#METABOLOMICS WORKBENCH RCulpHill_20210318_142251 DATATRACK_ID:2536 STUDY_ID:ST001732 ANALYSIS_ID:AN002819 PROJECT_ID:000000 VERSION 1 CREATED_ON March 24, 2021, 3:04 pm #PROJECT PR:PROJECT_TITLE Mitochondrial ATP fuels ABC transporter-mediated drug efflux in cancer PR:PROJECT_TITLE chemoresistance PR:PROJECT_SUMMARY Chemotherapy remains the standard of care for most cancers worldwide, however PR:PROJECT_SUMMARY development of chemoresistance due to the presence of the drug-effluxing ABC PR:PROJECT_SUMMARY transporters remains a significant problem. The development of safe and PR:PROJECT_SUMMARY effective means to overcome chemoresistance is critical for achieving durable PR:PROJECT_SUMMARY remissions in many cancer patients. We have investigated the energetic demands PR:PROJECT_SUMMARY of ABC transporters in the context of the metabolic adaptations of PR:PROJECT_SUMMARY chemoresistant cancer cells. Here we show that ABC transporters use PR:PROJECT_SUMMARY mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer PR:PROJECT_SUMMARY cells. We further demonstrate that the loss of MCJ (DnaJC15), an endogenous PR:PROJECT_SUMMARY negative regulator of mitochondrial respiration, in chemoresistant cancer cells PR:PROJECT_SUMMARY boosts their ability to produce ATP from mitochondria and fuel ABC transporters. PR:PROJECT_SUMMARY We have developed novel MCJ mimetics that can attenuate mitochondrial PR:PROJECT_SUMMARY respiration and safely overcome chemoresistance in vitro and in vivo. PR:PROJECT_SUMMARY Administration of MCJ mimetics in combination with standard chemotherapeutic PR:PROJECT_SUMMARY drugs could therefore become an new strategy for treatment of multiple cancers. PR:INSTITUTE University of Colorado, Denver PR:DEPARTMENT Biochemistry and Molecular Genetics PR:LABORATORY Angelo D'Alessandro PR:LAST_NAME Culp-Hill PR:FIRST_NAME Rachel PR:ADDRESS 12801 E 17th Ave L18-9403D PR:EMAIL rachel.hill@cuanschutz.edu PR:PHONE 3037245798 #STUDY ST:STUDY_TITLE Mitochondrial ATP fuels ABC transporter-mediated drug efflux in cancer ST:STUDY_TITLE chemoresistance (part-III) ST:STUDY_SUMMARY Chemotherapy remains the standard of care for most cancers worldwide, however ST:STUDY_SUMMARY development of chemoresistance due to the presence of the drug-effluxing ABC ST:STUDY_SUMMARY transporters remains a significant problem. The development of safe and ST:STUDY_SUMMARY effective means to overcome chemoresistance is critical for achieving durable ST:STUDY_SUMMARY remissions in many cancer patients. We have investigated the energetic demands ST:STUDY_SUMMARY of ABC transporters in the context of the metabolic adaptations of ST:STUDY_SUMMARY chemoresistant cancer cells. Here we show that ABC transporters use ST:STUDY_SUMMARY mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer ST:STUDY_SUMMARY cells. We further demonstrate that the loss of MCJ (DnaJC15), an endogenous ST:STUDY_SUMMARY negative regulator of mitochondrial respiration, in chemoresistant cancer cells ST:STUDY_SUMMARY boosts their ability to produce ATP from mitochondria and fuel ABC transporters. ST:STUDY_SUMMARY We have developed novel MCJ mimetics that can attenuate mitochondrial ST:STUDY_SUMMARY respiration and safely overcome chemoresistance in vitro and in vivo. ST:STUDY_SUMMARY Administration of MCJ mimetics in combination with standard chemotherapeutic ST:STUDY_SUMMARY drugs could therefore become an new strategy for treatment of multiple cancers. ST:INSTITUTE University of Colorado Anschutz Medical Campus ST:DEPARTMENT Biochemistry and Molecular Genetics ST:LABORATORY Angelo D'Alessandro ST:LAST_NAME Culp-Hill ST:FIRST_NAME Rachel ST:ADDRESS 12801 E 17th Ave L18-9403D ST:EMAIL rachel.hill@cuanschutz.edu ST:PHONE 3037245798 #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 - crtl1 Factor:Control