemura, A., Morimoto, S., et al. (2010). Virus-infection or 50ppp-RNA activates antiviral signal through redistribution of IPS-1 mediated by MFN1. PLoS Pathogens, 6, e1001012. Opitz, B., Vinzing, M., van Laak, V., Schmeck, B., Heine, G., Gunther, S., et al. (2006). Legionella pneumophila induces IFN-beta in lung epithelial cells via IPS-1 and IRF3, which also control bacterial replication. Journal of Biological Chemistry, 281, 36173. Oshiumi, H., Matsumoto, M., Hatakeyama, S., & Seya, T. (2009). Riplet/RNF135, a RING finger protein, ubiquitinates RIG-I to promote interferon-beta induction during the early phase of viral infection. Journal of Biological Chemistry, 284, 807. Oshiumi, H., Miyashita, M., Inoue, N., Okabe, M., Matsumoto, M., & Seya, T. (2010). The ubiquitin ligase Riplet is essential for RIG-I-dependent innate immune responses to RNA virus infection. Cell Host & Microbe, 8, 496. Palm, N. W., & Medzhitov, R. (2009). Pattern recognition receptors and control of adaptive immunity. Immunological Reviews, 227, 221. Pichlmair, A., Schulz, O., Tan, C. P., Naslund, T. I., Liljestrom, P., Weber, F., et al. (2006). RIG-I-mediated antiviral responses to single-stranded RNA bearing 50-phosphates. Science, 314, 997. Pichlmair, A., Schulz, O., Tan, C. P., Rehwinkel, J., Kato, H., Takeuchi, O., et al. (2009). Activation of MDA5 requires higher-order RNA structures generated during virus infection. Journal of Virology, 83, 10761. Platanias, L. C. (2005). Mechanisms of type-I- and type-II-interferon-mediated signalling. Nature Reviews. Immunology, 5, 375. RIG-I-Like Receptor Signaling 123

Plumet, S., Herschke, F., Bourhis, J. M., Valentin, H., Longhi, S., & Gerlier, D. (2007). Cytosolic 50-triphosphate ended viral leader transcript of measles virus as activator of the RIG I-mediated interferon response. PLoS One, 2, e279. Poeck, H., Bscheider, M., Gross, O., Finger, K., Roth, S., Rebsamen, M., et al. (2010). Rec- ognition of RNA virus by RIG-I results in activation of CARD9 and inflammasome signaling for interleukin 1 beta production. Nature Immunology, 11, 63. Roberts, T. L., Idris, A., Dunn, J. A., Kelly, G. M., Burnton, C. M., Hodgson, S., et al. (2009). HIN-200 proteins regulate caspase activation in response to foreign cytoplasmic DNA. Science, 323, 1057. Roth-Cross, J. K., Bender, S. J., & Weiss, S. R. (2008). Murine coronavirus mouse hepatitis virus is recognized by MDA5 and induces type I interferon in brain macrophages/ microglia. Journal of Virology, 82, 9829. Rothenfusser, S., Goutagny, N., DiPerna, G., Gong, M., Monks, B. G., Schoenemeyer, A., et al. (2005). The RNA helicase Lgp2 inhibits TLR-independent sensing of viral replication by retinoic acid-inducible gene-I. Journal of Immunology, 175, 5260. Roux, C. M., Rolan, H. G., Santos, R. L., Beremand, P. D., Thomas, T. L., Adams, L. G., et al. (2007). Brucella requires a functional Type IV secretion system to elicit innate immune responses in mice. Cellular Microbiology, 9, 1851. Saito, T., Hirai, R., Loo, Y. M., Owen, D., Johnson, C. L., Sinha, S. C., et al. (2007). Reg- ulation of innate antiviral defenses through a shared repressor domain in RIG-I and LGP2. In: Proceedings of the National Academy of Sciences of the United States of America, 104, 582. Saito, T., Owen, D. M., Jiang, F., Marcotrigiano, J., & Gale, M., Jr. (2008). Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA. Nature, 454, 523. Samanta, M., Iwakiri, D., Kanda, T., Imaizumi, T., & Takada, K. (2006). EB virus-encoded RNAs are recognized by RIG-I and activate signaling to induce type I IFN. EMBO Jour- nal, 25, 4207. Satoh, T., Kato, H., Kumagai, Y., Yoneyama, M., Sato, S., Matsushita, K., et al. (2010). LGP2 is a positive regulator of RIG-I- and MDA5-mediated antiviral responses. In: Pro- ceedings of the National Academy of Sciences of the United States of America, 107, 1512. Schlee, M., & Hartmann, G. (2010). The chase for the RIG-I ligand—Recent advances. Molecular Therapy, 18, 1254. Schlee, M., Roth, A., Hornung, V., Hagmann, C. A., Wimmenauer, V., Barchet, W., et al. (2009). Recognition of 50 triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative-strand virus. Immunity, 31, 25. Schmidt, A., Schwerd, T., Hamm, W., Hellmuth, J. C., Cui, S., Wenzel, M., et al. (2009). 50-Triphosphate RNA requires base-paired structures to activate antiviral signaling via RIG-I. Proceedings of the National Academy of Sciences of the United States of America, 106, 12067. Schulz, O., Diebold, S. S., Chen, M., Naslund, T. I., Nolte, M. A., Alexopoulou, L., et al. (2005). Toll-like receptor 3 promotes cross-priming to virus-infected cells. Nature, 433, 887. Seth, R. B., Sun, L., Ea, C. K., & Chen, Z. J. (2005). Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3. Cell, 122, 669. Stanley, S. A., Johndrow, J. E., Manzanillo, P., & Cox, J. S. (2007). The Type I IFN response to infection with Mycobacterium tuberculosis requires ESX-1-mediated secretion and contributes to pathogenesis. Journal of Immunology, 178, 3143. Stetson, D. B., & Medzhitov, R. (2006). Recognition of cytosolic DNA activates an IRF3- dependent innate immune response. Immunity, 24, 93. 124 Evelyn Dixit and Jonathan C. Kagan

