#METABOLOMICS WORKBENCH yz819_20240117_101310 DATATRACK_ID:4589 STUDY_ID:ST003106 ANALYSIS_ID:AN005085 VERSION 1 CREATED_ON 03-06-2024 #PROJECT PR:PROJECT_TITLE FGFR inhibition blocks NF-ĸB-dependent glucose metabolism and confers metabolic PR:PROJECT_TITLE vulnerabilities in cholangiocarcinoma PR:PROJECT_SUMMARY Genomic alterations that activate FGFR2 are common in intrahepatic PR:PROJECT_SUMMARY cholangiocarcinoma (ICC) and confer sensitivity to FGFR inhibitor treatment. PR:PROJECT_SUMMARY However, the depth and duration of responses are often limited. Here, we PR:PROJECT_SUMMARY conducted integrative transcriptomics, metabolomics, and phosphoproteomics PR:PROJECT_SUMMARY analysis of patient-derived models to define the pathways that fuel tumor growth PR:PROJECT_SUMMARY downstream of oncogenic FGFR2 signaling in ICC and to uncover compensatory PR:PROJECT_SUMMARY mechanisms associated with pathway inhibition. We find FGFR2-mediated activation PR:PROJECT_SUMMARY of NF-kB maintains a highly glycolytic phenotype. Conversely, FGFR inhibition PR:PROJECT_SUMMARY blocks glucose uptake and glycolysis while inciting a series of adaptive PR:PROJECT_SUMMARY changes, including switching fuel source utilization to favor fatty acid PR:PROJECT_SUMMARY oxidation and increasing mitochondrial fusion and autophagy. Accordingly, FGFR PR:PROJECT_SUMMARY inhibitor efficacy is potentiated by combined mitochondrial targeting, an effect PR:PROJECT_SUMMARY enhanced in xenograft models by intermittent fasting. Thus, we show that PR:PROJECT_SUMMARY oncogenic FGFR2 signaling drives NF-kB-dependent glycolysis in ICC and that PR:PROJECT_SUMMARY metabolic reprogramming in response to FGFR inhibition confers new targetable PR:PROJECT_SUMMARY vulnerabilities. PR:INSTITUTE mgh PR:LAST_NAME Zhen PR:FIRST_NAME Yuanli PR:ADDRESS 185 cambridge street, room 4100 PR:EMAIL yzhen1@mgh.harvard.edu PR:PHONE 4698792279 #STUDY ST:STUDY_TITLE 13C-palmitate labeling experiment in ICC13-7 treated with DMSO or Infigratinib ST:STUDY_SUMMARY Genomic alterations that activate FGFR2 are common in intrahepatic ST:STUDY_SUMMARY cholangiocarcinoma (ICC) and confer sensitivity to treatment with FGFR ST:STUDY_SUMMARY inhibitors. However, the depth and duration of responses are often limited. ST:STUDY_SUMMARY Here, we conducted integrative transcriptomic and metabolomic analysis of ST:STUDY_SUMMARY patient-derived models to define the pathways that fuel tumor growth downstream ST:STUDY_SUMMARY of oncogenic FGFR2 signaling in ICC and to uncover compensatory mechanisms ST:STUDY_SUMMARY associated with pathway inhibition. We find FGFR2-mediated activation of NF-B ST:STUDY_SUMMARY maintains a highly glycolytic phenotype. Conversely, FGFR inhibition blocks ST:STUDY_SUMMARY glucose uptake and glycolysis while inciting a series of adaptive changes, ST:STUDY_SUMMARY including switching fuel source utilization to favor fatty acid oxidation and ST:STUDY_SUMMARY increasing mitochondrial fusion and autophagy. Accordingly, FGFR inhibitor ST:STUDY_SUMMARY efficacy is potentiated by combined mitochondrial targeting, an effect enhanced ST:STUDY_SUMMARY in xenograft models by intermittent fasting. Thus, we show that oncogenic FGFR2 ST:STUDY_SUMMARY signaling drives NF-kB-dependent glycolysis in ICC and that metabolic ST:STUDY_SUMMARY reprogramming in response to FGFR inhibition confers new targetable ST:STUDY_SUMMARY vulnerabilities. ST:INSTITUTE Massachusetts General Hospital ST:LAST_NAME Zhen ST:FIRST_NAME Yuanli ST:ADDRESS 185 cambridge street, room 4100 ST:EMAIL yzhen1@mgh.harvard.edu ST:PHONE 4698792279 ST:SUBMIT_DATE 2024-01-17 #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 - SUB13607p4_SPL07 Treatment:DMSO | Sample source:ICC13-7 cells RAW_FILE_NAME(Raw file name)=SUB13607p4_SPL07 SUBJECT_SAMPLE_FACTORS - SUB13607p4_SPL08 Treatment:DMSO | Sample source:ICC13-7 cells RAW_FILE_NAME(Raw file name)=SUB13607p4_SPL08 SUBJECT_SAMPLE_FACTORS - SUB13607p4_SPL09 Treatment:DMSO | Sample source:ICC13-7 cells RAW_FILE_NAME(Raw file name)=SUB13607p4_SPL09 SUBJECT_SAMPLE_FACTORS - SUB13607p4_SPL10 Treatment:Infigratinib | Sample source:ICC13-7 cells RAW_FILE_NAME(Raw file name)=SUB13607p4_SPL10 SUBJECT_SAMPLE_FACTORS - SUB13607p4_SPL11 Treatment:Infigratinib | Sample source:ICC13-7 cells RAW_FILE_NAME(Raw file name)=SUB13607p4_SPL11 SUBJECT_SAMPLE_FACTORS - SUB13607p4_SPL12 