EZH2i EPZ-6438 and HDACi vorinostat synergize with ONC201/TIC10 to activate integrated stress response, DR5, reduce H3K27 methylation, ClpX and promote apoptosis of multiple tumor types including DIPG

Abstract

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.

Keywords: Breast cancer; EZH2; Glioma; HDAC; Histone acetylation; Histone methylation; ONC201.

Fig. 1

Synergistic interaction between EZH2i and ONC201 in the suppression of cell viability of tumor cell lines. (A) CellTiterGlo assay (CTG) analysis of the viability of tumor cell lines treated with EPZ-6438 and ONC201 for 72 H. Drug doses and cell lines are as indicated. (B) The plot of the combination index (CI) as the function of cell viability for tumor cell lines treated with EPZ-6438 and ONC201 combinations in tested tumor cell lines. (C) CTG analysis of the viability of tumor cell lines treated with PF-06821497 and ONC201 for 72H. Drug doses and cell lines are as indicated. (D) The plot of the combination index (CI) as a function of cell viability for tumor cell lines treated with PF-06821497 and ONC201 combinations in tested tumor cell lines.

Fig. 1

Synergistic interaction between EZH2i and ONC201 in the suppression of cell viability of tumor cell lines. (A) CellTiterGlo assay (CTG) analysis of the viability of tumor cell lines treated with EPZ-6438 and ONC201 for 72 H. Drug doses and cell lines are as indicated. (B) The plot of the combination index (CI) as the function of cell viability for tumor cell lines treated with EPZ-6438 and ONC201 combinations in tested tumor cell lines. (C) CTG analysis of the viability of tumor cell lines treated with PF-06821497 and ONC201 for 72H. Drug doses and cell lines are as indicated. (D) The plot of the combination index (CI) as a function of cell viability for tumor cell lines treated with PF-06821497 and ONC201 combinations in tested tumor cell lines.

Fig. 2

Combination of EZH2i plus ONC201 enhances tumor cell line apoptosis. Immunoblotting of cleaved-PARP (clvd-PARP) in (A) MCF7 treated with EPZ-6438 (0, 20, 40, 80 μM) plus ONC201 (0, 2.5, 5 μM) and (C) U251 treated with EPZ-6438 (0, 20, 40, 80 μM) plus ONC201 (0, 4, 8 μM) for 48 H. Densitometric analysis of clvd-PARP/PARP ratios is as indicated in (B) for MCF7 and (D) for U251. (F) Flow cytometric analysis of cell death distribution of MDA-MB-468 cell lines treated with EPZ-6438 (0, 20, 50, 100 μM) plus ONC201 (0, 2.5, 5, 10 μM) for 72 H. The observed % sub-G1 fraction for each experimental condition is as indicated. (G) Immunoblotting of cleaved-PARP (clvd-PARP) and cleaved-caspase 3 in U251 treated with PF-06821497 (0, 50, 100 μM) plus ONC201 (0, 5 μM).

Fig. 3

The combination of EZHi and ONC201 enhances the up-regulation of AFT4 and DR5 induced by ONC201. (A) Immunoblotting of ATF4 and DR5 in MCF7 treated with EPZ-6438 (0, 20, 40, 80 μM) plus ONC201 (0, 2.5, 5 μM). Densitometric analysis of ATF4/Ran and DR5/Ran ratios in MCF7 is as indicated in (B) and (C). (D) Immunoblotting of ATF4 and DR5 in U251 treated with EPZ-6438 (0, 20, 40, 80 μM) plus ONC201 (0, 4, 8 μM) for 48 H. Densitometric analysis of ATF4/ran and DR5/Ran ratios in U251 is as indicated in (E) and (F).

Fig. 4

The combination of EPZ-6438, vorinostat and, ONC201 is synergistic in the suppression of cell viability in multiple human tumor cell lines. (A) CTG analysis of cell viability of tumor cell lines treated with the combination of EPZ-6438, vorinostat, and ONC201 for 72 H. Cell line and drug doses are as indicated. (B) The plot of the combination index (CI) as a function of cell viability for tumor cell lines treated with the combination of EPZ-6438, vorinostat, and ONC201.

Fig. 5

The combination of EPZ-6438, vorinostat and ONC201 enhances apoptosis in human tumor cell lines. Immunoblotting of clvd-PARP in (A) MCF7 treated with the combination of EPZ-6438 (0, 25, 50 μM), vorinostat (0, 0.25 μM) and ONC201 (0, 2.5 μM), and (C) Hep3B treated with EPZ-6438 (0, 25, 50 μM), vorinostat (0, 0.25 μM) and ONC201 (0, 2.5 μM) for 48 H. Densitometric analysis of clvd-PARP/PARP ratios is as indicated in (B) for MCF7 and (D) for U251. (E) Flow cytometric analysis of cell death distribution of the MCF7 cell line treated with combination of EPZ-6438 (0, 25, 50 μM), vorinostat (0, 0.25 μM) and ONC201 (0, 2.5 μM) for 72 H. The observed % sub-G1 fraction for each experimental condition is as indicated.

Fig. 6

EPZ-6438 and vorinostat enhance expression of ATF4 and DR5 induced by ONC201 in MCF7 and Hep3B cells. Immunoblotting of ATF4 and DR5 in MCF7 (A) and Hep3B (C) cells treated with the combination of EPZ-6438 (0, 25, 50 μM), vorinostat (0, 0.25 μM) and ONC201 (0, 2.5 μM) for 48 H. Densitometric analysis of ATF4/ran and DR5/Ran ratios in MCF7 is as indicated in (B) and (C) and Hep3B in (E) and (F).

