#METABOLOMICS WORKBENCH cendalfr_20240411_082827 DATATRACK_ID:4772 STUDY_ID:ST003170 ANALYSIS_ID:AN005203 PROJECT_ID:PR001970 VERSION 1 CREATED_ON April 11, 2024, 8:36 am #PROJECT PR:PROJECT_TITLE Releasing the mitochondrial respiration brake MCJ/DnaJC15 enhances CD8 CAR-T PR:PROJECT_TITLE cell therapy efficacy PR:PROJECT_SUMMARY Metabolism of chimeric antigen receptor (CAR) T cells is emerging as an PR:PROJECT_SUMMARY important area to improve CAR-T cell therapy in cancer treatment. Mitochondrial PR:PROJECT_SUMMARY respiration is essential for survival and function of CAR-T cells, but PR:PROJECT_SUMMARY developing strategies to specifically enhance mitochondrial respiration has been PR:PROJECT_SUMMARY challenging. Here we identify MCJ/DnaJC15, an endogenous negative regulator of PR:PROJECT_SUMMARY mitochondrial Complex I, as a metabolic target to enhance mitochondrial PR:PROJECT_SUMMARY respiration in CD8 CAR-T cells. Loss of MCJ in CD8 CAR-T cells increases their PR:PROJECT_SUMMARY in vitro and in vivo efficacy against mouse B cell leukemias. MCJ deficiency in PR:PROJECT_SUMMARY TCR- specific CD8 cells also increases their efficacy against solid tumors in PR:PROJECT_SUMMARY vivo. Furthermore, we reveal that human CD8 cells express MCJ and that silencing PR:PROJECT_SUMMARY MCJ expression increases mitochondrial metabolism and anti-tumor activity of PR:PROJECT_SUMMARY human CAR-T cells. Thus, targeting MCJ to enhance mitochondrial metabolism is a PR:PROJECT_SUMMARY promising therapeutic strategy to improve the efficacy of adoptive T cell PR:PROJECT_SUMMARY therapies. PR:INSTITUTE University of Colorado School of Medicine PR:LABORATORY Laboratory of Angelo D'Alessandro in collaboration with Mercedes Rincon PR:LAST_NAME Cendali PR:FIRST_NAME Francesca PR:ADDRESS 13199 East Montview Boulevard, Aurora, CO, 80045, USA PR:EMAIL francesca.cendali@cuanschutz.edu PR:PHONE 3037246131 #STUDY ST:STUDY_TITLE Releasing the mitochondrial respiration brake MCJ/DnaJC15 enhances CD8 CAR-T ST:STUDY_TITLE cell therapy efficacy ST:STUDY_SUMMARY Metabolism of chimeric antigen receptor (CAR) T cells is emerging as an ST:STUDY_SUMMARY important area to improve CAR-T cell therapy in cancer treatment. Mitochondrial ST:STUDY_SUMMARY respiration is essential for survival and function of CAR-T cells, but ST:STUDY_SUMMARY developing strategies to specifically enhance mitochondrial respiration has been ST:STUDY_SUMMARY challenging. Here we identify MCJ/DnaJC15, an endogenous negative regulator of ST:STUDY_SUMMARY mitochondrial Complex I, as a metabolic target to enhance mitochondrial ST:STUDY_SUMMARY respiration in CD8 CAR-T cells. Loss of MCJ in CD8 CAR-T cells increases their ST:STUDY_SUMMARY in vitro and in vivo efficacy against mouse B cell leukemias. MCJ deficiency in ST:STUDY_SUMMARY TCR- specific CD8 cells also increases their efficacy against solid tumors in ST:STUDY_SUMMARY vivo. Furthermore, we reveal that human CD8 cells express MCJ and that silencing ST:STUDY_SUMMARY MCJ expression increases mitochondrial metabolism and anti-tumor activity of ST:STUDY_SUMMARY human CAR-T cells. Thus, targeting MCJ to enhance mitochondrial metabolism is a ST:STUDY_SUMMARY promising therapeutic strategy to improve the efficacy of adoptive T cell ST:STUDY_SUMMARY therapies. ST:INSTITUTE University of Colorado School of Medicine ST:LABORATORY Laboratory of Angelo