Role of Microrna-29 in the Pathogenesis of B-Cell Chronic Lymphocytic Leukemia

Role of microRNA-29 in the Pathogenesis of B-Cell Chronic Lymphocytic Leukemia

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Urmila Santanam

Graduate Program in Molecular, Cellular and Developmental Biology

The Ohio State University

2010

Dissertation Committee:

Dr. Carlo M. Croce, Advisor

Dr. Kay Huebner

Dr. Denis Guttridge

Dr. Amanda Toland

Urmila Santanam

2010

Abstract

B-cell chronic lymphocytic leukemia (B-CLL), the most common human leukemia in the world, is a malignancy of mature B-lymphocytes. Deregulation of the

TCL1 oncogene is a causal event in the pathogenesis of the aggressive form of this disease as was verified by using animal models. To study the mechanism of TCL1 regulation in CLL, we carried out microRNA expression profiling of three types of CLL: indolent CLL, aggressive CLL, and aggressive CLL showing 11q deletion. We identified distinct microRNA signatures corresponding to each group of CLL. We further determined that TCL1 expression is regulated by miR-29 and miR-181, two microRNAs differentially expressed in CLL. Expression levels of miR-29 and miR-181 generally inversely correlated with TCL1 expression in the CLL samples we examined. Our results suggest that TCL1 expression in CLL is, at least in part, regulated by miR-29 and miR-

181 and that these microRNAs may be candidates for therapeutic agents in CLLs overexpressing TCL1.

Human B-CLL occurs in two forms: aggressive (showing high ZAP-70 expression and unmutated IgH VH) and indolent (showing low ZAP-70 expression and mutated IgH VH). We found that miR-29a is upregulated in indolent human B-CLL compared to aggressive B-CLL and normal CD19+ B-cells. To study the role of miR-

29 in B-CLL, we generated Eµ-miR-29 transgenic mice overexpressing miR-29 in mouse

B-cells. Flow cytometric analysis revealed a markedly expanded CD5+ population in the ii spleen of these mice starting at 2 months of age. 85% (34/40) of miR-29 transgenic mice exhibited an expanded population of CD5+ B-cells, a characteristic of the B-CLL phenotype. An average of 50% of the B-cell population in these transgenics was CD5 positive. At the age of 2 years these mice showed significantly enlarged spleens and an increase in CD5+ B-cell population of up to 100% of B-cells. Of 20 Eµ-miR-29 transgenic mice followed up to the age of 24-26 months, 4 (20%) developed frank leukemia and prematurely died from the disease. The expanded CD5+ B-cell population was found to be proliferative, with an increased number of cells in the S-phase of the cell cycle, compared to wild type CD19+ B-cells. These results suggest that deregulation of miR-29 can cause, or at least significantly contribute to the pathogenesis of indolent B-

CLL.

Although many microRNAs are up- or down-regulated in a number of solid tumors and hematological malignancies and several are postulated to function as tumor suppressors or oncogenes, there have been only two reports demonstrating that up- regulation of a single microRNA can cause malignancy. Here we demonstrate that up- regulation of miR-29 in human indolent CLL is an important initiating event in the pathogenesis of this disease. These results show that overexpression of miR-29 is sufficient to cause the development of indolent CLL with high penetrance and provide a new mouse model for indolent CLL.

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Dedication

This document is dedicated to my parents.

Acknowledgments

First, I would like to thank my advisor Dr Carlo Croce for having me as a student in his lab and for his continuous support and encouragement. I thank Dr. Yuri Pekarsky for guiding me through my work, and for his valuable counsel.

I would like to thank my committee members, Dr. Kay Huebner, Dr. Denis

Guttridge and Dr. Amanda Toland, for their time and guidance. I am grateful to my program director, Dr. David Bisaro, and the MCDB program co-ordinator Jan Zinaich. I also thank our lab administrative staff, Sharon Palko, Dorothee Wernicke-Jameson,

Susan Lutz, and John Nisbit for their help and patience.

I thank Alex Palamarchuk, Vadim Maximov and Alexey Efanov for helping me learn new techniques initially, and for their assistance. I thank Natalya Nazaryan and

Shruthi Sampath for helping me when I injured my foot. I also thank all my friends, and other members of the Croce lab for their help, and for making my years in the lab and in

Columbus pleasant and memorable. I‟d like to thank Vidhya Ramachandran for being a great friend throughout my stay in Columbus.

I would like to thank my aunt and uncle, Drs. Shobha and Manohar Ratnam, and my brother-in-law Sunil Iyengar, for helping me upon my arrival to Columbus, and for their constant advice and encouragement.

I thank my fiancé, Anand Ramanathan for preventing me from procrastinating the writing of my dissertation. I also thank him for dialing my number when he got to the

US, and for his continued love and support.

Finally, I would like to thank my parents, Poornima and K. Santanam, and sister,

Dr. Kavita Iyengar, who have made me the person I am today. I owe all my accomplishments to them.

Thank you.

Urmila Santanam

Vita

1997...... Sophia High School, Bangalore, India

1999...... Pre-University, Mount Carmel College,

Bangalore, India.

2002...... B.S. Microbiology, Chemistry and Zoology

St. Joseph‟s College, Bangalore, India.

2004 ...... Graduate Research Associate, MCDB

Program, The Ohio State University

Publications

“Chronic lymphocytic leukemia modeled in mouse by targeted miR-29 expression.” Urmila Santanam, Nicola Zanesi, John P Hagan, Stefano Volinia, Hansjuerg Alder, Laura Rassenti, Thomas Kipps, Carlo M Croce and Yuri Pekarsky; PNAS. 2010 June 21; epub ahead of print.

“Tcl1 expression in chronic lymphocytic leukemia is regulated by miR-29 and miR-181.” Yuri Pekarsky*, Urmila Santanam*, Amelia Cimmino, Alexey Palamarchuk, Alexey Efanov, Vadim Maximov, Stefano Volinia, Hansjuerg Alder, Chang-Gong Liu, Laura Rassenti, George Calin, John Hagan, Thomas Kipps and Carlo M. Croce; Cancer Res. 2006 Dec 15; 66(24):11590-3. (* Equal contribution)

“13q14 deletions in CLL involve cooperating tumor suppressors.” Alexey Palamarchuk, Alexey Efanov, Natalya Nazaryan, Urmila Santanam, Hansjuerg Alder, Laura Rassenti, Thomas Kipps, Carlo M. Croce and Yuri Pekarsky; Blood 2010 May 13; 115(19):3916- 22.

“Tcl1 functions as a transcriptional regulator and is directly involved in the pathogenesis of CLL.” Yuri Pekarsky, Alexey Palamarchuk, Vadim Maximov, Alexey Efanov, vii

Natalya Nazaryan, Urmila Santanam, Laura Rassenti, Thomas Kipps and Carlo M Croce; PNAS 2008 Dec 16; 105(50):19643-8.

“Tal1 transgenic expression reveals absence of B-Lymphocytes.” Alexey Palamarchuk, Nicola Zanesi, Rami Aqeilan, Alexey Efanov, Vadim Maximov, Urmila Santanam, John Hagan, Carlo M. Croce and Yuri Pekarsky; Cancer Res 2006 Jun 15; 66:6014-17.

Fields of Study

Major Field: Molecular, Cellular and Developmental Biology

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Table of Contents

Abstract ...... ii

Dedication ...... iv

Acknowledgments...... v

Vita ...... vii

List of Tables ...... xi

List of Figures ...... xii

Chapter 1: Introduction ...... 1

B-cell chronic lymphocytic leukemia ...... 1

Tcl1 oncogene in B-CLL ...... 12

microRNA ...... 14

Chapter 2: TCL1 expression in B-cell chronic lymphocytic leukemia is possibly regulated by miR-29 and miR-181 ...... 28

Intruduction ...... 29

Materials and Methods ...... 31

Results and Discussion ...... 33

Chapter 3: B-Cell Chronic Lymphocytic Leukemia modeled in mouse by targeted miR-29 expression ...... 47

Introduction ...... 48

Results ...... 50

Discussion ...... 59

Experimental Procedures ...... 62

Chapter 4: Synopsis and Conclusion ...... 93

References ...... 99

List of Tables

Table 1. CLL sample information...... 37

Table 2. CLL sample information...... 38

Table 3. CLL sample information...... 39

Table 4. Statistically significant microRNAs differentiating CLL subtypes...... 40

List of Figures

Figure 1. Production of blood cells from pluripotent stem cells in the bone marrow...... 3

Figure 2. B-cell Maturation...... 4

Figure 3. B-cell surface molecules...... 5

Figure 4. microRNA Biogenesis...... 6

Figure 5. miRs as Oncogenes and Tumor Suppressors...... 7

Figure 6. TCL1 expression in B-CLL...... 41

Figure 7. Sequence alignment of miR-29b and miR-181b with 3‟ UTR of TCL1...... 42

Figure 8. Real time RT-PCR analysis of representative CLL samples...... 43

Figure 9. miR-29 and miR181 target TCL1 expression in luciferase assays...... 44

Figure 10. Effect of miR-29b and miR-181b on TCL1 protein expression...... 45

Figure 11. Inverse correlation of Tcl1 protein expression with miR-181b and miR-29b expression in B-CLL samples by microarray...... 46

Figure 12. miR-29a and miR-29b expression in aggressive and indolent CLL...... 67

Figure 13. Eµ-miR-29 construct ...... 68

Figure 14. Expression of miR-29a and miR-29b in splenic lymphocytes of Eµ-miR-29 founders...... 69

Figure 15. Expression of GFP in splenic lymphocytes of Eµ-miR-29 founders...... 70

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Figure 16. Flow cytometric analysis of miR-29 transgenic and control lymphocytes isolated from spleen...... 71

Figure 17. Flow cytometric analysis of miR-29 transgenic and control lymphocytes isolated from peripheral blood...... 72

Figure 18. Flow cytometric analysis of miR-29 transgenic and control lymphocytes isolated from bone marrow ...... 73

Figure 19. Expanded population of CD5+ B-cells in spleens of transgenic mice relative to

WT spleens...... 74

Figure 20. Analysis of CD5+ B-cell populations in miR-29 transgenic mice...... 75

Figure 21. CD5+ B-cell population size expanded linearly with age of transgenic mice. 76

Figure 22. Leukemic transgenic mouse spleen...... 77

Figure 23. Analysis of IgH gene configuration by Southern blot ...... 78

Figure 24. Histopathological analysis of Eµ-miR-29 mice...... 79

Figure 25. Histopathological analysis of CLL invasion in liver and kidney of Eµ-miR-29 mice ...... 80

Figure 26. Cell cycle analysis of leukemic cells from Eμ-miR-29 transgenic mice...... 81

Figure 27. Serum immunoglobulin levels of WT and transgenic animals...... 82

Figure 28. Immune response in transgenic and WT mice ...... 83

Figure 29. Flow cytometric analysis of Eµ-TCL1/ Eµ-miR-29 and Eµ-TCL1 transgenic lymphocytes from spleen...... 84

Figure 30. Percentage of CD5+ B-cells in Eµ-TCL1/ Eµ-miR-29 and Eµ-TCL1 transgenic spleen lymphocytes...... 85

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Figure 31. Spleen weight from Eμ-TCL1/ Eμ-miR-29 and Eμ-TCL1 transgenic mice. ... 86

Figure 32. Western blot analysis of CDK6, DNMT3A, PTEN and MCL1 expression in

CD19+ B-cells of miR-29 transgenic and WT mice...... 87

Figure 33. Microaray expression data for PXDN, BCL7A and ITIH5 in CD19+ B-cells of miR-29 transgenic and WT mice...... 88

Figure 34. Sequence alignments of miR-29a and 3‟ UTRs of PXDN, BCL7A and ITIH5.

...... 89

Figure 35. miR-29 targets PXDN but not BCL7A and ITIH5 expression in luciferase reporter assays...... 90

Figure 36. Effect of miR-29 on PXDN protein expression...... 91

Figure 37. PXDN expression in CLL...... 92

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CHAPTER 1

Introduction

B-cell Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the

Western world. (Sgambati, Linet, & Devesa, 2001) It is also the prototype of B-cell chronic lymphoid malignancies. (F. Caligaris-Cappio & R. Dalla-Favera, 2005) CLL has become the focus of major interest for many reasons. First, both ethnic disparity and a strong familial tendency provide compelling evidence for a CLL genetic susceptibility.

Second, the CLL cell has numerous features that cause uncertainties about its cellular origin. CLL lymphocytes are frequently self-reactive and produce polyreactive natural autoantibodies. They present many genomic abnormalities, especially in the advanced phases of the disease. Still, a molecular or cytogenetic abnormality unique to CLL has yet to be identified, and the CLL-gene(s) has become a sort of Holy Grail in hematological oncology. CLL differs from other B-cell chronic lymphoid malignancies because reciprocal translocations that juxtapose immunoglobulin (Ig) loci to protooncogenes are exceedingly rare. Further, authentic CLL tumor cell lines have never been obtained unless infected in vitro or in vivo by Epstein-Barr virus (EBV). EBV induced CLL cell lines are also extremely rare, as CLL B-cells show a characteristic resistance to EBV-

1 induced immortalization. Finally, only very recently, animal models that reproduce or mimic human CLL have been obtained. (F. Caligaris-Cappio & R. Dalla-Favera, 2005)

Remarkable clinical properties likewise parallel these unusual biological features.

