Summary of Study ST002408

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001550. The data can be accessed directly via it's Project DOI: 10.21228/M8BM6G This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Show all samples | Perform analysis on untargeted data
Download mwTab file (text) | Download mwTab file(JSON) | Download data files (Contains raw data)

Study ID	ST002408
Study Title	Multi-omic Analysis of ClpP Activation in Triple-Negative Breast Cancer Cells
Study Summary	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.
Institute	University of North Carolina at Chapel Hill
Last Name	Graves
First Name	Lee
Address	4111 Genetic Medicine Building, 120 Mason Farm Rd, Chapel Hill, NC 27514
Email	lmg@med.unc.edu
Phone	(919) 966-0915
Submit Date	2022-12-13
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2023-12-18
Release Version	1

Select appropriate tab below to view additional metadata details:

Sample Preparation:

Sampleprep ID:	SP002503
Sampleprep Summary:	SUM159 (WT and ClpP-KO) cells were plated as described for proteomics analysis. Cells were washed 3 x 5 mL ice-cold DPBS. 1 mL -20°C acetonitrile and 750 μL ice cold H2O were added to washed plates and cells were mechanically scraped and transferred to 15 mL conical tubes and stored at -80°C until extraction was performed. To extract metabolites, ~five 2mm zirconia beads were added to each 15 mL conical followed by 500 μL chloroform (-20°C) and samples were vortexed 3 x 30 seconds. Samples were then centrifuged in a 4°C swing-bucket centrifuge (3,700g, 60 minutes). The aqueous layer was then transferred to a 2 mL Lo-Bind tube and the organic layer to a glass vial. Remaining samples were transferred to a 1.5 mL Lo-Bind tube and 15 mL conicals were washed with 300 μL 2:1 chloroform:methanol solution (-20°C) and transferred to corresponding 1.5 mL Lo-Bind tube and centrifuged at 4°C (15,000g, 20 minutes). Aqueous and organic layers were transferred to corresponding 2 mL Lo-Bind tube and glass vial for each sample and frozen at -80°C. Aqueous fractions of cell extracts were dried by speed vac and reconstituted using 200 μL of reconstitution solution (95:5 water:methanol), and 150 μL of the reconstituted extract was transferred to new tubes. An aliquot of 20 μL was taken from each extract and combined to make a total study pool (SP). All samples and the SP were centrifuged at 4 °C and 16,000 x g for 10 minutes, and the supernatants were transferred to LC-MS vials. An injection volume of 5 μL was used for LC-MS analysis.

Sampleprep ID:

SP002503

Sampleprep Summary:

SUM159 (WT and ClpP-KO) cells were plated as described for proteomics analysis. Cells were washed 3 x 5 mL ice-cold DPBS. 1 mL -20°C acetonitrile and 750 μL ice cold H2O were added to washed plates and cells were mechanically scraped and transferred to 15 mL conical tubes and stored at -80°C until extraction was performed. To extract metabolites, ~five 2mm zirconia beads were added to each 15 mL conical followed by 500 μL chloroform (-20°C) and samples were vortexed 3 x 30 seconds. Samples were then centrifuged in a 4°C swing-bucket centrifuge (3,700g, 60 minutes). The aqueous layer was then transferred to a 2 mL Lo-Bind tube and the organic layer to a glass vial. Remaining samples were transferred to a 1.5 mL Lo-Bind tube and 15 mL conicals were washed with 300 μL 2:1 chloroform:methanol solution (-20°C) and transferred to corresponding 1.5 mL Lo-Bind tube and centrifuged at 4°C (15,000g, 20 minutes). Aqueous and organic layers were transferred to corresponding 2 mL Lo-Bind tube and glass vial for each sample and frozen at -80°C. Aqueous fractions of cell extracts were dried by speed vac and reconstituted using 200 μL of reconstitution solution (95:5 water:methanol), and 150 μL of the reconstituted extract was transferred to new tubes. An aliquot of 20 μL was taken from each extract and combined to make a total study pool (SP). All samples and the SP were centrifuged at 4 °C and 16,000 x g for 10 minutes, and the supernatants were transferred to LC-MS vials. An injection volume of 5 μL was used for LC-MS analysis.