#METABOLOMICS WORKBENCH ly259_20220212_185155 DATATRACK_ID:3080 STUDY_ID:ST002085 ANALYSIS_ID:AN003403 PROJECT_ID:PR001324 VERSION 1 CREATED_ON February 15, 2022, 5:44 am #PROJECT PR:PROJECT_TITLE Proline metabolism in T cells PR:PROJECT_SUMMARY T cell metabolites detection after PRODH2 overexpression. PR:INSTITUTE Yale University PR:LAST_NAME Ye PR:FIRST_NAME Lupeng PR:ADDRESS 850 West campus drive PR:EMAIL lupeng.ye@yale.edu PR:PHONE 2035436568 #STUDY ST:STUDY_TITLE A genome-scale gain-of-function CRISPR screen in CD8 T cells identifies proline ST:STUDY_TITLE metabolism as a means to enhance CAR-T therapy(Part 1) ST:STUDY_SUMMARY Chimeric antigen receptor (CAR)-T cell-based immunotherapy for cancer and ST:STUDY_SUMMARY immunological diseases has made great strides, but it still faces multiple ST:STUDY_SUMMARY hurdles. Finding the right molecular targets to engineer T cells toward a ST:STUDY_SUMMARY desired function has broad implications for the armamentarium of T cell-centered ST:STUDY_SUMMARY therapies. Here, we developed a dead-guide RNA (dgRNA)-based CRISPR activation ST:STUDY_SUMMARY screen in primary CD8+ T cells, and identified gain-of-function (GOF) targets ST:STUDY_SUMMARY for CAR-T engineering. Targeted knock-in or overexpression of a lead target, ST:STUDY_SUMMARY PRODH2, enhanced CAR-T-based killing and in vivo efficacy in multiple cancer ST:STUDY_SUMMARY models. Transcriptomics and metabolomics in CAR-T cells revealed that augmenting ST:STUDY_SUMMARY PRODH2 expression re-shaped broad and distinct gene expression and metabolic ST:STUDY_SUMMARY programs. Mitochondrial, metabolic and immunological analyses showed that PRODH2 ST:STUDY_SUMMARY engineering enhances the metabolic and immune functions of CAR-T cells against ST:STUDY_SUMMARY cancer. Together these findings provide a system for identification of GOF ST:STUDY_SUMMARY immune boosters, and demonstrate PRODH2 as a target to enhance CAR-T efficacy. ST:INSTITUTE Yale University ST:LAST_NAME Ye ST:FIRST_NAME Lupeng ST:ADDRESS 850 West campus drive ST:EMAIL lupeng.ye@yale.edu ST:PHONE 2035436568 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #FACTORS #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 - 20181221_hProdh2_OE_1_M62 Genotype:PRODH2_OE | Treatment:PRODH2 RAW_FILE_NAME=20181221_hProdh2_OE_1_M62.d SUBJECT_SAMPLE_FACTORS - 20181221_hProdh2_OE_2_M62 Genotype:PRODH2_OE | Treatment:PRODH2 RAW_FILE_NAME=20181221_hProdh2_OE_2_M62.d SUBJECT_SAMPLE_FACTORS - 20181221_hProdh2_OE_3_M62 Genotype:PRODH2_OE | Treatment:PRODH2 RAW_FILE_NAME=20181221_hProdh2_OE_3_M62.d SUBJECT_SAMPLE_FACTORS - 20181221_hProdh2_OE_4_M62 Genotype:PRODH2_OE | Treatment:PRODH2 RAW_FILE_NAME=20181221_hProdh2_OE_4_M62.d SUBJECT_SAMPLE_FACTORS - 20181221_hProdh2_OE_5_M62 Genotype:PRODH2_OE | Treatment:PRODH2 RAW_FILE_NAME=20181221_hProdh2_OE_5_M62.d SUBJECT_SAMPLE_FACTORS - 20181221_hProdh2_OE_Vector1_M62 Genotype:Vector_OE | Treatment:Control RAW_FILE_NAME=20181221_hProdh2_OE_Vector1_M62.d SUBJECT_SAMPLE_FACTORS - 20181221_hProdh2_OE_Vector2_M62 Genotype:Vector_OE | Treatment:Control RAW_FILE_NAME=20181221_hProdh2_OE_Vector2_M62.d SUBJECT_SAMPLE_FACTORS - 20181221_hProdh2_OE_Vector3_M62 Genotype:Vector_OE | Treatment:Control RAW_FILE_NAME=20181221_hProdh2_OE_Vector3_M62.d SUBJECT_SAMPLE_FACTORS - 20181221_hProdh2_OE_Vector4_M62 Genotype:Vector_OE | Treatment:Control RAW_FILE_NAME=20181221_hProdh2_OE_Vector4_M62.d SUBJECT_SAMPLE_FACTORS - 20181221_hProdh2_OE_Vector5_M62 Genotype:Vector_OE | Treatment:Control RAW_FILE_NAME=20181221_hProdh2_OE_Vector5_M62.d #COLLECTION CO:COLLECTION_SUMMARY cell were counted, washed twice with PBS then used for metabolite extraction. CO:SAMPLE_TYPE T-cells #TREATMENT TR:TREATMENT_SUMMARY T cells were transduced with lentivirus, then collected for metabolite TR:TREATMENT_SUMMARY extraction. