#METABOLOMICS WORKBENCH rushingb_20221213_083239 DATATRACK_ID:3643 STUDY_ID:ST002408 ANALYSIS_ID:AN003925 VERSION 1 CREATED_ON 09-06-2023 #PROJECT PR:PROJECT_TITLE Multi-omic Analysis of ClpP Activation in Triple-Negative Breast Cancer Cells PR:PROJECT_SUMMARY The ClpP activators ONC201 and related small molecules (TR compounds, Madera PR:PROJECT_SUMMARY Therapeutics), have demonstrated significant anti-cancer potential in an array PR:PROJECT_SUMMARY of in vitro cell models and in vivo studies, including clinical trials for PR:PROJECT_SUMMARY refractory solid tumors. Though progress has been made in identifying specific PR:PROJECT_SUMMARY phenotypic outcomes following ClpP activation, the exact mechanism by which ClpP PR:PROJECT_SUMMARY activation leads to broad anti-cancer activity has yet to be fully elucidated. PR:PROJECT_SUMMARY In this study, we utilized a multi-omics approach to identify the ClpP-dependent PR:PROJECT_SUMMARY proteomic, transcriptomic, and metabolomic changes resulting from ONC201 or the PR:PROJECT_SUMMARY TR compound TR-57 in triple-negative breast cancer cells (TNBC). Applying mass PR:PROJECT_SUMMARY spectrometry- based methods of proteomics and metabolomics, we identified ~8000 PR:PROJECT_SUMMARY proteins and 588 metabolites, respectively. From proteomics data, approximately PR:PROJECT_SUMMARY 3400 (ONC201) and 3000 (TR-57) proteins increased and ~4600 (ONC201) and ~4800 PR:PROJECT_SUMMARY (TR-57) proteins decreased in this study. Additionally, gene ontological PR:PROJECT_SUMMARY analysis revealed strong similarities between proteins up- or downregulated by PR:PROJECT_SUMMARY ONC201 or TR-57 treatment. Notably, this included the downregulation of many PR:PROJECT_SUMMARY mitochondrial processes and proteins, including mitochondrial translation and PR:PROJECT_SUMMARY mitochondrial matrix proteins. We also performed a large-scale transcriptomic PR:PROJECT_SUMMARY analysis of WT SUM159 cells, identifying ~7700 transcripts (~3600 and 3800 PR:PROJECT_SUMMARY increasing, ~4000 and 3900 decreasing in ONC201 and TR-57 treated cells, PR:PROJECT_SUMMARY respectively). Less than 21% of these genes were affected by these compounds in PR:PROJECT_SUMMARY ClpP null cells. Gene ontological analysis of these data demonstrated additional PR:PROJECT_SUMMARY similarity of response to ONC201 and TR-57. Many of the same gene ontology PR:PROJECT_SUMMARY processes and cellular components were identified, including a decrease in PR:PROJECT_SUMMARY transcripts related to the mitochondrial inner membrane and matrix, the cell PR:PROJECT_SUMMARY cycle, and the nucleus, as well as increases in other nuclear transcripts and PR:PROJECT_SUMMARY transcripts related to metal-ion binding. Comparative analysis demonstrated a PR:PROJECT_SUMMARY highly analogous response in all -omics datasets. Analysis of metabolites also PR:PROJECT_SUMMARY revealed significant similarities between ONC201 and TR-57 with increases in PR:PROJECT_SUMMARY α-ketoglutarate and 2-hydroxyglutaric acid and decreased levels of PR:PROJECT_SUMMARY ureidosuccinic acid, L-ascorbic acid, L-serine, and cytidine observed following PR:PROJECT_SUMMARY ClpP activation in TNBC cells. Further analysis identified multiple pathways PR:PROJECT_SUMMARY that were specifically impacted by ClpP activation, including ATF4 activation, PR:PROJECT_SUMMARY heme biosynthesis, and the citrulline/urea cycle. In summary the results of our PR:PROJECT_SUMMARY studies demonstrate that ONC201 and TR-57 induce highly similar and broad PR:PROJECT_SUMMARY effects against multiple mitochondrial processes required for cell PR:PROJECT_SUMMARY proliferation. PR:INSTITUTE University of North Carolina at Chapel Hill PR:LAST_NAME Rushing PR:FIRST_NAME Blake PR:ADDRESS 500 Laureate Way, Kannapolis, NC, 28081, USA PR:EMAIL blake_rushing@unc.edu