D'Alessandro in collaboration with Mercedes Rincon ST:LAST_NAME Cendali ST:FIRST_NAME Francesca ST:ADDRESS 13199 East Montview Boulevard, Aurora, CO, 80045, USA ST:EMAIL francesca.cendali@cuanschutz.edu ST:PHONE 3037246131 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - A Factor:WT CART med | Sample source:Cells RAW_FILE_NAME(Raw File Name)=1 SUBJECT_SAMPLE_FACTORS - B Factor:WT CART med | Sample source:Cells RAW_FILE_NAME(Raw File Name)=2 SUBJECT_SAMPLE_FACTORS - C Factor:WT CART med | Sample source:Cells RAW_FILE_NAME(Raw File Name)=3 SUBJECT_SAMPLE_FACTORS - D Factor:MCJ KO CART med | Sample source:Cells RAW_FILE_NAME(Raw File Name)=4 SUBJECT_SAMPLE_FACTORS - E Factor:MCJ KO CART med | Sample source:Cells RAW_FILE_NAME(Raw File Name)=5 SUBJECT_SAMPLE_FACTORS - F Factor:MCJ KO CART med | Sample source:Cells RAW_FILE_NAME(Raw File Name)=6 #COLLECTION CO:COLLECTION_SUMMARY Murine CD8 cells were isolated from lymph nodes and spleen using negative CO:COLLECTION_SUMMARY selection with CD8+ T cell isolation kit (for generation of CAR-T cells) CO:COLLECTION_SUMMARY (Miltenyi, order# 130-104-075) or positive selection (for OT-I CD8 cells) with CO:COLLECTION_SUMMARY CD8 (Ly-2) Microbeads (Miltenyi, order# 130-117-044). Mouse CD8 cells were CO:COLLECTION_SUMMARY activated with plate-bound anti-CD3 (5 g/ml) and soluble anti-CD28 mAbs (1 CO:COLLECTION_SUMMARY g/ml) for 48h for WT and cMCJ KO CD8 studies and OT-I CD8 cells studies. CO:SAMPLE_TYPE T-cells #TREATMENT TR:TREATMENT_SUMMARY Metabolomics differences in this experiment are observed at baseline #SAMPLEPREP SP:SAMPLEPREP_SUMMARY CAR-T cells were isolated as described above either from in vitro culture or SP:SAMPLEPREP_SUMMARY from bone marrow harvested in an in vivo study. The cells were washed in PBS and SP:SAMPLEPREP_SUMMARY frozen at -80C until the assay is ready to run. Metabolites from cells were SP:SAMPLEPREP_SUMMARY extracted at 2x106 cells/ml at 4°C (30 min) in the presence of 5:3:2 SP:SAMPLEPREP_SUMMARY MeOH:MeCN:water (v/v/v). The samples were spun down and the resulting SP:SAMPLEPREP_SUMMARY supernatant was transferred to new tubes and dried under a vacuum. The resulting SP:SAMPLEPREP_SUMMARY residue was reconstituted in 0.1% formic acid at a 3x concentration, then SP:SAMPLEPREP_SUMMARY analyzed on a Thermo Vanquish UHPLC coupled to a Thermo Q Exactive MS as SP:SAMPLEPREP_SUMMARY previously described in detail. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Positive Ion Mode CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH: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 CH:FLOW_GRADIENT 95-5% B, 3-5 min hold at 5% B. CH:FLOW_RATE 0.450ml/min CH:COLUMN_TEMPERATURE 45 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, MS:MS_COMMENTS microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, MS:MS_COMMENTS capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas MS:MS_COMMENTS 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were MS:MS_COMMENTS annotated and integrated using Maven in conjunction with the KEGG database. