ERK1/2 signalling and protein ubiquitylation in the control of apoptosis Kate Ann Stuart Girton College, University of Cambridge The Babraham Institute This dissertation is submitted for the degree of Doctor of Philosophy September 2018 Summary Title: ERK1/2 signalling and protein ubiquitylation in the control of apoptosis. Name: Kate Stuart Programmed cell death, or apoptosis, is critical for normal developmental processes that involve cell turnover including embryogenesis and development and function of the immune system. It is preceded by classical changes in cell morphology, driven by biochemical changes including caspase activation. Apoptosis is deregulated in multiple human diseases, with suppression of apoptosis being critical for carcinogenesis. As such, proteins that regulate apoptosis are tightly regulated by cell fate signalling pathways. The ERK1/2 signalling pathway is a key regulator of cell intrinsic apoptosis, in part through regulation of the pro-apoptotic protein ‘BCL2-interacting mediator of cell death’ (BIM). BIM is phosphorylated by ERK1/2 and this serves to drive its K48-linked polyubiquitylation and proteasome-dependent degradation, thereby promoting cell survival. βTrCP, an F-box protein that acts in a larger SCF complex, is one of several E3 ligases that have been proposed to polyubiquitinate BIM. This study demonstrated that of the major isoforms of BIM only BIMEL interacts with βTrCP. ERK1/2-driven phosphorylation of BIMEL is essential for this interaction, leading to BIMEL destabilisation and degradation. As a consequence, tumour cells that are addicted to ERK1/2 signalling undergo BIM-dependent cell death in response to MEK1/2 inhibitors when combined with BH3-mimetics such as ABT263, small molecules that inhibit pro-survival proteins of the apoptotic pathway. The RSK1/2 protein kinases, immediate downstream targets of ERK1/2, have also been implicated in the destabilisation of BIMEL. Specifically phosphorylation of BIMEL by RSK1/2 is proposed to be required for βTrCP binding. This study revealed that whilst the putative RSK1/2 phosphorylation sites in BIMEL may be required for βTrCP binding, inhibition of RSK activity by three distinct RSK inhibitors does not block BIMEL:βTrCP binding or BIMEL turnover. Furthermore, tumour cells that are addicted to ERK1/2 signalling for survival are not addicted to RSK activity, arguing against a role for RSK in the regulation of BIMEL. This suggests that ERK1/2 and an as yet unidentified kinase cooperate to drive BIMEL degradation. Deubiquitylating enzymes (DUBs) remove ubiquitin from target proteins. In the context of BIMEL, DUB activity might oppose E3 ligases and thus cause its accumulation. Until recently the DUB for BIM was unknown however, USP27x has now been suggested. Follow-up validation of the reported interaction I between BIMEL and USP27x was challenging but loss of BIMEL polyubiquitination was observed following overexpression of USP27x, suggesting that USP27x may serve as a DUB for BIM. Numerous DUBs control cellular processes that are dysregulated in cancer, including proliferation and apoptosis, making them attractive therapeutic targets. Recent interest in the DUB USP30 has increased as it has been shown to inhibit parkin-mediated mitophagy, with defective mitophagy being linked to Parkinson’s disease. USP30 has also been suggested to play a role in apoptosis and its depletion was shown to sensitise cells to BH3 mimetics. These findings suggest that USP30 depletion or inhibition could provide a means for inducing tumour cell death. Indeed, combining a novel USP30 inhibitor (MTX32), provided by Mission Therapeutics, with the BH3 mimetic, ABT-263, induced apoptotic tumour cell death that required BAX and caspase activation. However, this was not replicated by a more selective USP30 inhibitor (MTX48), suggesting that the observed apoptotic cell death reflected the off-target effects of MTX32 rather than specific inhibition of USP 30. Finally, an RNAi screen, targeting 94 DUBs, and 8 sentrin/SUMO-specific proteases (SENPs), in the human genome, was performed to identify DUBs that modulate cell death induced by MEK1/2 or mTOR inhibition. As such, inhibitors of identified ‘hit’ DUBs might be suitable as a combinatorial therapy with MEK1/2 or mTOR inhibitors in the treatment of cancer. The RNAi screen identified several DUBs, including USP10, YOD1, and VCIP135, that, when knocked down, sensitised HCT116 cells to MEK1/2 inhibitor treatment and enhanced MEK1/2 inhibitor induced cell death in a BAX-dependent fashion. This work is discussed in the context of the role of ERK1/2 signalling as a pro-survival pathway, its specific role in BIM regulation, the potential for co-targeting DUBs and the ERK1/2 pathway to inhibit the growth of ERK1/2 