Targeting cyclin-dependent kinase 9 and myeloid cell leukaemia 1 in MYC-driven B-cell lymphoma Gareth Peter Gregory ORCID ID: 0000-0002-4170-0682 Thesis for Doctor of Philosophy September 2016 Sir Peter MacCallum Department of Oncology The University of Melbourne Doctor of Philosophy Submitted in total fulfilment of the degree of Abstract Aggressive B-cell lymphomas include diffuse large B-cell lymphoma, Burkitt lymphoma and intermediate forms. Despite high response rates to conventional immuno-chemotherapeutic approaches, an unmet need for novel therapeutic by resistance to chemotherapy and radiotherapy. The proto-oncogene MYC is strategies is required in the setting of relapsed and refractory disease, typified frequently dysregulated in the aggressive B-cell lymphomas, however, it has proven an elusive direct therapeutic target. MYC-dysregulated disease maintains a ‘transcriptionally-addicted’ state, whereby perturbation of A significant body of evidence is accumulating to suggest that RNA polymerase II activity may indirectly antagonise MYC activity. Furthermore, very recent studies implicate anti-apoptotic myeloid cell leukaemia 1 (MCL-1) as a critical survival determinant of MYC-driven lymphoma. This thesis utilises pharmacologic and genetic techniques in MYC-driven models of aggressive B-cell lymphoma to demonstrate that cyclin-dependent kinase 9 (CDK9) and MCL-1 are oncogenic dependencies of this subset of disease. The cyclin-dependent kinase inhibitor, dinaciclib, and more selective CDK9 inhibitors downregulation of MCL1 are used to demonstrate efficient apoptosis induction conferred at least in part by other transcriptional cyclin-dependent kinases that are required for viability of transcription. Furthermore, a genetic screen identifies MYC-driven lymphoid disease. Finally, having established MCL-1 as a critical oncogenic dependency of MYC-driven direct pharmacologic antagonism of MCL-1 using a small molecule BH3-mimetic lymphoma, this thesis demonstrates the significant activity that is conferred by dependency involving CDK9 regulated RNA polymerase II-mediated transcription inhibitor of MCL-1. These findings confirm a druggable pathway of oncogenic cMYC of MCL-1, and proposes pharmacologic inhibition of CDK9 and MCL-1 as novel anti-lymphoma strategies. i Declaration This is to certify that: 1. This thesis comprises only my original work towards the degree of Doctor of Philosophy except where indicated in the preface. 2. Due acknowledgement has been made in the text to all other material used. 3. The thesis is fewer than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices. Gareth Gregory 24 September 2016 ii Preface The following generous contributions of experimental work depicted in this thesis are acknowledged: Figure 3.4 Lymphoma transplantation performed by Ms Eva Vidacs Figure 4.8 Real-time PCR assisted by Mr Joshua Hilton Figure 4.11 Mouse retro-orbital bleeds performed by Ms Eva Vidacs Figure 4.15 Experiment performed in conjunction with Mr Simon Hogg Figures 4.17-4.19 Experiments performed in conjunction with Mr Zheng Fan Figures 5.11-5.12 AZ-MCL1 administered by Ms Eva Vidacs Gregory GP, Hogg SJ, Kats LM, Vidacs E, Baker AJ, Gilan O, Lefebure M, Martin BP, Dawson MA, Johnstone RW, Shortt J. 2015. CDK9 inhibition by dinaciclib potently suppresses Mcl-1 to induce durable apoptotic responses in aggressive MYC-driven B-cell lymphoma in vivo. Leukemia 29:1437-41. Figure 1c Experiment performed in conjunction with Mr Simon Hogg Figure 1d Experiment assisted by Dr Omer Gilan Figures 2a-d Lymphoma transplantation performed by Ms Eva Vidacs iii Acknowledgements I would like to acknowledge the incomparable supervision and mentoring provided by my doctorate supervisors, Professor Ricky Johnstone and Associate Professor Jake Shortt. Beyond their supervision, they have fostered an environment in which I have been inspired and nurtured to thrive as a Clinician-Scientist and I value the ongoing roles that they will play in my path beyond the scope of these studies. Furthermore, I would like to thank my research mentor, Professor Joe Trapani, and the incredible training, assistance and support offered by my colleagues in the Johnstone laboratory. Particular mention must go to Ben Martin, Eva Vidacs, Marcus Lefebure, Simon Hogg, Lev Kats, Leonie Cluse, Joshua Hilton, Zheng Fan, core facilities. Research was funded by the Leukaemia Foundation of Australia, the Izabela Todorovski, Linda Stevens and members of the animal and flow cytometry Royal Australasian College of Physicians and Cancer Therapeutics CRC. I would also like to acknowledge the extraordinary contributions from my family: the patience and support offered by my wife Amy and the unquestionable support and opportunities provided by my parents, Peter