RAW_FILE_NAME=crtl1 SUBJECT_SAMPLE_FACTORS - crtl2 Factor:Control RAW_FILE_NAME=crtl2 SUBJECT_SAMPLE_FACTORS - crtl3 Factor:Control RAW_FILE_NAME=crtl3 SUBJECT_SAMPLE_FACTORS - crtl4 Factor:Control RAW_FILE_NAME=crtl4 SUBJECT_SAMPLE_FACTORS - treat1 Factor:Treatment RAW_FILE_NAME=treat1 SUBJECT_SAMPLE_FACTORS - treat2 Factor:Treatment RAW_FILE_NAME=treat2 SUBJECT_SAMPLE_FACTORS - treat3 Factor:Treatment RAW_FILE_NAME=treat3 SUBJECT_SAMPLE_FACTORS - treat4 Factor:Treatment RAW_FILE_NAME=treat4 #COLLECTION CO:COLLECTION_SUMMARY NCI/ADR-RES cells were verified to be of ovarian origin by genotyping. All CO:COLLECTION_SUMMARY cancer cell lines were maintained in RPMI 1640 (Sigma R8758) containing glucose CO:COLLECTION_SUMMARY (2 mg/ml) and glutamine (0.6 mg/ml) but no pyruvate and was supplemented with 5% CO:COLLECTION_SUMMARY Fetal calf serum. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY Cells were cultured under normal conditions, detached using trypsin-EDTA (0.05 TR:TREATMENT_SUMMARY %), counted, normalized to 0.5 x 106 in each sample, and then cell pellets were TR:TREATMENT_SUMMARY snap frozen in liquid nitrogen prior to analysis. To determine the effect of TR:TREATMENT_SUMMARY N-MCJ mimetic treatment, equal numbers of NCI/ADR-RES cells were plated and TR:TREATMENT_SUMMARY allowed grow for 2 d followed by the addition of vehicle or MITOx30 (25 μM) for TR:TREATMENT_SUMMARY 12 h. Cells were then collected and processed as above. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolomics and flux analyses were performed as previously reported 31,61. SP:SAMPLEPREP_SUMMARY Briefly, 2 x 106 cells and 20 μl of cell media were extracted in 1 mL and 980 SP:SAMPLEPREP_SUMMARY μL of cold lysis and extraction buffer (methanol : acetonitrile : water, SP:SAMPLEPREP_SUMMARY 5:3:2), respectively. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Positive Mode 10 μL of water and methanol soluble fractions were run through a CH:CHROMATOGRAPHY_SUMMARY Kinetex C18 1.7 μm, 100 x 2.1 mm (Phenomenex) reversed phase column (Positive CH:CHROMATOGRAPHY_SUMMARY ion mode - phase A: water, 0.1 % formic acid; B: acetonitrile, 0.1 % formic CH:CHROMATOGRAPHY_SUMMARY acid) via an ultra-high performance chromatographic system (UHPLC - Vanquish, CH:CHROMATOGRAPHY_SUMMARY Thermo Fisher). UHPLC was coupled in line with a high-resolution quadrupole CH:CHROMATOGRAPHY_SUMMARY Orbitrap instrument run in positive polarity mode (QExactive, Thermo Fisher) at CH:CHROMATOGRAPHY_SUMMARY 70,000 resolution (at 200 m/z). Gradients and other technical parameters and the CH:CHROMATOGRAPHY_SUMMARY variants employed herein have been extensively described. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um) #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 UHPLC was coupled in line with a high-resolution quadrupole Orbitrap instrument MS:MS_COMMENTS run in positive polarity mode (QExactive, Thermo Fisher) at 70,000 resolution MS:MS_COMMENTS (at 200 m/z). Gradients and other technical parameters and the variants employed MS:MS_COMMENTS herein have been extensively described. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Relative Abundance MS_METABOLITE_DATA_START Samples crtl1 crtl2 crtl3 crtl4 treat1 treat2 treat3 treat4 Factors Factor:Control Factor:Control Factor:Control Factor:Control Factor:Treatment Factor:Treatment Factor:Treatment Factor:Treatment L-alanine 2383551 2537523 2554244 2885529 2896081 2909667 2898561 3024482 L-arginine 1468725 1275449 1221978 1392397 1747953 1697368 1809966 1856333 L-asparagine 209155 247497 246260 289495 252155 286508 231209 277536 L-aspartate 6965506 9232811 9383328 10993120 7835236 9367476 8177130 9065245 L-cysteine 330528 360811 394219 459652 645621 691689 658243 741879 L-glutamate 38161030 46857410 49899760 58786100 48523810 55501110 55269070 54282090 L-glutamine 