CLL has a considerable clinical heterogeneity. Some patients present with an aggressive disease and a poor prognosis, others have an indolent course and a virtually normal life expectancy. Irrespective of these clinical differences, the cells from all patients have a common phenotype [CD5+, surface (s)Iglow], which is quite unique within the broad spectrum of chronic B-cell malignancies. In addition, microarray analysis shows the cells to be surprisingly similar, confirming that CLL cells have the phenotypic profile of activated memory B-cells. CLL patients present a severe immunodeficiency with hypogammaglobulinaemia, which progresses with advancing disease. The disease course may be characterized also by the occurrence of autoimmune manifestations caused by polyclonal auto-antibodies restricted to blood cell self-antigens that cause autoimmune cytopenias. These features suggest a complex disturbance of immunoregulation brought about by the malignancy. (F. Caligaris-Cappio & R. Dalla-Favera, 2005)

All events that mark the natural history of CLL occur in tissues where the balance between proliferation and reduced apoptosis influences the clonal accumulation. CLL cell proliferation and extended survival are favored by the leukemic cell capacity to respond to the proliferative and anti-apoptotic microenvironmental signals provided by tissue bystander cells, such as T-cells and stromal cells, through cellular contacts and soluble factors. Focal clusters of proliferating prolymphocytes and paraimmunoblasts

(pseudofollicles) can be identified in the lymph nodes or the bone marrow. These focal

B-cell aggregates are infiltrated by T-cells most belonging to the CD4+ subset and represent the tissue proliferating reservoir of the downstream accumulation compartment that spills over and circulates in the peripheral blood. (F. Caligaris-Cappio & R. Dalla-

Favera, 2005)

The analysis of Ig variable heavy chain (IgVH) somatic mutations which allow us to track the history of individual malignant clones to an in vivo activation triggered by the stimulation of the B cell receptor (BCR) discriminates two distinct CLL subsets, one with somatically mutated (M) and one with unmutated (U) IgVH genes. The clinical importance of this biological observation is that the two subsets have a markedly different prognosis; U-CLL patients have a considerably shorter survival. This correlation links prognosis to biology and raises the question of whether and how the features of

IgVH genes and the properties of BCR may relate to the pathogenesis of the disease. The biased use of certain IgVH genes, the existence of similarities among Ig rearrangements and the expression of gene products typically associated with B-cell activation raise the possibility that an antigenic stimulation may be instrumental in the malignant cell growth.

M-CLLs are typically unresponsive to BCR stimulation in vitro similar to B cells that have undergone receptor desensitization following chronic stimulation by antigen. On the other hand, though expressing germline IgVH genes, U-CLL strongly respond in vitro to anti-IgM stimulation, suggesting that they carry a more competent BCR that remains able to receive signals for maintenance or proliferation. (F. Caligaris-Cappio & R. Dalla-

Favera, 2005)

B-lymphocyte Development and Function

B-lymphocytes (B-cells) are a population of cells, produced in the bone marrow

(Figure 1), that express clonally diverse cell surface immunoglobulin (Ig) receptors recognizing specific antigenic epitopes. Mammalian B-cell development encompasses a continuum of stages that begin in primary lymphoid tissue (eg, human fetal liver and fetal/adult marrow), with subsequent functional maturation in secondary lymphoid tissue

(eg, human lymph nodes and spleen), (Figure 2). The functional/protective end point is antibody production by terminally differentiated plasma cells. (LeBien & Tedder, 2008)

B-cell development in mice (Hardy, Kincade, & Dorshkind, 2007) and humans

(LeBien, 2000) has been extensively studied, and the functional rearrangement of the Ig loci is a sine qua non. (LeBien & Tedder, 2008) This occurs via an error-prone process involving the combinatorial rearrangement of the V, D, and J gene segments in the H chain locus and the V and J gene segments in the L chain loci. (Brack, Hirama, Lenhard-

Schuller, & Tonegawa, 1978) In mice and humans, this occurs primarily in fetal liver and adult marrow, culminating in the development of a diverse repertoire of functional VDJH and VJL rearrangements encoding the B-cell receptor (BCR). (LeBien & Tedder, 2008)

Following their identification using mAbs, the structures of B-cell restricted target molecules have been determined and gene-targeted mice lacking expression of these CDs

(clusters of differentiation) have been generated. Most of these target molecules regulate

B-cell development and function, facilitate communication with the extracellular environment, or provide a cellular context in which to interpret BCR signals. CD19 is expressed by essentially all B-lineage cells and regulates intracellular signal transduction

4 by amplifying Src-family kinase activity (Figure 3). Among other surface molecules are

B220 and IgM. B220 is expressed in all B-lineage cells, and IgM is expressed in all mature B-cells. (LeBien & Tedder, 2008)

In addition to their essential role in humoral immunity, B-cells also mediate or regulate many other functions essential for immune homeostasis. B-cells are required for the initiation of T-cell immune responses (Ron, De Baetselier, Gordon, Feldman, &

Segal, 1981), normal immune system development and maintenance, regulation of lymphoid tissue organization and neogenesis, regulation of wound healing and transplanted tissue rejection, and influence tumor development and tumor immunity. B- cells can also function as polarized cytokine-producing effector cells that influence T-cell differentiation. (Harris et al., 2000)

Abnormalities in B-cell development and regulation may lead to immunodeficiencies and autoimmunity respectively. The normal B-cell developmental stages have malignant counterparts that reflect the expansion of a dominant subclone leading to development of leukemia and lymphoma. (LeBien & Tedder, 2008)

CD5+ B-cells

B-cells that bear the pan-T cell glycoprotein CD5 (originally known as "Ly-1")

(Figure 3) show a variety of novel features that distinguish them from the bulk of

IgD++CD5- "conventional" B cells (Hardy & Hayakawa, 1994; Hardy, Carmack, Li, &

Hayakawa, 1994; Hayakawa & Hardy, 1988; Kantor & Herzenberg, 1993; Kipps, 1989).

Among these are distinctive surface phenotype (IgM++ /IgDlow /B220low), novel

5 anatomical localization (enrichment in the peritoneal cavity), early appearance in ontogeny, secretion of certain autoreactive antibodies and increased frequency in several autoimmune mouse strains. (Hardy et al., 1994) Further work has demonstrated predisposition to unregulated growth or even lymphoma. The demonstration of a homologous population in human ("CD5+ B-cells") with many similar features, including early appearance and novel biases in specificity together with the long-recognized presence of CD5 on B-lineage chronic lymphocytic leukemia cells (Boumsell et al.,

1978) has stimulated interest in defining more precisely the relationship of this B-cell subpopulation to the majority of conventional B-cells. CD5+ B-cells as the remnant of a distinct fetal B-cell differentiation pathway suggests that the selection of cells from this fetal/neonatal population into the adult long-lived pool results in the over-expression of certain germline-encoded autoreactivities, such as antibody to bromelain-treated mouse red blood cells and intact thymocytes. (Hardy et al., 1994) (Bannerji & Byrd, 2000;

Hisada, Biggar, Greene, Fraumeni, & Travis, 2001)

B-CLL Pathogenesis

Chronic lymphocytic leukemia (CLL) is a malignancy of mature B-lymphocytes of unknown etiology. (Bannerji & Byrd, 2000; Hisada et al., 2001) B-CLL has been characterized by the monoclonal expansion of mature, resting B-lymphocytes that are present in the peripheral blood, bone marrow, and lymphoid organs, and by an indolent and chronic disease course that ultimately becomes lethal (Caligaris-Cappio & Hamblin,

1999). It predominantly originates through malignant transformation of marginal zone B

6 cells. The observation that B-CLL is more related to memory or marginal zone B-cells than to any other known normal B-cell subset suggests that the process leading to the clonal expansion may initiate in these cells. (U. Klein & R. Dalla-Favera, 2005)

B-CLL is characterized by only a few common chromosome abnormalities including an association with 13q14 deletions that is present in around 50% of cases, depending on the panel studied (Corcoran et al., 1998; Dohner, Stilgenbauer, Dohner,

Bentz, & Lichter, 1999; Mabuchi et al., 2001; Migliazza et al., 2001). These deletions are thought to reflect the inactivation of an as-yet-unknown tumor-suppressor gene.

Generally, the homogeneous gene expression profile of B-CLL suggests that its pathogenesis is associated with a largely common mechanism of transformation. (U.

Klein & R. Dalla-Favera, 2005)

Recombination of the three genetic components of the immunoglobulin heavy chain (H) to create the VH-DH-JH transcriptional unit is a unique feature of B cells.

Subsequent events of VL-JL recombination (immunoglobulin light chain), somatic mutation, isotype switching, and antigen selection all leave evidence in the V-gene sequences of the stage of differentiation reached by the B-cell of origin. This clonal history is maintained in the neoplastic B-cell and provides insight into B-cell differentiation and tumor development. (Levy et al., 1987; Ottensmeier et al., 1998;

Schroeder & Dighiero, 1994). VH gene analysis has shown that chronic lymphocytic leukemia (CLL) may be heterogeneous, with one subset derived from naive B-cells with unmutated sequences, and another from cells that have acquired somatic mutations.

(Oscier, Thompsett, Zhu, & Stevenson, 1997) After somatic mutation, a normal B-cell

7 either undergoes selection by antigen or dies by apoptosis. (Liu et al., 1989) It remains possible that antigen may play a role in driving growth of neoplastic B-cells, but tumor cells have various devices to escape apoptotic death. (Bahler et al., 1991)

A number of recent large-scale IgV gene repertoire analyses strongly imply the role for antigen in the development of B-CLL. Not only are particular IgVH gene segments over-represented in antibodies expressed by B-CLLs (Chiorazzi & Ferrarini,

2003), but there is also strong evidence for selection of particular IgVH and light chain combinations (Ghiotto et al., 2004; Kolar & Capra, 2004), and subgroups of B-CLL show similar CDRIII regions (complementarity determining regions) of light and/or heavy chains (Widhopf et al., 2004). These observations may suggest that the binding of specific antigen receptors to particular foreign antigens or autoantigens provides a continuous stimulatory signal through the BCR that keeps the B-CLL precursor in cell cycle for long periods of time and eventually allows for the acquisition of genomic aberrations. (U. Klein & R. Dalla-Favera, 2005)

Aggressive and Indolent CLL

B-CLL may occur in one of two forms, namely Aggressive and Indolent.

Aggressive CLL generally describes intermediate, high grade and fast growing lymphomas. Sometimes, perhaps rarely, types of lymphomas expected to be aggressive can progress slowly and behave indolently. Indolent CLL generally describes low grade, slow growing lymphomas. Indolent lymphomas can progress steadily and behave aggressively. Treatment is often deferred until the patient becomes symptomatic. The

8 goal of treatment is often management as indolent lymphomas are rarely cured, unless it is diagnosed when still localized. Treatment options are varied and there is no standard treatment. (2008)

Aggressive B-CLL is characterized by a high ZAP-70 positivity and an unmutated

VH status, whereas Indolent B-CLL is characterized by a low ZAP-70 positivity and a mutated VH status. (Herling et al., 2006)

Immunodeficiency in CLL

Patients with chronic lymphocytic leukaemia (CLL) are all to a degree immunodeficient. (A. D. Hamblin & Hamblin, 2008) The most obvious and well-known abnormality is hypogammaglobulinaemia, which is present in up to 85% of patients. (T.

J. Hamblin, 1987) Serum immunoglobulin levels may be suppressed in other lymphoid malignancies, but in CLL the suppression is far greater (A. D. Hamblin & Hamblin,

2008).