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY After normalizing cell counts, 800 μL of 80 % (vol / vol) HPLC-grade methanol SP:SAMPLEPREP_SUMMARY (Sigma) (precooled to -80 oC on dry ice) was added to fresh cells in a 1.5-mL SP:SAMPLEPREP_SUMMARY microcentrifuge tube, then tubes were put on dry ice for 30 minutes. The tubes SP:SAMPLEPREP_SUMMARY were then incubated on ice for 20 minutes and centrifuged at 15,000 x g for 15 SP:SAMPLEPREP_SUMMARY min at 4 oC to pellet the cell debris. The metabolite-containing supernatant was SP:SAMPLEPREP_SUMMARY transferred to a new 1.5-mL microcentrifuge tube on dry ice. Metabolite SP:SAMPLEPREP_SUMMARY extraction was repeated with 400 μL of 80 % (vol / vol) HPLC-grade methanol. SP:SAMPLEPREP_SUMMARY The cell lysate / methanol mixtures were dried by Speedvac at room temperature. SP:SAMPLEPREP_SUMMARY The metabolites were dissolved again with 80 % (vol / vol) methanol, then SP:SAMPLEPREP_SUMMARY centrifuged at 18,000 x g for 10 min to remove any particulates, and the SP:SAMPLEPREP_SUMMARY metabolite mixtures were stored at -80 oC until LC-MS analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 6550 CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6550 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Two metabolomics strategies were adopted, i.e. untargeted metabolomics (aiming MS:MS_COMMENTS to unbiasedly detect all detectable metabolites) and targeted approaches (aiming MS:MS_COMMENTS to detect specifically defined metabolites, such as related metabolites in the MS:MS_COMMENTS proline metabolism and T cell metabolism). For untargeted metabolomics analysis, MS:MS_COMMENTS the optimized workflow consists of automated peak detection and integration, MS:MS_COMMENTS peak alignment, background noise subtraction, and multivariate data analysis. MS:MS_COMMENTS These steps were carried out for comprehensive metabolite phenotyping of the two MS:MS_COMMENTS groups using Agilent Mass Hunter Qualitative Analysis Software (Version MS:MS_COMMENTS B.07.0.0, build 7.0.7024.0) and Agilent Mass Profiler Professional (Version MS:MS_COMMENTS 14.5-Build 2772). The metabolites were first putatively identified based on MS:MS_COMMENTS accurate mass match (accurate mass ± 30 ppm error) and fragmentation pattern MS:MS_COMMENTS match. Putative structural annotation was carried out by searching the MS:MS_COMMENTS metabolite databases HMDB (http://www.hmdb.ca/) and METLIN (http:// MS:MS_COMMENTS metlin.scripps.edu) using the mass-to-charge ratio of the metabolic features. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ppm MS_METABOLITE_DATA_START Samples 20181221_hProdh2_OE_Vector1_M62 20181221_hProdh2_OE_Vector2_M62 20181221_hProdh2_OE_Vector3_M62 20181221_hProdh2_OE_Vector4_M62 20181221_hProdh2_OE_Vector5_M62 20181221_hProdh2_OE_1_M62 20181221_hProdh2_OE_2_M62 20181221_hProdh2_OE_3_M62 20181221_hProdh2_OE_4_M62 20181221_hProdh2_OE_5_M62 Factors Genotype:Vector_OE | Treatment:Control Genotype:Vector_OE | Treatment:Control Genotype:Vector_OE | Treatment:Control Genotype:Vector_OE | Treatment:Control Genotype:Vector_OE | Treatment:Control Genotype:PRODH2_OE | Treatment:PRODH2 Genotype:PRODH2_OE | Treatment:PRODH2 Genotype:PRODH2_OE | Treatment:PRODH2 Genotype:PRODH2_OE | Treatment:PRODH2 Genotype:PRODH2_OE | Treatment:PRODH2 Glutathione 921413 604123 487883 1620478 1019804 