#METABOLOMICS WORKBENCH imssr_20250602_203722 DATATRACK_ID:5982 STUDY_ID:ST003964 ANALYSIS_ID:AN006516 PROJECT_ID:PR002483 VERSION 1 CREATED_ON June 12, 2025, 10:24 am #PROJECT PR:PROJECT_TITLE Metabolomics of CAR-T cells and their products in leukemia patients PR:PROJECT_TYPE Untargeted metabolomics PR:PROJECT_SUMMARY Although most patients with relapsed/refractory B-cell acute lymphoblastic PR:PROJECT_SUMMARY leukemia (B-ALL) receiving CD19-targeted chimeric antigen receptor (CAR) T cell PR:PROJECT_SUMMARY therapy achieve remission, loss of CAR T cell functionality and subsequent PR:PROJECT_SUMMARY relapse remains an unmet therapeutic need. We applied an integrative approach to PR:PROJECT_SUMMARY study the immunometabolism of pre- and post-infusion CD19-CAR T cells of PR:PROJECT_SUMMARY patients with relapsed/refractory B-ALL. Pre-infusion CAR T cells of long-term PR:PROJECT_SUMMARY responders (LTR) had increased oxidative phosphorylation, fatty acid oxidation, PR:PROJECT_SUMMARY and pentose phosphate pathway activities, higher mitochondrial mass, tighter PR:PROJECT_SUMMARY cristae, and lower mTOR expression compared to products of short-term PR:PROJECT_SUMMARY responders. Post-infusion CAR T cells in bone marrow (BM) of LTR had high PR:PROJECT_SUMMARY immunometabolic plasticity and mTOR-pS6 expression supported by the BM PR:PROJECT_SUMMARY microenvironment. Transient inhibition of mTOR during manufacture induced PR:PROJECT_SUMMARY metabolic reprogramming and enhanced anti-tumor activity of CAR T cells. Our PR:PROJECT_SUMMARY findings provide insight into immunometabolic determinants of long-term response PR:PROJECT_SUMMARY and suggest a therapeutic strategy to improve long-term remission. PR:INSTITUTE Translational Genomics Research Institute PR:LAST_NAME Pirrotte PR:FIRST_NAME Patrick PR:ADDRESS 445 N 5TH STREET PR:EMAIL ims@tgen.org PR:PHONE 6023438454 PR:FUNDING_SOURCE NIH K12 grant no. 5K12CA001727–29, The Hyundai Hope on Wheels Young PR:FUNDING_SOURCE Investigator Award, NCI grant no.P30CA033572 PR:PROJECT_COMMENTS Study 2 of 4 PR:CONTRIBUTORS Lior Goldberg, Eric R. Haas, Jiaqi Wu, Bryan Garcia, Ryan Urak, Vibhuti Vyas, PR:CONTRIBUTORS Ruby Espinosa, Tamara Munoz, Shirley Bierkatz, Jamie R. Wagner, Jinny Paul, Mary PR:CONTRIBUTORS C. Clark, Dat Ngo, Ibrahim Aldoss, Stephen J. Forman & Xiuli Wang #STUDY ST:STUDY_TITLE Immunometabolism determinants of enhanced antileukemic activity upon rapamycin ST:STUDY_TITLE mediated mTOR inhibition in CAR-T cells of short-term responder leukemia ST:STUDY_TITLE patients receiving CD19 CAR T cell therapy ST:STUDY_TYPE Untargeted metabolomics ST:STUDY_SUMMARY To investigate role of mTOR activity on the efficacy of CAR-T cells of long-term ST:STUDY_SUMMARY responders, we used generated CAR-T cells from the leukaphresis products of ST:STUDY_SUMMARY short-term responder treated with or without mTOR inhibitor rapamycin to ST:STUDY_SUMMARY transiently inhibit mTOR activity. We performed metabolomics on the CAR T cells ST:STUDY_SUMMARY from short-term responders with or without rapamycin. We saw higher levels of ST:STUDY_SUMMARY glycerphospholipids, amino acids, sphioglipids and lower levels of metabolites ST:STUDY_SUMMARY of pentose phosphate pathway suggesting higher lipid metabolism and decreased ST:STUDY_SUMMARY non-oxidative pentose phosphate pathway metabolism upon rapamycin treatment ST:STUDY_SUMMARY which could provide insight in role of mTOR in metabolic activation and efficacy ST:STUDY_SUMMARY of CAR-T cell therapy. ST:INSTITUTE Translational Genomics Research Institute ST:LAST_NAME Pirrotte ST:FIRST_NAME Patrick ST:ADDRESS 445 N 5TH STREET, Phoenix, Arizona ST:EMAIL kpathak@tgen.org ST:PHONE 12137931730 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Male and female #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 strCT10x543WO strCT10x543WO_HILICneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01A_R_strCT10x543WOxHILICneg_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT10x543WO strCT10x543WO_HILICpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01A_R_strCT10x543WOxHILICpos_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT11x549W strCT11x549W_HILICneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01A_R_strCT11x549WxHILICneg_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT11x549W strCT11x549W_HILICpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01A_R_strCT11x549WxHILICpos_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT12x549WO strCT12x549WO_HILICneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01A_R_strCT12x549WOxHILICneg_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT12x549WO strCT12x549WO_HILICpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01A_R_strCT12x549WOxHILICpos_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT1x209W strCT1x209W_HILICneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01A_R_strCT1x209WxHILICneg_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT1x209W