#METABOLOMICS WORKBENCH yz819_20240117_122523 DATATRACK_ID:4593 STUDY_ID:ST003107 ANALYSIS_ID:AN005086 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 Characterization of metabolic changes upon FGFR inhibition in ICC13-7 Xenograft 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-B-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 Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - SUB13746p4_SPL06 treatment:Pemigatinib | Sample source:ICC13-7 Xenograft RAW_FILE_NAME(Raw file name)=SUB13746p4_SPL06 SUBJECT_SAMPLE_FACTORS - SUB13746p4_SPL07 treatment:Pemigatinib | Sample source:ICC13-7 Xenograft RAW_FILE_NAME(Raw file name)=SUB13746p4_SPL07 SUBJECT_SAMPLE_FACTORS - SUB13746p4_SPL08 treatment:Pemigatinib | Sample source:ICC13-7 Xenograft RAW_FILE_NAME(Raw file name)=SUB13746p4_SPL08 SUBJECT_SAMPLE_FACTORS - SUB13746p4_SPL02 treatment:Vehicle | Sample source:ICC13-7 Xenograft RAW_FILE_NAME(Raw file name)=SUB13746p4_SPL02 SUBJECT_SAMPLE_FACTORS - SUB13746p4_SPL03 treatment:Vehicle | Sample source:ICC13-7 Xenograft RAW_FILE_NAME(Raw file name)=SUB13746p4_SPL03 SUBJECT_SAMPLE_FACTORS - SUB13746p4_SPL04 treatment:Vehicle | Sample source:ICC13-7 Xenograft RAW_FILE_NAME(Raw file name)=SUB13746p4_SPL04 #COLLECTION CO:COLLECTION_SUMMARY Tumors were collected and snap-frozen in liquid N2 CO:SAMPLE_TYPE ICC13-7 xenograft #TREATMENT TR:TREATMENT_SUMMARY ICC13-7 xenografts were treated with vehicle vs Pemigatinib(1mg/kg) for 7 days. TR:TREATMENT_SUMMARY On the day7, collect tumor 4h after last dose of treatment (vehicle vs 1mg/kg TR:TREATMENT_SUMMARY pemigatinib). #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/7% A; 40% B/60% A in 19 min; 100% A in 9 min; 100% A for 5 min; CH:FLOW_GRADIENT back to 93% B/7% A in 3 min and re-equilibration at 93% B/7% A for 9 min. CH:FLOW_RATE 0.05 to 0.15 mL/min in 30 seconds uniformly; then held at 0.15 mL/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 MS:MS_COMMENTS 3.3 (CD, ThermoFisher Scientific). A mix of standard of each target was run MS:MS_COMMENTS along the samples and used as the unlabeled reference for the CD labelling MS:MS_COMMENTS workflow. Isotopomer distribution is found in the exchange column, as % of 0 MS:MS_COMMENTS labelled carbon, 1 labelled carbon, 2 labelled carbon, etc. Those are corrected MS:MS_COMMENTS for 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 SUB13746p4_SPL06 SUB13746p4_SPL07 SUB13746p4_SPL08 SUB13746p4_SPL02 SUB13746p4_SPL03 SUB13746p4_SPL04 Factors treatment:Pemigatinib | Sample source:ICC13-7 Xenograft treatment:Pemigatinib | Sample source:ICC13-7 Xenograft treatment:Pemigatinib | Sample source:ICC13-7 Xenograft treatment:Vehicle | Sample source:ICC13-7 Xenograft treatment:Vehicle | Sample source:ICC13-7 Xenograft treatment:Vehicle | Sample source:ICC13-7 Xenograft 2-deoxyribose-5P 495000.0000 347000.0000 412000.0000 1200000.0000 498000.0000 596000.0000 3-Aminoisobutyric acid 44600000.0000 32700000.0000 31200000.0000 43500000.0000 54400000.0000 73200000.0000 3-phosphoglycerate 23600000.0000 25600000.0000 22900000.0000 28300000.0000 26100000.0000 33200000.0000 6-phosphogluconate 10100000.0000 8540000.0000 9500000.0000 16300000.0000 17000000.0000 19100000.0000 AcetylCarnitine 27200000000.0000 30800000000.0000 32600000000.0000 26000000000.0000 29200000000.0000 