1470537 911867 1597407 762426 1431081 Sphinganine 32621 25332 30326 39073 31656 43878 22062 20403 35196 39059 Argininosuccinate 3149 1984 1603 3472 2733 1235 3082 2542 3363 3183 Homocystine 473 449 875 1629 1120 288 2058 1325 984 1394 Cystine 3314 490 1438 9123 3205 6809 7638 10820 10351 5970 Anserine 6567 1036 2801 3031 1164 1072 2752 1595 2533 792 Cystathionine 2440 1871 2619 2800 1226 1167 1907 2336 3702 1116 Tryptophan 14206 17648 19085 16338 11956 19105 16018 17618 16552 26498 Spermine 42471 36872 159882 221133 87789 265372 139276 76612 48855 43190 3-Phosphoserine 2051 722 2442 2808 646 1968 3001 878 6420 2976 Tyrosne 264182 42504 61903 73028 43442 75233 44268 47975 46778 49910 Citrulline 1853 2570 1890 2845 1435 2259 9008 7373 2752 3765 Arginine 1254486 888562 1063438 1210823 756655 824313 864002 1063356 782954 980753 Phenylalanine 339919 198992 187418 194564 160759 122000 143264 157312 149922 182668 4-Hydroxy-Glutamate 6300 3382 2946 6041 2656 951 1208 3492 4155 3755 Histidine 1771187 1079282 1450365 1706363 1125901 816556 964912 997551 922590 1012593 Guanine 20808 24335 13872 13608 10681 25574 38572 18967 24089 25096 Methionine 28378 14648 44762 55402 20079 9992 31879 34112 32449 17776 2-Hydroxy-Glutarate 43202 3879 25582 43366 6616 19140 20065 17383 27650 5763 Glutamate 355124 141338 253678 447163 509152 1135091 1417242 1676107 1776951 1031736 Lysine 1537 1637 2687 3865 2384 1739 4982 2612 1182 1913 Glutamine 2177009 887833 1772151 2489780 985467 817893 998139 1343620 1379413 701559 Oxo-glutarate 67864 21720 48780 72103 31016 24202 21136 34484 38906 18712 Spermidine 107887 111647 330073 548680 215443 206822 515459 327648 93700 273430 Malate 3476 5557 5757 5807 5391 2466 1588 3451 4448 2989 Aspartic acid 669462 550116 692076 423341 457646 262024 188968 206615 307343 248102 Oxaloacetate 51981 44469 72558 97556 69089 44026 45214 34558 28376 36482 Ornithine 74512 39949 77101 96890 37660 29229 18443 23510 21021 19829 Leucine 925260 654986 1314280 2172796 1108789 729055 698269 498362 465365 626195 4-Hydroxyproline 111449 156937 116312 138532 175782 47082 60066 68003 51383 65504 PHC 1401 2604 1719 2412 1132 3957 3094 3554 4512 3806 Oxoproline 1735415 1454747 983490 1352858 816916 445316 96070 214330 230874 136518 Taurine 285448 501681 55922 192604 575081 25812 52821 676736 145371 255438 Cysteine 20403 6595 1866 23348 6201 8145 5658 9692 17265 10735 Threonine 1129 2326 3357 2629 399 580 1550 3087 923 1124 Succinate 10719 12032 8978 3968 4207 14492 10502 14282 7773 11305 Valine 435192 414758 168393 170461 166457 117614 123922 582183 286282 388072 Fumarate 124135 75114 198665 291479 92950 371169 206622 146301 155752 103204 Proline 2823719 3638626 4286561 6440219 3087870 2543357 4455294 3259016 3462500 2358333 Choline 18755 16472 26950 25612 37119 48604 94374 40526 58028 79363 Lactate 1774 993 1183 749 762 2297 1858 1654 2148 1805 Pyruvate 3121 2563 1871 1473 2384 1167 858 1160 805 825 Glyine 2772 1545 1595 2009 1133 669 1552 1107 1336 994 Glyoxylate 1430 1022 1717 1959 2434 3193 1450 1596 1384 1564 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Glutathione Sphinganine Argininosuccinate Homocystine Cystine Anserine Cystathionine Tryptophan Spermine 3-Phosphoserine Tyrosne Citrulline Arginine Phenylalanine 4-Hydroxy-Glutamate Histidine Guanine Methionine 2-Hydroxy-Glutarate Glutamate Lysine Glutamine Oxo-glutarate Spermidine Malate Aspartic acid Oxaloacetate Ornithine Leucine 4-Hydroxyproline PHC Oxoproline Taurine Cysteine Threonine Succinate Valine Fumarate Proline Choline Lactate Pyruvate Glyine Glyoxylate METABOLITES_END #END