Gain-of-function and dominant-negative effects of distinct p53 mutations in lung tumours Frances Kathryn Turrell Clare College, University of Cambridge This dissertation is submitted for the degree of Doctor of Philosophy December 2017 2 Gain-of-function and dominant-negative effects of distinct p53 mutations in lung tumours Frances Kathryn Turrell Lung cancer is the most common cause of cancer-related mortality worldwide with current treatments providing limited therapeutic benefit in most cases. TP53 (Trp53, p53) mutations occur in approximately 50% of lung adenocarcinoma cases and are associated with poor prognosis and so novel therapies that target these p53 mutant lung tumours are urgently needed. Despite the high frequency of p53 mutations in lung tumours, the impact these mutations have on response to therapy remains unclear in this cancer type. The aim of my project is to characterise the gain-of-function and dominant- negative effects of p53 mutations in lung tumours and to identify ways of therapeutically targeting these p53 mutant tumours based on dependencies and susceptibilities that our analysis uncovers. To characterise the gain-of-function and dominant-negative effects of p53 mutations I compared p53 mutant murine lung tumour cells that endogenously express either a contact (R270H, equivalent to R273H in humans) or conformational (R172H, equivalent to R175H in humans) p53 mutant protein and p53 null lung tumour cell lines; both in the presence and absence of wild-type p53. Interestingly, transcriptional and functional analysis uncovered metabolic gain-of-functions that are specific to the type of p53 mutation. Upregulation of mevalonate pathway expression was observed only in R270H lung tumours and consequently R172H and R270H lung tumours displayed distinct sensitivities to simvastatin, a mevalonate pathway inhibitor widely used in the clinic. Furthermore, the transcriptional signature underlying this sensitivity to simvastatin was also present in human lung tumours with contact p53 mutations, indicating that these findings may be clinically relevant. On the other hand, our analysis of the potential dominant-negative effects of the p53 mutants on wild-type p53 demonstrated that wild-type p53 was able to induce typical p53 target genes to a similar level in p53 null and mutant cells. Furthermore, wild-type p53 restoration resulted in comparable tumour suppressive responses in p53 mutant and null tumours and thus, p53-restoration therapy will likely be of benefit to patients with p53 mutations in lung cancer. Hence, I have demonstrated that lung tumours harbouring contact and conformational p53 mutations display common and distinct therapeutic susceptibilities. 3 Preface Declaration of originality This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except where indicated in the text. I confirm that no part of this dissertation has been submitted for any other degree or qualification. Statement of length This dissertation does not exceed 60,000 words in length. i Publications Turrell FK, Kerr EM, Gao M, Thorpe H, Doherty G, Cridge J, Shorthouse D, Speed A, Samarajiwa S, Hall BA, Meryl Griffiths M and Martins CP (2017). Lung tumors with distinct p53 mutations respond similarly to p53-targeted therapy but exhibit genotype- specific statin sensitivity. Genes and Development 2017 Aug 8. Kerr EM, Gaude E, Turrell FK, Frezza C, Martins CP. Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities. Nature. 2016 March 03; (531):110–113 (work not included in the thesis). Talks and poster presentations F. K. Turrell, E. M. Kerr, M. Gao, H. Thorpe and C. P. Martins. Lung tumours with distinct p53 mutations display genotype-specific metabolic profiles and differential sensitivities to statins. Short ‘poster-taster’ talk and poster presentation - Beatson International Cancer Conference, 2nd-5th July 2017. F. K. Turrell Determining the impact of distinct p53 mutations on lung tumour evolution and therapy. Talk - MRC Cancer Unit annual symposium, 2nd December 2016 F. K. Turrell, M. Gao, E. M. Kerr and C. P. Martins Determining the impact of distinct p53 mutations in lung tumours. Poster - MRC Cancer Unit annual symposium, 20th November 2015. F. K. Turrell, M. Gao, E. Gaude, E. M. Kerr, C. Frezza and C. P. Martins. Determining the metabolic impact of p53 mutations in lung cancer. Poster - MRC Cancer Unit annual symposium, 21st November 2014. ii Acknowledgements Firstly, I would like to thank my supervisor Dr Carla Martins for the opportunity to pursue a PhD in her laboratory and her continued guidance. I would also like to thank Dr Emma Kerr for all of her scientific input and support over the years, which have been instrumental to this work, and for being a