Targeting the mitochondria for the treatment of MLH1-deficient disease ! A thesis submitted in partial fulfillment of the requirements of the Degree of Doctor of Philosophy at the University of London Dr. Sukaina Rashid Clinical Research Fellow Centre for Molecular Oncology Barts Cancer Institute Queen Mary University of London EC1M 6BQ UK ! ! ! 1! Declaration!! I, Sukaina Rashid confirm that the research included within this thesis is my own work or that where it has been carried out in collaboration with, or supported by others, that this is duly acknowledged below and my contribution indicated. Previously published material is also acknowledged below. I attest that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge break any UK law, infringe any third party’s copyright or other Intellectual Property Right, or contain any confidential material. I accept that the College has the right to use plagiarism detection software to check the electronic version of the thesis. I confirm that this thesis has not been previously submitted for the award of a degree by this or any other university. The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author. Signature: Date: 14/9/2016 ! 2! Supervisors!! Primary Supervisor: Dr Sarah Martin Senior Lecturer, Deputy Centre Lead, Centre for Molecular Oncology, Barts Cancer Institute. Secondary Supervisor: Dr Michelle Lockley Clinical Senior Lecturer Centre Lead and Honorary Consultant in Medical Oncology, Centre for Molecular Oncology, Barts Cancer Institute. Collaborations!! Professor Gyorgy Szabadkai Professor of Physiology Cell and Developmental Biology, Division of Biosciences, University College London Professor Andrew Silver Professor of Cancer Genetics Centre for Genomics and Child Health, Blizzard Institute, Queen Mary University of London ! Funding!! This work was supported by a grant from Cancer Research UK. ! ! ! 3! With!special!thanks!! First and foremost I would like to thank my PhD supervisor, Dr Sarah Martin, for taking me under her wing, guiding me from a complete scientific novice and facilitating my exploration of the wonderful world of mitochondria and mismatch repair. I am grateful for all of her advice and encouragement. I would like to extend special thanks Dr Melissa Phillips, who has always been helpful and supportive, and without whose flexibility for covering my clinical commitments, I would not have been able to finish writing this thesis. I would like to extend my gratitude to my lab-mates, especially Dr Rumena Begum, Dr Gemma Bridge, Dr Delphine Guilotin, Dr Matthew Locke and Hannah Shailes. Thank you for being patient and encouraging – particularly at the beginning, where I am sure supervising my use of the equipment was like teaching a toddler calculus. A special thanks should also go to the other members of the molecular oncology department, especially Dr Dan Foxler, Ramsay Khadeir and Dr Katherine Bridge for all of their support and what I guess can only be described as “scientific banter,” throughout my time in the lab. To my comrades in arms, Dr Louise Lim, Dr Laura Tookman and Dr Kate Smith, thank you for helping me get through the tough times, and being there when we all needed to mutually despair at our ineptitudes. Finally, I would like to extend my gratitude to Prof Ian McNeish for believing in me, and introducing me to Dr Sarah Martin. To my family, Mummy, Papa, and my sisters, Zahida and Zohra. Thank you for giving me everything I’ve ever needed, without ever having to ask for it. To my Darling Husband, thank you for your unconditional love and support. Our first year of marriage has been overtaken by this PhD – thank you for believing in me, especially during the times when I didn’t quite believe in myself. ! 4! Dedication!! I would like to dedicate this work to my parents, Shiraz and Noorjehan Rashid. Thank you for instilling the confidence and work ethic needed to achieve my ambitions.! ! ! 5! Abstract! The DNA Mismatch repair (MMR) pathway is responsible for the repair of base-base mismatches and insertion/deletion loops that arise during DNA replication. MMR deficiency is currently estimated to be present in 15-17% of colorectal cancer cases and 30% of endometrial cancers. MLH1 is one of the key proteins involved in the MMR pathway. MMR deficient tumours are often resistant to standard chemotherapies, therefore there is a critical need to identify new therapeutic strategies to treat MMR deficient disease. This study demonstrates that MLH1 deficient tumours are synthetically lethal with the mitochondrial-targeted agent Parthenolide which is known to induce reactive oxygen species (ROS) as one of its main mechanisms of action. Upon functional analysis, I show for the first time that loss of MLH1 is associated with deregulated mitochondrial function evidenced by a reduction in complex I expression and activity, reduced basal oxygen consumption rate and reduced spare respiratory capacity. This mitochondrial phenotype in the MLH1-deficient cell lines is accompanied by a reduction in mitochondrial biogenesis as evidenced by down regulation of pgc1β and decreased mitochondrial copy number. Furthermore, MLH1-deficient cancer cells have a decreased antioxidant defence capacity with reduced expression of the antioxidant genes NRF1, NRF2, Catalase, Glutathione peroxidase and SOD1 as well as increased ROS production when treated with Parthenolide. I further demonstrate that both MSH2- and MSH6-deficient cell lines also display deficiencies in complex I compared to their MMR-proficient counterparts. Taken together, the results of this study show a novel role for MLH1 in mitochondrial function and biogenesis. The MMR proteins MSH2 and MSH6 are also likely to have a role in the mitochondria. My results suggest that targeting the mitochondria may be a potential therapeutic strategy for the treatment of MMR and specifically MLH1 deficient disease. ! ! ! 6! Table!of!contents! ! List!of!Figures!.................................................................................................................................................!12! List!of!Tables!..................................................................................................................................................!13! Abbreviations!.................................................................................................................................................!14! 1! Introduction!.................................................................................................................................!20! 1.1! DNA!damage!.........................................................................................................................................!20! 1.2! The!mismatch!repair!(MMR)!pathway!........................................................................................!23! 1.2.1! The!canonical!role!of!the!MMR!pathway!............................................................................................!23! 1.2.2! The!role!of!MLH1!in!the!MMR!pathway!..............................................................................................!28! 1.3! Non!Canonical!roles!of!the!MMR!system!....................................................................................!30! 1.3.1! Meiotic!and!mitotic!recombination!and!MMR!.................................................................................!30! 1.3.1.1! !MMR!and!meiotic!recombination!...................................................................................................................!30! 1.3.1.2! !MMR!and!mitotic!recombination!....................................................................................................................!31! 1.3.2! MMR!and!immunoglobulin!diversification!.......................................................................................!31! 1.3.3! MMR!and!chromatin!assembly!...............................................................................................................!32! 1.4! MMR!activity!in!the!mitochondria!................................................................................................!33! 1.5! MMR!deficiency!and!cancer!............................................................................................................!34! 1.5.1! Treatment!of!MMR!cancers!......................................................................................................................!38! 1.5.1.1! !5FFU!and!dMMR!.....................................................................................................................................................!39! 1.5.1.2! !Platinums!and!dMMR!..........................................................................................................................................!42! 1.5.1.3! !Topoisomerase!inhibitors!and!anthracyclines!in!!dMMR!....................................................................!43! 1.4.1.4! !Alkylating!agent!and!dMMR!..............................................................................................................................!43! 1.6.1!! The!mitochondrial!genome!.....................................................................................................................!44! 1.6.2! Heteroplasmy!and!homoplasmy!............................................................................................................!44!