Infections are the major cause of death in between a quarter and a half of patients with CLL. (T. J. Hamblin, 1987) Bacterial infection of the respiratory tract, skin or urinary tract is the commonest problem, and before the use of purine analogues for treatment, the usual organisms were Streptococcus pneumoniae, Staphylococcus aureus,

Streptococcus pyogenes and Escherichia coli. Protection against these organisms is provided principally by antibody, but only 15% of patients with CLL have completely normal serum immunoglobulins. (T. J. Hamblin, 1987)

The extent of hypogammaglobulinaemia depends on the stage and duration of the disease. It occurs in patients with mutated and unmutated immunoglobulin heavy chain

(IgHV) genes. Although older papers described serum IgA as the first immunoglobulin to be reduced followed by IgM and IgG, (T. J. Hamblin, 1987) this is by no means invariable and most patients have or will have depression of all classes of immunoglobulin. It should be stressed that the hypogammaglobulinaemia is not confined to patients who have been treated. In one study of an untreated patient with stage B disease without a detectable paraprotein, the hypogammaglobulinaemia was so profound that over 90% of the detectable immunoglobulin in the serum was idiotypic, and thus derived from the tumour. (Stevenson, Hamblin, Stevenson, & Tutt, 1980)

The proportion and overall number of non-malignant B-cells are often significantly reduced, and clearly their function is impaired as the normal immunoglobulins are suppressed. What is not clear is whether they are directly suppressed by the tumour or indirectly as a consequence of inhibitory effects elsewhere in the immune response. Although this is most obviously manifested as hypogammaglobulinaemia, the interpolation of tumour cells within all secondary immunological organs interferes with almost all aspects of immune function. Exactly how this is mediated is still unclear, but it seems to be multifactorial and involves both cell to cell contact and the secretion of cytokines. This interaction is bi-directional, and while it inhibits immunity, it sustains and invigorates the tumour. (A. D. Hamblin &

Hamblin, 2008)

There is no obvious remedy for the immunodeficiency. Treatment of the CLL usually makes the immunodeficiency worse. IvIg has some benefit but is very expensive.

Prophylactic antibiotics have specific roles. Vaccination against infections is generally ineffective. (A. D. Hamblin & Hamblin, 2008)

Tumors after CLL

Prior studies have reported that site-specific excesses of second cancer may exist among patients with CLL. (Greene, Hoover, & Fraumeni, 1978; Hisada et al., 2001;

Travis, Curtis, Hankey, & Fraumeni, 1992) Data indicates that the overall risk of developing a second cancer is modestly but significantly elevated, independent of initial treatment, in persons with CLL compared with those in the general population. Although the role of shared etiologic factors remains unclear, the pattern of excess cancers in CLL survivors suggests an influence of immunodeficiency associated with CLL. (Hisada et al.,

2001; Linet MS, 1996)

Tcl1 oncogene in B-CLL

The TCL1 oncogene was discovered as a target of frequent chromosomal rearrangements at 14q31.2 in mature T-cell leukemias (Virgilio et al., 1994). Previously, we reported that transgenic mice expressing TCL1 in B-cells develop B-CLL (Bichi et al.,

2002). These results suggested that deregulation of TCL1 may be a causal event in the pathogenesis of B-CLL. We and others also have shown that Tcl1 is a co-activator of the

Akt oncoprotein, a critical molecule in the transduction of anti-apoptotic signals in B- and

T-cells (Laine, Kunstle, Obata, Sha, & Noguchi, 2000; Pekarsky et al., 2000). A recent report suggested that high Tcl1 expression in human B-CLL correlates with unmutated

VH status and ZAP70 positivity suggesting that Tcl1-driven B-CLL is an aggressive form of B-CLL (Herling et al., 2006). One of the most significant genetic factors associated with poor prognosis in human B-CLL is the chromosome 11q deletion (Dohner et al.,

2000). Interestingly, B-CLL samples showing 11q deletion also display higher Tcl1 expression levels (Herling et al., 2006).

Tcl1 mouse model of B-CLL

Since expression of Tcl1 is detected at various levels in almost all stages of B-cell development and is activated in AIDS IBLP, a B cell malignancy, it is possible that Tcl1 may also play a role in other B-cell leukemias and lymphomas (Pekarsky Y., Calin G. A.,

Aqeilan R., & Croce C. M., 2005). To investigate this possibility, we and others recently created transgenic mouse models showing deregulated expression of Tcl1 in B-cells

(Bichi et al., 2002; Hoyer et al., 2002). We generated a transgenic mouse line expressing the human TCL1 gene under the control of a VH promoter IgH-Eμ enhancer to target transgene expression to immature and mature B-cells (Bichi et al., 2002; Pekarsky Y. et al., 2005)(Bichi et al., 2002). At the age of 10–15 months these transgenic animals became visibly sick. Pathological analysis revealed enlarged liver and spleen and high white blood cell counts, on average 180×106 cells/ml compared with 2.8×106 cells/ml in wild-type animals (Bichi et al., 2002). The predominant leukemic cell type was represented in large lymph nodes and enlarged spleen. Histological analysis also revealed infiltration in lymph nodes, liver, and spleen by malignant lymphocytes; these cells as expected were human TCL1 positive (Bichi et al., 2002). Flow cytometric analysis showed that these leukemic cells were CD5 and IgM positive, suggesting that these mice developed mature B-cell leukemia, a disease very similar to human B-CLL (Bichi et al.,

2002). A significantly enhanced CD5+ cell population in the peritoneal cavity of these transgenic animals was observed by the age of 2 months, in spleen by the age of 3 to 5 months, and in bone marrow by the age of 5 to 8 months. Southern analysis of immunoglobulin gene rearrangements revealed the presence of pre-leukemic and leukemic clones, confirming the resemblance to human B-CLL (Bichi et al., 2002;

Pekarsky Y. et al., 2005).

microRNA

MicroRNAs (miRNAs) are a class of non-coding RNAs whose final product is a

~22 nucleotide functional RNA molecule (Griffiths-Jones, Grocock, van Dongen,

Bateman, & Enright, 2006). They are as abundant as 50,000 copies per cell (Kim, 2005a;

Lim et al., 2003) and play important roles in the regulation of target genes by binding to complementary regions of messenger transcripts to repress their translation or regulate degradation (Bartel, 2004; Filipowicz, Jaskiewicz, Kolb, & Pillai, 2005; Sontheimer &

Carthew, 2005). miRNAs have been implicated in cellular roles as diverse as developmental timing in worms, cell death and fat metabolism in flies, hematopoiesis in mammals, and leaf development and floral patterning in plants (Ambros, 2004; Kidner &

Martienssen, 2005). Recent reports have suggested that miRNAs control pathways including apoptosis, cell proliferation and tumorigenesis and may play roles in human cancers (Griffiths-Jones et al., 2006; He et al., 2005; Lu et al., 2005; O'Donnell, Wentzel,

Zeller, Dang, & Mendell, 2005).

microRNA Biogenesis

microRNA Biogenesis is depicted in Figure 4, and is described below.

Early annotation of the genomic position of miRNAs indicated that most miRNAs are located in intergenic regions (>1 kb away from annotated/predictedgenes), although a sizeable minority was found in the intronic regions of known genes in the sense or antisense orientation (Kim, 2005a; Lagos-Quintana, Rauhut, Lendeckel, & Tuschl, 2001;

Lau, Lim, Weinstein, & Bartel, 2001). The expression of miRNA and protein-coding

14 genes might be coordinated, especially when a miRNA and a protein-coding region both reside in a single transcript. About 50% of known miRNAs are found in close proximity to other miRNAs (Kim, 2005a; Lagos-Quintana et al., 2001; Lau et al., 2001; Mourelatos et al., 2002). A detailed analysis of miRNA gene expression showed that miRNA genes can be transcribed from their own promoters (Cai, Hagedorn, & Cullen, 2004; Y. Lee et al., 2004), and that the clustered miRNAs are generated as polycistronic primary transcripts (pri-miRNAs) (Y. Lee, Jeon, Lee, Kim, & Kim, 2002). miRNA genes can be grouped on the basis of their genomic locations: first, exonic miRNA in non-coding transcription units; second, intronic miRNA in non-coding transcription units; and third, intronic miRNA in protein-coding transcription units. Mixed miRNA genes can be assigned to one of the above groups depending on the given splicing pattern (Kim,

2005a).

Transcription of miRNA genes is mediated by RNA polymerase II (pol II) (Cai et al., 2004; Y. Lee et al., 2004) although the possibility that a small number of miRNA genes might be transcribed by other RNA polymerases cannot be excluded (Kim, 2005a).

Transcription of miRNA genes yields primary transcripts, pri-miRNAs, that are usually several kilobases (~70) long and that contain a local hairpin structure. The stem-loop structure is cleaved by the nuclear RNase III Drosha (a large protein of ~160 kDa, conserved in animals (Filippov, Solovyev, Filippova, & Gill, 2000; Fortin, Nicholson, &

Nicholson, 2002; Wu, Xu, Miraglia, & Crooke, 2000) to release the precursor of miRNA

(pre-miRNA) (Y. Lee et al., 2003). The tertiary structure of pri-miRNAs is the primary determinant for Drosha substrate specificity (Y. Lee et al., 2003; Zeng & Cullen, 2003;

Zeng, Yi, & Cullen, 2005). Interestingly, it seems that the Drosha complex can measure the length of the stem, because the cleavage site is located approximately two helical turns (~22 nucleotides) from the terminal loop (Zeng et al., 2005). The remnants (the flanking fragments) are thought to be degraded in the nucleus, although it remains to be seen whether the 5′ and 3′ fragments that surround the stem-loop have their own functions (Y. Lee et al., 2004).

Pre-miRNA stem-loops typically comprise a stem of ~22 bp, a terminal loop and a 3′ overhang of about 2 nucleotides (Basyuk, Suavet, Doglio, Bordonne, & Bertrand,

2003; Y. Lee et al., 2003; Lund, Guttinger, Calado, Dahlberg, & Kutay, 2004; Zeng &

Cullen, 2004). Following nuclear processing by Drosha, pre-miRNAs are exported to the cytoplasm. Export of pre-miRNA is mediated by one of the nuclear transport receptors, exportin-5 (Bohnsack, Czaplinski, & Gorlich, 2004; Lund et al., 2004; Yi, Qin, Macara,

& Cullen, 2003).

Following their export from the nucleus, pre-miRNAs are subsequently processed into ~22-nucleotide miRNA duplexes by the cytoplasmic RNase III Dicer (Bernstein,

Caudy, Hammond, & Hannon, 2001; Grishok et al., 2001; Hutvagner et al., 2001; Ketting et al., 2001; Knight & Bass, 2001). The cleavage products (~22-nucleotide miRNA duplexes) do not persist in the cell for long. Usually, one strand of this short-lived duplex disappears, whereas the other strand remains as a mature miRNA (Kim, 2005a).

Mature miRNAs are incorporated into effector complexes that are known as

„miRNP‟ (miRNA-containing ribonucleoprotein complex), „mirgonaute‟ or „miRISC‟

(miRNA-containing RNA-induced silencing complex). Studies on siRNA duplexes

16 indicate that the relative thermodynamic stability of the two ends of the duplex determines which strand is to be selected (Khvorova, Reynolds, & Jayasena, 2003;

Schwarz et al., 2003). The strand with relatively unstable base pairs at the 5′ end typically remains (for example, G:U pair versus G:C pair) (Khvorova et al., 2003; Schwarz et al.,

2003). The same rule is thought to be applicable to miRNA (Kim, 2005a).

Functions of microRNAs

Most animal miRNAs are imprecisely complementary to their mRNA targets

(Ambros, 2004). As regulators of gene expression, miRNAs can work by essentially two modes (Doench, Petersen, & Sharp, 2003; Hutvagner & Zamore, 2002; Rhoades et al.,

2002; Tang, Reinhart, Bartel, & Zamore, 2003; Zeng, Wagner, & Cullen, 2002; Zeng, Yi,

& Cullen, 2003). MiRNAs base pair with messenger RNA targets and direct cleavage and destruction of the target mRNA (Rhoades et al., 2002; Tang et al., 2003). They also inhibit protein synthesis through an unknown mechanism that preserves the stability of the mRNA target; some studies even suggest that the translationally repressed target mRNAs remain associated with ribosomes (Ambros, 2004; Olsen & Ambros, 1999;

Seggerson, Tang, & Moss, 2002).

Out of hundreds of miRNAs, only a handful of miRNAs are known for their biological functions. The paradigm for the function of miRNAs has been originally provided by lin-4 and let-7, which were identified by genetic analysis of C. elegans developmental timing (Kim, 2005b; R. C. Lee, Feinbaum, & Ambros, 1993; Reinhart et al., 2000). They were initially called small temporal RNAs (stRNAs) because of their

17 temporal expression pattern and their roles in temporal regulation. Lin-4 and let-7 act as post-transcriptional repressors of their target genes when bound to their specific sites in the 3′ untranslated region of the target mRNA (R. C. Lee et al., 1993; Moss, Lee, &

Ambros, 1997; Olsen & Ambros, 1999; Slack et al., 2000; Wightman, Ha, & Ruvkun,

1993). The level of target mRNA does not change, suggesting that the inhibition occurs at the level of translation. Other animal miRNAs act similarly in various pathways

(Ambros, 2004; Bartel, 2004). Another nematode miRNA, lsy-6 RNA, was identified in a gene screening process for left/right asymmetry of neuronal chemoreceptor expression

(Chang, Johnston, Frokjaer-Jensen, Lockery, & Hobert, 2004). Lsy-6 RNA targets the cog-1 transcription factor (Chang et al., 2004). The bantem RNA from Drosophila suppresses apoptosis and stimulates cell proliferation by inhibiting translation of hid mRNA during development (Brennecke, Hipfner, Stark, Russell, & Cohen, 2003). In mammals, miR-181 is involved in the control of hematopoiesis through as yet unknown target(s) (Chen, Li, Lodish, & Bartel, 2004). Mouse miR-196 miRNAs represses the expression of the HOXB8 gene that is a transcription factor important in developmental regulation (Yekta, Shih, & Bartel, 2004). MiR-196 RNAs are the first examples of animal miRNAs that cause target mRNA cleavage rather than translational repression (Kim,

2005b; Yekta et al., 2004).

