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The N-Truncated C/Ebpα Isoform Correlates to the Expression Of The Role of MicroRNA-181a in Acute Leukemia: Biological, Clinical, and Therapeutic Implications Dissertation Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By Christopher Jon Hickey, B.S. The Molecular, Cellular, and Developmental Biology Graduate Program The Ohio State University 2011 Dissertation Committee: Guido Marcucci, M.D.; Advisor Danilo Perrotti, M.D., Ph.D. Lai-Chu Wu, D.Phil. Kenneth Chan, Ph.D. Copyright by Christopher Jon Hickey 2011 ABSTRACT Recent cytogenetic and molecular characterization along with gene expression profiling of acute myeloid leukemia (AML) patients have revealed previously unknown features associated with distinct subsets of the disease. Among these newly discovered moleclar features were mutations within the CCAAT/Enhancer Binding Protein Alpha (CEBPA) gene found in approximately 15% of patients lacking chromosomal abnormalities, thus characterized as cytogenetically normal AML. These mutations were shown to favorably impact the outcome of AML patients through unknown mechanisms. Notably, patients harboring CEBPA mutations also present with increased expression of microRNA-181a (miR-181a). High levels of miR-181a expression have been suggested to have tumor suppressor qualities in accord with CEBPA mutations. Therefore, it is logical to hypothesize that a mechanistic correlation exists between miR-181a expression levels and CEBPA mutations which contribute to favorable outcomes associated to this subset of AML patients. In this body of work, the correlation between different mutant isoforms of C/EBPα and miR-181a was identified and the potential therapeutic implications described. Experimentally, miR-181a expression was found to be higher in those ii cells expressing the N-truncated C/EBPα isoform. As a result of elevated miR- 181a expression, targeted transcripts coding for innate immune effector molecules, Toll-Like Receptor 4 (TLR4) and Interleukin-1 Beta (IL-1β), were down modulated due to direct interaction of miR-181a. The miR-181a repression of these effector molecules ultimately contributed to the repression of the characteristic tumorigenic activity associated to NF-κB. Furthermore, smaller tumors were observed in mouse models xenografted with leukemia cells supplemented with miR-181a. In an effort to recapitulate the therapeutic benefits involving miR-181a, an immunomodulatory compound, lenalidomide, was investigated as an agent to induce miR-181a expression in leukemia cells. Lenalidomide was found to induce the expression of the N-truncated C/EBPα isoform similar to the N- mutant isoforms observed in AML patients. The mechanism favoring the expression of the N-truncated C/EBPα isoform was identified and involved the activity of the translational subunit eIF4E. Following the lenalidomide treatment and subsequent induction of the N-truncated C/EBPα isoform, miR-181a expression was increased. The in vitro data implicating lenalidomide to increased miR-181a expression were validated in xenograft mouse models. These tumors were found to have higher miR-181a expression and the tumors were physically smaller in size and weight. Finally, the in vivo mouse data were validated following analyses of miR-181a expression of individual leukemia iii patients who participated in two separate clinical trials utilizing lenalidomide as an anti-leukemia therapy. The therapeutic benefits associated to lenalidomide were also found to be applicable to acute lymphoblastic leukemia (ALL) cells which harbor the tumorigenic chimera protein, TEL/AML1. In two different ALL cell lines, the lenalidomide induction of miR-181a expression was found to lower the expression of TEL/AML1, subsequently lowered the expression of the pro- survival protein SURVIVIN, and ultimately induced apoptosis in those cells treated with the therapeutic drug. Taken together, increasing the expression of miR-181a has therapeutic benefits in acute leukemias. Indeed, this work served as a paradigm for designing and initiating clinical trials with lenalidomide followed by chemotherapy in distinct cohorts of AML patients with previously untreated or relapsed/refractory disease. iv DEDICATION Dedicated to my family. v ACKNOWLEDGEMENTS I would like to extend my gratitude to my advisor Dr. Guido Marcucci. With his guidance I was able to develop as a scientist and contribute to the ongoing efforts with resolving some of the enigmas surrounding acute leukemias. At the onset of my endeavors, Dr. Marcucci first told me that he wanted me to teach him something about acute leukemia. This dissertation is to serve as a medium of instruction and insight thereby fulfilling his request bestowed upon me years ago when we initially embarked on our collaborative journey. As being his first graduate student and graduating with a doctorate, I am forever grateful for his willingness to accept me into his lab and to work in a collaborative fashion toward the same goal. vi VITA October 4, 1977....