The Role of EZH2 in the Induction and Maintenance of Acute Myeloid Leukaemia Faisal Basheer Downing College University of Cambridge This dissertation is submitted for the degree of Doctor of Philosophy September 2017 Declaration The work presented in this dissertation was carried out under the supervision of Professor Brian Huntly from November 2013 to November 2016 in the Department of Haematology, University of Cambridge. This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except as declared in the Preface and specified in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. It does not exceed the prescribed word limit set by the Clinical Medicine and Clinical Veterinary Medicine Degree Committee. Faisal Basheer September 2017 i Acknowledgements First and foremost, I would like to thank my supervisor, Professor Brian Huntly, for providing me with the opportunity to work towards this thesis - for his help, encouragement and support over the last four years of my PhD. His dedicated high-level supervision, meticulous attention to detail and excellent direction throughout this time has undoubtedly helped me to maximise my potential in the lab throughout a highly interesting and rewarding project. I would also like to thank all of my colleagues and friends in the Huntly laboratory over the past four years - working together with all of you as a team has been an unforgettable and rewarding experience. Firstly, George Giotopoulos for all the technical training he has given me regarding most aspects of this project, in particular with the planning and setting up of the numerous mouse models required, but also for his constant help, supervision and general troubleshooting outwith this over the course of the entire four years. I am grateful to Eshwar Meduri for his help and assistance with analysis and referencing of the large genomic datasets generated during this project, using his bioinformatic expertise. Haiyang Yun and Daniel Sasca have been very supportive through the latter stages of this project, assisting not only in mouse work but also with helpful detailed discussions and their observations when analysing the genomic datasets. I would specifically like to thank Haiyang for his teaching and supervision when performing all the chromatin immunoprecipitation and subsequent necessary steps for high throughput sequencing. Olivia Sheppard and Sarah Horton have been constantly helpful with large parts of the mouse work and I would like to express my gratitude toward them. I would also like to thank Hikari Osaki for her help and teaching during the early parts of the project involving retroviral protocols and tissue culture training. Paolo Gallipoli has been helpful in collating and working with primary samples and inhibitors. The Vassiliou Group at the Sanger Centre, in particular Milena Mazan and Monika Dudek were very helpful for overseeing the breeding and maintenance of the Ezh2 conditional mice and thank you to their group leader, Dr George Vassiliou for his collaboration. The CIMR Flow cytometry core have also been very accommodating throughout this project and I am grateful to them. I would also like to thank the Wellcome Trust PhD Programme for Clinicians for awarding me with a junior clinical research fellowship which has allowed me to carry out this research, and all the patients with AML who have generously donated samples for this project. Finally, I would like to thank my wife, parents and family for their patience, understanding and support throughout everything. ii Work done and assistance received by others Dr George Giotopoulos: assistance with setting up in vitro and in vivo mouse models and analysing data assistance with setup and then overseeing functional validation mouse experiment Dr Eshwar Meduri: bioinformatic analysis of RNA-sequencing and ChIP-sequencing data Dr Haiyang Yun: supervision and assistance with ChIP-sequencing experiments Dr Daniel Sasca: assistance with library preparation of ChIP samples Genomics Core, CRUK RNA-sequencing and high throughput sequencing of ChIP samples Cambridge Institute: iii Abbreviations AML Acute Myeloid Leukaemia AML1-ETO Acute Myeloid Leukaemia 1 - Eight Twenty One BM Bone Marrow BSA Bovine Serum Albumin ChIP Chromatin immunoprecipitation cDNA Complementary DNA Ct Threshold cycle DLBCL Diffuse Large B Cell Lymphoma DMEM Dulbecco’s Modified Eagle’s Medium DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic acid EED Embryonic Ectoderm Development ES Embryonic Stem EZH1 Enhancer of Zeste Homologue 1 EZH2 Enhancer of Zeste Homologue 2 FBS Foetal Bovine Serum FCS Foetal Calf Serum FL Follicular Lymphoma GFP