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak Areas MS_METABOLITE_DATA_START Samples A B C D E F Factors Factor:WT CART med | Sample source:Cells Factor:WT CART med | Sample source:Cells Factor:WT CART med | Sample source:Cells Factor:MCJ KO CART med | Sample source:Cells Factor:MCJ KO CART med | Sample source:Cells Factor:MCJ KO CART med | Sample source:Cells L-alanine 2899601.25 1423708 2347212.25 2074696 1722981 1278456 L-arginine 4378797.5 2704065 4461140.5 4934806 4223950.5 6972328 L-asparagine 703490.31 544056.44 833265.69 567966.38 395946.41 376895.38 L-aspartate 5977391.5 5584386.5 7831730.5 4871708 3764491.75 2684774.25 L-cysteine 4048.84 0 8091.44 0 4655.7 5678.73 L-glutamate 6848666.5 10564893 12352985 7319765.5 6202989.5 5458377.5 L-glutamine 4403248.5 2513811.75 4662096 2399477.75 2220893.25 1995128 L-histidine 1053737.62 319320.62 526109.19 940233.31 1038589.25 901908 L-leucine/L-isoleucine 18551056 5101423.5 10462659 23616624 26848600 26839890 L-lysine 1556454.62 723165 1213436.62 1772048 1616108.5 1824667.5 L-methionine 2163834 655633.06 1371486.62 3615653.25 4054355 4977298.5 L-phenylalanine 9279409 2495139.75 5557782.5 12300485 12438457 14553895 L-proline 3687341 1669128.5 2985754.75 4483041.5 4816232.5 5357467.5 L-serine 3460968.25 1172575.5 2322425.75 2751616.75 2296694.75 1262677.88 L-threonine 2266535 1505199.38 2384315.75 1677710.38 1276456 902260.44 L-tryptophan 4972201.5 1271265.5 2854987 6331887.5 5395861.5 6540048.5 L-tyrosine 9781055 2439037.75 5448181.5 12511029 12843873 14191539 L-valine 7786622.5 3211052.75 4734124 9403460 10358271 9624791 L-cystine 17054.77 40252.64 43958.91 26954.08 14206.53 15569.73 Adenosine 744224.25 90820.98 263896.41 972697.19 587653.5 675454.56 GMP 140813.75 26505.21 52639.27 519133.22 128707.75 200740.3 CMP 245513.5 179945.58 221565.33 347736.5 321683.28 330805.59 Cytidine 268586.66 104568.44 268844.16 258058.48 238843.33 219376.11 Cytosine 755998.81 471160.28 811429.31 691954.5 834295.56 873001.31 Thymine 61353.93 9672.73 17846.94 54683.97 65031.56 47290.68 Uracil 141063.98 43676.78 104610.27 101250.62 151095.73 192534.17 Hypoxanthine 616633.38 167498.89 420965.59 703927.44 751096.19 810906.75 Xanthine 279047.47 41528.81 158168.95 351033.34 367374.09 347689.12 Pyridoxal 26197.57 29369.78 26379.74 26285.33 28834.73 27215.24 Nicotinamide 50033.61 41158.32 31674.04 1599020 1582243.88 1941346 NAD+ 169119.92 97853.34 159148.12 318612.53 266344.31 311710.59 Glutathione disulfide 308701.34 68639.66 239406.64 125322.65 183067.95 215601.55 5-Oxoproline 8317864 748918.06 2611579 12060173 12192916 8213134.5 Cys-Gly 259763.73 119837.75 195899.56 591315.12 574105.25 599195.06 gamma-L-Glutamyl-L-cysteine 43406.39 0 34799.02 87423.65 75943.65 80581.55 5-10-Methenyltetrahydrofolate 6786.34 17884.08 1984.07 2326.09 0 7652.7 Ornithine 1176229.38 199346.05 502341.03 1649606.12 1402677.38 1003263.5 L-Citrulline 918793.5 252538.55 650328.69 882434.25 745029.56 358113.59 Spermidine 160086.17 62881.47 168013.23 7178412 789808.81 430480.09 Creatine 4041169 3179826.75 4313000.5 5077945.5 5148315.5 5679788.5 Creatinine 251845.62 152382.91 189605.12 372297.34 322271.19 257966.98 Guanidinoacetate 37512.12 24503.65 34844.28 30042.47 40801.73 34055.34 Pantothenol 411571.5 172507.56 313711.78 488571.88 542859.81 635116.62 Taurine 5265075.5 5270539 6398993.5 6138257.5 5176429.5 4157402 Hypotaurine 256271.69 258927.19 317501.28 195892.12 196788.69 131817.86 Indole-3-acetate 356172.97 150644.39 288192.38 295362.75 436574.47 747935.5 kynurenine 10813.21 3551.59 4352.9 4409.37 6276.17 14509.63 Sphingosine 3161847.75 1051735 2036684.62 658386 1846358.62 2768849.25 Sphingosine 1-phosphate 147247.88 5067.22 32281.68 