Sumpter, R., Jr., Loo, Y. M., Foy, E., Li, K., Yoneyama, M., Fujita, T., et al. (2005). Regulating intracellular antiviral defense and permissiveness to hepatitis C virus RNA replication through a cellular RNA helicase, RIG-I. Journal of Virology, 79, 2689. Sun, W., Li, Y., Chen, L., Chen, H., You, F., Zhou, X., et al. (2009). ERIS, an endoplasmic reticulum IFN stimulator, activates innate immune signaling through dimerization. In: Proceedings of the National Academy of Sciences of the United States of America, 106, 8653. Sun, L., Wu, J., Du, F., Chen, X., & Chen, Z. J. (2012). Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science, 339, 786. Suthar, M. S., Ramos, H. J., Brassil, M. M., Netland, J., Chappell, C. P., Blahnik, G., et al. (2012). The RIG-I-like receptor LGP2 controls CD8( ) T cell survival and fitness. Immunity, 37, 235. þ Takahasi, K., Kumeta, H., Tsuduki, N., Narita, R., Shigemoto, T., Hirai, R., et al. (2009). Solution structures of cytosolic RNA sensor MDA5 and LGP2 C-terminal domains: Identification of the RNA recognition loop in RIG-I-like receptors. Journal of Biological Chemistry, 284, 17465. Takahasi, K., Yoneyama, M., Nishihori, T., Hirai, R., Kumeta, H., Narita, R., et al. (2008). Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses. Molecular Cell, 29, 428. Takeda, K., & Akira, S. (2005). Toll-like receptors in innate immunity. International Immu- nology, 17, 1. Tal, M. C., Sasai, M., Lee, H. K., Yordy, B., Shadel, G. S., & Iwasaki, A. (2009). Absence of autophagy results in reactive oxygen species-dependent amplification of RLR signaling. In: Proceedings of the National Academy of Sciences of the United States of America, 106, 2770. Tattoli, I., Carneiro, L. A., Jehanno, M., Magalhaes, J. G., Shu, Y., Philpott, D. J., et al. (2008). NLRX1 is a mitochondrial NOD-like receptor that amplifies NF-kappaB and JNK pathways by inducing reactive oxygen species production. EMBO Reports, 9, 293. Unterholzner, L., Keating, S. E., Baran, M., Horan, K. A., Jensen, S. B., Sharma, S., et al. (2010). IFI16 is an innate immune sensor for intracellular DNA. Nature Immunology, 11, 997. Uzri, D., & Gehrke, L. (2009). Nucleotide sequences and modifications that determine RIG- I/RNA binding and signaling activities. Journal of Virology, 83, 4174. Vance, J. E. (1990). Phospholipid synthesis in a membrane fraction associated with mitochondria. Journal of Biological Chemistry, 265, 7248. Venkataraman, T., Valdes, M., Elsby, R., Kakuta, S., Caceres, G., Saijo, S., et al. (2007). Loss of DExD/H box RNA helicase LGP2 manifests disparate antiviral responses. Journal of Immunology, 178, 6444. Wu, J., Sun, L., Chen, X., Du, F., Shi, H., Chen, C., et al. (2012). Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science, 339, 826. Xu, L. G., Wang, Y. Y., Han, K. J., Li, L. Y., Zhai, Z., & Shu, H. B. (2005). VISA is an adapter protein required for virus-triggered IFN-beta signaling. Molecular Cell, 19, 727. Yamamoto, M., Sato, S., Mori, K., Hoshino, K., Takeuchi, O., Takeda, K., et al. (2002). Cutting edge: A novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-beta promoter in the toll-like receptor signaling. Journal of Immunology, 169, 6668. Yoneyama, M., Kikuchi, M., Matsumoto, K., Imaizumi, T., Miyagishi, M., Taira, K., et al. (2005). Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity. Journal of Immunology, 175, 2851. RIG-I-Like Receptor Signaling 125

Yoneyama, M., Kikuchi, M., Natsukawa, T., Shinobu, N., Imaizumi, T., Miyagishi, M., et al. (2004). The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nature Immunology, 5, 730. Zeng, W., Sun, L., Jiang, X., Chen, X., Hou, F., Adhikari, A., et al. (2010). Reconstitution of the RIG-I pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity. Cell, 141, 315. Zhong, B., Yang, Y., Li, S., Wang, Y. Y., Li, Y., Diao, F., et al. (2008). The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation. Immunity, 29, 538. Zhou, S., Cerny, A. M., Zacharia, A., Fitzgerald, K. A., Kurt-Jones, E. A., & Finberg, R. W. (2010). Induction and inhibition of type I interferon responses by distinct components of lymphocytic choriomeningitis virus. Journal of Virology, 84, 9452. Intentionally left as blank INDEX

Note: Page numbers followed by “f ” indicate figures, and “t” indicate tables. A B Activation-induced cytidine deaminase B lymphocytes (AID) AID targets for DSBs initiation, 53–54 binding, 54–55 leukemia, 3, 43 DSBs and translocations RAG1/2 translocation, 53 description, 43 Bromodomain (BRD)-containing protein expression, 43–44 family off-target activity, 44 description, 24 transcription of mRNA, 43 development of MM, 24 expression and CSR induction, 51–52 inhibitors, 24 HTGTS and TC-Seq translocation, 53–54 in oncogene, 62–63 C physiologic mechanisms, 40 Chromatin Acute lymphoblastic leukemia (ALL) epigenetic marks, 26 and B-ALL, 3, 43 in RAG activity, 42 description, 3 remodeling complexes, 25 and T-ALL, 4, 15–16, 43 Chromosomal translocations treatment, 4 DNA DSB formation (see DNA Acute myeloid leukemia (AML) double-strand breaks (DNA DSBs)) chromosomal translocations, 57 DNA-repair mechanisms description, 3 C-NHEJ, 46–47 DNMT3A mutations, 7 DDR pathway, 45 MOZ gene, 22 NHEJ, 45 myelodysplastic syndromes high-throughput methods (MDS)/-neoplasms (MPN), 3 HTGTS, 51–52 subtypes, 3 TC-Seq, 52 TET proteins, 10–12 HTGTS and TC-Seq AID. See Activation-induced cytidine AID targets, B cells, 53–54 deaminase (AID) gene density, transcription, and ALL. See Acute lymphoblastic leukemia translocations, 54–55 (ALL) nuclear positioning and chromosomal AML. See Acute myeloid leukemia (AML) structure, 55–57 Antiviral immunity RAG1/2 translocation, pro-B cells, 53 host cell’s metabolic pathways, 99–100 mechanisms, 40 innate immune response, 100 spatial organization of the genome LGP2 role, 104 chromosome territories, 48–49 nucleic acid detection, 100–101 description, 48, 48f PRRs and PAMPs, 100 and DNA repair, 49 Arginine methyltransferases, 19–20 genome-wide contact analysis, 49–50 Autoimmune liver disease “transcription factories”, 49 PBC, 81 structural landscape PSC, 80–81 chromothripsis, 58–62

127 128 Index

Chromosomal translocations (Continued) fragile sites, 44–45 driver translocations, 57 nonprogrammed pathologic DSBs, 44–45 intra/interchromosomal physiologic/pathologic mechanisms, 40 rearrangements, 57–58 RAG-initiated DSBs and translocations, oncogenes, 62–63 41–43 territories (see Chromosome territories) topoisomerases, 45 TET proteins, 10–11 DNA methylation Chromosome territories aberrant methylation patterns, 6–7 functions of, 48–49 CpG islands, 6 and gene proximity, 47–48 hypomethylation, 6–7 in situ hybridization approaches, 48 IDH1 and IDH2 proteins, 12–14 Chromothripsis mutations, DNMT3a, 7 cancer types, 58 PHD-containing proteins, 24–25 chromosomal rearrangements, 57–58 TET proteins, 10–12 genomic disorders, 61–62 DNA methyltransferase (DNMT) implications, 60–61 DNMT3A mutations mechanisms, in normal and cancer cells, in hematopoietic malignancies, 7, 8f 59–60, 60f molecular consequence, 7–9 progressive rearrangement model, 58–59 mouse models, 9 Chronic liver disease. See Intestinal prognostic marker, 9 microbiota, chronic liver disease inhibitors, 9–10 Chronic lymphocytic leukemia (CLL), 4, 58 DNMT. See DNA methyltransferase Chronic myeloid leukemia (CML) (DNMT) chromosomal proximity, 47–48 Dysbiosis description, 2–3 with innate immune deficiency, 87–89 treatment with Imatinib, 2–3 probiotic interventions, 89–90 Cirrhosis, intestinal microbiota description, 78–79 E and HE, 79–80 Epigenetic modulators liver fibrogenesis, 78–79 arginine methyltransferases, 19–20 Classical NHEJ (C-NHEJ) BRD-containing protein family, 24 in knock-out mouse models/in human chromatin remodeling complexes, 25 patients, 46 DNA methylation (see DNA methylation) multistep DNA-repair process, 46 HATs, 22–23 sequence homology, 58–59 HDACs, 23 in translocations and chromosomal histone demethylases inhibitors integrity, 46–47 (KDMi), 22 CLL. See Chronic lymphocytic leukemia histone-modifying complexes and MLL, (CLL) 14–19 CML. See Chronic myeloid leukemia lysine demethylases (KDMs), 21 (CML) C-type lectin (CTL) receptors, 86–87 H HATs. See Histone acetyl transferases D (HATs) DNA-damage response (DDR) pathway, 45 HCC. See Hepatocellular carcinoma (HCC) DNA double-strand breaks (DNA DSBs) HDACs. See Histone deacetylases (HDACs) AID-initiated DSBs and translocations, Hematopoietic malignancies. 43–44 See also Leukemia Index 129

