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Genome-Wide DNA Methylation in Chronic Myeloid Leukaemia

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Genome-Wide DNA Methylation in Chronic Myeloid Leukaemia Genome-wide DNA Methylation in Chronic Myeloid Leukaemia Alexandra Bazeos Imperial College London Department of Medicine Centre for Haematology Thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy of Imperial College London 2015 1 Abstract Epigenetic alterations occur frequently in leukaemia and might account for differences in clinical phenotype and response to treatment. Despite the consistent presence of the BCR-ABL1 fusion gene in Philadelphia-positive chronic myeloid leukaemia (CML), the clinical course of patients treated with tyrosine kinase inhibitors (TKI) is heterogeneous. This might be due to differing DNA methylation profiles between patients. Therefore, a validated, epigenome-wide survey in CML CD34+ progenitor cells was performed in newly diagnosed chronic phase patients using array-based DNA methylation and gene expression profiling. In practice, the CML DNA methylation signature was remarkably homogeneous; it differed from CD34+ cells of normal persons and did not correlate with an individual patient’s response to TKI therapy. Using a meta-analysis tool it was possible to demonstrate that this signature was highly enriched for developmentally dynamic regions of the human methylome and represents a combination of CML-unique, myeloid leukemia- specific and pan-cancer sub-signatures. The CML profile involved aberrantly methylated genes in signaling pathways already implicated in CML leukaemogenesis, including TGF-beta, Wnt, Jak-STAT and MAPK. Furthermore, a core set of differentially methylated promoters were identified that likely have a role in modulating gene expression levels. In conclusion, the findings are consistent with the notion that CML starts with the acquisition of a BCR-ABL1 fusion gene by a haematopoietic stem cell, which then either causes or cooperates with a series of DNA methylation changes that are specific for CML. 2 Declarations All clinical diagnoses referred to in this thesis were made by other physicians and not myself. Dr. Rob Lowe performed all bioinformatic analyses. Unless otherwise stated or referenced, all work presented in this thesis is my own. ------------------ Alexandra Bazeos The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they no do use it for the commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. 3 Acknowledgements On December 24th 2013, the day my dearest friend and mentor Professor John Goldman died, his daughter Cassie gave me her father’s MD thesis “Leukaemic Stem Cells”. On the first page in faded typewriter print was the epigraph “Felix, qui potuit rerum cognoscere causas” literally translated “Happy is he who has been able to understand the causes of things”. This is certainly true; the complexities of the epigenetic code in chronic myeloid leukaemia had provided the backdrop for hours of animated debate, usually late into the night with John diligently commenting on yet another paper I had presented him with. His enthusiasm was infectious, his intellect superhuman and his ability to interpret scientific data extraordinary. Moreover, John guided me, placed opportunities in front of me and showed me the doors that might be useful to open. I know with certainty that I will never have the privilege of meeting anyone like him again. I am grateful to my supervisors Professors Vardhman Rakyan, Jane Apperley and Letizia Foroni. Vardhman, thanks for allowing me to spend so much time with your group at the Blizard and equipping me with the molecular techniques necessary to carry out this study. Letizia, without your practical support, I am not sure that this thesis would have ever reached its conclusion. I would especially like to thank my friend and collaborator Dr. Rob Lowe for not only providing his bioinformatic expertise but also for contributing many excellent ideas during our time working together. It is true to say that without his analyses (and re- analyses), endless assistance and kindness; my project would genuinely not have been possible. I must also express my greatest appreciation and respect to the patients many alive, some no longer, who in the face of adversity donated the blood and bone marrow samples that form the basis of this work. Thanks also, to my medical and nursing staff colleagues at The Catherine Lewis Centre, Hammersmith Hospital. This work was generously supported by the Medical Research Council’s Clinical Research Training Fellowship and Leuka (Registered charity no. 1154856). 4 Finally, I acknowledge my close friends and amazing family whose boundless love, encouragement and faith will forever inspire my human experience. Dad, Mum, Anna and Sophia, as long as we have each other, everything is possible. I dedicate my thesis to John Goldman’s delectable grandchildren Bodecia (aged 7), Juno (aged 7), Zephyrus (aged 6) and Penn (aged 6). 5 1 INTRODUCTION................................................................................................ 13 1.1 Milestones in CML ..................................................................................................................... 13 1.2 Aetiology and epidemiology of CML ......................................................................................... 18 1.3 Clinical aspects of CML ............................................................................................................. 19 1.4 Treatment and treatment related outcomes ............................................................................. 25 1.5 Resistance to imatinib and other TKIs ..................................................................................... 30 1.6 Molecular biology of CML ......................................................................................................... 34 1.7 Epigenetics ................................................................................................................................... 41 1.8 Hypothesis and aims of this thesis ............................................................................................. 57 2 MATERIALS AND METHODS ........................................................................ 58 2.1 Materials ...................................................................................................................................... 58 2.2 Methods ....................................................................................................................................... 65 3 IDENTIFICATION OF A UNIQUE DNA METHYLATION SIGNATURE IN CML CELLS ......................................................................................................... 94 3.1 Background ................................................................................................................................. 94 3.2 Hypothesis ................................................................................................................................... 97 3.3 Objectives .................................................................................................................................... 97 3.4 Results .......................................................................................................................................... 98 3.5 Discussion .................................................................................................................................. 182 4 THE EFFECTS OF BCR-ABL1 CONDITIONAL EXPRESSION ON THE GENOME-WIDE DNA METHYLATION PATTERNS OF U937P210BCR- ABL/C6 CELLS ....................................................................................................... 187 6 4.1 Introduction .............................................................................................................................. 187 4.2 Hypothesis ................................................................................................................................. 189 4.3 Aims ........................................................................................................................................... 189 4.4 Methods and overall experimental design .............................................................................. 190 4.5 Results ........................................................................................................................................ 194 4.6 Discussion .................................................................................................................................. 202 5 INVESTIGATING THE IMPACT OF ABERRANT DNA METHYLATION ON GENE EXPRESSION IN CML ....................................................................... 205 5.1 Introduction .............................................................................................................................. 205 5.2 Hypothesis ................................................................................................................................. 208 5.3 Aims ........................................................................................................................................... 208 5.4 Methods ..................................................................................................................................... 209 5.5
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