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Investigating the Impact of Telomere Dysfunction on the Chronic Lymphocytic Leukaemia Genome

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INVESTIGATING THE IMPACT OF TELOMERE DYSFUNCTION ON THE CHRONIC LYMPHOCYTIC LEUKAEMIA GENOME Laura Escudero-Monreal A thesis submitted for the degree of Doctor of Philosophy September 2017 Institute of Cancer and Genetics School of Medicine Cardiff University Funded by Cancer Research Wales and Cardiff University I dedicate this Ph.D. thesis to everyone who suffers from cancer, for I hope one day, science can fight and win this battle. Laura Escudero-Monreal NOTICE OF SUBMISSION DECLARATION This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other award. Signed: Date: STATEMENT 1 This thesis is being submitted in partial fulfilment of the requirements for the degree of PhD. Signed: Date: STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated, and the thesis has not been edited by a third party beyond what is permitted by Cardiff University’s Policy on the Use of Third Party Editors by Research Degree Students. Other sources are acknowledged by explicit references. The views expressed are my own. Signed: Date: STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed: Date: STATEMENT 4: PREVIOUSLY APPROVED BAR ON ACCESS I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter-library loans after expiry of a bar on access previously approved by the Academic Standards & Quality Committee. Signed: Date: III ACKNOWLEDGEMENTS Firstly, I would like to thank my supervisors Professor Duncan Baird and Dr Kate Liddiard for the opportunity to undertake this PhD. I am very grateful for your support and guidance throughout the project. I would also like to thank Professor Chris Pepper for your advice, to the CLL patients and to Professor Chris Fegan for providing the patient samples. Thank you to Dr Kate Liddiard, Dr Rhiannon Robinson and Mrs Julia Grimstead for teaching me the techniques and for always being willing to help; to Dr Kevin Norris for facilitating the identification of the CLL patient samples with short telomeres; and to Dr Kez Cleal for your bioinformatics input. To everyone in the lab, I will always remember the lunch time laughs and the fun nights out. Particularly the events to raise money for charity including the CGB Bake Off, the 5K pretty muddy races with The GenetChicks and the Christmas secret Santa. It has been the best working environment and I will miss you all. Especially Sam Hyatt and Melanie Hurtz, thank you for being by my side during this journey; as well as my colleagues and my housemates, you became my best friends. Sobre todo, quiero dar las gracias a mi familia, por apoyar mis decisiones y ayudarme durante el camino a convertirme en investigadora. Sin vuestro cariño, ayuda y entendimiento, hoy no estaría aquí. A mis amigas más cercanas, gracias por recordarme que hay vida más allá de la academia y ayudarme a mantener un equilibrio en mi vida. Junto con mi familia, gracias especialmente por darme ánimos cuando más los necesitaba. To my partner Jahn, thank you for your understanding, for supporting my decisions and for your unconditional love. Thank you to your family, for always making me feel welcome. Thank you to Cancer Research Wales (CRW) and Cardiff University for the funding that has enabled this project. I am grateful to the European Association for Cancer Research (EACR), Cancer Research UK (CRUK), Keystone Symposia and The Genetics Society for the travel awards that allowed me to present my work at several conferences. It helped me to broaden my horizons, get new ideas and establish collaborations. To all of you, thank you. During these past 4 years I have grown as a person but especially as a scientist. Now, more than ever, I want to become an excellent researcher and help in the fight against cancer. I know it will be hard, but with passion and dedication, everything is possible. IV SUMMARY Short dysfunctional telomeres can result in chromosome fusions that drive genomic rearrangements and ultimately malignant progression. In Chronic Lymphocytic Leukaemia (CLL), telomere length (TL) is a powerful predictor of patient survival. The aim of this project is to understand the role that telomere dysfunction plays in driving the evolution of the CLL genome. Telomere fusions were detected in 71% of 276 CLL patients with short telomeres (TL<3.81Kb). From 9 CLL patient samples with the highest fusion frequency (>4.20x10- 5/diploid genome), 914 telomere fusions were characterised using Illumina HiSeq paired- end sequencing. In addition to intra- and inter-chromosomal recombinations, telomere fusions