DNA methylation, 6–7 Inhibitors DNMT3A mutations, 7 BRD, 24 Hepatic encephalopathy (HE) DNMT, 9–10 intestinal microbiota, 78–79 HAT (HATi), 22–23 nonculture-based methods, 79–80 HDAC, 23 pathogenesis, 79 histone methyltransferase, 17 Hepatocellular carcinoma (HCC), 80 KDMi, 22 High-throughput genomic translocation Innate immunity sequencing (HTGTS) and intestinal microbiota AID targets, 53–54 CTL receptors, 86–87 analysis of SNPs, 52–53 dysbiosis, 87–89 application, 55–56 inflammasomes, 84–86 clone translocation junctions, 51–52 PRRs, 81–82 gene density, transcription, and receptors expression, 82 translocations, 54–55 TLRs, 82–84 normal mature B cells and pro-B cells, PRRs and PAMPs, 100 51–52, 51f Interferon (IFN) RAG1/2 translocation, 53 IFN-a, 101 TC-Seq, 52 IFN-b Histone acetyl transferases (HATs) IRF3 and IRF7, 102–103 family, 22 LGP2 role, antiviral immunity, 104 inhibitors (HATi), 22–23 IRF (see Interferon regulatory factor monocytic leukemia zinc-finger protein (IRF)) (MOZ), 22 type I IFNs, TLR9-associated liver MOZ-related factor (MORF), 22 damage, 83–84 Histone deacetylases (HDACs) Interferon regulatory factor (IRF) classes, 23 dimerization, 112–113 inhibitors (HDACi), 23 IRF3 and IRF7, 102–103 transcription role, 23 LGP2 role, antiviral immunity, 104 Histone demethylases inhibitors (KDMi), 22 ubiquitination system-activated IRF3, Histone-modifying complexes, leukemia 112–113 description, 14–17 Intestinal microbiota, chronic liver disease MLL function, 17–19 autoimmune liver disease, 80–81 PRC1, 17 cirrhosis and associated comorbidities, PRC2, 14 78–80 HTGTS. See High-throughput genomic gastrointestinal tract, 74 translocation sequencing (HTGTS) HCC, 80 hepatic artery, 74 I and innate immune system (see Innate IDH. See Isocitrate dehydrogenase (IDH) immunity) IFN. See Interferon (IFN) liver (see Liver) Inflammasomes NAFLD (see Nonalcoholic fatty liver components, 85 disease (NAFLD)) intestinal tracts of mice deficient, 87–88 probiotics, 89–90 NLRP3, 86 IRF. See Interferon regulatory factor (IRF) NLR proteins, 84–85 Isocitrate dehydrogenase (IDH) response against tissue damage, 85–86 animal models, 14 sequential stimuli, 84–85 as homodimers, 12–13 130 Index

Isocitrate dehydrogenase (IDH) (Continued ) MAVS signal, 115–117 2-hydroxyglutarate (2-HG), 13 NLRX1, 114 IDH1 and IDH2 mutations, 12–13 ubiquitination, RIG-I activation, 112 oncometabolites, role, 13 Mitochondria-associated membranes wild-type, 13 (MAM), 113, 115–116, 117–118 Mixed-lineage leukemia (MLL) L COMPASS complexes, 17–18 Leukemia description, 17–18 ALL, 3–4 fusion proteins, 18–19 AML, 3 genetic perturbations, 14–15, 15f B-ALL/T-ALL, chromosomal MLL-rearranged leukemias, 18 translocations, 43 role of CBX8, 19 chromosomal proximity, 47–48 MLL. See Mixed-lineage leukemia (MLL) chronic variants, 2 CLL, 4 N CML, 2–3 NAFLD. See Nonalcoholic fatty liver disease DNA methylation, 6–14 (NAFLD) epigenetic modifiers, 19, 20f NHEJ. See Nonhomologous DNA end epigenetics joining (NHEJ) bivalent domains, 25 NOD-like receptor (NLR) proteins classes of genes, 5 development and progression, NASH, 86 combinatorial chromatin marks, 26 Kupffer cells and sinusoidal endothelial combinatorial histone marks, 25–26 cells, 85 definition, 4 types, 84–85 DNA methylation, 5 Nonalcoholic fatty liver disease (NAFLD) long noncoding RNAs (lncRNAs), 26 calorie intake, Western society diets, modulators (see Epigenetic modulators) 76–77 nuclear architecture, 26–27 obesity, 75–76 perturbations, 5–6 prevalence, 75 phenomena, 4,5 prevalence of SIBO, 78 histone-modifying complexes, 14–19 primary and secondary, 75 IDH1 and IDH2 proteins, 12–14 for progression, gut-derived factors, 77–78 TET proteins, 10–12 regulation, 75–76, 76f types, 2 “two-hit” mechanism, 75 Liver Nonhomologous DNA end joining (NHEJ) chronic disease (see Intestinal microbiota, C-NHEJ (see Classical NHEJ (C-NHEJ)) chronic liver disease) description, 45 and gastrointestinal tract, 74 Lysine demethylases (KDMs) P amine oxidation and dioxygenases, 21 PAMPs. See Pathogen-associated molecular hydroxylation, 21 patterns (PAMPs) in tumorigenesis, 21 Pathogen-associated molecular patterns (PAMPs) M detection of viruses, 100–101 Microbiota. See Intestinal microbiota, innate immune system, 100 chronic liver disease Pattern recognition receptors (PRRs) Mitochondria homeostatic extrahepatic expression, 89 IDH mutations, leukemia, 13 host and indigenous microflora, 81–82 MAM, 113, 115–116, 117–118 innate immune response, 100 Index 131

innate receptors expression, 82 DSBs and translocations nucleic acid detection, 100–101 aberrant RAG activity, 42–43 TLRs, 82 mechanisms, 42 PBC. See Primary biliary cirrhosis (PBC) off-target RAG activity, 42–43 Peroxisomes recurrent translocations, B-cell MAVS, 113, 115–116 lymphomas, 43 RIG-I pathway, 112 V(D)J recombination, 41–42 Plant homeodomain (PHD)-containing in pro-B lymphocytes, 53, 55–56 proteins RIG-I-like receptors (RLRs) JARID1C, 24 activation translocation, 24–25 adapter protein MAVS, 113 Polycomb repressive complex 1 (PRC1) ATPase activity, 110–111 histone methyltransferase inhibitors, 17 CARDs and helicase domain, 110 HSCs maintenance and transformation crystallographic structures, 111 in vivo, 17 description, 109–110 Polycomb repressive complex 2 (PRC2) downstream signaling, 111–112 components, 14–15 TRIM25 and K63-linked ubiquitin, EZH2, 14–15, 15f 112–113 gene silencing, 14–15 ubiquitination, 112 loss-of-function mutation, 15–16 adapter protein MAVS, 102–103 mutations, protein ASXL1, 17 domain architecture, 101–102, 102f and PRC1, 17 IFN-b transcription, 102–104 T-ALL mutations, 15–16 IFN role, bacterial infections, 108 as tumor suppressor, 16 IRF family, 102–103, 103f PRC1. See Polycomb repressive complex 1 LGP2 role, 104 (PRC1) ligand specificities, 102–103 PRC2. See Polycomb repressive complex 2 MDA5, 104 (PRC2) nucleic acid detection, 100–101 Primary biliary cirrhosis (PBC) nucleic acid-specific endosomal TLRs, 101 antimitochondrial antibodies (AMAs), 81 regulators, RLR signaling autoimmune liver disorder, 81 adapter protein MAVS, 115–116 TLR4 expression, 82–83 MFN2 and MFN1, 115–117 Primary sclerosing cholangitis (PSC) NLRX1, 114 CARD9, 86–87 STING, 114–115 microbiota, 80–81 ZAPS, 113–114 pathogenesis, 80 RIG-I, MDA5, and LGP2, 101–102 TLR4 expression, 82–83 structural characteristics, 104–106 Probiotics type II IFN-g, Shigella flexneri, 109 description, 89 viruses detection, 106–107, 107t interventions, 89–90 RLRs. See RIG-I-like receptors (RLRs) and prebiotics, 89 PRRs. See Pattern recognition receptors T (PRRs) T-cell acute lymphoblastic leukemia PSC. See Primary sclerosing cholangitis (PSC) (T-ALL) mutations, 15–16 Ten-eleven translocation (TET) proteins R J-binding proteins, 10 RAG. See Recombination-activating genes mutations analysis, TET1, 10 (RAG) TET2 mouse models, 12 Recombination-activating genes (RAG) TET2 mutations, 11–12 132 Index

