CHARACTERIZATION OF TCL1-MURINE B-1A CELL TRANSCRIPTOME DYNAMICS REVEALS NOVEL INSIGHTS INTO CHRONIC LYMPHOCYTIC LEUKEMIA ONSET DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yuntao Dai, B.S., M.Sc Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2015 Dissertation Committee: Carlo Croce, MD; Advisor Jeffrey Parvin, MD, PhD Qianben Wang, PhD Flavia Pichiorri, PhD Copyright by Yuntao Dai 2015 ABSTRACT B-cell chronic lymphocytic leukemia (B-CLL or CLL) is the most common leukemia in adults in western countries. This disease seems to arise from genetic lesions that block differentiation of normal B lymphocytes. Patients with CLL (both aggressive and indolent) are at risk for development of invasive over-proliferation of malignant CD5 B lymphocytes, caused by an immature expansion of B cell precursors (B-1a). B-1a cells are thus ideal for the study of CLL disease initiation steps. From previous studies it is known that uncontrolled T-Cell Leukemia/Lymphoma 1A (TCL1) signaling is involved in aggressive CLL development. In order to substantiate the pathogenic effect of TCL1 and to provide paths to study CLL in vivo, EuTCL1-transgenic mice (TCL1 mice), with targeted human TCL1 overexpression in B cells, have been generated. TCL1 mice, which consistently develop aggressive CLL symptoms, represent a good model to screen for novel factors that may play significant roles in CLL disease initiation by studying their transcriptome profiling. This will lead to the discovery of new prognostic markers and/or therapeutic targets for clinical use. Next Generation Sequencing RNA sequencing (RNA seq) provides comprehensive overviews of transcriptome dynamics and thus is ideal for the genomic profiling. Therefore, as described in Chapter 2, RNA seq was performed to compare the B-1a cell transcriptome of early age (1-4mo) TCL1-transgenic mice to the wild-type (WT) ii counterparts. We found that: i) the expression levels of several coding and non-coding genes are deregulated; ii) the number of deregulated genes increases with age; and iii) certain oncogenic pathways such as NF-kB are stimulated due to the targeted TCL1 overexpression in mice. We focused on the top 15 up/down regulated genes in most genotype/age categories to perform further studies. As shown in Chapter 3 we validated the selected genes and picked the most promising candidates to focus on. In particular, quantitative real time PCR (qRT-PCR) was performed to validate the transcriptional deregulation of protein coding genes in transgenic mice vs WTs, specifically. Neto2 (Neuropilin and Tolloid-like 2) and Hbegf (Which resulted to be upregulated and downregulated, respectively), while the transcriptional deregulation of the noncoding genes AI427909 and 1700097N02Rik (which resulted to be upregulated and downregulated, respectively) was not validated. For the protein coding genes Neto2 and Hbegf, western blots confirmed their expression changes at the protein level. Validation of the changes in Neto2 and Hbegf was performed in human samples and cell lines. qRT-PCR on 31 human samples revealed that both NETO2 and HBEGF showed collinear relationship with TCL1 expression levels (positive and negative, respectively) confirmed at the protein level by carrying out western blot experiments on TCL1-transfected human cell lines. Moreover, the correlation between NETO2 and TCL1 was verified by western blot also in randomly selected patients. Therefore, we decided to focus on the potential oncogene NETO2. NETO2 encodes a transmembrane protein that regulates glutamate receptor function and modulates glutamate signaling in the central nervous system (CNS). Glutamate iii receptor, in addition to its primarily reported role in CNS, was recently published to be deregulated in several cancer types. Given that our results show NETO2/Neto2 is upregulated in CLL-related samples from both human and mouse as well as TCL1- transfected cell lines, it is reasonable to conclude that NETO2 can be associated with CLL, therefore representing a potential prognostic marker or therapeutic targets for future clinical uses. To validate this, NETO2 transgenic mouse models are proposed in the discussion on future directions in Chapter 4. Taken together, our findings not only continue to decode the mechanism of CLL through appreciation of the signaling network, but also help us understand NETO2 and its potential prognostic and therapeutic values. iv DEDICATION This document is dedicated to my family. v ACKNOWLEDGEMENTS First and foremost, I would like to express my deep and sincere gratitude to my advisor, Dr. Carlo Croce, for his mentorship, support, understanding and patience at all times. His wide knowledge, illuminating insight and persistent passion for science