MetaboLights

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11048/FILES/T0_EV2.D/AcqData/Contents.xmlftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11048/FILES/Leu_WT_3_T24.D/checksum.xmlftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11048/FILES/Leu_RES_2_SS.D/AcqData/sample_info.xmlftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11048/FILES/Q_T60_KO3.D/AcqData/sample_info.xmlftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11048/FILES/Leu_WT_3_T24.D/AcqData/worklist.xmlprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11048Metabolite identification was conducted as per [Gravel et al., 2016]MetaboLightsPublicGas Chromatography MS - positiveGC-MS methods and mass isotopomer distribution analyses are as previously described [McGuirk et al., 2013; Gravel et al., 2016; Papadopoli et al., 2021].GC-MS instrumentation and software were all from Agilent.Mitochondrial ETF insufficiency drives neoplastic growth by selectively optimizing cancer bioenergetics. 10.7554/elife.106587.1.David PapadopoliMcGill UniversityMus musculusSolvent blankHomo sapiensmass spectrometry assayBoth steady state and tracing samples were washed in cold saline solution (9g/L NaCl) and scraped off with 80% methanol. Samples were sonicated at 4-C for 10 minutes (high setting, 30 seconds on/30 seconds off cycles) to rupture cells. Lysates were centrifuged at 4-C (14000g,10 minutes), and supernatants were collected with addition of an internal standard (750ng myristic acid-D27). Samples were dried overnight at 4-C by speed-vac (Labconco). Dried pellets are resuspended in methoxyamine hydrochloride (10mg/ml), sonicated, and cleared for 10 minutes. Samples are heated 70-C for 30 minutes then applied with N-tert-butyldimethylsilyl-N- methyltrifluoroacetamide (MTBSTFA) at 70-C for 1 hour. Derivatized samples are injected into an Agilent 5975C GC/MS (Agilent Technologies, CA, USA).Mus musculusSolvent blankHomo sapienshttps://www.ebi.ac.uk/metabolights/MTBLS11048Ivan Topisirovic. McGill University. ivan.topisirovic@mcgill.ca.MassHunter software (Agilent) was used to carry out mass isotopomer distribution analyses, with metabolites normalized to myristic acid-D27 and cell number. Isotopic distributions were corrected for naturally occurring isotopes using an in-house algorithm [McGuirk et al., 2013]Cell linedavid.papadopoli@mail.mcgill.caCells were seeded in 6-well dishes. Prior to 13C-glutamine SITA experiments, culture media was replaced with one whose composition contains 2mM unlabelled glutamine for 2 hours. Afterwards, media was replaced with media containing 2mM labelled ([U-13C])-glutamine media (Cambridge Isotope Laboratories, MA, USA; CLM-1822; L-glutamine ([U-13C5]), 99%) for 5, 15, 30, and 60 minutes. For 13C-leucine SITA experiments, culture media was replaced with one containing unlabelled leucine (0.105g/L) for 2 hours, then incubated in media containing labelled (0.105g/L) ([U-13C])-leucine media (Cambridge Isotope Laboratories, MA, USA; CLM-2262-H-0.1; L-leucine ([U-13C6]), 99%) for 24 hours. MetabolomicsTranslationMus musculusSolvent blankuntargeted analysisEukaryotic Translation Initiation Factor 4E-Binding Protein 1Agilent 5975CHomo sapiensexperimental blankmTORC1Electron Transfer Flavoprotein Subunit Alpha, Mitochondrialenergy metabolismTranslationMus musculusSolvent blankuntargeted analysisEukaryotic Translation Initiation Factor 4E-Binding Protein 1Agilent 5975CHomo sapiensexperimental blankmTORC1Electron Transfer Flavoprotein Subunit Alpha, Mitochondrialenergy metabolismGC-MS methods and mass isotopomer distribution analyses are as previously described [Gravel et al., 2016].GC-MS instrumentation and software were all from Agilent.Glutamate m+5falseMitochondrial ETF insufficiency drives neoplastic growth by selectively optimizing cancer bioenergeticsMitochondrial electron transport flavoprotein (ETF) insufficiency causes metabolic diseases known as a multiple acyl-CoA dehydrogenase deficiency (MADD). Although essential in muscle, we identified ETF dehydrogenase (ETFDH) as one of the most dispensable metabolic genes in neoplasia, and its expression is reduced across human cancers. ETF insufficiency caused by decreased ETFDH expression limits flexibility of OXPHOS fuel utilization, but paradoxically increases cancer cell bioenergetics and accelerates neoplastic growth by retrograde activation of the mTORC1/BCL-6/4E-BP1 axis. Collectively, these findings reveal that while ETF insufficiency is rare and has detrimental effects in non-malignant tissues, it is common in neoplasia, where ETFDH downregulation leads to bioenergetic and signaling reprogramming that accelerate neoplastic growth.2026-05-022025-01-06MTBLS11048