383647.84 275147.69 175304.27 Acetylcholine 341317.66 165527.55 300137.22 459234.47 533833.88 540494.5 Choline 16901946 9498761 13981761 25780640 26762016 25263662 L-Carnitine 1296800 312192.47 798093.06 1923007.38 1906532.38 1680421.38 acyl-C2 (acetyl-carnitine) 0 0 2395.08 5016.06 5124.45 3883.29 acyl-C3 (propionyl-carnitine) 88985.52 43960.79 59945.25 151073 169809.11 163341.39 acyl-C4 (butanoyl-l-carnitine) 31386.37 11047.66 21979.24 54573.37 59169.31 56681.35 acyl-C4-OH (Hydroxybutyrylcarnitine) 2786.45 0 2465.62 6528.4 6092.74 3931.44 acyl-C5 (isovalerylcarnitine) 115314.91 25418.54 46752.33 171694.3 211221.27 125328.62 acyl-C5-OH 2651.68 0 2306.64 3251.13 3092 9224.33 acyl-C6 (hexanoyl-l-carnitine) 5302.28 0 5888.43 12565.7 8557.76 0 acyl-C12:1 (O-dodecenoyl-carnitine) 233369.12 202824.5 229522.17 232325.06 322816.88 276151.25 acyl-C14 (O-tetradecanoyl-L-carnitine) 80482.43 15904.25 52073.17 24801.63 61691.72 96589.43 acyl-C14:1 (Tetradecenoyl Carnitine) 111206.41 21080.8 27550.81 18586.01 81515.98 56359.87 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name CmpdID parent medRt L-alanine C00041 90.055 0.683 L-arginine C00062 175.119 0.661 L-asparagine C00152 133.061 0.674 L-aspartate C00049 134.045 0.684 L-cysteine C00097 122.028 0.598 L-glutamate C00025 148.060 0.688 L-glutamine C00064 147.076 0.679 L-histidine C00135 156.077 0.638 L-leucine/L-isoleucine C00123 132.102 0.991 L-lysine C00047 147.113 0.642 L-methionine C00073 150.058 0.904 L-phenylalanine C00079 166.086 1.481 L-proline C00148 116.071 0.710 L-serine C00065 106.050 0.674 L-threonine C00188 120.066 0.687 L-tryptophan C00078 205.097 1.799 L-tyrosine C00082 182.081 0.933 L-valine C00183 118.086 0.745 L-cystine C00491 241.031 0.674 Adenosine C00212 268.104 0.930 GMP C00144 364.065 0.805 CMP C00055 324.059 0.712 Cytidine C00475 244.093 0.729 Cytosine C00380 112.051 0.697 Thymine C00178 127.049 0.737 Uracil C00106 113.035 0.950 Hypoxanthine C00262 137.046 0.917 Xanthine C00385 153.041 0.926 Pyridoxal C00250 168.065 1.079 Nicotinamide C00153 123.055 1.010 NAD+ C00003 664.117 0.789 Glutathione disulfide C00127 613.160 0.912 5-Oxoproline C01879 130.050 0.926 Cys-Gly C01419 179.048 0.896 gamma-L-Glutamyl-L-cysteine C00669 251.070 0.931 5-10-Methenyltetrahydrofolate C00445 457.170 0.743 Ornithine C01602 133.097 0.633 L-Citrulline C00327 176.103 0.688 Spermidine C00315 146.165 0.602 Creatine C00300 132.077 0.696 Creatinine C00791 114.066 0.702 Guanidinoacetate C00581 118.061 0.689 Pantothenol C00864 220.118 1.746 Taurine C00245 126.022 0.690 Hypotaurine C00519 110.027 0.685 Indole-3-acetate C00954 176.071 1.981 kynurenine C00328 209.092 1.475 Sphingosine C00319 300.290 2.131 Sphingosine 1-phosphate C06124 380.253 2.197 Acetylcholine C01996 146.118 0.733 Choline C00114 104.107 0.675 L-Carnitine C00318 162.112 0.688 acyl-C2 (acetyl-carnitine) C02571 204.123 0.930 acyl-C3 (propionyl-carnitine) C03017 218.139 1.079 acyl-C4 (butanoyl-l-carnitine) C02862 232.154 1.834 acyl-C4-OH (Hydroxybutyrylcarnitine) HMDB13127 248.149 0.937 acyl-C5 (isovalerylcarnitine) C20826 246.170 1.856 acyl-C5-OH ac107 262.165 1.118 acyl-C6 (hexanoyl-l-carnitine) HMDB00756 260.186 1.848 acyl-C12:1 (O-dodecenoyl-carnitine) HMDB13326 342.269 2.738 acyl-C14 (O-tetradecanoyl-L-carnitine) HMDB05066 372.311 2.172 acyl-C14:1 (Tetradecenoyl Carnitine) HMDB13329 370.300 3.146 METABOLITES_END #END