26900000000.0000 Aconitate 160000000.0000 144000000.0000 158000000.0000 154000000.0000 159000000.0000 177000000.0000 Acteyl-CoA 61300.0000 172000.0000 602000.0000 564000.0000 469000.0000 286000.0000 Adenosine 9830000000.0000 9070000000.0000 8940000000.0000 9370000000.0000 5460000000.0000 6870000000.0000 ADP 188000000.0000 202000000.0000 183000000.0000 217000000.0000 204000000.0000 227000000.0000 Alanine 808000000.0000 743000000.0000 688000000.0000 773000000.0000 799000000.0000 857000000.0000 Alphaketoglutarate 265000000.0000 270000000.0000 302000000.0000 276000000.0000 315000000.0000 301000000.0000 AMP 500000000.0000 451000000.0000 523000000.0000 384000000.0000 370000000.0000 183000000.0000 Arginine 2610000000.0000 2340000000.0000 3000000000.0000 2340000000.0000 2350000000.0000 2910000000.0000 Asparagine 93300000.0000 88000000.0000 91100000.0000 156000000.0000 134000000.0000 139000000.0000 Asparate 710000000.0000 598000000.0000 500000000.0000 800000000.0000 827000000.0000 926000000.0000 ATP 125000000.0000 129000000.0000 75700000.0000 91000000.0000 140000000.0000 141000000.0000 Beta-alanine 23900000.0000 32900000.0000 22500000.0000 23500000.0000 26000000.0000 31600000.0000 CDP 17900000.0000 16900000.0000 10600000.0000 21700000.0000 14900000.0000 15200000.0000 Citrate 4640000000.0000 4260000000.0000 4520000000.0000 3290000000.0000 3650000000.0000 4510000000.0000 Citrulline 375000000.0000 436000000.0000 538000000.0000 792000000.0000 594000000.0000 603000000.0000 CMP 32700000.0000 30700000.0000 24000000.0000 34100000.0000 32000000.0000 32700000.0000 Creatine 21400000000.0000 20900000000.0000 17800000000.0000 19400000000.0000 22400000000.0000 22600000000.0000 CTP 35700000.0000 29000000.0000 11700000.0000 20000000.0000 32800000.0000 47200000.0000 Cysteine Cystine cytosine 190000000.0000 228000000.0000 215000000.0000 265000000.0000 202000000.0000 244000000.0000 d-ADP d-AMP 2280000.0000 1730000.0000 1270000.0000 6090000.0000 2530000.0000 1870000.0000 dATP 37100.0000 105000.0000 12100.0000 141000.0000 234000.0000 320000.0000 d-CDP 119000.0000 323000.0000 102000.0000 237000.0000 d-CMP 3490000.0000 596000.0000 1470000.0000 1330000.0000 1200000.0000 1940000.0000 dCTP 330000.0000 265000.0000 169000.0000 361000.0000 363000.0000 500000.0000 d-GDP d-GMP 45000000.0000 38800000.0000 31600000.0000 36400000.0000 40300000.0000 43400000.0000 dGTP Dihydrouracil 6480000.0000 6180000.0000 6500000.0000 11000000.0000 9430000.0000 11900000.0000 dTTP 390000.0000 410000.0000 302000.0000 827000.0000 1020000.0000 1000000.0000 Erythrose 4-P 1480000.0000 1020000.0000 823000.0000 1200000.0000 1320000.0000 1260000.0000 Farnesyl-PP Fructose-1.6-bisphosphate 38900000.0000 29100000.0000 28300000.0000 43200000.0000 43200000.0000 45000000.0000 Fumarate 243000000.0000 143000000.0000 193000000.0000 186000000.0000 211000000.0000 189000000.0000 GDP 49300000.0000 45200000.0000 37200000.0000 43600000.0000 52300000.0000 63800000.0000 GlcNAc-1-P 12400000.0000 12700000.0000 12600000.0000 17800000.0000 17100000.0000 15800000.0000 Glucosamine-6-P 3290000.0000 2570000.0000 1520000.0000 35900000.0000 3590000.0000 3320000.0000 Glucose 57700000.0000 33900000.0000 18800000.0000 19400000.0000 25500000.0000 19400000.0000 Glucuronic acid 10300000.0000 10600000.0000 12700000.0000 13800000.0000 