Role of Microrna-155 in B-Cell Leukemias/Lymphomas

ROLE OF MICRORNA-155 IN B-CELL LEUKEMIAS/LYMPHOMAS

DISSERTATION

Presented in Partial Fulfilment of the Requirements for the Degree Doctor of Philosphy in the Graduate School of The Ohio State University

By Sukhinder Sandhu, MS *******

Graduate Program in Molecular, Cellular and Developmental Biology

The Ohio State University 2011

Dissertation Committee: Dr. Carlo M. Croce, Advisor Dr. Kay Huebner Dr. Denis Guttridge Dr. Mark Parthun Dr. E. Antonio Chiocca

ABSTRACT

MicroRNAs (miRNAs) are a class of non-coding RNAs which regulate gene expression at transcriptional and translational levels. Similar to genes and their products, dysregulation of miRNA expression is associated with initiation and progression of several cancers. miR-155 is one of the most over-expressed miRNAs in various solid and hematological malignancies. In order to better understand the role of miR-155 in B-cell disorders, our group generated a transgenic miR-155 mouse model (Eµ-miR-155) where its expression was driven in B-cells under the immunoglobulin heavy chain promoter and

Eµ enhancer. The main goal of my research has been to understand the molecular mechanisms of miR-155 induced leukemias using this mouse model.

Eµ-miR-155 mice develop pre-B cell proliferation followed by high-grade lymphoma/leukemia by 9-12 months of age. These leukemias are transplantable and show variable clinical presentation and are often associated with polyclonal pre–B-cell proliferation. Analysis of these leukemias showed that the B-cell precursors in the transgenic mice have the highest miR-155 transgene expression and are at the origin of the disease. Mechanistic analysis to determine the basis of this phenotype led to Src homology 2 domain–containing inositol-5-phosphatase (SHIP1) and CCAAT enhancer- binding protein β (C/EBPβ). SHIP1 and C/EBPβ are two important regulators of the interleukin-6 (IL6) signaling pathway, were found to be direct targets of miR-155, being ii gradually down-regulated during the transition from pre-leukemic to leukemic state. Our results showed that by down-modulating Ship1 and C/EBPβ, miR-155 initiates a chain of events that leads to the accumulation of large pre-B cells and acute lymphoblastic leukemia/lymphoma.

Genome-wide mRNA analysis of miR-155 transgenic mice B-cells, showed downregulation of a key transcriptional repressor and proto-oncogene, B-cell leukemia-6

(Bcl6). This was unlike the established role of this transcription factor in various human

B-cell diseases such as diffuse large B-cell lymphoma (DLBCL), where this is frequently activated by translocation. It is possible that downregulation of Bcl6 in Eµ-miR-155 mice contributes to leukemogenesis due to de-repression of some of its targets which play role in inhibition of B-cell differentiation and increased proliferation. Some of these targets include ID2, overexpression of which has been shown to cause B-cell arrest and proinflammatory IL6 associated with increased proliferation. Investigation into the causes of

Bcl6 downregulation by miR-155 led to identification of another interesting, direct target of miR-155, HDAC4 (histone deacetylase 4). In addition to the canonical role in deacetylating histones to regulate global gene expression, some HDACs can regulate transcription factors such as BCL6, p53 and PLZF by removing the acetyl group from their lysine residues. Our results show that miR-155 directly regulates HDAC4, resulting in accumulation of acetylated BCL6 that is inactive. Further, we show that the inactive

BCL6 is a target of ubiquitin-mediated degradation. In addition, Bcl6 transcription is regulated by miR-155 mediated activation of Mad1/Mxd1 regulatory complex, which has been known to suppress Bcl6 by direct interaction with its promoter. Interestingly,

iii ectopic expression of HDAC4 in human activated B-cell type diffuse large B-cell lymphoma cell line induces Bcl6 expression and results in reduced miR-155 induced proliferation and clonogenic potential and increased apoptosis. These studies indicate that downregulation of BCL6 and HDAC4 by miR-155 may contribute to the development of pre-B acute leukemia in Eµ-miR-155 mice, by causing a differentiation arrest at the pre-B cell stage of B-cell development and increased proliferation.

Dedicated to My parents, Mrs Jeet K. Sandhu and late Mr. Darshan S. Sandhu

My Aunt, Ms. Pritpal K. Brar for dedicating her life to my upbringing, education and making me the person I am today.

My Husband, Mr. Yadwinder Deol, for his love, patience and help with everything and the motivation for constant striving for perfection.

v ACKNOWLEDGEMENTS

I would like to sincerely thank my advisor Dr. Carlo M. Croce for providing me the opportunity to work in his lab and giving me the great experience I had learning from the independence and faith he gives us all. I cannot imagine another place where I would have learnt as much as I did working in his lab at this crucial stage of my career.

I am very grateful to my advisory committee members, Dr. Kay Huebner, Dr.

Antonio E. Chiocca, Dr. Denis Guttridge and Dr. Mark Parthun for their valuable time and guidance. I thank our lab administration staff, Ms Sharon Palko, Dr. Dorothee

Wernicke, and Ms. Susan Lutz for their help in everyday things.

I would like to thank Dr. Yuri Pekarsky for his help and advice in variety of lab techniques, intellectual discussions and mice projects. I would like to thank Dr. Ramiro

Garzon for his scientific critiques on part of my thesis work.

I also acknowledge my Master’s program mentor, Dr. Parwinder Grewal, who helped me learn the basis of scientific research, hypothesis development and encouragement to pursue PhD in Biomedical research. I would also like to thank MCDB program Director Dr. David Bisaro for his time to listen and talk to me about my progress in the program and future plans, and the program co-ordinator Ms. Jan Zinaich for her help in everything else. I am very grateful to Dr. Helen Chamberlin and Dr. Harold Fisk

vi for their help during initial transitioning to the MCDB program.

I would like to thank all members of the Croce lab who have been a great help when I needed advice or reagents and who make this lab a great place to work in. I would like to thank the Flow Cytometry Core lab and their members for their help in data analysis and answering all my questions. I would like to thank past and present members of the University Lab Animal and Resources (ULAR) and Vet technicians who have constantly helped and trained me with the mice techniques and colony maintenance. In particular I thank Dr. Carrie Freed, Dr. Judy Hickman-Davis, Amanda Leber and former members, Amber McPherson and Dr. Daise Da Cunha. I would like to thank Ms.

Deborah Devor-Henneman of Comparative Pathology and Mouse Phenotyping Shared

Resource from whom I learnt the impressive mouse necropsy techniques, anatomy and common clinical features of unhealthy mice.

vii VITA

2003……….….…………...... ……….BS, Punjab Agri. University, Ludhiana, India

2005……….…………………..……..MS, The Ohio State University, Columbus, USA

2005-present…………….……..…….PhD Candidate, The Ohio State University,

Columbus, USA

PUBLICATIONS:

1. Sandhu SK, Volinia S, Costinean S, Neinast R, Santhanam R, Parthun MR, Garzon R, Croce CM. By targeting HDAC4 and impairing BCL6 transcriptional activity, miR-155 induces block in B-cell differentiation and development of acute leukemia. (Submitted to JEM 2011)

2. Sandhu SK, Croce CM, Garzon R. 2011. Micro-RNA Expression and Function in Lymphomas. Advances in Hematology. v2011, Article ID 347137.

3. Valeri N, Gasparini P, Fabbri M, Braconi C, Veronese A, Lovat F, Adair B, Vannini I, Fanini F, Bottoni A, Costinean S, Sandhu SK, Nuovo GJ, Alder H, Gafa R, Calore F, Ferracin M, Lanza G, Volinia S, Negrini M, McIlhatton MA, Amadori D, Fishel R, Croce CM. 2010. Modulation of mismatch repair and genomic stability by miR-155. Proc Natl Acad Sci U S A. Apr 13;107 (15): 6982- 7.

4. Costinean* S, Sandhu* SK, Pedersen IM, Tili E, Trotta R, Perrotti D, Ciarlariello D, Neviani P, Harb J, Kauffman LR, Shidham A, Croce CM. 2009. Src homology 2 domain-containing inositol-5-phosphatase and CCAAT enhancer-binding protein beta are targeted by miR-155 in B cells of Eµ-MiR-155 transgenic mice. Blood. 114 (7): 1374-82. *Equal contribution.

viii 5. Sandhu SK, Jagdale GB, Hogenhout SA and Grewal PS. 2006. Comparative analysis of the expressed genome of the infective juvenile entomopathogenic nematode, Heterorhabditis bacteriophora. Molecular & Biochemical Parasitology. 145: 239–244.

PUBLISHED ABSTRACTS

1. P Ranganathan, C Heaphy, N Stauffer, S Costinean, R Santhanam, S Sandhu, J Nuovo, G He, M Hamadani, G Hadley, G Marcucci, C Croce, S Devine, R Garzon. Regulation of Acute graft versus host disease by microRNAs. 16th Congress of the European Hematology Association, London, Jun 9-12, 2011.

2. Sandhu SK, Neinast R, Balatti V, Lovat F, Volinia S, Garzon R, Perkarsky Y, and Croce CM. B-cell lymphoma in eµ-miR-17~92 transgenic mice. Abstract No. 774, Blood, v116, no. 21, 2010.

3. Stauffer N, Hamadani M, Ramasamy S, Heaphy CE, Sandhu SK, Costinean S, Nuovo G, Perrotti D, Croce CM, Hadley G, Marcucci G, Devine S, and Garzon R. Involvement of miR-155 in Acute Graft-versus-Host Disease. Abstract No. 245, Blood, v116, no. 21, 2010.

4. Sandhu SK, Costinean S, Neinast R, Parthun M, Garzon R, Croce CM. MiRNA- 155 targets BCL6 and HDAC4 in a murine B-cell leukemia model: A paradigm shift in the oncogenic mechanisms of microRNAs. 15th Congress of the European Hematology Association, Barcelona, Spain. Jun 12, 2010.

5. Sandhu SK, Costinean S, Neinast R, Parthun M, Garzon R, Croce CM. OncomiR-155 targets oncogenes HDAC4 and BCL6 in a murine B cell leukemia model: A paradigm shift in the oncogenic mechanisms of microRNAs. Cancer Research, Abstract No. 3: April 15, 2010; Volume 70, Issue 8, Supplement 1

6. Sandhu SK and Grewal PS. Genetic and molecular analysis of infective juvenile longevity in the entomopathogenic nematode Heterorhabditis bacteriophora. 44th Society of Nematologists Annual Meeting, Fort Lauderdale, Florida, Jul 9-13, 2005.

FIELDS OF STUDY

Major field: Molecular and Cellular Developmental Biology Area of emphasis: MicroRNAs in Hematological Diseases

ix TABLE OF CONTENTS

ABSTRACT………...……………………………………………………………………ii DEDICATION…………………………………………………………………………...v ACKNOWLEDGEMENTS…………..………………………………………………...vi VITA…….……………………………………………………………………………...viii LIST OF TABLES…….………………………………...……………………………..xii LIST OF FIGURES….…………………………………………..……………………xiii LIST OF ABBREVIATIONS…….………………………………………..………….xiv

CHAPTER 1: MicroRNAs and their role in B-cell lymphomas/leukemias...... 1

1.1: Introduction…………………………………………………………………...1 1.2: The miRNA-155……………………………………………………………...4 1.3: miR-155 in B-cell development and function……………………………...... 6 1.4: miR-155 in B-cell malignancies……………………………………………...7 1.5. miRNAs in lymphomas: miR-17~92 cluster…………………………..……10 1.6: Potential for miRNAs in prognosis and outcome prediction………………..12

CHAPTER 2: Ship and CEBP/β are targeted by miR155 in B cells of Eµ-miR155 transgenic mice………………………………………………………………………….18

2.1. Introduction……………………………………………………………….....18 2.2. Eµ-miR-155 Transgenic mouse model……………………………………...19 2.3. Characterization of Eµ-miR-155 mice ...……………………………………21 2.4. miR-155 targeting of Ship1 and C/ebpβ…………………………………….22 2.5. Material and Methods……………………………………………………….23 2.6. Results……………………………………………………………………….29 2.6.1. miR-155 leukemias are transplantable…………………………….29 2.6.2. miR-155 induced mice leukemias have mixed immunophenotype ...... 30 2.6.3. miR-155 transgene is highly expressed in Bone marrow and pre- B cells of Eµ-miR-155 mice……………………………………..31 2.6.4. A Subset of Eµ-miR-155 B-cell tumors are clonal………………..32 2.6.5. miR-155 directly targets Ship1 and C/ebpβ……………………….33 2.6.6. SHIP1 and C/EBPβ are significantly downregulated in Eµ-MiR- 155 mice………………………………...... 34 x 2.7. Discussion………………………………...... 36

CHAPTER 3: By targeting HDAC4 and impairing BCL6 transcriptional activity, miR-155 induces a block in B cell differentiation and development of acute leukemia…………………………………………………………………………………54

3.1. Introduction………………………………...... 54 3.2. Materials and Methods………………………………...... 55 3.3. Results.………………………………...... 61 3.3.1. Genome profile of E-miR-155 mouse B-cells………………….....61 3.3.2. Downregulation of Bcl6 and consequent upregulation of its transcriptional targets in E-miR-155 leukemic mouse model……62 3.3.3. Transcriptional regulation of Bcl6 by miR-155 is indirectly mediated through Mad1 upregulation………………………...... 63 3.3.4. miR-155 targeting of HDAC4 enhances acetylation-mediated BCL6 downregulation through ubiquitination…………………...64 3.3.5. Restoration of HDAC4 expression is apoptotic and anti- proliferative………………………………………………………65 3.3.6. Bic/miR-155 expression is negatively correlated with Hdac4 and Bcl6 levels in DLBCL patients…………………………………..66

