Oncolytic viruses (OVs) are viruses which selectively replicate within and directly lyse cancer cells whilst sparing host tissue. This selectivity not only helps their safety profiles and minimise toxicities but also allows OVs to potentially increase their therapeutic dose over time as they replicate and spread throughout tumours. Whilst tumour lysis helps reduce tumour burden, a secondary purpose of OVs as immunotherapies is to stimulate the host's immune system culminating in a systemic anti-tumour response. To do so relies on OVs ability to trigger to two distinct forms of immunity simultaneously: anti-tumour and anti-viral.

OV-induced anti-viral immunity typically refers to release of virions, cytokines and damage- or pathogen-associated molecular patterns (DAMPs and PAMPs) to the tumour microenvironment (TME) upon tumour lysis. Doing so helps to recruit innate and adaptive immune cells to the TME, helping to overcome its naturally immunosuppressive state and stimulate the generation, recruitment and activation of tumour antigen-specific T cell responses. In theory, this combination of anti-viral and anti-tumour immunities has the potential to build to a systemic anti-tumour T cell response that targets distant, secondary tumour sites. In reality, although effective in reducing local tumour burden in the short-term, OVs struggle to produce sustainable anti-tumour responses in the long-term as monotherapies. Recent research into improving OV efficacy has focused on enhancing anti- tumour immunity by combining OVs with other immunotherapies such as immune checkpoint inhibitors (ICI). However, to ultimately achieve a sustainable systemic anti-tumour response against future tumour relapse, a greater understanding of OV-induced anti-viral immunity is needed. In particular, little is known about cancer cell’s intrinsic ability to sense and respond to OV infection and how this may impact OV efficacy.

The purpose of this study was to gain a mechanistic understanding of how tumour cells may sense infection by a vaccinia (VACV)-derived OV, superior killing virus (SKV), through cytoplasmic DNA sensing pathways and how manipulation of this pathway may impact OV efficacy. Here, I screened several syngeneic murine cancer cell lines for their expression of cytoplasmic DNA sensing proteins and susceptibility to OV infection. After establishing the BRAF-mutant melanoma cell line 4434 as our model system, I found that deletion of cytoplasmic DNA sensor cyclic GMP-AMP Synthase (cGAS) resulted in loss of STING-dependent signalling in response to DNA stimulation and SKV infection whilst deletion of DNA-dependent protein kinase (DNA-PK) complex protein Ku80 did not. Despite these differences in STING-dependent signalling, loss of either cGAS or DNA-PK resulted in loss of pro-inflammatory signalling and significantly increased SKV replication in vitro. This was also true in vivo, where cGAS-/- tumours had significantly increased SKV load in comparison to WT tumours. Interestingly, deleting cGAS also had an impact on tumour growth and immune cell infiltration to the TME prior to SKV treatment. Altogether, this work highlights the importance of investigating the underlying activity of cytoplasmic DNA sensing pathways in tumours prior to OV therapy. In particular, these results indicate that VACV-derived OV treatment efficacy of tumours expressing cGAS and DNA-PK may be enhanced by incorporating cGAS and DNA-PK inhibitors to their design.