{ "METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003169","ANALYSIS_ID":"AN005201","VERSION":"1","CREATED_ON":"April 11, 2024, 8:27 am"}, "PROJECT":{"PROJECT_TITLE":"Releasing the mitochondrial respiration brake MCJ/DnaJC15 enhances CD8 CAR-T cell therapy efficacy","PROJECT_SUMMARY":"Metabolism of chimeric antigen receptor (CAR) T cells is emerging as an important area to improve CAR-T cell therapy in cancer treatment. Mitochondrial respiration is essential for survival and function of CAR-T cells, but developing strategies to specifically enhance mitochondrial respiration has been challenging. Here we identify MCJ/DnaJC15, an endogenous negative regulator of mitochondrial Complex I, as a metabolic target to enhance mitochondrial respiration in CD8 CAR-T cells. Loss of MCJ in CD8 CAR-T cells increases their in vitro and in vivo efficacy against mouse B cell leukemias. MCJ deficiency in TCR- specific CD8 cells also increases their efficacy against solid tumors in vivo. Furthermore, we reveal that human CD8 cells express MCJ and that silencing MCJ expression increases mitochondrial metabolism and anti-tumor activity of human CAR-T cells. Thus, targeting MCJ to enhance mitochondrial metabolism is a promising therapeutic strategy to improve the efficacy of adoptive T cell therapies.","INSTITUTE":"University of Colorado School of Medicine","LABORATORY":"Laboratory of Angelo D''Alessandro in collaboration with Mercedes Rincon","LAST_NAME":"Cendali","FIRST_NAME":"Francesca","ADDRESS":"13199 East Montview Boulevard, Aurora, CO, 80045, USA","EMAIL":"francesca.cendali@cuanschutz.edu","PHONE":"3037246131"}, "STUDY":{"STUDY_TITLE":"Releasing the mitochondrial respiration brake MCJ/DnaJC15 enhances CD8 CAR-T cell therapy efficacy","STUDY_SUMMARY":"Metabolism of chimeric antigen receptor (CAR) T cells is emerging as an important area to improve CAR-T cell therapy in cancer treatment. Mitochondrial respiration is essential for survival and function of CAR-T cells, but developing strategies to specifically enhance mitochondrial respiration has been challenging. Here we identify MCJ/DnaJC15, an endogenous negative regulator of mitochondrial Complex I, as a metabolic target to enhance mitochondrial respiration in CD8 CAR-T cells. Loss of MCJ in CD8 CAR-T cells increases their in vitro and in vivo efficacy against mouse B cell leukemias. MCJ deficiency in TCR- specific CD8 cells also increases their efficacy against solid tumors in vivo. Furthermore, we reveal that human CD8 cells express MCJ and that silencing MCJ expression increases mitochondrial metabolism and anti-tumor activity of human CAR-T cells. Thus, targeting MCJ to enhance mitochondrial metabolism is a promising therapeutic strategy to improve the efficacy of adoptive T cell therapies.","INSTITUTE":"University of Colorado School of Medicine","LABORATORY":"Laboratory of Angelo D''Alessandro in collaboration with Mercedes Rincon","LAST_NAME":"Cendali","FIRST_NAME":"Francesca","ADDRESS":"13199 East Montview Boulevard, Aurora, CO, 80045, USA","EMAIL":"francesca.cendali@cuanschutz.edu","PHONE":"3037246131"}, "SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Mus