ONC201/TIC10 plus TLY012 anti-cancer effects via apoptosis inhibitor downregulation, stimulation of integrated stress response and death receptor DR5 in gastric adenocarcinoma

Gastric adenocarcinoma typically presents with advanced stage when inoperable. Chemotherapy options include non-targeted and toxic agents, leading to poor 5-year patient survival outcomes. Small molecule ONC201/ TIC10 (TRAIL-Inducing Compound #10) induces cancer cell death via ClpP-dependent activation of the integrated stress response (ISR) and up-regulation of the TRAIL pathway. We previously found in breast cancer, pancreatic can-cer and endometrial cancer that ONC201 primes tumor cells for TRAIL-mediated cell death through ISR-dependent upregulation of ATF4, CHOP and TRAIL death receptor DR5. We investigated the ability of ONC201 to induce apop-tosis in gastric adenocarcinoma cells in combination with recombinant human TRAIL (rhTRAIL) or PEGylated tri-meric TRAIL (TLY012). AGS (caspase 8-, KRAS-, PIK3CA-mutant, HER2-amplified), SNU-1 (KRAS-, MLH1-mutant, microsatellite unstable), SNU-5 (p53-mutant) and SNU-16 (p53-mutant) gastric adenocarcinoma cells were treated with ONC201 and TRAIL both in cell culture and in vivo. Gastric cancer cells showed synergy following dual therapy with ONC201 and rhTRAIL/TLY012 (combination indices < 0.6 at doses that were non-toxic towards normal fibro-blasts). Synergy was observed with increased cells in the sub-G1 phase of the cell cycle with dual ONC201 plus TRAIL therapy. Increased PARP, caspase 8 and caspase 3 cleavage after ONC201 plus TRAIL further documented apoptosis. Increased cell surface expression of DR5 with ONC201 therapy was observed by flow cytometry, and immunoblotting revealed ONC201 upregulation of the ISR, ATF4, and CHOP. We observed downregulation of anti-apoptotic cIAP-1 and XIAP in all cells except AGS, and cFLIP in all cells except SNU-16. We tested the regimen in an organoid model of human gastric cancer, and in murine sub-cutaneous xenografts using AGS and SNU-1 cells. Our results suggest that ONC201 in combination with TRAIL 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. Our results demonstrate in vivo anti-tumor effects of ONC201 plus TLY012 against gastric cancer that could be further investigated in clinical trials.