TLRs. See Toll-like receptors (TLRs) and RLRs, 101 Toll-like receptors (TLRs) TLR9, 83–84 concanavalin A (ConA) model, 84 TLR4–MyD88–NF-kB signaling, 82–83 expression, 82 TLR4 pathway, 117–118 microbial translocation, 84 Translocation-capture sequencing (TC-Seq) nucleic acid detection, 100–101 AID targets, 53–54 PRRs, 82 description, 52 CONTENTS OF RECENT VOLUMES

Volume 85 Volume 87 Role of the LAT Adaptor in T-Cell Cumulative Subject Index Volumes 66–82 Development and Th2 Differentiation Bernard Malissen, Enrique Aguado, and Marie Malissen Volume 86 The Integration of Conventional and Adenosine Deaminase Deficiency: Unconventional T Cells that Metabolic Basis of Immune Characterizes Cell-Mediated Responses Deficiency and Pulmonary Daniel J. Pennington, David Vermijlen, Inflammation Emma L. Wise, Sarah L. Clarke, Michael R. Blackburn and Robert E. Tigelaar, and Adrian C. Hayday Rodney E. Kellems Negative Regulation of Cytokine and TLR Mechanism and Control of V(D)J Signalings by SOCS and Others Recombination Versus Class Switch Tetsuji Naka, Minoru Fujimoto, Hiroko Recombination: Similarities Tsutsui, and Akihiko Yoshimura and Differences Pathogenic T-Cell Clones in Autoimmune Darryll D. Dudley, Jayanta Chaudhuri, Diabetes: More Lessons from the NOD Craig H. Bassing, and Frederick W. Alt Mouse Isoforms of Terminal Kathryn Haskins Deoxynucleotidyltransferase: The Biology of Human Lymphoid Developmental Aspects and Malignancies Revealed by Gene Function Expression Profiling To-Ha Thai and John F. Kearney Louis M. Staudt and Sandeep Dave Innate Autoimmunity New Insights into Alternative Mechanisms Michael C. Carroll and V. Michael Holers of Immune Receptor Diversification Formation of Bradykinin: A Major Gary W. Litman, John P. Cannon, and Contributor to the Innate Jonathan P. Rast Inflammatory Response The Repair of DNA Damages/ Kusumam Joseph and Allen P. Kaplan Modifications During the Maturation of Interleukin-2, Interleukin-15, and the Immune System: Lessons from Their Roles in Human Natural Human Primary Immunodeficiency Killer Cells Disorders and Animal Models Brian Becknell and Michael A. Caligiuri Patrick Revy, Dietke Buck, Franc¸oise le Deist, and Jean-Pierre de Villartay Regulation of Antigen Presentation and Cross-Presentation in the Dendritic Antibody Class Switch Recombination: Cell Network: Facts, Hypothesis, Roles for Switch Sequences and and Immunological Implications Mismatch Repair Proteins Nicholas S. Wilson and Jose A. Villadangos Irene M. Min and Erik Selsing

Index Index

133 134 Contents of Recent Volumes

Volume 88 The Surprising Diversity of Lipid Antigens for CD1-Restricted T Cells CD22: A Multifunctional Receptor That D. Branch Moody Regulates B Lymphocyte Survival and Signal Transduction Lysophospholipids as Mediators Thomas F. Tedder, Jonathan C. Poe, and of Immunity Karen M. Haas Debby A. Lin and Joshua A. Boyce Tetramer Analysis of Human Autoreactive Systemic Mastocytosis CD4-Positive T Cells Jamie Robyn and Dean D. Metcalfe Gerald T. Nepom Regulation of Fibrosis by the Regulation of Phospholipase C-g2 Immune System Networks in B Lymphocytes Mark L. Lupher, Jr. and Masaki Hikida and Tomohiro Kurosaki W. Michael Gallatin Role of Human Mast Cells and Basophils in Immunity and Acquired Alterations in Bronchial Asthma Cognition and Emotion: Lessons from Gianni Marone, Massimo Triggiani, Arturo SLE Genovese, and Amato De Paulis Betty Diamond, Czeslawa Kowal, Patricio T. Huerta, Cynthia Aranow, A Novel Recognition System for Meggan Mackay, Lorraine A. DeGiorgio, MHC Class I Molecules Constituted Ji Lee, Antigone Triantafyllopoulou, by PIR Joel Cohen-Solal Bruce, and T. Volpe Toshiyuki Takai Immunodeficiencies with Dendritic Cell Biology Autoimmune Consequences Francesca Granucci, Maria Foti, and Luigi D. Notarangelo, Eleonora Gambineri, Paola Ricciardi-Castagnoli and Raffaele Badolato The Murine Diabetogenic Class II Histocompatibility Molecule I-Ag7: Index Structural and Functional Properties and Specificity of Peptide Selection Anish Suri and Emil R. Unanue Volume 90 RNAi and RNA-Based Regulation of Cancer Immunosurveillance and Immune System Function Immunoediting: The Roles of Dipanjan Chowdhury and Carl D. Novina Immunity in Suppressing Tumor Development and Shaping Tumor Index Immunogenicity Mark J. Smyth, Gavin P. Dunn, and Robert D. Schreiber Volume 89 Mechanisms of Immune Evasion by Tumors Posttranscriptional Mechanisms Regulating Charles G. Drake, Elizabeth Jaffee, and the Inflammatory Response Drew M. Pardoll Georg Stoecklin Paul Anderson Development of Antibodies and Negative Signaling in Fc Chimeric Molecules for Cancer Receptor Complexes Immunotherapy Marc Dae¨ron and Renaud Lesourne Thomas A. Waldmann and John C. Morris Contents of Recent Volumes 135