3.4. Discussion………………………………...... 67

CHAPTER 4: Conclusions and Future Directions…………………………...81

BIBLIOGRAPHY………………………………...... 88

xi LIST OF TABLES

Table 2.1: List of microRNAs up (A) and down-regulated (B) in Eμ-miR-155 mice splenocytes…………………………………………………………………………………………42 Table 3.1: Ingenuity pathway analysis of Eµ-miR-155 mice B-cell mRNA profile…….69

xii LIST OF FIGURES

Figure 1.1: MicroRNA Biogenesis pathway……………………………………………..14 Figure 1.2: Genomic organization of mammalian microRNAs………………………….15 Figure 1.3: miRNA regulation of B-cell development and function…………………….16 Figure 1.4: Tumor suppressor and oncogeneic miRNAs in DLBCL………………………...17 Figure 2.1. Eµ-MiR-155 transgenic mice………………………………………………..43 Figure 2.2: Phenotypic and immunochemical characterization of Eµ-MiR-155 mice…..44 Figure 2.3: Flow cytometry analysis reveals pre-B cell leukemia……………………….45 Figure 2.4: Kaplan meier survival curve…………………………………………………46 Figure 2.5: Flow cytometry analysis of leukemic and preleukemic spleens…………….47 Figure 2.6: Flow cytometry analysis of T-cells………………………………………….48 Figure 2.7: Differential miR-155 expression in various hematopoietic tissues…………49 Figure 2.8: Eµ-miR-155 mice tumors are oligoclonal…………………………………...50 Figure 2.9: Ship1 and C/ebpβ are direct targets of miR-155…………………………….51 Figure 2.10: Ship1 and C/ebpβ are down-regulated in Eµ-miR-155 mice……………….52 Figure 2.11: Immunoblot analysis of SHIP1 and C/EBPβ levels in B cell-subsets……...53 Figure 3.1: Bcl6 is downregulated in Eµ-miR-155 mice………………………………...70 Figure 3.2: BCL6 targets upregulated in Eµ-miR-155 mice…………………………….71 Figure 3.3: Bcl6 is not a direct target of miR-155……………………………………….72 Figure 3.4: Trancriptional regulation of BCL6…………………………………………..73 Figure 3.5: Translational regulation of BCL6……………………………………………74 Figure 3.6: HDAC4 is a direct target of miR-155……………………………………….75 Figure 3.7: HDAC4 is significantly downregulated in Eu-miR-155 mice………………76 Figure 3.8: Immunoblot analysis of HDAC4 levels in bic-/- mice………………………77 Figure 3.9: HDAC4 inhibits colony formation, proliferation, and induce apoptosis of OCI-Ly3 cells…………………………………………………………….78 Figure 3.10: HDAC4 impairs miR-155 induced proliferation in OCI-Ly1 cells………...79 Figure 3.11: HDAC4, BCL6 mRNA levels are negatively correlated with miR-155 expression in human DLBCLs…………………………………………...80 Figure 3.12: Proposed model of miR-155 induced leukaemogenesis……………………81

xiii LIST OF ABBREVIATIONS

miRNA MicroRNA UTR Untranslated Region BCL6 B-cell leukemia 6 HDAC4 Histone deacetylase 4 SHIP Src homology 2 domain–containing inositol-5-phosphatase C/EBPβ C/CAAT enhancer-binding protein β CLL Chronic Lymphocytic Leukemia NHL Non-Hodgkin Lymphoma DLBCL Diffuse large B-cell lymphoma ABC-DLBCL Activated B-cell Diffuse large B-cell lymphoma GCB-DLBCL Germinal Center B-cell Diffuse large B-cell lymphoma DC Dendritic cells AID activation-induced cytidine deaminase MZ or MZB Marginal Zone B-cells UTR UnTranslated Region MZ Marginal Zone B-cells Fo-B Follicular B-cells HSC Hematopoietic Stem Cells

xiv CHAPTER 1

MicroRNAs and their role in B-cell lymphomas/leukemias

1.1. Introduction

Recently, a class of non-coding RNAs called microRNAs (miRNAs) has emerged as critical gene regulators in cell growth, disease and development. MiRNAs are 18-24 nucleotide long non-coding RNAs, which regulate gene expression by pairing with 3’ untranslated region (UTR) of target mRNA and inhibiting protein translation and/or inducing mRNA degradation. MiRNAs modulate critical cell processes including cell growth, development and differentiation. MiRNAs constitute approximately 1-3% of the genome and are predicted to regulate 30% of human genes. Currently there are 940 miRNAs identified in humans and 590 in mice based on evolutionary conservation

(miRBase 15, http://www.mirbase.org/). MiRNAs are transcribed by RNA polymerase II as long primary transcripts (pri-miRNA), which are processed into ~70 nucleotide long precursor miRNAs (pre-miR) by an RNAse-III-like enzyme, Drosha, together with

DGCR8 (DiGeorge Syndrome Critical Region Gene 8), an RNA binding protein in the nucleus (Lee et al., 2003; Lee et al., 2002). Export of pre-miR to cytoplasm is mediated by Exportin-5 via GTP-dependent export, where it is further cleaved by another RNAse-

III enzyme, called Dicer, into a mature dsRNA duplex. After strand-selection mature

1 miRNA is assembled into the RNA-induced silencing complex (RISC) where it ultimately performs regulatory function (Gregory et al., 2005). The process of miRNA biogenesis is summarized (Figure 1.1).

MiRNA-mRNA interactions are characterized by perfect or nearly perfect

Watson-Crick base-pairing involving 5’ miRNA seed region (typically bases 2-8) that binds the target mRNA (Doench and Sharp, 2004). A single miRNA is predicted to target about 300 mRNAs just as a single mRNA can be targeted by multiple miRNAs. mRNA targets for specific miRNA are predicted by various bioinformatics’ algorithms

(Targetscan, Pictar, Miranda), but validation must be achieved through luciferase reporter assays, quantitative real-time PCR and immunoblotting.

In addition to the canonical mechanisms of miRNA gene regulation through 3′

UTR interactions, other 'non-canonical' miRNA-mediated mechanisms of mRNA expression modulation are emerging (reviewed in (Garzon et al., 2010)). Some miRNAs have been shown to bind to the open reading frame or to the 5′ UTR of the target genes and, in some cases, activate rather than inhibit gene expression (Garzon et al., 2010).

Recently it was reported that miRNAs also exhibit decoy activity and bind to ribonucleoproteins in a seed sequence and a RISC-independent manner and interfere with their RNA binding functions (Eiring et al., 2010; Garzon et al., 2010). A few studies have reported that miRNAs can also regulate gene expression at the transcriptional level by binding directly to the DNA (Garzon et al., 2010). Overall, these data show the

2 complexity and widespread regulation of gene expression by miRNAs that should be taken into consideration when developing miRNA-based therapies.

Analyses of miRNA localization in the genome have yielded useful information into their mode of transcription (Olena and Patton, 2010; Rodriguez et al., 2004). Based on their location, miRNAs can be intergenic, intronic or exonic and can be transcribed as a single miRNA from its own promoter, (monocistronic) or several miRNAs as a cluster from a shared promoter, (polycistronic) (Figure 1.2). Intergenic miRNAs are found in between genes in distinct transcription units. miRNAs can be intronic of coding or non- coding genes, where they may be transcribed from the same promoter as the host gene. A special class of intronic miRNAs, mirtrons, which bypass the drosha mediated processing into pre-miRNAs and comprise the whole intron spliced during mRNA splicing. Mirtrons have been discovered in C. elegans, Drosophila and mammals (Berezikov et al., 2007;

Okamura et al., 2007; Ruby et al., 2007). The exonic miRNAs are rare and are found in exons of coding or non-coding genes. A study from our lab showed that in mouse, miRNA genes are frequently located near cancer susceptibility loci, which are often subjected to genomic alterations leading to activation by translocations, amplifications or inactivation due to deletions, insertions or mutations (Sevignani et al., 2007).

In addition to these structural genetic alterations, various epigenetic events can also lead to miRNA expression deregulation. miRNA promoter hypermethylation and/or histone hypoacetylation has been described in solid tumors and hematological malignancies (Hackanson et al., 2008; Lujambio et al., 2007; Saito et al., 2006).

3 Furthermore, treatment with methylating agents or histone deacetylases inhibitor drugs leads to activation of tumor suppressor miRNAs like miR-127 which targets protooncogene BCL6 in human bladder cancer cells (Saito et al., 2006). Aberrant miRNA expression may also result from downstream miRNA processing. For example, short hairpin mediated silencing of Dicer and Drosha (RNAses involved in miRNA processing) can lead to global repression of miRNA expression promoting cellular transformation and tumorigenesis in vivo (Kumar et al., 2009). Finally, miRNA activation can result from increased transcription from the respective host genes due to aberrant transcription factor activity. For example, activation of miR-34a, miR-34b and miR-34c, a family of miRNAs by tumor suppressor p53 may in part contribute to its tumor suppression activity (Chang et al., 2007; He et al., 2007).

1.2. The microRNA-155

miR-155 was initially discovered as a non-coding RNA called bic for B-cell integration cluster, from a common retroviral integration site in avian leukosis virus

(ALV)-induced B-cell lymphomas, and later shown to cooperate with c-MYC to induce lymphomagenesis (Tam et al., 1997). It is one of the most frequently de-regulated miRNAs in solid and hematological cancers. It is also one of the few miRNAs for which both, gain- and loss-of-function mouse models have been developed which help provide important insight into its functions. A miR-155 transgenic (gain-of-function) mouse model was developed in Dr. Carlo Croce’s lab with overexpression driven in B-cells under Eµ enhancer and IgH promoter. These mice develop pre-B cell leukemia followed

4 by high grade lymphoma (Costinean et al., 2006). This model has also helped us understand the role of miR-155 in gene regulation which may contribute to disease development (Chapter 2 and 3), and DNA repair (Tili et al., 2011; Valeri et al., 2010).

Recently, our lab has also generated a conditional knock-in model of miR-155 where miR-155 is expressed under the control of Rosa26 locus and crossing these mice to tissue specific Cre transgenic mice will allow overexpression of miR-155 in respective tissue.

This model will help us study role of miR-155 in cancers of most tissues and understand tissue-specific gene regulation.

Loss-of-function models of miR-155/bic has independently been developed by two groups (Rodriguez et al., 2007; Thai et al., 2007) and showed its requirement in immune cell development and function. miR-155/bic-/- mice are viable and fertile, but their immune cells (T, B and DCs) have impaired functionality and these mice die within a month when challenged with Salmonella. These mice have reduced numbers of B-cells in their guts where they help fight antigens that can enter through food, and have structural abnormalities in lung airways, similar to asthma. Since a miRNA targets multiple genes, its loss can be a bigger blow to the cell than losing a single gene.

Even with two knock-outs and a transgenic model available, targets of miR-155 have not all been identified and hence the mechanisms defining its roles in oncogenesis are ill-defined and warrant further research.

5 1.3. miR-155 in B-cell development and function

The immune cells express more than 100 different miRNAs which have the potential to broadly influence the developmental and functional pathways of innate and adaptive immune responses (O'Connell et al., 2010). miRNAs are known to regulate every step of hematopoiesis starting from development of hematopoietic stem cells (HSC) to common progenitors (CLP or CMP) to development of mature cells of innate (Granulocyte,

Monocyte, and Dendritic cells) and adaptive immune response (T and B-cells) and eventually their function (Figure 1.3).

miRNAs are essential for survival and maturation of developing B-cells, as Dicer- deficient mice have a block at the progenitor (pro) stage of B-cell development (Koralov et al., 2008). B-cell development occurs in two-phases, antigen-independent phase in bone marrow and antigen-dependent in the peripheral tissues like spleen or lymph node.

In order for the successful initial phase of development, B-cells undergo many somatic rearrangements at the immunoglobulin (Ig) heavy (H) (Human: 1250 kb, Chr 14, Mouse:

2300 kb, Chr 12) (Abdul K. Abbas, 2005) and Ig light (L) chain loci to express membrane-bound antigen receptor (BCR) (reviewed in (Rajewsky, 1996)). During this process, DHJH followed by VHDHJH segments are assembled from the germline IgH locus to form the successful pre-BCR expressing IgH chain of class µ (Igµ) by the pro-B cells.

The cells with VHDHJH in-frame, undergo clonal expansion to precursor (pre) B cells to rearrange their L loci. L-chains are encoded by two loci called Igκ (kappa) (Human: 1820 kb, Chr 2, Mouse: 3200 kb, Chr 6) and Igλ (lamda) (Human: 1050 kb, Chr 22, Mouse:

6 240 kb, Chr 16) which undergo VL and JL joining in that order to yield a IgL chain expressed along with the Igµ from H-chain rearrangement to form immature B-cells with the functional BCR. Some of the miRNAs which have been shown to affect early B-cell development include miR-150 and miR-17~92 and their targets Myb and Bim, respectively.

The next phase is the antigen-dependent B-cell development which results in the differentiation to mature and functional B-cells occurs in lymph nodes after interaction with antigen-presenting cells such as, T and DC cells. The three functional subtypes of mature B-cells include, B1, MZ and Fo-B cells. miR-155 has been shown to play an important role in peripheral B-cell development, maturation and function through some of its targets like activation-induced cytidine deaminase (AID) (Dorsett et al., 2008; Teng et al., 2008) and PU.1 (Vigorito et al., 2007) which promote antibody class switching and antibody production.

1.4. miRNA-155 in B-cell malignancies

The expression of certain miRNAs is markedly dysregulated in various cancers of immunological origin. These miRNAs are thought to have tumor promoting or tumor suppressing roles depending on the target mRNAs regulated. Earlier studies showed miR-

155 to be upregulated in pediatric Burkitt’s lymphoma (Eis et al., 2005), aggressive activated B-cell-like (ABC) subtype of DLBCL (Rai et al., 2008), primary mediastinal B- cell lymphoma (PMBL) and Hodgkin’s lymphoma (Kluiver et al., 2005) and CLL (Calin

7 et al., 2005). Transgenic mice that overexpress miR-155 in B cells under the Eµ promoter

(as mentioned above) develop a B-cell lymphoprolifeation similar to some of these lymphomas and hence provide a proof-of-principle that a single miRNA deregulation can cause cancer (Costinean et al., 2006). The oncogenic nature of this miRNA was reinforced by identification of miR-155 orthologue (miR-K12-11) in the Kaposi sarcoma- associated herpesvirus (KSHV) associated with B- cell tumors (Gottwein et al., 2007) and recently, from Marek’s disease virus of chickens (Zhao et al., 2009b). The mechanisms involved in miR-155 induced lymphomagenesis are still under intensive investigation.

However, two recent independent studies indicate that miR-155 represses SH2-domain containing inositol-5-phosphatase 1 (SHIP-1), which is a critical phosphatase that negatively down-modulates the AKT pathway and has functions during normal B cell development (Costinean et al., 2009) (Chapter 2). Thus, sustained over-expression of miR-155 in B-cells may unblock AKT activity, inducing B-cell proliferation. Similar results were reported in the myeloid cells, resulting from myeloproliferation in mice transplanted with miR-155 transduced murine bone marrow HSCs (O'Connell et al.,

2009). Hematopoietic reconstitution assays showed extensive myeloproliferation with associated splenomegaly and morphological dysplastic changes. In addition to SHIP-1,

C/EBPβ, PU.1 and CSFR are also validated miR-155 targets.

Physiologically miR-155 is upregulated during B-cell activation in the germinal centers upon antigen stimulation and hence plays a role in antibody class switching and plasma cell formation, both of which are impaired in the miR-155 deficient mice

(Rodriguez et al., 2007; Thai et al., 2007). In another study miR-155/BIC was shown to

8 be transcriptionally regulated during normal B cell receptor activation through the AP-1 transcription factor and extracellular signaling-regulated kinase (ERK) and c-Jun N- terminal kinase JNK pathways (Yin et al., 2008b). Above findings show critical role of miR-155 in immune cell development, differentiation, function and immune response regulation, dysregulation of which can lead to malignancies like lymphoma.

DLBCL is the most common type of NHL that represents over one third of new diagnoses (~20,000 new cases per year) (Westin and Fayad, 2009). Since DLBCL can be considered as a prototype of human lymphomas, role of miRNAs and their respective targets in lymphomagenesis are discussed in detail and depicted in Figure 1. Gene expression profiling is used to classify DLBCL into three major histologically indistinguishable subgroups: germinal center B-cell-like DLBCL (GCB-DLBCL), activated B-cell-like (ABC-DLBCL), and primary mediastinal DLBCL (PMBCL)

(Rosenwald et al., 2002). Recently even miRNA expression profiling have been shown to successfully classify DLBCL into the above subcategories. Cytogenetically, GCB lymphomas are characterized by t(14;18) translocation, deletion of tumor suppressor

PTEN, and amplification of microRNA cluster miR- 17~92 and p53 mutations (Lenz and

Staudt, 2010). ABC lymphomas are known for anti-apoptotic BCL2 amplification, and majority of them have deletion of tumor suppressor loci like INK4-ARF which encodes p16, an inhibitor of senescence and p14ARF, an inhibitor of p53 activation (Lenz and

Staudt, 2010). Loss of these tumor suppressors contributes to poor prognosis and resistance to chemotherapy in ABC type of lymphomas.

