Imipridones ONC201/ONC206 + RT/TMZ triple (IRT) therapy reduces intracranial tumor burden, prolongs survival in orthotopic IDH-WT GBM mouse model, and suppresses MGMT

Abstract

Glioblastoma remains a lethal brain tumor in adults with limited therapeutic options. TIC10/ONC201, a first-in-class imipridone we discovered, achieved meaningful therapeutic effects in phase I/II trials in patients with diffuse gliomas (DG's) harboring H3K27M mutations, and currently the drug is in randomized phase III testing (ACTION trial; NCT05580562). ONC201 targets mitochondrial protease ClpP to disrupt oxidative phosphorylation and trigger the integrated stress response (ISR), TRAIL/DR5, and tumor cell death. While ONC201 and its analog ONC206 are undergoing clinical trials as single agents, there is limited information on their interactions with stand-of-care therapy. We show that ONC201 and ONC206 synergize with temozolomide (TMZ) and Radiotherapy (RT). ONC201 enhances TMZ- or RT-induced apoptosis, ISR and cytotoxicity. ClpP-silencing suppresses ONC201-induced cytotoxicity but not TMZ. Both ONC201 and ONC206 reduce expression of TMZ-resistance mediator MGMT observed in H3K27M-mutated DG cells following treatment with imipridones+TMZ. Cytokine profiling indicates distinct effects of ONC201 relative to TMZ treatment. These results suggest mechanisms underlying ONC201's anti-tumoral activity are distinct from those associated with TMZ or RT with potential for synergy between these three treatments. Triple ONC201+RT+TMZ (IRT) therapy prolonged median survival to 123 days with tail on survival curve (3-of-7 mice alive beyond 200-days) in orthotopic U251 GBM model versus ONC201 (44-days; p = 0.000197), RT (63-days; p = 0.0012), TMZ (78-days; p = 0.0354), ONC201+RT (55-days; p = 0.0004), ONC201+TMZ (80-days; p = 0.0041) and RT+TMZ (103-days; p > 0.05). By 231-days, the only surviving mice were in IRT group. Our results support investigation of ONC201/ONC206 in combination with RT/TMZ (IRT) in GBM or H3K27M mutated DG therapy.

Keywords: IDH; MGMT; ONC201; ONC206; glioblastoma multiforme; radiotherapy; temozolomide.

Figure 1. ONC201 synergizes with RT.

(A) Combination of ONC201 and RT treated GBM cell lines SNB19, T98G, and U251, and atypical teratoid rhabdoid tumor (ATRT: BT-12, BT-16) cell lines show treatment effects on cell viability. (B) Colony formation assays with ONC201 up to 4 μM alone or in combination with radiotherapy up to 8 Gy. Blue boxes are dose combinations that show synergy between ONC201 and radiation therapy. Combenefit was used to evaluate the interaction of multiple treatments on short term cell viability and long-term colony formation assay - synergistic combinations are denoted by a synergy score greater than zero. (C) Western blots were utilized to assess induction of PKA substrate phosphorylation, induction of ATF4 as a marker of ISR activation, and multiple markers of cell death such as cleaved PARP and cleaved Caspase 3 in treated brain tumor cells.

Figure 2. ONC201 synergizes with TMZ.

(A) Cell viability assay diffuse intrinsic pontine glioma (DIPG) cell line SF8628 treated with the combination of ONC201 and TMZ. CellTiter Glo assays were performed to determine the cell viability after treatment. Combination indices (CI) were calculated by the method of Chou and Talalay using the CompuSyn software. Highlighted in boxes are dose combinations that show synergy. Data demonstrate synergy between ONC201 up to 5 μM and TMZ up to 400 μM. (B) Colony formation assays were performed to determine long-term cell viability after treatment. Combenefit was used to evaluate the interaction of two treatments on colony formation assay - synergistic combinations are denoted by a synergy score greater than zero. Data demonstrate the strongest synergy between 5 μM ONC201 and 200 μM TMZ. (C) Western blotting was utilized to assess induction of ATF4 and CHOP as markers of ISR activation, and cleaved PARP as a marker of cell death in treated brain tumor cells. The loading control in Figure 2C came from the same experiment and same gel for 0 Gy condition in Figure 3D. Data demonstrate synergy between ONC201 and TMZ. (D) Mitochondrial dysfunction induced by ONC201 is reflected in reduction of maximal cell respiration in the SU-DIPG 36 cell line. Treatment conditions shown in different colors as indicated. (E) Effect of ONC201 on oxygen consumption rate (OCR) tested in SU-DIPG 36 cell line using single-agent therapy with ONC201 or TMZ or doublet therapy using ONC201 in combination with TMZ. OCR of treated cells was measured by Seahorse Analyzer. Treatment conditions as in panel D. (F) siRNA knockdown of ClpP in GBM cell line U251 shows that ONC201 induced cytotoxicity depends on ClpP whereas siRNA knockdown of ClpP has no effect on TMZ-induced cytotoxicity.

