#METABOLOMICS WORKBENCH kelleno_20220302_151332 DATATRACK_ID:3110 STUDY_ID:ST002115 ANALYSIS_ID:AN003514 VERSION 1 CREATED_ON 02-08-2024 #PROJECT PR:PROJECT_TITLE A ferroptosis defense mechanism mediated by glycerol 3-phosphate dehydrogenase 2 PR:PROJECT_TITLE in mitochondria PR:PROJECT_SUMMARY Mechanisms of defense against ferroptosis (an iron-dependent form of cell death PR:PROJECT_SUMMARY induced by lipid peroxidation) in cellular organelles remain poorly understood, PR:PROJECT_SUMMARY hindering our ability to target ferroptosis in disease treatment. In this study, PR:PROJECT_SUMMARY metabolomic analyses revealed that treatment of cancer cells with glutathione PR:PROJECT_SUMMARY peroxidase 4 (GPX4) inhibitors results in intracellular glycerol 3-phosphate PR:PROJECT_SUMMARY (G3P) depletion. We further showed that supplementation of cancer cells with G3P PR:PROJECT_SUMMARY attenuates ferroptosis induced by GPX4 inhibitors in a G3P dehydrogenase 2 PR:PROJECT_SUMMARY (GPD2)-dependent manner; GPD2 deletion sensitizes cancer cells to GPX4 PR:PROJECT_SUMMARY inhibition-induced mitochondrial lipid peroxidation and ferroptosis, and PR:PROJECT_SUMMARY combined deletion of GPX4 and GPD2 synergistically suppresses tumor growth by PR:PROJECT_SUMMARY inducing ferroptosis in vivo. Mechanistically, inner mitochondrial PR:PROJECT_SUMMARY membrane-localized GPD2 couples G3P oxidation with ubiquinone reduction to PR:PROJECT_SUMMARY ubiquinol, which acts as a radical-trapping antioxidant to suppress ferroptosis PR:PROJECT_SUMMARY in mitochondria. Taken together, these results reveal that GPD2 participates in PR:PROJECT_SUMMARY ferroptosis defense in mitochondria by generating ubiquinol. PR:INSTITUTE University of Texas MD Anderson Cancer Center PR:LAST_NAME Gan PR:FIRST_NAME Boyi PR:ADDRESS 6565 MD Anderson Blvd, Houston, TX 77030 PR:EMAIL bgan@mdanderson.org PR:PHONE 713-792-8653 PR:DOI http://dx.doi.org/10.21228/M8HD8Q #STUDY ST:STUDY_TITLE LC-MS analysis of metabolic changes induced by GPX4 inhibitor treatment in ST:STUDY_TITLE cultured HT1080 cells ST:STUDY_SUMMARY HT1080 cells were treated with vehicle (DMSO), RSL3 (10 micromolar), ML210 (10 ST:STUDY_SUMMARY micromolar), or ML162 (10 micromolar) for 2 hours. Cellular metabolites were ST:STUDY_SUMMARY then extracted and analyzed by LC-MS. ST:INSTITUTE University of Texas MD Anderson Cancer Center ST:LAST_NAME Gan ST:FIRST_NAME Boyi ST:ADDRESS 6565 MD Anderson Blvd, Houston TX, 77030 ST:EMAIL bgan@mdanderson.org ST:PHONE 713-792-8653 ST:SUBMIT_DATE 2022-03-02 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_STRAIN_DETAILS HT1080 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - HT1080_DMSO_01 Treatment:DMSO RAW_FILE_NAME=HT1080_DMSO_01.mzXML; RAW_FILE_NAME=HT1080_DMSO_01_HILIC.mzXML SUBJECT_SAMPLE_FACTORS - HT1080_DMSO_02 Treatment:DMSO RAW_FILE_NAME=HT1080_DMSO_02.mzXML; RAW_FILE_NAME=HT1080_DMSO_02_HILIC.mzXML SUBJECT_SAMPLE_FACTORS - HT1080_DMSO_03 Treatment:DMSO RAW_FILE_NAME=HT1080_DMSO_03.mzXML; RAW_FILE_NAME=HT1080_DMSO_03_HILIC.mzXML SUBJECT_SAMPLE_FACTORS - HT1080_ML162_01 Treatment:ML162 RAW_FILE_NAME=HT1080_ML162_01.mzXML; RAW_FILE_NAME=HT1080_ML162_01_HILIC.mzXML SUBJECT_SAMPLE_FACTORS - HT1080_ML162_02 Treatment:ML162 RAW_FILE_NAME=HT1080_ML162_02.mzXML; RAW_FILE_NAME=HT1080_ML162_02_HILIC.mzXML SUBJECT_SAMPLE_FACTORS - HT1080_ML162_03 Treatment:ML162 RAW_FILE_NAME=HT1080_ML162_03.mzXML; RAW_FILE_NAME=HT1080_ML162_03_HILIC.mzXML SUBJECT_SAMPLE_FACTORS - HT1080_ML210_01 Treatment:ML210 RAW_FILE_NAME=HT1080_ML210_01.mzXML; RAW_FILE_NAME=HT1080_ML210_01_HILIC.mzXML