musculus"}, "SUBJECT_SAMPLE_FACTORS":[ { "Subject ID":"-", "Sample ID":"1", "Factors":{"Factor":"WT CART in vivo","Sample source":"Cells"}, "Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"1"} }, { "Subject ID":"-", "Sample ID":"2", "Factors":{"Factor":"WT CART in vivo","Sample source":"Cells"}, "Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"2"} }, { "Subject ID":"-", "Sample ID":"3", "Factors":{"Factor":"WT CART in vivo","Sample source":"Cells"}, "Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"3"} }, { "Subject ID":"-", "Sample ID":"4", "Factors":{"Factor":"MCJ KO CART in vivo","Sample source":"Cells"}, "Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"4"} }, { "Subject ID":"-", "Sample ID":"5", "Factors":{"Factor":"MCJ KO CART in vivo","Sample source":"Cells"}, "Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"5"} }, { "Subject ID":"-", "Sample ID":"6", "Factors":{"Factor":"MCJ KO CART in vivo","Sample source":"Cells"}, "Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"6"} } ], "COLLECTION":{"COLLECTION_SUMMARY":"The E2a cells (106) were cultured in CMM for no more than 2 passages before intravenous tail vail injection to B6 mice 3 days before CAR-T cell injection. The E2a-bearing mice were irradiated with 500cGy sublethal dosage the day before CAR-T cell injection. The murine CD8 CAR-T cells were generated and expanded with rhIL-2 (60 IU/ml) as described above. The expanded CD8 CAR-T cells were washed with PBS, and 106 CAR+ cells were injected into the E2a-bearing mice via intravenous tail vail injection. The mice were followed for survival with the humane end point of development of hind limb paralysis, or weight loss of more than 20% of the original weight, or signs of major discomfort as determined by the institutional veterinarian.","SAMPLE_TYPE":"T-cells"}, "TREATMENT":{"TREATMENT_SUMMARY":"Metabolomics differences in this experiment are observed at baseline"}, "SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"CAR-T cells were isolated as described above either from in vitro culture or from bone marrow harvested in an in vivo study. The cells were washed in PBS and frozen at -80C until the assay is ready to run. Metabolites from cells were extracted at 2x106 cells/ml at 4°C (30 min) in the presence of 5:3:2 MeOH:MeCN:water (v/v/v). The samples were spun down and the resulting supernatant was transferred to new tubes and dried under a vacuum. The resulting residue was reconstituted in 0.1% formic acid at a 3x concentration, then analyzed on a Thermo Vanquish UHPLC coupled to a Thermo Q Exactive MS as previously described in detail."}, "CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Positive Ion Mode","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Exploris120","COLUMN_NAME":"Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)","SOLVENT_A":"100% water; 0.1% formic acid","SOLVENT_B":"100% acetonitrile; 0.1% formic acid","FLOW_GRADIENT":"0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min 95-5% B, 3-5 min hold at 5% B.","FLOW_RATE":"0.450ml/min","COLUMN_TEMPERATURE":"45"}, "ANALYSIS":{"ANALYSIS_TYPE":"MS"}, "MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database."