Green Fluorescent Protein GSK GlaxoSmithKline HEPES 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid HSC Haematopoietic Stem Cell iv HSPC Haematopoietic Stem and Progenitor Cell IC50 Half maximal inhibitory concentration IRES Internal Ribosomal Entry Site LB Luria-Bertani LT-HSC Long Term HSC MCS Multiple Cloning Site MDS Myelodysplasia MLL Mixed Lineage Leukaemia MPN Myeloproliferative Neoplasm MPP Multipotent progenitor MOZ-TIF2 Monocytic Leukaemia Zinc finger – Transcriptional Intermediary Factor 2 MTA Materials Transfer Agreement NPM1 Nucleophosmin PI Propidium Iodide PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction PRC1 Polycomb Repressive Complex 1 PRC2 Polycomb Repressive Complex 2 RE Restriction Enzyme RNA Ribonucleic Acid RPMI Roswell Park Memorial Institute RUNX1 Runt-related transcription factor 1 SCF Stem Cell Factor v SDS Sodium Dodecyl Sulphate SDS-PAGE SDS-Poly Acrylamide Gel Electrophoresis SEM Standard Error of the Mean ST-HSC Short Term HSC SUZ12 Suppressor of Zeste 12 TSS Transcriptional Start Site UV Ultraviolet WHO World Health Organisation WT Wild Type YFP Yellow Fluorescent Protein vi The role of EZH2 in the induction and maintenance of Acute Myeloid Leukaemia Faisal Basheer, Downing College Summary Acute myeloid leukaemia (AML) is an aggressive haematological cancer that remains an unmet medical need, with over 70% of patients still succumbing to this disease. Despite this, a deeper understanding of AML biology has allowed for the identification of novel agents with efficacy against specific molecular subtypes. Recently, a better understanding of aberrant epigenetic control in AML has led to the development of promising small molecule inhibitors of epigenetic regulators. The work described in this thesis details the results of genetic and pharmacological experiments designed to perturb the histone methyltransferase EZH2, a critical regulator of gene silencing during development and cell fate decisions and a potential therapeutic target, in the context of AML. The core aims were to assess its role during the development and subsequent maintenance of AML both in vitro and in vivo using mouse models, and to subsequently determine mechanisms by which its activity may facilitate leukaemia and how inhibition may abrogate leukaemic growth. Through utilising a conditional Ezh2 knock-out murine model together with bone marrow retroviral transduction and transplantation assays, Ezh2 was shown to have markedly differing roles during the induction and maintenance of three well-characterised subtypes of AML (MLL-AF9, AML1-ETO9a, MOZ- TIF2) both in vitro and in vivo, serving to highlight its complex nature. Whilst there were no significant effects in vitro, prior Ezh2 loss led to a significant acceleration in the induction of MLL-AF9 and AML1- ETO9a leukaemias in vivo, whilst significantly decelerating induction of MOZ-TIF2 leukaemias in vivo. This finding of a single gene demonstrating both tumour-suppressive and oncogenic roles, clearly dependent upon subtype of AML during the same induction phase of disease was particularly surprising. Contrary to the induction experiments, Ezh2 loss in vitro completely abolished the growth of MLL-AF9, AML1-ETO9a and MOZ-TIF2 transformed murine cell lines, strongly suggesting that it was an oncogenic facilitator of the transformed state. When examined in vivo, Ezh2 loss caused a significant prolongation in the latency of disease development of MLL-AF9 and AML1-ETO9a, suggesting that it was required for the maintenance of both as per the in vitro assays. Though a similar trend was established for secondary MOZ-TIF2 leukaemias in vivo, this was not statistically significant. vii Thus, completely contrasting, stage-specific requirements of Ezh2 during the evolution of AML were demonstrated. The diametrically opposite effects of Ezh2 loss between the induction and maintenance phases of MLL-AF9 and AML1-ETO9a leukaemias in vivo, was a novel finding, suggesting that this gene was unexpectedly capable of fulfilling both tumour-suppressive and oncogenic roles within the same subtypes of AML, but at different phases of disease. Whilst the maintenance experiments provided evidence for targeting EZH2 therapeutically, the acceleration seen upon Ezh2 loss during AML induction raised caution over EZH2 inhibition. This prompted a detailed mechanistic analysis to reconcile these disparate effects. Utilising RNA-sequencing, further genomic characterisation of differential gene expression changes following Ezh2 loss in murine HSPCs was made. This demonstrated a significant upregulation of genes - consistent with its role as a