#METABOLOMICS WORKBENCH xn0016_20231025_063752 DATATRACK_ID:4416 STUDY_ID:ST002962 ANALYSIS_ID:AN004863 PROJECT_ID:PR001843 VERSION 1 CREATED_ON November 1, 2023, 7:50 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 Yanxiang PR:ADDRESS Room 3028, 195 Little Albany Street PR:EMAIL yanxiang@cinj.rutgers.edu PR:PHONE 17327632262 PR:FUNDING_SOURCE R01CA237347-01A1, ACS 134036-RSG-19-165-01-TBG, GO2 Foundation for Lung Cancer, PR:FUNDING_SOURCE and Ludwig Princeton Branch of the Ludwig Institute for Cancer Research to PR:FUNDING_SOURCE J.Y.G. R01CA163591 and Ludwig Princeton Branch of the Ludwig Institute for PR:FUNDING_SOURCE Cancer Research to E.W. NIH P30 CA072720 to Rutgers Cancer Institute of New PR:FUNDING_SOURCE Jersey #STUDY ST:STUDY_TITLE LC/MS detection for NADPH and NADP+ levels in KRAS-driven lung tumors with LKB1 ST:STUDY_TITLE or 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 NADPH ST:STUDY_SUMMARY and NADP+ 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_SIM Scan Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=892-T-7_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 892-T-8_SIM Scan Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=892-T-8_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 892-T-9_SIM Scan Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=892-T-9_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 709-T-7_SIM Scan Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=709-T-7_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 709-T-8_SIM Scan Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=709-T-8_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 709-T-9_SIM Scan Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=709-T-9_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 710-T-7_SIM Scan Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=710-T-7_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 710-T-8_SIM Scan Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=710-T-8_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 710-T-9_SIM Scan Genotype:G6pd-wild type LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=710-T-9_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 4744-T-7_SIM Scan Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=4744-T-7_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 4744-T-8_SIM Scan Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=4744-T-8_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 4744-T-9_SIM Scan Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=4744-T-9_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 4746-T-7_SIM Scan Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=4746-T-7_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 4746-T-8_SIM Scan Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=4746-T-8_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 4746-T-9_SIM Scan Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=4746-T-9_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 5784-T-7_SIM Scan Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=5784-T-7_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 5784-T-8_SIM Scan Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=5784-T-8_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 5784-T-9_SIM Scan Genotype:G6pd-Knockout LSL-KrasG12D/+ p53-/- RAW_FILE_NAME=5784-T-9_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 3094-T-7_SIM Scan Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=3094-T-7_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 3094-T-8_SIM Scan Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=3094-T-8_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 3094-T-9_SIM Scan Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=3094-T-9_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 3095-T-7_SIM Scan Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=3095-T-7_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 3095-T-9_SIM Scan Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=3095-T-9_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 3096-T-7_SIM Scan Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=3096-T-7_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 3096-T-8_SIM Scan Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=3096-T-8_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 3096-T-9_SIM Scan Genotype:G6pd-wild type LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=3096-T-9_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 1515-T-8_SIM Scan Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=1515-T-8_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 1515-T-9_SIM Scan Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=1515-T-9_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 1516-T-8_SIM Scan Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=1516-T-8_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 1516-T-9_SIM Scan Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=1516-T-9_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 1517-T-7_SIM Scan Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=1517-T-7_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 1517-T-8_SIM Scan Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=1517-T-8_SIM Scan.mzXML SUBJECT_SAMPLE_FACTORS - 1517-T-9_SIM Scan Genotype:G6pd-Knockout LSL-KrasG12D/+ LKB1-/- RAW_FILE_NAME=1517-T-9_SIM Scan.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_SIM Scan 892-T-8_SIM Scan 892-T-9_SIM Scan 709-T-7_SIM Scan 709-T-8_SIM Scan 709-T-9_SIM Scan 710-T-7_SIM Scan 710-T-8_SIM Scan 710-T-9_SIM Scan 4744-T-7_SIM Scan 4744-T-8_SIM Scan 4744-T-9_SIM Scan 4746-T-7_SIM Scan 4746-T-8_SIM Scan 4746-T-9_SIM Scan 5784-T-7_SIM Scan 5784-T-8_SIM Scan 5784-T-9_SIM Scan 3094-T-7_SIM Scan 3094-T-8_SIM Scan 3094-T-9_SIM Scan 3095-T-7_SIM Scan 3095-T-9_SIM Scan 3096-T-7_SIM Scan 3096-T-8_SIM Scan 3096-T-9_SIM Scan 1515-T-8_SIM Scan 1515-T-9_SIM Scan 1516-T-8_SIM Scan 1516-T-9_SIM Scan 1517-T-7_SIM Scan 1517-T-8_SIM Scan 1517-T-9_SIM Scan 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-/- NADP+ 214128.0471 206278.4409 104293.2651 117076.5753 133082.5335 63223.19595 101447.1642 117789.5483 104496.4177 108194.1599 98352.21248 123936.9033 86614.80495 60480.87409 121129.0976 159922.4274 130432.2658 100913.0099 63812.46023 65586.30387 74113.91023 86870.51592 69723.3619 87924.17292 92461.50998 66430.87478 100900.4953 70136.67792 224750.3619 136903.2011 98862.97589 56927.92953 123110.3417 NADPH 50347.26402 52010.84353 94319.49201 63607.47296 68903.65364 75039.97789 82835.16501 77516.89503 102124.6521 39016.24613 74903.62221 52282.87257 41220.55565 40962.4597 88161.10607 46298.21745 110482.3168 61133.03181 34363.61977 24029.53669 47219.30303 39420.01182 46724.69997 63442.66953 68091.64663 49157.64725 24161.30422 32037.39316 40919.81669 29880.28099 40787.4931 17946.88447 39649.19818 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Rentetion time quantified m/z PubChem ID KEGG ID NADPH 11.225 744.084229 5884 C00005 NADP+ 12.608 743.069092 5886 C00006 METABOLITES_END #END