Induction of Tumor Immunity Accessibility Control of V(D)J Following Allogeneic Stem Cell Recombination Transplantation Robin Milley Cobb, Kenneth J. Oestreich, Catherine J. Wu and Jerome Ritz Oleg A. Osipovich, and Eugene M. Oltz Vaccination for Treatment and Prevention of Cancer in Animal Models Targeting Integrin Structure and Function Federica Cavallo, Rienk Offringa, in Disease Sjoerd H. van der Burg, Guido Forni, Donald E. Staunton, Mark L. Lupher, and Cornelis J. M. Melief Robert Liddington, and W. Michael Gallatin Unraveling the Complex Relationship Between Cancer Immunity and Endogenous TLR Ligands and Autoimmunity: Lessons from Autoimmunity Melanoma and Vitiligo Hermann Wagner Hiroshi Uchi, Rodica Stan, Mary Jo Turk, Genetic Analysis of Innate Manuel E. Engelhorn, Gabrielle Immunity A. Rizzuto, Stacie M. Goldberg, Kasper Hoebe, Zhengfan Jiang, Jedd D. Wolchok, and Alan N. Houghton Koichi Tabeta, Xin Du, Immunity to Melanoma Antigens: Philippe Georgel, Karine Crozat, From Self-Tolerance to and Bruce Beutler Immunotherapy TIM Family of Genes in Immunity Craig L. Slingluff, Jr., and Tolerance Kimberly A. Chianese-Bullock, Vijay K. Kuchroo, Jennifer Hartt Meyers, Timothy N. J. Bullock, William W. Grosh, Dale T. Umetsu, and David W. Mullins, Lisa Nichols, Walter Rosemarie H. DeKruyff Olson, Gina Petroni, Mark Smolkin, and Victor H. Engelhard Inhibition of Inflammatory Responses by Leukocyte Ig-Like Receptors Checkpoint Blockade in Cancer Howard R. Katz Immunotherapy Alan J. Korman, Karl S. Peggs, and Index James P. Allison Combinatorial Cancer Immunotherapy Volume 92 F. Stephen Hodi and Glenn Dranoff Systemic Lupus Erythematosus: Multiple Immunological Phenotypes in a Index Complex Genetic Disease Anna-Marie Fairhurst, Amy E. Wandstrat, and Volume 91 Edward K. Wakeland A Reappraisal of Humoral Immunity Based Avian Models with Spontaneous on Mechanisms of Antibody-Mediated Autoimmune Diseases Protection Against Intracellular Georg Wick, Leif Andersson, Karel Pathogens Hala, M. Eric Gershwin,Carlo Selmi, Arturo Casadevall and Gisela F. Erf, Susan J. Lamont, and Liise-anne Pirofski Roswitha Sgonc 136 Contents of Recent Volumes

Functional Dynamics of Naturally Regulation of Immune Responses and Occurring Regulatory T Cells in Health Hematopoiesis by the Rap1 Signal and Autoimmunity Nagahiro Minato, Kohei Kometani, and Megan K. Levings, Sarah Allan, Eva Masakazu Hattori d’Hennezel, and Ciriaco A. Piccirillo Lung Dendritic Cell Migration BTLA and HVEM Cross Talk Hamida Hammad and Bart N. Lambrecht Regulates Inhibition and Costimulation Maya Gavrieli, John Sedy, Index Christopher A. Nelson, and Kenneth M. Murphy Volume 94 The Human T Cell Response to Melanoma Discovery of Activation-Induced Cytidine Antigens Deaminase, the Engraver of Antibody Pedro Romero, Jean-Charles Cerottini, and Memory Daniel E. Speiser Masamichi Muramatsu, Hitoshi Nagaoka, Antigen Presentation and the Reiko Shinkura, Nasim A. Begum, and Ubiquitin-Proteasome System in Tasuku Honjo Host–Pathogen Interactions DNA Deamination in Immunity: AID in the Joana Loureiro and Hidde L. Ploegh Context of Its APOBEC Relatives Silvestro G. Conticello, Marc-Andre Langlois, Index Zizhen Yang, and Michael S. Neuberger The Role of Activation-Induced Deaminase Volume 93 in Antibody Diversification and Chromosome Translocations Class Switch Recombination: A Almudena Ramiro, Bernardo Reina Comparison Between Mouse San-Martin, Kevin McBride, and Human Mila Jankovic, Vasco Barreto, Qiang Pan-Hammarstro¨m, Yaofeng Zhao, Andre´ Nussenzweig, and and Lennart Hammarstro¨m Michel C. Nussenzweig Anti-IgE Antibodies for the Treatment of Targeting of AID-Mediated Sequence IgE-Mediated Allergic Diseases Diversification by cis-Acting Tse Wen Chang, Pheidias C. Wu, Determinants C. Long Hsu, and Alfur F. Hung Shu Yuan Yang and David G. Schatz Immune Semaphorins: Increasing Members AID-Initiated Purposeful Mutations in and Their Diverse Roles Immunoglobulin Genes Hitoshi Kikutani, Kazuhiro Suzuki, and Myron F. Goodman, Matthew D. Scharff, Atsushi Kumanogoh and Floyd E. Romesberg Tec Kinases in T Cell and Mast Evolution of the Immunoglobulin Cell Signaling Heavy Chain Class Switch Martin Felices, Markus Falk, Yoko Kosaka, Recombination Mechanism and Leslie J. Berg Jayanta Chaudhuri, Uttiya Basu, Ali Zarrin, Integrin Regulation of Lymphocyte Catherine Yan, Sonia Franco, Thomas Trafficking: Lessons from Structural and Perlot, Bao Vuong, Jing Wang, Signaling Studies Ryan T. Phan, Abhishek Datta, Tatsuo Kinashi John Manis, and Frederick W. Alt Contents of Recent Volumes 137

Beyond SHM and CSR: AID and Related Volume 96 Cytidine Deaminases in the Host Response to Viral Infection New Insights into Adaptive Immunity Brad R. Rosenberg and in Chronic Neuroinflammation F. Nina Papavasiliou Volker Siffrin, Alexander U. Brandt, Josephine Herz, and Frauke Zipp Role of AID in Tumorigenesis Il-mi Okazaki, Ai Kotani, and Regulation of Interferon-g During Innate Tasuku Honjo and Adaptive Immune Responses Jamie R. Schoenborn and Christopher Pathophysiology of B-Cell Intrinsic B. Wilson Immunoglobulin Class Switch Recombination Deficiencies The Expansion and Maintenance of Anne Durandy, Nadine Taubenheim, Antigen-Selected CD8þ T Cell Sophie Peron, and Alain Fischer Clones Douglas T. Fearon Index Inherited Complement Regulatory Protein Deficiency Predisposes to Human Disease in Acute Injury and Chronic Inflammatory States Volume 95 Anna Richards, David Kavanagh, Fate Decisions Regulating Bone Marrow and John P. Atkinson and Peripheral B Lymphocyte Fc-Receptors as Regulators of Immunity Development Falk Nimmerjahn and Jeffrey V. Ravetch John G. Monroe and Kenneth Dorshkind Tolerance and Autoimmunity: Index Lessons at the Bedside of Primary Immunodeficiencies Magda Carneiro-Sampaio and Antonio Coutinho Volume 97 T Cell Activation and the Cytoskeleton: B-Cell Self-Tolerance in Humans You Can’t Have One Without Hedda Wardemann and Michel the Other C. Nussenzweig Timothy S. Gomez and Daniel Manipulation of Regulatory T-Cell D. Billadeau Number and Function with HLA Class II Transgenic Mice Mimic CD28-Specific Monoclonal Human Inflammatory Diseases Antibodies Ashutosh K. Mangalam, Govindarajan Thomas Hu¨nig Rajagopalan, Veena Taneja, and Osteoimmunology: A View from Chella S. David the Bone Roles of Zinc and Zinc Signaling in Jean-Pierre David Immunity: Zinc as an Intracellular Mast Cell Proteases Signaling Molecule Gunnar Pejler, Magnus A˚brink, Toshio Hirano, Masaaki Murakami, Maria Ringvall, and Sara Wernersson Toshiyuki Fukada, Keigo Nishida, Satoru Yamasaki, and Index Tomoyuki Suzuki 138 Contents of Recent Volumes

