ONC201 is a first-in-class imipridone molecule currently in clinical trials for the treatment of multiple cancers. Despite enormous clinical potential, the mechanism of action is controversial. To investigate the mechanism of ONC201 and identify compounds with improved potency, we tested a series of novel ONC201 analogues (TR compounds) for effects on cell viability and stress responses in breast and other cancer models. The TR compounds were found to be ∼50–100 times more potent at inhibiting cell proliferation and inducing the integrated stress response protein ATF4 than ONC201. Using immobilized TR compounds, we identified the human mitochondrial caseinolytic protease P (ClpP) as a specific binding protein by mass spectrometry. Affinity chromatography/drug competition assays showed that the TR compounds bound ClpP with ∼10-fold higher affinity compared to ONC201. Importantly, we found that the peptidase activity of recombinant ClpP was strongly activated by ONC201 and the TR compounds in a dose- and time-dependent manner with the TR compounds displaying a ∼10–100 fold increase in potency over ONC201. Finally, siRNA knockdown of ClpP in SUM159 cells reduced the response to ONC201 and the TR compounds, including induction of CHOP, loss of the mitochondrial proteins (TFAM, TUFM), and the cytostatic effects of these compounds. Thus, we report that ClpP directly binds ONC201 and the related TR compounds and is an important biological target for this class of molecules. Moreover, these studies provide, for the first time, a biochemical basis for the difference in efficacy between ONC201 and the TR compounds.

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ClpP activators ONC201 and related small molecules (TR compounds, Madera Therapeutics), have demonstrated significant anti-cancer potential 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. Applying mass spectrometry-based methods of proteomics and metabolomics, we identified ∼8,000 proteins and 588 metabolites, respectively. From proteomics data, 113 (ONC201) and 191 (TR-57) proteins significantly increased and 572 (ONC201) and 686 (TR-57) proteins significantly decreased in this study. Gene ontological (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. We performed a large-scale transcriptomic analysis of WT SUM159 cells, identifying ∼7,700 transcripts (746 and 1,100 significantly increasing, 795 and 1,013 significantly decreasing in ONC201 and TR-57 treated cells, respectively). Less than 21% of these genes were affected by these compounds in ClpP null cells. GO analysis of these data demonstrated additional similarity of response to ONC201 and TR-57, including a decrease in transcripts related to the mitochondrial inner membrane and matrix, cell cycle, and nucleus, and increases in other nuclear transcripts and transcripts related to metal-ion binding. Comparison of response between both compounds demonstrated a highly similar response in all -omics datasets. Analysis of metabolites also revealed significant similarities between ONC201 and TR-57 with increases in α-ketoglutarate and 2-hydroxyglutaric acid and decreased ureidosuccinic acid, L-ascorbic acid, L-serine, and cytidine observed following ClpP activation in TNBC cells. Further analysis identified multiple pathways that were specifically impacted by ClpP activation, including ATF4 activation, heme biosynthesis, and the citrulline/urea cycle. In summary the results of our studies demonstrate that ONC201 and TR-57 induce highly similar and broad effects against multiple mitochondrial processes required for cell proliferation.

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Imipridones are a novel class of anticancer drugs with promising antiproliferative effects in several cancer cell types, including breast cancer. Recent studies identified the mitochondrial ATP-dependent caseinolytic peptidase P (ClpP) as the target for imipridones and related analogs. Despite these findings, the specific processes by which ClpP activators inhibit cancer cell growth remain poorly understood. Here we report that two structurally distinct ClpP activators, ONC201 and TR-57, promote senescence in SUM159 and MDA-MB-231 triple-negative breast cancer (TNBC) cell lines. Induction of senescence was measured through β-galactosidase assays and confirmed by the increase of H2A.X phosphorylation, hypophosphorylation of retinoblastoma protein (Rb), upregulation of multiple interleukin mRNAs and other markers. The level of senescence induced by these compounds was equivalent to that observed with the CDK4/6 inhibitor and positive control Abemaciclib. To confirm the crucial role of ClpP activation in senescence induction, we generated ClpP null TNBC cell lines using CRISPR interference (CRISPRi). Neither ONC201 nor TR-57 induced senescence in the ClpP null models. Incubation of WT cells with ClpP activators led to a reduction in the levels of apoptosis-related proteins like XIAP, SMAC/DIABLO, Survivin, DR4 and DR5, which correlated with the lack of apoptosis observed in these cells. Interestingly, treatment with TR-57 strongly reduced apoptosis induced by staurosporine but increased sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). To investigate the enhanced effects of TRAIL, we examined the expression of Wee1 in senescent cells and found that both TR-57 and Abemaciclib down-regulated Wee1. Addition of a Wee1 inhibitor partially sensitized cells to TRAIL suggesting the importance of Wee1 in this process. In summary, we show that ClpP activators induce senescence in a ClpP-dependent manner and that combined treatment of ClpP activators with TRAIL provides an effective approach to eliminate malignant senescent cells in vitro.