SUBJECT_SAMPLE_FACTORS - HT1080_ML210_02 Treatment:ML210 RAW_FILE_NAME=HT1080_ML210_02.mzXML; RAW_FILE_NAME=HT1080_ML210_02_HILIC.mzXML SUBJECT_SAMPLE_FACTORS - HT1080_ML210_03 Treatment:ML210 RAW_FILE_NAME=HT1080_ML210_03.mzXML; RAW_FILE_NAME=HT1080_ML210_03_HILIC.mzXML SUBJECT_SAMPLE_FACTORS - HT1080_RSL3_01 Treatment:RSL3 RAW_FILE_NAME=HT1080_RSL3_01.mzXML; RAW_FILE_NAME=HT1080_RSL3_01_HILIC.mzXML SUBJECT_SAMPLE_FACTORS - HT1080_RSL3_02 Treatment:RSL3 RAW_FILE_NAME=HT1080_RSL3_02.mzXML; RAW_FILE_NAME=HT1080_RSL3_02_HILIC.mzXML SUBJECT_SAMPLE_FACTORS - HT1080_RSL3_03 Treatment:RSL3 RAW_FILE_NAME=HT1080_RSL3_03.mzXML; RAW_FILE_NAME=HT1080_RSL3_03_HILIC.mzXML #COLLECTION CO:COLLECTION_SUMMARY Metabolites were extracted from cells in 35 mm culture plates by rapidly CO:COLLECTION_SUMMARY aspirating the culture medium and incubating the plates with 0.6 ml of an 80% CO:COLLECTION_SUMMARY methanol: 20% water mixture on a cold block on dry ice for 15 min. Next, the CO:COLLECTION_SUMMARY cell material was scraped into Eppendorf tubes pre-chilled on ice. After CO:COLLECTION_SUMMARY centrifugation at 13,000 RCF for 5 min at 4 °C, the supernatant was collected CO:COLLECTION_SUMMARY into a fresh tube and stored on dry ice until analysis. CO:SAMPLE_TYPE Cultured cells CO:STORAGE_CONDITIONS -80? #TREATMENT TR:TREATMENT_SUMMARY Cells were seeded in 35-mm culture plates. When the cell confluence reached TR:TREATMENT_SUMMARY 70-80%, cells were treated with RSL3, ML210, or ML162 for 2 hours. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For analysis by reverse phase chromatography, just before analysis, 500 µL of SP:SAMPLEPREP_SUMMARY extract was dried under a nitrogen gas flow and then resuspended in 100 µL of SP:SAMPLEPREP_SUMMARY water. For analysis by HILIC chromatography, the extracts were analyzed SP:SAMPLEPREP_SUMMARY directly. SP:PROCESSING_STORAGE_CONDITIONS 4? SP:EXTRACT_STORAGE 4? #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The gradient was 0 min, 85% B; 2 min, 85% B; 3 min, 80% B; 5 min, 80% B; 6 min, CH:CHROMATOGRAPHY_SUMMARY 75% B; 7 min, 75% B; 8 min, 70% B; 9 min, 70% B; 10 min, 50% B; 12 min, 50% B; CH:CHROMATOGRAPHY_SUMMARY 13 min, 25% B; 16 min, 25% B; 18 min, 0% B; 23 min, 0% B; 24 min, 85% B; 30 min, CH:CHROMATOGRAPHY_SUMMARY 85% B. The injection volume was 5 µL. CH:INSTRUMENT_NAME Thermo Accela 1250 CH:COLUMN_NAME Waters XBridge BEH Amide (150 x 2.1mm,2.5um,100A) CH:COLUMN_TEMPERATURE 40 CH:FLOW_RATE 150 CH:SOLVENT_A 95% water/5% acetonitrile; 20 mM ammonium acetate, pH 9.4 CH:SOLVENT_B 100% acetonitrile CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS The scan range was 80-1000 m/z. Raw data files were converted to mzXML format MS:MS_COMMENTS using msconvert (ProteoWizard). Data was analyzed in the MAVEN software suite MS:MS_COMMENTS and metabolite assignments were made using a previously generated list of MS:MS_COMMENTS retention times derived from pure standard solutions. MS:ION_MODE NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area (top) MS_METABOLITE_DATA_START Samples HT1080_DMSO_01 HT1080_DMSO_02 HT1080_DMSO_03 HT1080_ML162_01 HT1080_ML162_02 HT1080_ML162_03 HT1080_ML210_01 HT1080_ML210_02 HT1080_ML210_03 HT1080_RSL3_01 HT1080_RSL3_02 HT1080_RSL3_03 Factors Treatment:DMSO Treatment:DMSO Treatment:DMSO Treatment:ML162 Treatment:ML162 Treatment:ML162 Treatment:ML210 Treatment:ML210 Treatment:ML210 Treatment:RSL3 Treatment:RSL3 Treatment:RSL3 alanine 101551.9000 97807.1500 103232.4000 78917.6200 85744.7700 90654.8100 113286.1000 114503.8000 119594.4000 