9 miR-155 is one of the most commonly deregulated miRNAs in DLBCL, and interestingly it is over-expressed in ABC-DLBCL as compared to GCB-DLBCL. Some of its validated targets, which have roles in B cell homeostasis include PU.1, AID and

SOCS1. miR-155 overexpression has also been shown to induce resistance in some

DLBCLs to the growth-inhibitory effects of both TGFβ1 and bone morphogenetic protein

(BMP), via defective induction of p21 and impaired cell cycle arrest through targeting

SMAD5 (Rai et al., 2010).

1.5. miRNAs in lymphomas: miR-17~92 cluster

Another miRNA which is reported to have a major role in lymphomagenesis is the miR-17~92 polycistron located in 13q31-32, a region commonly amplified in B-cell lymphomas and upregulated in 65% of B-cell lymphoma patients (He et al., 2005; Ota et al., 2004). The miR-17~92 cluster consists of miR-17, 18a, 19a, 19b-1, 20a and 92a-1 and is encoded from the last exon of non-coding RNA, C13orf25. The cluster has two paralogs in the genome, miR-106a~363 on chromosome X in mice and humans consisting of six miRNAs, and miR-106b~25 on chromosome 5 in mice (chromosome 7 in humans) consisting of three miRNAs encoded from the 13th intron of the DNA- replication gene Mcm7 (Ventura et al., 2008).

Gain and loss-of-function studies of miR-17~92 polycistron have provided an important insight into its mechanism of action and its targets. He et al., demonstrated that virus-mediated over-expression of miR-17~92 in lymphocytes of eµ-MYC (B-cell)

10 transgenic mice accelerated tumor development (He et al., 2005). O’Donnell et al., simultaneously reported that MYC binds to and activates expression of the miR-17~92 cluster (O'Donnell et al., 2005). Two members of the polycistron: miR-17-5p and miR-

20a downregulate E2F1, which is a direct target of MYC that promotes cell cycle progression. More recently, Xiao and colleagues (Xiao et al., 2008) reported that mice with sustained expression of miR-17~92 in lymphocytes exhibit a lymphoproliferative disorder, autoimmunity, and premature death. These mice have decreased levels of the pro-apoptotic BIM and the tumor suppressor PTEN. Both genes were further confirmed as targets of the miR-17~92 polycistron members (Xiao et al., 2008). Ventura et al.,

(Ventura et al., 2008) confirmed these findings and showed that targeted deletion of miR-

17~92 polycistron (but not its paralogs) in mice is embryonically lethal and critical for lung and B-cell development. In particular, these mice exhibited a block in B cell differentiation at the pro-B to pre-B transition caused by high levels of the pro-apoptotic protein BIM. These experiments suggest that the miR-17~92 cluster acts specifically during the transition from pro-B to pre-B lymphocyte development, enhancing the survival of the B-cells at this stage by targeting the pro-apoptotic BIM. Further dissection of the genetic complexity of the cluster was demonstrated by generating conditional knockout alleles of the four seed regions represented in the cluster: miR-17, miR-20a; miR-18a; miR-19a, miR-19b-1 and miR-92-1 (Mu et al., 2009). Mu and colleagues (Mu et al., 2009) found that deletion of the whole miR-17~92 cluster slows c-Myc-induced oncogenesis. This phenotype was rescued by reintroduction of the full cluster, but not by the cluster lacking miR-19a and miR-19b, thereby suggesting miR-19 as the most important miRNA of the cluster. Using a different approach, Olive and colleagues (Olive

11 et al., 2009) overexpressed individual miRNAs in the Eµ-MYC mice model. They found that overexpression of the whole cluster, without miR-92, but not the miR-19a or miR-19b promotes oncogenesis. In summary, both studies indicate that miR-19 is critical for the oncogenic activities of this cluster. Recently miR-17~92 was also shown to downregulate

TGFβ signaling pathway leading to clusterin downregulation and hence stimulating angiogenesis and tumor cell growth in glioblastomas (Dews et al., 2010).

1.6. Potential for miRNAs in prognosis and outcome prediction

miRNA expression profiling studies have been successfully used not only to differentiate normal from cancer tissues but also to classify tumor types and grades. In addition, miRNA expression profiling can also provide a unique miRNA expression signature, which could be related to response to therapy and survival (Navarro et al.,

2008; Zhang et al., 2009). Li et al., (Li et al., 2009), Roehle et al., (Roehle et al., 2008) and others have successfully used miRNA expression profiles to determine unique miRNA signatures, which can classify lymphomas into categories with distinct treatment response and outcome in the patients (Table 1). A recent report by Lawrie et al., (Lawrie et al., 2008) showed miR-21 serum levels to be associated with relapse-free survival in patients with DLBCL. Similarly, serum levels of miR-141 have been shown to distinguish between prostate cancer patients and healthy individuals (Mitchell et al., 2008). In Mantle cell lymphoma, miR-20b expression is related to poor survival and its lack of expression, distinguished cases with a survival probability of 56% at 60 months (Di Lisio et al.,

2010). One of the miRNAs consistently downregulated in most lymphomas is miR-150,

12 owing to its important role as tumor suppressor as shown in a loss-of-function mouse model (Xiao et al., 2007). Mice lacking miR-150 has increased expression of its target transcription factor, c-Myb oncogene which plays an important role in lymphocyte development and maturation (Xiao et al., 2007). Therefore, miRNA expression profiling provides a useful tool for prognosis, diagnosis and outcome prediction in lymphoma patients.

13 Figure 1.1: MicroRNA Biogenesis pathway: (adapted from Winter et al., 2009 NCB) (Winter et al., 2009)

14 Figure 1.2: Genomic organization of mammalian microRNAs (adopted from Olena and Patton, 2010) (Olena and Patton, 2010). Legend: Black arrows indicate promoter. (A) Intergenic miRNAs are located in regions different from known transcription units, and can be monocistronic with their individual promoter (top) or polycistronic, with several miRNAs transcribed as a cluster with shared promoter (bottom). (B) Intronic miRNAs are found in introns of protein coding or non-coding genes, can exist individually (top) or in clusters (bottom). A new class called mirtrons (middle), share their pre-miR sequence with the complete intron and bypass the canonical miRNA- processing pathway. (C) Exonic miRNAs are rare and normally found in exons of non- coding genes. These are transcribed by host gene promoter and their maturation is independent of host gene function.

15 Figure 1.3: miRNA regulation of B-cell development and function. (a) Survival and maturation of B-cells requires miRNAs and Dicer activity. Mice with Dicer deficiency at early B-cell stages have block at pro-B stage, miR-150 and miR-17~92 are specifically required and regulate MYB and BIM. (b) miR-155 regulates peripheral B-cell development through targeting AID and PU.1, which promote antibody class switching and antibody production (O’Connell et al., 2010).

O’Connell, RM et al., 2010

16 Figure 1.4: Tumor suppressor and oncogenic miRNAs in DLBCL and their target genes implicated in various processes involved in malignant transformation.

Tumor Suppressor miRNAs Targets Cellular Processes

miR-127 BCL6 Apoptosis, DNA damage response

miR-15a-16 BCL2, CCND2, CCND3, Cell Cycle, Proliferation CCNE, CDK4, CDK6

Oncogenic miRNAs

miR-17~92 PTEN, BIM, CLU, TGFβRII Apoptosis, Angiogenesis, Proliferation

miR-125b IRF4, PRDM1/Blimp1 B-cell Differentiation

miR-223 LMO2, MYBL1 Proliferation

miR-155 SHIP1, SOCS1, AID, Differentiation, Proliferation

17 CHAPTER 2

Ship and CEBP/β are targeted by miR155 in B cells of Eµ-miR155 transgenic mice

2.1. Introduction

B-cell lymphomas represent the majority of the lymphoid neoplasms, and despite important steps towards the understanding of their molecular basis, little is known about the initiating factors. We and other groups have identified alterations of miRNA expression in various cancers ranging from leukemias (Calin et al.) to solid tumors

(Volinia et al.). Whole genome expression studies have revealed that microRNA deregulations are tumor specific, and that they could predict the clinical course and outcome. Therefore, it seems that microRNA profiling could constitute a “signature” useful to differentiate various types of tumors and predict their possible clinical course

(Calin et al.). Since a single miRNA can target multiple mRNAs simultaneously, a modification of their expression could reverberate on various biological levels.

Among the many miRNAs involved in various cancers, some seem to be more relevant. These include miR15a/16-1, one of the very few miRNAs down-regulated in.

18 chronic lymphocytic leukemia (CLL) and believed to have a tumor suppressor function

(Calin et al.). Others are miR-21 and cluster miR-17~92, overexpressed in many solid and hematological malignancies (Iorio et al.; Volinia et al.), (He et al., 2005). In addition,

MiR-155 was found overexpressed in several types of B-cell leukemias/lymphomas

(Calin et al.), (Eis et al.), (Kluiver et al.) with the highest level of expression in activated

B-cell type of Diffuse large B-cell lymphoma (ABC-DLBCL) (Eis et al.).

2.2. The Eµ-miR-155 Transgenic mouse model

In order to understand the miR-155 deregulated B-cell malignancies our lab generated an

Eµ-miR-155 transgenic mouse model (Costinean et al.). Briefly, mmu-miR155 (mouse miR155) was cloned into the EcoRV and SalI sites under the control of a VH promoter-Ig heavy chain Eµ enhancer, which becomes active at the late pro-B cell stage of the B cell development. The linearized DNA construct was injected into one of the pro-nuclei of the oocytes and transplanted into pseudo-pregnant females. Fifteen transgenic founders were obtained as identified by Southern Blot hybridization, seven on C57BL/B6 and eight on

FVB/N backgrounds. These were bred to wild-type mice of the same strain to produce 5 independent transgenic lines with highest miR expression as determined by Northern Blot analysis on total RNA extracted from transgenic and wild-type spleens (Figure 2.1).

These mice exhibit splenomegaly as early as 3 weeks of age. Histologically, the spleens from 3-week-old mice featured a consistent atypical lymphoid population invading the red pulp and expanding it; the lymphoid follicles were unaffected, and there

19 were multiple foci of secondary hematopoiesis (Figure 2.2A). Mice at 6 months of age presented histologically a greatly increased malignant lymphoid population with marked atypia and blastic appearance, proliferating in the vascular channels of the red pulp and gradually replacing the white pulp. The number of lymphoid follicles was decreased, and the overall architecture of the spleen was distorted by lymphoid proliferation (Figure

2.2B).

Flow Cytometry Analysis of Eµ-mmu-miR155 spleens and bone marrows at 3, 6, and 7 weeks and 6 months of age, revealed an expansion of the B220lo/CD10lo/IgM-/CD5-

/TCR-/CD43- population in transgenic mice. This phenotype resembles the phenotype of proliferating lymphocytes observed in human acute lymphoblastic leukemia or lymphoblastic lymphoma. Further, these B220lo/CD10lo/IgM-/CD5-/TCR-/CD43- lymphoid population in the spleens of transgenic mice varied from 9% at 3-weeks age to

6.6 ± 1.4% in the 6-week to 4.7 ± 0.3% at 7 weeks of age while wild type spleens were always at 1.65%. Bone marrow of 6 month old transgenic mice had expansion of the pre-

B cell population as defined by B220lo/IgM- expression, compared with the wild type

(Figure 2.3).

miRNA microarray analysis of total RNA extracted from the splenic white cells of five transgenics and six wild-type littermates revealed a 10- to 20-fold increase in expression of miR-155, miR-194, miR-224, miR-217, and miR-151 and a 2- to 3-fold decrease in miR-146 and miR-138 (Table 2.1). Total mRNA profiling (Affymetrix) of the same group of mice showed 200 proliferation genes were up-regulated, and 50 genes

20 were down-regulated in the Eµ-miR155 mice. Interestingly, the VpreB1 mRNA was upregulated, and represents the pre-B cell expansion in these mice. Overall it was shown that these mice present a block at the pre-B stage of B-cell differentiation at an early age

(3-4 weeks), followed by a pre–B-cell proliferation that later translates into frank acute lymphoblastic leukemia/high grade lymphoma.

2.3. Characterization of Eµ-miR-155 mice

Next we present a detailed characterization of the immunophenotype of these mice and describe a possible mechanism for these leukemias. As mentioned above, all transgenic mice showed an initial increase of the B220+ IgM- population; which later on, loose some of the B220 surface antigen expression and become B220lo; thus, the leukemias studied exhibit a mixed immunophenotype characterized by B220lo IgM- and

B220loIgM+ (most likely due to some degree of differentiation of some of the malignant clones of the pre-B cells of origin). Moreover, all transgenic mice exhibit an increase of the myeloid line and a reduced T-cell population with no significant changes in the

CD4/CD8 ratio (Figure 2.6).

MiR-155 expression analysis across various hematopoietic tissues showed significant expression in the bone marrow and spleen and in pre-B-cell subpopulations of

B-cells identified by B220+IgM-. To understand the role of MiR-155 in the B-cell differentiation and leukemogenesis, we focused on two of the predicted targets Ship1

(Inpp5d) and C/ebpβ, which may have key role in B-cell maturation/activation.

21 2.4. miR-155 targeting of Ship1 and C/ebpβ

Src homology 2 domain–containing inositol-5-phosphatase (SHIP or SHIP1 or

INPP5D) is a negative regulator of the cell signaling in the immune system (Nakamura et al., 2002). This phosphatase has been implicated in B-cell maturation because it exhibits differential expression in the pro-B compared with the pre-B stage (Helgason et al.; Liu et al.). Irradiated mouse bone marrow reconstituted with Ship-/- hematopoietic cells shows a reduction in the immature and mature forms of B cells (Helgason et al.), (Liu et al.).

Moreover, Ship-/- cells were more viable and had better survival due to the activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase

(PI3K)/protein kinase B (AKT) pathways (Helgason et al.).

CCAAT enhancer-binding protein β (C/EBPβ; nuclear factor–interleukin, IL-6) is a mediator of the IL-6 signaling pathway; IL-6 activates C/EBPβ through the phosphorylation of MAPK (Nakajima et al.), (Trautwein et al.). In addition, C/EBPβ expression varies during differentiation of myeloid and plasma cells, (Cooper et al., 1992;

Natsuka et al., 1992; Scott et al., 1992) suggesting its involvement in myeloid and lymphoid maturation. Mice lacking C/EBPβ showed a lymphoproliferative disorder nearly identical to a rare and aggressive form of human Castelman disease, characterized by lymphoid follicular hyperplasia due to an acceleration of the B-cell maturation and accumulation of IgG1-secreting lymphoid subpopulation (Screpanti et al., 1995). Since loss-of-function mouse models of these two genes had a phenotype similar to the miR-

155 transgenic mice, we focused on miR-155 targeting of these genes.

22 In this study, we show that SHIP and C/EBPβ protein levels are diminished in the

MiR-155 transgenic pre-B lymphocytes. SHIP is gradually down-regulated in the preleukemic and leukemic pre-B cells, whereas C/EBPβ, even though it maintains the same level of expression in the preleukemic mice, is markedly diminished in the leukemic pre-B cells. Thus, we conclude that MiR-155 transgene has a maximum expression in the pre-B stage, in which it down-regulates both Ship1 and C/ebpβ, the two inhibitors of IL-6. This blocks the B-cell differentiation and might also induce a reactive proliferation of the myeloid line. The B-cell precursors, arrested in their development, proliferate and are at the origin of malignant leukemias with a mixed immunophenotype

B220lo/IgM- and B220lo/IgM+.