PR:PHONE +1 (704) 250-5000 PR:DOI http://dx.doi.org/10.21228/M8BM6G #STUDY ST:STUDY_TITLE Multi-omic Analysis of ClpP Activation in Triple-Negative Breast Cancer Cells ST:STUDY_SUMMARY The ClpP activators ONC201 and related small molecules (TR compounds, Madera ST:STUDY_SUMMARY Therapeutics), have demonstrated significant anti-cancer potential in an array ST:STUDY_SUMMARY of in vitro cell models and in vivo studies, including clinical trials for ST:STUDY_SUMMARY refractory solid tumors. Though progress has been made in identifying specific ST:STUDY_SUMMARY phenotypic outcomes following ClpP activation, the exact mechanism by which ClpP ST:STUDY_SUMMARY activation leads to broad anti-cancer activity has yet to be fully elucidated. ST:STUDY_SUMMARY In this study, we utilized a multi-omics approach to identify the ClpP-dependent ST:STUDY_SUMMARY proteomic, transcriptomic, and metabolomic changes resulting from ONC201 or the ST:STUDY_SUMMARY TR compound TR-57 in triple-negative breast cancer cells (TNBC). Applying mass ST:STUDY_SUMMARY spectrometry- based methods of proteomics and metabolomics, we identified ~8000 ST:STUDY_SUMMARY proteins and 588 metabolites, respectively. From proteomics data, approximately ST:STUDY_SUMMARY 3400 (ONC201) and 3000 (TR-57) proteins increased and ~4600 (ONC201) and ~4800 ST:STUDY_SUMMARY (TR-57) proteins decreased in this study. Additionally, gene ontological ST:STUDY_SUMMARY analysis revealed strong similarities between proteins up- or downregulated by ST:STUDY_SUMMARY ONC201 or TR-57 treatment. Notably, this included the downregulation of many ST:STUDY_SUMMARY mitochondrial processes and proteins, including mitochondrial translation and ST:STUDY_SUMMARY mitochondrial matrix proteins. We also performed a large-scale transcriptomic ST:STUDY_SUMMARY analysis of WT SUM159 cells, identifying ~7700 transcripts (~3600 and 3800 ST:STUDY_SUMMARY increasing, ~4000 and 3900 decreasing in ONC201 and TR-57 treated cells, ST:STUDY_SUMMARY respectively). Less than 21% of these genes were affected by these compounds in ST:STUDY_SUMMARY ClpP null cells. Gene ontological analysis of these data demonstrated additional ST:STUDY_SUMMARY similarity of response to ONC201 and TR-57. Many of the same gene ontology ST:STUDY_SUMMARY processes and cellular components were identified, including a decrease in ST:STUDY_SUMMARY transcripts related to the mitochondrial inner membrane and matrix, the cell ST:STUDY_SUMMARY cycle, and the nucleus, as well as increases in other nuclear transcripts and ST:STUDY_SUMMARY transcripts related to metal-ion binding. Comparative analysis demonstrated a ST:STUDY_SUMMARY highly analogous response in all -omics datasets. Analysis of metabolites also ST:STUDY_SUMMARY revealed significant similarities between ONC201 and TR-57 with increases in ST:STUDY_SUMMARY α-ketoglutarate and 2-hydroxyglutaric acid and decreased levels of ST:STUDY_SUMMARY ureidosuccinic acid, L-ascorbic acid, L-serine, and cytidine observed following ST:STUDY_SUMMARY ClpP activation in TNBC cells. Further analysis identified multiple pathways ST:STUDY_SUMMARY that were specifically impacted by ClpP activation, including ATF4 activation, ST:STUDY_SUMMARY heme biosynthesis, and the citrulline/urea cycle. In summary the results of our ST:STUDY_SUMMARY studies demonstrate that ONC201 and TR-57 induce highly similar and broad ST:STUDY_SUMMARY effects against multiple mitochondrial processes required for cell ST:STUDY_SUMMARY proliferation. ST:INSTITUTE University of North Carolina at Chapel Hill ST:LAST_NAME Graves ST:FIRST_NAME Lee ST:ADDRESS 4111 Genetic Medicine Building, 120 Mason Farm Rd, Chapel Hill, NC 27514 ST:EMAIL lmg@med.unc.edu ST:PHONE (919) 966-0915 ST:SUBMIT_DATE 2022-12-13 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - S_10 Cell line:ClpP