80702.5200 86634.9300 87881.4400 arginine 94936.0400 96383.1500 92794.4600 132622.4000 127992.3000 109805.5000 107545.1000 112496.3000 116352.2000 112874.1000 132357.3000 115729.6000 aspartate 52178.5900 54141.8600 60268.8700 74655.2800 72813.8300 76963.9600 78907.9500 74708.0600 75020.9400 57969.2700 56438.7800 61164.6600 Glucose 666613.1000 660280.6000 683906.8000 791808.9000 805293.0000 732017.0000 710907.2000 715462.2000 801161.0000 731017.2000 863660.2000 763524.8000 glutamate 967403.2000 854600.5000 838844.3000 635458.8000 544856.1000 525247.0000 713587.8000 672064.3000 606847.3000 611834.3000 546255.7000 541195.2000 glutamine 1654169.0000 2001391.0000 2311045.0000 1458132.0000 1688058.0000 1822691.0000 1806233.0000 1885333.0000 2128476.0000 1372038.0000 1627212.0000 1855053.0000 glutathione 19303.8200 24635.5700 29883.5800 15068.0900 15508.2100 19411.5400 25475.0500 22068.3700 19003.9200 20193.2600 15984.2800 19175.4300 glycine 89332.6000 82858.0400 90238.8500 63316.2900 68282.8700 59567.2400 75291.3400 75085.2400 80861.6600 60361.9300 69885.1600 66789.2000 histidine 94736.2900 114969.7000 130209.1000 108791.2000 125013.2000 113392.1000 123607.8000 129195.1000 150443.6000 92060.7700 131388.9000 132900.8000 isoleucine 1376633.0000 1439873.0000 1489161.0000 1543555.0000 1772399.0000 1436135.0000 1468195.0000 1498275.0000 1785272.0000 1420464.0000 1853383.0000 1509861.0000 leucine 2064003.0000 1976074.0000 2087769.0000 2191078.0000 2241593.0000 2004691.0000 2027068.0000 2115165.0000 2254332.0000 2053952.0000 2513751.0000 2145853.0000 lysine 161918.0000 169353.7000 177281.5000 227189.1000 232995.3000 205409.5000 189375.4000 204237.1000 223788.8000 195278.4000 235075.5000 215910.9000 methionine 94855.7300 94607.1500 103346.7000 94532.8800 101688.9000 92447.2100 96885.0400 104997.3000 115198.8000 87474.7900 104949.0000 98759.5500 phenylalanine 1089198.0000 1018819.0000 1083265.0000 1156538.0000 1198397.0000 1068988.0000 1096370.0000 1128335.0000 1252671.0000 1050801.0000 1247629.0000 1156215.0000 proline 17408.5500 22775.8800 22381.4800 14828.8000 12604.6500 15489.3300 26748.0500 24471.3800 23461.4800 17913.9400 17478.3500 23370.4200 serine 210768.4000 177742.0000 168468.5000 135593.3000 111803.8000 96815.1900 152248.0000 133635.4000 115592.1000 135994.1000 118238.9000 104438.7000 threonine 979744.9000 941010.0000 997598.5000 556799.9000 547907.8000 492506.6000 776963.2000 768381.2000 831801.5000 522066.0000 641436.6000 557558.5000 tryptophan 267201.5000 260168.2000 275271.2000 282037.0000 295680.1000 254781.7000 265732.9000 285808.9000 299895.8000 264185.0000 315798.8000 284527.3000 tyrosine 670291.3000 627341.8000 661427.6000 707496.0000 718016.0000 648223.0000 679079.2000 665386.6000 728373.2000 657806.2000 737360.6000 686001.2000 valine 809639.2000 760725.2000 781467.9000 882378.4000 921539.6000 812134.8000 813278.4000 824712.8000 868177.9000 825705.6000 932994.5000 857564.2000 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant alanine 5950 14.3 88.03951 arginine 6322 18.5 173.1037 aspartate 5960 15.5 132.0297 Glucose 5793 12.2 179.056 glutamate 33032 15.3 146.0452 glutamine 5961 15.0 145.0609 glutathione 124886 15.4 306.0767 glycine 750 14.8 74.0238 histidine 6274 15.3 154.0615 isoleucine 6306 10.3 130.0866 leucine 6106 9.7 130.0866 lysine 5962 18.5 145.0974 methionine 6137 11.0 148.0432 phenylalanine 6140 9.5 164.0711 proline 145742 12.4 114.0553 serine 5951 15.3 104.0349 threonine 6288 14.5 118.0501 tryptophan 6305 9.7 203.082 tyrosine 6057 12.2 180.0661 valine 6287 11.9 116.0708 METABOLITES_END #END