Abstract 3312: Oncolytic HSV (34.5ENVE) sensitizes bortezomib-induced cancer cell killing through induction of RIP1 dependent necroptosis.
Authors: Hurwitz Brian S. , Yoo Ji Young , Bolyard Chelsea , Yu Jun-Ge , Wojton Jeffery , Old Matthew , Kaur Balveen 
Published: August 2014

Abstract
The aim of this study is to evaluate the combination of oncolytic herpes simplex virus (34.5ENVE) and bortezomib in the treatment of solid tumors. Although phase 1 clinical trials of oncolytic viruses have established the relative safety of this approach, evidence for efficacy has remained elusive. Therefore, strategies to enhance the efficacy of this treatment are being explored. We investigated the impact of 34.5ENVE in combination with bortezomib, a potent and selective proteosome inhibitor, in the treatment of multiple solid tumor models. In vitro, various human cancer cells were treated with bortezomib alone, 34.5ENVE alone, or the combination of the two. We analyzed the treatments for synergism in cancer cell killing using Chou-Talalay analysis (CI index &lt;1: synergy; CI index &lt; 0.8: strong synergy). Combination treatment of bortezomib and 34.5ENVE displayed strong synergism in multiple cell lines from ovarian cancer, head & neck cancer, and glioma (CI index &lt; 0.8). In vivo, subcutaneous and intracranial tumor xenografts were utilized to study the impact of bortezomib+34.5ENVE on anti-tumor efficacy. Survival was analyzed by the Kaplan-Meier method and evaluated with a two-sided log-rank test. In vivo, the combination of bortezomib and 34.5ENVE significantly enhanced anti-tumor efficacy and prolonged mouse survival. The impact of bortezomib on 34.5ENVE replication was measured by quantifying virus titer. Sub lethal doses of bortezomib increased virus replication (p value &lt;0.001), and treatment of cells with higher doses did not change the absolute viral titer. To understand the mechanism of the synergistic interaction between 34.5ENVE and bortezomib, we analyzed western blotting for cellular markers of ER stress and the JNK signaling pathway. Bortezomib treatment induced ER stress with strong induction of Grp78, CHOP, PERK and IRE1α (western blot analysis). Additionally, the combination of bortezomib and 34.5ENVE increased phosphorylation of JNK and c-Jun more so than the single treatments alone. Production of reactive oxygen species (ROS) was then measured by FACS analysis of cells treated with CellROX. Consistent with JNK activation, increased ROS was observed in both bortezomib and 34.5ENVE treated cells. Furthermore, cells treated with both bortezomib and 34.5ENVE displayed much higher ROS levels compared to cells treated with either agent alone. Both N-acetylcysteine, a ROS scavenger, and dipheylene iodide, an NADPH oxidase inhibitor, rescued the increased synergistic killing. We also treated SP0600125, a selective JNK inhibitor, and necrostatin-1, an inhibitor of RIP1, to investigate the initiation of cell killing. Pretreatment with both inhibitors was able to rescue synergy (CI&gt;0.8). These findings suggest that the combination of bortezomib and 34.5ENVE leads to synergistic cell killing mediated by RIP1 dependent necroptosis induction.
Citation Format: Brian S. Hurwitz, Ji Young Yoo, Chelsea Bolyard, Jun-Ge Yu, Jeffery Wojton, Matthew Old, Balveen Kaur. Oncolytic HSV (34.5ENVE) sensitizes bortezomib-induced cancer cell killing through induction of RIP1 dependent necroptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3312. doi:10.1158/1538-7445.AM2013-3312