MECHANISMS OF TRAIL RESISTANCE Neelam Kumar University College London A thesis submitted for the degree of Doctor of Philosophy 2018 1 DECLARATION I, Neelam Kumar, confirm that the work presented in this thesis is my own. Where information has been derived from other sources I confirm that this has been indicated and acknowledged in the thesis. 2 ABSTRACT Malignant pleural mesothelioma (MPM) is a devastating disease for which limited effective therapies are currently available. Tumour necrosis factor- related apoptosis-inducing ligand (TRAIL) and other death receptor (DR) agonists are pro-apoptotic agents that trigger the extrinsic apoptotic pathway selectively in cancer cells. Previous work has shown that supports that loss of function of the nuclear deubiquitinase BRCA associated protein-1 (BAP1) augments sensitivity to recombinant TRAIL (rTRAIL) in MPM cells. This study shows that BAP1 is a candidate biomarker for rTRAIL sensitivity in cell line, early passage culture and tumour explant models of MPM. In addition, BAP1 is a potential biomarker for sensitivity to other DR agonists and for additional cancer types with BAP1 mutations. I have also identified other novel candidate biomarkers for DR agonist sensitivity, notably ASXL1/2, through exploration of the mechanism underlying the BAP1-rTRAIL association. I present data supporting the clinical relevance and utility of the BAP1-TRAIL association. I have shown that loss of BAP1 function occurs in a significant proportion of MPM tumours in the UK and that loss of BAP1 function augments sensitivity to TRAIL in primary tumour tissue. I describe in vitro data supporting the hypothesis that loss of BAP1 function augments sensitivity to rTRAIL in MPM. I have shown that loss of BAP1 function augments sensitivity to other DR agonists and that BAP1 can act as a biomarker for DR agonist sensitivity in other cancers with BAP1 mutations. Finally, I explore the mechanism underlying the BAP1-TRAIL association. I present data supporting the notion that BAP1 binds to the ASXL1/2 proteins to form the polycomb repressor deubiquitinase complex which underlies BAP1-induced TRAIL resistance. Loss of this function results in a change in the expression of proteins of the extrinsic apoptotic pathway, which may favour apoptosis upon DR agonist binding. 3 IMPACT STATEMENT The findings presented in this thesis have significant potential clinical impact. I demonstrate the potential utility of loss of function of BAP1 and ASXL1/2 as biomarkers for sensitivity to DR agonists. Several DR agonists exist that have already completed phase I/II clinical testing in this context but so far have not demonstrated significant efficacy over standard therapy in several different malignancies when tested in unselected populations. Notably there have been no trials of DR agonists in MPM to date. Within completed trials however, a few patients were noted to exhibit partial or complete response, yet to date no means by which to identify such responders has been found. The data presented within this thesis supports the loss of function of BAP1, and potentially ASXL1/2, as candidate biomarkers for DR agonist sensitivity. BAP1 loss of function has been observed in 42-67% of MPM tumours, which would therefore be amenable to DR agonist therapy. ASXL1 mutations are highly prevalent in myeloid malignancies (34-45%) and ASXL2 mutations are present in 4% of pancreatic and 6% of prostate cancers. These malignancies would also be potentially amenable to targeted DR agonist therapy. Insights gained into the mechanism suggest that BAP1 loss of function alters expression of proteins of the extrinsic apoptotic pathway. This could be exploited to sensitise BAP1 wild-type tumours to DR agonists either by inhibiting anti-apoptotic proteins or even by inhibiting BAP1 itself. 4 ACKNOWLEDGMENTS There are two people without whom I simply could not have completed this PhD. Firstly, my supervisor Professor Sam Janes. His faith and encouragement have kept me afloat throughout and his pragmatism and perspective have been lessons for life. Secondly, my ‘unofficial supervisor’ Dr Krishna Kolluri. It is no a platitude to say that he has taught me everything I know about science and has done so with an unlimited patience. Developing scientific skills from a clinical background has been a challenge, but the Lungs for Living scientists have indulged and tolerated my many questions and mistakes, and the clinical fellows have provided much needed solidarity in unfamiliar terrain. The ‘BAP1 team’ of Yuki Ishii and Doraid Alrifai deserve particular mention. Collaborators have been integral to completion of this thesis and I have had the pleasure and honour to work with Costi Alifrangis, Ultan McDermott, Sara Busacca, Dean Fennell, Alan Holmes, Richard Angell, Naomi Guppy, Mary Falzon, and Elaine Borg all of whom have been incredibly generous with their time and expertise. As with all of my endeavours, I am beyond fortunate to have had the support of my friends and family. In particular I would like to thank Sally Whitehill and Rajni Aldridge for being my crisis management team, Cordelia Coltart and Richard Tacon for regularly relieving mid-day ennui, Foad Rouhani for his unique blend of scientific and social sustenance, and Niko Algieri for strengthening my physical and emotional reserve. Especial thanks and love to my niece and nephew, Arjun and Arya, for always brightening my day, and to my brothers-in-law Suneil Shukla and Kam Punia for joining the ride. This thesis is dedicated to the warriors who face every battle with kindness, humour and the obligate touch of crazy. Mum, Dad, Sal and Pod – thank you, for everything. 5 TABLE OF CONTENTS 1 INTRODUCTION ............................................................................................... 12 1.1 Malignant Pleural Mesothelioma .................................................................................................. 12 1.1.1 Pathogenesis of MPM ...................................................................................................................... 13 1.1.2 Diagnosis and staging of MPM ................................................................................................... 14 1.1.3 Management of MPM ...................................................................................................................... 15 1.2 BRCA associated protein-1 .............................................................................................................. 19 1.2.1 Deubiquitinases ................................................................................................................................. 20 1.2.2 BAP1 structure .................................................................................................................................. 20 1.2.3 BAP1 function .................................................................................................................................... 22 1.3 The polycomb group proteins ........................................................................................................ 25 1.3.1 The polycomb repressor complexes .......................................................................................... 25 1.3.2 The polycomb repressive deubiquitinase complex ............................................................. 26 1.3.3 The ASXL Proteins ............................................................................................................................ 28 1.4 BAP1 and ASXL1/2 in cancer ......................................................................................................... 31 1.4.1 BAP1 in cancer ................................................................................................................................... 31 1.4.2 ASXL proteins in cancer ................................................................................................................. 34 1.5 TNF related apoptosis inducing ligand (TRAIL) .................................................................... 37 1.5.1 TRAIL receptors ................................................................................................................................ 37 1.5.2 TRAIL apoptotic pathway ............................................................................................................. 37 1.5.3 TRAIL and cancer cells ................................................................................................................... 38 1.5.4 TRAIL and DR agonists as anti-cancer therapies ............................................................... 39 1.6 BRCA associated protein-1 and TRAIL ....................................................................................... 43 1.6.1 Loss of function of BAP1 as biomarker for TRAIL sensitivity ........................................ 43 1.6.2 The deubiquitinase function of BAP1 mediates TRAIL resistance .............................. 46 1.6.3 BAP1 function affects transcription of extrinsic apoptotic proteins ......................... 48 1.6.4 Loss of BAP1 function augments sensitivity to rTRAIL in mouse MPM xenograft models 49 1.7 Hypothesis .............................................................................................................................................. 50 1.8 Aims ........................................................................................................................................................... 50 2 METHODS ......................................................................................................