}, "MS_METABOLITE_DATA":{ "Units":"Peak Areas", "Data":[{"Metabolite":"L-alanine","1":"1197023.88","2":"1327983.88","3":"1022429.81","4":"1568968.62","5":"1427793.12","6":"1062461"},{"Metabolite":"L-arginine","1":"6863230.5","2":"7222230.5","3":"6243684.5","4":"7980496","5":"7930764","6":"6132052.5"},{"Metabolite":"L-asparagine","1":"535468.31","2":"476826.5","3":"450014.75","4":"631193.06","5":"505875","6":"375585"},{"Metabolite":"L-aspartate","1":"1372252.62","2":"1483275.12","3":"1116382.38","4":"1451198.62","5":"1424129","6":"1146679.62"},{"Metabolite":"L-glutamate","1":"3973126.75","2":"3948598.75","3":"3736170","4":"4412249.5","5":"4241764.5","6":"3648352"},{"Metabolite":"L-glutamine","1":"8488004","2":"8887393","3":"7065745.5","4":"10065109","5":"9675385","6":"7004350"},{"Metabolite":"L-histidine","1":"453154.34","2":"577390.25","3":"269786.47","4":"415080.84","5":"422430","6":"382865.53"},{"Metabolite":"L-leucine/L-isoleucine","1":"24789086","2":"27747430","3":"24184186","4":"28680718","5":"26054418","6":"20049822"},{"Metabolite":"L-lysine","1":"775457.31","2":"877422.56","3":"377104.09","4":"806658.81","5":"727064.5","6":"675014.25"},{"Metabolite":"L-methionine","1":"7392514.5","2":"8478231","3":"7065848","4":"8697247","5":"8380496","6":"6430578.5"},{"Metabolite":"L-phenylalanine","1":"6978496.5","2":"8475595","3":"6852887.5","4":"7940340","5":"7643455.5","6":"6032851.5"},{"Metabolite":"L-proline","1":"6982688","2":"6567523.5","3":"7047762","4":"7652542.5","5":"7620930.5","6":"6172780"},{"Metabolite":"L-serine","1":"427444.22","2":"485046.16","3":"345151.25","4":"514835.25","5":"394962.66","6":"229377.73"},{"Metabolite":"L-threonine","1":"485082.16","2":"524086.28","3":"511597.22","4":"528426.88","5":"403367.66","6":"419341.84"},{"Metabolite":"L-tryptophan","1":"3562216.75","2":"4013098.75","3":"3605524.75","4":"3124009.75","5":"3073252.75","6":"2051719.88"},{"Metabolite":"L-tyrosine","1":"10081315","2":"10097824","3":"9413260","4":"9469005","5":"9446284","6":"8265036"},{"Metabolite":"L-valine","1":"5042666.5","2":"5201343.5","3":"4247850.5","4":"5328159.5","5":"5261880","6":"4673793.5"},{"Metabolite":"AMP","1":"456886.5","2":"351903.41","3":"297473.53","4":"348723.72","5":"443815.5","6":"500947.84"},{"Metabolite":"Adenosine","1":"707023.75","2":"664739.56","3":"720749.81","4":"745050.44","5":"850719.25","6":"1729506"},{"Metabolite":"Guanine","1":"323993.69","2":"358170.78","3":"292883.28","4":"359655.97","5":"364053.09","6":"299765.22"},{"Metabolite":"CMP","1":"71679.34","2":"66506.85","3":"50993.55","4":"79041.96","5":"74343.79","6":"85925.59"},{"Metabolite":"Hypoxanthine","1":"358667.34","2":"377833.62","3":"363256.12","4":"335150.09","5":"354565.38","6":"366094.12"},{"Metabolite":"Urate","1":"759133.5","2":"815769.31","3":"771022.94","4":"702023.81","5":"665949.5","6":"692983.06"},{"Metabolite":"Nicotinamide","1":"1961781.12","2":"1801942.62","3":"1629010.38","4":"1850226.88","5":"1589012","6":"1885181.5"},{"Metabolite":"5-Oxoproline","1":"10309443","2":"12489417","3":"8682290","4":"10292717","5":"11624371","6":"8675300"},{"Metabolite":"S-Glutathionyl-L-cysteine","1":"224014.3","2":"296579.34","3":"169744.86","4":"320