2.5. Material and Methods

Transgenic mice

MiR-155 transgenic mice were generated on both backgrounds, C57BL/6 and FVB, as described above. Transgenic mice were housed and euthanized in accordance with protocols approved by the Institutional Animal Care and Use Committee of The Ohio

State University.

Transplants

Fresh splenocytes were collected from transgenic leukemic mice and resuspended in phosphate-buffered saline (Elmen et al.). Dilutions of 106 cells/ml were prepared and injected intraperitoneally in syngeneic mice (on average, 5 syngeneic mice were injected

23 for each leukemic mouse). An equal number of wild-type mice, strain-, age-, and sex- matched, were then injected the same way with PBS only. Mice were kept under observation until all of the transplanted mice got sick and died.

Histology, immunohistochemistry, and flow cytometry

All sick mice were sacrificed and weighed, together with littermate controls; spleens, liver, kidneys, lungs, and lymph nodes were collected; spleens were weighed. Fragments of spleens were fixed in 10% buffered formalin, paraffin included, stained with hematoxylin and eosin, and studied with a Olympus BX41 light microscope. Images were acquired with the Olympus software. Unstained slides were prepared and later immunohistochemically stained with in-house manufactured antibodies: CD20, CD79a,

CD3, CD43, IgM, and κ and λ light chains (Children's Hospital). Single-cell suspension of splenocytes was depleted of mature red blood cells by hypotonic lysis (0.165 M

NH4Cl) and stained with the following conjugated antibodies: anti-B220, anti-CD19, anti-CD138, anti-IgM, anti-IgD, anti-CD3, anti-CD4, anti-CD8, anti-CD11b, and anti-IL-

4 (all antibodies were from BD Pharmingen). Flow cytometry was carried out on a BD

FACSCalibur, and data were analyzed using the BD FACS Convert 1.0 for Mac software.

For cell sorting, the splenocytes were stained with anti-B220 and anti-IgM; the cell sorting was carried out on a BD Aria. Three populations were selected: B220+ only,

B220+/IgM+, and B220−/IgM−.

24 Southern blot for VDJ rearrangement

A probe was amplified in the JH4 fragment of the IgH region on the mouse genomic, using the following primers: forward, 5′-TGAAGGATCTGCCAGAACTGAA-3′; reverse, 5′-TGCAATGCTCAGAAAACTCCAT-3′, and then cloned into a TOPO TA vector (Invitrogen).

Spleens of the transgenic and wild-type mice were dissociated between frosted slides in

PBS, treated with ammonium chloride for the erythrocyte lysis, centrifuged, and resuspended in PBS; DNA was extracted from the splenocytes with phenol-chloroform and digested with EcoRI and PvuII. Digested DNA was then separated on a 1% agarose gel, blotted on a HyBond N+ membrane (GE Healthcare), hybridized overnight with the

JH4 probe radioactively labeled with 32P (with Klenow enzyme from PrimeIT II

Random Primer Labeling kit; Stratagene), and exposed to a phosphor-image screen and processed using a Typhoon scanner (GE Healthcare).

Western blots

For Western blot on total splenocytes, cells were isolated from sick and wild-type mice, by dissociating the spleens in between 2 frosted slides; red blood cells were lysed with ammonium chloride, and proteins were extracted from the remaining white blood cells with a lysis buffer containing Tris-HCl, pH 7.5 (30 mM), NaCl (150 mM), 10% glycerol, and 1% Triton X-100 and protease inhibitors (Roche; 1 dose/7 mL buffer). A total of 80

µg proteins was then loaded, separated on a 4% to 10% sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS-PAGE) gel in an electrophoretic X-Cell Sure

25 Lock machine, and blotted on polyvinylidene difluoride membranes (Bio-Rad); membranes were incubated overnight with a polyclonal anti–rabbit SHIP-1 primary antibody at a final concentration of 1/100 (Millipore) and polyclonal anti–rabbit C/EBPβ

1/200 (Santa Cruz Biotechnology). The following day, membranes were incubated with an anti–rabbit secondary antibody for 1 hour. Signal was detected with an enhanced chemiluminescence kit (Amersham).

For the immunoblot on sorted splenocytes, cells were harvested, washed once with ice- cold PBS, and directly lysed in Laemmli buffer (Bio-Rad; 106 cells/50 µl). Western blotting was performed according to previously published protocols (Trotta et al., 2005).

Antibody-reactive proteins were detected with horseradish peroxidase-labeled sheep anti– rabbit, –mouse, and/or –goat Ig sera and enhanced chemiluminescence (Amersham).

Proteins were analyzed in 4% to 15% SDS-PAGE (Bio-Rad) using reducing conditions.

Monoclonal and polyclonal antibodies used were as follows: polyclonal rabbit sera anti–

SHIP-1 from Millipore; polyclonal rabbit anti-C/EBPβ antibodies and polyclonal goat anti-actin antibodies from Santa Cruz Biotechnology; and mouse monoclonal anti-GRB2 antibodies from Santa Cruz Biotechnology.

All Western blot films were scanned with a Personal Densitometer SI (Molecular

Dynamics) scanner, and protein expression bands were analyzed with Image Quant

(Molecular Dynamics) software as follows: scanned bands were selected and analyzed in terms of quantity of the black hue. Results were displayed on graphics. Then the intensity

26 values of the bands for the proteins of interest were divided by the intensity values of the normalizing bands. The ratios obtained this way were then compared.

Luciferase reporter assay

A fragment of 584 basepairs of the mouse C/ebpβ 3′-untranslated region (UTR) and another one of 1295 basepairs of the mouse Ship1 3′-UTR, both containing complementary sequences to mmu-MiR-155, were amplified from mouse genomic DNA and cloned in the luciferase pGL3 control vector (Promega) in the XbaI restriction site.

These constructs were then used to synthesize a MiR-155 mutant seed, with the help of the Stratagene QuikChange XL Site-Directed Mutagenesis kit. The mutagenic primers used for Ship1 were as follows: 5′-

GGTGGGTCCTGAGATGTTTTTAAAAAGCAAATAAGAAAACCATCGG- 3′ for the forward and 5′-CCGATGGTTTTCTTATTTGCTTTTTAAAAACATCTCAGGACCC-

ACC-3′ for the reverse; for C/EBPβ the mutating primers were as follows: 5′-

GTATATTTTGAGAACCTTTTCCGTTTCGAGCAAATAAGT-GAAGACA- 3′ for the forward and 5′-

TGTCTTCACTTATTTGCTCGAAACGGAAAAGGTTCTCAAAATATAC-3′ for the reverse. We used the same mutation as described by (Vigorito et al., 2007) All mutations were confirmed by sequencing.

To test whether Ship1 is a target for miR-155, we cotransfected 293 cells using

Lipofectamine 2000 (Invitrogen), as follows: 6 samples were transfected with 0.8 µg

Ship1 3′-UTR cloned in pGL3 control vector and 100 mM mmu-miR-155 precursor

27 (Ambion), and 6 other samples with 0.8 µg Ship1 3′-UTR cloned in pGL3 control vector and 100 mM miR negative control I (Ambion) instead of miR-155. We also cotransfected

6 samples with 0.8 µg mutant Ship1 3′-UTR and 100 mM mmu-miR-155, and 6 samples with 0.8 µg mutant Ship1 3′-UTR and 100 mM miR negative control I instead of miR-

155 (Ambion); the same experiment was run for CEBPβ. All wells were also transfected with 0.05 µg/well control Renilla luciferase vector pRL-TK (Renilla luciferase;

Promega). Cells were lysed after 24 hours and analyzed for luciferase and Renilla luciferase activities using the Dual Luciferase Reporter Assay kit (Promega) on a

GLOMAX (Promega) microplate luminometer.

TaqMan assays

TaqMan assays were run in triplicate with mouse miR-155 TaqMan probe (Applied

Biosystems) and 1× Universal Master Mix in 20 µL final volume on Bio-Rad ICycler under universal cycling conditions. RNA was extracted with TRIzol (Invitrogen).

Expression was normalized to sno-RNA-135, and fold changes were calculated by q-ΔCt method (Schmittgen and Livak, 2008).

TargetScan

TargetScan (Whitehead Institute for Biomedical Research) predicts biologic targets of miRNAs based on the search for the presence of conserved 8-basepair and 7-basepair sites that match the seed region of each miRNA (Lewis et al., 2005). The program also identifies sites with mismatches in the seed region that are compensated by conserved 3′ pairing (Friedman et al., 2009). Predictions in mammals are ranked based on the

28 predicted targeting efficacy as calculated using the context scores of the sites (Grimson et al., 2007).

2.6. Results

2.6.1. miR-155 leukemias are transplantable

Eµ/VHmiR-155 transgenics of both strains (FVB and C57BL/6) develop leukemias/lymphomas transplantable into syngeneic mice. We expanded the colony to

500 transgenics and observed it for more than 2 years. As reported, all transgenics display enlarged spleens as early as 3 to 4 weeks of age; some of them start showing clinical signs of malignant disease at 4 to 6 months; however, we noted that more often the onset of the disease was at 9 months; in most cases, the mice were dead before 1 year of age.

Malignant lymphocytes from both strains were isolated from leukemic spleens and injected in 40 syngeneic mice that showed signs of disease approximately 4 weeks after the injection (survival chart for the FVB strain transplanted mice Figure 2.4B; the

C57BL/6 exhibited a very similar profile; data not shown); the leukemias in the syngeneic mice were clinically, histologically, and immunohistochemically identical to the primary ones. There were some differences between the FVB (400 mice taken into the study) and C57BL/6 transgenic mice (∼100 mice considered for our study) in terms of the phenotype exhibited. The FVB transgenics showed signs of disease slightly earlier

(by 13 months, only 26% of the group was still alive; see survival chart, Figure 2.4A) and 29 presented from a very early age an important increase in the size of the spleen, whereas the C57BL/6 initially had less prominent splenomegaly and presented signs of disease a few months later than the FVB mice (60% of the group was alive at 13 months, and 20% at 18 months; see survival chart, Figure 2.4).

2.6.2. miR-155 induced leukemias have a mixed immunophenotype

Leukemias in the miR-155 transgenic mice have a mixed B220loIgM-IgD- and

IgM+/IgD+ phenotype. Histologically, the transgenic spleens exhibited the effacement of their architecture by a diffuse lymphoid proliferation composed mainly of large cells with marked atypia and numerous atypical mitoses. This lymphoproliferation was also present extensively in the bone marrow with the complete obliteration of their histology, and in a significant number of cases also in other organs. All sick mice exhibited marked lymphadenopathy.

Spleens, livers, and lymph nodes of sick mice immunohistochemically stained showed marked lymphoproliferation positive for pre–B-cell immunomarkers, such as

CD20 and CD79a, and negative for T-cell markers, such as CD3 (unpublished observation SC). The proliferating lymphocytes also stained positively for µ intracytoplasmic chains and negative for surface IgM (unpublished observation SC). Igκ and Igλ chains were also absent.

30 Flow cytometry showed an increase in the pre–B-cell population of approximately

7.45% ± 0.14% (marked B220+/IgM-) in the preleukemic mice, whereas the leukemic cells exhibited a mixed phenotype B220lo/IgM- and B220lo/IgM+ (Figure 2.5A). The percentages of B and pre-B cells did not change when we marked them for CD19 and

IgD instead of B220 and IgM (data not shown). The T-cell population was diminished in all transgenics in various degrees of 5% to 30% (Figure 2.6). The T helper cell 1 population stained CD4+ and IL-2+ was unchanged (data not shown). Surprisingly enough, the myeloid line (CD11b+) was also increased in the transgenics (to 16.52% ±

2.4%) compared with the wild-types (Figure 2.5B).

2.6.3. The miR-155 transgene is highly expressed in Bone marrow and pre-B cells of

Eµ-miR-155 mice

TaqMan assay detected the highest levels of miR-155 transgene expression in the bone marrow and the B-cell precursors. TaqMan assay was used to quantify the miR-155 transgene expression in various hematopoietic organs as well as in different flow cytometry-sorted B-cell subpopulations. We determined that miR-155 is more highly expressed in the bone marrow (∼ 2.5 times more) compared with the spleen, a possible indication of preferential transcription of this transgene in the precursor rather than the mature lymphoid cells (Figure 2.7A). The splenocytes of leukemic, preleukemic, and wild-type mice were sorted by flow cytometry in 2 subpopulations: pre-B (B220+/IgM-) and mature B (B220+/IgM+) cells. TaqMan assay performed on RNA extracted from these 2 cell populations indicated miR-155 transgene to be approximately 10 times more 31 expressed in the precursors (B220+/IgM-) compared with the mature lymphocytes

(B220+/IgM+; Figure 2.7B).

2.6.4. A Subset of Eµ-miR-155 B-cell tumors are clonal

B-cell development involves successful rearragement of the three regions of immunoglobulin genes viz., V (variable), D (diversity) and J (joining) in the order of DJ, followed by VDJ (as discussed in detail in Chapter 1). This process involves DNA breaking and joining at a very high rate. Only the cells with successful or complete VDJ rearrangement are able to survive and express their B-cell receptor. In case of B-cell malignancies, there can be expansion of expansion of B-cells arrested at incomplete VDJ rearrangements. In a normal individual (or mouse) Southern blot analysis for the VDJ rearrangement using immunoglobulin heavy-chain J-region probe (JH) will show a single germline band around 6 kb in mouse or 10 kb in humans. While in case of tumors which have arisen from single (monoclonal) or few (oligoclonal) malignant B-cells, JH probe will show multiple bands. Southern blot on PvuI digested diseased splenocytes DNA from transgenic for the VDJ rearrangement of IgH chains (to assess the clonality) showed oligo- and monoclonal rearranged bands (Figure 2.8A, B) as compared to wild type mice.

In lane 1, the extra band of 4 kb, and lane 4 had two rearranged bands, of 6 kb and 10 kb, with 5-kb being the germline heavy chain locus (red arrow Figure 2.8A); an additional faint band of 3 kb observed in all samples is a cross-hybridization band (blue arrow

Figure 2.8A). Because the intensity of the germline bands is much stronger than that of the rearranged bands, it is quite likely that the disease for the most part is polyclonal, with

32 the emergence of one or few dominant clones. In Figure 2.8B, (EcoRI digested DNA), there is a case of leukemia with a rearranged band of approximately 8 kb (lane 3, red asterisk) of equal intensity to that of the germline that is of approximately 6 kb (blue asterisk), suggesting that this lymphoproliferation is monoclonal.

2.6.5. miR-155 directly targets Ship1 and C/ebpβ

MiR-155 targets SHIP and C/EBPβ, the two negative regulators of IL-6 lymphoid signaling pathway. SHIP1 (Inpp5d) is a phosphatase differentially expressed in B-cell precursors; SHIP-/- knockout mice display a block in B-cell differentiation at the pre-BI stage, whereas SHIP1-/- splenocytes have an enhanced proliferative responsiveness to cytokine stimulation (Helgason et al., 2000). They also exhibit prolonged survival and increased viability, supposedly due to the activation of PI3K/AKT and MAPK pathways

(Helgason et al., 2000). Ship1 is a predicted target for MiR-155 (TargetScan) and has at least one site of perfect complementarity with the seed of this microRNA in its 3′-UTR sequence (Figure 2.9A, top).

