Abstract 1156: The integrated stress response (ISR) is involved in the synergistic combinatorial efficacy of ONC201 and epigenetic modulators in brain tumor cell lines

Abstract

Abstract First-in-class small molecule ONC201 is an anti-cancer agent that activates the TRAIL pathway through ATF4 and the integrated stress response (ISR). ONC201 demonstrated durable tumor regressions and prolonged disease stabilization in some patients with histone H3K27M-mutated diffuse midline glioma (DMG). The H3K27M-mutation reduces H3K27 methylation. EZH2 inhibitors (EZH2i) reduce global H3K27 methylation. We hypothesized that ONC201 sensitivity and tumor cell death may be enhanced by reducing H3K27 methylation with EZH2i as a mimic of the H3K27M-mutation, particularly in tumor types beyond DMG. We treated cancer cells from different tissue origins including breast cancer, pancreatic cancer, colorectal cancer, glioblastoma, and DMG with single agent ONC201, EZH2 inhibitor EPZ-6438 or PF-06821497, or the combination of ONC201 plus EPZ-6438 or ONC201 plus PF-06821497. Cell viability was determined with the Cell Titer-Glo assay. Integrated stress response (ISR) activation widely observed in ONC201-treated tumor cells was evaluated using Western blot analysis of ATF4. We determined TRAIL pathway activity by analysis of DR5, and apoptosis by analysis of PARP cleavage. The effect of EPZ-6438 or PF-06821497on the target was validated by immunoblotting of H3K27me3. We knocked down ATF4 with siRNA to evaluate the role of ISR in cell death. Our results demonstrate that ONC201 synergistically reduced cell viability in combination with EZH2i, induced integrated stress response and apoptosis in combination with EZH2i. There was greater induction of ATF4 and DR5 in combinatorial therapy-treated tumor cells versus monotherapy. Knockdown of ATF4 reduced the apoptosis induced by combination therapy. EPZ-6438 or PF-06821497 decreased H3K27 tri-methylation. Our study results unravel potent synergy between ONC201 and EZH2i, highlight the role of ISR in the synergy, and provide further insights into the role of H3K27me3 in ONC201 drug sensitivity. Citation Format: Yiqun Zhang, Lanlan Zhou, Varun V. Prabhu, Joshua E. Allen, Attila A. Seyhan, Wafik S. El-Deiry. The integrated stress response (ISR) is involved in the synergistic combinatorial efficacy of ONC201 and epigenetic modulators in brain tumor cell lines [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 1156.