Fig. 7

Knock-down of ATF4 reduces cell death induced by the combination of ONC201 and EPZ-6438 in U251 glioma cells that do not harbor an H3K27M mutation. (A) CellTiterGlo assay (CTG) analysis of cell viability in U251 cells treated with the combination of EPZ-6438 and ONC201 for 72 hours after ATF4 knockdown. (B) Cell viability assessment of U251 cells treated with the combination of EPZ-6438 and ONC201 for 72 H after ATF4 knockdown. (C) ATF4 was knocked down in U251 for cell viability assessment. (D) Immunoblotting of ATF4, DR5 and clvd-PARP in U251 treated with the combination of EPZ-6438 and ONC201 for 72 H after ATF4 knockdown. Densitometric analysis of DR5/Ran and clvd-PARP/PARP ratios in U251 cells is as indicated in (E) and (F).

Fig. 8

Post-translational modification of H3K27 following treatment of tumor cells with EPZ-6438 or vorinostat. EPZ-6438 or EPZ-6438 plus vorinostat reduce H3K27 tri-methylation in (A) U251 cell lines treated with the combination of EPZ-6438 (0, 40, 80 μM) and ONC201 (0, 8 μM), (B) MCF7 cells treated with the combination of EPZ-6438 (0, 25, 50 μM), vorinostat (0, 0.25 μM), and ONC201 (0, 2.5 μM) and (C) Hep3B cells treated with the combination of EPZ-6438 (0, 25, 50 μM), vorinostat (0, 0.25 μM), and ONC201 (0, 2.5 μM) for 72 H.

Fig. 9

ONC201 sensitivity is not associated with basal expression of dopamine receptors or with the ratio of DRD2/DRD5 in a panel of 12 tumor cell lines. (A–D) Lack of correlation between dopamine receptor expression (as indicated) determined by RT-PCR with ONC201 sensitivity in a panel of 12 human cancer cell lines. (E) The ratio of DRD2 expression versus DRD5 expression in multiple cancer cell lines. ΔCt was calculated by (Ct value of dopamine receptor - Ct value of GAPDH). The 12 cell lines are HCT116, HT29, SW480, MCF7, MDA-MB-361, MDA-MB-468, T98G, SF8628, U251, SNU16, DU145, and PC3 and the ΔCt values are shown in Table 1.

Fig. 10

Change of ONC201 sensitivity was not associated with modulation of dopamine receptor mRNA expression by vorinostat in multiple human tumor cell lines. The change of dopamine receptor mRNA expression was depicted by 2^−ΔΔCt and is shown for the 12-cell line panel as indicated. The 12 cell lines are HCT116, HT29, SW480, MCF7, MDA-MB-361, MDA-MB-468, T98G, SF8628, U251, SNU16, DU145, and PC3 and the ΔCt values are shown in Table 1. ΔIC50 of ONC201 was generated by (IC50 of ONC201 with simultaneous treatment of vorinostat - IC50 of ONC201 without vorinostat treatment).

Fig. 11

Reduced H3K27me3 methylation in multiple tumor cell types including H3K27M-mutated DIPG cells treated with EPZ-6438 or vorinostat and ONC201. (A) H3K27M-mutated DIPG-13 cells were treated with combinations of ONC201 plus either vorinostat or EPZ-6438. PARP cleavage, H3K27 methylation and H3K27 acetylation is shown by western blot along with loading controls. (B) H3K27M-mutated DIPG-25 cells were treated with combinations of ONC201 plus either vorinostat or EPZ-6438. PARP cleavage, H3K27 methylation and H3K27 acetylation is shown by western blot along with loading controls. (C) A second experiment with H3K27M-mutated DIPG-25 cells were treated with combinations of ONC201 plus either vorinostat or EPZ-6438. PARP cleavage, H3K27 methylation and H3K27 acetylation is shown by western blot along with loading controls. (D–G). DIPG-29, Hep3B, U251, and T98G cells were treated with combinations of ONC201 plus either vorinostat or EPZ-6438. PARP cleavage, H3K27 methylation and H3K27 acetylation is shown by western blot along with loading controls.

Fig. 12

Effects of dopamine on cell viability of multiple human tumor cell lines treated with ONC201. (A) MCF7 breast, (B) A549 lung, (C) H460 lung, (D) HCT116 colorectal, and (E) HPAF II pancreatic cancer cell lines were treated with drug doses as indicated and CellTiterGlo assays were performed at 72 H.

Fig. 13

Effects of dopamine on cell viability of multiple human brain tumor cell lines treated with ONC201. (A) U251 glioma, (B) T98G glioma, (C) DIPG4, (D) DIPG13, and (E) DIPG25 cell lines were treated with drug doses as indicated and CellTiterGlo assays were performed at 72 H. Dopamine and ONC201 concentrations are in μM.

Fig. 14

Effects of DRD2 agonist sumarinole on cell viability of multiple human brain tumor cell lines treated with ONC201. (A) T98G glioma, (B) U251 glioma, (C) DIPG25, (D) DIPG13, (E) DIPG4 and (F) DIPG29 cell lines were treated with drug doses as indicated and CellTiterGlo assays were performed at 72 H. ONC201 and sumanirole concentrations are in μM.

Fig. 15

Dopamine or the DRD2 agonist sumanirole do not rescue ONC201 mediated loss of cell viability in multiple human tumor cell lines including DIPG. Dopamine effects on ONC201-induced increase in PKA substrate in DIPG13 (A), DIPG25 (D). Effects of dopamine on apoptosis by ONC201 in different tumor cell lines on ATF4 induction, DR5 activation or apoptosis (B,C,E,F,G). In panels A, and D, there are multiple bands that are impacted (increased intensity) by the positive control Forskolin, reflecting phosphorylation of cellular PKA substrates.