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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Mitochondria are multifaceted organelles which are important for bioenergetics, biosynthesis and signaling in metazoans. Mitochondrial functions are frequently altered in cancer to promote both the energy and the necessary metabolic intermediates for biosynthesis required for tumor growth. Cancer stem cells (CSCs) contribute to chemotherapy resistance, relapse, and metastasis. Recent studies have shown that while non-stem, bulk cancer cells utilize glycolysis, breast CSCs are more dependent on oxidative phosphorylation (OxPhos) and therefore targeting mitochondria may inhibit CSC function. We previously reported that small molecule ONC201, which is an agonist for the mitochondrial caseinolytic protease (ClpP), induces mitochondrial dysfunction in breast cancer cells. In this study, we report that ClpP agonists inhibit breast cancer cell proliferation and CSC function in vitro and in vivo. Mechanistically, we found that OxPhos inhibition downregulates multiple pathways required for CSC function, such as the mevalonate pathway, YAP, Myc, and the HIF pathway. ClpP agonists showed significantly greater inhibitory effect on CSC functions compared with other mitochondria-targeting drugs. Further studies showed that ClpP agonists deplete NAD(P)+ and NAD(P)H, induce redox imbalance, dysregulate one-carbon metabolism and proline biosynthesis. Downregulation of these pathways by ClpP agonists further contribute to the inhibition of CSC function. In conclusion, ClpP agonists inhibit breast CSC functions by disrupting mitochondrial homeostasis in breast cancer cells and inhibiting multiple pathways critical to CSC function.

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.

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.

We recently described the identification of a new class of small molecule activators of the mitochondrial protease ClpP. These compounds synthesized by Madera Therapeutics showed increased potency of cancer growth inhibition over the related compound ONC201. In this study, we describe chemical optimization and characterization of the next generation of highly potent and selective small molecule ClpP activators (TR compounds) and demonstrate their efficacy against breast cancer models in vitro and in vivo. One of these compounds (TR-107) was selected for further studies because of excellent potency, specificity and drug-like properties. Examination of TR-107 effects in Triple Negative Breast Cancer (TNBC) cell models showed growth inhibition in the low nanomolar range, equipotent to paclitaxel, in a ClpP-dependent manner. TR-107 reduced specific mitochondrial proteins including OXPHOS and TCA cycle components, in a time, dose and ClpP-dependent manner. Seahorse XF Analysis and glucose deprivation experiments confirmed inactivation of OXPHOS and demonstrated an increased dependence on glycolysis following TR-107 exposure. The pharmacokinetic properties of TR-107 were compared to other known ClpP activators including ONC201 and ONC212. TR-107 displayed excellent exposure and serum t1/2 after oral administration. Using an orthotopic xenograft murine model of MDA-MB-231 cells, the anti-tumor response to TR-107 was investigated. Oral administration of TR-107 resulted in reduction in tumor volume and extension of survival in the treated vs. vehicle control groups. In summary, these studies describe the identification of highly potent new ClpP agonists with improved efficacy against TNBC, through targeted inactivation of OXPHOS and disruption of mitochondrial metabolism.

Mitochondria are multifaceted organelles which are important for bioenergetics, biosynthesis and signaling in metazoans. Mitochondrial functions are frequently altered in cancer to promote both the energy and the necessary metabolic intermediates for biosynthesis required for tumor growth. Cancer stem cells (CSCs) contribute to chemotherapy resistance, relapse, and metastasis. Recent studies have shown that while non-stem, bulk cancer cells utilize glycolysis, breast CSCs are more dependent on oxidative phosphorylation (OxPhos) and therefore targeting mitochondria may inhibit CSC function. We previously reported that small molecule ONC201, which is an agonist for the mitochondrial caseinolytic protease (ClpP), induces mitochondrial dysfunction in breast cancer cells. In this study, we report that ClpP agonists inhibit breast cancer CSC function in vitro and in vivo. Mechanistically, we found that OxPhos inhibition downregulates multiple pathways required for CSC function, such as the mevalonate pathway, YAP, Myc, and the HIF pathway. ClpP agonists showed significantly greater inhibitory effect on CSC functions compared with other mitochondria-targeting drugs. Further studies showed that ClpP agonists deplete NAD(P)+ and NAD(P)H and induce redox imbalance, and dysregulate one-carbon metabolism and proline biosynthesis. Downregulation of these pathways by ClpP agonists further contribute to the inhibition of CSC function. In conclusion, ClpP agonists inhibit breast CSC functions by disrupting mitochondrial homeostasis in breast cancer cells and inhibiting multiple pathways critical to CSC function.

Transcription of the mitochondrial genome is essential for the maintenance of oxidative phosphorylation (OXPHOS) and other functions directly related to this unique genome. Considerable evidence suggests that mitochondrial transcription is dysregulated in cancer and cancer metastasis and contributes significantly to cancer cell metabolism. Recently, inhibitors of the mitochondrial DNA-dependent RNA polymerase (POLRMT) were identified as potentially attractive new anti-cancer compounds. These molecules (IMT1, IMT1B) inactivate cancer cell metabolism through reduced transcription of mitochondrially-encoded OXPHOS subunits such as ND1-5 (Complex I) and COI-IV (Complex IV). Studies from our lab have discovered small molecule regulators of the mitochondrial matrix caseinolytic protease (ClpP) as probable inhibitors of mitochondrial transcription. These compounds activate ClpP proteolysis and lead to the rapid depletion of POLRMT and other matrix proteins, resulting in inhibition of mitochondrial transcription and growth arrest. Herein we present a comparison of POLRMT inhibition and ClpP activation, both conceptually and experimentally, and evaluate the results of these treatments on mitochondrial transcription, inhibition of OXPHOS, and ultimately cancer cell growth. We discuss the potential for targeting mitochondrial transcription as a cancer cell vulnerability.