To confirm that Ship1 is a direct target of MiR-155, a fragment of the Ship1 3′-

UTR containing the binding seed sequence for MiR-155 was cloned into the luciferase reporter plasmid pGL3, and cotransfected in HEK-293 cells, together with a miR-155 mimic. MiRNA mimics are synthetic mature miRNA molecules which function similar to biologically occuring miRNAs and serve as readily available reagents. The relative luciferase activity decreased by 42% in the presence of the miR-155 mimic compared

33 with the control (Figure 2.9A, bottom). The repressive effect of miR-155 was abolished in the presence of the mutated 3′-UTR, demonstrating the direct interaction between the microRNA and the binding site. Thus, Ship1 is a direct target of miR-155 and, therefore, we expect its expression to be reduced in the Eµ-miR-155 B lymphocytes.

C/EBPβ acts as a negative regulator of the IL-6–related signaling pathway in B cells; on the other hand, IL-6 phosphorylates MAPK that activate C/EBPβ (Nakajima et al., 1993). C/ebpβ is a predicted direct target of MiR-155 (TargetScan; Figure 2.9B, top).

To verify whether this is biologically true, luciferase activity assays were performed on

HEK-293 cells cotransfected with C/ebpβ 3′-UTR, cloned in the luciferase reporter pGL3 vector, and a miR-155 mimic; we recorded a decrease of approximately 53% of the relative luciferase activity in the presence of the miR-155 mimic compared with the control (Figure 2.9B, bottom); after mutating 3 bases in the 3′-UTR seed sequence, this decrease was only of approximately 14%, indicating that the binding site is operational.

Therefore, C/ebpβ is a direct target of miR-155, and we expect it to be diminished in the

MiR-155 transgenic B cells.

2.6.6. SHIP1 and C/EBPβ are significantly downregulated in Eµ-MiR-155 mice

We hypothesized that Ship1 expression could be under the control of MiR-155 and thus down-regulated in the transgenic B-cell precursors. Indeed immunohistochemical staining of leukemic spleens indicated a sharp reduction of SHIP1

34 expression in MiR-155 transgenic leukemias (Figure 2.10A), confirming that, indeed,

Ship1 is down-regulated in the context of MiR-155 overexpression.

SHIP1 and C/EBPβ expression in the leukemic lymphocytes by Western blot analysis on protein extracts from leukemic and wild-type total splenocytes also showed significant reduction (Figure 2.10B). Infact SHIP1 was almost absent in the transgenic samples, whereas C/EBPβ was not expressed in 2 transgenics and had diminished levels in 2 others (Figure 2.10C). Interestingly, 2 T-cell leukemias arisen in the MiR-155 transgenics had normal levels of CEPB/β (Figure 2.10C; the T-cell leukemia samples are marked with “T”). To study the correlation between SHIP1 and C/EBPβ expression level and the degree of malignant lymphoid transformation, we sorted 3 spleen subpopulations by flow cytometry, as follows: immature B cells (B220+ IgM-), mature B cells (B220+,

IgM+), and non-B cells (B220- IgM-). We then assessed SHIP1 and C/EBPβ expression by Western blot in all these 3 subpopulations in the preleukemic, leukemic, and wild-type mice (Figure 2.11A-D). Densitometry on the SHIP1 expression bands in the pre-B cells indicated that this protein is the least expressed in the leukemic mice, less expressed in the preleukemic, and the most expressed in the wild-type mice (Figure 2.11 A, B). The mature B cells displayed the same gradual reduction of the SHIP1 expression, ranging from the best expressed in the wild-type to the least expressed in the leukemic. C/EBPβ, on the other hand, did not seem to be less expressed in the preleukemic compared with the wild-type pre-B cells. However, the leukemic pre-B cells exhibited decreased levels of C/EBPβ compared with both the wild-type and preleukemic, as shown by the densitometric analysis of the protein bands on the immunoblot (Figure 2.11 C-D). Unlike

35 SHIP1, C/EBPβ seems to maintain the same low level of expression in all mature B cells, without any significant difference between the wild-type, preleukemic, and leukemic mice (Figure 2.11C). Western blot on the same subpopulations of B cells failed to identify any expression of IL-6, indicating that, most likely, the IL-6 intracellular pathway and not the IL-6 transcription is influenced by SHIP and C/EBPβ downregulation (data not shown).

2.7. Discussion

The Eµ-MiR-155 mice were followed up for approximately 2 years. All transgenic mice developed polyclonal lymphoproliferation, followed by malignant leukemias and lymphomas. All leukemias were transplantable into syngeneic mice with a latency of 1 to 1.5 months. There were only minor differences between the 2 strains

(FVB and C57BL/6) in the sense in which C57BL/6 exhibited a slightly longer latency and a milder onset of the disease. Immunohistochemistry showed the malignant lymphoproliferation to be composed of B-cell precursors B220+/CD79a+/IgM-, irrespective of the strain. Flow cytometry unveiled a more nuanced picture: we, thus, could see that initially the lymphoproliferation was with B220+/IgM-/IgD- cells, whereas the mature B-cell population did not seem to be affected; in the leukemic mice, however, most of the B cells had diminished expression of the surface antigen B220 (becoming

B220lo), and many of them did not display IgM or IgD. The B220+/IgM+ mature population was drastically diminished in the leukemic spleens compared with the preleukemic ones. All transgenics showed an unexpected and consistent increase of the

36 myeloid lineage. T-cell population was variably decreased in all transgenics compared with the wild-type mice. TaqMan showed MiR-155 transgene to be expressed at higher levels in the bone marrow compared with the spleen. The pre-B cells exhibited the highest levels of MiR-155 expression. VDJ rearrangement analyses of malignant splenocytes revealed the existence of rearranged bands consistent with the emergence of dominant clones. Noteworthily, Ship-/- knockout mice present a block in the B-cell differentiation at the stage of pre-BI (lymphoblast), similar to the MiR-155 transgenic mice, together with a decrease of the bone marrow mature B lymphocytes. Ship1 is known to be differentially expressed in the pro- and pre-B stages of B-cell maturation

(Helgason et al., 2000). Recently, Nakamura et al demonstrated that the B-cell–defective development in Ship-/- mice is due to exogenous IL-6 signaling pathway activation

(Nakamura et al., 2002). Experiments with IL-6 recombinant in stromal-free cultures of wild-type bone marrow cells show that this proinflammatory cytokine acts on hematopoietic cells and promotes myelopoiesis while suppressing B lymphoid development (Nakamura et al., 2002). This suggests that SHIP1 has an important role in the early stages of B lymphoid development, and its action might be due in part to the suppression of the IL-6–initiated cell signaling. Interestingly, Helgason et al demonstrated that Ship1-/- B cells have an increased phosphorylation and activation of

MAPK and AKT, and consequently, are resilient to apoptosis and show a prolonged survival (Helgason et al., 2000). Moreover, splenocytes from double-knockout mice for

PTEN-/- and Ship-/- have even lower apoptotic rates and longer survival than Ship-/- single mutants, demonstrating that, indeed, SHIP is an inhibitor of the AKT pathway (Moody et al., 2004). The luciferase assays performed by us indicated that Ship1 is down-regulated

37 by miR-155. Recently, other groups also have identified Ship1 as a target of miR-155

(O'Connell et al., 2009; Petersen et al., 2009). O'Connell et al (O'Connell et al., 2009) showed that repression of Ship1 by miR-155 might be at the origin of the myeloproliferative disorder phenotype expressed by hematopoietic MiR-155– overexpressing mice. Moreover, Pedersen et al determined that MiR-155–SHIP1 interaction is part of the tumor necrosis factor–dependent B-cell lymphoma growth pathway (Pedersen et al., 2009). Immunhistochemical staining showed the absence of

Ship1 expression in the MiR-155–related murine leukemias. Western blot on total splenocytes found SHIP1 to be drastically decreased in transgenics compared with wild- types. Western blot on flow cytometry-sorted B cells showed SHIP1 to be downregulated mainly in the pre-B and mature B cells. The Eµ/VH promoter/enhancer is activated at the early pre-B stage (pre-BI). We think that once MiR-155 transgene is overexpressed in the B-cell lineage, SHIP1 levels diminish drastically, resulting in a block of the B-cell differentiation at the pre-BI stage. In these SHIP1-deficient B-cell precursors, PI3K/AKT and MAPK remain activated. Thus, the incompletely differentiated lymphocytes fail to undergo apoptosis. Instead, they survive and accumulate. This causes the initial polyclonal lymphoproliferation that we find in all of our transgenics. Ulteriorly, this lymphoproliferation becomes oligo/monoclonal and displays a mixed immunophenotype of B220lo/IgM- and B220lo/IgM+ cells, due to the partial loss of B220 surface antigen by the malignant B cells. Another protein downregulated by miR-155 is C/EBPβ, also known as a negative regulator of the IL-6 signaling pathway (Screpanti et al., 1995). In B cells, C/EBPβ is activated by the MAPK after their phosphorylation by exogenous IL-6 (Nakajima et al., 1993). Both the C/ebpβ

38 knockout and IL-6 transgenic mice (that overexpress this protein in all hematopoietic cells) mimic an aggressive form of human Castleman disease characterized by polyclonal hypergammaglobulinemia and plasma cell hyperplasia (Brandt et al., 1990; Screpanti et al., 1995). The inactivation of the IL-6 gene in T cells reverses the abnormal B and plasma cell expansion of the C/ebpβ-/- mice, supposedly by lack of induction of the IL-6– dependent B-cell signaling pathway (Screpanti et al., 1995).

Similar to the C/ebpβ-/- mice and IL-6 transgenics, the CD21cre-MiR-155 knockin mice presented an augmentation of the IgG1-synthesizing cells in spleen and lymph nodes (He et al., 2005). The phenotype of both MiR-155 knockout and knockin mice seems to suggest that MiR-155 could cause an accelerated B-cell maturation and increased IgG1 cell accumulation at later stages of B-cell differentiation; this could occur in humans as well, because up-regulation of MiR-155 is commonly observed in diffuse large B-cell lymphoma, especially in the ABC subtype (with activated mature B cells)

(Eis et al., 2005); Vigorito et al recently suggested that one possible mechanism for the

MiR-155 role in the B lymphocyte maturation might be down-regulation of PU.1

(Vigorito et al., 2007). We believe that MiR-155 regulates the IL-6–mediated signaling pathway (implicated in B-cell differentiation) by targeting C/EBPβ. The down-regulation of C/EBPβ causes an IL-6–dependent accumulation of IgG1-positive cells in the knockin

MiR-155 murine model, similar to the PU.1 down-modulation. A deletion of human chromosome 20q in the region where C/EBPβ maps has been reported in several cases of myeloproliferative disorders and myelodisplastic syndromes, suggesting that this gene could be a link between myelo- and lymphoproliferative disorders (Guerzoni et al.,

39 2006). Interestingly enough, the MiR-155 transgenic mice also exhibit invariably an increase of the myeloid lineage. We were able to demonstrate by luciferase assay experiments that C/EBPβ is a direct target of miR-155. Two different groups, O'Connell et al (O'Connell et al., 2009) and Yin et al, (Yin et al., 2008a) have also recently shown that C/EBPβ is directly regulated by miR155. Western blot on total splenocytes indicated a decrease of the C/EBPβ expression in 2 of our transgenic samples and its total abolition in 2 more others. The immunoblot on sorted splenocytes from wild-type, preleukemic, and leukemic animals indicated that, whereas there was no relevant difference in terms of

C/EBPβ expression between the various mature B cells, leukemic pre-B cells were exhibiting obviously decreased levels of C/EBPβ compared with the wild-type and leukemic B-cell precursors. We concluded that C/EBPβ down-regulation, similar to that of SHIP, is one of the early events induced by the MiR-155 overexpression occurring at the pre-BI stage. We believe that in the absence of C/EBPβ, cells proliferate faster in preparation for a differentiation that does not occur anymore due to the Ship1 downregulation.

In conclusion, we have shown that MiR-155 transgene expression reaches its peak at the pre–B-cell stage. High levels of MiR-155 down-regulate Ship1 and free the IL-6 pathway of its inhibitory control, generating a block in the B-cell differentiation and favoring the accumulation of predominantly apoptosis-resistant pre-BI cells. This initiates the leukemogenesis, malignant Ship-deficient lymphocytes proliferating preferentially over the ones that still maintain some Ship1 expression, as shown by the gradual decrease in intensity of the SHIP1 synthesis in leukemic pre-B cells compared with preleukemic

40 cells. MiR-155 overexpression also down-modulates C/EBPβ, liberating the IL-6– dependent signaling pathway of another inhibitory control. This also contributes to the lymphoproliferation identified in the MiR-155 transgenic mice.

41 Table 2.1. List of microRNAs up (A) and down-regulated (B) in Eμ-miR-155 mice splenocytes

(A) Up in Eμ-miR-155 mice Score Fold Change q-Value mmu-mir-151-prec 20.00526459 21.0581238 0 mmu-mir-217-precNo2 15.00875911 21.4448988 0 mmu-mir-224- 10.05109939 9.47394397 0 precformer175No1 mmu-mir-194-prec 9.244472641 6.12092971 0 mmu-mir-155-prec 8.266618234 7.09169563 0 mmu-mir-201-prec 7.755778599 13.3118393 0 mmu-mir-218-2-precNo2 5.103683557 20.9412313 0 mmu-mir-182-prec 3.854156301 6.34890613 0 mmu-mir-187-prec 2.373056618 2.02340409 0 mmu-mir-189-prec 2.206407297 3.09086724 8.767284 mmu-mir-183-precNo1 2.127834255 3.26358689 8.767284 mmu-mir-153-prec 1.867054579 2.7134042 16.07335

(B) Down in Eμ-miR-155 Score Fold Change q-Value mice mmu-mir-138-2No1 -2.476488478 0.57735797 18.08252 mmu-mir-146-prec -2.336995188 0.53811531 18.08252 mmu-mir-29b-1No1 -2.207943524 0.45956948 18.08252 mmu-mir-001d-prec -2.091975029 0.25934967 18.08252

42 Figure 2.1: Eµ-MiR-155 transgenic mice (a) Eµ-MiR-155 transgene construct, and (b).

Northern blot analysis of miR-155 expression in founder mice obtained from the pronuclear injected females showing five founder lines with miR-155 over-expression

(Costinean et al., 2006).

43 Figure 2.2: Phenotypic and immunochemical characterization of Eµ-MiR-155 mice. Transgenic mice, 6 months old, presented an enlarged abdomen and important splenomegaly. (A) Transgenic mice, 6 months old, had a considerably enlarged abdomen compared with wild-type mice, due to the clinically evident splenomegaly. (B) Spleens of the mice shown in A. The transgenic spleen is enlarged due to expansion of leukemic/lymphoma cells. (C) H&E of spleens showing overproliferation of malignant lymphocytes staining dark purple in transgenic spleen (left panel) versus the normal spleen (right panel).

44 Figure 2.3: Flow cytometry analysis reveals pre-B cell leukemia: (A) Spleen analysis showing expansion of B220+/IgM- precursor B-cell population, in transgenic (74 & 156 TG) as compared to wild type (68 & 157 WT) mice, and in (B) Bone marrow, (8 TG) and (24 WT).

A B220

B B220

45 Figure 2.4: Kaplan meier survival curve: Comparative Kaplan Meier survival curve for MiR-155 transgenic mice. (A) Survival of FVB and C57BL/6 MiR-155 transgenics compared with their wild-type counterparts, showing shorter lifespan for the transgenic mice. (B) Survival of transplanted FVB mice compared with the wild-type controls; all transplanted mice were dead after 6 weeks, whereas all the wild-type controls were alive.