great friend during my time here in Cambridge. I would like to thank Dr Meiling Gao for providing the samples for the microarray analysis and therefore, her work towards initiating the project. I am also grateful to all members of the Martins and Shields laboratories, past and present, who have all helped me in some way over the years. In particular, I would like to thank Dr Angela Riedel, Dr Luisa Pedro, Dr Ilya Kotov, Dr Gary Doherty, Jennifer Harris, Sarah Davidson and Alyson Speed for their continuous friendship and support. I would also like to thank Dr Emma Kerr, Dr Hannah Thorpe, Dr Gary Doherty and Jake Cridge for their help addressing reviewer comments. Furthermore, I am grateful to Ellie Gregson, who has been pursuing her PhD alongside me from interview day until submission, for supporting me through it. I thank all the staff at the Biological Resources Unit and Cambridge Biomedical Services for their help with the in vivo work. In particular, I am grateful to Alyson Speed and Mike Mitchell for their invaluable help with the in vivo experiments. In addition, I would like to thank Dr Christian Frezza and members of his laboratory, particularly Edo Gaude, for running the mass spectrometry and for their advice on the metabolism side of the project. I am also grateful to Dr Shamith Samarajiwa and Dr David Shorthouse for their work on the microarray data normalisation and advice on the analysis, respectively. Finally, I would like to thank all my family and friends as without their support none of this would have been possible. iii Abstract Lung cancer is the most common cause of cancer-related mortality worldwide with current treatments providing limited therapeutic benefit in most cases. TP53 (Trp53, p53) mutations occur in approximately 50% of lung adenocarcinoma cases and are associated with poor prognosis and so novel therapies that target these p53 mutant lung tumours are urgently needed. Despite the high frequency of p53 mutations in lung tumours, the impact these mutations have on response to therapy remains unclear in this cancer type. The aim of my project is to characterise the gain-of- function and dominant-negative effects of p53 mutations in lung tumours and to identify ways of therapeutically targeting these p53 mutant tumours based on dependencies and susceptibilities that our analysis uncovers. To characterise the gain-of-function and dominant-negative effects of p53 mutations I compared p53 mutant murine lung tumour cells that endogenously express either a contact (R270H, equivalent to R273H in humans) or conformational (R172H, equivalent to R175H in humans) p53 mutant protein and p53 null lung tumour cell lines; both in the presence and absence of wild-type p53. Interestingly, transcriptional and functional analysis uncovered metabolic gain-of-functions that are specific to the type of p53 mutation. Upregulation of mevalonate pathway expression was observed only in R270H lung tumours and consequently R172H and R270H lung tumours displayed distinct sensitivities to simvastatin, a mevalonate pathway inhibitor widely used in the clinic. Furthermore, the transcriptional signature underlying this sensitivity to simvastatin was also present in human lung tumours with contact p53 mutations, indicating that these findings may be clinically relevant. On the other hand, our analysis of the potential dominant-negative effects of the p53 mutants on wild-type p53 demonstrated that wild-type p53 was able to induce typical p53 target genes to a similar level in p53 null and mutant cells. Furthermore, wild- type p53 restoration resulted in comparable tumour suppressive responses in p53 mutant and null tumours and thus, p53-restoration therapy will likely be of benefit to patients with p53 mutations in lung cancer. Hence, I have demonstrated that lung tumours harbouring contact and conformational p53 mutations display common and distinct therapeutic susceptibilities. iv List of abbreviations 2D/3D Two-dimensional/ three-dimensional 4OHT 4-hydroxytamoxifen 14-3-3 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein sigma Acat2 Acetyl-coA acetyltransferase 2 Actb Beta-actin Adeno-Cre Cre recombinase-expressing adenovirus Aif Apoptosis-inducing factor ALK Anaplastic lymphoma kinase AMPK 5’-adenosine monophosphate-activated protein kinase ANOVA Analysis of variance Apaf1 Apoptotic peptidase activating factor 1 APC Allophycocyanin ATP Adenosine triphosphate AWERB Animal welfare and ethical review body Bax Bcl-2-associated X protein Bbc3 Bcl2 binding component 3 BCA Bicinchoninic acid Birc5 Baculoviral inhibitor of apoptosis repeat-containing 5 BrdU Bromodeoxyuridine BSA Bovine serum albumin Btg2 B cell translocation gene 2 CCCP Carbonyl cyanide m-chlorophenyl