Abstract 361: Allosteric ClpP agonist ONC206 alters mitochondrial metabolism, stress response, chromatin accessibility and cell cycle to elicit apoptosis in pheochromocytoma

Abstract Dordaviprone (ONC201), an allosteric caseinolytic protease P (ClpP) agonist and dopamine receptor D2/3 (DRD2/3) antagonist, has demonstrated durable responses in recurrent H3 K27M-mutant glioma patients. ONC206, a derivative of dordaviprone with nanomolar potency for DRD2/3 and ClpP, is in Phase I trials. Dordaviprone demonstrated responses in patients with neuroendocrine pheochromocytoma-paraganglioma (PC-PG) including tumors with SDHB/FH loss-of-function driver mutations. We characterized ClpP binding, in vitro efficacy, comparison to standard of care (SOC) treatments, downstream signaling and response determinants for ONC206 in PC. Co-crystallization with ClpP that assembles as a tetradecameric complex revealed an allosteric ligand interaction with distinctions in the ONC206-ClpP resolved crystal structure relative to the dordaviprone-bound or apo complex, including an increase in the resolved pore size and decreased complex height. ONC206 was more potent than dordaviprone in cell-free human ClpP casein/peptide degradation assays. Accordingly, PC lines (hPheo1, PC12, MPC10) exhibited increased nanomolar sensitivity to ONC206 (∼6 fold) relative to dordaviprone in cell viability assays. ONC206 induced dose- and time-dependent apoptosis in PC but not fibroblast (HFF-1, MRC-5) cells as measured with caspase Glo and/or annexin V assays. ONC206 generally required a shorter duration of exposure (∼24h) and induced enhanced apoptosis in PC cell lines relative to dordaviprone at therapeutically relevant concentrations. ONC206 also demonstrated superior cell viability inhibition and apoptosis induction in PC cell lines relative to temozolomide and sunitinib at equivalent concentrations. CRISPR-mediated SDHB or FH knockout hPheo1 cells were equally sensitive to ONC206 as parental cells. Unlike DRD2, CRISPR-mediated ClpP knockout in hPheo1 cells impaired ONC206 sensitivity. Proteomics indicated inhibition of mitochondrial metabolism including OXPHOS and TCA cycle in hPheo1 cells. Accordingly, metabolomics revealed ONC206 elevated α-ketoglutaric acid, 2-hydroxyglutaric acid and reduced succinic acid, fumaric acid in a ClpP-dependent manner in hPheo1 cells. Consistent with epigenetic regulation by metabolites, ATACseq revealed ONC206 altered chromatin accessibility in hPheo1 cells. RNAseq demonstrated ONC206 upregulated stress response, apoptosis and downregulated cell cycle, metabolism-related pathways in hPheo1 cells. Accordingly, western blot showed ONC206 downregulated mitochondrial proteins (ClpX, SDHB), neuroendocrine markers (tyrosine hydroxylase, enolase-2, synaptophysin) and upregulated stress response (ATF4) in hPheo1 cells. Thus, ONC206 is a potent novel agent that is superior to SOC and dordaviprone that have shown clinical activity in PC-PG. Citation Format: Varun V. Prabhu, Cristina Maranto, Andrew Lee, Scott Foster, Joshua E. Allen. Allosteric ClpP agonist ONC206 alters mitochondrial metabolism, stress response, chromatin accessibility and cell cycle to elicit apoptosis in pheochromocytoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 361.