Dataset Information

Proteomics and phosphoproteomics analysis of SUM159 cells upon ClpP activation with ONC201 and TR57 compounds

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Laura Herring

LAB HEAD: Lee Graves

PROVIDER: PXD038990 | Pride | 2023-05-10

REPOSITORIES: Pride

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Publications

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

Fennell Emily M J EMJ Aponte-Collazo Lucas J LJ Pathmasiri Wimal W Rushing Blake R BR Barker Natalie K NK Partridge Megan C MC Li Yuan-Yuan YY White Cody A CA Greer Yoshimi E YE Herring Laura E LE Lipkowitz Stanley S Sumner Susan C J SCJ Iwanowicz Edwin J EJ Graves Lee M LM

Frontiers in pharmacology 20230331

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 approa ...[more]

PMID: 37063293

Similar Datasets

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.

Project description:The ClpP activators ONC201 and related small molecules (TR compounds, Madera Therapeutics), have demonstrated significant anti-cancer potential in an array of in vitro cell models 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 (TNBC). 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. Additionally, gene ontological 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 also 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. Gene ontological analysis of these data demonstrated additional similarity of response to ONC201 and TR-57. Many of the same gene ontology processes and cellular components were identified, including a decrease in transcripts related to the mitochondrial inner membrane and matrix, the cell cycle, and the nucleus, as well as increases in other nuclear transcripts and transcripts related to metal-ion binding. Comparative analysis demonstrated a highly analogous 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 levels of 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.

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)

Project description:GOAL: Identification of genes and/or pathways involved in the pathogenesis of animal models of Rheumatoid Arthritis (RA). Total RNA samples were extracted from synovial fibroblast ex vivo cultures or whole joints (J), isolated from arthritic mice (197, DARE) and their corresponding normal (F1, wt) littermates. Fluorescent-labeled cRNA probes were utilized to hybridize (in duplicates for 197, indicated by "new") the Affymetrix Mu11K oligonucleotide DNA chipset (A+B). In detail the samples are: <table> <tr> <td> Animal model </td> <td> Sample source </td> <td> Sample name </td> </tr> <tr> <td> Tg197 </td> <td> Tg197 ex-vivo cultured synoviocyte populations </td> <td> 197 </td> </tr> <tr> <td> Tg197 </td> <td> Tg197 ex-vivo cultured synoviocyte populations </td> <td> 197new </td> </tr> <tr> <td> Tg197 </td> <td> wt ex-vivo cultured synoviocyte populations </td> <td> F1 </td> </tr> <tr> <td> Tg197 </td> <td> wt ex-vivo cultured synoviocyte populations </td> <td> F1new </td> </tr> <tr> </tr> <tr> <td> Tg197 </td> <td> Tg197 whole joints </td> <td> 197J </td> </tr> <tr> <td> Tg197 </td> <td> Tg197 whole joints </td> <td> 197Jnew </td> </tr> <tr> <td> Tg197 </td> <td> wt whole joints </td> <td> F1J </td> </tr> <tr> <td> Tg197 </td> <td> wt whole joints </td> <td> F1Jnew </td> </tr> <tr> </tr> <tr> <td> DARE </td> <td> DARE ex-vivo cultured synoviocyte populations </td> <td> DARE </td> </tr> <tr> <td> DARE </td> <td> wt ex-vivo cultured synoviocyte populations </td> <td> wt </td> </tr> <tr> <td> DARE </td> <td> DARE whole joints </td> <td> DAREJ </td> </tr> <tr> <td> DARE </td> <td> wt whole joints </td> <td> wtJ </td> </tr> </table> Submitted files carry the extension A or B, corresponding to the subArrays of chipset Mu11K. Arthritic samples were always compared with the corresponding wt sample and not to a common reference chip.

Dataset Information

Proteomics and phosphoproteomics analysis of SUM159 cells upon ClpP activation with ONC201 and TR57 compounds

Publications

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

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