ONC201/TIC10 activates TRAIL signaling through ATF4 and the integrated stress response (ISR). ONC201 demonstrated tumor regressions and disease stability in patients with histone H3K27M-mutated midline-glioma. H3K27M-mutation prevents H3K27-methylation on the mutated allele. EZH2 inhibitors (EZH2i) reduce H3K27 methylation and have anti-tumor effects. We hypothesized ONC201 sensitivity and tumor apoptosis may increase by reducing H3K27-methylation with EZH2i or HDACi as mimics of H3K27M-mutation. EZH2i EPZ-6438 (tazemetostat) or PF-06821497 and HDACi vorinostat were combined with ONC201 to treat multiple cancer cell lines and cell viability and histone modifications were analyzed. We observed synergistic effects towards cell viability in multiple cancers by EPZ-6438 or PF-06821497 plus ONC201 or triple therapy with vorinostat, EPZ-6438, and ONC201. EPZ-6438 and vorinostat synergized with ONC201 to enhance apoptosis. Activation of the ISR and TRAIL-DR5 were observed in cells treated with ONC201 -/+ epigenetic modulators. Knockdown of ATF4 reduced DR5 induction and apoptosis following EZH2i and ONC201 treatment of U251 glioma cells. mRNA expression of dopamine-receptors did not correlate with ONC201 sensitivity in the tumor cell lines tested (N = 12), including changes after epigenetic drugs. Dopamine did not rescue apoptosis by ONC201 in different tumor cell lines (N = 10) including 2 GBM, 3 DIPG and did not prevent DR5 activation or apoptosis. DRD2 agonist sumanirole did not protect brain tumor cells (N = 6 including 4 DIPG cell lines) from ONC201 reduction in viability. Although synergy was observed with ONC201 and vorinostat, there was no significant increase in H3K27 acetylation in cell lines including DIPG as compared to vorinostat alone, and in some cases the acetylation was less than vorinostat alone at 72 H. H3K27 methylation reduction correlated with synergy from combinations of either EPZ-6438 or vorinostat with ONC201 or triple combination. Our findings provide a rationale for combination of ONC201 and epigenetic modulators including triple therapy for in vivo and clinical testing in treatment of human malignancies including brain tumors and DIPG.

ONC201/TIC10 通过 ATF4 和综合应激反应 (ISR) 激活 TRAIL 信号。ONC201 在组蛋白 H3K27M 突变的中线神经胶质瘤患者中表现出肿瘤消退和疾病稳定性。H3K27M 突变可防止突变等位基因上的 H3K27 甲基化。EZH2 抑制剂 (EZH2i) 减少 H3K27 甲基化并具有抗肿瘤作用。我们假设 ONC201 敏感性和肿瘤细胞凋亡可能通过用 EZH2i 或 HDACi 减少 H3K27 甲基化作为 H3K27M 突变的模拟物来增加。EZH2i EPZ-6438 (tazemetostat) 或 PF-06821497 和 HDACi 伏立诺他与 ONC201 联合治疗多种癌细胞系并分析细胞活力和组蛋白修饰。我们观察到 EPZ-6438 或 PF-06821497 加 ONC201 或伏立诺他、EPZ-6438 和 ONC201 三联疗法对多种癌症细胞活力的协同作用。EPZ-6438 和伏立诺他与 ONC201 协同作用以增强细胞凋亡。在用 ONC201 -/+ 表观遗传调节剂处理的细胞中观察到 ISR 和 TRAIL-DR5 的激活。在 EZH2i 和 ONC201 处理 U251 胶质瘤细胞后，ATF4 的敲低减少了 DR5 的诱导和细胞凋亡。多巴胺受体的 mRNA 表达与测试的肿瘤细胞系 (N = 12) 中的 ONC201 敏感性无关，包括表观遗传药物后的变化。多巴胺在不同的肿瘤细胞系 (N = 10) 中没有通过 ONC201 挽救细胞凋亡，包括 2 个 GBM、3 个 DIPG，并且没有阻止 DR5 激活或细胞凋亡。DRD2 激动剂 sumaniro 不能保护脑肿瘤细胞（N = 6，包括 4 个 DIPG 细胞系）免受 ONC201 活力降低的影响。尽管观察到 ONC201 和伏立诺他有协同作用，与单独的伏立诺他相比，包括 DIPG 在内的细胞系中 H3K27 乙酰化没有显着增加，并且在某些情况下，在 72 H 时乙酰化低于单独的伏立诺他。H3K27 甲基化减少与 EPZ-6438 或伏立诺他与ONC201 或三重组合。我们的研究结果为 ONC201 和表观遗传调节剂（包括三联疗法）的组合提供了理论依据治疗人类恶性肿瘤（包括脑肿瘤和 DIPG ）的体内和临床试验。