KO | Treatment:0.1% DMSO sample type=study sample; RAW_FILE_NAME=S_10 SUBJECT_SAMPLE_FACTORS - S_16 Cell line:ClpP KO | Treatment:0.1% DMSO sample type=study sample; RAW_FILE_NAME=S_16 SUBJECT_SAMPLE_FACTORS - S_4 Cell line:ClpP KO | Treatment:0.1% DMSO sample type=study sample; RAW_FILE_NAME=S_4 SUBJECT_SAMPLE_FACTORS - S_11 Cell line:ClpP KO | Treatment:10 uM ONC201 sample type=study sample; RAW_FILE_NAME=S_11 SUBJECT_SAMPLE_FACTORS - S_17 Cell line:ClpP KO | Treatment:10 uM ONC201 sample type=study sample; RAW_FILE_NAME=S_17 SUBJECT_SAMPLE_FACTORS - S_5 Cell line:ClpP KO | Treatment:10 uM ONC201 sample type=study sample; RAW_FILE_NAME=S_5 SUBJECT_SAMPLE_FACTORS - S_12 Cell line:ClpP KO | Treatment:150 nM TR57 sample type=study sample; RAW_FILE_NAME=S_12 SUBJECT_SAMPLE_FACTORS - S_18 Cell line:ClpP KO | Treatment:150 nM TR57 sample type=study sample; RAW_FILE_NAME=S_18 SUBJECT_SAMPLE_FACTORS - S_6 Cell line:ClpP KO | Treatment:150 nM TR57 sample type=study sample; RAW_FILE_NAME=S_6 SUBJECT_SAMPLE_FACTORS - SP_1_1 Cell line:- | Treatment:- sample type=Study Pool; RAW_FILE_NAME=SP_1_1 SUBJECT_SAMPLE_FACTORS - SP_1_2 Cell line:- | Treatment:- sample type=Study Pool; RAW_FILE_NAME=SP_1_2 SUBJECT_SAMPLE_FACTORS - SP_1_3 Cell line:- | Treatment:- sample type=Study Pool; RAW_FILE_NAME=SP_1_3 SUBJECT_SAMPLE_FACTORS - SP_1_4 Cell line:- | Treatment:- sample type=Study Pool; RAW_FILE_NAME=SP_1_4 SUBJECT_SAMPLE_FACTORS - S_1 Cell line:WT | Treatment:0.1% DMSO sample type=study sample; RAW_FILE_NAME=S_1 SUBJECT_SAMPLE_FACTORS - S_13 Cell line:WT | Treatment:0.1% DMSO sample type=study sample; RAW_FILE_NAME=S_13 SUBJECT_SAMPLE_FACTORS - S_7 Cell line:WT | Treatment:0.1% DMSO sample type=study sample; RAW_FILE_NAME=S_7 SUBJECT_SAMPLE_FACTORS - S_14 Cell line:WT | Treatment:10 uM ONC201 sample type=study sample; RAW_FILE_NAME=S_14 SUBJECT_SAMPLE_FACTORS - S_2 Cell line:WT | Treatment:10 uM ONC201 sample type=study sample; RAW_FILE_NAME=S_2 SUBJECT_SAMPLE_FACTORS - S_8 Cell line:WT | Treatment:10 uM ONC201 sample type=study sample; RAW_FILE_NAME=S_8 SUBJECT_SAMPLE_FACTORS - S_15 Cell line:WT | Treatment:150 nM TR57 sample type=study sample; RAW_FILE_NAME=S_15 SUBJECT_SAMPLE_FACTORS - S_3 Cell line:WT | Treatment:150 nM TR57 sample type=study sample; RAW_FILE_NAME=S_3 SUBJECT_SAMPLE_FACTORS - S_9 Cell line:WT | Treatment:150 nM TR57 sample type=study sample; RAW_FILE_NAME=S_9 #COLLECTION CO:COLLECTION_SUMMARY The human triple-negative breast cancer (TNBC) cell line SUM159 was cultured in CO:COLLECTION_SUMMARY Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (DMEM/F-12, Gibco, CO:COLLECTION_SUMMARY 11330-032) supplemented with 5% fetal bovine serum (VWR-Seradigm, 97068-085), 1% CO:COLLECTION_SUMMARY antibiotic/antimycotic (ThermoFisher Scientific, 15240062), 5 μg/mL insulin CO:COLLECTION_SUMMARY (Gibco, 12585014), and 1 μg/mL hydrocortisone. CRISPRi was used to generate CO:COLLECTION_SUMMARY CLPP-knockout SUM159 cells. WT MDA-MB-231 cells were cultured in RPMI 1640 media CO:COLLECTION_SUMMARY (Gibco, 11875-093) supplemented with 10% FBS and 1% antibiotic/antimycotic. CO:COLLECTION_SUMMARY Cells were incubated at 5% CO2 and 37°C and periodically tested for mycoplasma. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY SUM159 (WT and ClpP-KO) cells were plated in a 10 cm2 dish (Corning) and allowed TR:TREATMENT_SUMMARY to adhere overnight. Cells were then treated with 0.1% DMSO, 10 μM ONC201, or TR:TREATMENT_SUMMARY 150 nM TR-57 for 24 hours. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY SUM159 (WT and ClpP-KO) cells were plated as described for proteomics analysis. SP:SAMPLEPREP_SUMMARY Cells were washed 3 x 5 mL ice-cold DPBS. 1 mL -20°C acetonitrile and 750 μL SP:SAMPLEPREP_SUMMARY ice cold H2O were added to washed plates and cells were mechanically scraped and SP:SAMPLEPREP_SUMMARY transferred to 15 mL conical tubes and stored at -80°C until extraction was SP:SAMPLEPREP_SUMMARY performed. To extract metabolites, ~five 2mm zirconia beads were added to each SP:SAMPLEPREP_SUMMARY 15 mL conical followed by 500 μL chloroform (-20°C) and samples were vortexed SP:SAMPLEPREP_SUMMARY 3 x 30 seconds. Samples were then centrifuged in a 4°C swing-bucket centrifuge SP:SAMPLEPREP_SUMMARY (3,700g, 60 minutes). The aqueous layer was then transferred to a 2 mL Lo-Bind SP:SAMPLEPREP_SUMMARY tube and the organic layer to a glass vial. Remaining samples were transferred SP:SAMPLEPREP_SUMMARY to a 1.5 mL Lo-Bind tube and 15 mL conicals were washed with 300 μL 2:1 SP:SAMPLEPREP_SUMMARY chloroform:methanol solution (-20°C) and transferred to corresponding 1.5 mL SP:SAMPLEPREP_SUMMARY Lo-Bind tube and centrifuged at 4°C (15,000g, 20 minutes). Aqueous and organic SP:SAMPLEPREP_SUMMARY layers were transferred to corresponding 2 mL Lo-Bind tube and glass vial for SP:SAMPLEPREP_SUMMARY each sample and frozen at -80°C. Aqueous fractions of cell extracts were dried SP:SAMPLEPREP_SUMMARY by speed vac and reconstituted using 200 μL of reconstitution solution (95:5 SP:SAMPLEPREP_SUMMARY water:methanol), and 150 μL of the reconstituted extract was transferred to new SP:SAMPLEPREP_SUMMARY tubes. An aliquot of 20 μL was taken from each extract and combined to make a SP:SAMPLEPREP_SUMMARY total study pool (SP). All samples and the SP were centrifuged at 4 °C and SP:SAMPLEPREP_SUMMARY 16,000 x g for 10 minutes, and the supernatants were transferred to LC-MS vials. SP:SAMPLEPREP_SUMMARY An injection volume of 5 μL was used for LC-MS analysis. #CHROMATOGRAPHY CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) CH:COLUMN_TEMPERATURE 50 ℃ CH:FLOW_GRADIENT Time(min) Flow Rate %A %B Curve 1. 0 0.4 99.0 1.0 5 2. 1.00 0.4 99.0 1.0 5 3. CH:FLOW_GRADIENT 16.00 0.4 1.0 99.0 5 4. 19.00 0.4 1.0 99.0 5 5. 19.50 0.4 99.0 1.0 5 6. 22.00 CH:FLOW_GRADIENT 0.4 99.0 1.0 5 CH:FLOW_RATE 0.4 mL/min CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% methanol; 0.1% formic acid CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF-X Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS Metabolomics data were acquired on a Vanquish UHPLC system coupled to a Q MS:MS_COMMENTS Exactive™ HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher MS:MS_COMMENTS Scientific, San Jose, CA). Metabolites were separated via an HSS T3 C18 column MS:MS_COMMENTS (2.1 × 100 mm, 1.7 µm, Waters Corporation) at 50 °C with binary mobile MS:MS_COMMENTS phase of water (A) and methanol (B), each containing 0.1% formic acid (v/v). The MS:MS_COMMENTS UHPLC linear gradient started from 2% B, and increased to 100% B in 16 min, then MS:MS_COMMENTS held for 4 min, with the flow rate at 400 µL/min. The untargeted data was MS:MS_COMMENTS acquired from 70 to 1050 m/z using the data-dependent acquisition mode. Method MS:MS_COMMENTS blanks and SP injections were placed after every 6 samples (n=3 each). MS:MS_COMMENTS Progenesis QI (version 2.1, Waters Corporation) was used for peak picking, MS:MS_COMMENTS alignment, and normalization. Background signals were filtered out by removing MS:MS_COMMENTS peaks with a fold change less than 3 in the total SP vs the blank injections. MS:MS_COMMENTS Samples were then normalized in Progenesis QI using the “normalize to all” MS:MS_COMMENTS feature. Coefficient of variation (CV) values were calculated across the total MS:MS_COMMENTS SP replicates for each peak and those with CV >30% were removed. Filtered, MS:MS_COMMENTS normalized data was exported and multivariate analysis was performed using SIMCA MS:MS_COMMENTS 16. MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST002408_AN003925_Results.txt UNITS:Normalized intensity Has RT:Yes RT units:Minutes #END