Abstract A37: Increased efficacy of oncolytic vaccinia virus, an emerging therapy for pancreatic adenocarcinoma, due to intratumoral hypoxia and vascular endothelial growth factor A.

Abstract

Pancreatic adenocarcinoma (PDAC) is an aggressive malignancy which is resistant to conventional treatments. PDAC typically contains areas that are profoundly hypoxic which both drives early epithelial to mesenchymal transition, with subsequent metastasis, and is an important determinant of resistance to chemotherapy and radiotherapy. Oncolytic Vaccinia virus (VV) is an enveloped DNA virus from the poxvirus family and has demonstrated efficacy as an anti-tumor therapy in clinical trials. It is currently the only oncolytic viral therapy to show efficacy on systemic administration, which would be crucial for the treatment of metastatic PDAC (1). No unique host cell surface receptor has been identified for VV and the reasons for its tropism for PDAC and other cancers are unclear. We have investigated VV as a vector for targeting the hypoxic regions in PDAC, as other viral vectors have been found to replicate poorly in hypoxia. We found that cytotoxicity was enhanced in hypoxia in PDAC cells up to 10 fold (2). This increase in cytotoxicity was only seen in PDAC cell lines where there was hypoxic induction of vascular endothelial growth factor-A (VEGF). Stable MiaPaca2 cell lines expressing VEGF (MPVe-165) and a paired vector control (MPVC) were used to demonstrate that VEGF can augment viral transgene expression, cytotoxicity and replication of oncolytic VV. SUIT2 cells treated with VEGF specific siRNA or control were used to demonstrate that conversely, VEGF suppression reduces viral transgene expression, cytotoxicity and replication in vitro. MPVe-165 and MPVC xenografts were established in BALB/c nude mice before treatment with systemic oncolytic VV. Increased viral replication and gene expression in MPVe-165 xenografts demonstrates this novel finding is relevant in vivo. Using qPCR, confocal microscopy of a fluorescently tagged VV (VVL-488) and specific tyrosine kinase inhibition we found that VEGF acts in an autocrine fashion to increase VV internalization, via Akt phospohorylation in PDAC. These results show that VEGF is a novel factor involved in the tropism and pathogenesis of VV for PDAC, and other tumor types, and has relevance for potential combination therapy regimes. Given these findings we then constructed an oncolytic VV to target hypoxic PDAC cells using the HIF-1α oxygen degradation domain, encephalomyocarditis virus internal ribosomal entry site and the VEGF 3’ un-translated region to regulate gene expression in hypoxia. A conventional hypoxia response element promoter driven construct was not feasible given that the VV life cycle is located in the host cell cytoplasm and gene expression does not rely on eukaryotic transcription factors. We have shown a dose-, time- and oxygen-dependent effect using this construct in hypoxic cell culture, on exposure to hypoxia mimetics (DMOG/CoCl) and using a proteasomal inhibitor (MG132). We propose this concept may be adapted to regulate therapeutic genes, or produce a conditionally replicating VV, in hypoxic conditions, and enhance the specificity of this targeted therapy.