{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002586","ANALYSIS_ID":"AN004258","VERSION":"1","CREATED_ON":"05-22-2023"},

"PROJECT":{"PROJECT_TITLE":"Alanine supplementation exploits glutamine dependency induced by SMARCA4/2-loss","PROJECT_SUMMARY":"SMARCA4 (BRG1) and SMARCA2 (BRM) are the two paralogous ATPases of the SWI/SNF chromatin remodeling complexes frequently inactivated in cancers.we uncover that SMARCA4/2-loss represses expression of the glucose transporter GLUT1, causing reduced glucose uptake and glycolysis accompanied with increased dependency on oxidative phosphorylation (OXPHOS); adapting to this, these SMARCA4/2-deficient cells rely on elevated SLC38A2, an amino acid transporter, to increase glutamine import for fueling OXPHOS. Consequently, SMARCA4/2-deficient cells and tumors are highly sensitive to inhibitors targeting OXPHOS or glutamine metabolism. Furthermore, supplementation of alanine, also imported by SLC38A2, restricts glutamine uptake through competition and selectively induces death in SMARCA4/2-deficient cancer cells. At a clinically relevant dose, alanine supplementation synergizes with OXPHOS inhibition or conventional chemotherapy eliciting marked antitumor activity in patient-derived xenografts. Our findings reveal multiple druggable vulnerabilities of SMARCA4/2-loss exploiting a GLUT1/SLC38A2-mediated metabolic shift. Particularly, unlike dietary deprivation approaches, alanine supplementation can be readily applied to current regimens for better treatment of these aggressive cancers.","INSTITUTE":"McGill University","DEPARTMENT":"Biochemistry","LABORATORY":"Sidong Huang Lab","LAST_NAME":"Fu","FIRST_NAME":"Zheng","ADDRESS":"McIntyre Medical Sciences Building","EMAIL":"zheng.fu2@mail.mcgill.ca","PHONE":"5145869072","DOI":"http://dx.doi.org/10.21228/M8R72W"},

"STUDY":{"STUDY_TITLE":"Stable isotope tracer analysis (SITA) using 13C6-glucose and 13C5-glutamine measured by GC/MS in BIN-67 cells ± SMARCA4 restoration","STUDY_SUMMARY":"SMARCA4/2-loss drives the metabolic shift preferring glutamine than glucose to sustain the TCA cycle. SITA using 13C6-glucose confirmed that glucose uptake was increased in BIN-67 cells upon SMARCA4-restoration, leading to overall increased metabolic flux through glucose (lactate) and TCA cycle intermediates (citrate, fumarate, malate, aspartate) via pyruvate carboxylation.Conversely, the 13C5-glutamine SITA in BIN-67 cells revealed that SMARCA4-restoration decreased glutamine uptake and utilization via glutaminolysis to fuel the TCA cycle as indicated by reduced glutamine metabolites (glutamate, α-KG, m+5 metabolites) and TCA cycle intermediates (succinate, fumarate, malate, aspartate, m+4 metabolites)","INSTITUTE":"McGill University","DEPARTMENT":"Biochemistry","LABORATORY":"Sidong Huang Lab","LAST_NAME":"Fu","FIRST_NAME":"Zheng","ADDRESS":"McIntyre Medical Sciences Building, 3655 promenade Sir-William-Osler","EMAIL":"zheng.fu2@mail.mcgill.ca","PHONE":"5143985446","SUBMIT_DATE":"2023-04-25"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"21_Ala_C13_Glutamine_1",
"Factors":{"Treatment":"Alanine 18h and C13 Glutamine 60min"},
"Additional sample data":{"RAW_FILE_NAME":"21_ALA_C13_Q_1_151221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"22_Ala_C13_Glutamine_2",
"Factors":{"Treatment":"Alanine 18h and C13 Glutamine 60min"},
"Additional sample data":{"RAW_FILE_NAME":"22_ALA_C13_Q_2_151221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"23_Ala_C13_Glutamine_3",