with non-telomeric loci were detected, including the ancestral telomere at Chr2q13, mitochondrial DNA, and loci associated with copy number aberrations in CLL. Telomere fusions also incorporated genes expressed in CLL-B lymphocytes and other oncogenes, suggesting that active chromatin is more prone to damage and aberrant repair. These events were potentially mediated by A-NHEJ that requires microhomology. Translocations involving hTERT, proximal to the 5p telomere, have previously been detected in CLL and associated with telomerase upregulation. In this study, 5p telomere fusions were identified in 22.6% patient samples and 172 fusion events that involved 5p were characterised, which may explain mechanisms of telomerase reactivation in cancer. Surprisingly, 67% of the 9 patients presented bimodal TL distributions compared to the overall cohort (4% of 276), consistent with intra-tumour heterogeneity, which was confirmed for one patient. For this patient with indolent disease, chromosomal rearrangements were detected, in addition to a novel mutation in REV3L implicated in translesion synthesis that may negatively impact cancer cells fitness and be a potential therapeutic target. This study shows that telomere dysfunction in CLL initiates genome-wide instability providing a source for genetic variability that allows intra-tumour heterogeneity and tumour progression. However, therapeutically-exploiting this instability could prove beneficial for patient outcome. V List of abbreviations LIST OF ABBREVIATIONS ADMIRE Does the ADdition of Mitoxantrone Improve Response to FCR chemotherapy in patients with CLL? (ADMIRE) trial AID APOBEC-Induced cytosine Deamination (c: canonical; nc: non-canonical) ALC Absolute Lymphocyte Count ALT Alternative Lengthening of Telomeres AML Acute Myeloid Leukaemia ARCTIC Attenuated dose Rituximab with ChemoTherapy In Chronic lymphocytic leukaemia (ARCTIC) trial A-NHEJ Alternative Non-Homologous End Joining ANOVA ANalysis OF VAriance APBs ALT-associated promyelocytic leukaemia (PML) nuclear bodies ATM Ataxia Telangiectasia Mutated ATR Ataxia telangiectasia and Rad3 related ATRX Alpha Thalassemia/Mental Retardation Syndrome X-Linked BAI Indexed BAM file BAM Binary version of a SAM file BFB Breakage-Fusion-Bridge BIR Break-Induced Replication BL Baseline BLAST Basic Local Alignment Search Tool BML Bloom syndrome protein bp Base Pairs BRCA1 Breast Cancer Type 1 Susceptibility BRCA2 Breast Cancer Type 2 Susceptibility BSA Bovine Serum Albumin BTK Bruton Tyrosine Kinase BWA Burrows-Wheeler Aligner CBS Central Biotechnology Service (CBS) CDK Cyclin-Dependent Kinase CF Chromosome Fusion CFS Common Fragile Site CHK1 Checkpoint Kinase 1 CHK2 Checkpoint Kinase 2 CIN Chromosomal INstability CLL Chronic Lymphocytic Leukaemia C-NHEJ Classical Non-Homologous End Joining CN-LOH Copy-Neutral Loss Of Heterozygosity CNV Copy Number Variation CTCF CCCTC-binding factor CtIP CtBP-interacting protein DAPI 4',6-diamidino-2-phenylindole VI List of abbreviations DAVID Database for Annotation, Visualization and Integrated Discovery DAXX Death-Domain Associated DDR DNA-Damage Response DKC Dyskerin DLBCL Diffuse Large B-Cell Lymphoma D-Loop Displacement Loop DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic acid DNA-PKcs DNA-Dependent Protein Kinase DN-hTERT Dominant Negative Human Telomerase Reverse Transcriptase dNTP Deoxyribonucleotide DSB Double-Strand Break ECTR Extrachromosomal telomeric repeats EDTA Ethylenediaminetetraacetic Acid EtBr Ethidium Bromide ETS E-twenty-six-domain EXO1 Exonuclease 1 FACS Fluorescence-Activated Cell Sorting FBS Fetal Bovine Serum FCS Fetal Calf Serum FCR Fluradabine, Chlorabucil and Rituximab FISH Fluorescence In situ Hybridisation FJ Fusion Junction (m: mapped; um: unmapped) FoSTeS Fork Stalling and Template Switching GABP GA-Binding Protein GRCh37 Genome Reference Consortium human build 37 GSEA Gene Set Enrichment Analysis GWAS Genome-Wide Association Studies H2AFX H2A histone Family, member X HCl Hydrochloric Acid HCT1080 Human fibrosarcoma cell line HCT116 Human colon cancer cell line HDR Homology Directed Repair HEK293 Human Embryonic Kidney 293 cell line hg19 Human genome version 19 HJ Holliday Junction hnRNPA1 heterogeneous nuclear RiboNucleoProtein A1 HR Homologous Recombination HR Hazard Ratio HSCT Haematopoietic Stem Cell Transplantation HT Q-FISH High Throughput Quantitative Fluorescent In situ Hybridisation ICGC International Cancer Genome Consortium Ig Immunoglobulin IGHV ImmunoGlobulin Heavy-chain Variable region gene (m:mutated; u: unmutated) IGV Integrative Genome Viewer VII List of abbreviations kb Kilobases Ku Ku70-Ku80
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