11100000.0000 14700000.0000 Glutamate 5090000000.0000 4860000000.0000 4380000000.0000 5120000000.0000 5360000000.0000 6300000000.0000 Glutamine 4270000000.0000 4130000000.0000 3320000000.0000 4980000000.0000 5450000000.0000 5180000000.0000 Glutathione oxidized 117000000.0000 108000000.0000 154000000.0000 140000000.0000 154000000.0000 146000000.0000 Glutathione reduced 2590000000.0000 1500000000.0000 927000000.0000 978000000.0000 1780000000.0000 1830000000.0000 glyceraldehyde-3phosphate 675000.0000 474000.0000 657000.0000 876000.0000 673000.0000 781000.0000 Glycerol 66200000.0000 87500000.0000 72100000.0000 52100000.0000 66900000.0000 77900000.0000 Glycine 142000000.0000 133000000.0000 89300000.0000 197000000.0000 178000000.0000 197000000.0000 Glyoxylic acid 3190000.0000 1590000.0000 2470000.0000 2590000.0000 3170000.0000 2750000.0000 GMP 39400000.0000 33200000.0000 37600000.0000 40200000.0000 33100000.0000 22200000.0000 GTP 24200000.0000 22200000.0000 9200000.0000 14900000.0000 24900000.0000 39800000.0000 Guanosine 159000000.0000 153000000.0000 129000000.0000 184000000.0000 90700000.0000 121000000.0000 Hexose-P - Glucose-P 209000000.0000 168000000.0000 124000000.0000 90300000.0000 187000000.0000 171000000.0000 Histidine 558000000.0000 516000000.0000 565000000.0000 588000000.0000 682000000.0000 596000000.0000 Inosinic acid IMP 6520000.0000 6610000.0000 7180000.0000 4480000.0000 25000000.0000 7760000.0000 Inositol 262000000.0000 320000000.0000 162000000.0000 173000000.0000 173000000.0000 186000000.0000 Isoleucine 1540000000.0000 1490000000.0000 1650000000.0000 1730000000.0000 1270000000.0000 1970000000.0000 Itaconate 94100000.0000 102000000.0000 88200000.0000 123000000.0000 109000000.0000 119000000.0000 Lactate 52800000000.0000 25700000000.0000 40300000000.0000 38400000000.0000 38800000000.0000 34100000000.0000 Leucine 2170000000.0000 2260000000.0000 2360000000.0000 2530000000.0000 2000000000.0000 2900000000.0000 Lysine 522000000.0000 396000000.0000 462000000.0000 564000000.0000 584000000.0000 647000000.0000 Malate 2690000000.0000 1580000000.0000 2030000000.0000 2110000000.0000 2330000000.0000 2140000000.0000 Methionine 1670000000.0000 1540000000.0000 1870000000.0000 2590000000.0000 1870000000.0000 2490000000.0000 Mevalonate 8480000.0000 9600000.0000 9720000.0000 20500000.0000 12600000.0000 12100000.0000 Mevalonic acid 5-P 27300.0000 N-Acetylneuraminic acid 30900000.0000 27100000.0000 21200000.0000 29500000.0000 28500000.0000 39200000.0000 NAD+ 52000000.0000 55600000.0000 42200000.0000 47600000.0000 46600000.0000 52200000.0000 NADH NADP 897000.0000 1020000.0000 1660000.0000 1090000.0000 1280000.0000 1090000.0000 NADPH 1010000.0000 1750000.0000 1840000.0000 666000.0000 1830000.0000 1180000.0000 nicotinic acid 1950000.0000 941000.0000 236000.0000 33800000.0000 2640000.0000 3270000.0000 Ornithine 69200000.0000 68700000.0000 75300000.0000 121000000.0000 83300000.0000 127000000.0000 Oxaloacetate Pantothenic acid 107000000.0000 128000000.0000 82200000.0000 97500000.0000 113000000.0000 128000000.0000 Pentose-P - Ribose-P 22200000.0000 19700000.0000 19200000.0000 22500000.0000 20300000.0000 20800000.0000 Phenylalanine 4370000000.0000 4250000000.0000 4850000000.0000 4560000000.0000 3850000000.0000 5120000000.0000 Phenylpyruvate 21000000.0000 9010000.0000 20500000.0000 