Onc201 As A Novel Anti-Cancer Therapeutic Via Modulation Of Inhibitors Of Apoptosis And Upregulation Of Dr5 In Gastric Adenocarcinoma.

Abstract Gastric adenocarcinoma is commonly asymptomatic early in its course, leading to late-stage diagnosis at a time when patients are inoperable. Currently available chemotherapy options are non-targeted and highly toxic, leading to a poor 5-year survival of only 31.5%. ONC201 is a small molecule imipridone anti-cancer therapy discovered by our lab that induces cell death in a variety of malignant tumors via a multitude of mechanisms including activation of the integrated stress response (ISR) leading to up-regulation of TNF-related apoptosis-inducing ligand (TRAIL), which induces apoptosis after binding to the death receptor 5 (DR5). We investigated the ability of ONC201 to induce cell death in gastric adenocarcinoma cells when used in combination with recombinant human TRAIL (rhTRAIL), and its affect on DR5 cell surface expression and expression of inhibitors of apoptosis (IAP) in order to further establish mechanism of action. AGS (caspase 8, KRAS, PIK3CA mutant, HER2 amplified), SNU-1 (KRAS and MLH1 mutant, microsatellite instable), SNU-5 (p53 mutant) and SNU-16 (p53 mutant) gastric adenocarcinoma cell lines were treated with ONC201 and rhTRAIL and viability assays were performed to determine cell line sensitivities. All lines were then treated with combination therapy based on sensitivities and viability assays were performed in order to determine combination indices. Cell death was verified with sub-G1 analysis via flow cytometry and protein expression was established via western blotting. All cell lines exhibited strong synergy in response to dual therapy with ONC201 and rhTRAIL, with combination indices <0.6 at doses that did not induce cell death in normal fibroblast cells. Synergy was confirmed via flow cytometry analysis with increased cells in the sub-G1 phase of the cell cycle with dual therapy in excess of what would be expected from an additive effect. On western blot analysis, apoptosis was confirmed with increased cleavage of PARP, caspase 8 and caspase 3 after dual treatment with ONC201 and rhTRAIL. Flow cytometry revealed increased cell surface expression of DR5 with ONC201 therapy, and western blot analysis showed that ONC201 lead to up-regulation of ATF-4 and CHOP, indicating activation of the ISR, and down-regulation of anti-apoptotic CIAP-2 and XIAP in all cell lines except AGS, as well as FLIP in all cell lines except SNU-16. These in vitro results suggest that ONC201 in combination with rhTRAIL may be an effective and non-toxic option for the treatment of gastric adenocarcinoma by inducing apoptosis via activation of the ISR, increased cell surface expression of DR5 and down-regulation of inhibitors of apoptosis. We are currently applying this regimen to an organoid model, as well as a murine sub-cutaneous xenograft model using AGS and SNU-1 cells. Citation Format: Cassandra Susan Parker, Lanlan Zhou, Varun Prabhu, Josh Allen, Wafik El-Deiry. ONC201 as a novel anti-cancer therapeutic via modulation of inhibitors of apoptosis and up-regulation of DR5 in gastric adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1044.