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Modeling and Analysis of Acute Leukemia Using Human Hematopoietic Stem and Progenitor Cells

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Modeling and Analysis of Acute Leukemia Using Human Hematopoietic Stem and Progenitor Cells Modeling and analysis of acute leukemia using human hematopoietic stem and progenitor cells A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirement for the degree of Doctor of Philosophy in the Molecular &Developmental Biology Graduate Program of the College of Medicine by Shan Lin BS, Tsinghua University, 2009 Dissertation Committee: James C. Mulloy, PhD (Chair) Geraldine Guasch, PhD Ashish R. Kumar, MD, PhD Daniel T. Starczynowski, PhD Yi Zheng, PhD Abstract For decades, elegant mouse models have yielded important insights into the complex biology of acute leukemia development. However, species differences between human and mouse could have significant influences on biological and translational applications. Therefore, human primary hematopoietic cells and xenograft mouse models have become important research tools in the field. In this report, we briefly review the methodologies that use human primary hematopoietic cells to model acute leukemia and examine the effects of leukemic oncogenes. The advantages and limitations of the human model system compared to syngeneic mouse models are discussed. The species-related complexity in human disease modeling is highlighted in the study establishing a faithful model of proB-ALL caused by MLL-AF4, the fusion product of the t(4;11). MLL-AF4 proB-ALL has poor prognosis, the lack of an accurate model hampers the study of disease pathobiology and therapeutic testing. We find human AF4 cDNA inhibits retroviral production and efficient transduction, this limitation can be overcome by fusing MLL with murine Af4, highly conserved with human AF4. Whereas MLL-Af4-transduced murine cells induce only AML, transduced human CD34+ cells produce proB-ALL faithful to t(4;11) disease, fully recapitulating the immunophenotypic and molecular aspects of the disease. We find that MLL-fusion leukemia shows genetic heterogeneity driven by differential DNA binding of the fusion proteins. We report lineage plasticity as a new mechanism of resistance to CD19-directed therapy in t(4;11) patients that exemplifies the clinic relevance of our model. This model can provide unique insight for targeting t(4;11) ALL. In addition, by using a pre-leukemia model of AML1-ETO, the fusion protein generated by t(8;21) and associated with AML, we identify FOXO1 as an essential self-renewal factor in this disease. FOXO1 is consistently upregulated in t(8;21) AML and functions as a critical oncogenic mediator rather than a tumor suppressor. Expression of FOXO1 in human CD34+ cells promotes a pre-leukemic state, partially phenocopying the cellular and transcriptional effects of AML1-ETO expression. These functions are specific to FOXO1 and are not elicited by FOXO3, and the DNA binding ability of FOXO1 is required. AML1-ETO and FOXO1 co-occupy the majority of their binding sites, whereby FOXO1 binds to i multiple crucial self-renewal genes and is necessary for their activation. In agreement with this observation, loss of FOXO1 inhibits the long-term proliferation and clonogenicity of AML1-ETO cells. Thus, increased FOXO1 represents a new mechanism for acquiring aberrant self-renewal by pre-leukemia stem cells and provides a novel target for therapeutic intervention. This study also serves as an example to demonstrate the usefulness and capacity of the human model system to dissect critical targets and pathways for acute leukemia development. ii iii Acknowledgements I would like to thank my advisor Dr. James Mulloy. It is really fortunate for a graduate student to have a great mentor as you. Thank you for providing me with constant support not only for research work but also helping solve many non-scientific issues, so that I can focus on science, which is the more interesting part of the Ph.D life. You are always optimistic and encouraging, leading me to see the promising things during the struggling, and to overcome all the difficulties. Thank you for providing me with an open environment to develop scientifically and independently. I also want to thank other committee members Drs. Yi Zheng, Ashish Kumar, Daniel Starczynowski and Guasch Geraldine for their invaluable advice and patience through my doctoral training. They are always ready for help with broad knowledge and critical thinking, which benefit me a lot in the pursuit of my graduate degree and future studies. Thanks the Division of Experimental Hematology and Cancer Biology for having such a nice collaborating atmosphere, everybody is willing to help. I appreciate Drs. Lee Grime, Gang Huang, Mohammad Azam and other faculty members, whose brilliant ideas and suggestions have broadened my view of my own