"Factors":{"Treatment":"Alanine 18h and C13 Glutamine 60min"},
"Additional sample data":{"RAW_FILE_NAME":"23_ALA_C13_Q_3_171221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"24_Ala_C13_Glutamine_4",
"Factors":{"Treatment":"Alanine 18h and C13 Glutamine 60min"},
"Additional sample data":{"RAW_FILE_NAME":"24_ALA_C13_Q_4_171221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"25_Ala_Glutamine",
"Factors":{"Treatment":"Alanine 18h and Glutamine 60min"},
"Additional sample data":{"RAW_FILE_NAME":"25_ALA_Q_171221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"1_C13_Glucose_1",
"Factors":{"Treatment":"C13 Glucose 30min"},
"Additional sample data":{"RAW_FILE_NAME":"1_C13_GLUC_1_151221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"2_C13_Glucose_2",
"Factors":{"Treatment":"C13 Glucose 30min"},
"Additional sample data":{"RAW_FILE_NAME":"2_C13_GLUC_2_171221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"3_C13_Glucose_3",
"Factors":{"Treatment":"C13 Glucose 30min"},
"Additional sample data":{"RAW_FILE_NAME":"3_C13_GLUC_3_171221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"4_C13_Glucose_4",
"Factors":{"Treatment":"C13 Glucose 30min"},
"Additional sample data":{"RAW_FILE_NAME":"4_C13_GLUC_4_151221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"11_C13_Glutamine_1",
"Factors":{"Treatment":"C13 Glutamine 60min"},
"Additional sample data":{"RAW_FILE_NAME":"11_C13_Q_1_171221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"12_C13_Glutamine_2",
"Factors":{"Treatment":"C13 Glutamine 60min"},
"Additional sample data":{"RAW_FILE_NAME":"12_C13_Q_2_151221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"13_C13_Glutamine_3",
"Factors":{"Treatment":"C13 Glutamine 60min"},
"Additional sample data":{"RAW_FILE_NAME":"13_C13_Q_3_171221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"14_C13_Glutamine_4",
"Factors":{"Treatment":"C13 Glutamine 60min"},
"Additional sample data":{"RAW_FILE_NAME":"14_C13_Q_4_151221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"5_Glucose",
"Factors":{"Treatment":"Glucose 30min"},
"Additional sample data":{"RAW_FILE_NAME":"5_GLUC_151221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"15_Glutamine",
"Factors":{"Treatment":"Glutamine 60min"},
"Additional sample data":{"RAW_FILE_NAME":"15_Q_151221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"6_A4_C13_Glucose_1",
"Factors":{"Treatment":"SMACA4 repstration + C13 Glucose 30min"},
"Additional sample data":{"RAW_FILE_NAME":"6_A4_C13_GLUC_1_151221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"7_A4_C13_Glucose_2",
"Factors":{"Treatment":"SMACA4 repstration + C13 Glucose 30min"},
"Additional sample data":{"RAW_FILE_NAME":"7_A4_C13_GLUC_2_171221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"8_A4_C13_Glucose_3",
"Factors":{"Treatment":"SMACA4 repstration + C13 Glucose 30min"},
"Additional sample data":{"RAW_FILE_NAME":"8_A4_C13_GLUC_3_151221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"9_A4_C13_Glucose_4",
"Factors":{"Treatment":"SMACA4 repstration + C13 Glucose 30min"},
"Additional sample data":{"RAW_FILE_NAME":"9_A4_C13_GLUC_4_171221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"16_A4_C13_Glutamine_1",
"Factors":{"Treatment":"SMACA4 repstration + C13 Glutamine 60min"},
"Additional sample data":{"RAW_FILE_NAME":"16_A4_C13_Q_1_171221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"17_A4_C13_Glutamine_2",
"Factors":{"Treatment":"SMACA4 repstration + C13 Glutamine 60min"},
"Additional sample data":{"RAW_FILE_NAME":"17_A4_C13_Q_2_151221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"18_A4_C13_Glutamine_3",