Treatment:Infigratinib | Sample source:ICC13-7 cells RAW_FILE_NAME(Raw file name)=SUB13607p4_SPL12 #COLLECTION CO:COLLECTION_SUMMARY media was completely aspirated, and cells were washed with ice-cold saline CO:COLLECTION_SUMMARY quickly. After washing, fully remove saline and cells can be scrapped in 1 ml CO:COLLECTION_SUMMARY pre-cooled methanol (-20°C) with internal standards (Cambridge Isotope CO:COLLECTION_SUMMARY Laboratories, MSK-A2-1.2), and transferred to glass vials, stored at -80°C CO:COLLECTION_SUMMARY until extraction. CO:SAMPLE_TYPE ICC13-7 #TREATMENT TR:TREATMENT_SUMMARY Cells were pretreated with DMSO or 100nM Infigratinib for 24h in lipid-depleted TR:TREATMENT_SUMMARY media. Then they were changed to 13C-Plamitate labeling media for 8h (with DMSO TR:TREATMENT_SUMMARY or infigratinib) before harvesting cells. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY extractions were conducted following the biphasic extraction protocol. SP:SAMPLEPREP_SUMMARY Basically, add cold chloroform to samples (in methanol) with a ratio of 2:1. SP:SAMPLEPREP_SUMMARY Vortex samples for 1 minute to homogenize. Add 1 fraction of water, and vortex SP:SAMPLEPREP_SUMMARY samples again. Glass vials were centrifuged at 3000 rcf for 10 minutes for phase SP:SAMPLEPREP_SUMMARY separation. The aqueous phase was transferred to a new glass vial for SP:SAMPLEPREP_SUMMARY metabolomics and the remaining interphase was used for protein quantification. SP:SAMPLEPREP_SUMMARY The aqueous phase was evaporated under nitrogen flow. Samples were resuspended SP:SAMPLEPREP_SUMMARY in 50% acetonitrile, and the volume was scaled according to the protein amounts. SP:SAMPLEPREP_SUMMARY 15ul was used for the lowest biomass, and all others were scaled accordingly. SP:SAMPLEPREP_SUMMARY Standard mixes were prepared at 100 uM and run after the samples to allow for SP:SAMPLEPREP_SUMMARY the identification of the targets. #CHROMATOGRAPHY CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um) CH:COLUMN_TEMPERATURE 40 CH:FLOW_GRADIENT Started at 93% B and 7% A ; 40% B/60% A in 19 min; 100% A in 9 min; 100% A for 5 CH:FLOW_GRADIENT min; back to 93% B/7% A in 3 min; re-equilibration at 93% B/7% A for 9 min. CH:FLOW_RATE 0.05 to 0.15 mL/min in 30 sec; then held at 0.15mL/min CH:SOLVENT_A 100% Water; 20 mMAmmonium Carbonate, 0.1% Ammonium hydroxide CH:SOLVENT_B Acetonitrile 97%, 3% water CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap ID-X tribrid MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS Data was acquired on the ID-X in switching polarities at 120000 resolution, with MS:MS_COMMENTS an AGC target of 1e5, and a m/z range of 65 to 1000. MS1 data is acquired in MS:MS_COMMENTS switching polarities for all samples.Data is analyzed in Compound Discoverer 3.3 MS:MS_COMMENTS (CD, ThermoFisher Scientific). A mix of standard of each target was run along MS:MS_COMMENTS the samples and used as the unlabeled reference for the CD labelling workflow. MS:MS_COMMENTS Isotopomer distribution is found in the exchange column, as % of 0 labelled MS:MS_COMMENTS carbon, 1 labelled carbon, 2 labelled carbon, etc. Those are corrected for MS:MS_COMMENTS natural abundance. For the compounds that couldn’t be analyzed by CD, the MS:MS_COMMENTS areas have been extracted with Tracefinder manually. MS:ION_MODE UNSPECIFIED #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS area analyzed by Compound discoverer (counts x seconds) MS_METABOLITE_DATA_START Samples SUB13607p4_SPL07 SUB13607p4_SPL08 SUB13607p4_SPL09 SUB13607p4_SPL10 SUB13607p4_SPL11 SUB13607p4_SPL12 Factors Treatment:DMSO | Sample source:ICC13-7 cells Treatment:DMSO | Sample source:ICC13-7 cells Treatment:DMSO | Sample source:ICC13-7 cells Treatment:Infigratinib | Sample source:ICC13-7 cells Treatment:Infigratinib | Sample source:ICC13-7 cells Treatment:Infigratinib | Sample source:ICC13-7 cells Acetyl-CoA 1.0000 0.3508 alpha-KG 0.2321 0.2342 0.2485 0.2753 0.2800 0.2486 Citrate 0.3349 0.2922 0.3090 0.3096 0.3579 0.3590 Fumarate 0.1563 0.1451 0.1288 0.1476 0.1601 0.1382 Isocitrate Malate 0.1531 0.1496 0.1567 0.1626 0.1636 0.1737 Oxaloacetate Succinate 0.1579 0.1242 0.1713 0.1519 0.1580 0.1435 SuccinylCoA 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 Acetyl-CoA alpha-KG Citrate Fumarate Isocitrate Malate Oxaloacetate Succinate SuccinylCoA METABOLITES_END #END