Potent preclinical sensitivity to imipridone-based combination therapies in oncohistone H3K27M-mutant diffuse intrinsic pontine glioma is associated with induction of the integrated stress response, TRAIL death receptor DR5, reduced ClpX and apoptosis

Abstract

The H3K27M oncohistone mutation, identified in approximately 80% of diffuse intrinsic pontine gliomas (DIPG), is a potential target for therapy. Imipridone ONC201/TIC10 (TRAIL-Inducing Compound #10) induces apoptosis of cancer cells, and has clinical efficacy against H3K27M-mutant DIPG. We demonstrate synergy between ONC201, ONC206 and ONC212, and targeted therapies with known preclinical activity against DIPG. We hypothesized that imipridone combinations with HDAC or proteasome inhibitors may be superior to single agent ONC201 treatment in H3K27M mutant DIPG. Six patient-derived DIPG cell lines (SU-DIPG-IV, SU-DIPG-13, SU-DIPG-25, SU-DIPG-27, SU-DIPG-29, SU-DIPG-36) were exposed to imipridones alone or combinations with histone de-acetylase inhibitors [HDACi], marizomib, etoposide, and temozolomide. Dose-dependent response to imipridones was observed in DIPG cells with half-maximal inhibitory concentration (IC₅₀) of 1.46 µM, 0.11 µM, and 0.03 µM, for ONC201, ONC206, and ONC212, respectively. Upon treatment with the imipridones, DIPG cell lines engaged CLpP/CLPX, the integrated stress response with ATF4 activation, and TRAIL death receptor 5 (DR5) induction. Strong synergy was identified between ONC201 and HDACi panobinostat (combination index [CI] 0.01), romidepsin (CI 0.08) and proteasome inhibitor marizomib (CI 0.19). Synergy was demonstrated between ONC201 and etoposide (CI 0.54), although to a lesser degree than with panobinostat, romidepsin, and marizomib. ONC206 and ONC212 showed similar synergistic effects with panobinostat, romidepsin, and marizomib. Induction of apoptosis was demonstrated with imipridones and panobinostat or romidepsin combinations. Our results suggest increased sensitivity of H3K27M-mutant DIPG cell lines to second generation imipridone therapies, as compared to ONC201. Additionally, there is synergistic cell death with combination of imipridones and panobinostat, romidepsin, or marizomib, which may be further tested in vivo and in clinical trials.

Keywords: DIPG; Diffuse intrinsic pontine glioma; ONC201; ONC206; ONC212; imipridones; pediatric cancer.

Figure 1

Dose dependent response demonstrates that imipridone analogs ONC206 and ONC212 are more potent cytotoxic agents than ONC201 against H3K27M mutant DMG. A. Single treatment viability assay as measured by CellTiter-Glo (CTG) of ONC201, ONC206, and ONC212 in cell line SU-DIPG-IV. B. Graphic representation and table of IC₅₀ values of imipridones effect on cell line SU-DIPG-IV. C. Single treatment viability assay as measured by CTG of ONC201, ONC206, and ONC212 in cell line SU-DIPG-13. D. Graphic representation and table of IC₅₀ values of the effect of imipridones on cell line SU-DIPG-13. Cells were treated for 96 hours before assessment of cell viability.

Figure 2

Combination of ONC201 and HDAC inhibitors reveal synergistic activity against H3K27M mutant DIPG. Synergy analyses as measured by CTG, demonstrating the combination of (A) ONC201 and panobinostat in cell line SU-DIPG-13, with best combination index (CI) of 0.01 (B) ONC201 and panobinostat in cell line SU-DIPG-IV, with best CI of 0.01 (C) ONC201 and romidepsin in cell line SU-DIPG-13, with best CI of 0.02 (D) ONC201 and romidepsin in cell line SU-DIPG-29, with best CI of 0.18. Combinations were tested for 96 hours before assessment of cell viability.

Figure 3

Combination of ONC201 and proteasome inhibitor marizomib demonstrates promising synergy against H3K27M mutant DIPG. Synergy analysis of ONC201 and marizomib in cell line SU-DIPG-IV as measured by CTG, with best combination index of 0.19. Combinations were tested for 96 hours before assessment of cell viability.

Figure 4

Mild synergy revealed via combination analysis of ONC201 and etoposide or temozolomide. A. Combination of ONC201 and etoposide with best combination index of 0.46 in cell line SU-DIPG-IV, and 0.53 in cell line SU-DIPG-25. B. Combination of ONC201 and temozolomide with best combination index of 0.35 in cell line SU-DIPG-IV and 0.44 in cell line SU-DIPG-13. Combinations were tested for 96 hours before assessment of cell viability.

Figure 5

Second generation imipridones synergize with HDAC and proteasome inhibitors. A. Combination analysis of ONC206 and panobinostat in cell line SU-DIPG-IV, with best combination index of 0.54. B. Synergy analysis of ONC206 and marizomib has best CI of 0.07 in cell line SU-DIPG-36. C. Synergy analysis of ONC212 and marizomib in cell line SU-DIPG-IV with best combination index of 0.29. D. Combination of ONC212 and romidepsin in cell line SU-DIPG-IV shows best combination index of 0.24. All combinations were tested for 96 hours before assessment of cell viability.

Figure 6

Imipridone therapies induce tumor cell apoptosis. Sub-G1 DNA content of SU-DIPG-13 cells treated with ONC201, ONC206, ONC212, Panobinostat, and etoposide for 96 hours at equitoxic doses. All combinations were tested for 96 hours before assessment of cell viability.

Figure 7

Imipridone induced apoptosis is dependent on timeframe of incubation period. Sub-G1 DNA content of SU-DIPG-13 cells treated with equitoxic doses of ONC201 and ONC206 at both 48 and 96 hours.

Figure 8

Imipridone single agent therapy induces integrated stress response as demonstrated by upregulation of ATF4 and DR5. A. Western blot analysis of SU-DIPG-IV cells treated with both high and low dose ONC206 monotherapy incubated for 48 hours. B. Western blot analysis of SU-DIPG-36 cells treated with high and low dose ONC206 as single agent therapy, incubated for 48 hours. C. Western blot analysis of SU-DIPG-36 cells treated with high and low dose ONC212, incubated for 48 hours.

Figure 9

Integrated stress response and apoptosis induced by imipridones as monotherapy and in combination with other therapeutic agents. Western blot analysis of SU-DIPG-13 cells treated with (A) ONC201 at varying doses, both alone and in combination with panobinostat or romidepsin (B) ONC206 at both low and high doses, alone and in combination with panobinostat or romidepsin (C) Low and high dose ONC212, as single agent therapy and in combination with panobinostat or romidepsin.