EXTH-86. BH3 PROFILING IDENTIFIES ONC201/TIC10 AS A PROMISING PARTNER OF ABT-263 IN MEDULLOBLASTOMA IN VITRO

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Abstract OBJECTIVE Medulloblastoma represents one of the most common brain tumors in children. In this study, we identified by BH3 profiling that ONC201/TIC10 sensitizes for Bcl-xL/Bcl-2 inhibition in medulloblastoma and performed a preclinical testing of a combined treatment with ONC201/TIC10 and the Bcl-xL/Bcl-2 inhibitor ABT-263. METHODS BH3 profiling was performed to examine ONC201/TIC10-mediated dependencies on anti-apoptotic Bcl-2 family proteins. The combination treatment with ONC201/TIC10 and ABT-263 was tested in different medulloblastoma cells using MTT assays. Isobolograms were calculated to characterize the drug-drug interaction. Tumor spheroid and chorioallantoic membrane assays were used to examine the effects of the combination therapy in a 3D setting. Annexin V/PI staining and flowcytometry were used to detect pro-apoptotic effects. Western blot analyses and knockdown experiments with siRNA were performed for molecular analysis. Extracellular flux analyses served at examining effects on the tumor cell metabolism. RESULTS BH3 profiling showed that ONC201/TIC10 sensitizes medulloblastoma cells to Bcl-xL/Bcl-2 inhibition. In line with this finding, combined treatment with ONC201/TIC10 and ABT-263 led to a predominantly synergistic inhibitory effect on the cell viability of established (D425, D458, DAOY, HD-MB03), primary cultured (PC322) and stem-like (SC322) medulloblastoma cells in 2D and 3D models. The response towards the combination therapy was independent of baseline c-myc expression. On the molecular level, treatment with ONC201/TIC10 led to a dose-dependent decrease of Mcl-1. Moreover, the combination caused enhanced cleavage of caspases 9 and 3. On the metabolic level, the combination therapy led to a reduction in both, oxidative phosphorylation and the glycolytic rate and a reduced expression of respiratory chain proteins. CONCLUSION Combined treatment with ONC201/TIC10 and ABT-263 had a predominantly synergistic inhibitory effect on the cell viability of medulloblastoma cells. This effect was associated with downregulation of Mcl-1. Moreover, the combination treatment resulted in a metabolic reprogramming which likely creates a state of energy deprivation.