Determining mechanisms of ONC212-resistance in uveal melanoma to develop combination therapy strategies

Uveal melanoma (UM) is the most common primary eye cancer in adults, affecting ~2,500 people annually in USA. Metastasis in ~50% of UM patients is predominantly to the liver (>95%), resulting in poor prognosis. Only two FDA-approved therapies exist for metastatic UM (mUM), with poor response rates. There is an urgent need for developing new therapeutic strategies to achieve effective response. Our preliminary studies indicated that mUM has high Oxidative Phosphorylation (OXPHOS). Indirect targeting of OXPHOS with the imipridone compound ONC212, significantly inhibited UM cell survival, reduced tumor burden and improved survival in orthotopic liver mUM mouse models. However, extended treatment resulted in tumor recurrence, suggesting acquired resistance. Thus, successful translation of the growth inhibitory effect of ONC212 in mUM depends on identification of ONC212 resistance mechanisms and new targets. In this study, we generated and characterized multiple ONC212-resistant UM cell clones. More than thirty ONC212-resistant clones were generated from MM28 and MP46 parental UM cells via prolonged treatment with high-dose ONC212 (0.3 μM). Cell viability assays were done to calculate IC 50 and resistance index (RI) for ONC212, and mitochondrial function was assessed using the Seahorse Mito Stress Test. Whole-genome sequencing (WGS) was used to identify acquired mutations in resistant clones, while proteomic profiling via Reverse Phase Protein Array (RPPA) was used to determine alterations in cell signaling pathways. A high throughput drug screen was completed to identify sensitivities of ONC212-resistant cells to approved anticancer drugs. Orthotopic liver-mUM mouse models were generated with resistant cells and growth of tumors monitored by bioluminescence imaging. ONC212-resistant clones exhibited higher IC 50, and RI compared to parental cells and showed cross-resistance to other imipridones (ONC201, ONC206), suggesting shared mechanisms of resistance. Resistant clones displayed reduced sensitivity to ONC212-mediated OXPHOS inhibition. WGS of each resistant clone confirmed mutations in CLPP, the target of ONC212 action in UM cells. RPPA data revealed alterations in multiple cell signaling pathways. Bioluminescence imaging of liver mUM in vivo models revealed rapid proliferative capacity of ONC212-resistant clones. Using these preclinical models and the compounds identified in drug screen, combination therapy strategies will be tested and validated for clinical studies in future. Importantly, ONC201, the first imipridone was recently approved for therapy of midline gliomas. Therefore, lessons learned from this study may benefit patients acquiring resistance to ONC201 in future as well.