strCT1x209W_HILICpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01A_R_strCT1x209WxHILICpos_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT2x209WO strCT2x209WO_HILICneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01A_R_strCT2x209WOxHILICneg_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT2x209WO strCT2x209WO_HILICpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01A_R_strCT2x209WOxHILICpos_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT3x293W strCT3x293W_HILICneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01A_R_strCT3x293WxHILICneg_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT3x293W strCT3x293W_HILICpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01A_R_strCT3x293WxHILICpos_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT4x293WO strCT4x293WO_HILICneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01A_R_strCT4x293WOxHILICneg_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT4x293WO strCT4x293WO_HILICpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01A_R_strCT4x293WOxHILICpos_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT5x379W strCT5x379W_HILICneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01A_R_strCT5x379WxHILICneg_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT5x379W strCT5x379W_HILICpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01A_R_strCT5x379WxHILICpos_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT6x379WO strCT6x379WO_HILICneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01A_R_strCT6x379WOxHILICneg_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT6x379WO strCT6x379WO_HILICpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01A_R_strCT6x379WOxHILICpos_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT7x466W strCT7x466W_HILICneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01A_R_strCT7x466WxHILICneg_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT7x466W strCT7x466W_HILICpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01A_R_strCT7x466WxHILICpos_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT8x466WO strCT8x466WO_HILICneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01A_R_strCT8x466WOxHILICneg_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT8x466WO strCT8x466WO_HILICpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01A_R_strCT8x466WOxHILICpos_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT9x543W strCT9x543W_HILICneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01A_R_strCT9x543WxHILICneg_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT9x543W strCT9x543W_HILICpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01A_R_strCT9x543WxHILICpos_28JUN24_WAT_WAT_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT10x543WO strCT10x543WO_RPneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01B_R_strCT10x543WOxRPneg_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT10x543WO strCT10x543WO_RPpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01B_R_strCT10x543WOxRPpos_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT11x549W strCT11x549W_RPneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01B_R_strCT11x549WxRPneg_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT11x549W strCT11x549W_RPpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01B_R_strCT11x549WxRPpos_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT12x549WO strCT12x549WO_RPneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01B_R_strCT12x549WOxRPneg_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT12x549WO strCT12x549WO_RPpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01B_R_strCT12x549WOxRPpos_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT1x209W strCT1x209W_RPneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01B_R_strCT1x209WxRPneg_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT1x209W strCT1x209W_RPpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01B_R_strCT1x209WxRPpos_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT2x209WO strCT2x209WO_RPneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01B_R_strCT2x209WOxRPneg_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT2x209WO strCT2x209WO_RPpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01B_R_strCT2x209WOxRPpos_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT3x293W strCT3x293W_RPneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01B_R_strCT3x293WxRPneg_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT3x293W strCT3x293W_RPpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01B_R_strCT3x293WxRPpos_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT4x293WO strCT4x293WO_RPneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01B_R_strCT4x293WOxRPneg_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT4x293WO strCT4x293WO_RPpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01B_R_strCT4x293WOxRPpos_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT5x379W