Hundreds of miRNA genes have been found in diverse animals, and many of these are phylogenetically conserved. With miRNA roles identified in developmental timing, cell death, cell proliferation, hematopoiesis and patterning of the nervous system, evidence is mounting that animal miRNAs are more numerous, and their regulatory

18 impact more pervasive, than was previously suspected. A single miRNA species can bind to many different mRNA targets and, conversely, several different miRNAs can cooperatively control a single mRNA target (Ambros, 2004). Thus, miRNAs and their targets seem to constitute remarkably complex regulatory networks. (Kim, 2005b)

microRNAs and Cancer

MicroRNAs control a wide array of biological processes, including differentiation, proliferation, and apoptosis. As the deregulation of these very same processes is a hallmark of cancer, there has been speculation that mutations affecting miRNAs or their functional interactions with oncogenes and tumor suppressor genes might also contribute to tumorigenesis. (Ventura & Jacks, 2009)

Overexpression, amplification, or loss of epigenetic silencing of a gene encoding a miRNA that targets one or more tumor suppressor genes could inhibit the activity of an anti-oncogenic pathway. By contrast, the physical deletion or epigenetic silencing of a miRNA that normally represses expression of one or more oncogenes might lead to increased protein expression and a gain of oncogenic potency (Figure 5). More subtle mutations affecting the sequence of the mature miRNA could reduce or eliminate binding to key targets or even drastically change its specificity, thereby altering the balance of critical growth regulatory proteins. Seed-match sequences of target mRNAs could also be the sites of mutation, rendering them free from the repression of a given miRNA or subject to the effects of another. (Ventura & Jacks, 2009)

Evidence strongly supports an important role of these tiny RNAs in controlling cell transformation and tumor progression. Beginning with the work of Carlo Croce and 19 colleagues in 2002, who showed that a pair of neighboring miRNAs, miR-15 and miR-16, are frequently deleted in human chronic lymphocytic leukemia (CLL), there are now additional examples in which miRNA genes are either lost or amplified in tumors (Calin

& Croce, 2006).

microRNAs as Oncogenes

miR-155 is upregulated in several hematopoietic malignancies and tumors of the breast, lung, and pancreas (Kluiver, Kroesen, Poppema, & van den Berg, 2006). Ectopic expression of miR-155 in the bone marrow of mice has been reported to induce either polyclonal pre-B cell proliferation followed by full-blown B-cell leukemia (Costinean et al., 2006) or myeloproliferation (O'Connell et al., 2008), depending on the system used to drive expression of the transgene.

Another notable member of the family of oncogenic miRNAs is the miR-17~92 cluster (Mendell, 2008). This cluster, which consists of six miRNAs that are processed from a single primary transcript, was initially linked to cancer based on the observation that it maps to a chromosomal region that is frequently amplified in a subset of human B- cell lymphomas (Ota et al., 2004) and overexpressed in a variety of other human cancers such as lung cancers (Hayashita et al., 2005). Although deregulation of miR-17~92 does not appear to be sufficient to initiate tumorigenesis per se, transgenic mice overexpressing this cluster in lymphocyte progenitor cells develop a lymphoproliferative disorder affecting both B and T cells that eventually results in autoimmunity (Xiao et al.,

2008).

microRNAs as Tumor Suppressors

Several miRNAs have been implicated as tumor suppressors based on their physical deletion or reduced expression in human cancer (Ventura & Jacks, 2009). There is strong circumstantial evidence that miR-15a~miR-16-1 is a bona fide tumor suppressor. miR-15a~16-1 is located in the minimally deleted region in CLL (Calin et al., 2002), and a germline point mutation (a single base change) immediately downstream of the pre- miR-16-1 sequence has been observed in a few CLL patients (Calin et al., 2005).

Inhibition of miR-15a and miR-16 activity leads to hyperplasia of the prostate in mice and promotes survival, proliferation, and invasion of primary prostate cells in vitro (Bonci et al., 2008).

Consistent with a role in inhibiting tumor development in humans, reduced expression of multiple members of the let-7 family is frequently observed in lung cancers, where they correlate with poor prognosis (Yanaihara et al., 2006). In addition, various let-7 genes are located at chromosomal sites deleted in a variety of human cancers. Overexpression of let-7 miRNAs can suppress tumor development in mouse models of breast and lung cancer (Kumar et al., 2008).

miR-34 family as an important mediator of p53 activity, a tumor suppressor gene that is frequently inactivated in human cancers (He, He, Lowe, & Hannon, 2007).

Reduced expression of miR-34b/miR-34c has been reported in breast and non-small cell lung cancer cell lines. (Ventura & Jacks, 2009)

Despite remarkable recent progress, the field of miRNAs and cancer is still in its infancy and many important questions remain to be addressed. Sophisticated in vivo models will likely help determine the oncogenic and tumor suppressor potential of individual miRNAs and miRNA families. (Ventura & Jacks, 2009)

Figure 1. Production of blood cells from pluripotent stem cells in the bone marrow.

(Goldman A. S. & Prabhakar B. S., 1996)

Figure 2. B-cell Maturation. B-cells are produced in the bone marrow and mature in peripheral lymphoid tissue (spleen). CD-19 is a B-cell surface marker present on immature and mature B-cells. (Dalakas, 2008)

Figure 3. B-cell surface molecules. The B-cell has a number of surface molecules like

CD-19 and B-220. CD5 is typically expressed on the surface of malignant B-cells, and is indicative of B-cell Leukemia. ()

Figure 4. microRNA Biogenesis. A miRNA precursor (pri-miRNA) is transcribed in the nucleus, forms a stem−loop structure, is processed to form another precursor (pre- miRNA), and is exported to the cytoplasm. Further processing by Dicer protein creates the mature miRNA, one strand of which is incorporated into the RNA-induced silencing complex (RISC). Base pairing between the miRNA and its target directs RISC to either destroy the mRNA or impede its translation into protein. (Meltzer, 2005)

Figure 5. miRs as Oncogenes and Tumor Suppressors. Over-expression of an oncogenic miR may result in under-expression of a target tumor suppressor gene. Under- expression of a tumor suppressor miR may result in over-expression of a target oncogene.

This could result in cancer.

CHAPTER 2

TCL1 expression in B-cell chronic lymphocytic leukemia is possibly regulated by miR-29 and miR-181

B-cell chronic lymphocytic leukemia (B-CLL) is the most common human leukemia in the world. Deregulation of the TCL1 oncogene is a causal event in the pathogenesis of the aggressive form of this disease as was verified by using animal models. To study the mechanism of TCL1 regulation in CLL, we carried out microRNA expression profiling of three types of CLL: indolent CLL, aggressive CLL, and aggressive CLL showing 11q deletion. We identified distinct microRNA signatures corresponding to each group of CLL. We further determined that TCL1 expression is regulated by miR-29 and miR-181, two microRNAs differentially expressed in CLL.

Expression levels of miR-29 and miR-181 generally inversely correlated with TCL1 expression in the CLL samples we examined. Our results suggest that TCL1 expression in

CLL is, at least in part, regulated by miR-29 and miR-181 and that these microRNAs may be candidates for therapeutic agents in CLLs overexpressing TCL1.

Introduction

The TCL1 (T-cell leukemia/lymphoma 1) oncogene was discovered as a target of frequent chromosomal rearrangements at 14q31.2 in mature T-cell leukemias (Virgilio et al., 1994). It was previously reported in our lab that transgenic mice expressing TCL1 in

B-cells develop B-CLL (Bichi et al., 2002). These results suggested that deregulation of

TCL1 may be a causal event in the pathogenesis of B-CLL. Our lab and others also have shown that TCL1 is a co-activator of the Akt oncoprotein, a critical molecule in the transduction of anti-apoptotic signals in B and T cells (Laine et al., 2000; Pekarsky et al.,

2000). A report suggested that high TCL1 expression in human B-CLL correlates with unmutated VH status and ZAP-70 positivity, suggesting that TCL1-driven B-CLL is an aggressive form of B-CLL (Herling et al., 2006). Another study showed that the TCL1 transgenic model replicates the immunoglobulin V region rearrangements characteristic of the aggressive, treatment-resistant form of human B-CLL (Yan et al., 2006). One of the most significant genetic factors associated with poor prognosis in human B-CLL is the chromosome 11q deletion (Dohner et al., 2000). Interestingly, B-CLL samples showing 11q deletion also display higher TCL1 expression levels (Herling et al., 2006).

MicroRNAs are a large family of highly conserved noncoding genes thought to be involved in temporal and tissue specific gene regulation (Ambros, 2004). Our lab showed that microRNA expression profiles can be used to distinguish normal B cells from malignant B-CLL cells and that microRNA signatures are associated with prognosis and progression of chronic lymphocytic leukemia (Calin et al., 2004; Calin et al., 2005). To

29 determine whether TCL1 expression is regulated by microRNAs in B-CLL, we studied microRNA expression patterns and TCL1 protein expression in 80 B-CLL samples of three types of B-CLL: indolent B-CLL, aggressive B-CLL with normal chromosome 11, and aggressive B-CLL showing 11q deletion. We analyzed these three types of B-CLL because a recent study suggested a differential expression of TCL1 in these three groups

(Herling et al., 2006).

Materials and Methods

CLL samples and microRNA microchip experiments.

Eighty B-CLL samples were obtained with informed consent from patients diagnosed with B-CLL from CLL Research Consortium institutions. Research was done with the approval of the Institutional Review Board of The Ohio State University.

Briefly, blood was obtained from CLL patients, and then lymphocytes were isolated through Ficoll/Hypaque gradient centrifugation (Amersham, Piscataway, NJ) and processed for RNA extraction using the standard Trizol method. Protein extraction was carried out as previously described (Palamarchuk et al., 2005). MicroRNA microchip experiments were done as previously described (Calin et al., 2005). Each microRNA microchip contained duplicate probes, corresponding to 326 human and 249 mouse microRNA genes. Statistical analysis was carried out as previously described (Volinia et al., 2006). To identify statistically significant differentially expressed microRNA, class prediction analyses were done using BRB ArrayTools developed by Dr. Richard Simm and Amy Peng Lam.

DNA constructs, transfection, Western blotting, and luciferase assay.

Full-length TCL1 cDNA including 5‟ and 3‟ untranslated region (UTR) was cloned into a pUSEamp vector (Upstate Biotechnology, Chicago, IL; used in Fig. 2B).

MiR-29b and miR-181b RNA duplexes were purchased from Ambion (Austin, TX). For miR-29 luciferase assays, a fragment of the 3‟ UTR of TCL1 cDNA, including a region

31 complimentary to miR-29, was inserted using the XbaI site immediately downstream from the stop codon of luciferase into pGL3 vector (Promega, Madison, WI). For miR-

181 assays, full-length TCL1 cDNA was inserted into pGL3 vector in sense (Tcl1FL) or antisense (Tcl1FLAS) orientation. Transfections were carried out as previously described

(Cimmino et al., 2005). Firefly and renilla luciferase activities were assayed with the dual luciferase assay system (Promega) and firefly luciferase activity was normalized to renilla luciferase activity. Cell lysate preparations and Western blot analyses were carried out using anti-TCL1 monoclonal antibody (clone 27D6) as previously described

(Pekarsky et al., 2000). Each Western filter contained reference sample. TCL1 protein expression was assessed using this sample as a reference. P values were two tailed and calculated by Fisher‟s exact test.

Results and Discussion

High expression of TCL1 correlates with aggressive B-CLL phenotype.

To evaluate TCL1 and microRNA expression in B-CLL samples, we chose three groups of B-CLL: 23 samples of indolent B-CLL, 25 samples of aggressive B-CLL, and

32 samples of aggressive B-CLL showing 11q deletion. Detailed description of the samples can be found in Tables 1-3. MicroRNA microchip experiments revealed that three groups of B-CLL show significant characteristic differences in microRNA expression pattern (Table 4). To determine TCL1 protein expression in three groups of

B-CLL, we carried out Western blot analysis using TCL1 monoclonal antibody (clone

27D6). Results of these experiments are shown in Figure 6A and Figure 6B. TCL1 expression was assessed as low, medium, high, and very high. Our experiments revealed low levels in 15 of 23 (65%) indolent B-CLLs, in 11 of 25 (44%) aggressive B-CLLs, and in 1 of 32 (3%) aggressive B-CLLs with 11q deletions, whereas high and very high

TCL1 expression was observed in 1 of 23 (4%) indolent B-CLLs, in 14 of 25 (56%) aggressive B-CLLs, and in 24 of 32 (75%) aggressive B-CLLs with 11q deletions

(Figure 6B). This finding suggests that TCL1 overexpression correlates with aggressive

B-CLL phenotype (P < 10-6) and 11q deletions (P = 10-4). Our results are consistent with the recently published study showing that high TCL1 expression in human B-CLL correlates with unmutated VH status and ZAP-70 positivity (Herling et al., 2006).