"Factors":{"Treatment":"SMACA4 repstration + C13 Glutamine 60min"},
"Additional sample data":{"RAW_FILE_NAME":"18_A4_C13_Q_3_151221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"19_A4_C13_Glutamine_4",
"Factors":{"Treatment":"SMACA4 repstration + C13 Glutamine 60min"},
"Additional sample data":{"RAW_FILE_NAME":"19_A4_C13_Q_4_171221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"10_A4_Glucose",
"Factors":{"Treatment":"SMACA4 repstration + Glucose 30min"},
"Additional sample data":{"RAW_FILE_NAME":"10_A4_GLUC_171221_01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20_A4_Glutamine",
"Factors":{"Treatment":"SMACA4 repstration + Glutamine 60min"},
"Additional sample data":{"RAW_FILE_NAME":"20_A4_Q_171221_01.mzXML"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"BIN-67 with or without SMARCA4 restoration cells were plated in RPMI supplemented with 6% dialyzed FBS (Wisent Bio, Cat# 080-950) at ~80% confluency the day before experiments. For isotope metabolic tracing, media was replaced with glutamine- or glucose-free RPMI supplemented with 6% dialyzed FBS and 13C5-glutamine or 13C6-glucose (Cambridge Isotopes Laboratories, Tewksbury, MA) for 1 hr or 0.5 hr, respectively. In addition, dishes were kept in unlabeled media as control. Cells were washed twice in cold saline solution (NaCl, 0.9 g/l) and metabolites were extracted with 1 ml 80% ice-cold methanol (GC/MS grade).","SAMPLE_TYPE":"Ovarian cancer cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"BIN-67 with or without SMARCA4 restoration cells were plated in RPMI supplemented with 6% dialyzed FBS (Wisent Bio, Cat# 080-950) at ~80% confluency the day before experiments. For isotope metabolic tracing, media was replaced with glutamine- or glucose-free RPMI supplemented with 6% dialyzed FBS and 13C5-glutamine or 13C6-glucose (Cambridge Isotopes Laboratories, Tewksbury, MA) for 1 hr or 0.5 hr, respectively. In addition, dishes were kept in unlabeled media as control. Cells were washed twice in cold saline solution (NaCl, 0.9 g/l) and metabolites were extracted with 1 ml 80% ice-cold methanol (GC/MS grade)."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Cells were washed twice in cold saline solution (NaCl, 0.9 g/l) and metabolites were extracted with 1 ml 80% ice-cold methanol (GC/MS grade). After 2 rounds 10 min sets of sonication (30 seconds on/30 seconds off at high intensity) on slurry ice using a Bioruptor UCD-200 sonicator, the homogenates were centrifuged at 14,000 × g at 4 °C for 10 min. Supernatants were collected and supplemented with internal control (800 ng myristic acid-D27) and dried in a cold vacuum centrifuge (Labconco) overnight. The dried pellets were reconstituted with 30 μL of 10 mg/mL methoxyamine-HCl in pyridine, and incubated for 30 min at room temperature. Samples were then derivatized with MTBSTFA for 30 min at 70 °C. A volume of 1 μL of sample was injected splitless with an inlet temperature of 280 oC into the GC (7890, Agilent)/MS (5975C, Agilent) instrument."},