Figure 1. Comparative Kaplan Meier survival curve for MiR-155 transgenic mice.

46 Figure 2.5: Flow cytometry analysis of leukemic and preleukemic spleens showing pre-B (A) and Cd11b myeloid (B) cell expansion in pre-leukemic and leukemic Eµ-miR- 155 mice.

47 Figure 2.6: Flow cytometry analysis of T-cells: showing reduced total number of T- cells in Eµ-miR-155 mice (TG), but similar CD4:CD8 as compared to wild type (WT).

48 Figure 2.7: Differential miR-155 expression of various hematopoietic tissues showing (A) Bone marrow with the highest expression followed by spleen. (B) miR-155 expression in FACS sorted pre-B cells showing more than 100-fold higher expression than mature B-cells as compared to wild type mice.

49 Figure 2.8: Eµ-miR-155 mice tumors are oligoclonal: (A) Oligo/monoclonal rearrangement of the B-cell miR 155 leukemias (small black arrows indicate bands of oligo/monoclonal rearrangement in the transgenics compared to the wild types; red arrow indicates germ line band; blue arrow indicates a cross hybridizing normal band) the genomic DNA was digested with PvuII (see materials and methods); (B) Same analysis as in (A) shows in lane 1 a leukemia with a few rearranged clones while in lane 3 there is only one rearranged band (see blue asterisk); leukemia in lane 2 is still polyclonal; the red asterisk indicates the germ line; the genomic DNA was digested with EcoRI; M = DNA molecular marker. A B

50 Figure 2.9: Ship1 and C/ebpβ are direct targets of miR-155. (A) Sequence alignment of miR-155 and Ship1 3’UTR (top panel, http://www.targetscan.org/) showing conserved miR-155 binding site across Mouse (Mm), Human (Hs), Rat (Rn) and Dog (Cf). Luciferase activity assay showing miR-155 regulation of Ship1 (bottom panel) (B) Sequence alignment of miR-155 and C/ebpβ 3’UTR (top panel) and Luciferase activity assay showing miR-155 regulation of C/ebpβ-3′-UTRC/ebpβ (bottom panel).

51 Figure 2.10: SHIP and C/EBPβ are down-regulated in Eµ-miR-155 mice: (A) Comparison of transgenic and wild-type splenocytes showed B-cell precursors had lower expression than the mature B cells. Spleen, immunohistochemistry, 200×. SHIP-1 is highly expressed in normal B and T lymphocytes (left panel) (Olympus BX41 microscope and software; the bar represents 200 µm). Malignant lymphocytes do not stain for SHIP-1 due to the loss of its expression (right panel). (B-C) Western blots on proteins extracted from total splenocytes show the lack of expression of SHIP1 (B) and C/EBPβ (C) in the leukemic compared with the wild-types. Note: The samples marked “T” are 2 T-cell leukemias arisen in the miR-155 transgenic mice.

52 Figure 2.11: Immunoblot analysis of B-cell subsets for SHIP1 and C/EBPβ show that miR-155 leukemic pre-B cells have lower expression of these 2 proteins compared with the wild-type counterparts. (A) Immunoblot shows a stepwise down-regulation of the SHIP expression in the pre-B and mature B cells in the leukemic mice compared with their preleukemic and wild-type counterparts. (B) Densitometry for SHIP expression in pre-B cells calculated on the previous immunoblot confirms that SHIP is expressed the most in the wild-type, less in the preleukemic, and the least in the leukemic cells. (C) Immunoblot on sorted splenocytes shows a down-regulation of C/ebpβ in the pre-B cells of leukemic mice compared with their preleukemic and wild-type counterparts. (D) Densitometry for the C/ebpβ expression in different pre-B cells (wild-type, preleukemic, and leukemic), calculated on the previous immunoblot, confirms that C/ebpβ has higher expression in the leukemic pre-B cells than in the preleukemic and wild-type ones.

53 Chapter 3

By targeting HDAC4 and impairing BCL6 transcriptional activity, miR-155 induces

a block in B cell differentiation and development of acute leukemia

3.1. Introduction

MicroRNAs (miRNAs) are 18-24 nucleotide long non-coding RNA molecules that regulate gene expression in many cellular processes, including proliferation, differentiation, and development. Recent studies have established that miRNA expression is widely altered in cancer (Calin and Croce, 2006). Notably, miR-155 is one of the most frequently over-expressed miRNAs in various solid and hematological malignancies

(Calin and Croce, 2006; Sandhu et al., 2011). MiR-155 is embedded in a host non-coding

RNA named the B cell integration cluster (Calin et al.), which is a common retroviral integration site in avian leucosis virus-induced lymphomas and is located at Chr. 21q23.

Causative role for miR-155 in leukemias has been shown by various approaches: in-vitro in combination with c-Myc (Tam et al., 2002) and independently by in-vivo transgenic mouse model where ectopic overexpression of miR-155 in B cells causes aggressive pre-

B cell lymphoma/leukemia (Costinean et al., 2006). In addition, sustained miR-155 expression in hematopoietic stem cells induced a myeloproliferative disease in the

54 transplanted mice (O'Connell et al., 2008). Loss-of-function mouse model has shown that miR-155 plays an important role in immune system development and function

(Rodriguez et al., 2007; Thai et al., 2007). In addition, amplification of miR-155 has been found in the ABC (activated B-cell) sub-type of human DLBCL (Diffuse large B-cell lymphoma) (Rai et al., 2008).

MiRNAs regulate gene expression in sequence specific manner whereby perfect complimentarity of the miRNA seed region to the target mRNA 3’ UTR results in mRNA degradation, while imperfect base-pairing leads to translation inhibition (Bartel, 2004).

About 30% of human genes are predicted to be regulated by miRNAs which constitute about 3% of the total genes (Lewis et al., 2005). Previously, few targets of miR-155, including Src homology-2 containing inositol phosphatase-1 (SHIP-1) have been shown to play a role in miR-155 induced leukemogenesis (Costinean et al., 2009; O'Connell et al., 2009). Since a single miRNA targets several distinct gene products, it is likely that multiple other genes are involved in this process. Therefore, in order to discover novel mRNA targets involved in miR-155 induced leukemogenesis we performed genome wide mRNA expression analysis of B-cells from the Eµ-miR-155 transgenic mice, followed by target validation and functional studies both in-vivo and in-vitro. We found that well known protooncogene Bcl6 and a histone deacetylase, HDAC4 are significantly downregulated in these mice. While HDAC4 is targeted directly by miR-155, Bcl6 is indirectly regulated by miR-155 at the transcriptional level through MAD1/MXD1 and post-transcriptional level through HDAC4 mediated acetylation. To corroborate our findings of transcriptionally inactive BCL6 in Eµ-miR-155 mice, we found significant

55 enrichment of its transcriptional targets including Id2, Il6, Mip1α and Ccnd1. Further, we show that overexpression of HDAC4 in lymphoma cells reduces miR-155 induced proliferation and colony formation ability due to increased apoptosis. Additionally, since

BCL6 plays an important role in germinal center (GC) B-cell development, its downregulation in Eµ-miR-155 mice may in part contribute to the impaired innate immunity of these mice in response to external antigens and enhance the development of leukemia. Indeed we found Eµ-miR-155 mice have reduced number of GC B-cells in response to T-cell dependent antigen immunization. Hence we provide mechanistic evidence for the involvement of two key genes, BCL6 and HDAC4 in the pathogenesis of miR-155 induced lymphoma/leukemia.

3.2. Materials and Methods

Mice, cell lines, DNA Constructs and Retrovirus

Eµ-miR-155 (B-cell miR-155 transgenic) mice have been described before

(Costinean et al., 2006). Bic/miR-155 deleted mice (B6.Cg-Mirn155tm1.1Rsky/J) were obtained from the Jackson Laboratories and have been described elsewhere (Thai et al.,

2007). All mice were housed in the BRT vivarium as per the IACUC and University lab animal research (ULAR) approved protocols. For GC B-cell analysis, 6-9 week old eµ- miR-155 and WT mice were immunized with 100µg NP-CG via i.p and spleens were analyzed after two weeks. Total splenocytes were stained with antibodies against B220,

PNA, and CD138 followed by flow cytometry analysis on BD FACS LSR II, and data analysis by Flow Jo. 56 Human diffuse large B-cell lymphoma (DLBCL) cell lines, OCI-Ly1 and OCI-

Ly3 (Gift from Dr. Ricardo C. T. Aguiar) were maintained in IMDM, 20% FBS and 1%

Penicillin and Streptomycin. Full length cDNA of Hdac4 and Bcl6 was cloned into

Murine Stem Cell Virus (MSCV) containing IRES and GFP (pMIG). Retrovirus was produced in Phoenix cells which were maintained in DMEM with 10% FBS. DLBCL cell lines were transduced with 2 mls of virus per 1 X 106 by spin-infection with 10 ug/ml polybrene (SCBT). Briefly, the cells containing virus were centrifuged at 37°C at 2000 rpm for 90 minutes, and incubated with virus for additional 4-5 hours, after which it was replaced with fresh media for recovery overnight. After 3-4 days, GFP positive cells were sorted using BD FACS Aria and cultured for colony formation, apoptosis and proliferation assays.

Luciferase Reporter Assay

miR-155 target prediction was according to the Targetscan (2006) and RNA22 algorithms. The 3’UTR of the human and mouse Hdac4 and Bcl6 was PCR amplified with XbaI flanked primers (Primers available upon request). The PCR products were purified, digested and cloned downstream of the luciferase coding region in the pGL3 control vector (Promega). Human HEK293T cells (and four other cell lines: mouse NIH-

3T3; Human HeLa, Jurkat) were co-transfected with respective luciferase constructs and miR-155 encoding plasmid along with Renilla luciferase expression plasmid (pRL-TK) as a transfection control using Lipofectamine (Invitrogen). Deletion and mutant HDAC4 luciferase constructs were prepared by deleting and mutating the miR-155 binding sites in the original 3’UTR construct, respectively. After 48 hours cells were lysed and analyzed

57 for relative luciferase activity using Dual Luciferase assay kit (Promega). Results are representative of three independent experiments.

Microarray and Quantitative RT-PCR

Total mRNA was isolated using Trizol (Invitrogen) from splenic B-cells isolated from Eu-miR-155 transgenic and wild type mice using B cell Isolation kit (Miltenyi

Biotech) and analyzed on Mouse Genome 430_2 arrays. The data were analyzed using

BRB array tools. GC-RMA normalized data was used to identify “canonical pathways” affected by miR-155 using Ingenuity Pathway Analysis (IPA).

Total RNA from naïve B-cells from Eµ-miR-155 transgenic (n=3) and wild type littermates (n=4) was reverse transcribed using TaqMan microRNA reverse transcription kit (for miR-155 quantification) or iScript cDNA synthesis kit (Biorad) (for Taqman

Gene expression assays and sybr green mRNA quantification), using manufacturers protocol. The respective cDNA was amplified using miRNA Taqman probes (ABI) for miR-155 and sno-135 (normalizer) and mouse BCL6, HDAC4 and beta-actin

(normalizer). BCL6 target genes were detected using sybr green based real time PCR

(picked from RTprimer database available at http://medgen.ugent.be/rtprimerdb/). All real time PCR assays were performed on the Biorad IQ5 or CFX96. Data were analyzed

- Ct - Ct using 2 ΔΔ for fold change results as compared to wild type controls or 2 Δ method for relative amount/expression as compared to an internal control gene like actin or gapdh

(Schmittgen and Livak, 2008). For calculating the overlap between upregulated genes in mice and known BCL6 targets (Polo et al., 2007) in humans, these genes were clustered

58 using Gene Cluster 3.0. Genes were mean centered and average linkage was performed using Euclidean distance, after filtering the genes with SD >1.

DLBCL Patient Sample Analysis

A total of 84 DLBCL patient samples (frozen biopsies) from two Gene

Expression Omnibus (GEO, NCBI) datasets were analyzed for Hdac4, Bcl6 and bic/miR-

155 mRNA expression correlation studies. 11 samples from GSE12453 and 73 from

GSE12195 series were RMA normalized, managed and analyzed by BRB-ArrayTools

Version 3.8.1 (Affymetrix HG U133 Plus2.0). Genes whose expression differed by at least 1.5 fold from the median in at least 20% of the arrays were used. Using Spearman correlation, which measures the correlation of rank ordering between two values, at p<0.01 stringency, expression of Bcl6 and Hdac4 negatively correlated with BIC (miR-

155).

Cell proliferation, Apoptosis and colony formation

For apoptosis analysis, cells were stained with Annexin (BD Pharmingen) and 7-

AAD (Biolegend). The cells were analyzed on BD FACS LSR-II using the FACS DiVa

software (BD Biosciences). Cell proliferation was assayed using CellTiter 96® AQueous

Non-Radioactive Cell Proliferation Assay (Promega) after transfection with miR-

(inhibitors, mimics or control, Ambion) or full length cDNAs (HDAC4, BCL6, empty vector pCMV6-KN). HDAC4, or Empty retrovirus transduced OCI-Ly3 cells were assessed for their colony formation ability using Methocult media (Stem Cell).

59 Immunoblotting and Co-immunoprecipitations (Co-IP)

Mouse B-cells or total splenocytes were lysed in RIPA (Radio- immunoprecipitation Assay Buffer, Sigma), lysates run on 4-20% SDS/PAGE (Lonza,

Walkersville) gel and transferred to nitrocellulose membrane using wet transfer (Biorad).

Membranes were blocked for 1 hour in 5% non-fat dry milk and incubated in primary antibodies overnight at 4°C. Following primary antibodies were used: rabbit HDAC4

(sc-11418); rabbit BCL6 (sc-368); rabbit Acetylated-lysine (Cell Signaling Technology,

9441); goat ACTIN (sc-1616). For in-vivo detection of acetylated BCL6, 40 X 106 total mice splenocytes (after erythrocyte lysis) were lysed in 1 ml RIPA buffer (Sigma) supplemented with protease inhibitors cocktail (Calbiochem) and phosphatase inhibitors

(Davis et al.). About 1 mg of total cell (nuclear) lysate was precleared using A/G beads

(Santa Cruz Biotechnology) and incubated with rabbit BCL6 antibody (C-19) and A/G beads for four hours for immunoprecipitation (IP). Immobilized complexes were washed three times with RIPA buffer, denatured and analyzed by western blot on 4-20% SDS-

PAGE gel using nitrocellulose membrane for immobilizing proteins and ECL reagent for detection (GE Amersham). For detection of BCL6 ubiquitination in TG and WT mice, total splenocyte lysates were incubated with anti-ubiquitin antibody (Upstate Signaling) for IP and complexes were immobilized on nitrocellulose membrane and probed with anti-BCL6. For in-vitro ubiquitination analysis, HEK-293T cells transfected with miR-

155 and pCMV6-BCL6.