The SLAM and SAP Gene Families Control Volume 99 Innate and Adaptive Immune Responses Cis-Regulatory Elements and Epigenetic Silvia Calpe, Ninghai Wang, Xavier Romero, Changes Control Genomic Scott B. Berger, Arpad Lanyi, Pablo Engel, Rearrangements of the IgH Locus and Cox Terhorst Thomas Perlot and Frederick W. Alt Conformational Plasticity and Navigation of DNA-PK: The Means to Justify the Ends? Signaling Proteins in Antigen-Activated Katheryn Meek, Van Dang, and Susan B Lymphocytes P. Lees-Miller Niklas Engels, Michael Engelke, and Ju¨rgen Thymic Microenvironments for T-Cell Wienands Repertoire Formation Takeshi Nitta, Shigeo Murata, Tomoo Ueno, Index Keiji Tanaka, and Yousuke Takahama Pathogenesis of Myocarditis and Dilated Volume 98 Cardiomyopathy Daniela Cihakova and Noel R. Rose Immune Regulation by B Cells and Antibodies: A View Towards Emergence of the Th17 Pathway and Its the Clinic Role in Host Defense Kai Hoehlig, Vicky Lampropoulou, Toralf Roch, Darrell B. O’Quinn, Matthew T. Palmer, Patricia Neves, Elisabeth Calderon-Gomez, Yun Kyung Lee, and Casey T. Weaver Stephen M. Anderton, Ulrich Steinhoff, and Peptides Presented In Vivo by HLA-DR in Simon Fillatreau Thyroid Autoimmunity Cumulative Environmental Changes, Laia Muixı´, In˜aki Alvarez, and Dolores Skewed Antigen Exposure, and the Jaraquemada Increase of Allergy Tse Wen Chang and Ariel Y. Pan Index New Insights on Mast Cell Activation via the High Affinity Receptor for IgE Volume 100 Juan Rivera, Nora A. Fierro, Ana Olivera, Autoimmune Diabetes Mellitus—Much and Ryo Suzuki Progress, but Many Challenges Hugh O. McDevitt and Emil R. Unanue B Cells and Autoantibodies in the Pathogenesis of Multiple Sclerosis and CD3 Antibodies as Unique Tools to Restore Related Inflammatory Demyelinating Self-Tolerance in Established Diseases Autoimmunity: Their Mode of Action Katherine A. McLaughlin and and Clinical Application in Type 1 Kai W. Wucherpfennig Diabetes Sylvaine You, Sophie Candon, Chantal Human B Cell Subsets Kuhn, Jean-Franc¸ois Bach, and Lucienne Stephen M. Jackson, Patrick C. Wilson, Chatenoud Judith A. James, and J. Donald Capra GAD65 Autoimmunity—Clinical Studies Index Raivo Uibo and A˚ke Lernmark Contents of Recent Volumes 139

CD8 T Cells in Type 1 Diabetes Volume 102 Sueþ Tsai, Afshin Shameli, and Pere Santamaria Antigen Presentation by CD1: Lipids, T Cells, and NKT Cells in Microbial Dysregulation of T Cell Peripheral Immunity Tolerance in Type 1 Diabetes Nadia R. Cohen, Salil Garg, and Michael R. Tisch and B. Wang B. Brenner Gene–Gene Interactions in the NOD How the Immune System Achieves Mouse Model of Type 1 Diabetes Self–Nonself Discrimination William M. Ridgway, Laurence B. Peterson, During Adaptive Immunity John A. Todd, Dan B. Rainbow, Barry Hong Jiang and Leonard Chess Healy, and Linda S. Wicker Cellular and Molecular Mechanisms in Index Atopic Dermatitis Michiko K. Oyoshi, Rui He, Lalit Kumar, Juhan Yoon, and Raif S. Geha Volume 101 Micromanagers of Immune Cell Fate TSLP in Epithelial Cell and Dendritic and Function Cell Cross Talk Fabio Petrocca and Judy Lieberman Yong-Jun Liu Immune Pathways for Translating Viral Natural Killer Cell Tolerance: Licensing Infection into Chronic Airway and Other Mechanisms Disease A. Helena Jonsson and Wayne M. Yokoyama Michael J. Holtzman, Derek E. Byers, Loralyn A. Benoit, John T. Battaile, Biology of the Eosinophil Yingjian You, Eugene Agapov, Chaeho Carine Blanchard and Marc E. Rothenberg Park, Mitchell H. Grayson, Edy Y. Kim, Basophils: Beyond Effector Cells of Allergic and Anand C. Patel Inflammation John T. Schroeder Index DNA Targets of AID: Evolutionary Link Between Antibody Somatic Volume 103 Hypermutation and Class Switch The Physiological Role of Lysyl tRNA Recombination Synthetase in the Immune System Jason A. Hackney, Shahram Misaghi, Hovav Nechushtan, Sunghoon Kim, Kate Senger, Christopher Garris, Gillian Kay, and Ehud Razin Yonglian Sun, Maria N. Lorenzo, and Ali A. Zarrin Kill the Bacteria … and Also Their Messengers? Interleukin 5 in the Link Between the Robert Munford, Mingfang Lu, and Alan Innate and Acquired Immune Varley Response Kiyoshi Takatsu, Taku Kouro, and Yoshinori Role of SOCS in Allergic and Innate Nagai Immune Responses Suzanne L. Cassel and Paul Index B. Rothman 140 Contents of Recent Volumes

Multitasking by Exploitation of Intracellular The Family of IL-10-Secreting CD4þ Transport Functions: The Many Faces T Cells of FcRn Keishi Fujio, Tomohisa Okamura, E. Sally Ward and Raimund J. Ober and Kazuhiko Yamamoto Artificial Engineering of Secondary Index Lymphoid Organs Jonathan K. H. Tan and Volume 104 Takeshi Watanabe Regulation of Gene Expression in AID and Somatic Hypermutation Peripheral T Cells by Runx Robert W. Maul and Patricia Transcription Factors J. Gearhart Ivana M. Djuretic, Fernando Cruz-Guilloty, and Anjana Rao BCL6: Master Regulator of the Germinal Center Reaction and Long Noncoding RNAs: Implications Key Oncogene in B Cell for Antigen Receptor Diversification Lymphomagenesis Grace Teng and F. Nina Papavasiliou Katia Basso and Riccardo Pathogenic Mechanisms of Allergic Dalla-Favera Inflammation: Atopic Asthma as a Paradigm Index Patrick G. Holt, Deborah H. Strickland, Anthony Bosco, and Frode L. Jahnsen The Amplification Loop of the Volume 106 Complement Pathways The Role of Innate Immunity in Peter J. Lachmann B Cell Acquisition of Antigen Within LNs Index Santiago F. Gonzalez, Michael P. Kuligowski, Lisa A. Pitcher, Volume 105 Ramon Roozendaal, and Michael Learning from Leprosy: Insight into the C. Carroll Human Innate Immune Response Nuclear Receptors, Inflammation, Dennis Montoya and Robert L. Modlin and Neurodegenerative The Immunological Functions of Saposins Diseases Alexandre Darmoise, Patrick Maschmeyer, Kaoru Saijo, Andrea Crotti, and and Florian Winau Christopher K. Glass OX40–OX40 Ligand Interaction in Novel Tools for Modulating Immune T-Cell-Mediated Immunity and Responses in the Host— Immunopathology Polysaccharides from the Capsule Naoto Ishii, Takeshi Takahashi, of Commensal Bacteria Pejman Soroosh, and Suryasarathi Dasgupta and Kazuo Sugamura Dennis L. Kasper Contents of Recent Volumes 141