33 miR-29 and miR-181 target TCL1.

To determine which micro-RNA(s) target TCL1, we used RNAhybrid software offered by Bielefeld University Bioinformatics Server and miRBase database (Griffiths-

Jones et al., 2006). Among miR candidates targeting TCL1, we found that miR-29b and miR-181b (Figure 7; several other sites with lower homology not shown) are also down- regulated in aggressive B-CLLs with 11q deletions (Table 1). The expression of these miRs was confirmed by real-time reverse transcription-PCR in a representative set of samples (Figure 8). Furthermore, it was previously shown that expression of members of miR-29 family could discriminate between CLL samples with good and bad prognosis

(Calin et al., 2005). We thus proceeded to determine if these miRs indeed target TCL1 expression using the TCL1 3‟ UTR inserted downstream of luciferase open reading frame, as previously described (Cimmino et al., 2005). HEK293 cells were cotransfected with the miR-29b or scramble negative control, as indicated, and pGL3 construct containing a part of TCL1 cDNA, including a region homologous to miR-29, or pGL3 vector alone as indicated. For miR-181 assays, full-length TCL1 cDNA was inserted into pGL3 vector in sense (Tcl1FL) or antisense (Tcl1FLAS) orientation. Figure 9 shows that

TCL1 mRNA expression is inhibited by miR-29 and miR-181. To confirm these findings, we cloned full-length TCL1 cDNA, including 5‟ and 3‟ UTRs, into cytomegalovirus mammalian expression vector and investigated whether miR-29b and miR181b affect

TCL1 protein expression levels. We co-transfected this construct with miR-29b, miR-

181b, and pre-miR negative control (scramble) into 293 cells as indicated in Figure 10.

These experiments revealed that co-expression of TCL1 with miR-29 and miR-181

34 significantly decreased TCL1 expression (Figure 10, lane 2 and 4 versus lanes 1 and 3).

We therefore concluded that miR-29b and miR-181b target TCL1 expression at mRNA and protein levels. Interestingly, we found an inverse correlation between miR-29b and miR-181b expression and TCL1 protein expression in B-CLL samples (Figure 11). For samples with highest miR-29b expression (top 20%), 10 of 12 had low or medium TCL1 expression, whereas in samples with highest miR-181b expression (top 20%), 11 of 12 had low or medium TCL1 expression. Likewise, for samples with high expression of both miR-29b and miR-181b, 4 of 4 had low or medium TCL1 expression. In summary, for samples with high miR-29b and/or miR-181b expression, 17 of 20 showed low or medium

TCL1 expression (P = 0.04). In addition, none of the samples with high miR-29b and/or miR-181b expression showed high TCL1 expression (P = 0.05). These results suggest that TCL1 expression in B-CLL is, at least in part, regulated by miR-29 and miR-181.

In this report, we show that TCL1 expression is regulated by miR-29 and miR-181 and this regulation is relevant to the three groups of B-CLL we studied. Although we observed a reverse correlation between TCL1 protein expression and these two miRs, a significant proportion of B-CLL samples show low TCL1 expression and low expression of miR-29 and miR-181 (Figure 11). This suggests that, in these samples, TCL1 expression is down-regulated transcriptionally or by other microRNAs. The fact that neither miR-29 nor miR-181 is located at 11q suggests that the region may contain an important regulator of the expression of these two miRs. Previously, a micro-RNA signature was published with 13 microRNAs that differentiate aggressive and indolent B-

CLL (Calin et al., 2004; Calin et al., 2005). Intriguingly, of the four down-regulated

35 microRNAs in aggressive B-CLL, three are different isoforms of miR-29 (miR-29a-2, miR-29b-2, and miR-29c), strongly suggesting that miR-29 and TCL1 interactions play an important role in the pathogenesis of aggressive B-CLL. Interestingly, miR-181 is differentially expressed in B-cells and TCL1 is mostly a B-cell–specific gene

(Ramkissoon et al., 2006). This suggests that TCL1 might be a target of miR-181 not only in B-CLL cells but also in normal B-lymphocytes. Additional studies are necessary to determine whether there is an inverse correlation between TCL1 and miR-181 expression at different stages of B-cell maturation. Because miR-29 and miR-181 are natural TCL1 inhibitors, these miRs may be candidates for therapeutic agents in B-CLL-overexpressing

TCL1.

AGGRESSIVE CLL N=25 ID CRC ID % VH % ZAP 1 CLL1 100 70.1 2 CLL2 99.6 71.5 3 CLL3 100 86.8 4 CLL4 100 71.3 5 CLL5 100 75.2 82 CLL82 98.6 65.2 7 CLL7 100 78.1 8 CLL8 99.3 57 9 CLL9 99.3 50.1 10 CLL10 99.6 67.4 11 CLL11 99.3 74.4 12 CLL12 99.3 92.2 13 CLL13 100 67.7 14 CLL14 100 76.5 15 CLL15 100 77.7 16 CLL15 99.6 66.9 17 CLL17 100 84.3 18 CLL18 100 83.2 19 CLL19 100 70.1 20 CLL20 99.6 87.1 21 CLL21 100 91.1 22 CLL22 100 92.5 23 CLL23 100 80.9 24 CLL24 100 84.5 25 CLL25 100 92.7

Table 1. CLL sample information.

INDOLENT CLL N=23 ID CRC ID % VH % ZAP 26 CLL26 92.9 0.6 27 CLL27 92.6 0.5 28 CLL28 92.9 0.1 29 CLL29 94.7 0.2 30 CLL30 91.8 7.1 31 CLL31 94.7 10.9 32 CLL32 91.8 1 33 CLL33 97.2 7.6 34 CLL34 90.2 9.2 35 CLL35 91 9.4 36 CLL36 92.2 8.3 37 CLL37 96.8 7 38 CLL38 92.2 6.7 39 CLL39 92.3 6.4 40 CLL40 89.8 9.3 41 CLL41 96.5 6.9 42 CLL42 96.1 9.3 43 CLL43 95.4 8.5 44 CLL44 92.7 7.3 45 CLL45 97.5 9.8 46 CLL46 93.9 7.3 47 CLL47 90.4 9.7 49 CLL49 95.2 9.8

Table 2. CLL sample information.

AGGRESSIVE CLL with 11q del N= 32 ID CRC ID % VH % ZAP FISH 50 CLL50 99.6 45.5 11q deletion 51 CLL51 100 5.9 11 deletion 52 CLL52 99.6 ND 11q deletion 53 CLL53 100 25.4 11q deletion 54 CLL54 ND ND 11q deletion 55 CLL55 100 59.1 11q deletion 56 CLL56 99.6 36.2 in 61% cells 57 CLL57 100 31.7 in 11% cells 58 CLL58 100 20.7 in 83% cells 59 CLL59 ND ND in 81% cells 60 CLL60 100 47.8 in 10% cells 61 CLL61 99.3 23.6 11q deletion 62 CLL62 100 51.2 11q deletion 63 CLL63 98.9 37.8 11q deletion 64 CLL64 95.7 26.6 in 90% cells 65 CLL65 100 39.1 in 43% cells 66 CLL66 98 39.8 in 55% cells 67 CLL67 99.3 20.3 in 90% cells 68 CLL68 99.6 68.9 in 54% cells 69 CLL69 100 38.1 in 64% cells 70 CLL70 ND 26.7 in 93% cells 71 CLL71 100 34.7 in 14% cells 72 CLL72 100 23.3 in 24% cells 73 CLL73 ND 23.2 11q deletion 74 CLL74 100 14.3 11q deletion 75 CLL75 100 10.3 11q deletion 76 CLL76 98 3.7 11q deletion 77 CLL77 100 27.3 in 50% cells 78 CLL78 99.6 3.4 11q deletion 79 CLL79 100 16.5 11q deletion 80 CLL80 ND 0.6 in 96% cells 81 CLL81 98.9 22.1 11q deletion

Table 3. CLL sample information.

Table 4. Statistically significant microRNAs differentiating CLL subtypes.

Figure 6. TCL1 expression in B-CLL. A, Lanes 1 to 8, B-CLL samples. Lanes 2 and

6, TCL1 expression was rated as low. For all other lines, TCL1 expression was rated as high. B, TCL1 expression in three groups of B-CLL. Columns, relative number of indicated B-CLL samples.

Figure 7. Sequence alignment of miR-29b and miR-181b with 3’ UTR of TCL1.

Figure 8. Real time RT-PCR analysis of representative CLL samples. Three samples with high expression (25, 37 and 41) and four samples with low expression (55, 56, 72 and 81) of both miR-181 and miR-29 were chosen. Real time RT-PCR analysis (ABI) was carried out for miR-181a, miR-181b, miR-181c, miR-181d, miR-29a, miR-29b and miR-

29c according to manufacturer‟s protocol. All experiments were carried out in triplicates.

Figure 9. miR-29 and miR181 target TCL1 expression in luciferase assays. 293 cells were co-transfected with the miR-29b or scramble negative control, as indicated, and pGL3 construct containing a part of TCL1 cDNA, including a region homologous to miR-

29 (Tcl1), or pGL3 vector alone as indicated. For miR-181 assays, Tcl1FL or Tcl1FLAS were cotransfected with miR-181. Firefly and renilla luciferase activities were assayed with the dual luciferase assay system (Promega) and firefly luciferase activity was normalized to renilla luciferase activity, as suggested by the manufacturer. All experiments were carried out in triplicate.

Figure 10. Effect of miR-29b and miR-181b on TCL1 protein expression. 293 cells were transfected with pcDNA3TCL1fl (a mammalian expression vector containing full- length TCL1 cDNA) alone (lane 1) or co-transfected with pcDNA3-TCL1fl and miR-29b

(lane 2), pre-miR negative control (lane 3), or miR-181b (lane 4). TCL1 expression was detected by Western blot with anti-TCL1 antibody.

Figure 11. Inverse correlation of Tcl1 protein expression with miR-181b and miR-

29b expression in B-CLL samples by microarray. The values represent microRNA microarray hybridization signal. Also refer to Fig. 8 for RT-PCR analysis.

CHAPTER 3

B-Cell Chronic Lymphocytic Leukemia modeled in mouse by targeted miR-29 expression

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the western world. Human B-CLL occurs in two forms: aggressive (showing high ZAP-

70 expression and unmutated IgH VH) and indolent (showing low ZAP-70 expression and mutated IgH VH). We found that miR-29a is upregulated in indolent human B-CLL compared to aggressive B-CLL and normal CD19+ B-cells. To study the role of miR-

29 in B-CLL, we generated Eµ-miR-29 transgenic mice overexpressing miR-29 in mouse

B-cells. Flow cytometric analysis revealed a markedly expanded CD5+ population in the spleen of these mice starting at 2 months of age. 85% (34/40) of miR-29 transgenic mice exhibited an expanded population of CD5+ B-cells, a characteristic of the B-CLL phenotype. An average of 50% of the B-cell population in these transgenics was CD5 positive. At the age of 2 years these mice showed significantly enlarged spleens and an increase in CD5+ B-cell population of up to 100% of B-cells. Of 20 Eµ-miR-29 transgenic mice followed up to the age of 24-26 months, 4 (20%) developed frank leukemia and prematurely died from the disease. The expanded CD5+ B-cell population was found to be proliferative, with an increased number of cells in the S-phase of the cell cycle, compared to wild type CD19+ B-cells. These results suggest that deregulation

47 of miR-29 can cause, or at least significantly contribute to the pathogenesis of indolent B-

CLL.

Introduction

Chronic lymphocytic leukemia (CLL) is the most common human leukemia, accounting for ~30% of all cases (Sgambati et al., 2001), with ~10,000 new cases observed each year in the United States. Characteristically, CLL is a disease of elderly people, with the incidence increasing linearly with each decade above age 40 (Bullrich &

Croce, 2001; Sgambati et al., 2001). It is known that this disease is characterized by the clonal expansion of CD5 positive B-cells (Bullrich & Croce, 2001) and occurs in two forms, aggressive and indolent (Sgambati et al., 2001).

MicroRNAs, representing between 1 and 3% of all eukaryotic genes, are a class of endogenous non-coding RNAs, 19-25 nucleotides in size, which regulate gene expression at the transcriptional or translational level (Bartel, 2004). Recent studies have shown that microRNAs are of significant importance in development and in various processes, such as cellular growth, differentiation, cell death (Zhang & Chen, 2009), and

DNA methylation (Fabbri, Ivan, Cimmino, Negrini, & Calin, 2007). Approximately half of human microRNAs are located at fragile sites and genomic regions involved in alterations in cancers (Calin et al., 2004), and alteration of microRNA expression profiles occurs in most cancers, suggesting that individual microRNAs could function as tumor suppressors or oncogenes (Volinia et al., 2006).