Figure 3. IRT triple combination of ONC201 with RT and TMZ synergizes in brain tumor cells.

(A) IRT triple combination of ONC201, RT, and TMZ treated BT16 cells was assessed using cell viability. Combination indices are shown for rhabdoid tumor cell line BT16 treated at different radiation doses, TMZ and ONC201 as indicated. (B) Colony formation assays of GBM U251 cells treated with ONC201 alone or in combination with TMZ at indicated concentrations of each drug are shown. (C) Quantification of colony formation of GBM U251 cells after IRT triple combination treatment. Combination indices (CI) were calculated using the CompuSyn software with the lowest CI of 0.34 between 25 μM TMZ and 1.25 μM ONC201. (D) Western blotting to assess induction of ATF4 and CHOP as markers of ISR activation, cleaved PARP as marker of cell death and suppression of Rad51, a selective DNA repair target to radiosensitize glioma stem cells in treated GBM U251 cells. Data demonstrate synergy with IRT therapy with ONC201, RT, and TMZ.

Figure 4. IRT triple combination of ONC201, TMZ and RT shows strong synergy, induces ATF as marker of ISR activation, and inhibits ClpX to unleash ClpP in SNB19, T98G, U138, and U251 GBM cells.

IRT combination treatment effects with ONC201, RT, and TMZ on BT16, SNB19, and U251 GBM cell lines as assessed by Western blotting. IR combination treatment induces ATF4 and inhibited ClpX. Microtubule-associated protein light chain 3 (LC3) used as a specific marker to monitor autophagy shows no significant changes with treatments. Cells treated with ONC201, TMZ, and RT at indicated drug concentrations or radiation doses at 37^°C for 48 hours.

Figure 5. Differential cytokine profiling of IRT triple combinations of ONC201 or ONC206 with RT and TMZ for 48 hr. in U251 GBM cell line in vitro.

U251 tumor cells treated with ONC201 or ONC206, TMZ, and RT at indicated concentrations or radiation doses for 48 hr. and cell culture supernatant analyzed with the Luminex 200. Fold-change is shown where red indicates a positive fold-change and green indicates a negative fold-change.

Figure 6. IRT triple combination of ONC201, RT, and TMZ for 4 weeks significantly prolongs mouse survival and reduces tumor burden in an orthotopic brain tumor model.

(A) Randomized treatment group mice received weekly treatment of ONC201 (100 mg/kg p.o.) and/or radiotherapy (2 Gy local irradiation) and/or TMZ (20 mg/kg i.p.) for four weeks for long-term survival and tumor monitoring. (B) Long-term survival studies show that IRT triple combination of ONC201, RT, and TMZ significantly prolongs survival and reduces tumor burden as compared to single- and dual-treatments. (C) Tumors were growing at the time just before treatment was initiated: tumor images at baseline and day -3. (D) IHC assessment of IRT triple combination of ONC201, radiotherapy, and TMZ for 2 weeks shows reduced Ki67 expression and induction of cleaved caspase 3. (E) Quantification of short-term biomarkers demonstrating IRT triple combination treatment decreases tumor cell proliferation (Ki67) and induces more apoptosis (cleaved caspase 3).

Figure 7. IRT triple combination of ONC206 with TMZ and RT for two weeks inhibits proliferation and induces apoptosis in the orthotopic U251 GBM model in vivo.

A group of three mice received weekly treatment of ONC206 (100 mg/kg p.o.) and/or radiotherapy (2 Gy local irradiation) and/or TMZ (20 mg/kg i.p.) for two weeks for short-term biomarker studies. Short-term biomarker studies demonstrate that IRT triple combination of ONC206, TMZ, and RT for two weeks decreases tumor cell proliferation (Ki67) and induces apoptosis (cleaved Caspase 3). Quantification is shown from three mice per group.

Figure 8. Imipridones (ONC201, ONC206 and ONC212) modulate MGMT and ClpX expression in DIPG cell lines.

(A) ONC201 reduced MGMT expression in SF8628 cell line. (B) ONC201 inhibited ClpX expression in multiple DIPG cell lines (SU-DIPG-13, SU-DIPG-25, SU-DIPG-27 and SU-DIPG-29). Imipridones inhibited ClpX expression in a dose-dependent manner but did not reduce MGMT in SU-DIPG-13 (C) and SU-DIPG-25 (D) cell lines. (E) Imipridones decrease ClpX and MGMT expression in a dose-dependent manner in the SU-DIPG-IV cell line. (F) Imipridones (ONC201 and ONC206) inhibit ClpX expression and reduce MGMT in SU-DIPG-36 and SF8628 cell lines.