Sensitivity to ONC201 correlates with mitochondrial markers, while potent activation of PI3K/Akt drives resistance in diffuse intrinsic pontine glioma.

Abstract

Background:Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive childhood brain cancer with a median overall survival of 9 months. Remarkably, 80-90% of patients harbor a recurring point mutation in histone H3 encoding genes, resulting in a lysine-to-methionine substitution (H3K27M). Currently, there are no therapies targeting H3K27M mutant tumors. The DRD2 receptor antagonist, ONC201, has been reported to have selective efficacy in adult H3K27M mutant glioblastoma, prompting clinical investigations in pediatric DIPGs (NCT03416530). However, preclinical studies to determine the drug's mechanism of action, efficacy, and resistance in DIPG have not been performed. Aim(s): This project aims to identify the mechanism of action ofONC201 and to further determine signaling pathways involved in treatment resistance, thereby, uncovering combination strategies to increase the survival of DIPG patients. Method(s): Ten DIPG patient-derived cell lines were treated with ONC201, and cell growth and survival were determined. Gene and protein expression levels were determined via RNAseq, proteomics, phosphoproteomics, and western blotting assays. Pearson's regression analysis was used to correlate protein expression and ONC201 sensitivity. Result(s): Seven of 10 (70%) patient-derived H3K27M DIPG cell lines tested were sensitive to ONC201 treatment. No correlation was observed between ONC201 sensitivity and DRD2 protein expression (R2 = 0.003775, P = .8661). However, expression of the electron transport chain complex II subunit succinate dehydrogenase (SDHA) was significantly inversely correlated with sensitivity to ONC201 (R2 = 0.7542, P = .0011), providing clues into the mechanism of action. DIPG cells grown in hypoxic conditions were resistant to ONC201, and those that showed inherent resistance (30%) were driven by potent activation of the PI3K/Akt signaling axis. Combined inhibition of PI3K/Akt using paxalisib and SDHA with ONC201 resulted in synergistic cell death in ONC201 resistant cell lines (Bliss score hypoxic = 12.336; normoxic = 9.599). Conclusion(s): In DIPGs,ONC201 primarily targetsmitochondrial pathways. ONC201 resistance may be driven by reprograming to anaerobic glycolysis replacing dependency on oxidative phosphorylation. Reprogramming of core metabolic activity is underpinned by PI3K/Akt, driving anaerobic glycolysis to confer a selective survival advantage. This metabolic plasticity sees tumor cells evade apoptosis and enhance DIPG cell proliferation, which can be overcome by using targeted combination therapies ofONC201 and paxalisib. Translational aspect: These studies highlight the potential of combined administration of two safe, CNS penetrant, orally administered therapeutics and support clinical trial utility. Consumer engagement: ONC201 (NCT03416530) and paxalisib (NCT03696355) are both currently in clinical trials for DIPG. The direct translational benefit to using both for combination treatment is an exciting novel therapeutic potential.