#METABOLOMICS WORKBENCH ly259_20220214_085334 DATATRACK_ID:3084 STUDY_ID:ST002084 ANALYSIS_ID:AN003401 PROJECT_ID:PR001324 VERSION 1 CREATED_ON February 14, 2022, 6:27 pm #PROJECT PR:PROJECT_TITLE Proline metabolism in T cells PR:PROJECT_SUMMARY T cell metabolites detection after PRODH2-CD22-CAR knock-in. 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 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 - 20190125 CarT_Prodh2_KI_Vector1 M105 Genotype:PRODH2(Stop)-CAR | Treatment:PRODH2(Stop) RAW_FILE_NAME=20190125 CarT_Prodh2_KI_Vector1 M105.d SUBJECT_SAMPLE_FACTORS - 20190125 CarT_Prodh2_KI_Vector2 M105 Genotype:PRODH2(Stop)-CAR | Treatment:PRODH2(Stop) RAW_FILE_NAME=20190125 CarT_Prodh2_KI_Vector2 M105.d SUBJECT_SAMPLE_FACTORS - 20190125 CarT_Prodh2_KI_Vector3 M105 Genotype:PRODH2(Stop)-CAR | Treatment:PRODH2(Stop) RAW_FILE_NAME=20190125 CarT_Prodh2_KI_Vector3 M105.d SUBJECT_SAMPLE_FACTORS - 20190125 CarT_Prodh2_KI_Vector4 M105 Genotype:PRODH2(Stop)-CAR | Treatment:PRODH2(Stop) RAW_FILE_NAME=20190125 CarT_Prodh2_KI_Vector4 M105.d SUBJECT_SAMPLE_FACTORS - 20190125 CarT_Prodh2_KI_Vector5 M105 Genotype:PRODH2(Stop)-CAR | Treatment:PRODH2(Stop) RAW_FILE_NAME=20190125 CarT_Prodh2_KI_Vector5 M105.d SUBJECT_SAMPLE_FACTORS - 20190125 CarT_Prodh2_KI_1 M105 Genotype:PRODH2-CAR | Treatment:PRODH2 RAW_FILE_NAME=20190125 CarT_Prodh2_KI_1 M105.d SUBJECT_SAMPLE_FACTORS - 20190125 CarT_Prodh2_KI_2 M106 Genotype:PRODH2-CAR | Treatment:PRODH2 RAW_FILE_NAME=20190125 CarT_Prodh2_KI_2 M105.d SUBJECT_SAMPLE_FACTORS - 20190125 CarT_Prodh2_KI_3 M107 Genotype:PRODH2-CAR | Treatment:PRODH2 RAW_FILE_NAME=20190125 CarT_Prodh2_KI_3 M105.d SUBJECT_SAMPLE_FACTORS - 20190125 CarT_Prodh2_KI_4 M108 Genotype:PRODH2-CAR | Treatment:PRODH2 RAW_FILE_NAME=20190125 CarT_Prodh2_KI_4 M105.d SUBJECT_SAMPLE_FACTORS - 20190125 CarT_Prodh2_KI_5 M109 Genotype:PRODH2-CAR | Treatment:PRODH2 RAW_FILE_NAME=20190125 CarT_Prodh2_KI_5 M105.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 electroporated with Cas9-RNP, then transduced with CD22-CAR;PRODH2 TR:TREATMENT_SUMMARY or PRODH2(Stop) AAV, CAR-T cells were collected for metabolite 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 6490 CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6490 QQQ MS:INSTRUMENT_TYPE Triple quadrupole 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 20190125 CarT_Prodh2_KI_1 M105 20190125 CarT_Prodh2_KI_2 M106 20190125 CarT_Prodh2_KI_3 M107 20190125 CarT_Prodh2_KI_4 M108 20190125 CarT_Prodh2_KI_5 M109 20190125 CarT_Prodh2_KI_Vector1 M105 20190125 CarT_Prodh2_KI_Vector2 M105 20190125 CarT_Prodh2_KI_Vector3 M105 20190125 CarT_Prodh2_KI_Vector4 M105 20190125 CarT_Prodh2_KI_Vector5 M105 Factors Genotype:PRODH2-CAR | Treatment:PRODH2 Genotype:PRODH2-CAR | Treatment:PRODH2 Genotype:PRODH2-CAR | Treatment:PRODH2 Genotype:PRODH2-CAR | Treatment:PRODH2 Genotype:PRODH2-CAR | Treatment:PRODH2 Genotype:PRODH2(Stop)-CAR | Treatment:PRODH2(Stop) Genotype:PRODH2(Stop)-CAR | Treatment:PRODH2(Stop) Genotype:PRODH2(Stop)-CAR | Treatment:PRODH2(Stop) Genotype:PRODH2(Stop)-CAR | Treatment:PRODH2(Stop) Genotype:PRODH2(Stop)-CAR | Treatment:PRODH2(Stop) 4-Hydroxyproline 99747 60908 68952 52355 65804 139154 171550 172539 175270 110744 Oxoproline 41089 35687 36483 32225 42952 44431 42964 45227 28773 31479 Arginine 93427 109275 93223 66985 108574 156709 148219 119704 188062 128518 Citrulline 6427 2408 2529 3669 5456 2734 5196 7104 5501 7628 Glutamine 92860 83693 115716 128067 129830 160878 127948 127592 124887 144343 PHC 13752 20673 14267 16313 14229 8981 9030 6611 7152 7945 Proline 106041 98838 99133 103108 115949 199656 106756 153108 145377 125636 Spermine 43704 44973 58035 62606 93644 66104 82068 73033 73818 101843 Alanine 97554 82285 79439 48237 71257 112927 91452 77415 79204 85497 Argininosuccinate 825 570 595 693 774 852 727 1288 1045 1047 Asparagine 4274 3285 4039 2278 4457 6002 4522 5441 4972 6143 Cystathionine 5112 3146 4166 1692 3993 6595 2674 1981 1829 5099 Cysteine 6356 9843 10023 6033 12392 14578 12829 14408 12946 15042 Glutamate 34351 8278 49529 32363 15144 42341 13936 26117 34291 37529 Glutathione 11697 13666 16355 19829 14757 19901 13191 13409 13503 25492 Glyine 900 170 159 782 901 755 248 818 1107 709 Histidine 47429 42907 56008 71447 43208 130693 42545 60815 46591 75250 Isoleucine 9628 13182 15335 10697 11705 15807 10881 10964 21891 12417 Methionine 4703 5626 10227 11286 8314 8041 9109 7535 7914 11756 Phenylalanine 95595 96438 38118 37972 40203 69225 43412 41910 48612 52481 Threonine 682 854 371 231 375 428 525 325 298 387 Tryptophan 6042 4955 4866 4029 3943 7084 6150 5602 5851 6209 Tyrosine 7963 6665 6234 5583 10983 9551 6385 8381 7975 6309 Valine 135368 111816 281141 98582 88940 391851 338146 110874 94992 127660 2-Keto-Glutaramate 18686 13024 46146 52314 53902 43223 60647 70400 80702 82083 3-Phosphoserine 461 619 1217 672 717 659 449 305 666 1316 4-Aminobutyraldehyde 22756 22075 20435 19367 20973 21213 21148 20441 19554 20858 4-Hydroxy-Glutamate 1529 1639 2546 602 2183 735 2691 516 2055 679 6-Phosphogluconic acid 6906 17634 6599 8055 22927 9723 26437 27706 9983 17196 Aminoadipic acid 30235 12537 30865 22564 30072 51131 26583 28019 32547 25950 Inosine 3971 2313 2589 2029 2245 4442 1713 1007 1685 2041 Me-His 6213 6825 4661 5333 9098 8352 6041 7768 7977 6236 N-Acetylglutamic acid 10643 8767 8796 6101 7965 19889 12381 12027 13917 8688 Taurine 25275 15936 8448 7384 15112 35736 19505 18750 19668 22001 3-Hydroxybutyric acid 408 479 634 1436 981 1025 871 911 555 635 Acetoacetic acid 5742 4221 6363 6643 5310 7484 7302 7168 9535 7441 Fructose bisphosphate 1008 721 1004 422 770 517 1126 761 653 1786 Glucose 12841 8593 11537 10544 8784 10418 8776 16370 16259 17542 Glucose-phosphate 9406 9043 6017 9186 8852 18070 15886 14383 14509 15811 Glyceraldehyde-phosphate 18820 11370 17804 8882 8546 12053 8207 8091 14580 7696 Glyoxylate 6818 6833 8681 4712 5023 11981 7294 9409 8006 9646 Lactate 2544 2803 3245 2649 1768 3823 2440 