Proteomics and phosphoproteomics analysis of SUM159 cells upon ClpP activation with ONC201 and TR57 compounds

ClpP activators ONC201 and related small molecules (TR compounds, Madera Therapeutics), have demonstrated significant anti-cancer potential in an array of in vitro and in vivo studies, including clinical trials for refractory solid tumors. Though progress has been made in identifying specific phenotypic outcomes following ClpP activation, the exact mechanism by which ClpP activation leads to broad anti-cancer activity has yet to be fully elucidated. In this study, we utilized a multi-omics approach to identify the ClpP-dependent proteomic, transcriptomic, and metabolomic changes resulting from ONC201 or the TR compound TR-57 in triple-negative breast cancer cells. In this portion of the study, we applied mass spectrometry-based proteomics, and quantified ~8000 proteins. From the proteomics data, approximately 3400 (ONC201) and 3000 (TR-57) proteins increased and ~4600 (ONC201) and ~4800 (TR-57) proteins decreased. Gene ontology (GO) analysis revealed strong similarities between proteins up- or downregulated by ONC201 or TR-57 treatment. Notably, this included the downregulation of many mitochondrial processes and proteins, including mitochondrial translation and mitochondrial matrix proteins. Additionally, phosphoproteomics analysis was performed, quantifying ~17,000 phosphopetides, of which >800 were significantly upregulated and >1000 were significantly downregulated phosphopeptides across both ONC201 and TR-57 treatments. Between both treatments, 245 upregulated and 477 downregulated phosphopeptides were shared. Multiple kinases were predicted to be activated including ATM, ATR, AMPK and others, while other kinases were predicted to be inactivated including CDK2, CDK4 and AurB.