addicted tumour cells and suggestions for future work are outlined. II Declaration This thesis contains original work that has not previously been submitted for a degree or diploma or other qualifications at the University of Cambridge or any other University or similar institution. It is the result of my own work and contains nothing which is the outcome of work done in collaboration with others, except as specified in the text and acknowledgements. The length does not exceed the limit as stated in the Memorandum to Graduate Students. The dissertation does not exceed the limit of 60 000 words imposed by the Cambridge University Faculty of Biology Degree Committee. Kate Stuart III Acknowledgements I would first like to thank my supervisor Simon Cook for giving me the opportunity to work in his laboratory. Without his guidance, patience (I know I worry a lot) and support my PhD would not have been possible. A big thank you to my co-supervisor at Mission Therapeutics, Yaara Ofir-Rosenfeld, for supporting me and providing me with practical advice whilst performing the RNAi screens. In addition, I would like to thank Jeanine Harrigan at Mission Therapeutics mostly for her time and for the helpful comments/revisions during the final stages of my thesis writing. Thank you to everyone in the Cook lab, it has been a privilege to work with you all over the last four years. I am particularly grateful to my mentor Becky, without her reassurance and guidance I would not be the scientist I am today. Kathy (Bob), thank you so all of your support, endless knowledge and for helping me out on numerous occasions. I have made many friends at Babraham who have kept me sane over the last four years, in particular Katherine and Ine thank you for all of the chats, lunchtime walks and for generally knowing how to make me laugh. I would like thank Victoria, who has seen me at my worst and best during this process and who has provided me with unwavering support. Thank you for making me believe that I can do this even when I thought could not. I could not wish for a better person to share my future with. Finally, I would like to thank my family, Peter, Sally and Sarah for their endless love and support. IV Acknowledgement of assistance received during the course of this thesis 1. Initial Training in techniques and laboratory practice and subsequent mentoring Flow cytometry training – Rachel Walker General laboratory practice – Kathy Balmanno and Rebecca Gilley Immunoprecipitation training – Anne Ashford Molecular biology training – Anne Ashford and Rebecca Gilley RNAi screening – Yaara Ofir-Rosenfield Tissue culture training - Rebecca Gilley Western blotting training – Rebecca Gilley 2. Data obtained from a technical service provider None 3. Data produced jointly Chapter 5 was performed at Mission Therapeutics particularly with help of Nadia Arnaudo, Aurelie Le Feuvre Yaara Ofir-Rosenfeld and Aleksandra Zemia. 4. Data/materials provided by someone else For materials provided by others, see Materials and Methods. Figure 3.5F was performed by Ceri Wiggins, a previous member of the Cook Lab. Publications associated with this thesis COOK, S. J., STUART, K., GILLEY, R. & SALE, M. J. 2017. Control of cell death and mitochondrial fission by ERK1/2 MAP kinase signalling. Febs j, 284, 4177-4195. V Abbreviations 4E-BP eIF (eukaryotic initiation factor) 4E-binding protein A1/BFL BCL2-related protein A1 A Alanine aa Amino acid ADP Adenosine diphosphate AIF Apoptosis-inducing factor AKT v-akt murine thymoma viral oncogene homologue 1 ANOVA Analysis of variance AML Acute myeloid leukaemia AMSH Associated molecule with the SH3 domain AMSH-LP AMSH-like protease AP1 Activator protein 1 APC/C Anaphase promoting complex or cyclosome APAF1 Apoptotic protease activating factor1 ARAF v-raf murine sarcoma 3611 viral oncogene homolog ARTS Sept4_i2 ATM Ataxia-telangiectasia mutated ATP Adenosine triphosphate ATR Ataxia telangiectasia and Rad3 related βTrCP Beta-transducin repeat-containing protein BAD BCL2/BCL-XL associated death promoter BAK BCL2 homologous antagonist killer BAP1 BRCA1 Associated Protein 1 BAX BCL2-associated X protein BC (groove) BH3 and C-terminus-binding groove BCL2 B-cell leukaemia/lymphoma 3 BCL-XL B-cell leukaemia/lymphoma extra large BCL-w B-cell leukaemia/lymphoma-like protein 2 BH BCL2-homology BH3 BCL2-homology 3 BI-1 BAX inhibitor 1 BID BH3-interacting domain death agonist BIK BCL2-interacting killer BIM BCL2-interacting mediator BIMEL BIM-extra long BIML BIM-long BIMS BIM-short BMF BCL2- modifying factor BOK BCL2-related ovarian killer BOP BH3-only protein BRAF v-raf murine sarcoma viral oncogene homologue B1 BRAFi BRAF inhibitor BRCA Breast cancer type 1 BRcat Benign-catalytic BRUCE/Apollon BIRC6 – baculoviral IAP repeat containing