The 13q14 deletion is the most common CLL aberration and is detected by cytogenetic analysis in approximately half of the cases (Dohner et al., 2000). Analysis of a deletion at 13q14.3 and chromosomal breakpoint mapping of a translocation t(2;13)(q32;q14) led to the discovery of two physically linked microRNAs, miR-15a and miR-16-1, as targets of these deletions (Calin et al., 2002). Consequently, miR-15a and miR-16-1 expression is reduced in the majority of CLL cases (Calin et al., 2002), and further studies indicated that miR-15a/miR-16-1 negatively regulate BCL2 expression

(Cimmino et al., 2005). These findings indicated that microRNAs play important roles in

CLL and that down-regulation of miR-15/16 and subsequent BCL2 up-regulation contribute to CLL pathogenesis (Calin et al., 2002; Cimmino et al., 2005). Since miR-

15/16 was identified as a tumor suppressor in indolent CLL, the microRNA expression profile in CLL has been studied extensively and a signature profile was reported, describing 13 microRNAs that differentiate aggressive and indolent CLL (Calin et al.,

2004).

We and others observed that microRNA-29 expression is down-regulated in aggressive CLL versus indolent CLL (Herling et al., 2006; Pekarsky et al., 2006), and several reports indicated that miR-29 might function as a tumor suppressor by targeting several oncogenes, including TCL1, MCL1 and CDK6 (Mott, Kobayashi, Bronk, &

Gores, 2007; Pekarsky et al., 2006; Zhao et al., 2010). On the other hand, one report showed that miR-29 expression is up-regulated in metastatic breast cancer and a very recent study reported that miR-29 over-expression can cause AML in mice (Gebeshuber,

Zatloukal, & Martinez, 2009; Han et al., 2010). To clarify the role of miR-29 in B-cell

49 leukemias we generated transgenic mice over-expressing miR-29 in B-cells and now report the phenotype of this new mouse model.

Results

miR-29 expression in CLL and production of Eμ-miR-29 transgenic mouse model.

As noted above, we have previously reported that miR-29 expression is downregulated in aggressive versus indolent CLL (Herling et al., 2006; Pekarsky et al.,

2006), but data showing miR-29 expression in CLL versus normal CD19+ B-cells was not available. To determine expression levels of miR-29 in CLL and normal CD19+ B-cells, we studied expression of miR-29a and miR-29b in 29 aggressive CLL samples, 33 indolent CLL samples and two normal CD19+ B-cell controls. Figure 12 shows real time

RT-PCR results in these samples. MiR-29a expression was 4.5 fold higher in indolent

CLL, when compared with normal CD19+ B-cells while aggressive CLL samples showed a 3.2 fold increase. Similarly, miR-29b expression was increased 4 fold in indolent CLL and 3.5 fold in aggressive CLL when compared with normal CD19+ B-cells. Both miR-

29a and miR-29b, were downregulated in aggressive versus indolent CLL confirming previous observations (Pekarsky et al., 2006), although in the case of miR-29b this difference was not statistically significant. Interestingly, in all samples miR-29a expression level was over 20 fold higher than that of miR-29b (Figure 12).

Since expression levels of miR-29a and miR-29b were significantly higher in indolent CLL than in normal CD19+ B-cells, we hypothesized that miR-29 could contribute to the pathogenesis of CLL. To investigate this possibility we generated transgenic mice in which expression of the mouse miR-29a/b cluster was controlled by a

VH promoter - IgH-Eμ enhancer (Bichi et al., 2002), along with the hrGFP (humanized renilla green fluorescent protein), and the SV40 (Simian virus 40) poly(A) site; this promoter/enhancer combination drives expression of miR-29a/b in immature and mature

B-cells (Figure 13). The miR-29a/b cluster sequence was inserted within the intron of this construct (Figure 13). Two founders on FVB/N background, designated F1 and F2, were generated and bred to establish the transgenic lines. Expression of miR-29a and miR-29b was examined by Northern Blot analysis, using RNAs isolated from spleens of transgenic animals. Figure 14 shows overexpression of miR-29a and miR-29b in both transgenic lines (F1 and F2) compared to nontransgenic littermates (WT). To confirm that the transgene is expressed in B-cells we performed flow cytometry using CD19 as a

B-cell marker. Figure 15 shows that all CD19+ cells in both transgenic lines (F1 and F2) also express GFP, while no GFP expression was detected in wild type littermates (WT).

Eμ-miR-29 transgenics develop CLL phenotype.

We used flow cytometry to determine the immunophenotypic profile of spleen lymphocytes from miR-29 transgenics. Juvenile (2-4 months) mice showed no increase in spleen size or other gross pathological abnormalities, although some animals showed increased populations of CD5+CD19+IgM+ B-cells. At the age of 12-24 months, flow

51 cytometric analysis revealed a markedly expanded CD5+ B-cell population in the spleen of 34 of 40 (85%) miR-29 transgenic mice, a characteristic of CLL; ~50% of B-cells in these transgenics were CD5 positive. Figure 16 (left) shows a representative example.

Although almost all spleen B-cells from this animal were CD5+CD19+IgM+, these cells represented only 25-30% of all spleen lymphocytes. A more advanced CLL case is shown in Figure 16 (middle). Almost all normal lymphocytes in the spleen of this animal were replaced by malignant CD5+CD19+IgM+ B-cells. As expected, almost no

CD5+CD19+IgM+ B-cells were detected in spleens of WT littermates (Figure 16, right).

The expanded population of CD5+CD19+ B-cells was also detected in peripheral blood and bone marrow from miR-29 transgenic mice, but not from WT littermates (Figure 17 and Figure 18). Figure 19 shows the number of animals with increased

CD5+CD19+IgM+ populations in spleen. While only 7 of 40 (17%) miR-29 transgenic mice showed 0-20% CD5+ B-cells, 16 of 40 (40%) showed 60% or more

CD5+CD19+IgM+ cells (Figure 20). In addition, miR-29 transgenic mice showed significant increases in the percentage of CD5+ splenic B-cells with age (Figure 21). In animals younger than 15 months, CD5+ B-cells represented only ~20% of total B-cells; by 15-20 months of age that increased to ~40% (Figure 21). At the age of 20-26 months, on average, >65% of all B-cells were CD5+ (Figure 21). These data suggest gradual progression of indolent CLL in miR-29 transgenic mice. We followed 20 Eμ-miR-29 mice to the age of 24-26 months. Almost all these mice showed significantly enlarged spleens, and 4 of 20 (20%) developed frank leukemia and died of disease. Figure 22 shows a

52 representative case of frank leukemia presenting with an enlarged spleen, liver and advanced lymphadenopathy.

Clonal IgH gene rearrangements are typical in human CLL cases (Sgambati et al.,

2001). These rearrangements were also observed in the Tcl1 driven mouse model of CLL

(Bichi et al., 2002). To determine whether CD5+ B-cells from Eμ-miR-29 transgenic mice show clonality, we carried out southern blot hybridization using spleen B-lymphocyte

DNA isolated from cases showing at least 50% CD5+CD19+IgM+ B-cells. Figure 23 shows clonal rearrangements of the IgH gene in 3 of 5 cases analyzed. These results further indicate that the expansion of CD5+ B-cells in Eμ-miR-29 mice resembles human

CLL.

To further confirm that Eμ-miR-29 mice develop CLL-like disease, we carried out histological and immunohistological analysis. Figure 24A-C shows representative smears from blood of Eμ-miR-29 transgenic mice and WT control. The smear from a WT mouse showed few rare lympho-monocytes with a normal appearance (Figure 24A). In contrast, the smear from a Eμ-miR-29 mouse with low grade CLL exhibited an increased number of atypical lymphoid cells (Figure 24B, black arrows), while the smear from a miR-29 transgenic with advanced CLL presented numerous malignant lymphoid cells

(Figure 24C), including smudge cells, typical of CLL (Figure 24C inset, smudge cells indicated by arrowheads). Figure 24D-L shows representative histological images of Eμ- miR-29 transgenics and a WT control. The spleen of the WT mouse shows preserved architecture and several normal looking lymphoid follicles (Figure 24D, green arrow). In contrast, the spleen of a diseased miR-29 transgenic mouse with CLL, exhibits distorted

53 architecture, with ill-defined lymphoid follicles and lymphoid proliferation invading the red pulp (Figure 24E), while the spleen of a miR-29 mouse with advanced CLL shows total obliteration of the normal architecture by malignant lymphoid proliferation (Figure

24F). B220 staining of the same sections shows a lymphoid follicle of a WT mouse presenting a normal B-cell disposition (Figure 24G). In contrast, transgenic spleens show lymphoid follicles in disarray due to the low grade malignant lymphoid proliferation

(Figure 24H), or CLL with diffuse distribution of a malignant B-cell population (Figure

24I). Figure 24J-L shows low expression of Cyclin D1 in a WT spleen and moderate to high Cyclin D1 expression in low grade and advanced CLL. In summary, histological and immunohistological examination confirmed that Eμ-miR-29 mice develop CLL like disease.

As noted above, only 20% of Eμ-miR-29 transgenic mice developed advanced leukemia and died from the disease. Figure 25 shows a representative advanced case of

CLL that invaded liver and kidney. Histological examination showed total obliteration of the normal spleen architecture with high expression of B220, Cyclin D1 and Ki67

(Figure 25 A- D). These B220+ malignant B-cells infiltrated the liver (Figure 25 E-H) and kidney (Figure 25 I-L).

Recent investigations showed that accumulation of CLL lymphocytes can result not only from prolonged survival, but also from proliferating CD5+B220+ cells originating in the bone morrow, lymph nodes or spleen (Chiorazzi, 2007; Messmer et al.,

2005; Sieklucka et al., 2008). To determine whether CLL cells from Eμ-miR-29 mice proliferate, we performed cell cycle analyses based on BrdU incorporation. We assessed

54 the proliferative capacity of CD19+CD5+, as well as CD19+CD5- transgenic splenic lymphocytes in comparison with WT CD19+ splenic lymphocytes. Figure 26 A-J shows that CD19+CD5+ B-cells from Eμ-miR-29 mice proliferate, while no proliferation was detected for CD19+ WT lymphocytes (2.7% and 5.6% cells in S-phase for transgenic B- cells versus 0.3% and 0.5% for WT B-cells, Figure 26 I,J versus Figure 26 C,D).

Interestingly, even CD19+CD5- transgenic lymphocytes showed increased proliferation compared to CD19+ WT B-cells (1.0% and 0.95% cells in S-phase versus 0.3% and 0.5% for WT B-cells, Figure 26 G,H versus Figure 26 C,D). This data suggests that miR-29 over-expression promotes B-cell proliferation, even in CD5 negative cells.

Human CLL is characterized by immune incompetence and progressive severe hypogammaglobulinemia that eventually develops in almost all patients (Caligaris-

Cappio & Hamblin, 1999). To determine if this is the case in Eμ-miR-29 mice, we compared levels of serum immunoglobulins in transgenic mice of approximately 18 months of age versus WT littermates. Figure 27 shows that levels of IgG1, IgG2a and

IgG2b were decreased 2-4 folds in Eμ-miR-29 transgenic mice versus WT controls. On the other hand, no significant differences in levels of IgA and IgM were found (data not shown). To determine if Eμ-miR-29 mice show impaired immune response, we measured levels of anti-SRBC antibodies after injection of SRBC (sheep red blood cells) in miR-29 transgenics and WT siblings. Figure 28 shows that serum levels of anti-SRBC antibodies were decreased ~4 fold in serum of miR-29 transgenics compared to age-matched WT mice. This data clearly indicates that, similar to human CLL, the CLL-like disease in Eμ- miR-29 mice is characterized by hypogammaglobulinemia and immune incompetence.

Previously, our laboratory reported development and characterization of a TCL1 driven mouse model of CLL (Bichi et al., 2002; Efanov et al., 2010; N. Zanesi et al.,

2006), and showed that miR-29 can target TCL1 expression (Pekarsky et al., 2006). In this mouse model, the TCL1 ORF (lacking 3‟ UTR) was under the control of a VH promoter - IgH-Eμ enhancer (Bichi et al., 2002). Because of absence of the 3‟ UTR in the transgenic construct, miR-29 could not inhibit TCL1 expression in these mice. Eμ-TCL1 transgenic mice develop aggressive CLL and all mice die of the disease at 12-15 months of age (Bichi et al., 2002). To determine if transgenic miR-29 expression can accelerate

CLL in Eμ-TCL1 transgenic mice, we crossed Eμ-miR-29 and Eμ-TCL1 transgenic mice.