"CHROMATOGRAPHY":{"INSTRUMENT_NAME":"Agilent 7890A","COLUMN_NAME":"Agilent DB5-MS (30m x 0.25mm, 0.25um)","COLUMN_TEMPERATURE":"60-320","FLOW_GRADIENT":"None","FLOW_RATE":"0.69 ml/min","SOLVENT_A":"None","SOLVENT_B":"None","CHROMATOGRAPHY_TYPE":"GC"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Agilent 5975C","INSTRUMENT_TYPE":"Single quadrupole","MS_TYPE":"EI","MS_COMMENTS":"A volume of 1 μL of sample was injected splitless with an inlet temperature of 280 oC into the GC (7890, Agilent)/MS (5975C, Agilent) instrument. Metabolites were resolved by separation on DB-5MS+DG (30 m x 250 µm x 0.25 µm) capillary column (Agilent J&W, Santa Clara, CA, USA). Helium was used as the carrier gas with a flow rate such that myristic-d27 acid eluted at approximately 18 min. The quadrupole was set at 150 ˚C, the source was at 230 °C and the GC/MS interface was at 320 ˚C. The oven program started at 60 ˚C held for 1 min, then increased at a rate of 10 ˚C/min until 320 ˚C. Bake-out was at 320 ˚C for 9 min. Metabolites were ionized by electron impact at 70 eV. All samples were injected three times: twice using scan (50-1000 m/z) mode (1x and 25x dilution for steady-state samples or 1x and 24x dilution for tracer samples) and once using selected ion monitoring (SIM) mode. All of the metabolites described in this study were validated against authentic standards confirming mass spectra and retention times. Integration of ion intensities was accomplished using Mass Hunter Quant (Agilent). Generally, M-57+. Ions (and isotopes) were analyzed. Mass isotopomer distribution analysis was carried out using in-house software using an in-house algorithm adapted from Nanchen et al as previously described.","ION_MODE":"POSITIVE"},

"MS_METABOLITE_DATA":{
"Units":"intensity",

"Data":[{"Metabolite":"2-hydroxyglutaric (433_245)","21_Ala_C13_Glutamine_1":"1948.4825","22_Ala_C13_Glutamine_2":"1637.1375","23_Ala_C13_Glutamine_3":"400.5270","24_Ala_C13_Glutamine_4":"1357.4110","25_Ala_Glutamine":"792.6740","1_C13_Glucose_1":"4478.7545","2_C13_Glucose_2":"5258.6275","3_C13_Glucose_3":"8389.0895","4_C13_Glucose_4":"14460.1925","11_C13_Glutamine_1":"5977.3565","12_C13_Glutamine_2":"5006.5735","13_C13_Glutamine_3":"6080.4990","14_C13_Glutamine_4":"4758.5380","5_Glucose":"11214.5270","15_Glutamine":"6549.5915","6_A4_C13_Glucose_1":"6075.2485","7_A4_C13_Glucose_2":"12033.5525","8_A4_C13_Glucose_3":"7231.5350","9_A4_C13_Glucose_4":"12453.4140","16_A4_C13_Glutamine_1":"14862.8130","17_A4_C13_Glutamine_2":"8943.5730","18_A4_C13_Glutamine_3":"14571.2000","19_A4_C13_Glutamine_4":"11868.3790","10_A4_Glucose":"14979.0510","20_A4_Glutamine":"9517.7050"},{"Metabolite":"2-hydroxyglutaric M+1","21_Ala_C13_Glutamine_1":"824.9975","22_Ala_C13_Glutamine_2":"682.0445","23_Ala_C13_Glutamine_3":"146.8005","24_Ala_C13_Glutamine_4":"663.3555","25_Ala_Glutamine":"444.8870","1_C13_Glucose_1":"1439.4850","2_C13_Glucose_2":"1695.8250","3_C13_Glucose_3":"3853.4045","4_C13_Glucose_4":"5287.9025","11_C13_Glutamine_1":"2291.2840","12_C13_Glutamine_2":"1816.8860","13_C13_Glutamine_3":"2432.6275","14_C13_Glutamine_4":"1821.2765","5_Glucose":"3902.6920","15_Glutamine":"2096.2460","6_A4_C13_Glucose_1":"2197.8060","7_A4_C13_Glucose_2":"4504.6900","8_A4_C13_Glucose_3":"2446.6750","9_A4_C13_Glucose_4":"4189.0695","16_A4_C13_Glutamine_1":"5650.4025","17_A4_C13_Glutamine_2":"3142.4955","18_A4_C13_Glutamine_3":"5161.9710","19_A4_C13_Glutamine_4":"4589.4780","10_A4_Glucose":"5142.5505","20_A4_Glutamine":"3823.8935"},{"Metabolite":"2-hydroxyglutaric M+2","21_Ala_C13_Glutamine_1":"285.4635","22_Ala_C13_Glutamine_2":"314.5540","23_Ala_C13_Glutamine_3":"","24_Ala_C13_Glutamine_4":"308.8340","25_Ala_Glutamine":"104.2385","1_C13_Glucose_1":"807.2825","2_C13_Glucose_2":"941.2345","3_C13_Glucose_3":"1951.6460","4_C13_Glucose_4":"2763.7770","11_C13_Glutamine_1":"1073.0100","12_C13_Glutamine_2":"723.0490","13_C13_Glutamine_3":"941.6690","14_C13_Glutamine_4":"783.5490","5_Glucose":"1916.9590","15_Glutamine":"1116.0105","6_A4_C13_Glucose_1":"996.1170","7_A4_C13_Glucose_2":"2794.5980","8_A4_C13_Glucose_3":"1281.5130","9_A4_C13_Glucose_4":"2547.5400","16_A4_C13_Glutamine_1":"2637.8610","17_A4_C13_Glutamine_2":"1447.3770","18_A4_C13_Glutamine_3":"2836.9945","19_A4_C13_Glutamine_4":"2434.5470","10_A4_Glucose":"2610.6430","20_A4_Glutamine":"1568.3900"},{"Metabolite":"2-hydroxyglutaric