#METABOLOMICS WORKBENCH xn0016_20231026_022759 DATATRACK_ID:4419 STUDY_ID:ST002963 ANALYSIS_ID:AN004864 PROJECT_ID:PR001843 VERSION 1 CREATED_ON November 1, 2023, 7:51 am #PROJECT PR:PROJECT_TITLE G6PD Maintains Redox Homeostasis and Biosynthesis in LKB1-Deficient KRAS-Driven PR:PROJECT_TITLE Lung Cancer PR:PROJECT_SUMMARY Cancer cells depend on nicotinamide adenine dinucleotide phosphate (NADPH) to PR:PROJECT_SUMMARY combat oxidative stress and support reductive biosynthesis. One major NAPDH PR:PROJECT_SUMMARY production route is the oxidative pentose phosphate pathway (committed step: PR:PROJECT_SUMMARY glucose-6-phosphate dehydrogenase, G6PD). Alternatives exist and can compensate PR:PROJECT_SUMMARY in some tumors. Here, using genetically-engineered lung cancer model, we show PR:PROJECT_SUMMARY that ablation of G6PD significantly suppresses KrasG12D/+;Lkb1-/- (KL) but not PR:PROJECT_SUMMARY KrasG12D/+;p53-/- (KP) lung tumorigenesis. In vivo isotope tracing and PR:PROJECT_SUMMARY metabolomics revealed that G6PD ablation significantly impaired NADPH PR:PROJECT_SUMMARY generation, redox balance and de novo lipogenesis in KL but not KP lung tumors. PR:PROJECT_SUMMARY Mechanistically, in KL tumors, G6PD ablation caused p53 activation that PR:PROJECT_SUMMARY suppressed tumor growth. As tumor progressed, G6PD-deficient KL tumors increased PR:PROJECT_SUMMARY an alternative NADPH source, serine-driven one carbon metabolism, rendering PR:PROJECT_SUMMARY associated tumor-derived cell lines sensitive to serine/glycine depletion. Thus, PR:PROJECT_SUMMARY oncogenic driver mutations determine lung cancer dependence on G6PD, whose PR:PROJECT_SUMMARY targeting is a potential therapeutic strategy for tumors harboring KRAS and LKB1 PR:PROJECT_SUMMARY co-mutations. PR:INSTITUTE Rutgers Cancer Institute of New Jersey PR:LAST_NAME Guo PR:FIRST_NAME Jessie PR:ADDRESS Room 3028, 195 Little Albany Stree (Rutgers Cancer Institute of New Jersey) PR:EMAIL yanxiang@cinj.rutgers.edu PR:PHONE 17327632262 #STUDY ST:STUDY_TITLE LC/MS detection for GSH and GSSG levels in KRAS-driven lung tumors with LKB1 or ST:STUDY_TITLE p53 deficiency, comparing cases with G6PD wild-type and G6PD knockout ST:STUDY_SUMMARY In this study, LC/MS was employed to assess whether G6PD loss affects the GSH ST:STUDY_SUMMARY and GSSG pool in Lkb1-deficient KRAS-driven lung tumors with LKB1 or p53 ST:STUDY_SUMMARY deficiency. ST:INSTITUTE Rutgers Cancer Institute of New Jersey ST:LAST_NAME Guo ST:FIRST_NAME Jessie ST:ADDRESS Room 3020, 195 Little Albany Stree (Rutgers Cancer Institute of New Jersey) ST:EMAIL yanxiang@cinj.rutgers.edu ST:PHONE 17327632262 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENDER Male and female #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - 892-T-7 Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=892-T-7.mzXML SUBJECT_SAMPLE_FACTORS - 892-T-8 Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=892-T-8.mzXML SUBJECT_SAMPLE_FACTORS - 892-T-9 Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=892-T-9.mzXML SUBJECT_SAMPLE_FACTORS - 709-T-7 Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=709-T-7.mzXML SUBJECT_SAMPLE_FACTORS - 709-T-8 Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=709-T-8.mzXML SUBJECT_SAMPLE_FACTORS - 709-T-9 Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=709-T-9.mzXML SUBJECT_SAMPLE_FACTORS - 710-T-7 Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=710-T-7.mzXML SUBJECT_SAMPLE_FACTORS - 710-T-8 Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=710-T-8.mzXML SUBJECT_SAMPLE_FACTORS - 710-T-9 Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=710-T-9.mzXML SUBJECT_SAMPLE_FACTORS - 4744-T-7 Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=4744-T-7.mzXML SUBJECT_SAMPLE_FACTORS - 4744-T-8 Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=4744-T-8.mzXML SUBJECT_SAMPLE_FACTORS - 4744-T-9 Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=4744-T-9.mzXML SUBJECT_SAMPLE_FACTORS - 4746-T-7 Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=4746-T-7.mzXML SUBJECT_SAMPLE_FACTORS - 4746-T-8 Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=4746-T-8.mzXML SUBJECT_SAMPLE_FACTORS - 4746-T-9 Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=4746-T-9.mzXML SUBJECT_SAMPLE_FACTORS - 5784-T-7 Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=5784-T-7.mzXML SUBJECT_SAMPLE_FACTORS - 5784-T-8 Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=5784-T-8.mzXML SUBJECT_SAMPLE_FACTORS - 5784-T-9 Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=5784-T-9.mzXML SUBJECT_SAMPLE_FACTORS - 3094-T-7 Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=3094-T-7.mzXML SUBJECT_SAMPLE_FACTORS - 3094-T-8 Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=3094-T-8.mzXML SUBJECT_SAMPLE_FACTORS - 3094-T-9 Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=3094-T-9.mzXML SUBJECT_SAMPLE_FACTORS - 3095-T-7 Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=3095-T-7.mzXML SUBJECT_SAMPLE_FACTORS - 3095-T-9 Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=3095-T-9.mzXML SUBJECT_SAMPLE_FACTORS - 3096-T-7 Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=3096-T-7.mzXML SUBJECT_SAMPLE_FACTORS - 3096-T-8 Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=3096-T-8.mzXML SUBJECT_SAMPLE_FACTORS - 3096-T-9 Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=3096-T-9.mzXML SUBJECT_SAMPLE_FACTORS - 1515-T-8 Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=1515-T-8.mzXML SUBJECT_SAMPLE_FACTORS - 1515-T-9 Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=1515-T-9.mzXML SUBJECT_SAMPLE_FACTORS - 1516-T-8 Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=1516-T-8.mzXML SUBJECT_SAMPLE_FACTORS - 1516-T-9 Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=1516-T-9.mzXML SUBJECT_SAMPLE_FACTORS - 1517-T-7 Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=1517-T-7.mzXML SUBJECT_SAMPLE_FACTORS - 1517-T-8 Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=1517-T-8.mzXML SUBJECT_SAMPLE_FACTORS - 1517-T-9 Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=1517-T-9.mzXML #COLLECTION CO:COLLECTION_SUMMARY At 6-8 weeks of age, mice were intranasally infected with Lenti-Cre (University CO:COLLECTION_SUMMARY of Iowa Viral Vector Core) at 5×106 plaque-forming units (pfu) per mouse to CO:COLLECTION_SUMMARY induce lung tumors. At 12 weeks post tumor induction, mice were euthanized for CO:COLLECTION_SUMMARY rapid lung tumors collection. CO:SAMPLE_TYPE Lung tumors #TREATMENT TR:TREATMENT_SUMMARY Samples were taken from the mouse model. This model was generated as described TR:TREATMENT_SUMMARY in the collection section. No additional treatment was done. