We propose to investigate the role of autophagy in facilitating synergistic increase in oncolytic activity of vesicular stomatitis virus (VSV) in combination with the FDA-approved histone deacetylase inhibitor vorinostat. Vorinostat reversibly sensitizes hormone-refractory prostate cancer cells to oncolysis; transcriptome and biochemical analysis of PC3 prostate cancer cells identified NF-kB-mediated upregulation of the autophagic pathway as a potential mechanism responsible for suppression of the antiviral response and potentiation of VSV oncolysis (Shulak et al, J. Virol, 2014). Our hypothesis is that host genes involved in distinct stages of autophagosome formation contribute to vorinostat-mediated blocking of antiviral signalling, thus facilitating increased VSV replication and stimulation of adaptive immune responses during oncolysis.
Our specific aims are: 1. To evaluate autophagosome formation and examine the role of key autophagic proteins in vorinostat-dependent potentiation of VSV oncolysis. Genes that regulate canonical stages of autophagosome formation - a) initiation, b) nucleation, c) elongation and closure, and d) recycling - will be knocked-down with siRNA to determine which steps of the autophagic pathway contribute to synergy. Following gene knockdown, cells will be treated with vorinostat and infected with VSV; mRNA levels of various pro-inflammatory cytokines and other molecules with roles in innate responses and autophagic pathway will be measured by a nanofluidic BioMarkTM assay. In addition, the crosstalk between autophagy and apoptosis will be assessed by examining the stability of the BCL-2/BECLIN1 complex using immunoprecipitation and by determining the effect of either knockdown or overexpression of BCL-2 and BECLIN1 on VSV oncolysis and innate immune responses. 2. To examine the contribution of autophagy to innate and adaptive immune responses in a murine model of prostate cancer. The contribution of vorinostat-induced autophagy to immune responses will be examined in the immunocompetent TRAMP-C2 model of prostate cancer. Co-administration of the autophagy inhibitor hydroxychloroquine (HCQ) will be used to inhibit autophagy in vivo. Immune responses to vorinostat + VSV +/- HCQ will be assessed by enumeration of tumor infiltrating immune cells and by measuring mRNA levels of antiviral proteins. Changes in adaptive immune responses will be assessed using tumor cell lysate- pulsed dendritic cells. Such cells will be used to measure T cell priming to tumor antigens in CD4+ T cells (IFNg production) and to measure cytolytic activity in CD8+ T cells by assessing the intracellular levels of perforin and granzyme B in treated mice. Altogether this project will define the role autophagy plays in potentiation of VS oncolysis and in modulation of innate and adaptive responses to vorinostat + VSV combination.
This project will investigate the mechanisms underlying the synergistic oncolytic effects of a FDA approved epigenetic modulator - Vorinostat (SAHA) - in combination with vesicular stomatitis virus (VSV) in pre-clinical models of oncolysis-resistant prostate cancer. This study will contribute to our understanding of how autophagy manipulates the host antiviral pathway to increase therapeutic potential of oncolytic viruses in therapy-resistant cancers.