2904 1423 2096 Phosphoenolpyruvate 8719 10241 10901 12055 11267 13556 10240 8202 8901 13880 Pyruvate 12319 15472 12579 16489 14993 18085 14570 16601 17453 17726 Dihydroxyacetone phosphate 5637 3935 1988 5263 4909 3344 3843 1444 5867 5202 Hydroxypyruvic acid 19532 13264 19757 10034 8010 9061 8339 14447 11487 8457 PRPP 3215 2863 3367 9491 4513 1296 965 1943 3604 1791 UDP-glucose 3357 4968 3238 5256 6100 1261 1159 1310 1025 1821 Xylose 1073 898 376 737 456 726 466 638 537 493 Xylulose-phosphate 166845 103481 165843 179350 104250 117565 84509 83953 70992 137146 Adenylosuccinic acid 377 594 525 303 370 295 216 308 299 320 Malate 2167 2484 1932 2411 1600 1103 3045 2055 1998 2697 Citrate-Isocitrate 8400 6892 14790 1806 6074 12679 7642 4948 2027 9861 Fumarate 4086 5828 4531 5228 6956 9212 7716 6271 7054 5747 Oxaloacetate 30698 35041 24834 18329 15707 23757 11600 11632 21934 16740 Succinate 3950 2988 4573 3971 4507 4329 4369 4521 5508 5811 Carnitine 3787 1273 2539 3226 1877 4859 3669 5549 5759 7984 Carnosine 5038 2967 3784 1941 2982 5449 2015 2888 2650 3755 Creatine 16984 13750 13720 11023 10581 16904 10323 18147 13907 12353 Creatinine 139 300 126 178 420 149 109 120 148 133 Guanine 29610 18799 21294 14949 17093 23562 8738 22431 18622 19655 NAD+ 3785 6259 4249 4919 3470 272 5451 6317 2558 2393 NADH 477 1114 392 2058 899 599 580 1131 1346 644 NADP+ 18388 32224 27033 27929 27321 166 31546 33112 18488 19706 NADPH 201 298 447 119 189 191 120 123 196 188 ADP 54683 47319 50504 70409 43480 27176 47055 49249 45138 51578 AMP 14643 8129 16878 17474 7511 10129 15122 13651 16165 11581 ATP 2500 2236 2416 2834 3111 2042 2174 2269 2153 2453 cAMP 509 289 421 200 203 274 193 82 126 214 GDP 11331 12321 12790 12111 10364 19010 11427 10141 12062 11425 GMP 10807 20708 20929 20987 19760 20475 24628 22921 13599 19492 GTP 626 781 1788 1152 775 544 504 667 1197 319 Acetoacetyl-CoA 599 1054 1644 1693 911 145 1173 1679 538 137 HMG-CoA 4495 1138 2722 1158 1878 2259 1468 835 246 4856 Malonyl-CoA 334 581 734 585 447 375 592 680 506 347 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name 4-Hydroxyproline Oxoproline Arginine Citrulline Glutamine PHC Proline Spermine Alanine Argininosuccinate Asparagine Cystathionine Cysteine Glutamate Glutathione Glyine Histidine Isoleucine Methionine Phenylalanine Threonine Tryptophan Tyrosine Valine 2-Keto-Glutaramate 3-Phosphoserine 4-Aminobutyraldehyde 4-Hydroxy-Glutamate 6-Phosphogluconic acid Aminoadipic acid Inosine Me-His N-Acetylglutamic acid Taurine 3-Hydroxybutyric acid Acetoacetic acid Fructose bisphosphate Glucose Glucose-phosphate Glyceraldehyde-phosphate Glyoxylate Lactate Phosphoenolpyruvate Pyruvate Dihydroxyacetone phosphate Hydroxypyruvic acid PRPP UDP-glucose Xylose Xylulose-phosphate Adenylosuccinic acid Malate Citrate-Isocitrate Fumarate Oxaloacetate Succinate Carnitine Carnosine Creatine Creatinine Guanine NAD+ NADH NADP+ NADPH ADP AMP ATP cAMP GDP GMP GTP Acetoacetyl-CoA HMG-CoA Malonyl-CoA METABOLITES_END #END