117165 162087 175352 173664 220321 281731 248505 244172 glycine 953591 1095557 1175349 1272263 1368542 1654741 1507054 1526638 L-histidine 387317 431680 463205 422331 417180 508083 447129 440668 L-leucine 7162086 6769176 8023406 8809751 9402797 10529820 10640100 10749730 L-lysine 625806 643796 774067 932923 984835 919215 1018774 1185621 L-methionine 799387 1011965 1107585 1276611 1277670 1281224 1253040 1369610 L-phenylalanine 1553348 1475581 1494889 1705198 1919981 1863619 1868085 1941728 L-proline 15783660 16346490 16687700 19751050 16835360 15601570 15854970 19041930 L-serine 269879 413491 420908 461715 436690 503788 432007 461704 L-threonine 451693 384219 431770 542410 563624 677845 585713 546744 L-tryptophan 319218 390203 399417 431229 413103 515881 423263 523283 L-tyrosine 1473286 1783856 1975523 2116760 2066136 2342529 2113011 2379783 L-valine 7088030 6576432 6902530 7474938 8298390 7069502 8067876 8698493 L-cystine 176512 148422 146930 163715 194842 204608 193723 157151 AMP 3505766 4975971 4201656 3641990 11085510 7423978 8765207 10743340 dAMP 403775 447102 507398 398040 452321 429352 472989 485008 Guanine 69055 55277 63587 47761 132196 154057 134121 127368 Cytosine 81385 69398 91073 112872 87256 95877 93169 84282 Uracil 580340 381653 488480 530526 450329 485775 424343 438177 IMP 208851 184568 210640 179405 287937 303763 291219 298786 Xanthine 270196 278136 316956 425336 323860 226363 312229 333614 Allantoate 68798 63577 34954 74189 81879 73056 91846 92144 5-6-Dihydrothymine 1892138 1968967 2235196 2437387 1421284 1383298 1538757 1551436 Nicotinamide 5257542 5741416 6045452 6296714 7729276 7441614 7211492 8044942 NAD+ 2380196 2686330 3167592 3269284 2511281 2643190 2551854 2492171 Phosphate 1562450 1485664 1551390 1978047 2862806 2710518 3200722 3503811 Glutathione 52950870 77143020 72497910 82713370 79803620 75436730 81993340 82065630 Glutathione disulfide 43156 52281 52531 50689 41325 34268 44211 41483 Cys-Gly 159686 194800 189574 196321 208184 198609 217791 223485 gamma-L-Glutamyl-D-alanine 262938 292939 274327 349841 309847 300458 280965 345005 gamma-Glutamyl-gamma-aminobutyrate 52448 48493 43414 65152 77170 37341 26948 35715 (5-L-Glutamyl)-L-glutamine 15042 17131 23180 19343 17928 15633 12354 18445 Dimethylglycine 927796 1033687 972910 1117580 747254 763185 580830 730350 Phosphoserine 324000 526424 407776 466360 315888 347801 400804 356343 S-Adenosyl-L-methionine 402759 306499 403850 403689 326505 382882 375038 424630 L-Citrulline 572057 723371 833282 935221 941863 1120405 942830 1027849 Putrescine 3391246 2271276 1998148 2472062 2809821 2312378 2680531 3073016 Spermidine 3755185 2514234 1455976 2869817 1529952 727417 1944664 1818782 Spermine 2795492 2885859 1515638 2397127 1036035 152409 1691276 1097160 alpha-D-Glucosamine 1-phosphate 44513 68904 52124 66721 65856 48221 43869 71822 Creatine 326881300 343072900 341989400 388269400 303123600 280706100 304081800 330623800 Creatinine 827207 876096 936784 1135963 967764 932235 906194 1010572 Pantothenol 158360 132778 119822 127426 151479 150662 140332 148657 Taurine 1968923 1907741 2287479 2517612 968172 1154962 979858 962107 Hypotaurine 120286 201507 185085 195895 100283 129886 96721 98478 L-Methionine S-oxide 335996 326675 332774 333533 398766 389559 356076 441314 3-Methyleneoxindole 75561 72077 78424 71899 92924 81701 86179 89332 g-Oxalo-crotonate 7814022 6496004 7385372 8392155 8031650 7583850 7806650 7782324 L-Adrenaline 255019 146785 137800 67253 261808 156741 255756 125868 Inositol 1-2-3-5-6-pentakisphosphate 362834 223093 153354 132842 136483 128640 121718 87244 N-Methylethanolamine phosphate 418144 441574 529385 442690 357646 424238 383353 425205 Acetylcholine 3594262 3112679 3216796 2994502 2429072 2311890 2375953 2593385 CDP-choline 147287 150450 151810 112101 163689 198179 185288 129602 