Investigating the anti-apoptotic role of EBV in endemic Burkitt lymphoma by LEAH FITZSIMMONS A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Cancer Sciences College of Medical and Dental Sciences The University of Birmingham September 2014 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties.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 Epstein-Barr virus (EBV) has been etiologically associated with Burkitt lymphoma (BL) since its discovery 50 years ago, but despite this long-standing association the precise role of the virus in the pathogenesis of BL remains enigmatic. EBV can be lost spontaneously from EBV-positive BL cell lines, and these EBV-loss clones have been reported to exhibit increased sensitivity to apoptosis. We have confirmed and extended those observations and report that sporadic loss of EBV from BL cells is consistently associated with enhanced sensitivity to apoptosis-inducing agents and conversely, reduced tumorigenicity in vivo. Importantly, reinfection of EBV-loss clones with EBV can restore apoptosis protection, although surprisingly, individual Latency I genes cannot. We also used inducible pro-apoptotic BH3 ligands to investigate Bcl-2-family dependence in BL clones as well as profiling gene expression changes in response to apoptosis induction in EBV- positive versus EBV-loss clones. We found that EBV-loss was consistently associated with enhanced sensitivity to BH3-ligand-induced death and increased activation of apoptosis signalling pathways, although no individual apoptosis-related gene was responsible. Instead we find that Latency I EBV genes co-operate to co-ordinately repress the BH3-only proteins Bim, Puma and Noxa to inhibit apoptosis in BL. Acknowledgements This PhD would not have been possible without the support and guidance of my supervisors, Martin Rowe and Gemma Kelly, to whom I am very grateful. I also owe special thanks to Dr Rosemary Tierney for discussion and guidance, as well as technical, intellectual and emotional support. I would like to thank the members of the Rowe and Strasser research groups who contributed their time, expertise, patience and advice to me particularly; Andreas Strasser, Marco Herold, Nikki Smith, Andrew Bell, and Claire Shannon-Lowe. I would also like to thank Alan Rickinson for all the helpful advice and discussion. I am grateful to several members of other research groups, who kindly contributed reagents and advice to this project, which rescued several sets of experiments when they ground to a halt. Additionally, I could not have undertaken this degree without financial support from the Kay Kendall Leukaemia Fund and Cancer Research UK. I owe enormous thanks to my friends and family who have all been forced to endure endless tirades about viruses, cancer, immunology and non-coding RNAs. I am particularly grateful to Holly Joynes, Kathryn Warder, Tommy Morrison and Kipp Jones who have borne the worst of the brunt and will probably spend the rest of their lives avoiding scientists in order to recover. Most of all, I would like to thank Andrew Holland for keeping me together and forgiving me denying us a holiday for the last two years as well as putting up with me constantly organising our lives around my cell culture commitments. I promise we can go on holiday now, although I can’t promise that you’re not going to end up in tissue culture on New Year’s Eve again. Contents 1. Introduction............................................................................................................................... 1 1.1 The early history of EBV research ................................................................................. 1 1.1.1 ‘More than a curiosity’ .......................................................................................... 1 1.1.2 ‘A cell filled with herpesvirus!’ .............................................................................. 2 1.1.3 A ‘stalking horse’ ................................................................................................... 2 1.2 EBV ................................................................................................................................ 3 1.2.1 Taxonomy .............................................................................................................. 3 1.2.2 Structure ................................................................................................................ 3 1.2.3 Viral genome ......................................................................................................... 4 1.2.4 Strain variation ...................................................................................................... 4 1.2.5 EBV infection and growth transformation in vitro ................................................ 6 1.2.6 EBV infection and persistence in vivo ................................................................... 9 1.2.7 Reactivation and lytic cycle ................................................................................. 10 1.2.8 Tissue tropism ..................................................................................................... 12 1.2.9 Viral latency ......................................................................................................... 12 1.2.10 Disease associations ............................................................................................ 16 1.3 Burkitt lymphoma........................................................................................................ 19 1.3.1 Incidence and clinical characteristics .................................................................. 19 1.3.2 The role of cofactors in BL pathogenesis ............................................................ 22 1.3.3 Genetic factors .................................................................................................... 24 1.3.4 Treatment and prognosis .................................................................................... 26 1.3.5 The role of EBV in BL ........................................................................................... 27 EBNA1 .................................................................................................................................. 29 EBER RNAs ........................................................................................................................... 30 BART microRNAs ................................................................................................................. 31 1.4 Apoptosis ..................................................................................................................... 33 1.4.1 Overview of apoptosis ......................................................................................... 33 1.4.2 The Bcl-2 family ................................................................................................... 38 1.4.3 Regulation of the Bcl-2 family ............................................................................. 43 1.4.4 The Bcl-2 family and lymphomagenesis .............................................................. 44 1.4.5 Targeting the Bcl-2 family in human disease ...................................................... 45 1.5 Aims and Objectives .................................................................................................... 46 2. Materials and Methods ........................................................................................................... 47 2.1 Cell culture .................................................................................................................. 47 2.1.1 Cell lines used in this study ................................................................................. 47 2.1.2 Cell line maintenance .......................................................................................... 48 2.1.3 Single cell cloning of BL cell lines ........................................................................ 49 2.1.4 Preparation of lentivirus stocks........................................................................... 49 2.1.5 Generation of stable lentivirus-transduced cell lines ......................................... 50 2.1.6 Preparation of infectious stocks of recombinant EBV ........................................ 51 2.1.7 Reinfection of EBV-loss clones with recombinant EBV ....................................... 52 2.2 Cell death, cell growth and cell survival assays ........................................................... 53 2.2.1 Cell viability assays .............................................................................................. 53 2.2.2 Proliferation assays ............................................................................................. 53 2.2.3 Apoptosis assays.................................................................................................. 54 2.2.4 Data analysis for apoptosis assays .....................................................................