projects as well as leukemia research. Huge Thanks to our wonderful collaborators, Dr. Michael Thirman and his colleagues in University of Chicago, and Dr. Constanze Bonifer and her group from University of Birmingham. Thanks all current and previous members in Mulloy lab, I would not be able to finish my study without their selfless support and hard work. Kevin gave me a great orientation when I joined the lab, preparing me with all necessary techniques for leukemia research. He, Susumu and Fu-sheng spent a lot of their private time to revise my proposal for qualifier examination, that is why I can survive. Mark, Christina and Mahesh have taken care most of my mice experiments. Andy and Janet make really delicious dessert. Ben and Eric, like other people, never say no when I need a help. Also to all visiting scholars and students and supporting staff that I have worked with, Shuhong, Vase, Dino, Alba, Sam, Rushi, Bargav, Ann, Courtney, Mahima, Navin and Ryan, thank you for making lab fun and I appreciate all I learn from you. Special thanks to Alba for hard work to gather data for me even when she was in a stressful period. And iv thanks rotating students Jordan and David for giving me an opportunity to be a mentor, and your contributions really help my project, I hope you will make a great success for your Ph.D study. I would also thank colleagues from other labs, with you I can quickly learn new skills and get my experiments cooking. Last but not least are my dear family, without whom I would never become who I am now. Many thanks to my dad and mom, Xiaohui Lin and Manli Peng, who are my greatest supporters. Even though you do not know too much about the research, your unwavering moral support and understanding carry me through all difficulties and challenges of the doctoral training, and your life experiences will also be invaluable reference for my future life. Thanks to my lovely girlfriend, Liu Yang, you are the sunshine, wiping out all my stress and depression. Thanks all friends, Bo Liu, Xuan Zhou, Sha Wang, Qin Wang, Rui Wang, MukYan Wong, Kuan-shen Wong, Jiang Jiang Yang, Zhen Zhang, Jieqing Fan, Jia You, Cindy Whitaker, Shaun Rudolph and Tamala Whitaker, who help me to settle down in Cincinnati, from renting an apartment to fixing a car, you are always there when I am in trouble. Thanks for all the people whose names are not mentioned due to limited page space but whose help will never be forgot. v Contents List of figures……………………………………………………………………………………...1 Chapter 1. General introduction…………………………………………………………………...2 1.1 Acute leukemia…………………………………………………………………………2 1.2 Mouse model for leukemia study………………………………………………………….2 1.3 Human cell model system for acute leukemia study……………………….……………....7 1.4 Limitation of the human cell model system…………………………………………...11 1.5 References.…………………………………………………………………………..14 Chapter 2. Establishment of a faithful MLL-AF4 proB-ALL model using human cells………...19 2.1 Introduction………………………………………………………………………………19 2.2 Results………...….………………………………………………………………………29 2.3 Discussion………....………...…………………………………………………………..44 2.4 Experimental procedures…….…………………………………………………………...48 2.5 References………………………………………………………………………………..55 2.6 Appendix Tables….………………………………………………………………………61 Chapter 3. FOXO1-induced self-renewal defines the AML1-ETO pre-leukemic program………68 3.1 Introduction………………………………………………………………………………68 3.2 Results….…………………………………………………...……………………………76 3.3 Discussion………………………………………………………………………………..87 3.4 Experimental procedures…….…………………………………………………………...90 3.5 Reference………………………………………………………………………………....97 Chapter 4. Future implications………………………………………………………………….103 vi 4.1 Future directions………………………………………………………………………...103 4.2 References………………………………………………………………………………106 vii List of figures Figure 1-1 Experimental models for leukemia study………………………………..………………………3 Figure 2-1 Schematic representation of the MLL protein and MLL fusions……………………………….20 Figure 2-2 Stably expressed MLL-Af4 in mouse HSPCs induces AML…………………………………...31 Figure 2-3 Human CD34+ cells expressing MLL-Af4 initiate proB ALL in NSG mice…………………...33 Figure 2-4 MLL-Af4 proB-ALL is transplantable…………………………………………………………34 Figure 2-5 Gene signatures derived from t(4;11) patients are enriched in MLL-Af4 proB-ALL…………..35 Figure 2-6 MLL-Af4 and MLL-AF9 lead to block at distinct B cell development stage…………………...37 Figure 2-7 MLL-Af4 gene expression profile recapitulates t(4;11) specific molecular signature………….39 Figure 2-8 MLL-fusions promote distinct gene expression profiles via differential DNA binding………...40 Figure 2-9. MLL-Af4 maintains lymphoid potential after myeloid priming……………………………….41 Figure 2-10 MLL-Af4 phenotypic myeloid cells keep an active lymphoid program………………………42 Figure 2-11 Phenotypic flexibility of t(4;11) ALL contributes to resistance to CD19-taregeted therapy…..45 Figure 3-1 t(8;21)
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