564.03","5":"339876.75","6":"191665.62"},{"Metabolite":"gamma-L-Glutamyl-L-cysteine","1":"184416.39","2":"189766.45","3":"110685.09","4":"78813.71","5":"104129.03","6":"163364.39"},{"Metabolite":"Ornithine","1":"180562.64","2":"259328.88","3":"27569.28","4":"77610.2","5":"115839.06","6":"36205.36"},{"Metabolite":"L-Citrulline","1":"180369.12","2":"164223.02","3":"203855.31","4":"113525.44","5":"134043.44","6":"139997.25"},{"Metabolite":"Creatine","1":"4770057","2":"8793808","3":"4054201","4":"4360741.5","5":"3005996.75","6":"10805841"},{"Metabolite":"Creatinine","1":"2846940","2":"3534683.25","3":"2355705","4":"2754858","5":"2667432.25","6":"3342950.75"},{"Metabolite":"N-Acetyl-L-citrulline","1":"12353771","2":"13820144","3":"11396885","4":"15381347","5":"12215839","6":"10168484"},{"Metabolite":"Guanidinoacetate","1":"117404.18","2":"72310.2","3":"98977.09","4":"56083.73","5":"84498.34","6":"67815.17"},{"Metabolite":"Pantothenol","1":"440249.59","2":"518334.91","3":"404414.91","4":"529658.81","5":"479921.66","6":"395565.91"},{"Metabolite":"Taurine","1":"1408399.12","2":"1351138.62","3":"821823.5","4":"1320948.75","5":"1256985.25","6":"1125754.62"},{"Metabolite":"Indole-3-acetaldehyde","1":"285553.47","2":"277493.47","3":"263842.72","4":"8207.73","5":"5372.33","6":"25682.28"},{"Metabolite":"Indole-3-acetate","1":"1448145.5","2":"1526587.38","3":"1911843.12","4":"1680266.88","5":"1729845.5","6":"1299315.88"},{"Metabolite":"6-Hydroxykynurenic acid","1":"1486375.88","2":"1398272.62","3":"1111398","4":"737258.19","5":"744064.31","6":"398366.47"},{"Metabolite":"kynurenine","1":"23525.04","2":"34734.91","3":"31615.96","4":"68574","5":"65982.12","6":"42198.25"},{"Metabolite":"N-formyl kynurenine","1":"7078.12","2":"5688.14","3":"8759.02","4":"36287.55","5":"37959.28","6":"22354.58"},{"Metabolite":"Anthranilate","1":"230236.42","2":"208531.12","3":"234244.56","4":"180784.58","5":"203353.31","6":"150428.47"},{"Metabolite":"g-Oxalo-crotonate","1":"124694.13","2":"104803.19","3":"104126.43","4":"135527.92","5":"135116.95","6":"78721.22"},{"Metabolite":"2-Oxoadipate","1":"123582.88","2":"135552.73","3":"192910.25","4":"185970.05","5":"162221.08","6":"104098.6"},{"Metabolite":"L-Adrenaline","1":"215566","2":"391056.41","3":"542457.81","4":"226354.56","5":"263924.34","6":"458769.59"},{"Metabolite":"Ethanolamine phosphate","1":"337839.84","2":"338737.34","3":"214072.86","4":"291651.41","5":"360397.53","6":"314050.91"},{"Metabolite":"Sphingosine","1":"631332.44","2":"368950.78","3":"351429.72","4":"546680.56","5":"492058.91","6":"650668.81"},{"Metabolite":"Acetylcholine","1":"139253.92","2":"115964.66","3":"184844.95","4":"113749.4","5":"213390.48","6":"370313.41"},{"Metabolite":"Choline","1":"10643782","2":"11372032","3":"12102527","4":"10968815","5":"11590809","6":"10520196"},{"Metabolite":"L-Carnitine","1":"4897933.5","2":"5952352.5","3":"3980325.25","4":"4253042.5","5":"3873179.25","6":"6830796"},{"Metabolite":"acyl-C2 (acetyl-carnitine)","1":"4928999.5","2":"5844600","3":"3997628","4":"4427018.5","5":"5025709.5","6":"5826114.5"},{"Metabolite":"acyl-C3 (propionyl-carnitine)","1":"404599.66","2":"445941.59","3":"396862.59","4":"369899.28","5":"389751.72","6":"449780.78"},{"Metabolite":"acyl-C4 (butanoyl-l-carnitine)","1":"144729.33","2":"152380.14","3":"132910.02","4":"127478.52","5":"166861.56","6":"101832.57"},{"Metabolite":"acyl-C4-OH (Hydroxybutyrylcarnitine)","1":"194528.62","2":"234728.06","3":"143606.22","4":"179798.36","5":"199170.86","6":"222721.3"},{"Metabolite":"acyl-C4-DC","1":"22472.05","2":"40887.15","3":"17242.7","4":"27167.21","5":"27819.52","6":"50310.48"},{"Metabolite":"acyl-C6 (hexanoyl-l-carnitine)","1":"78703.78","2":"68030.15","3":"64582.07","4":"74810.56","5":"57538.34","6":"75826.67"},{"Metabolite":"acyl-C14 (O-tetradecanoyl-L-carnitine)","1":"474356.84","2":"60020.62","3":"18004.3","4":"152064.39","5":"235384.45","6":"44302.54"},{"Metabolite":"acyl-C18 (Octadecanoyl-L-carnitine)","1":"4398604.5","2":"539934.06","3":"98986.34","4":"1182649.38","5":"1317746.25","6":"566479.94"},{"Metabolite":"acyl-C20","1":"947354.69","2":"1089732.12","3":"851443.69","4":"692495.62","5":"740909.75","6":"695332.75"},{"Metabolite":"acyl-C20:4","1":"288275.88","2":"595105.12","3":"273191.84","4":"126742.95","5":"218085.8","6":"130637.21"}], "Metabolites":[{"Metabolite":"L-alanine","CmpdID":"C00041","parent":"90.055092","medRt":"0.656"},{"Metabolite":"L-arginine","CmpdID":"C00062","parent":"175.118912","medRt":"0.637"},{"Metabolite":"L-asparagine","CmpdID":"C00152","parent":"133.060074","medRt":"0.661"},{"Metabolite":"L-aspartate","CmpdID":"C00049","parent":"134.044876","medRt":"0.671"},{"Metabolite":"L-glutamate","CmpdID":"C00025","parent":"148.060455","medRt":"0.667"},{"Metabolite":"L-glutamine","CmpdID":"C00064","parent":"147.076462","medRt":"0.664"},{"Metabolite":"L-histidine","CmpdID":"C00135","parent":"156.076797","medRt":"0.63"},{"Metabolite":"L-leucine/L-isoleucine","CmpdID":"C00123","parent":"132.102005","medRt":"1.005"},{"Metabolite":"L-lysine","CmpdID":"C00047","parent":"147.112869","medRt":"0.636"},{"Metabolite":"L-methionine","CmpdID":"C00073","parent":"150.058319","medRt":"0.895"},{"Metabolite":"L-phenylalanine","CmpdID":"C00079","parent":"166.086288","medRt":"1.416"},{"Metabolite":"L-proline","CmpdID":"C00148","parent":"116.070793","medRt":"0.689"},{"Metabolite":"L-serine","CmpdID":"C00065","parent":"106.050072","medRt":"0.659"},{"Metabolite":"L-threonine","CmpdID":"C00188","parent":"120.065681","medRt":"0.662"},{"Metabolite":"L-tryptophan","CmpdID":"C00078","parent":"205.097153","medRt":"1.761"},{"Metabolite":"L-tyrosine","CmpdID":"C00082","parent":"182.081131","medRt":"0.899"},{"Metabolite":"L-valine","CmpdID":"C00183","parent":"118.086433","medRt":"0.668"},{"Metabolite":"AMP","CmpdID":"C00020","parent":"348.070404","medRt":"0.741"},{"Metabolite":"Adenosine","CmpdID":"C00212","parent":"268.104034","medRt":"0.895"},{"Metabolite":"Guanine","CmpdID":"C00242","parent":"152.05452","medRt":"0.897"},{"Metabolite":"CMP","CmpdID":"C00055","parent":"324.059174","medRt":"0.685"},{"Metabolite":"Hypoxanthine","CmpdID":"C00262","parent":"137.045883","medRt":"0.883"},{"Metabolite":"Urate","CmpdID":"C00366","parent":"169.035522","medRt":"0.894"},{"Metabolite":"Nicotinamide","CmpdID":"C00153","parent":"123.055458","medRt":"0.904"},{"Metabolite":"5-Oxoproline","