Dysregulated ClpP Activation Depletes AML Cells and Restores T-cell Function via Metabolic Competition
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE306851 | GEO | 2026/03/01
REPOSITORIES: GEO
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Project description: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. Applying mass spectrometry- based methods of proteomics and metabolomics, we identified ~8000 proteins and 588 metabolites, respectively. From proteomics data, approximately 3400 (ONC201) and 3000 (TR-57) proteins increased and ~4600 (ONC201) and ~4800 (TR-57) proteins 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 ~7700 transcripts (~3600 and 3800 increasing, ~4000 and 3900 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.
2023-04-01 | GSE221327 | GEO
Project description:Dysregulated ClpP Activation Depletes AML Cells and Restores T-cell Function via Metabolic Competition
| PRJNA1312529 | ENA
Project description:Systematic genetic interaction profiles can reveal the mechanism-of-action of bioactive compounds. The imipridone ONC201, which is currently in cancer clinical trials, has been ascribed a variety of different targets. To investigate the genetic dependencies of imipridone action, we screened a genome-wide CRISPR knockout library in the presence of either ONC201 or its more potent analog ONC212. Loss of the mitochondrial matrix protease CLPP or the mitochondrial intermediate peptidase MIPEP conferred strong resistance to both compounds. Biochemical and surrogate genetic assays showed that impridones directly activate CLPP and that MIPEP is necessary for proteolytic maturation of CLPP into a catalytically competent form. Quantitative proteomic analysis of cells treated with ONC212 revealed degradation of many mitochondrial as well as non-mitochondrial proteins. Prompted by the conservation of ClpP from bacteria to humans, we found that the imipridones also activate ClpP from Escherichia coli, B. subtilis and Staphylococcus aureus in biochemical and genetic assays. ONC212 and acyldepsipeptide (ADEP)-4, a known activator of bacterial ClpP, caused similar proteome-wide degradation profiles in S. aureus. ONC212 suppressed the proliferation of a number of Gram-positive (S. aureus, B. subtilis, Enterococcus faecium) and Gram-negative species (E. coli, Neisseria gonorrhoeae). Moreover, ONC212 enhanced the ability of rifampin to eradicate antibiotic-tolerant S. aureus persister cells. These results reveal the genetic dependencies of imipridone action in human cells and identify the imipridone scaffold as a new entry point for antibiotic development.
2020-02-23 | GSE138894 | GEO
Project description: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.
2023-05-10 | PXD038990 | Pride
Project description:Ectopic activation of the conserved ClpP protease by chemical activators causes toxicity in bacteria and human cells due to unrestrained proteolysis. The imipridone ONC201 has been ascribed multiple mechanisms of action and is currently in cancer clinical trials. To systematically investigate the genetic dependencies of imipridone action, we screened a genome-wide CRISPR knockout library in the presence of ONC201 and its more potent analog ONC212. Loss of the mitochondrial matrix protease CLPP conferred strong resistance to both compounds, consistent with recent reports that ONC201 directly activates CLPP in cancer cells. Biochemical assays and surrogate genetic assays in yeast confirmed activation of CLPP in the absence of its regulatory subunits. Imipridone toxicity was bypassed by loss of only one other gene, the mitochondrial intermediate peptidase MIPEP, which we showed is necessary for proteolytic maturation of a CLPP precursor form. Quantitative proteomic analysis of cells treated with ONC212 revealed degradation of many mitochondrial proteins as well as cell cycle regulators. Prompted by the conservation of ClpP across kingdoms, we showed that the imipridones activate Escherichia coli ClpP in vitro and Staphylococcus aureus ClpP in a surrogate yeast assay. ONC212 and acyldepsipeptide (ADEP)-4, a known activator of bacterial ClpP, caused similar proteomic degradation profiles in S. aureus. ONC212 suppressed the proliferation of a number of Gram-positive (S. aureus, Bacillus subtilis, Enterococcus faecium) and Gram-negative species (E. coli, Neisseria gonorrhoeae). Moreover, a combination of ONC212 and rifampicin eradicated antibiotic-tolerant S. aureus persister cells. These results reveal the genetic dependencies of imipridone action in human cells and identify the imipridone scaffold as a new entry point for antibiotic development.