60 3.3. Results

3.3.1. Genome profile of Eµ-miR-155 mouse B-cells

To identify potential miR-155 targets involved in the pathogenesis of pre-B-cell leukemia in the Eµ-miR-155 transgenic mice, we performed mRNA expression profiling of isolated splenic B-cells from these mice. We found that 268 genes were down- and

1077 were up-regulated in the Eµ-miR-155-expressing mice B-cells. Ingenuity pathway analysis (IPA, Ingenuity Systems Inc., Redwood City, CA, USA) of differentially expressed genes yielded Cell cycle G1/S transition as the most significant functional pathway (P<0.00001). Interestingly, the two most significant canonical pathways represented by the upregulated genes include, Aryl-Hydrocarbon Receptor signaling which has been linked to B-cell differentiation by modulating sleuth of genes (De Abrew et al., 2010) and Communication between Innate and Adaptive Immune cells, which is at the root of various cancers (Table 3.1A). The canonical pathways represented by the downregulated genes point towards impaired hematopoietic progenitor cell signaling including kinases, like MAPK and bonafide miR-155 target, SHIP1 (Table 3.1B).

Interestingly, among the downregulated genes was the Bcl6 gene, which was 2- fold downregulated (Figure 3.1A, B). We also found decreased BCL6 protein levels in total splenocytes (Figure 3.1C, top panel) and B-cells (Figure 3.1C, bottom panel) of Eµ- miR-155 mice. BCL6 is a POK (BTB/POZ and Kruppel-like zinc finger) family transcription factor which acts as a transcriptional repressor of various gene promoters 61 involved in GC development, B-cell activation, DNA-damage response, cell cycle arrest, cytokine-, toll-like receptor, TGF-WNT-signaling and differentiation (Basso and Dalla-

Favera, 2010). Bcl6 is frequently upregulated in various non-Hodgkin lymphomas (NHL) due to translocations, deletions, or point mutations (Albagli-Curiel, 2003). Based on the significance of BCL6 in B-cell development and function we further investigated its role in miR-155 induced leukemogenesis.

3.3.2. Downregulation of Bcl6 and consequent upregulation of its transcriptional targets in Eµ-miR-155 leukemic mouse model

First we reasoned that BCL6 suppression by miR-155 may unblock the expression of BCL6 transcriptional targets, some of which may contribute to leukemogenesis.

Therefore we measured the expression of known BCL6 transcriptional targets that could act as oncogenes. Global analysis of upregulated transcripts in Eµ-miR-155 mice showed significant number of known BCL6 targets as reported by (Polo et al., 2007) (Figure

3.2A). Specifically, BCL6 targets such as Inhibitor of differentiation (Id2), Chemokine

Ligand 3 (Ccl3/Mip1α), Interleukin-6 (Il6) and Cyclin D1 (Ccnd1) were significantly upregulated (Figure 3.2B). Downregulation of Id2 is essential for B-cell commitment and its upregulation in mouse bone marrow blocks B-cells at pre-pro-B stage (Thal et al.,

2009). Mip-1α, is a cytokine belonging to the CC chemokine family that is involved in the acute inflammatory state in the recruitment and activation of polymorphonuclear leukocytes (Schall and Bacon, 1994) and often activated in various lymphomas (Sivina et al., 2011). Il-6 is a pro-inflammatory cytokine that promotes lymphoma and leukemia

62 proliferation. Likewise, Ccnd1, an anti-apoptotic gene with role in G1/S phase of cell cycle increases proliferation and is overexpressed in many tumors (de Boer et al., 1997).

Altogether our data suggest that known repressed targets of BCL6 that are upregulated in

Eµ-miR-155 B-cells including differentiation inhibitors like Id2 and cell proliferation mediators like Mip1α, IL6 and Ccnd1, could contribute to the miR-155 induced block in

B-cell differentiation and increased proliferation observed in our mouse model. To further assess the impact of Bcl6 downregulation in Eµ-miR-155 mice, we investigated

T-cell dependent GC formation which is dependent on Bcl6 expression. As expected, we found that Eµ-miR-155 mice have reduced number of GC B cells upon T-cell stimulation

(Figure 2C). These effects may contribute to an impaired functional B-cell adaptive immune response in miR-155 induced leukemias.

3.3.3. Transcriptional regulation of Bcl6 by miR-155 is indirectly mediated through

Mad1 upregulation

To gain insight into Bcl6 regulation by miR-155 we began with the hypothesis of canonical miRNA targeting. Only one algorithm (Miranda et al.) predicted Bcl6 to be a miR-155 target but luciferase reporter assays did not show its regulation by miR-155

(Figure 3.3A, B). As an alternative mechanism, we found that MAD1/MXD1 regulatory complex which is a known transcriptional repressor of Bcl6 is significantly upregulated in

Eµ-miR-155 mice (Figure 3.4A). Further, we show that ectopic overexpression of miR-

155 in mouse macrophage cell line Raw 264.7, resulted in time-dependent upregulation of Mad1 and subsequent downregulation of Bcl6 (Figure 3.4B). MAD1 has been 63 previously shown to downregulate Bcl6 in mouse immature B lymphoma cells (Lee et al.,

2006). MAD1 or MAX dimerization protein 1, is one of the key spindle checkpoint proteins (Maldonado and Kapoor, 2011), and is a MYC antagonist often co-expressed with MYC (Baudino and Cleveland, 2001). Interestingly, Myc is also a target of Bcl6 and is significantly upregulated in Eµ-miR-155 mice (Data not shown). Together these results indicate that miR-155-induced transcriptional repression of Bcl6 could be at least in part mediated by Mad1 upregulation.

3.3.4. miR-155 targeting of HDAC4 enhances acetylation-mediated BCL6 downregulation through ubiquitination

Since post-translational regulation of BCL6 involves acetylation and phosphorylation-mediated degradation (Bereshchenko et al., 2002; Niu et al., 1998), we also investigated the BCL6 acetylation status in the Eµ-miR-155 mice. Indeed we found significantly increased acetylation of BCL6 (Figure 3.5A) in these mice. Since acetylated

BCL6 is inactive, we reasoned it may be a target of ubiquitin-mediated degradation. To test this end we show that ectopic overexpression of miR-155 in HEK-293 cells resulted in increased Bcl6 ubiquitination (Figure 3.5B). These results demonstrate that miR-155 over-expression could indirectly modulate BCL6 expression by enhancing its acetylation, and targeting it for ubiquitin-mediated degradation. We then investigated the mechanism by which miR-155 exerts these effects. Interestingly, one of the deacetylases known to deacetylate and hence inactivate BCL6, HDAC4, is a predicted miR-155 target. Sequence alignment of HDAC4 3’UTR across phyla showed miR-155 binding sites at a 7-mer

64 (AGCATTA), and a bonafide 8-mer (AGCATTAA) (Figure 3.6A). Luciferase reporter assay of the HDAC4 3’UTR confirmed a significant downregulation of reporter activity by miR-155 mimic as compared to scrambled control (Figure 3.6B). Furthermore,

HDAC4 protein levels in Eµ-miR-155 splenocytes (Figure 3.7A, upper panel) and B-cells

(Figure 3.7A, middle panel) were significantly downregulated. Ectopic over-expression of miR-155 in HEK-293 cells resulted in significant reduction of HDAC4 protein (Figure

3.7A, lower panel) and mRNA in OCI-Ly1 cells (Figure 3.7B). Inhibition of miR-155 in

Eµ-miR-155 mice B-cells in-vitro resulted in significant recovery of HDAC4 after 72 hours (Figure 3.7C). In addition miR-155 inhibition using anti-miR-155 in OCI-Ly3 cells also resulted in significant recovery of HDAC4 mRNA expression (Figure 3.7D). To further corroborate these findings we found higher levels of HDAC4 protein in mature B- cells from miR-155 deficient bic-/-(miR-155-/-) mice (Figure 3.8). Altogether our results confirm that HDAC4 is a direct target of miR-155 and its downregulation may contribute to increased acetylation and ubiquitination of BCL6.

3.3.5. Restoration of HDAC4 expression is apoptotic and anti-proliferative

To evaluate the functional relevance of downregulation of HDAC4/BCL6 expression in miR-155 induced leukemogenesis, we used a human activated B-cell

(ABC) type DLBCL-derived cell line, OCI-Ly3 (Tweeddale et al., 1987), which served as a good model since it expresses high endogenous miR-155 (Rai et al., 2008) and low

HDAC4 and BCL6. We found that exogenous HDAC4 (without 3’ UTR) expression in

OCI-Ly3 cells resulted in reduced clonogenic potential and proliferation (Figure 3.9A, B)

65 and increased apoptosis (Figure 3.9C). Quantitative real time PCR showed increased expression of Bcl6 in HDAC4 overexpressing OCI-Ly3 cells (Figure 3.10D). Further,

HDAC4 also inhibited the miR-155 induced proliferation of low miR-155, OCI-Ly1 cells when co-transfected with miR-155 (Figure 3.10A) and Bjab cells (Figure 3.10B). In addition HDAC4 overexpression in human Burkitt lymphoma Bjab and mouse pre-B lymphoma Wehi-231 cells also induced apoptosis (Figure 3.10C). Collectively, these findings suggest that HDAC4 has the ability to dampen miR-155 induced proliferative signals in DBLCL and overexpression of HDAC4 alone has antitumoral effects in this model.

3.3.6. Bic/miR-155 expression is negatively correlated with Hdac4 and Bcl6 levels in

DLBCL patients

Finally to establish relevance of our findings to human disease, we investigated whether there is a correlation between miR-155 expression and Bcl6 and Hdac4 expression in NHL patients. To achieve this we obtained two microarray datasets from

GEO, and analyzed 84 DLBCL patient samples from GSE12195 (Pasqualucci et al.,

2011) and GSE12453 (Brune et al., 2008), respectively. Using Spearman correlation analysis we found that miR-155/bic expression levels correlated negatively with Bcl6 and

Hdac4 (Correlation coefficient, R= -0.51, p<0.001 & R= -0.33, p<0.001 respectively)

(Figure 3.11).

66 3.4. Discussion

We show that miR-155 suppresses HDAC4 and BCL6 expression during miR-155 induced leukemogenesis, resulting in the upregulation of gene products that may block B- cell development at the pre-B stage of differentiation and induce cell proliferation (Figure

3.12: Proposed model). Depending on the genetic context of the tumor, ambivalent roles in cell survival and transformation has been described for BCL6 (reviewed in (Albagli-

Curiel, 2003)). Supporting a tumor suppressor function, higher Bcl6 expression in GCB-

DLBCL is associated with better prognosis than ABC-DLBCL. Furthermore, independent studies have linked human ABC-DLBCL with high miR-155 (Eis et al.,

2005; Rai et al., 2008) and lower Bcl6 expression (Iqbal et al., 2007). Here we provide the first evidence linking miR-155 over-expression and BCL6 downregulation indirectly through HDAC4.

We also demonstrate an anti-tumor role for HDAC4 in DLBCL cells. Several studies have linked HDAC4 to proliferative and anti-proliferative pathways in different cell types (Chen and Cepko, 2009; Sun et al., 2009; Wilson et al., 2008; Yang et al.,

2011). Infact HDAC4 was identified as a mediator of p53 and p19ARF-dependent proliferation arrest and senescence (Berns et al., 2004); is frequently mutated in human cancers (Sjoblom et al., 2006) and is required for repair of ionizing radiation-induced

DNA damage (Kao et al., 2003). Together these findings may have significant clinical impact on the treatment of miR-155 induced tumors, especially with HDAC- or BCL6- inhibitors. HDAC-inhibitors have anti-tumor effects in a wide range of tumors (Sakajiri

67 et al., 2005), but a phase II clinical trial with SAHA in relapsed DLBCL patients showed limited benefits (Crump et al., 2008). Therefore, it will be important to assess miR-155 expression in DLBCL patients treated with HDAC inhibitors to assess if it correlates with response to the treatment.

68 Table 3.1: Ingenuity pathway analysis of Eμ-miR-155 mice B-cell mRNA profile (A) Top Canonical pathways represented by the genes upregulated in Eμ-miR-155 mice using Ingenuity pathway analysis. (B) Top Canonical pathways represented by the genes downregulated in Eμ-miR-155 mice using Ingenuity pathway analysis.

A Top Upregulated Canonical Pathways p-value Aryl Hydrocarbon Receptor Signaling 1.81E-05 Glutathione Metabolism 2.06E-05 Mitotic Roles of Polo-Like Kinase 9.75E-04 Extrinsic Prothrombin Activation Pathway 1.02E-03 Communication between Innate and Adaptive Immune Cells 2.64E-03

B Top Downregulated Canonical Pathways p-value SAPK/JNK Signaling 1.06E-03 Activation of IRF by Cytosolic Pattern Recognition Receptors 1.06E-03 4-1BB Signaling in T Lymphocytes 2.35E-03 Dendritic Cell Maturation 5.21E-03 Toll-like Receptor Signaling 9.26E-03

69 Figure 3.1: Bcl6 is downregulated in Eµ-miR-155 (TG) mice B-cells (A) Section of heatmap from microarray analysis showing Bcl6 downregulation, (B) confirmed by qRT- PCR and (C) immunoblot of total splenocytes and B-cells (C). A

1.2 B C 1 WT TG

0.8 WB: BCL6

0.6 * WB: ACTIN Splenocytes WT WT TG TG 0.4

Fold Change WB: BCL6 0.2

WB: ACTIN B-cells 0 TG (n=3) WT (n=3)

70 Figure 3.2: BCL6 targets are upregulated in Eµ-miR-155 mice B-cells (A) Heatmap of known BCL6 targets upregulated in Eµ-miR-155 mice B-cells mRNA profile. (B) Real time PCR showing mRNA expression of selected BCL6 targets significantly upregulated in Eµ-miR-155 mice B-cells. Expression is normalized to Gapdh. (C) Flow cytometry analysis of total splenocytes isolated from Eµ-miR-155 and WT mice 2 weeks after immunization with NPCG. Splenocytes were stained with PNA and B220 and GC B-cells are identified as PNAhiB220+ as seen in the square gate. Eµ-miR-155 mice had 31.5% GC B-cells as compared to 41.3% in WT.

25 A B 20

10 Fold change

105 105

41.3 31.5 104 104

103 103 : PNA : PNA

GC B-cells GC B-cells 102 102

0 0

0 102 103 104 105 0 102 103 104 105 : B220 : B220 WT Eµ-miR-155

71 Figure 3.3: Bcl6 is not a direct target of miR-155: Sequence alignment of 3’UTR of human and mouse BCL6 with respect to miR-155 sequence from Miranda, predicting BCL6 as miR-155 target (upper panel). Luciferase reporter assay showing no difference in pGL3-BCL6-3’UTR (mouse or human) reporter activity by miR-155 or empty vector.

3 m155 scr

2.5

1.5 RLU

0.5

0 mmu_3utrbcl6 hsa_3utrbcl6

72 Figure 3.4: Trancriptional regulation of Bcl6: (A) qRT-PCR showing transcriptional repressor of BCL6, MXD1/MAD1 significantly upregulated in Eμ-miR-155 mice B- cells. (B) HDAC4, BCL6, MXD1/MAD1, and miR-155 levels (mean±SEM) in miR- 155* transfected mouse Raw cell line after 24 and 48hr post-transfection. *miR-155 levels are log-transformed in order to maintain the scale.

A 2 1

-1 Bcl6

Mxd1 Fold Change -2

-3

-4

-5

B 10

8 pre-miR-155 24h pre-miR-155 48h 6

for miR) for 2

Hdac4 Bcl6 0 Mxd1 miR-155

-2 Fold Change relative to Actin (sno135Actin relativeChange to Fold

-4

73 Figure 3.5: Translational regulation of Bcl6: (A) Quantification of immunoprecipitated total BCL6 from splenocytes showing significantly increased acetylation in Eµ-miR-155 mice as compared to WT, p<0.05 (left panel). Immunoblot is shown in the right panel. (B) HEK-293T cells transfected with pCMV-BCL6 and miR-155 expression plasmid or empty plasmid showing increased ubiquitination in the presence of miR-155.