Eμ-miR-29/Eμ-TCL1 mice and their Eμ-TCL1 littermates were sacrificed at ~7 months of age and analyzed. Figure 29 shows representative FACS analysis of spleen lymphocytes of these genotypes. TCL1/miR-29 double transgenic mice showed significantly increased

CD5+CD19+ B-cell populations compared to Eμ-TCL1 mice (93.9% and 93.3% versus

48.3% and 50%). On average, Eμ-miR-29/Eμ-TCL1 mice had 40% more CD5+CD19+ splenic B-cells (Figure 30), and 3-fold increases in spleen weight compared to Eμ-TCL1 mice (Figure 31). This data suggests that miR-29 can contribute to the pathogenesis of

CLL alongside with TCL1.

Analysis of miR-29 targets.

To determine if miR-29 over-expression in mouse B-cells affects expression of its targets, we analyzed expression levels of several previously reported miR-29 targets,

CDK6 (Zhao et al., 2010), MCL1 (Mott et al., 2007), DNMT3A (Fabbri et al., 2007), in

56 sorted B220+ B-cells from miR-29 transgenics and WT controls. We found that two targets, CDK6 and DNMT3A are down-regulated in miR-29 transgenic mice, while no differences in MCL1 and PTEN were detected (Figure 32) (although PTEN is not a proven miR-29 target it was previously predicted as a potential target (Han et al., 2010)).

Since CDK6 and DNMT3A are not known as tumor suppressors, we used

Affymetrix gene expression arrays to determine potential miR-29 targets contributing to its oncogenic activity. Using microarray analysis we compared gene expression in sorted

B220+ B-cells from miR-29 transgenics and WT controls. We then cross-referenced genes downregulated in miR-29 transgenic B-cells with known or potential tumor suppressor function, with the list of potential miR-29 targets obtained from Targetscan software. This resulted in three potential targets: peroxidasin homolog (PXDN), a p53 responsive gene, down-regulated in AML (Desmond et al., 2007; Horikoshi, Cong, Kley, & Shenk, 1999);

BCL7A, a pro-apoptotic gene down-regulated in T-cell lymphomas (van Doorn et al.,

2005); and ITIH5, a member of the inter-alpha-trypsin inhibitor family, down-regulated in breast cancer (Himmelfarb et al., 2004). Figure 33 shows down-regulation of expression of these three genes in CD19+ B-cells of miR-29 transgenics versus WT littermates. Figure 34 shows the alignment of miR-29a and corresponding 3‟ UTRs of

PXDN, BCL7A and ITIH5. To determine if miR-29 indeed targets expression of PXDN,

BCL7A and ITIH5 we inserted 3‟ UTR fragments (including miR-29 homology regions) of these cDNAs downstream of luciferase ORF into pGL3 vector, as previously described

(Cimmino et al., 2005). HEK293 cells were co-transfected with miR-29a, miR-29b or scrambled negative control, and pGL3 construct containing fragments of PXDN, BCL7A

57 and ITIH5 cDNAs, including a region homologous to miR-29, as indicated in Figure 35.

Expression of miR-29a or miR-29b significantly (~ 3-fold) decreased luciferase expression of the construct containing the 3‟ UTR of PXDN (Figure 35, middle), while no significant effect was observed for BCL7A and ITIH5 (Figure 35). Thus, we concluded that peroxidasin homologue expression could be targeted by miR-29. To confirm these findings, we used full-length PXDN cDNA including 5‟ and 3‟ UTRs in a

CMV mammalian expression vector and investigated whether miR-29 expression affects

PXDN protein expression levels. We co-transfected this construct with miR-29a, miR-29b or pre-miR negative control (scrambled) into HEK293 cells, as indicated in Figure 36.

These experiments revealed that co-expression of PXDN with miR-29a or miR-29b almost completely inhibited PXDN expression (Figure 36). We therefore concluded that miR-29a and miR-29b target PXDN expression at mRNA and protein levels. To determine if PXDN could play a role in the pathogenesis of human CLL we studied expression of PXDN in 25 human CLL samples and normal CD19+ B-cell control.

Figure 37 shows real time RT-PCR results in these samples. PXDN expression was drastically down-regulated (50-fold or more) in CLL samples compared to normal CD19+

B-cells. These results suggest that the oncogenic role of miR-29 in B-cells might be, at least in part, dependent on targeting peroxidasin homologue, although additional functional studies are necessary to prove that this is indeed the case.

Discussion

Although many microRNAs are up- or down-regulated in a number of solid tumors and hematological malignancies and several are postulated to function as tumor suppressors or oncogenes (Volinia et al., 2006), there have been only three reports demonstrating that dysregulation of microRNAs can cause cancer by exploiting mouse models. The first report showed that over-expression of miR-155 in B-cells results in pre-

B-cell leukemia in mice (Costinean et al., 2006). The knockout of miR-15/16 led to development of CLL with low penetrance (Klein et al., 2010; N. Zanesi, Pekarsky,

Trapasso, Calin, & Croce, 2010). Finally, a very recent report showed that over- expression of miR-29 can cause AML in mice (Han et al., 2010). While nearly all published reports conclude that miR-155 functions as an oncogene and miR-15/16 is a tumor suppressor, the function of miR-29 in this respect has not been clearly defined.

Previous reports showed that miR-29 inhibited tumorigenicity in lung cancer (Fabbri et al., 2007), its expression was down-regulated and correlated with poor prognosis in mantle cell lymphoma (Zhao et al., 2010), and its expression caused apoptosis in AML

(Garzon et al., 2009). On the other hand, miR-29 expression was up-regulated in metastatic breast cancer (Gebeshuber et al., 2009), its overexpression in mouse myeloid cells caused AML (Han et al., 2010), and in this report we show that miR-29 over- expression in B-cells results in CLL. For AML, several reports defined miR-29 as a tumor suppressor that functions by targeting oncogenes such as MCL1 and CDK6 and promoting apoptosis in AML cells (Garzon et al., 2008; Garzon et al., 2009). A very recent study reported directly opposite results showing that miR-29 is over-expressed in 59 some AML, and its up-regulation causes AML in a mouse model (Han et al., 2010).

Although the role of miR-29 in AML requires further scrutiny, it is likely that miR-29 can function as a tumor suppressor or oncogene depending on cellular contexts.

We and others previously reported that miR-29 is down-regulated in aggressive

CLL versus indolent CLL; we also demonstrated that miR-29 is one of the microRNAs targeting TCL1, a critical oncogene in the pathogenesis of aggressive CLL (Herling et al.,

2006; Pekarsky et al., 2006). Here we report that miR-29 is over-expressed in indolent

CLL compared to normal B-cells. Since only 20% of Eμ-miR-29 transgenic mice died of leukemia in old age, but almost all mice showed expanded CD5+CD19+ B-cell populations, the phenotype of Eμ-miR-29 is similar to that of indolent CLL. Therefore up-regulation of miR-29 initiates or at least significantly contributes to the pathogenesis of indolent CLL. On the other hand, TCL1 is largely not expressed in indolent CLL

(Pekarsky et al., 2006), and likely does not play an important role in indolent CLL. Our current hypothesis is that miR-29 over-expression is not sufficient to initiate aggressive

CLL. In contrast, up-regulation of TCL1 is a critical event in the pathogenesis of the aggressive form of CLL. Since miR-29 targets TCL1, its down-regulation in aggressive

CLL (compared to the indolent form) contributes to upregulation of TCL1 and development of aggressive phenotype.

Since in all human CLL samples miR-29a expression levels were >20-fold higher compared to miR-29b (Figure 12) and both microRNAs share the same seed sequence, it is likely that most of effects of expression of miR-29ab cluster in CLL can be attributed to miR-29a. MiR-29 functions as an oncogene in B-cells by regulating its targets.

Targetscan software predicts over 800 targets for miR-29. This list contains multiple oncogenes and tumor suppressor genes. Previous reports showed that miR-29 can target

TCL1, MCL1 and CDK6 oncogenes (Garzon et al., 2009; Mott et al., 2007; Pekarsky et al., 2006); here we propose that miR-29 targets the possible tumor suppressor peroxidasin homologue. Clearly, interactions of miR-29 with these genes contribute to its function, but it is very likely that multiple interactions of miR-29 with its targets hold keys to its oncogenic role in CLL.

Experimental Procedures

Eμ-miR-29 transgenic mice and human CLL samples.

A 1.0 kb fragment containing mouse miR-29ab cluster cloned into the BamHI and

SalI sites of the plasmid containing a mouse VH promoter (V186.2), the IgH-Eµ enhancer

(Bichi et al., 2002), along with the hrGFP (humanized renilla green fluorescent protein), and the SV40 (Simian virus 40) poly(A) site; this promoter drives expression of miR-

29a/b in immature and mature B-cells. The miR-29a/b cluster sequence was inserted within the intron of this construct. Transgenic construct, free from vector sequences, was injected into fertilized oocytes from FVB/N animals. Transgenic mice were produced in

Ohio State University transgenic mouse facility. Mice were screened for the presence of the transgene by PCR analysis on tail DNAs. Two founders were obtained (F1 and F2) and bred in FVB/N strain. Transgenic heterozygote mice issued from these founders were studied and compared with non-transgenic siblings raised in identical conditions as previously described (Efanov et al., 2010). Genotyping was performed on tail DNAs by

PCR. Primers were: miR29d: gct gac gtt gga gcc aca ggt aag; miR29r: aca aat tcc aaa aat gac ttc cag. Expression of miR-29a and miR-29b was confirmed by Northern blot analysis. Spleen lymphocyte isolation and Northern blot analysis using anti- miR-29a and miR-29b were carried out as previously described (Costinean et al., 2006).

Human CLL samples were obtained after informed consent from patients diagnosed with CLL from the CLL Research Consortium. Research was performed with the approval of the Institutional Review Board of The Ohio State University. Briefly,

62 blood was obtained from CLL patients, lymphocytes were isolated through

Ficoll/Hypaque gradient centrifugation (Amersham, Piscataway, NJ) and processed for

RNA extraction using the standard Trizol method as previously described (Palamarchuk et al., 2006). Real-time PCR experiments were carried out using miR-29a, miR-29b and

PXDN assays for Real-time PCR (2112, 413 and Hs00395488_m1 respectively, Applied

Biosystems, Foster City, CA) according to manufacturer‟s protocol. Control human cord blood CD19+ B-cells were purchased from Allcells (Emeryville, CA) and Lonza

(Rockland, ME).

Characterization of miR-29 transgenic lymphocytes.

Lymphocytes from spleens and bone marrow were isolated as previously described (Costinean et al., 2006). For immunostaining, single-cell suspensions were made in PBS supplemented with 1% bovine calf serum (staining solution). Cells were washed in this solution, and incubated with antibodies for 30 min, at RT. Flow cytomertry was carried out on a LSR2 using BD FACSDiva software (BD Bioscience), and FACSCalibur (BD Bioscience) using CellQuestPro software (BD Bioscience).

Conjugated monoclonal antibodies to CD5(53-7.3), CD19(1D3), CD45R(RA3-6B2),

IgM(R6-60.2), were purchased from BD Biosciences.

For studies of the SRBC immune response, mice, 16 to 18 months old, were immunized intravenously with 2×108 to 3×108 sheep red blood cells (SRBCs; Colorado

Serum Company, Denver CO) to elicit SRBC-specific antibody immune responses.

Preimmune blood samples were obtained 2 days before immunization, blood samples

63 were drawn and analyzed one week after immunization. Serum levels of anti-SRBC were measured using enzyme-linked immunoassay kit (Life Diagnostics, West Chester PA) according to manufacturer‟s instructions. Briefly, 100 ml standards and mice sera were loaded to each well of a 96-well plate, incubated for 45 min at room temperature and washed 5 times by wash solutions. After washing, 100 ml of peroxidase-labeled anti-

SRBC antibody conjugate was added to each well, incubated for 45 min at room temperature, and washed 5 times. For visualization of the antigen antibody reaction, the substrate 3, 3‟,5, 5‟- tetrametilbenzidine (TMB) was added 100 ml/well and plates were left at room temperature (RT) for 20 minutes. To avoid an increase in the background, the reaction was stopped by adding 100 ml/ well of 1M HCl.

To measure immunoglobulin levels we used enzyme-linked immunoassay kit

(Immunology Consultant Laboratory, Newberg OR). Experiments were carried out according to manufacturer‟s instructions. Briefly, 100 ml standards and mice sera were loaded to each well of a 96-well plate and incubated for 30 min at RT. Following incubation, plate wells were washed 4 times by Wash Solutions then 100 ml of appropriately diluted peroxidase-labeled antibody conjugate was added to each well and incubated for 30 min at RT followed by 4 washes by Wash Solutions. For visualization of the antigen antibody reaction, 100 ml/well of TMB was applied and plates were left at room temperature for 10 minutes to allow color development. To avoid an increase in the background in control wells, reactions were stopped by adding 100 ml/ well of 0.3M sulfuric acid. The quantity of immunoglobulins was calculated from the standard curve constructed from standards, and corrected for the serum dilutions.

To detect proliferation of B-cell populations, 1mg of BrdU was injected in mice

24 hours before proliferation and cell cycle measurement. Cell cycle and proliferation was measured using BrdU Flow Kit (BD Biosciences) according to manufacturer‟s instructions.

