Multi-omics analyses reveal ClpP activators disrupt essential mitochondrial pathways in triple-negative breast cancer

Abstract

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.

Targeting Mitochondrial DNA Transcription by POLRMT Inhibition or Depletion as a Potential Strategy for Cancer Treatment

“…Two of the most potent TR compounds, TR-57 and TR-107, were found to have significant effects on POLRMT [34,38], which will be discussed in more detail below. Though ClpP activation results in the widespread loss of mitochondrial matrix proteins [38], identification of specific ClpP substrates is still being pursued. Recent studies identified multiple ClpP-interacting proteins and putative substrates through proximity ligation (Bio-ID) and N-terminome (HYTANE) profiling methods [35,76].…”

“…This difference in treatment response indicates that there are significant differences in the mechanism of action of these drug classes. Most notably, ClpP agonists are known to impact a number of metabolic pathways that are required for cancer cell proliferation (i.e., proline biosynthesis and heme biosynthesis pathways) [38,78]. Table 1 compares cell lines where IC 50 data for both IMT1 and ClpP agonists has been obtained.…”

“…These compounds include the imipridones ONC201 and ONC206 (currently in clinical trials), the more potent TR compounds, and other recently discovered molecules [ 33 , 34 , 35 , 36 , 37 ]. Studies from our laboratory and others show that ClpP agonists rapidly deplete POLRMT protein and other key proteins involved in mtDNA transcription [ 33 , 34 , 35 , 38 ]. Despite an incomplete understanding of the mechanism of action, ClpP agonists are currently under investigation as potential anti-cancer compounds due to their significant growth inhibitory effects on many different cancer cell lines and cancer models [ 35 , 39 , 40 , 41 , 42 ].…”

“…Though ClpP agonists were found to inhibit cell proliferation in breast cancer and other cancer cell models, the anti-cancer mechanism of action is just beginning to be elucidated [ 33 , 35 , 39 , 42 , 70 , 71 , 72 , 73 , 74 ]. Studies have shown that ClpP activation induces mitochondrial stress and glycolytic dependence, inhibits OXPHOS, global protein synthesis, and cell proliferation, and dysregulates multiple mitochondrial-related events including TCA cycle function and heme biosynthesis [ 33 , 34 , 38 , 75 ]. Two of the most potent TR compounds, TR-57 and TR-107, were found to have significant effects on POLRMT [ 34 , 38 ], which will be discussed in more detail below.…”

Molecular mechanisms of multi-omic regulation in breast cancer

“…Some of these interactions are indeed becoming interesting clues towards targeted therapy. Such is the case of the mechanisms by which the mitochondrial protease ClpP is activated by drugs that are able to breakdown essential mitochondrial pathways in triple-negative breast cancer ( 113 ). Similarly, the role of heat shock proteins (which may be either acting as oncogenes and onco-suppressor genes) has been recently discussed at the light of multi-omic analysis ( 114 ).…”