Abstract A014: ClpP drives efficacy and mediates acquired resistance with imipridones ONC201 and ONC206 in glioma in vitro
Authors: Maranto Cristina , Foster Scott , Lee Andrew , Sethna Phiroze , Allen Joshua E. , Prabhu Varun V. 
Published: March 2024

INTRODUCTION: Dordaviprone (ONC201) is the first dual dopamine receptor D2 (DRD2) antagonist and mitochondrial protease ClpP agonist. ONC201 is being tested in a Phase 3 study in H3 K27M-mutant glioma. ONC206, a derivative of ONC201 with nanomolar DRD2/ClpP potency, is in Phase I trials for adult and pediatric central nervous system tumors. Downstream of DRD2/ClpP, imipridones activate the integrated stress response (ISR) and impair mitochondrial metabolism. We evaluated the role of ClpP in innate in vitro sensitivity and acquired resistance to imipridones in glioma. METHODS: FITC-casein and peptide degradation ClpP assays, CRISPR-mediated ClpP knockout, and ClpP overexpression were used for target engagement/validation studies. Human glioblastoma (GBM) (T98G, A172, U87MG), H3.3 K27M mutant diffuse intrinsic pontine glioma (SF8628, SF7761) and astrocytoma (H4, U118MG) cell lines were commercially sourced. Cell viability was determined using CellTiter-Glo. Polyclonal resistant cells were generated through prolonged passaging in increasing imipridone concentrations (SF8628 and T98G cells). Parental and resistant cells (+/- imipridones) were profiled using whole genome sequencing, RNAseq, metabolomics and western blot analysis. RESULTS: ONC206 was more potent than ONC201 in ClpP casein/peptide assays (~7-fold) and cell viability assays for glioma cell lines (~5-9 fold IC50). ONC206 generally impaired cell viability faster than ONC201. SF8628 and T98G cells with ClpP knockout demonstrated resistance to both imipridones. Depleting glucose in the media enhanced glioma cell viability inhibition by imipridones, suggesting the potential influence of mitochondrial metabolism. Imipridones inhibited TCA cycle enzyme α-ketoglutarate dehydrogenase (KGDH) in a ClpP-dependent manner. Metabolomics analysis revealed imipridone treatment elevated α-ketoglutaric acid, α-hydroxyglutaric acid, and glutaric acid. Cell lines with acquired resistance to ONC201 or ONC206 showed cross resistance to both imipridones. RNAseq revealed upregulation of the ISR and amino acid metabolism while western blotting showed downregulated ClpX, induced ATF4/CHOP and H3 K27me3 with imipridone treatment in parental cells but not resistant cells. ClpP mutations were identified in resistant cells; the specific mutation(s) and prevalence differed between cell lines, imipridone, or concentration used to generate resistance. Mutations in ClpP were located at monomer interfaces and distal to the imipridone binding site or catalytic active site. These ClpP mutations impaired intrinsic and imipridone-induced proteolytic activity. Expression of ClpP mutants in parental glioma cells resulted in resistance to imipridones while expression of wild-type ClpP in resistant cells partially restored sensitivity. CONCLUSIONS: Our results indicate that ClpP agonism is directly involved in mediating response and acquired resistance to imipridones and is associated with altered mitochondrial metabolism, ISR, ClpX, and histone modifications in gliomas.

Citation Format: Cristina Maranto, Scott Foster, Andrew Lee, Phiroze Sethna, Joshua E. Allen, Varun V. Prabhu. ClpP drives efficacy and mediates acquired resistance with imipridones ONC201 and ONC206 in glioma in vitro [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A014.