Choline 28999270 24126260 26488220 32875530 28633480 26937320 28718640 29237070 L-Carnitine 9508419 8531369 7956552 9209931 8712171 8837314 8904646 7533728 Pentobarbital 69383 84350 73608 32332 87801 61567 64443 60065 Triacanthine 2592983 2382512 2102974 2042786 1958517 1592258 1891274 1882150 4-Aminobenzoate 133181 106427 112501 140677 140132 140683 149340 158439 Leucocyanidin 42926 44516 50124 55683 40173 40306 60560 48538 Methylenediurea 3555614 3794054 3658066 4236542 2429324 3060469 3291498 3569611 beta-Butoxyethyl nicotinate 1284929 1255401 997819 941966 1537445 1041622 1617685 1432241 cis-p-Coumarate 245631 248685 216961 313710 291271 325217 299543 326683 Shikimate 3-phosphate 39030 65679 65496 54019 69402 81509 81975 95282 Poly-gamma-D-glutamate 6710466 8103276 8772196 10205820 8599536 9796837 10160310 9773791 D-Ribose 5-diphosphate 15759 24372 23251 28763 19315 12881 19524 26692 Thioredoxin disulfide 96882 177652 144046 154665 65445 75239 43569 75359 N-Acyl-D-glutamate 19287 18297 24608 29273 21955 14732 16215 22637 N-Acyl-D-aspartate 65751 72483 75768 106684 74835 55183 86657 101821 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name CmpdID parent medRt L-alanine C00041 90.0557 1.17 L-arginine C00062 175.1191 1.12 L-asparagine C00152 133.0610 1.17 L-aspartate C00049 134.0449 1.17 L-cysteine C00097 122.0273 1.19 L-glutamate C00025 148.0604 1.16 L-glutamine C00064 147.0763 1.19 glycine C00037 76.0402 1.15 L-histidine C00135 156.0768 1.10 L-leucine C00123 132.1020 1.21 L-lysine C00047 147.1129 1.08 L-methionine C00073 150.0583 1.21 L-phenylalanine C00079 166.0863 1.31 L-proline C00148 116.0710 1.20 L-serine C00065 106.0503 1.17 L-threonine C00188 120.0658 1.18 L-tryptophan C00078 205.0973 1.30 L-tyrosine C00082 182.0807 1.19 L-valine C00183 118.0866 1.21 L-cystine C00491 241.0294 1.16 AMP C00020 348.0702 1.29 dAMP C00360 332.0776 1.20 Guanine C00242 152.0567 1.49 Cytosine C00380 112.0510 1.20 Uracil C00106 113.0350 1.22 IMP C00130 349.0540 1.48 Xanthine C00385 153.0394 1.07 Allantoate C00499 177.0610 1.10 5-6-Dihydrothymine C00906 129.0660 1.19 Nicotinamide C00153 123.0555 1.21 NAD+ C00003 664.1160 1.19 Phosphate C00009 98.9848 1.35 Glutathione C00051 308.0908 1.21 Glutathione disulfide C00127 613.1584 1.51 Cys-Gly C01419 179.0486 1.51 gamma-L-Glutamyl-D-alanine C03738 219.0976 1.29 gamma-Glutamyl-gamma-aminobutyrate C15767 233.1138 1.20 (5-L-Glutamyl)-L-glutamine C05283 276.1187 1.51 Dimethylglycine C01026 104.0711 1.18 Phosphoserine C02532 186.0166 1.19 S-Adenosyl-L-methionine C00019 399.1443 1.14 L-Citrulline C00327 176.1030 1.19 Putrescine C00134 89.1080 1.08 Spermidine C00315 146.1651 1.09 Spermine C00750 203.2231 1.09 alpha-D-Glucosamine 1-phosphate C06156 260.0532 1.25 Creatine C00300 132.0768 1.19 Creatinine C00791 114.0666 1.18 Pantothenol C00864 220.1179 1.31 Taurine C00245 126.0221 1.16 Hypotaurine C00519 110.0275 1.16 L-Methionine S-oxide C02989 166.0531 1.31 3-Methyleneoxindole C02796 146.0601 1.32 g-Oxalo-crotonate C03453 159.0278 1.20 L-Adrenaline C00788 184.0944 1.18 Inositol 1-2-3-5-6-pentakisphosphate C04579 580.9039 1.12 N-Methylethanolamine phosphate C01210 156.0422 1.12 Acetylcholine C01996 146.1176 1.22 CDP-choline C00307 489.1142 1.17 Choline C00114 104.1075 1.16 L-Carnitine C00318 162.1125 1.17 Pentobarbital C07422 227.1389 1.23 Triacanthine C08435 204.1231 1.21 4-Aminobenzoate C00568 138.0549 1.25 Leucocyanidin C05906 307.0830 1.51 Methylenediurea C06381 133.0738 1.20 beta-Butoxyethyl nicotinate C13138 224.1281 1.21 cis-p-Coumarate C06738 165.0546 1.21 Shikimate 3-phosphate C03175 255.0285 1.53 Poly-gamma-D-glutamate C05723 130.0500 1.18 D-Ribose 5-diphosphate C01095 310.9944 1.30 Thioredoxin disulfide C00343 317.0389 1.17 N-Acyl-D-glutamate C06379 175.0452 1.07 N-Acyl-D-aspartate C06380 161.0297 1.07 METABOLITES_END #END