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Mechanistic Insights Into the Effect of RUNX1/ETO Knockdown in T(8;21) AML

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Mechanistic Insights Into the Effect of RUNX1/ETO Knockdown in T(8;21) AML Mechanistic insights into the effect of RUNX1/ETO knockdown in t(8;21) AML Anna Pickin A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY Institute of Biomedical Research Birmingham Centre for Genomic Biology College of Medical and Dental Sciences University of Birmingham September 2016 1 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT The mutation of transcription factor genes is a main cause for acute myeloid leukaemia. RUNX1/ETO, the product of the t(8;21) chromosomal translocation, subverts normal blood cell development by impairing myeloid differentiation. RUNX1/ETO knockdown alleviates this block, with a global reprogramming of transcription factor binding and initiation of myeloid differentiation. Co-depletion of the myeloid transcription factor C/EBPα with RUNX1/ETO suppressed this differentiation response. Furthermore, C/EBPα overexpression largely phenocopied the effect of RUNX1/ETO knockdown. Our data show that low levels of C/EBPα are critical to the maintenance of t(8;21) AML and that C/EBPα drives the response to RUNX1/ETO depletion. To examine how changes in transcription factor binding impact on the activity of cis- regulatory elements we mapped genome wide promoter-distal-element interactions in a t(8;21) AML cell line, via Capture HiC, and found that hundreds of interactions were altered by RUNX1/ETO knockdown. Differentially interacting elements exhibited changes in C/EBPα binding and were enriched for the CTCF motif. Our results demonstrate that the presence or absence of RUNX1/ETO has a profound impact on the intra-nuclear organisation of t(8;21) AML cells, and indicate which transcription factors are driving these changes. This work provides a novel mechanism for the RUNX1/ETO mediated differentiation block in t(8;21) AML. 2 AKNOWLEDGEMENTS Foremost, I express my sincerest gratitude to Professor Constanze Bonifer for her constant support, encouragement and immense knowledge. She has been an excellent supervisor and, despite an extremely busy schedule, always found time for me. I thank Dr Cameron Osborne for his generosity, patience and stimulating scientific discussion during my stay in London to learn Capture HiC. I would also like to thank Professor Ruud Delwel and Dr Stefan Gröschel for giving me the opportunity to work in the Netherlands and for their very thorough training. These experiences have been invaluable. A special thank you goes to Dr Anetta Ptasinska. Not only has she been a great academic supervisor, she has also given me unwavering emotional support. It has been a pleasure working with her during these four years. Thank you also to Dr Salam Assi for all of her bioinformatic analysis. I really appreciate the time spent getting to grips with the complicated methods in this study. I thank the other members of the Bonifer-Cockerill laboratory and last, but by no means least, I would like to thank my friends and family. Although they could not always completely understand the trials and tribulations of laboratory life, they were always there for me. This work was funded by Cancer Research UK 3 TABLE OF CONTENTS Chapter 1. INTRODUCTOIN ....................................................................................................... 1 1.1 Chromatin structure ..................................................................................................................... 1 1.1.1 The nucleosome ............................................................................................................... 1 1.1.2 Histone variants ................................................................................................................ 4 1.1.3 Chromatin remodelers ..................................................................................................... 5 1.1.4 Chromatin modifications .................................................................................................. 6 1.2 Eukaryotic transcription and its regulation .................................................................................. 9 1.2.1 The Eukaryotic transcriptional machinery ..................................................................... 10 1.2.2 Role of transcription factors ........................................................................................... 13 1.2.3 Transcriptional regulatory elements .............................................................................. 15 1.3 Higher-order chromatin structure .............................................................................................. 18 1.3.1 Topologically associating domains ................................................................................. 18 1.3.2 Chromosome compartments ......................................................................................... 20 1.3.3 Chromosome territories ................................................................................................. 22 1.3.4 Transcription factories ................................................................................................... 22 1.3.5 The beta-globin locus and the HOX gene cluster ........................................................... 23 1.4 Interrogating nuclear organisation and chromosome conformation ......................................... 24 1.4.1 Assays based on microscopy .......................................................................................... 25 1.4.2 Chromosome conformation capture .............................................................................. 27 4 1.5 Haematopoiesis .......................................................................................................................... 31 1.5.1 Origin of haematopoietic stem cells .............................................................................. 32 1.5.2 The maintenance of haematopoietic stem cells - the stem cell niche ........................... 33 1.5.3 Hierarchical differentiation of haematopoietic stem cells ............................................. 33 1.5.4 Myelopoiesis is controlled by transcription factors ....................................................... 40 1.6 Acute myeloid leukaemia ........................................................................................................... 43 1.6.1 AML can be caused by mutations in haematopoietic transcription factors .................. 43 1.6.2 CEBPA mutations and expression levels in AML ............................................................ 46 1.6.3 Clonal evolution of leukaemia ........................................................................................ 49 1.6.4 AML with the t(8;21) translocation ................................................................................ 50 1.6.5 Secondary mutations are required for the development of t(8;21) AML ...................... 51 1.7 Molecular pathogenesis of t(8;21) leukaemia ............................................................................ 54 1.7.1 The function of RUNX1 transcription factor ................................................................... 54 1.7.2 RUNX1/ETO exhibits dominant inhibition of RUNX1 function ....................................... 56 1.7.3 RUNX1/ETO has effects distinct from RUNX1 inhibition ................................................ 57 1.7.4 RUNX1/ETO interferes with the activity of various transcription factors ...................... 59 1.7.5 RUNX1/ETO alters the epigenetic landscape ................................................................. 61 1.7.6 RUNX1/ETO functions in an oligomeric complex ........................................................... 63 1.7.7 The effect of RUNX1/ETO knockdown in t(8;21) AML ................................................... 64 1.7.8 The role of C/EBPα in t(8;21) AML ................................................................................. 69 1.8 The role of SP1 in t(8;21) AML .................................................................................................... 71 5 1.9 t(8;21) AML is dependent on wild type RUNX1 for survival ....................................................... 73 1.9.1 Targeting transcription factor interaction ...................................................................... 73 1.9.2 Targeting RUNX1 with small molecule inhibitors ........................................................... 75 1.10 Aims and Objectives ................................................................................................................. 77 Chapter 2. METHODS ............................................................................................................. 80 2.1 Cell line culture ........................................................................................................................... 80 2.2 Small molecule inhibitor treatment ............................................................................................ 80 2.3 siRNA and shRNA mediated RUNX1/ETO and C/EBPα depletion
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