To analyze IgH gene rearrangements, Southern blot of spleen lymphocyte DNA was carried out using EcoRI digestions and mouse JH4 probe as previously described

(Bichi et al., 2002).

Histology and Immunohistochemistry.

Mice were necropsied, and spleens, livers and kidneys were fixed in 10% buffered formalin, included in paraffin, and then cut at 4 μm as previously described (Costinean et al., 2006). Sections were stained with H&E according to standard protocols (Costinean et al., 2006). B220, Cyclin D1 and Ki-67 were used as primary anti-mouse antibody (BD

PharMingen). Secondary antibodies and diaminobenzidine were added according to the manufacturer‟s instruction.

Analysis of miR-29 targets.

B-cells were isolated using B-Cell Isolation kit (Miltenyi Biotec, Auburn CA) according to manufacturer‟s instructions. Proteins from spleens were extracted with

Nonidet P-40 lysis buffer as previously described (Palamarchuk et al., 2006). Western blot analysis was carried out using CDK6 (H-96, Santa Cruz Biotechnology, Santa Cruz,

CA), DNMT3A (2160, Cell Signaling Technology, Danvers, MA), PTEN (mmac1, Lab

Vision, Fremont, CA), MCL1 (S-19, Santa Cruz Biotechnology), PXDN (Novus,

Littelton, CO) and GAPDH (2118, Cell Signaling Technology) antibodies. Ponceau-S staining was used to verify equivalent protein loading.

For luciferase assays fragments of PXDN, BCL7A and ITIH5 cDNA including regions complimentary to miR-29 were inserted using the XbaI site immediately downstream from the stop codon of luciferase into pGL3 vector (Promega, Madison, WI) as previously described (Pekarsky et al., 2006). MiR-29a, miR-29b and scrambled control

RNA duplexes were purchased from Ambion (Austin, TX). Expression construct, containing full length human PXDN was purchased from OriGene (Rockville, MD).

Transfections were carried out as previously described (Pekarsky et al., 2008).

Figure 12. A,B. miR-29a and miR-29b expression in aggressive and indolent CLL.

Figure 13. Eµ-miR-29 construct.

Figure 14. Expression of miR-29a and miR-29b in splenic lymphocytes of Eµ-miR-29 founders.

Figure 15. Expression of GFP in splenic lymphocytes of Eµ-miR-29 founders.

Figure 16. Flow cytometric analysis of miR-29 transgenic and control lymphocytes isolated from spleen.

Figure 17. Flow cytometric analysis of miR-29 transgenic and control lymphocytes isolated from peripheral blood.

Figure 18. Flow cytometric analysis of miR-29 transgenic and control lymphocytes isolated from bone marrow.

Figure 19. Expanded population of CD5+ B-cells in spleens of transgenic mice relative to WT spleens.

Figure 20. Analysis of CD5+ B-cell populations in miR-29 transgenic mice. Majority mice had 40-80% CD5+ spleen B-cells.

Figure 21. CD5+ B-cell population size expanded linearly with age of transgenic mice.

Figure 22. Leukemic transgenic mouse spleen. Gross pathology of a representative Eμ- miR-29 transgenic mouse showing advanced CLL and a WT control of the same age.

Figure 23. Analysis of IgH gene configuration by Southern blot. Spleen lymphocyte

DNA isolated from 5 representative cases showing at least 50% CD5+CD19+ B-cells.

Clonal rearrangements indicated by asterisks.

Figure 24. Histopathological analysis of Eµ-miR-29 mice. Smudge cells indicated by arrowheads. Atypical lymphoid cells indicated by black arrows. A normal lymphoid follicle indicated by a green arrow.

Figure 25. Histopathological analysis of CLL invasion in liver and kidney of Eµ-miR-29 mice

Figure 26. Cell cycle analysis of leukemic cells from Eμ-miR-29 transgenic mice. A-

D, BrdU incorporation into DNA of WT CD19+ B-cells. E-J, BrdU incorporation into transgenic CD19+CD5+ and CD19+CD5- B-cell DNA.

Figure 27. Serum immunoglobulin levels of WT and transgenic animals.

Figure 28. Immune response in transgenic and WT mice. Levels of anti-SRBC specific antibodies in serum of WT and transgenic animals 7 days after SRBC injection.

Figure 29. Flow cytometric analysis of Eµ-TCL1/ Eµ-miR-29 and Eµ-TCL1 transgenic lymphocytes from spleen.

Figure 30. Percentage of CD5+ B-cells in Eµ-TCL1/ Eµ-miR-29 and Eµ-TCL1 transgenic spleen lymphocytes.

Figure 31. Spleen weight from Eμ-TCL1/ Eμ-miR-29 and Eμ-TCL1 transgenic mice.

Figure 32. Western blot analysis of CDK6, DNMT3A, PTEN and MCL1 expression in CD19+ B-cells of miR-29 transgenic and WT mice.

Figure 33. Microaray expression data for PXDN, BCL7A and ITIH5 in CD19+ B- cells of miR-29 transgenic and WT mice.

Figure 34. Sequence alignments of miR-29a and 3’ UTRs of PXDN, BCL7A and

ITIH5.

Figure 35. miR-29 targets PXDN but not BCL7A and ITIH5 expression in luciferase reporter assays.

Figure 36. Effect of miR-29 on PXDN protein expression.

Figure 37. PXDN expression in CLL.

CHAPTER 4

Synopsis and Conclusion

B-Cell Chronic Lymphocytic Leukemia

B-Cell Chronic Lymphocytic Leukemia (B-CLL) is the most common leukemia in the western world. It is characterized by an expansion of a rare population of B-cells bearing the CD5 surface marker. It is a chronic disease that develops over a period of time and affects older people, over the age of approximately 50 years. They develop hypogammaglobulinemia and are immunocompromised, ultimately resulting in mortality.

B-CLL may occur in one of two forms, namely Aggressive and Indolent. Aggressive B-

CLL is characterized by a high ZAP-70 positivity and an unmutated VH status, whereas

Indolent B-CLL is characterized by a low ZAP-70 positivity and a mutated VH status.

TCL1 Oncogene in B-CLL

T-Cell Leukemia Oncogene 1 (TCL1) is an oncogene that was discovered as a target of frequent chromosomal rearrangements at chromosome 14q31.2 in mature T-cell leukemias. Transgenic mice expressing TCL1 in B-cells develop B-CLL. Hence, deregulation of TCL1 could be a causal event in the pathogenesis of B-CLL. TCL1 shown to be a co-activator of Akt oncoprotein which is a critical molecule for the

93 transduction of anti-apoptotic signals in both B and T cells. One more point. Reports of high TCL1 expression in human B-CLL accompanied by high ZAP-70 positivity and unmutated VH status indicated that TCL1-driven B-CLL is the aggressive form of this disease.

microRNAs and Cancer

microRNAs are small RNA molecules that regulate the expression of imprecisely complementary target mRNAs by causing mRNA degradation or inhibition of translation.

Their deregulation has been implicated as an important causal factor of oncogenesis in a variety of cancers like breast cancer, liver cancer, and leukemias. They may function as oncogenes or tumor suppressor genes, as their over-expression could result in the down- regulation of target tumor suppressor genes, and their under-expression could result in the up-regulation of target oncogenes.

Regulation of TCL1 expression by miR-29 and miR-181

To study the mechanism of TCL1 regulation in CLL, we carried out microRNA expression profiling of three types of CLL: indolent CLL, aggressive CLL, and aggressive CLL showing 11q deletion. We identified distinct microRNA signatures corresponding to each group of CLL. We further determined that TCL1 expression is regulated by miR-29 and miR-181, two microRNAs differentially expressed in CLL.

Expression levels of miR-29 and miR-181 generally inversely correlated with TCL1 expression in the CLL samples we examined. Our results suggest that TCL1 expression in

CLL is, at least in part, regulated by miR-29 and miR-181 and that these microRNAs may be candidates for therapeutic agents in CLLs overexpressing TCL1.

miR-29 overexpression in B-CLL

miR-29a and miR-29b were found to be overexpressed in B-cells of human

Aggressive and Indolent CLL samples compared to normal CD5+ B-cells. Indolent CLL had the highest levels of miR-29. This was indicative of a possible oncogenic role of miR-

29 in CLL. To study this, transgenic mice were generated that expressed miR-29 ab- cluster specifically in the B-lymphocytes. Two founder lines were analyzed, and reported to develop an expanded population of rare CD5+ B-cells (a carnal indicator of the development of B-CLL) as early as two months of age, in the spleen, blood and bone marrow. 85% of all mice analyzed developed this expanded B-cell population, and on an average, about 55% of the B-cells were CD5+. The size of the CD5+ B-cell population increased with the age of the mice, typical of the chronic nature of CLL. 20% of the mice followed to the age of about 24-26 months were found to develop frank leukemia and die.

They had grossly enlarged spleens and 100% of the B-cells were CD5 positive.

Analysis of IgH gene configuration of spleenic B-cells in these mice by Southern

Blot revealed the presence of B-cell clonality in three out of five mice analyzed. These three mice were among those that developed a pronounced CLL phenotype, as expected.

Histopathological analysis of the Eµ-miR-29 transgenic mice revealed the development of a CLL-like disease, characterized by an expanded lympho-monocyte population in blood smear, distorted spleen architecture resulting in replacement of

95 spleen red pulp by the expanded B-lymphocytes, and increased B-cell proliferation as evidenced by cyclin-D1 and Ki-67 positivity. 10% of the transgenic mice analyzed beyond the age of 20 months developed malignant B-cell infiltration in the liver and kidney, besides having spleens histologically characteristic of advanced B-CLL.

miR-29 overepression was found to promote B-cell proliferation in miR-29 transgenic mice. These mice also displayed hypogammaglobunemia and a decreased immune response.

Transgenic co-expression of miR-29 and TCL1 in B-cells of mice was found to accelerate the development of B-CLL like disease, compared to single Eµ-TCL1 transgenics.

This data shows that overexpression of miR-29 in B-cells could result in the development of B-CLL like disease.

miR-29: Tumor Suppressor or Oncogene?

miR-29 has been implicated as a potential tumor suppressor and an oncogene. Our data presenting the regulation of TCL1 oncogene expression by miR-29 in CLL, hints at a possible role for miR-29 as a tumor suppressor. In addition, reports in literature mention possible miR-29 targeting of other oncogenes like MCL1 and CDK6 (Mott et al., 2007;

Zhao et al.).

On the other hand, miR-29 was reported to be upregulated in metastatic breast cancer (Gebeshuber et al., 2009). Over-expression of miR-29 in myloid cells of mice was shown to result in AML (Han et al.). Our data depicting a B-CLL like phenotype in

96 transgenic mice overexpressing miR-29 in B-cells also describes a potential oncogenic role of this microRNA.

Thus it appears that a single microRNA might function as a potential oncogene or tumor suppressor, and the type of role assumed could be dependent on the cellular context.

Role of miR-29 in B-CLL

Our data demonstrated that the over-expression of miR-29 in B-cells of mice results in the development of a phenotype resembling that of B-CLL. Since majority of the mice that developed this disease did not die from it, and only 20% of the mice followed to the age of about 2 years died from frank leukemia, we concluded that the phenotype observed was not aggressive, but similar to that of Indolent CLL.

Low levels of TCL1 expression characteristic of Indolent CLL, was partly due to its downregulation by microRNAs like miR-29 and miR-181 which are overexpressed.

Downregulation of miR-29 in Aggressive CLL compared to Indolent CLL probably prevents it from sufficiently downregulating TCL1. It is important to note the possible role of other regulators of TCL1, as TCL1 is also regulated transcriptionally. Aggressive

CLL therefore develops due to high expression levels of TCL1 oncogene. High miR-29 expression levels in Aggressive CLL compared to normal B-cells is unable to contribute to the aggressive phenotype observed. High miR-29 expression, when accompanied by low TCL1 levels (owing to its downregulation by miRs and other possible regulators), results in Indolent CLL.

Peroxidasin homolog is a target of miR-29 and is downregulated in B-CLL

PXDN (peroxidasin homolog (drosophila), a p53 responsive protein downregulated in AML), BCL7A (B-cell CLL lymphoma7A, a pro-apoptotic gene downregulated in T-cell lymphomas) and ITIH5 (inter-alpha (globulin) inhibitor H5, downregulated in breast cancer) were found to be downregulated in B-cells of Eµ-miR-29 transgenic mice compared to wild type littermates and also contain miR-29 binding sites in their 3‟ UTRs. However, we found only PXDN to be targeted by miR-29a and miR-29b at the mRNA and protein levels. This hinted towards the possibility of PXDN as a target of miR-29.

We also found PXDN to be drastically downregulated (~50-fold or more) in human B-CLL samples compared with normal CD19+ B-cells. While we acknowledge that miR-29 might not be the sole factor responsible for this differential expression of

PXDN, we cannot ignore the possibility of atleast a partial role for miR-29 in this observation.

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