M+3","21_Ala_C13_Glutamine_1":"97.4225","22_Ala_C13_Glutamine_2":"89.5660","23_Ala_C13_Glutamine_3":"40.6875","24_Ala_C13_Glutamine_4":"178.8685","25_Ala_Glutamine":"41.7197","1_C13_Glucose_1":"208.7385","2_C13_Glucose_2":"170.8611","3_C13_Glucose_3":"269.7485","4_C13_Glucose_4":"533.0995","11_C13_Glutamine_1":"294.0855","12_C13_Glutamine_2":"302.2425","13_C13_Glutamine_3":"361.5860","14_C13_Glutamine_4":"441.5940","5_Glucose":"304.5150","15_Glutamine":"222.7485","6_A4_C13_Glucose_1":"165.7605","7_A4_C13_Glucose_2":"628.9320","8_A4_C13_Glucose_3":"271.2295","9_A4_C13_Glucose_4":"558.8655","16_A4_C13_Glutamine_1":"1119.0045","17_A4_C13_Glutamine_2":"497.4740","18_A4_C13_Glutamine_3":"550.6665","19_A4_C13_Glutamine_4":"775.9420","10_A4_Glucose":"628.8555","20_A4_Glutamine":"233.7735"},{"Metabolite":"2-Hydroxyglutaric M+4","21_Ala_C13_Glutamine_1":"","22_Ala_C13_Glutamine_2":"","23_Ala_C13_Glutamine_3":"","24_Ala_C13_Glutamine_4":"","25_Ala_Glutamine":"","1_C13_Glucose_1":"","2_C13_Glucose_2":"","3_C13_Glucose_3":"","4_C13_Glucose_4":"","11_C13_Glutamine_1":"","12_C13_Glutamine_2":"","13_C13_Glutamine_3":"77.1960","14_C13_Glutamine_4":"","5_Glucose":"","15_Glutamine":"","6_A4_C13_Glucose_1":"123.4455","7_A4_C13_Glucose_2":"212.3735","8_A4_C13_Glucose_3":"","9_A4_C13_Glucose_4":"87.6295","16_A4_C13_Glutamine_1":"278.3880","17_A4_C13_Glutamine_2":"","18_A4_C13_Glutamine_3":"162.5680","19_A4_C13_Glutamine_4":"228.3105","10_A4_Glucose":"89.8995","20_A4_Glutamine":""},{"Metabolite":"2-hydroxyglutaric M+5","21_Ala_C13_Glutamine_1":"313.0435","22_Ala_C13_Glutamine_2":"291.4635","23_Ala_C13_Glutamine_3":"","24_Ala_C13_Glutamine_4":"272.2480","25_Ala_Glutamine":"","1_C13_Glucose_1":"","2_C13_Glucose_2":"","3_C13_Glucose_3":"","4_C13_Glucose_4":"","11_C13_Glutamine_1":"1324.8830","12_C13_Glutamine_2":"920.0400","13_C13_Glutamine_3":"1569.9720","14_C13_Glutamine_4":"927.0845","5_Glucose":"","15_Glutamine":"","6_A4_C13_Glucose_1":"","7_A4_C13_Glucose_2":"","8_A4_C13_Glucose_3":"","9_A4_C13_Glucose_4":"","16_A4_C13_Glutamine_1":"2334.3315","17_A4_C13_Glutamine_2":"793.1640","18_A4_C13_Glutamine_3":"1375.6805","19_A4_C13_Glutamine_4":"1161.8995","10_A4_Glucose":"","20_A4_Glutamine":""},{"Metabolite":"2P-Glycerol M+0","21_Ala_C13_Glutamine_1":"7768.6370","22_Ala_C13_Glutamine_2":"26694.1850","23_Ala_C13_Glutamine_3":"18334.5680","24_Ala_C13_Glutamine_4":"40362.9295","25_Ala_Glutamine":"30463.1335","1_C13_Glucose_1":"737.3810","2_C13_Glucose_2":"1664.6920","3_C13_Glucose_3":"1692.3260","4_C13_Glucose_4":"1073.7580","11_C13_Glutamine_1":"7422.8305","12_C13_Glutamine_2":"4762.4785","13_C13_Glutamine_3":"8064.2230","14_C13_Glutamine_4":"3344.5735","5_Glucose":"8654.8190","15_Glutamine":"8851.7875","6_A4_C13_Glucose_1":"1638.4845","7_A4_C13_Glucose_2":"3529.0140","8_A4_C13_Glucose_3":"3086.3735","9_A4_C13_Glucose_4":"3961.8165","16_A4_C13_Glutamine_1":"9976.1035","17_A4_C13_Glutamine_2":"4988.9495","18_A4_C13_Glutamine_3":"9296.3990","19_A4_C13_Glutamine_4":"12427.3288","10_A4_Glucose":"10274.1280","20_A4_Glutamine":"10831.7040"},{"Metabolite":"2P-Glycerol 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