#METABOLOMICS WORKBENCH cendalfr_20240411_071306 DATATRACK_ID:4769 STUDY_ID:ST003166 ANALYSIS_ID:AN005194 PROJECT_ID:PR001970 VERSION 1 CREATED_ON April 11, 2024, 1:33 pm #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 Metabolomics of Murine WT or MCJ KO CD19-BBz CD8 CAR-T cells ST:STUDY_SUMMARY MCJ/DnaJC15 is an endogenous negative regulator of Complex I and mitochondrial ST:STUDY_SUMMARY respiration. The goal of these experiments are to characterize the metabolic ST:STUDY_SUMMARY profile of murine WT or MCJ KO CD8 CD19-BBz CAR-T cells after 3 expansions (6 ST:STUDY_SUMMARY days) with IL-2 (60IU/ml). 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 - WT CD19-BBz CART_1 factor:WT | Sample source:T-Cells RAW_FILE_NAME(Raw File)=1 SUBJECT_SAMPLE_FACTORS - WT CD19-BBz CART_2 factor:WT | Sample source:T-Cells RAW_FILE_NAME(Raw File)=2 SUBJECT_SAMPLE_FACTORS - WT CD19-BBz CART_3 factor:WT | Sample source:T-Cells RAW_FILE_NAME(Raw File)=3 SUBJECT_SAMPLE_FACTORS - WT CD19-BBz CART_4 factor:WT | Sample source:T-Cells RAW_FILE_NAME(Raw File)=4 SUBJECT_SAMPLE_FACTORS - MCJ KO CD19-BBz CART_1 factor:MCJ KO | Sample source:T-Cells RAW_FILE_NAME(Raw File)=5 SUBJECT_SAMPLE_FACTORS - MCJ KO CD19-BBz CART_2 factor:MCJ KO | Sample source:T-Cells RAW_FILE_NAME(Raw File)=6 SUBJECT_SAMPLE_FACTORS - MCJ KO CD19-BBz CART_3 factor:MCJ KO | Sample source:T-Cells RAW_FILE_NAME(Raw File)=7 SUBJECT_SAMPLE_FACTORS - MCJ KO CD19-BBz CART_4 factor:MCJ KO | Sample source:T-Cells RAW_FILE_NAME(Raw File)=8 #COLLECTION CO:COLLECTION_SUMMARY Mouse CD8 cells were isolated with negative selection as described above, CO:COLLECTION_SUMMARY activated with anti-CD3/anti-CD28 beads (Dynabeads™ Mouse T-Activator CD3/CD28 CO:COLLECTION_SUMMARY for T-Cell Expansion and Activation, Gibco, Cat#11453D) with addition of CO:COLLECTION_SUMMARY recombinant human IL-2 (rhIL-2) (60 IU/ml) and human recombinant IL-7 (10 CO:COLLECTION_SUMMARY ng/ml). On the second and third day of activation, the supernatant containing CO:COLLECTION_SUMMARY the CD19-BBz CAR/hEGFR retrovirus was spun on a plate coated with retronectin CO:COLLECTION_SUMMARY (Takara Bio Inc.) (2000xg for 2.5h at 32C). The activated cells were added to CO:COLLECTION_SUMMARY the viral-coated plate for transduction. The transduced CD8 CAR-T cells were CO:COLLECTION_SUMMARY removed from the beads on the fourth day and expanded with rhIL-2 (60 IU/ml) or CO:COLLECTION_SUMMARY rhIL-7 (10 ng/ml) and rhL-15 (100 ng/ml). After 2 days (1st expansion) cells CO:COLLECTION_SUMMARY were split 1/3 with fresh cytokine-containing medium. After 2 more days (2nd CO:COLLECTION_SUMMARY expansion), cells were split again with fresh cytokine-containing medium. Two CO:COLLECTION_SUMMARY days later (3rd expansion) cells were used for experiment or incubated in CO:COLLECTION_SUMMARY cytokine free-medium. For CAR+ cells enrichment, the CAR-T cells expanded with CO:COLLECTION_SUMMARY IL-2 for 3 expansions were incubated with an anti-hEGFR-PE (BioLegend, CO:COLLECTION_SUMMARY Cat#352904, RRID: AB_10896794) antibody, followed by incubation with anti-PE CO:COLLECTION_SUMMARY microbeads (Miltenyi, order#130-048-801), and purified using the Miltenyi LS CO:COLLECTION_SUMMARY columns as recommended by the manufacturer (Miltenyi). The enrichment resulted CO:COLLECTION_SUMMARY in a 98-100% CAR+ population, as verified by flow cytometry. 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 Negative 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 95% water/5% acetonitrile; 1 mM ammonium acetate CH:SOLVENT_B 95% acetonitrile/5% water; 1 mM ammonium acetate CH:FLOW_GRADIENT 0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min CH:FLOW_GRADIENT 100-0% B, 3-5 min hold at 0% 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 NEGATIVE 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 Area MS_METABOLITE_DATA_START Samples WT CD19-BBz CART_1 WT CD19-BBz CART_2 WT CD19-BBz CART_3 WT CD19-BBz CART_4 MCJ KO CD19-BBz CART_1 MCJ KO CD19-BBz CART_2 MCJ KO CD19-BBz CART_3 MCJ KO CD19-BBz CART_4 Factors factor:WT | Sample source:T-Cells factor:WT | Sample source:T-Cells factor:WT | Sample source:T-Cells factor:WT | Sample source:T-Cells factor:MCJ KO | Sample source:T-Cells factor:MCJ KO | Sample source:T-Cells factor:MCJ KO | Sample source:T-Cells factor:MCJ KO | Sample source:T-Cells ATP 41731.19 6428.767 24878.19 35170.29 79503.05 81745.34 138619.4 85962.03 CDP 53948 47685.77 46709.7 53225.76 83453.38 80334.26 79512.63 66829.29 dTMP 11528.27 13090.34 15471.74 12250.69 8103.299 10143.27 9107.273 12798.74 UTP 33895.8 14083.71 21179.4 29137.43 79893.7 80497.16 85839.19 48459.85 4-Pyridoxate 53052.21 36336.4 38370.96 40768.04 53107.3 51397.92 67147.98 73606.19 UDP-glucose 109945.5 102305 106772.5 117610.4 173604.3 171099.8 153633.5 137282.2 ADP-D-ribose 80313.99 39212.17 48370.59 37083.43 26234.21 54659.27 40937.18 57243.11 Phosphate 14296980 8234016 13455610 11907920 12452870 12267700 13097670 11994820 Diphosphate 1350856 888204.6 1270134 1227957 1829761 1288761 1300646 1350158 D-Glucose 34903.93 33176.79 52589.67 54858.92 37811.08 55359.25 37016.07 37469.9 D-Glucose 6/1-phosphate 85955.66 67637.87 52940.54 77000.13 111030.3 77257.45 78838.41 79792.44 D-Glyceraldehyde 3-phosphate/Glycerone phosphate 287686 343037.8 372692.3 339469.2 438925.6 401145.6 437910.4 360800.9 2/3-Phospho-D-glycerate 20790.25 13434.66 14234.36 19509.62 38532.4 33113.87 25507.95 28076.16 Pyruvate 57872.79 52969.32 45175.14 60090.41 53257.15 50325.11 74337.44 49558.11 Lactate 4848126 3885394 5318762 5787558 5375694 5242856 6198282 5424948 Maltose 21164.09 15210.54 15643.46 24864.53 10121.72 13867.65 27094.81 16564.82 Mannitol/Sorbitol 40654.9 41214.98 40424.16 39911.29 52945.3 50903.8 36894.93 35577.58 Citrate 618699.8 191719 289149.5 357374 404766.8 414949.8 642812.4 595414.9 2-Oxoglutarate 12281.25 15227.17 15536.72 18042.83 28747.53 23252.08 33593.17 32512.49 Succinate 138719.9 129564 173048.6 147926.4 174430.2 178328.8 205234.1 244900 Fumarate 155572.1 90909.04 124985.8 130289.6 129184.5 166935.8 179206.8 177238.2 Malate 328446.8 247421.1 218096.7 297673.4 325895.5 336860.8 453008.4 273032.2 2-Hydroxyglutarate/Citramalate 39109.5 40886.45 46718.08 44507.89 74731.09 79205.07 82418.81 77209.23 Sedoheptulose 1/7-phosphate 40664.46 38187.89 35360.77 34914.26 50929.48 34809.28 41409.61 46787.02 alpha-D-Ribose 1-phosphate 169594.8 125331.4 116209 120834.6 142507.4 122830.7 135970.8 164456.7 Dimethylglycine 183660.9 121348.4 160857.4 152375.3 145719 193498.6 185511.5 220374.2 N-Acetylneuraminate 122704.1 62105.49 62626.44 68726.91 81198.21 100110.1 107409.4 115418.2 N-Glycoloyl-neuraminate 249265.9 173564 179054.4 179983 232159.2 201731.4 225946.6 213605.1 UDP-N-acetyl-D-glucosamine 229337.2 207586.8 248297.6 229223 367094 355713.3 327184.5 301008.9 Phosphocreatine 12416.68 15688.38 12999.27 13079.56 20042.31 18910.34 17525.09 14660.42 Sphinganine 1-phosphate 7634.625 6593.951 11418.5 9275.2 6087.835 6923.96 8480.534 9538.961 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name CmpdID parent medRt ATP C00002 505.99 0.609 CDP C00112 402.01 0.632 dTMP C00364 321.05 0.929 UTP C00075 482.96 0.630 4-Pyridoxate C00847 182.04 0.716 UDP-glucose C00029 565.05 0.620 ADP-D-ribose C01882 558.06 0.646 Phosphate C00009 96.97 0.670 Diphosphate C00013 176.93 0.613 D-Glucose C00031 179.06 0.691 D-Glucose 6/1-phosphate C02965 259.02 0.637 D-Glyceraldehyde 3-phosphate/Glycerone phosphate C00118 168.99 1.818 2/3-Phospho-D-glycerate C00631 184.98 0.615 Pyruvate C00022 87.01 0.811 Lactate C01432 89.02 0.789 Maltose C00208 341.11 0.729 Mannitol/Sorbitol C00392 181.07 0.699 Citrate C00158 191.02 0.627 2-Oxoglutarate C00026 145.01 0.807 Succinate C00042 117.02 0.808 Fumarate C00122 108.24 0.628 Malate C00149 133.01 0.612 2-Hydroxyglutarate/Citramalate C02630 147.03 0.811 Sedoheptulose 1/7-phosphate C06222 289.03 0.636 alpha-D-Ribose 1-phosphate C00620 229.01 0.637 Dimethylglycine C01026 102.05 0.639 N-Acetylneuraminate C00270 308.10 0.646 N-Glycoloyl-neuraminate C03410 324.09 0.643 UDP-N-acetyl-D-glucosamine C00043 606.07 0.636 Phosphocreatine C02305 210.03 0.622 Sphinganine 1-phosphate C01120 380.26 3.706 METABOLITES_END #END