43500000.0000 10500000.0000 12400000.0000 PhosphoCreatine 1180000000.0000 1040000000.0000 597000000.0000 903000000.0000 983000000.0000 1190000000.0000 phosphoenolpyruvate 1040000.0000 1250000.0000 1370000.0000 1810000.0000 3270000.0000 3650000.0000 Proline 7740000000.0000 7210000000.0000 7310000000.0000 9090000000.0000 7440000000.0000 8780000000.0000 Pyruvate 988000000.0000 647000000.0000 979000000.0000 939000000.0000 1060000000.0000 837000000.0000 Sedoheptulose 7-P 22400000.0000 21300000.0000 24800000.0000 24300000.0000 27200000.0000 25200000.0000 Serine 91100000.0000 79700000.0000 60300000.0000 126000000.0000 117000000.0000 108000000.0000 spermine Succinate 746000000.0000 452000000.0000 365000000.0000 886000000.0000 703000000.0000 936000000.0000 Theonine 429000000.0000 451000000.0000 266000000.0000 601000000.0000 524000000.0000 592000000.0000 Thymidine 2450000.0000 1930000.0000 1580000.0000 8410000.0000 2990000.0000 4990000.0000 Thymine 3660000.0000 6130000.0000 22400000.0000 4450000.0000 2010000.0000 TMP 957000.0000 473000.0000 397000.0000 4760000.0000 685000.0000 897000.0000 Tryptophane 1110000000.0000 1200000000.0000 1500000000.0000 1200000000.0000 1110000000.0000 1320000000.0000 Tyrosine 605000000.0000 634000000.0000 831000000.0000 835000000.0000 579000000.0000 837000000.0000 UDP 95200000.0000 85200000.0000 67100000.0000 93600000.0000 90000000.0000 82200000.0000 UDP-GlcNAc 77400000.0000 70700000.0000 80000000.0000 81700000.0000 84400000.0000 73900000.0000 UDP-Glucose 53600000.0000 56600000.0000 37900000.0000 52700000.0000 62500000.0000 66900000.0000 UMP 101000000.0000 79600000.0000 90000000.0000 84200000.0000 85700000.0000 41100000.0000 Uracil 251000000.0000 152000000.0000 672000000.0000 260000000.0000 201000000.0000 391000000.0000 Uridine 3270000.0000 4230000.0000 11100000.0000 4090000.0000 1930000.0000 UTP 134000000.0000 117000000.0000 65800000.0000 86900000.0000 133000000.0000 166000000.0000 Valine 7460000000.0000 5160000000.0000 4810000000.0000 6840000000.0000 6260000000.0000 5970000000.0000 xanthine 570000.0000 470000.0000 431000.0000 617000.0000 107000.0000 80100.0000 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 2-deoxyribose-5P 3-Aminoisobutyric acid 3-phosphoglycerate 6-phosphogluconate AcetylCarnitine Aconitate Acteyl-CoA Adenosine ADP Alanine Alphaketoglutarate AMP Arginine Asparagine Asparate ATP Beta-alanine CDP Citrate Citrulline CMP Creatine CTP Cysteine Cystine cytosine d-ADP d-AMP dATP d-CDP d-CMP dCTP d-GDP d-GMP dGTP Dihydrouracil dTTP Erythrose 4-P Farnesyl-PP Fructose-1.6-bisphosphate Fumarate GDP GlcNAc-1-P Glucosamine-6-P Glucose Glucuronic acid Glutamate Glutamine Glutathione oxidized Glutathione reduced glyceraldehyde-3phosphate Glycerol Glycine Glyoxylic acid GMP GTP Guanosine Hexose-P - Glucose-P Histidine Inosinic acid IMP Inositol Isoleucine Itaconate Lactate Leucine Lysine Malate Methionine Mevalonate Mevalonic acid 5-P N-Acetylneuraminic acid NAD+ NADH NADP NADPH nicotinic acid Ornithine Oxaloacetate Pantothenic acid Pentose-P - Ribose-P Phenylalanine Phenylpyruvate PhosphoCreatine phosphoenolpyruvate Proline Pyruvate Sedoheptulose 7-P Serine spermine Succinate Theonine Thymidine Thymine TMP Tryptophane Tyrosine UDP UDP-GlcNAc UDP-Glucose UMP Uracil Uridine UTP Valine xanthine METABOLITES_END #END