Abstract A83: Inhibiting mitochondrial ATP synthase with Oligomycin A enhances the kinetics of oncolytic reovirus-induced apoptosis in vitro

Abstract

Abstract Introduction: Here we consider a role for metabolic activity in regulating the efficiency of oncolytic reovirus infection in cancer cells. It is well established that (i) many cancer cells show metabolic dysfunction compared to non-transformed cells and (ii) oncolytic reovirus demonstrates strong selectivity toward cancer cells. How reovirus attains this selectivity and what underlies the efficiency of oncolysis is not fully elucidated. Oncolytic reovirus (Reolysin®) is currently in phase III clinical trials and targeted application of this therapy could benefit significantly from a better determination of prognostic indicators of therapeutic success. Methods: We used chemical inhibitors of glycolysis (2-deoxyglucose [2DG]-Hexokinase), respiration (Rotenone [RN]-Complex I; Antimycin A [AA]-Complex III; Sodium Azide [AZ]-Complex IV; ), and oxidative phosphorylation (Oligomycin A- mitochondrial FO-F1 ATP Synthase) to treat HCT116 colon cancer cells concurrently infected with reovirus, and cell morphology and viability were assessed. Viral plaque assays, flow cytometry for caspase activity and cell viability, Western Blots, caspase inhibition (z-VAD-fmk [ZVAD]- pan-caspase inhibitor) and BAX/BAK double knockout [DKO] HCT116 cells were used to assess the influence of Oligomycin A on reovirus replication and cytolysis. A panel of other cancer cell lines of various tissue origins was used to assess more broadly how Oligomycin A influences cell killing by reovirus. Results: Chemical inhibition of mitochondrial ATP synthase using Oligomycin A , but not general inhibition of respiration or glycolysis, significantly enhanced the speed of reovirus-induced cell killing, as assessed by observing cell morphology and viability in HCT116 cancer cells. Oligomycin A promoted earlier and more substantial caspase activation in reovirus infected cancer cells and corresponding increases in markers of caspase-dependent apoptosis such as cleaved poly-ADP-ribose polymerase (PARP). Reovirus-induced apoptosis was responsible for the enhanced cytolysis observed in the presence of Oligomycin A because the pan-caspase inhibitor ZVAD abolished the observed cell death. Reovirus replication paradoxically appeared to decrease in the presence of Oligomycin A despite, or possibly because of, the increased kinetics of cell death. Reovirus infection is known to induce apoptosis in an atypical manner that is independent of the pro-apoptotic proteins BAX and BAK. Using BAX/BAK double knockout HCT116 cells we determined that the enhancement of cell death induced by reovirus in the presence of Oligomycin was retained, consistent with the atypical mechanism of reovirus-induced apoptosis. Finally, combining Oligomycin A with reovirus in multiple cancer cell lines led to improved cell death, suggesting a generalized mechanism of enhanced cell death that might have therapeutic relevance. Conclusions: This work is the first to investigate whether manipulation of energy metabolism can modulate reovirus infection of cancer cells. We found that inhibition of mitochondrial ATP synthase with Oligomycin A, but not inhibition of certain other steps in cellular energy metabolism, enhanced the kinetics of induction of caspase-dependent apoptosis and cell death during reovirus infection in cancer cells. Furthermore, combining Oligomycin A with reovirus infection led to enhanced killing of various types of cancer cell lines, suggesting this may be a widely applicable strategy for improving reovirus oncolysis. Our data suggest for the first time that mitochondrial metabolism might regulate and be exploited to modulate reovirus infection and provide a framework for future study. Citation Format: Matthew Clarkson, Siyuan Yin, Mio Tsutsui, David Andrews, Randal Johnston. Inhibiting mitochondrial ATP synthase with Oligomycin A enhances the kinetics of oncolytic reovirus-induced apoptosis in vitro. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A83.