PHARMACOLOGICAL IMPROVEMENT OF ONCOLYTIC VIROTHERAPY Mohammed Selman Thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the Doctorate of Philosophy in Biochemistry Department of Biochemistry, Microbiology & Immunology Faculty of Medicine University of Ottawa © Mohammed Selman, Ottawa, Canada, 2018 Abstract Oncolytic viruses (OV) are an emerging class of anticancer bio-therapeutics that induce antitumor immunity through selective replication in cancer cells. However, the efficacy of OVs as single agents remains limited. We postulate that resistance to oncolytic virotherapy results in part from the failure of tumor cells to be sufficiently infected. In this study, we provide evidence that in the context of sarcoma, a highly heterogeneous malignancy, the infection of tumors by different oncolytic viruses varies greatly. Similarly, for a given oncolytic virus, productive infection of tumors across patient samples varies by many orders of magnitude. To overcome this issue, we hypothesize that the infection of resistant tumors can be achieved through the use of selected small molecules. Here, we have identified two novel drug classes with the ability to improve the efficacy of OV therapy: fumaric and maleic acid esters (FMAEs) and vanadium compounds. FMAEs are enhancing infection of cancer cells by several oncolytic viruses in cancer cell lines and human tumor biopsies. The ability of FMAEs to enhance viral spread is due to their ability to inhibit type I IFN production and response, which is associated with their ability to block nuclear translocation of transcription factor NF-κB. Vanadium-based phosphatase inhibitors enhance OV infection of RNA viruses in vitro and ex vivo, in resistant cancer cell lines. Mechanistically, this involves subverting the antiviral type I IFN response towards a death-inducing and proinflammatory type II IFN response, leading to improved OV spread, increased bystander killing of cancer cells, and enhanced anti-tumor immune-stimulation. Both FMAEs and vanadium compounds improve therapeutic outcomes of OV treatment in syngeneic tumor models, leading to durable responses, even in models otherwise refractory to OV and drug alone. Overall, we showcased novel avenues for the development of improved immunotherapy strategies. ii Acknowledgements This thesis would not have been possible without the aid and support of several people. First, I would like to thank my supervisor Dr. Jean-Simon Diallo for his guidance, motivation and continuous support during the duration of my PhD. Furthermore, I would like to thank to my thesis committee members, Dr. Mary-Ellen Harper, Dr. Tommy Alain and Dr. Douglas Gray for their helpful advice throughout the course of the program. I would also like to thank the many collaborators for their work and valuable input: Dr. Debbie C. Crans, Dr. Brian A. Keller, Dr. Larissa Pikor, Dr. Fabrice Le Boeuf, Dr. John C. Bell and Dr. Carolina Ilkow. A special thanks to Dr. Fanny Tzelepis for teaching me new skills and all her effort and help with the many immunological experiments, and Andrew Chen for all his help managing the numerous in vivo experiments. Sincere thanks for all the hard work from honors students that I had the privilege to supervise and work with: Chris Rousso, Paula Ou and Anabel Bergeron. Finally, thanks to family, friends and all current and former lab members of the Diallo lab for their ongoing support. iii Table of Contents Abstract ii Acknowledgements iii List of Abbreviations vi List of Figures ix Chapter 1 - General Introduction 1 Cancer 1 Oncolytic virotherapy 1 Mechanism of action of oncolytic viruses 2 Oncolytic virus platforms 4 Oncolytic herpesvirus 5 Oncolytic poxviruses 6 Oncolytic reovirus 7 Oncolytic rhabdoviruses 9 Type I and type II interferon pathways 11 The NF-κB pathway 13 Defects in type I IFN signaling in cancer 14 Resistance to OV 16 Breaking resistance to OV: Viral sensitizers 17 Fumaric acid esters 21 DMF as cancer treatment 23 Vanadium 25 Vanadium compounds are promising anticancer agents 27 Sarcoma: a rare cancer in need of new therapies 28 Rationale and aim I 29 Rationale and hypothesis II 30 Chapter 2 - Oncolytic Maraba virus MG1 as a treatment for Sarcoma 32 Abstract 33 Introduction 34 Materials and Methods 36 Results 40 iv Discussion 52 Chapter 3 - Dimethyl Fumarate Potentiates Oncolytic Virotherapy through NF-kB inhibition 54 Abstract 55 Introduction 56 Results 58 Discussion 79 Chapter 4 - Multi-Modal Potentiation of Oncolytic Virotherapy by Vanadium Compounds 91 Abstract 92 Introduction 93 Results 95 Discussion 119 Materials and Methods 123 Chapter 5 - General discussion 132 Crosstalk between type I and II interferon pathways 132 The antiapoptotic and proapoptotic effects of vanadate 135 