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

**Background:** ONC201 and related TR compounds are small-molecule activators of the mitochondrial protease ClpP that show anti-cancer activity in preclinical models and clinical trials. While ClpP activation is known to degrade mitochondrial proteins and impair oxidative phosphorylation, the full scope of downstream consequences across multiple molecular layers had not been systematically characterized. This study aimed to comprehensively map proteomic, transcriptomic, and metabolomic changes following ClpP activation in triple-negative breast cancer (TNBC) cells.

**Methods:** Wildtype (WT) and ClpP-knockout (ClpP-KO) SUM159 TNBC cells were treated with 10 μM ONC201 or 150 nM TR-57 (∼10-fold above IC50) for 24 hours. Mass spectrometry-based proteomics identified ∼8,000 proteins; RNAseq quantified ∼7,700 transcripts; and LC-MS metabolomics measured 588 metabolites (from ∼4,100 detected features). Phosphoproteomics identified ∼17,000 phosphopeptides. Individual -omics datasets were analyzed with volcano plots (Log2 fold-change ≥|0.5|, p<0.05) and gene ontology (DAVID). Multi-omics integration was performed using MetaCore pathway analysis software to identify perturbed endogenous metabolic networks (EMNs). ATF4 dependence was validated by siRNA knockdown and immunoblotting. ALAS1 degradation was assessed using bortezomib, epoxomicin, and LonP siRNA.

**Key Results:** Proteomics revealed 572 (ONC201) and 686 (TR-57) significantly downregulated proteins and 113 and 191 significantly upregulated proteins, respectively, in WT cells. In ClpP-KO cells, only 19 (ONC201) and 1 (TR-57) proteins were significantly changed. Transcriptomics showed 746/795 (ONC201) and 1,100/1,013 (TR-57) significantly up/downregulated transcripts in WT cells, with <21% of these changes observed in ClpP-KO cells. Metabolomics identified 30 upregulated and 47 downregulated metabolites common to both treatments. MetaCore analysis identified 42 of the top 50 EMNs as common to both ONC201 and TR-57, including TCA cycle, amino acid metabolism, (S)-citrulline/urea cycle, pyrimidine metabolism, and heme biosynthesis. Within the TCA cycle, citrate synthase, IDH2, αKGDH, SDHA, FH, and MDH were downregulated at the protein level; citrate and succinate decreased while α-ketoglutarate increased. The heme biosynthetic enzyme ALAS1 was rapidly degraded (within 30 minutes for TR-57) in a ClpP-dependent manner; this was not prevented by bortezomib, epoxomicin, or LonP siRNA. ATF4 and ATF4-dependent genes (ASNS, GDF15, PSAT1, PHGDH) were upregulated at both protein and transcript levels; ASNS induction was blocked by ATF4 siRNA. Hemin supplementation did not rescue cell growth inhibition.

**Clinical Implications:** This study establishes that ClpP activation produces broad, multi-nodal disruption of essential mitochondrial metabolic pathways in TNBC cells, rather than targeting a single vulnerability. The ClpP-dependent loss of heme biosynthesis, TCA cycle enzymes, and pyrimidine precursors, combined with ATF4-driven stress responses, suggests that combination strategies targeting these downstream effects may enhance efficacy. The high concordance between ONC201 and TR-57 confirms ClpP as the primary target. These findings support ongoing clinical development of ClpP activators and suggest that metabolic biomarkers (e.g., α-ketoglutarate, 2-hydroxyglutaric acid, serine) could be used to monitor pharmacodynamic response.