………………………………..………….Born in Columbus, Ohio June 1996..…………………………………….………Gahanna-Lincoln High School June 2001…………….……………………………..…..….……..B.S with Distinction. Molecular Biology and Biology, Otterbein College September 2003 to present……………..………......Graduate Research Associate, The Ohio State University December 2009………………………….…….……..…Oral Presentation, American Society of Hematology vii PUBLICATIONS 1. Liu, S., Wu, LC., Pang, J., Santhanam, R., Schwind, S., Wu, YZ, Hickey, C.J., Yu, J., Becker, H., Maharry, K., Radmacher, M.D., Li, C., Whitman, S.P. Mishra, A., Stauffer, N., Eiring, A.M., Briesewitz, R., Baiocchi, R.A., Chan, K.K., Paschka, P., Caligiuri, M.A., Byrd, J.C., Croce, C.M., Bloomfield, C.D., Perrotti, D., Garzon, R., Marcucci, G. Sp1/NFkappaB/HDAC/miR-29b Regulatory Network in KIT-driven Myeloid Leukemia. Cancer Cell. 2010. Apr 13; 17(4): 333-47. 2. Eiring, A.M., Harb, J.G., Neviani, P., Garton, C., Oaks, J.J., Spizzo, R., Liu, S., Schwind, S., Santhanam, R., Hickey, C.J., Becker, H., Chandler, J.C., Andino, R., Cortes, J., Hokland, P., Huettner, C.S., Bhatia, R., Roy, D.C., Liebhaber, S.A., Caligiuri, M.A., Marcucci, G., Garzon, R., Croce, C.M., Calin, G.A., Perrotti, D. miR-328 Functions as an RNA Decoy to Modulate hnRNP E2 Regulation of mRNA Translation in Leukemia Blasts. Cell. 2010 Mar 5;140(5):652-65. 3. Schwind, S., Maharry, K., Radmacher, M.D., Mrozek, K., Holland, K.B., Margeson, D., Whitman, S.P., Hickey, C.J., Becker, H., Metzeler, K.H., Paschka, P., Baldus, C.D., Liu, S., Garzon, R., Powell, B.L., Kolitz, J.E., Carroll, A.J., Caligiuri, M.A., Larson, R.A., Marcucci, G., Bloomfield, C.D. Prognostic Significance of Expression of a Single microRNA, miR-181a, in Cytogenetically Normal Acute Myeloid Leukemia: a Cancer and Leukemia Group B Study. J. Clin. Oncol. 2010 Dec 20;28(36):5257-64. 4. Hickey, C.J., Schwind, S., Becker, H., Alachkar, H., Garzon, R., Wu, YZ, Liu, S., Perrotti, D., Marcucci, G. MicroRNA-181a Targets TEL/AML1 Expression and Impairs Cell Proliferation in t(12;21) Acute Lymphocytic Leukemia (ALL) Cells. Blood (ASH Annual Meeting). Oral Presentation. 2009; 114:Abstract 766. FIELDS OF STUDY Major Field: Molecular, Cellular, and Developmental Biology viii TABLE OF CONTENTS ABSTRACT…………………………………………………….…………………………...ii DEDICATION……………………………………………………….………………………v ACKNOWLEDGEMENTS……………………………………………..……………………vi VITA…………………………………………………………….………………………...vii LIST OF TABLES…………………………………………………….…………………….xi LIST OF FIGURES………………………………………………………………………...xii CHAPTER 1: INTRODUCTION…………………………………………………………….. 1 1.1 Cancer and Leukemia Hallmarks and Statistics…………………………………...1 1.2 Leukemia and Hematopoiesis…………………………………………………….....4 1.3 Acute Myeloid Leukemia………..……………………………………………………9 1.4 Transcription Factors Involved in AML…………………………………………....15 1.5 Genetic Features in AML………….………………………………………………..17 CHAPTER 2:C/EBPα ISOFORMS ARE CORRELATED TO microRNA-181A EXPRESSION..21 2.1 CCAAT/Enhancer Binding Protein Superfamily………………………………….21 2.2 CCAAT/Enhancer Binding Protein Alpha…………………….……...……......….23 2.3 C/EBPα Post-Transcriptional Isoforms……………………………………………27 2.4 C/EBPα Post-Translational Modifications.........................................................32 2.5 C/EBPα Functional Characteristics...................................................................36 2.6 Experimental……...………………………………….………………………………40 2.7 Future Work…...…………………………………………………………….……….53 CHAPTER 3: MicroRNA-181A AND ITS ROLE IN AML…………………………………………...58 3.1 MicroRNA Discovery, Processing, and Characteristics in Hematopoiesis........58 3.2 MicroRNA in AML……………………………………………………………….…...63 3.3 MicroRNA-181 Family and Hematopoiesis……………………………….………65 3.4 MicroRNA-181a and Leukemia……………………………………………….……71 3.5 MicroRNA-181a and Targets Relating to the Innate Immune System…….…..73 3.6 Experimental……………………….………………………………………….……..77 3.7 Future Work…..………………………………..………………………………….…86 ix CHAPTER 4: LENALIDOMIDE INDUCES microRNA-181A THROUGH C/EBPα-p30 EXPRESSION………90 4.1 Lenalidomide: Second-Generation Immunomodulatory Compound….............90 4.2 Lenalidomide and Leukemia…………………..……………………………..........93 4.3 Experimental………………………...…..…………………………........................97 4.4 Future Work…………………………………………………………………………113 CHAPTER 5: ACUTE LYMPHOBLASTIC LEUKEMIA………..………………….………............118 5.1 Introduction and Background…………...…………….………..…..…….……….118 5.2 ALL Classification...…………….……………………………………….………….121 5.3 ALL Chromosomal Translocation: TEL/AML1………………..…………........…125 5.4 Experimental……………………………….……………….……………….………132
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