Endocrine Abstracts

Background: Imipridone ONC206, a derivative of dordaviprone (ONC201), is a caseinolytic protease P (ClpP) agonist and dopamine receptor D2/3 (DRD2/3) antagonist with nanomolar potency. Dordaviprone demonstrated responses in patients with neuroendocrine pheochromocytoma-paraganglioma (PC-PG) including tumors with SDHB/FH mutations. We characterized the mechanism of action, in vitro efficacy relative to standard of care (SOC) and dordaviprone, response determinants and acquired resistance for ONC206 in PC models.

Methods: ClpP was expressed in E. coli and purified by His tag affinity for biochemical assays. PC (hPheo1, PC12, MPC10) and fibroblast (HFF-1, MRC-5) cells were commercially sourced. Cell viability (CellTiter-Glo, CyQuant), apoptosis (Caspase Glo, annexin V) assays, seahorse analysis and multi-omics were conducted in PC cells treated with vehicle/ONC206. Resistant hPheo1 cells were generated by passaging with increasing ONC206 concentrations.

Results: Co-crystallization with ClpP revealed an allosteric ligand interaction with distinctions in the ONC206-ClpP resolved crystal structure relative to the dordaviprone-bound or apo complexes. ONC206 was a more potent agonist than dordaviprone in cell-free human ClpP casein/peptide assays. Adrenal gland tumors emerged as most sensitive when ONC206 cytotoxicity was assessed in a panel of 432 human cancer cell lines. Accordingly, PC lines 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 cells. ONC206 demonstrated superior cell viability inhibition and/or apoptosis induction in PC lines relative to dordaviprone, temozolomide, sunitinib and belzutifan at equivalent and/or therapeutically relevant concentrations. In hPheo1 cells, CRISPR-mediated SDHB or FH knockout and DRD2 overexpression did not impact ONC206 sensitivity while ClpP knockout impaired ONC206 sensitivity. Proteomics indicated inhibition of mitochondrial metabolism by ONC206, including OXPHOS/TCA cycle while metabolomics revealed elevated α-ketoglutaric acid, 2-hydroxyglutaric acid and reduced succinic acid, fumaric acid in a ClpP-dependent manner. Consistent with epigenetic regulation by metabolites, ATACseq revealed ONC206 altered chromatin accessibility while RNAseq demonstrated upregulated stress response, apoptosis and downregulated metabolism-related pathways. Western blot confirmed ONC206 downregulated mitochondrial proteins, neuroendocrine markers and upregulated stress response. Whole exome sequencing of acquired resistant cells revealed diverse ClpP missense and/or termination mutations, further confirmed using PCR and/or western blot. Seahorse analysis showed ONC206 inhibited mitochondrially-derived ATP in parental but not acquired resistant lines. Overexpression of wild-type ClpP restored ONC206 sensitivity in acquired resistant lines.

Conclusions: ONC206 is a potent novel agent that is superior to SOC and dordaviprone in PC. ClpP mediates response and acquired resistance to ONC206 in PC.

Abstract ID #33451