The Role of Mechanistic Factors in Host–Bacterial Symbiosis in Health and Promoting Chromosomal Disease Translocations Found in Lymphoid Janet Chow, S. Melanie Lee, Yue Shen, Arya and Other Cancers Khosravi, and Sarkis K. Mazmanian Yu Zhang, Monica Gostissa, Dominic G. Hildebrand, Michael S. Becker, Cristian Index Boboila, Roberto Chiarle, Susanna Lewis, and Frederick W. Alt Volume 108 Index Macrophage Proinflammatory Activation and Deactivation: A Question of Balance Volume 107 Annabel F. Valledor, Monica Comalada, Luis Functional Biology of the IL-22-IL-22R Santamarı´a-Babi, Jorge Lloberas, and Pathway in Regulating Immunity Antonio Celada and Inflammation at Barrier Natural Helper Cells: A New Player in the Surfaces Innate Immune Response against Gregory F. Sonnenberg, Lynette A. Fouser, Helminth Infection David Artis Shigeo Koyasu, Kazuyo Moro, Masanobu Innate Signaling Networks in Mucosal IgA Tanabe, and Tsutomu Takeuchi Class Switching Mapping of Switch Recombination Alejo Chorny, Irene Puga, and Andrea Junctions, a Tool for Studying Cerutti DNA Repair Pathways during Specificity of the Adaptive Immune Immunoglobulin Class Switching Response to the Gut Microbiota Janet Stavnezer, Andrea Bjo¨rkman, Daniel A. Peterson and Roberto A. Jimenez Likun Du, Alberto Cagigi, and Qiang Cardona Pan-Hammarstro¨m Intestinal Dendritic Cells How Tolerogenic Dendritic Cells Induce Maria Rescigno Regulatory T Cells Roberto A. Maldonado and Ulrich H. The Many Face-Lifts of CD4 T Helper von Andrian Cells Daniel Mucida and Hilde Cheroutre Index GALT: Organization and Dynamics Leading to IgA Synthesis Keiichiro Suzuki, Shimpei Kawamoto, Volume 109 Mikako Maruya, and Sidonia Dynamic Palmitoylation and the Role of Fagarasan DHHC Proteins in T Cell Activation Bronchus-Associated Lymphoid Tissue and Anergy (BALT): Structure and Function Nadejda Ladygina, Brent R. Martin, and Troy D. Randall Amnon Altman 142 Contents of Recent Volumes

Transcriptional Control of Natural “A Rose is a Rose is a Rose,” but Killer Cell Development and Function CVID is Not CVID: Common Variable David G. T. Hesslein and Lewis. L. Lanier Immune Deficiency (CVID), What do we Know in 2011? The Control of Adaptive Immune Responses Patrick F. K. Yong, James E. D. by the Innate Immune System Thaventhiran, and Bodo Grimbacher Dominik Schenten and Ruslan Medzhitov Role of Activation-Induced Cytidine The Evolution of Adaptive Immunity in Deaminase in Inflammation-Associated Vertebrates Cancer Development Masayuki Hirano, Sabyasachi Das, Hiroyuki Marusawa, Atsushi Takai, Peng Guo, and Max D. Cooper and Tsutomu Chiba T Helper Cell Differentiation: More Comparative Genomics and Evolution than Just Cytokines of Immunoglobulin-Encoding Beata Zygmunt and Marc Veldhoen Loci in Tetrapods Sabyasachi Das, Masayuki Hirano, Index Chelsea McCallister, Rea Tako, and Nikolas Nikolaidis Volume 110 Pax5: A Master Regulator of B Cell AID Targeting in Antibody Diversity Development and Leukemogenesis Rushad Pavri and Michel C. Nussenzweig Jasna Medvedovic, Anja Ebert, Hiromi Tagoh, and Meinrad The IgH Locus 30 Regulatory Region: Busslinger Pulling the Strings from Behind Eric Pinaud, Marie Marquet, Re´mi Fiancette, Index Sophie Pe´ron, Christelle Vincent-Fabert, Yves Denizot, and Michel Cogne´ Transcriptional and Epigenetic Regulation Volume 112 of CD4/CD8 Lineage Choice Stability of Regulatory T-cell Lineage Ichiro Taniuchi and Wilfried Ellmeier Shohei Hori Modeling a Complex Disease: Multiple Thymic and Peripheral Differentiation of Sclerosis Regulatory T Cells Florian C. Kurschus, Simone Wo¨rtge, and Ari Hyang-Mi Lee, Jhoanne Lynne Bautista, Waisman and Chyi-Song Hsieh Autoinflammation by Endogenous DNA Regulatory T Cells in Infection Shigekazu Nagata and Kohki Kawane Rick M. Maizels and Katherine A. Smith Index Biological Functions of Regulatory T Cells Ethan M. Shevach Volume 111 Extrathymic Generation of Regulatory Early Steps of Follicular Lymphoma T Cells—Chances and Challenges Pathogenesis for Prevention of Autoimmune Disease Sandrine Roulland, Mustapha Faroudi, Carolin Daniel, and Harald von Boehmer Emilie Mamessier, Ste´phanie Sungalee, Gilles Salles, and Bertrand Nadel Index Contents of Recent Volumes 143

Volume 113 Volume 114 Studies with Listeria monocytogenes Lead the Nucleic Acid Adjuvants: Toward an Way Educated Vaccine Emil R. Unanue and Javier A. Carrero Jasper G. van den Boorn, Winfried Barchet, and Gunther Hartmann Interactions of Listeria monocytogenes with the Autophagy System of Host Cells Structure-Based Design for High-Hanging Grace Y. Lam, Mark A. Czuczman, Vaccine Fruits Darren E. Higgins and John H. Brumell Jaap W. Back and Johannes P. M. Langedijk Virulence Factors That Modulate the Cell Mechanisms of Peptide Vaccination in Biology of Listeria Infection and the Mouse Models: Tolerance, Immunity, Host Response and Hyperreactivity Serge Mostowy and Pascale Cossart Thorbald van Hall and Sjoerd H. van der Burg Dendritic Cells in Listeria monocytogenes Experience with Synthetic Vaccines for Infection Cancer and Persistent Virus Infections Brian T. Edelson in Nonhuman Primates and Patients Esther D. Quakkelaar and Cornelis J. M. Probing CD8 T Cell Responses with Listeria Melief monocytogenes Infection Stephanie A. Condotta, Martin J. Richer, Malaria Vaccine Development Using Vladimir P. Badovinac and John Synthetic Peptides as a Technical T. Harty Platform Giampietro Corradin, Nora Ce´spedes, Listeria monocytogenes and Its Products as Antonio Verdini, Andrey V. Kajava, Agents for Cancer Immunotherapy Myriam Are´valo-Herrera, and So´crates Patrick Guirnalda, Laurence Wood and Herrera Yvonne Paterson Enhancing Cancer Immunotherapy by Monocyte-Mediated Immune Defense Intracellular Delivery of Cell-Penetrating Against Murine Listeria monocytogenes Peptides and Stimulation of Pattern- Infection Recognition Receptor Signaling Natalya V. Serbina, Chao Shi and Helen Y. Wang and Rong-Fu Wang Eric G. Pamer TLR Ligand–Peptide Conjugate Innate Immune Pathways Triggered by Vaccines: Toward Clinical Application Listeria monocytogenes and Their Role Gijs G. P. Zom, Selina Khan, Dmitri in the Induction of Cell-Mediated V. Filippov, and Ferry Ossendorp Immunity Chelsea E. Witte, Kristina A. Archer, Behavior and Function of Tissue-Resident Chris S. Rae, John-Demian Sauer, Memory T cells Josh J. Woodward and Silvia Ariotti, John B. Haanen, and Ton Daniel A. Portnoy N. Schumacher Mechanisms and Immunological Effects of Rational Design of Vaccines: Learning from Lymphocyte Apoptosis Caused by Immune Evasion Mechanisms of Listeria monocytogenes Persistent Viruses and Tumors Javier A. Carrero, and Emil R. Unanue Ramon Arens