2020-03-26 | PXD016119 | Pride
Project description:The peptidase ClpP is conserved from bacteria to human. In the mitochondrial matrix, it multimerizes and forms a macromolecular proteasome-like cylinder. Because of its known relevance for the mitochondrial unfolded protein response during cell stress, we characterized two ClpP knock-out mouse founder lines and documented similar phenotypes. Ubiquitously, ClpP absence led to accumulation of its interactor protein ClpX without transcript upregulation. Interestingly, most wild-type tissues with substantial ClpP amounts had no detectable ClpX. This inverse correlation suggests that ClpX levels and degradation are regulated by ClpP. The expectation of similar protein levels, in view of a reported association of heptameric ClpP rings with hexameric ClpX rings, was confirmed only in testis of wild-type animals. Germline tissue was exceptional also in its vulnerability to ClpP deletion, with both founder lines showing complete infertility for males and females. Otherwise, ubiquitous mitochondrial dysfunction was apparent from severe growth retardation and reduced spontaneous motor activity of the animals, and from a pronounced decrease in pre-/postnatal survival. Spermatogenesis was found aborted at the spermatid stage, acrosomes and axonemes were not formed. Overall, tissue-specific roles of ClpP were evident by this massive effect for germ cells, mild bioenergetic deficits in muscle and liver tissues, and excellent compensation in brain. ClpX was previously reported to chaperone unfolded proteins and also DNA condensation in mitochondria, so it is likely that this pathway is particularly susceptible in germ cells. In conclusion, our study indicates that the role of ClpP in quality control is indispensable during development for cells with rapid changes of mitochondrial numbers, and is relevant during aging for growth and survival of the organism. Factorial design comparing ClpP knock-out mice with wild type littermates in five different tissues (brain, testis, liver, skeletal and heart muscle)
2013-07-17 | E-GEOD-40207 | biostudies-arrayexpress
Project description:Targeting the mitochondrial caseinolytic protease P (ClpP) is a potential novel strategy for treating leukemia. However, little is known about the therapeutic effects of ClpP in solid tumors. In this study, we identify a strong activator of the mitochondrial ClpP for subsequent use in targeted pancreatic ductal adenocarcinoma (PDAC) therapy. Analysis of clinical data demonstrated a positive association between ClpP expression and prognosis in PDAC patients. We validated that aberrant ClpP activation can lead to growth arrest in PDAC cells and tumors. We then performed high-throughput screening and synthetic optimization to develop a potent ClpP activator, ZG111, which promoted ClpP to degrade respiratory chain complexes and occurred in a ClpP-dependent manner. This resulted in impaired oxidative phosphorylation in the mitochondria and activation of JNK/c-Jun pathway and endoplasmic reticulum stress response. ZG111 also produced significant antitumor effects on tumors in cell-derived and patient-derived xenografted mice models. Altogether, our identification of ZG111 demonstrated that the aberrant activation of ClpP hydrolysis can be used as a potential therapeutic strategy to treat PDAC cancer.
2021-10-21 | GSE186091 | GEO
Project description:The peptidase ClpP is conserved from bacteria to human. In the mitochondrial matrix, it multimerizes and forms a macromolecular proteasome-like cylinder. Because of its known relevance for the mitochondrial unfolded protein response during cell stress, we characterized two ClpP knock-out mouse founder lines and documented similar phenotypes. Ubiquitously, ClpP absence led to accumulation of its interactor protein ClpX without transcript upregulation. Interestingly, most wild-type tissues with substantial ClpP amounts had no detectable ClpX. This inverse correlation suggests that ClpX levels and degradation are regulated by ClpP. The expectation of similar protein levels, in view of a reported association of heptameric ClpP rings with hexameric ClpX rings, was confirmed only in testis of wild-type animals. Germline tissue was exceptional also in its vulnerability to ClpP deletion, with both founder lines showing complete infertility for males and females. Otherwise, ubiquitous mitochondrial dysfunction was apparent from severe growth retardation and reduced spontaneous motor activity of the animals, and from a pronounced decrease in pre-/postnatal survival. Spermatogenesis was found aborted at the spermatid stage, acrosomes and axonemes were not formed. Overall, tissue-specific roles of ClpP were evident by this massive effect for germ cells, mild bioenergetic deficits in muscle and liver tissues, and excellent compensation in brain. ClpX was previously reported to chaperone unfolded proteins and also DNA condensation in mitochondria, so it is likely that this pathway is particularly susceptible in germ cells. In conclusion, our study indicates that the role of ClpP in quality control is indispensable during development for cells with rapid changes of mitochondrial numbers, and is relevant during aging for growth and survival of the organism.
2013-07-17 | GSE40207 | GEO
Project description:We performed RNAseq analysis to determine the effect of ClpP-deletion on cumulus cells global gene expression profile in mature (3 month-old) and (6 month-old) mice. RNA sequencing revealed about 13,000 genes (FPKM > 0.1) expressed in cumulus cells. A total of 1025 genes were significantly differentially expressed (p<0.05) in 3-month ClpP-/- cumulus cells compared to WT (548 up-regulated and 477 down-regulated). At 6-month, a total of 555 genes were significantly differentially expressed in ClpP-/- cumulus cells compared to WT (464 up-regulated and 91 down-regulated). In addition, 1012 and 1034 genes were significantly differentially expressed in both 6 month ClpP-/- and WT cumulus cells compared to younger ClpP-/- cumulus cells, respectively. In this study, we assessed the effect of ClpP global deletion on cumulus cells gene expression. Our findings provide new insight into the role of CLPP in the cumulus cells, and may help understand the potential mechanism of infertility and reproductive aging associated with ClpP-deficiency.
2020-08-15 | GSE102756 | GEO
Project description:We performed RNAseq analysis to determine the effect of ClpP-deletion on cumulus cells global gene expression profile in mature (3 month-old) and (6 month-old) mice. RNA sequencing revealed about 13,000 genes (FPKM > 0.1) expressed in cumulus cells. A total of 1025 genes were significantly differentially expressed (p<0.05) in 3-month ClpP-/- cumulus cells compared to WT (548 up-regulated and 477 down-regulated). At 6-month, a total of 555 genes were significantly differentially expressed in ClpP-/- cumulus cells compared to WT (464 up-regulated and 91 down-regulated). In addition, 1012 and 1034 genes were significantly differentially expressed in both 6 month ClpP-/- and WT cumulus cells compared to younger ClpP-/- cumulus cells, respectively. In this study, we assessed the effect of ClpP global deletion on cumulus cells gene expression. Our findings provide new insight into the role of CLPP in the cumulus cells, and may help understand the potential mechanism of infertility and reproductive aging associated with ClpP-deficiency.
2017-12-01 | GSE102269 | GEO