CmpdID":"C01879","parent":"130.049942","medRt":"0.943"},{"Metabolite":"S-Glutathionyl-L-cysteine","CmpdID":"C05526","parent":"427.095123","medRt":"0.675"},{"Metabolite":"gamma-L-Glutamyl-L-cysteine","CmpdID":"C00669","parent":"251.069855","medRt":"0.895"},{"Metabolite":"Ornithine","CmpdID":"C01602","parent":"133.09726","medRt":"0.627"},{"Metabolite":"L-Citrulline","CmpdID":"C00327","parent":"176.102966","medRt":"0.669"},{"Metabolite":"Creatine","CmpdID":"C00300","parent":"132.076859","medRt":"0.674"},{"Metabolite":"Creatinine","CmpdID":"C00791","parent":"114.066383","medRt":"0.734"},{"Metabolite":"N-Acetyl-L-citrulline","CmpdID":"C15532","parent":"218.113541","medRt":"0.661"},{"Metabolite":"Guanidinoacetate","CmpdID":"C00581","parent":"118.061272","medRt":"0.674"},{"Metabolite":"Pantothenol","CmpdID":"C00864","parent":"220.117981","medRt":"1.711"},{"Metabolite":"Taurine","CmpdID":"C00245","parent":"126.022072","medRt":"0.666"},{"Metabolite":"Indole-3-acetaldehyde","CmpdID":"C00637","parent":"160.075653","medRt":"1.133"},{"Metabolite":"Indole-3-acetate","CmpdID":"C00954","parent":"176.07074","medRt":"1.969"},{"Metabolite":"6-Hydroxykynurenic acid","CmpdID":"C08480","parent":"206.048141","medRt":"0.904"},{"Metabolite":"kynurenine","CmpdID":"C00328","parent":"209.091934","medRt":"1.421"},{"Metabolite":"N-formyl kynurenine","CmpdID":"C02700","parent":"237.087021","medRt":"1.051"},{"Metabolite":"Anthranilate","CmpdID":"C00108","parent":"138.055038","medRt":"1.8"},{"Metabolite":"g-Oxalo-crotonate","CmpdID":"C03453","parent":"159.027679","medRt":"0.888"},{"Metabolite":"2-Oxoadipate","CmpdID":"C00322","parent":"161.046036","medRt":"1.97"},{"Metabolite":"L-Adrenaline","CmpdID":"C00788","parent":"184.095642","medRt":"0.662"},{"Metabolite":"Ethanolamine phosphate","CmpdID":"C00346","parent":"142.026413","medRt":"0.662"},{"Metabolite":"Sphingosine","CmpdID":"C00319","parent":"300.289764","medRt":"2.149"},{"Metabolite":"Acetylcholine","CmpdID":"C01996","parent":"146.117569","medRt":"0.739"},{"Metabolite":"Choline","CmpdID":"C00114","parent":"104.107201","medRt":"0.652"},{"Metabolite":"L-Carnitine","CmpdID":"C00318","parent":"162.112473","medRt":"0.666"},{"Metabolite":"acyl-C2 (acetyl-carnitine)","CmpdID":"C02571","parent":"204.123032","medRt":"0.886"},{"Metabolite":"acyl-C3 (propionyl-carnitine)","CmpdID":"C03017","parent":"218.138626","medRt":"1.061"},{"Metabolite":"acyl-C4 (butanoyl-l-carnitine)","CmpdID":"C02862","parent":"232.154358","medRt":"1.75"},{"Metabolite":"acyl-C4-OH (Hydroxybutyrylcarnitine)","CmpdID":"HMDB13127","parent":"248.149384","medRt":"0.897"},{"Metabolite":"acyl-C4-DC","CmpdID":"HMDB13133","parent":"262.128693","medRt":"0.893"},{"Metabolite":"acyl-C6 (hexanoyl-l-carnitine)","CmpdID":"HMDB00756","parent":"260.185669","medRt":"1.824"},{"Metabolite":"acyl-C14 (O-tetradecanoyl-L-carnitine)","CmpdID":"HMDB05066","parent":"372.311218","medRt":"2.05"},{"Metabolite":"acyl-C18 (Octadecanoyl-L-carnitine)","CmpdID":"HMDB00848","parent":"428.373535","medRt":"2.624"},{"Metabolite":"acyl-C20","CmpdID":"ac124","parent":"458.417389","medRt":"0.6"},{"Metabolite":"acyl-C20:4","CmpdID":"ac125","parent":"448.341461","medRt":"2.642"}] } }