and Sylvia. The pathway toward these doctoral studies was inspired by a number of Haematologists and Clinician- Researchers who fostered a supportive environment for pursuing this career. I would like to acknowledge Drs Paul Cannell, Matthew Wright and Julian Cooney (Royal Perth Hospital); Professors Hatem Salem and Andrew Spencer (Alfred Health & Monash University); and Dr George Grigoriadis (Monash Health and Monash University) for their advice and support toward pursuing a research pathway in haematology. Finally, I would like to acknowledge the incredible support, mentoring and opportunities offered by Associate Professor Stephen Opat (Monash Health & Monash University). Beyond all of these roles, Stephen, Jake and Ricky were instrumental in ensuring that the preclinical promise of dinaciclib described in studies herein was rapidly translated into an international phase I clinical trial and furthermore offered and encouraged me to take the role of lead investigator on this trial. I am indebted to them for such an opportunity. iv Publications arising from this thesis Gregory GP, Hogg SJ, Kats LM, Vidacs E, Baker AJ, Gilan O, Lefebure M, Martin BP, Dawson MA, Johnstone RW, Shortt J. 2015. CDK9 inhibition by dinaciclib potently suppresses Mcl-1 to induce durable apoptotic responses in aggressive MYC-driven B-cell lymphoma in vivo. Leukemia 29:1437-41. Waibel W, Gregory G, Shortt J, Johnstone RW. Rational combination therapies targeting survival signaling in aggressive B cell leukemia / lymphoma. 2014. Curr Opin Hematol. 21(4):297-308. Baker AJ, Gregory GP, Verbrugge I, Kats L, Hilton JJ, Vidacs E, Lee EM, Locke RB, Zuber J, Shortt J, Johnstone RJ. 2016. Apoptotic and therapeutic effects of dinaciclib in pre-clinical models of MLL-rearranged acute myeloid leukemia. Cancer Res 76(5):1158-69. Hogg SJ, Newbold A, Martin BP, Gregory GP, Lefebure M, Vidacs E, Tothill R, Bradner JE, Shortt J, Johnstone RW. 2016. BET-inhibition induces apoptosis in aggressive B-cell lymphoma via epigenetic regulation of BCL-2 family members. Mol Cancer Ther. 15(9):2030-41. Lefebure M, Tothill RW, Kruse E, Hawkins E, Shortt J, Mathews G, Gregory GP, Martin BP, Kelly MJ, Todorovski I, Doyle MA, Lupat R, Li J, Schroeder J, Wall M, Craig S, Poortinga G, Cameron D, Bywater M, Kats L, Gearhart MD, Bardwell V, Dickins RA, Hannan RD, Papenfuss T, Johnstone RW. Genomic characterisation of Eµ-Myc Bcor as a MYC co-operative tumor-suppressor gene [revision under review]. lymphomas by massively-parallel sequencing identifies v Table of Contents Abstract............................................................................................... i Declaration......................................................................................... ii Preface.................................................................................................. iii Acknowledgements........................................................................ iv Publications arising from this thesis....................................... v Table of Contents............................................................................. vi Table of Tables.................................................................................. xiv Table of Figures................................................................................ xv Abbreviations.................................................................................... xix Chapter 1: The role of cyclin-dependent kinases and apoptosis family members in MYC-driven lymphoid malignancy.......................................................... 1 1.1 MYC-driven B-cell lymphoma..................................................... 2 1.1.1 Conventional therapeutic approaches to aggressive B-cell lymphoma............................................................................... 2 1.1.2 Current novel therapeutic approaches to aggressive B-cell lymphoma............................................................................... 3 1.1.2.1 Chronic active BCR signalling..................................................... 6 1.1.2.1.1 Targeting chronic active BCR signalling................................. 6 1.1.2.2 Tonic BCR signalling........................................................................ 7 1.1.2.2.1 Targeting tonic BCR signalling................................................... 8 1.1.2.3 Non-BCR targeted approaches................................................... 8 1.1.2.3.1 EZH2 inhibition................................................................................. 8 1.1.2.3.2 BCL-6 inhibition................................................................................ 8 1.1.2.3.3 HDAC inhibition...............................................................................