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Approximately 20 mg of tumor samples were precisely weighed and placed into a SP:SAMPLEPREP_SUMMARY pre-cooled tube. The tissue samples were then pulverized using the Cryomill. SP:SAMPLEPREP_SUMMARY Pre-cooled extraction buffer consisting of methanol: acetonitrile: H2O SP:SAMPLEPREP_SUMMARY (40:40:20, V/V) with 0.5% formic acid (Sigma-Aldrich, # F0507-100ML) was added SP:SAMPLEPREP_SUMMARY to the resulting powder (40 μL of solvent per mg of tumors). The samples were SP:SAMPLEPREP_SUMMARY then vortexed for 15 seconds and incubated on ice for 10 minutes. Subsequently, SP:SAMPLEPREP_SUMMARY 15% NH4HCO3 solution (5% V/V of the extraction buffer) was used to neutralize SP:SAMPLEPREP_SUMMARY the samples. Then all samples were vortexed again for 10 seconds and centrifuged SP:SAMPLEPREP_SUMMARY at 4°C, 13,000 × g for 20 minutes. The resulting supernatant was transferred SP:SAMPLEPREP_SUMMARY to LC-MS vials for subsequent analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Scientific Vanquish HPLC CH:COLUMN_NAME Waters XBridge BEH Amide (150×2.1 mm, 2.5 μm) CH:SOLVENT_A 95% water; 5% acetonitrile; 20 mmol/L NH3AC; 20 mmol/L NH3OH, pH~9 CH:SOLVENT_B 80% acetonitrile; 20% water; 20 mmol/L NH3AC; 20 mmol/L NH3OH, pH~9 CH:FLOW_GRADIENT The composition of mobile phase B varied over time as follows: 0 min, 100%; 3 CH:FLOW_GRADIENT min, 100%; 3.2 min, 90%; 6.2 min, 90%; 6.5 min, 80%; 10.5 min, 80%; 10.7 min, CH:FLOW_GRADIENT 70%; 13.5 min, 70%; 13.7 min, 45%; 16 min, 45%; 16.5 min, 100%. CH:FLOW_RATE 0.3 mL/min CH:COLUMN_TEMPERATURE 25 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS - #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Ion count MS_METABOLITE_DATA_START Samples 892-T-7 892-T-8 892-T-9 709-T-7 709-T-8 709-T-9 710-T-7 710-T-8 710-T-9 4744-T-7 4744-T-8 4744-T-9 4746-T-7 4746-T-8 4746-T-9 5784-T-7 5784-T-8 5784-T-9 3094-T-7 3094-T-8 3094-T-9 3095-T-7 3095-T-9 3096-T-7 3096-T-8 3096-T-9 1515-T-8 1515-T-9 1516-T-8 1516-T-9 1517-T-7 1517-T-8 1517-T-9 Factors Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- Glutathione 22136775.61 18913313.22 27582636.61 35891583.06 27946528.27 44054171.15 27345781.11 36097001.82 34418041.86 23453357.29 37593515.65 20939938.4 28443901.35 24906367.85 31809196 22538723.17 22089196.45 24328503.01 23050771.92 23893407.81 33131238.41 24396670.47 17441819.6 32926117.67 32920247.31 21075306.3 37992197.42 12997277.63 20683264.98 22495941.29 28337135.63 17467688.48 23068608.66 Glutathione disulfide 806887.1184 1067750.053 446363.1877 815495.8578 387274.5735 307340.5433 510332.6879 593819.7775 439692.1081 465660.5561 239014.0411 300994.4525 784024.1209 229017.5807 512732.4326 835811.7782 354838.5796 415226.4156 295174.6628 134706.8401 299999.2667 384813.5085 93557.09935 474976.3035 188648.0254 148153.8892 797083.2873 238418.1449 769849.8561 436092.2828 680562.1367 141144.0573 366149.5818 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Rentetion time quantified m/z PubChem ID KEGG ID Glutathione 8.888 306.076233 124886 C00051 Glutathione disulfide 12.844 611.14502 65359 C00127 METABOLITES_END #END