Expanding OV tropism 137 Improving Oncolytic rhabdovirus therapy in the treatment of Sarcoma 137 Cancer therapy targeting the NF-κB pathway 138 Treatment delivery 139 Beyond oncolytic immunotherapy 140 Concluding thoughts 141 Appendices I – Chapter 2 Supplemental information 143 Appendices II – Chapter 3 Supplemental information 147 Appendices III – Chapter 3 Supplemental information 157 References 178 v List of Abbreviations 7-AAD 7-Aminoactinomycin D ANOVA Analysis of variance Bax BCL2 Associated X BMOV Bismaltolato oxovanadium BSO Buthionine sulfoximine CDK Cyclin-dependent kinase CTCL Cutaneous T-cell lymphoma CTLA-4 Cytotoxic T-lymphocyte-associated protein-4 DEF Diethyl fumarate DEM Diethyl maleate DMEM Dulbecco’s Modified Eagle’s Medium DMF Dimethyl fumarate DMM Dimethyl maleate DNA Deoxyribonucleic acid ECM Extracellular matrix eIF2a eukaryotic initiation factor 2 alpha FA Fumaric acid FAE Fumaric acid esters FDA U S Food and Drug Administration FMAE Fumaric and maleic acid esters G Glycoprotein GAF IFN-γ-regulated DNA-binding factor GAS IFN-γ activation sequence GCL Glutamate Cysteine Ligase GM-CSF Granulocyte-macrophage colony-stimulating factor GO Gene Ontology GSH Glutathione HDAC Histone deacetylase inhibitor HEPS 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HR Higher responders HSV-1 Herpes simplex virus 1 IAP Inhibitors of apoptosis proteins IFIT1 Interferon-induced protein with tetratricopeptide repeats 1 IFITM1 Interferon-induced transmembrane protein 1 IFN Interferon IFNGR1 IFN-γ receptor 1 IKK IκB kinase IRF IFN-regulatory factor vi IRSE Interferon-stimulated response element ISGF3 Interferon-stimulated gene factor 3 IT Intratumoral IV Intravenous IVIS in vivo imaging system JAK Janus kinase JC John Cunningham JX-594 Pexastimogene devacirepvec KEAP1 Kelch-like ECH-associated protein 1 LPS Lipopolysaccharides LR Lower responders M Matrix MG1 Marabex MMF Monomethyl fumarate MOI Multiplicity of infection MS Multiple sclerosis mTOR mammalian target of rapamycin MV Measles MxA Myxoma resistance protein A N Nucleoprotein NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells NK Natural killer NRF2 Nuclear factor (erythroid-derived 2)-like 2 OHSN-REB Ottawa Health Science Network Research Ethics Board OV Oncolytic Virus P Phosphoprotein PBS Phosphate-buffered saline PD-L1 Programmed death-ligand 1 PD1 Programmed cell death protein 1 PDA Pancreatic ductal adenocarcinomas PFU Plaque forming units PI3K Phosphoinositide 3-kinase PKR Protein kinase R PML Progressive multifocal leukoencephalopathy PRR Pattern recognition receptors PTP Protein tyrosine phosphatases PTP1B Protein-tyrosine phosphatase 1B qPCR Quantitative polymerase chain reaction RECIST Response Evaluation Criteria In Solid Tumors RIG-1 Retinoic Acid Inducible Gene 1 vii RIPK3 Receptor Interacting Protein Kinase 3 RNA Ribonucleic acid ROS Reactive oxygen species RPMI Roswell Park Memorial Institute SC Sub-cutaneous Smac Second mitochondrial activator of caspase SMC Smac mimetic compounds STAT1 Signal transducer and activator of transcription 1 STAT2 Signal transducer and activator of transcription 2 T-Vec Talimogene laherparepvec TBK1 TANK-binding kinase 1 TFNα Tumor necrosis factor alpha TGF-β Transforming growth factor-β TK Thymidine kinase TLR Toll-like receptors VOx Vvanadium(V) oxytriethoxyde VS Vanadium(IV) oxide sulphate VSV Vesicular stomatitis virus VVdd double-deleted vaccinia virus wt wild-type viii List of Figures Figure 2.1. Cytopathic effect of oncolytic viruses in sarcoma cells lines in vitro. Figure 2.2. Productive infection of human sarcoma explants by MG1 and VV. Figure 2.3. Maraba virus MG1 infects and replicates in murine sarcoma S180 tumors, leading to persistent anti-tumor effects Figure 3.1. Dimethyl fumarate promotes viral infection Figure 3.2. DMF enhances infection ex vivo and in human clinical samples. Figure 3.3. Fumaric and maleic acid esters promote infection by VSVΔ51. Figure 3.4. Dimethyl fumarate enhances VSV∆51 therapeutic efficacy in syngeneic and xenograft tumor models. Figure 3.5. FMAEs inhibit antiviral cytokine production and response to type I interferon. Figure 3.6. DMF inhibits NF-κB translocation upon infection. Figure 4.1. Vanadate enhances VSV∆51 infection in cancer cells but not normal cells. Figure 4.2. Viral enhancement is dependent on Vanadium. Figure 4.3. Vanadate facilitates virus-induced type I interferon and ROS mediated cell death Figure 4.4. Vanadate increases VSV∆51 efficacy in resistant syngeneic tumor models. Figure 4.5. Vanadate /VSV∆51 co-treatment triggers T-cell infiltration and anti-tumor immunity. Figure 4.6. Vanadate re-wires antiviral type I IFN signaling towards pro-inflammatory type II IFN response ix Chapter 1 - General Introduction