have inspired me all the way along my study. I would like to acknowledge my graduate committee members, Dr. Jeffrey Parvin, Dr. Qianben Wang, and Dr. Flavia Pichiorri for their insights, supports and suggestions. I want to especially thank my laboratory mentors Dr. Mario Acunzo, Dr. Giulia Romano, Dr. Nicola Zanesi, and Dr. Veronica Balatti for their excellent mentorship. I want to thank our lab manager Dr. Dorothee Wernicke-Jameson, our lab secretary Mrs Sharon R Palko, and our department staff members Mrs Tornik Colette and Mrs Erin Kimbrell for their assistance. I also want to thank my supportive friends Dr. Mei Zhang, Dr. Fabienne McClanahan, and Dr. Christopher Walker for their consistent effort and encouragement to support me to move towards my life goal. Thank you to all the past and present members of the Croce lab and friendship labs who helped me along the way: Dr. Dario Veneziano, Dr. Alessandro Lagana, Dr. Hui- Lung Sun, Mr. Douglas Cheung, Dr. Young-Jun Jeon, Dr. Pearlly Yan, Dr. Yuri Pekarsky, Dr. Stefano Volinia, Dr. Sukhinder Sandhu, Mr. Bryan McElwain, Dr. vi Giovanni Nigita, Dr. Lara Rizzotto, Dr. Ri Cui, Dr. Huijun Wei, Dr. Yong Peng, Dr. Zhenghua Luo, Dr. Taewan Kim, Dr. Sung Suk Suh, Miss. Pooja Josh, Dr. Jessica Consiglio, Dr. Pierluigi Gasparini, Dr. Jinghai Wu, Dr. Esmerina Tili, Dr. Francesca Lovat, Dr. Federica Calore, Dr. Alex Palamarchuk, Dr. Dario Palmieri, Dr. Anna Tessari, Mr. Timothy Richmond, Ms. Janae Dulaney, Ms. Prasanthi Kumchala, Dr. Dayong Wu, Dr. Hongtao Jia. Last but not least, I am grateful to my parents, my relatives and friends back in China for their constant love and support. vii VITA December 22, 1982 ........................................Born in Wuhan, China September 2001 to June 2005 ........................B.S., Biological Sciences, Huazhong Agriculture University, Wuhan January 2007 to July 2009 ...........................M.S., Plant Pathology University of Arkansas, Fayetteville, AR June 2010 to present ......................................Graduate Research Associate, The Ohio State University viii PUBLICATIONS 1. Nigita G, Acunzo M, Romano G, Lagana A, Veneziano D, Dai Y, Vitiello M, Wernicke D, Ferro A, Croce CM (2015) MicroRNA editing favors dynamic cellular changes in hypoxic conditions. Submitted. 2. Dai Y, Veneziano D, Lagana A, Balatti V, Zanesi N, McClanahan F, Nigita G, Sun HL, Walker C, Jeon YJ, Romano G, Yan P, Cheung D, Peng Y, Pekarsky Y, Acunzo M, Croce CM (2015) Characterization of TCL1-Murine B-1a cell transcriptome dynamics reveals novel insights into CLL onset. Submitted. 3. Srivastava AK, Han C, Zhao R, Cui T, Dai Y, Mao C, Zhao W, Zhang X, Yu J, Wang QE (2015) Enhanced expression of DNA polymerase eta contributes to cisplatin resistance of ovarian cancer stem cells. PNAS 112: 4411-4416. 4. Dai Y, Winston E, Correll JC, Jia Y (2014) Induction of avirulence by AVR- Pita1 in virulent U.S. field isolates of Magnaporthe oryzae. The Crop Journal 2: 1-9. 5. Peng Y, Dai Y, Hitchcock C, Yang X, Kassis ES, Liu L, Luo Z, Sun HL, Cui R, Wei H, Kim T, Lee TJ, Jeon YJ, Nuovo GJ, Volinia S, He Q, Yu J, Nana- Sinkam P, Croce CM (2013) Insulin growth factor signaling is regulated by micro-RNA 486, an underexpressed microRNA in lung cancer. PNAS 110: 15043-15048. 6. Dai Y, He H, Wise GE, Yao S (2011) Hypoxia promotes growth of stem cells in dental follicle cell populations. Journal of Biomedical Science and Engineering 4: 454-461. 7. Yao S, Gutierrez G, He H, Dai Y, Liu D, Wise GE (2011) Proliferation of dental follicle derived cell populations in heat-stress conditions. Cell Proliferation 44: 486-493. 8. Dai Y, Jia Y, Correll JC, Wang X, Wang Y (2010) Diversification and evolution of the avirulence gene AVR-Pita1 in field isolates of Magnaporthe oryzae. Fungal Genetics and Biology 47: 973-980. 9. Jia Y, Liu G, Costanzo S, Lee S, Dai Y (2009) Current progress on understanding of genetic interactions of rice with rice blast and sheath blight fungi. Frontier Research in China 3: 231-239. 10. Zhang L, Lu Q, Chen H, Pan G, Xiao S, Dai Y, Li Q, Zhang J, Wu X, Wu J, Tu J, Liu K (2007) Identification of a cytochrome P450 hydroxylase, CYO81A6, as the candidate for the bentazon and sulfonylurea herbicide resistance gene, Bel, in rice. Molecular Breeding 19: 59-68. ix FIELDS OF STUDY Major Field: Molecular, Cellular and Developmental Biology x TABLE OF CONTENTS ABSTRACT ........................................................................................................................ ii DEDICATION .................................................................................................................... v ACKNOWLEGEMENTS .................................................................................................