Mechanisms of Immunogenic Cell Death in Tumour Cells Infected with Oncolytic Vaccinia Virus
Oncolytic viruses (OVs) infect and kill cancer cells whilst generating anti-tumour immune responses. The anti-tumour effect of OVs is attributed to their ability to induce immunogenic cell death (ICD) during their infection of tumour cells. The mechanisms by which ICD occurs is not well defined and while OVs show immunotherapeutic promise, greater understanding of how specific viruses achieve ICD is needed to allow their oncolytic abilities to be better utilised. In this project, the cell death profile of oncolytic vaccinia virus (oVACV) is characterised in human tumour cell lines. It was observed that oVACV can cause lytic cell death of multiple human cancer cell lines but that the virus suppresses many pathways of programmed cell death prior to lysis. Cell death involves membrane permeabilisation, but not by activation of the classical forms of necroptosis, apoptosis nor pyroptosis. Evidence was however found for active caspase-3, gasdermin E-mediated pyroptosis in one cancer cell line. Several vaccinia genes have been shown to function in blocking different host cell death pathways. We identified another inhibitor candidate, *A47L*, and tested its function as a regulator of the inflammatory cell death pathway, pyroptosis. We generated recombinant vaccinia viruses which lack *A47L* as well as another major inhibitor of inflammatory cell death, *B13R*. We observed that removal of these inhibitors augmented cell lysis during infection. In addition to this demonstration of how the virus’ genetics impacts host cell death, we also examined how the genomic background of the tumour cell can affect sensitivity to onco- viral therapy. Specifically, we focused on the interaction between host DNA-repair capabilities and oVACV infection. We tested whether mutations in the DNA repair proteins DNA-PKcs, BRCA1 or BRCA2 had an impact on oVACV-induced death and found that BCRA2-deficient cells were more sensitive to oVACV killing than their homologous recombination (HR) repair- proficient counterparts. Additionally, we tested whether additive effects can be achieved with combinational therapies that target DNA repair proteins in paired, HR deficient and proficient cell lines. It was found that oncolysis may be quantitively enhanced by inhibition of DNA-PKcs in cells with BRCA2 mutation and by inhibition of PARP1 in cells with wild-type BRCA2. Overall, this thesis explores the mechanisms by which vaccinia virus causes cell death in vitro and outlines the factors which influence this process and contribute to the current clinical limitations of this immunotherapy. Here, we propose methods which precisely control these factors and enhance the immunogenicity of oncolysis.
Cell death, Immunogenic, Immunotherapy, Oncolytic, Vaccinia, Virus
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