Index Index 144 Contents of Recent Volumes

Volume 115 Volume 116 The Immunobiology of IL-27 Classical and Alternative End-Joining Aisling O’Hara Hall, Jonathan S. Silver, and Pathways for Repair of Christopher A. Hunter Lymphocyte-Specific and General DNA Double-Strand Breaks Autoimmune Arthritis: The Interface Cristian Boboila, Frederick W. Alt, and Between the Immune System Bjoern Schwer and Joints Noriko Komatsu and Hiroshi Takayanagi The Leukotrienes: Immune-Modulating Lipid Mediators of Disease Immunological Tolerance During Antonio Di Gennaro and Fetal Development: From Mouse Jesper Z. Haeggstro¨m to Man Jeff E. Mold and Joseph M. McCune Gut Microbiota Drives Metabolic Disease in Immunologically Altered Mice Mapping Lupus Susceptibility Benoit Chassaing, Jesse D. Aitken, Genes in the NZM2410 Andrew T. Gewirtz, and Mouse Model Matam Vijay-Kumar Laurence Morel What is Unique About the IgE Response? Functional Heterogeneity in the Basophil Huizhong Xiong, Maria A. Curotto de Cell Lineage Lafaille, and Juan J. Lafaille Mark C. Siracusa, Elia D. Tait Wojno, and David Artis Prostanoids as Regulators of Innate and Adaptive Immunity An Emerging Role of RNA-Binding Takako Hirata and Shuh Narumiya Proteins as Multifunctional Regulators of Lymphocyte Development and Lymphocyte Development: Integration of Function DNA Damage Response Signaling Martin Turner and Daniel J. Hodson Jeffrey J. Bednarski and Barry P. Sleckman Active and Passive Anticytokine Immune Index Therapies: Current Status and Development He´le`ne Le Buanec, Armand Bensussan, Martine Bagot, Robert C. Gallo, and Daniel Zagury

Index OH OH A O O O IDH1 or IDH2 Mutant OH O IDH1 or IDH2 HO O O

OH OH OH Isocitrate Oxoglutarate O

OH O OH

2-Hydroxyglutarate

NH NH NH 2 2 OH 2 CH3 N DNMTs N TETs N

N O N O N O H H H ++ Oxoglutarate Succinate

B (i) CpG CpG RNAP2 CpG

DNMT DNMT DNMT (ii) X CpG CpG RNAP2 CpG

(iii) TET2 TET2 TET2 ? CpG CpG Unmodified CpG island CpG RNAP2 CpG

CpG Methylated CpG island Hydroxy methylated CpG CpG island Chapter 1, Figure 1.1 (See page 8 of this volume.) A Polycomb repressive complex 2 B EZH2

EZH2 CDKN2A/B NH SANT SANTCXC SET SUZ12 2 EED Set HOXA JARID2 RBBP4/7

Nonsense/InDels T-ALL Y641 Missense Myeloid disorders DLBCL

AT Hooks CxxC AF-9

AT CxxC AF-4 H3K4me3 H3K79me2 H3K27me3 Hooks

AT Hooks CxxC ENL Chapter 1, Figure 1.2 (See page 15 of this volume.) A KMTi HATi DNMTi vidaza PRC2 DNMT decitabine HATs Set

CpG

Sirtuins HDACs

KDM HDACi PRMT5 JAK2 vorinostat SIRTi romidepsin (V617F) KDMi

JAKi Ruxolitinib

B Monoclonal antibodies

EGFR

Cell membrane

Lysine methylation Inhibitors of signal BCR-ABL transduction Lysine acetylation Nuclear envelope

Arginine methylation Epigenetic inhibitors Alkylating agents

Cisplatin phosphorylation DNMT

CpG CpG CpG

Chapter 1, Figure 1.3 (See page 20 of this volume.) Pathologic DNA double strand breaks (DSBs) – ROS Physiologic DNA double – Ionizing radiations strand breaks (DSBs) – Stalled replication forks – Common fragile sites and ERFSs – RAG1/2 induced V(D)J recombination – Oncogene-induced replication breaks – AID induced CSR and SHM – Topoisomerases – DNA damage in micronuclei – “Off-target” activity of RAG1/2 – “Off-target” activity of AID

DSB

Legitimate DNA repair Illegitimate DNA repair C-NHEJ (all cell cycle) C-NHEJ HR (S/G2 phases) A-EJ FoSTeS (collapsed replication fork) FoSTeS MMBIR (collapsed replication fork) MMBIR

DNA integrity Translocations–Duplications–Chromothripsis Chapter 2, Figure 2.1 (See page 41 of this volume.)

Chromosome territories Transcription factories Replication factories DNA repair centers

Active A domain Inactive B domain mRNA

mRNA DNA RNA polymerases polymerases PCNA C-NHEJ A-EJ

mRNA mRNA

Short chromosomes Long chromosomes

Chapter 2, Figure 2.2 (See page 48 of this volume.) AID-induced DSBs and translocations

Mature B cells IgM+

DNA isolation AID induction Translocations Linker-mediated PCR 4 days stimulation IL-4 + CD40 or LPS I-Scel-mediated DSBs

Proliferation CSR to IgG1+ B cells Next-generation Translocation sequencing maps

A-MuLV Pro-B cells G1 arrest

RAG1/2 induction Translocations DNA isolation STI-571 Linker-mediated PCR

I-Scel-mediated DSBs

RAG1/2-induced DSBs and translocations

Chapter 2, Figure 2.3 (See page 51 of this volume.) Mitosis Chromosome shattering

Micronucleus pulverization

Radiation MMBIR

Predisposition to cancer due to oncogenes and oncosuppressors’ alterations NHEJ DNA repair MMBIR Chromosome territory P53 deficiency Postmitotic cell with chromothripsis

Mitosis exit

Chapter 2, Figure 2.4 (See page 60 of this volume.) Type 2 diabetes • Endotoxemia • Insulitis • Insulin resistance Intestinal microbiota Steatosis

Obesity • Increased calorie extraction • Cleavage of dietary polysaccharides • Dyslipidemia

Decreased choline metabolism Chapter 3, Figure 3.1 (See page 76 of this volume.)

Steatosis Hepatocyte: TLR2-4 Kupffer cell: TLR2-4

Stellate cell: TLR1-9

TNF IL-6 Inflammatory response in the liver LSEC: TLR2

Flux of PRR ligands

Enterocytes: NLRP6

Intestinal dysbiosis Chapter 3, Figure 3.2 (See page 83 of this volume.) RIG-I CARD CARD ATPase/helicase RD/CTD 1 87 92 172 251 735 925

MDA5 CARD CARD ATPase/helicase RD-like 7 97 110 190 316 882 1025

LGP2 ATPase/helicase RD/CTD 11 476 678

MAVS CARD Pro TM 10 77 103 153 514 534 540 Chapter 4, Figure 4.1 (See page 102 of this volume.)

Influenza virus WNV EMCV NDV Dengue virus Theiler’s virus SeV Reovirus VSV HCV JEV PPP Cytoplasm TRIM25 Riplet RIG-I MDA-5 RNF125 PKC-a/b NLRX1 MAVS

IKK-i IKK-a MAPKs ? TBK1 IKK-b IKK-g

IRF3/7 NF-kB ATF2/c-Jun

Type I IFN Cytokines ISGs Nucleus

Chapter 4, Figure 4.2 (See page 103 of this volume.)