strCT5x379W_RPneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01B_R_strCT5x379WxRPneg_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT5x379W strCT5x379W_RPpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01B_R_strCT5x379WxRPpos_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT6x379WO strCT6x379WO_RPneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01B_R_strCT6x379WOxRPneg_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT6x379WO strCT6x379WO_RPpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01B_R_strCT6x379WOxRPpos_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT7x466W strCT7x466W_RPneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01B_R_strCT7x466WxRPneg_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT7x466W strCT7x466W_RPpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01B_R_strCT7x466WxRPpos_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT8x466WO strCT8x466WO_RPneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01B_R_strCT8x466WOxRPneg_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT8x466WO strCT8x466WO_RPpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Not treated control RAW_FILE_NAME(File name)=160_0003_01B_R_strCT8x466WOxRPpos_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT9x543W strCT9x543W_RPneg Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01B_R_strCT9x543WxRPneg_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS strCT9x543W strCT9x543W_RPpos Sample source:CAR-T cells | Outcome:Short term responders | Treatment:Rapamycin RAW_FILE_NAME(File name)=160_0003_01B_R_strCT9x543WxRPpos_02JUL24_CELL_HUM_LUM_0000_07AB_20MIN_KP_KP_01.raw #COLLECTION CO:COLLECTION_SUMMARY De-identified clinical samples were collected from a subset of adult patients CO:COLLECTION_SUMMARY with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) who CO:COLLECTION_SUMMARY received investigational CD19-specific CAR T cells in a phase 1/2 clinical trial CO:COLLECTION_SUMMARY (NCT02146924) at City of Hope. Patient or donor's Tcells were collected by CO:COLLECTION_SUMMARY leukapheresis. The leukapheresis Tcells were used for metabolomics profiling. CO:COLLECTION_SUMMARY For CAR T cells, PBMC were depleted of CD14+ monocytes and CD25+ Tregs, followed CO:COLLECTION_SUMMARY by positive selection of CD62L+ T cells. Tcm-derived CAR T cells were purified, CO:COLLECTION_SUMMARY transduced, formulated, and released. The CAR T cells treated with or without CO:COLLECTION_SUMMARY rapamycin were used for metabolomics profiling. After 28th day post CAR-T CO:COLLECTION_SUMMARY infusion bone marrow serum was collected from the patients for metabolomics CO:COLLECTION_SUMMARY profiling CO:SAMPLE_TYPE CAR-T cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Leukapheresis products from Short-term and long-term responders were treated TR:TREATMENT_SUMMARY with rapamycin or vehicle control. These products are used for CAR T cell TR:TREATMENT_SUMMARY productions. The CAR-T cells with or without rapamycin treatment were subjected TR:TREATMENT_SUMMARY to untargeted metabolomics profiling. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 1.5 million CAR-T cells with or without rapamycin treatment were used for SP:SAMPLEPREP_SUMMARY metabolite extraction. Metabolites were extracted using 0.4 mL of 0.4 mL of SP:SAMPLEPREP_SUMMARY solvent mixture (water: methanol: acetonitrile, 1:2.25:0.75, v/v/v) and 3 SP:SAMPLEPREP_SUMMARY freeze-thaw (30 seconds each) cycles. For bone marrow, 3-fold excess SP:SAMPLEPREP_SUMMARY acetonitrile: methanol (3:1) was added for protein removal. The resultant SP:SAMPLEPREP_SUMMARY metabolite extracts from CAR T cells were recovered by centrifuging at 13,226 xg SP:SAMPLEPREP_SUMMARY at 4°C for 15 min. For HILIC chromatography, the extract was directly used SP:SAMPLEPREP_SUMMARY LC-MS analysis. For reversed phase chromatography, the extract was dried under SP:SAMPLEPREP_SUMMARY vacuum centrifugation and the dried extract was reconstituted in 20% SP:SAMPLEPREP_SUMMARY Acetonitrile, 80% water and used used for LC-MS analysis. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY HILIC chromatography CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um) CH:SOLVENT_A 95% water/5% acetonitrile; 10mM ammonium acetate; 0.1% acetic acid CH:SOLVENT_B 95% acetonitrilie/5% water; 10mM ammonium acetate; 0.1% acetic acid CH:FLOW_GRADIENT 99% B for 1 min, 99–85% B for 3 min, 85–75% B for 3 min, 75–30% B CH:FLOW_GRADIENT for 3 min, 30% B for 1 min, 30-99%B for 1 min and 99%B for 4 min CH:FLOW_RATE 0.35 ml/min CH:COLUMN_TEMPERATURE 45 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap Fusion Lumos Tribrid MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Data dependent acquisition Xcalibur Compound Discoverer 3.2 MS:MS_RESULTS_FILE ST003964_AN006516_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END