3 A * WT TG 2.5

IP: BCL6 2 WB: ActLys

1.5

IP: ActLys WB: BCL6 1

Acetylation % 0.5 IgG

0 WT TG

B pCMV-Bcl6 + + miR-155 + - hrGFP! - +

IP=Anti-Ub BCL6 WB= Anti-Bcl6

74 Figure 3.6: Hdac4 is a direct target of miR-155: (A) Sequence alignment of 3’ UTR of mouse (Mmu), rat (Rno), human (Hsa) and rhesus (Mml) HDAC4 highlighting two miR- 155 binding sites, 8-mer and a more conserved 7-mer. (B) Luciferase reporter assay showing upto 50% reduction in reporter activity when transfected with miR-155 precursor plasmid in five cell lines: HEK-293T (Renal), Hela (Ovarian), NIH-3T3 (Fibroblast), U373 (Brain) and Jurkat (T-cell line) as compared to empty plasmid encoding GFP. Values in graphs represent mean±s.e.m from three independent experiments, *P<0.05.

A miR-155 7-mer

miR-155 8-mer

1.4 Scr B m155 1.2

0.8 * * * 0.6 * RLU 0.4

0.2

75 Figure 3.7: Hdac4 is significantly downregulated in Eu-miR-155 mice: (A) Immunoblot analysis of HDAC4 expression in Eµ-miR-155 mice splenocytes (upper panel) and B-cells (middle panel) as compared to WT mice showing significant downregulation, and ectopic miR-155 transfection into HEK293T (lower panel) significantly downregulates endogenous HDAC4. (B) Quantitative PCR analysis of miR- 155 mimic or scrambled (Scr) transfected OCI-Ly1 cells showing downregulation of HDAC4 mRNA. (C) Immunoblot of Eµ-miR-155 mice B-cells treated with anti-miR-155 showing recovery of HDAC4 expression with time. (D) Recovery of HDAC4 expression by anti-miR-155 treatment of OCI-Ly3 cells.

A WT TG B 0.25 WB: HDAC4 0.2

WB: ACTIN 0.15 * Splenocytes WT1 WT2 TG1 TG2

WB: HDAC4 0.1 RelativeExpression

WB: ACTIN B-cells 0.05

miR-155 UTC Empty Vector 0 WB: HDAC4 m155 Scr

WB: ACTIN HEK-293 5 Hdac4 4 miR-155 D 3 C miR155 inhibitor Con Inhibitor 2 24 48 72 24 48 72 1 WB: HDAC4 0 Con Inhibitor Fold Change WB: GAPDH -1 TG B-cells TG -2 -3 miR Inhibitor -4

76 Figure 3.8: Immunoblot analysis of HDAC4 levels in bic-/- mice: miR-155 deficient CD19 cells from bic/miR-155 knock-out mice showing increased expression. βactin was used as a loading control in all immunoblots unless indicated otherwise.

77 Figure 3.9: Hdac4 inhibits colony formation, proliferation, and induce apoptosis of OCI-Ly3 cells. (A) Clonogenic potential of OCI-Ly3 cells transduced with pMIG- HDAC4 (HRV), or pMIG-Empty (ERV). Results are average of three independent experiments. (B) Proliferation of HRV or ERV transduced OCI-Ly3 cells. (C) HDAC4 resulted in increased apoptosis of OCI-Ly3 cells as determined by Annexin-7AAD staining.

350 2 A B ERV 300 HRV 1.5 250 * 200 * 1 150 * Absorbance pMIG-Empty pMIG-Empty # Colonies/3000 Cells 100 0.5

pMIG-Hdac4 0 0 OCI-Ly3 Day 1 Day 2 Day 3 Day 4 C

Dead Dead Apoptotic Apoptotic

31.4 36

17.4 21.4 Annexin Annexin

PI PI Empty Vector Hdac4

78 Figure 3.10: HDAC4 impairs miR-155 induced proliferation in OCI-Ly1 cells (A), reduces proliferation of HRV transduced Bjab cells (B), induce apoptosis of Bjab and Wehi cells (C).

miR-155 2 A 2.5 B Hdac4 HRV ERV miR-155+Hdac4 2 1.5 Scr+Hdac4 *

1.5 1

1 Absorbance Absorbance 0.5 0.5

0 0 Day 1 Day 2 Day 3 Day 4 Day1 Day2 Day3 Day4

10 Hdac4 10000 C HRV BRV Empty Vector D 8 1000

100

% Apoptosis Apoptosis % 4

10 2 Fold Change (Relative to ERV) (RelativeERV) Change to Fold

0 1 Bjab Wehi 231 Hdac4 Bcl6

79 Figure 3.11: HDAC4, BCL6 mRNA levels are negatively correlated with miR-155 expression in human DLBCLs. HDAC4, BCL6 and bic/miR-155 mRNA correlation analysis in 84 DLBCL patients microarray data obtained from published GEO datasets, GSE12195 (Pasqualucci et al., 2011) and GSE12453 (Brune et al., 2008) showing negative correlation between bic/miR-155 and HDAC4 and bic/miR-155 and BCL6.

BCL6 and HDAC4 are negatively corrrelated to miR-155 in human DLBCL

0.9

0.8

0.7

0.6 miR-155

0.5 BCL6 !"#$#%&%%%'(#)*#+%&,'-##

0.4 HDAC4 Percentmax of expression !"#$#%&%%%'(#)*#+%&..-##

0.3

0.2

0.1

0 0 10 20 30 40 50 60 70 80 90 Patient samples

80 Figure 3.12: Proposed model of miR-155 induced leukaemogenesis in Eµ-miR-155 mouse model through downregulation of HDAC4 and indirectly of BCL6 through acetylation. BCL6 mRNA is downregulated by MXD1/MAD1 transcription factor leading to de-repression of its target genes like ID2, CCND1, Mip1α/CCL3 and IL6, which may contribute to leukaemogenesis by various processes as mentioned. Further, since HDAC4 is an important co-repressor recruited by BCL6 to repress some of the target genes expression, we propose that BCL6 needs HDAC4 for its transcriptional repressor activity. Most of the BCL6 transcriptional signature that is upregulated in Eµ- miR-155 mice constitutes genes which have role in cell cycle regulation, proliferation and impaired B-cell differentiation. Legend: Pointed arrows indicate positive and blunt arrows indicate negative interactions, which are also indicated by the red/upward or green/downwards pointed arrows indicating expression level; kinks in the arrows indicate indirect regulation.

81 CHAPTER 4

Conclusions and Future Directions

miRNA regulation of gene expression have added an extra level of complexity to the already complex gene regulatory networks. Similar to mRNAs, miRNA expression varies across tissues and infact control target mRNA expression owing to 3’ UTR homology. miRNA de-regulation is hence very closely linked to mRNA de-regulation and eventually disease. But the challenge is to identify the mode of tissue-specific mRNA regulation in order to better understand the miRNA driven pathogenesis.

miRNA transgenic and knock-out mouse models provide a useful tool to study miR-based gene regulation and their role in normal and disease development. Eµ-miR-

155 transgenic mouse model has proved to be a beneficial source in understanding its role in B-cell diseases to some extent. It is the first miRNA transgenic mouse model where overexpression of a single miRNA is enough to cause B-cell leukemia/lymphoma. Some of the key targets of miR-155 through which it can contribute to leukemogenesis have been discovered using this model. SHIP1 is an important phosphatase expressed in hematopoietic cells where it functions to regulate signaling between lymphoid and myeloid cells. Downregulation of SHIP1 by miR-155 in B and myeloid cells have been shown to contribute to malignant proliferation. Considering the complexities of gene

82 regulation, it may be useful to determine direct and indirect mechanisms of miR- mediated gene regulation. For instance, although miR-155 directly regulates SHIP1, it can also mediate its effect indirectly through Nfκb activation. Eµ-miR-155 mice have activated Nfκb (unpublished observation SKS), which has been shown to suppress SHIP1 expression (Lu et al., 2008; O'Connell et al., 2009; O'Connell et al., 2008).

The finding that miR-155 targets HDAC4 may at first resonate with the idea of using it as a specific HDAC-inhibitor (HDIs) considering many HDIs are in use or clinical trials for anticancer properties. But our study shows a paradigm shift where this

HDAC may be useful for B-cells and its dysregulation may be contributing to their malignant transformation by miR-155. More interestingly we found that indirect downregulation of transcriptional repressor BCL6 in this model can be oncogenic.

However, there is need of additional functional studies to determine the exact role of some of the relevant targets like ID2 which when upregulated contribute to B-cell differentiation arrest.

Another key experiment can be to determine the direct role of HDAC4 in B-cell differentiation by complementation studies in Eµ-miR-155 mice. We have recently cloned and purified full-length mouse TAT-conjugated HDAC4 protein which can efficiently be transduced into target cells and even mice as shown before. This can be a helpful tool since primary mouse B-cells are not very amenable to efficient gene delivery by other means. Murine stem cell promoter based retroviral delivery is the only other

83 means, but rate of transduction is sub-optimal. Together with a system of in-vitro B-cell differentiation assay, this can help understand the role of HDAC4 in B-cell development.

Understanding and characterizing roles of individual HDACs in various tissues and cancers can help design specific HDI for the most beneficial results. Especially sparing inhibition of HDACs like HDAC4, which have many hallmarks of a tumor suppressor. As discussed earlier, it was identified as a mediator of p53 and p19ARF- dependent proliferation arrest and senescence and is required for repair of ionizing radiation-induced DNA damage (Berns et al., 2004). In addition, Hdac4 is frequently mutated in certain human cancers (Sjoblom et al., 2006) and downregulated in various cancers including, lung and colon carcinomas (ME et al., 2006). But since HDACs more often bear a stigma of being oncogenic, there has to be a compelling evidence to prove them otherwise.

So there are few interesting hypothesis which if proved will characterize precise role of HDAC4. Since HDAC4 is used to repress target genes by deacetylation, some of the upregulated genes in miR-155 overexpressing mice may be HDAC4 targets. Two of such putative candidates which may have escaped HDAC4 mediated repression are

RUNX2 and MEF2B which are 7- and 2-fold upregulated in these mice. RUNX2 or runt- related transcription factor-2 has mainy been shown to have role in osteogenic differentiation and skeletal morphogenesis. There is compelling evidence of its role in the hematopoietic development and malignancies (reviewed in Blyth et al., 2010). HDAC4 has been known to interact with RUNX2 to regulate chondrocyte growth and

84 differentiation (Vega et al., 2004) and HDAC4 mediated deacetylation have role in

VEGF promoter activity in chondrosarcoma (Sun et al., 2009).

HDAC4 interaction with MEF2 or myocyte enhancer factor-2 family of transcription factors is known to regulate target gene expression in cellular differentiation and proliferation of hematopoietic, nervous and musculoskeletal systems (Liu et al.,

2006). The only MEF2 upregulated in Eµ-miR-155 mice is MEF2B, and we found that its frequently upregulated in Burkitt’s lymphoma in human.

Therapeutic potentials for miRNAs in lymphomas

From the past and ongoing work on miRNAs in lymphomas, it is evident that deregulation of miRNA expression is one of the critical steps in pathogenesis of several lymphomas. Similar to mRNA, miRNA expression profiling can successfully classify various lymphomas into therapeutic outcome or survival categories. Identification of novel miRNA genes by new technologies like deep sequencing can further enhance the scope of miRNA functions and therapeutic applications (Jima et al., 2010). In order to better understand the role of miRNAs in various lymphomas, future work may involve studying the targets involved in lymphomas and understanding the associated mechanisms. In addition, generation of transgenic and knock out mouse models of lymphoma associated miRNAs will help understand mechanisms underlying pathogenesis of these malignancies. Development of miRNA-based therapeutics faces similar challenges such as small interfering RNA therapeutics, potential off-target effects, safety and mode of delivery (Garzon et al., 2010). Hence, mouse models of gain- and

85 loss-of-function of various miRNAs can provide useful for respective miR-inhibitor or mimic based therapies. These experiments can also lead to development of optimal formulation and delivery techniques.

Since miRNA expression is deregulated in several lymphomas and unique miRNA signatures have been identified for prognosis and response to therapy, they become quiet appealing as therapeutic targets. Common therapeutic strategies involve antisense mediated inhibition of oncogenic miRNAs and miRNA mimetics or viral-vector encoded overexpression of tumor suppressor miRNAs. Some of the strategies in the former category involve oligonucleotides with chemical modifications, liposomes, polymers, hydrogels and nanoparticles (Garzon et al., 2010). Synthetic anti-miRNA oligonucleotides (AMOs), which have 2-O-methyl modification provide an effective inhibition of miRNAs in cell culture and xenograft mouse models but work only at high doses (Trang et al., 2010). Targeting of miR-21 by 2- O-methyl AMOs in glioblastoma and breast cancer has been achieved in-vitro and xenograft mice model, respectively

(Chan et al., 2005; Si et al., 2007). An alternative to AMOs is locked nucleic acid (Trang et al.)-based anti-miRs, which have been shown to be more stable and less toxic in inhibiting endogenous miRNAs in-vivo (Elmen et al., 2008; Vester and Wengel, 2004).

Elmen et al., (Elmen et al., 2008) used LNA-antimiR for efficient silencing of miR-122 in primates and a Phase 1 study is currently underway in humans. Another strategy recently developed to inhibit multiple miRNAs is called miR-sponge. miRNA sponges are transcripts that contain multiple tandem binding sites to specific miRNAs (Ebert et al.,

2007). Sponge mRNA can be expressed from stably integrated transgenes in-vivo to

86 silence the target miRNAs and can perform comparable to antisense oligonucleotides. In case of miRs which are encoded in cluster, for example miR-17~92 family (and its paralogs, together encoding 15 miRNAs) it will be more efficient to generate a transgenic mouse with miR-cluster sponge to inhibit than deleting each miR individually

(Hammond, 2007).

On the other hand, restoration of tumor suppressor miRNAs can be achieved by synthetic miRNA mimics, which are usually double stranded and chemically modified

(2’-O-methyl with phosphorothioate modifications). Use of miR-15a and miR-29 mimics in prostate and AML cell lines, respectively, induced apoptosis (Bonci et al., 2008;

Garzon et al., 2010). Another method to increase expression of tumor suppressor miRNAs is adenovirus-associated vectors (AAV). AAV mediated miR-26 delivery into a murine model of liver cancer shows promise for miR replacement therapy (Kota et al.,

2009). AAV based vectors do not integrate into the genome and are eliminated efficiently with minimal toxicity as seen in Phase I and Phase II clinical trials of about 200 patients

(Aagaard and Rossi, 2007; Michelfelder and Trepel, 2009).

Other formulations like cationic lipids or liposomes, polymers and nanoparticles have recently become popular to increase the efficiency of oligonucleotide uptake

(Garzon et al., 2010). Liposomes consist of an aqueous compartment enclosed by a phospholipid bilayer, and form stabilized complexes on electrostatic interactions with oligonucleotides (Zhao et al., 2009a). In the studies so far liposomes have been shown to induce hypersensitive reactions owing to toxicity and hence research efforts to maximize

87 their benefits are underway (Zuhorn et al., 2007). Polymers and nanoparticles on the other hand are promising because they provide improved delivery and stability with minimal in-vivo toxicity (Chirila et al., 2002; Garzon et al., 2010)

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