#METABOLOMICS WORKBENCH RCulpHill_20210318_142251 DATATRACK_ID:2536 STUDY_ID:ST001732 ANALYSIS_ID:AN002820 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 Negative 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 (Negative CH:CHROMATOGRAPHY_SUMMARY ion mode – phase A: 1 mM NH4Ac 95:5 water : acetonitrile; phase B: 1 mM NH4Ac CH:CHROMATOGRAPHY_SUMMARY 95:5 acetonitrile : water) via an ultra-high performance chromatographic system CH:CHROMATOGRAPHY_SUMMARY (UHPLC - Vanquish, Thermo Fisher). UHPLC was coupled in line with a CH:CHROMATOGRAPHY_SUMMARY high-resolution quadrupole Orbitrap instrument run in negative polarity mode CH:CHROMATOGRAPHY_SUMMARY (QExactive, Thermo Fisher) at 70,000 resolution (at 200 m/z). Gradients and CH:CHROMATOGRAPHY_SUMMARY other technical parameters and the variants employed herein have been CH:CHROMATOGRAPHY_SUMMARY 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 NEGATIVE MS:MS_COMMENTS UHPLC was coupled in line with a high-resolution quadrupole Orbitrap instrument MS:MS_COMMENTS run in negative 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 ATP 562155 2276707 3575964 4600647 2610371 1856621 2669446 2942927 ADP 299846 526054 1270409 2281248 3142269 2527469 3038756 3772886 Adenosine 25438 41694 40015 20379 12048 29836 14023 12455 Adenine 854709 789313 545184 546386 554335 520725 511748 432833 GTP 522552 256861 474706 822070 387917 229255 378227 409048 GDP 219169 344867 446422 553896 718186 385873 652345 666557 GMP 364690 155769 102008 88586 342397 462664 381040 431325 Guanosine 198526 126038 115901 125617 72448 68921 81136 43871 CTP 178921 401509 355271 595348 158663 139523 207417 235233 CDP 289407 336111 290848 376246 280099 217021 287474 321236 CMP 54556 73077 141446 179096 353818 327019 290410 297845 Cytidine 51447 92612 127352 192576 185273 198464 224031 250903 dTMP 49989 36548 30642 29561 33119 29325 24731 15554 UTP 589885 1112015 1042644 1611955 662429 491899 725048 791941 UDP 1087035 1163609 1076927 1129069 1206928 884320 1136394 1283504 UMP 303969 189267 289398 525009 2323791 2545880 2087116 2659668 Inosine 4024527 2615461 2291127 2946371 1886051 1683064 1842830 1247857 Hypoxanthine 7071726 6081204 5244402 6533380 4070954 4256920 4806040 3712912 Adenylosuccinic acid 2087 58632 69498 58390 119270 116132 104312 109231 UDP-glucose 876326 812609 774576 842520 789775 658968 718106 804392 Diphosphate 1426048 2118288 2571707 3398832 5016606 3344289 4693084 5036494 D-Glucose 492549 654630 659937 726505 503457 545838 536269 513795 D-Fructose 1-6-bisphosphate 250018 175116 146432 208354 197756 133174 186256 198156 2/3-Phospho-D-glycerate 282517 331176 285034 198805 208228 233094 152545 199856 Phosphoenolpyruvate 30912 37488 33844 27402 21078 13729 16611 21182 Lactate 7464352 5032902 5013994 5970794 4992770 5657106 4923384 4706302 Mannitol 228970 323522 8591931 15541680 69791 3249002 240306 68601 Citrate 1846863 4409074 3655197 3778303 2842788 2656545 2503550 2703950 2-Oxoglutarate 24812 188785 98737 74929 73296 56968 59353 46537 Succinate 1222143 870143 1947234 1290847 1769108 1503498 1839673 1889803 Fumarate 953759 1037213 711438 818233 727979 608803 662216 720437 Malate 10275070 11326760 8356116 10043580 8533479 6538596 7931722 7634942 Oxaloacetate 368313 329946 326624 316337 349493 269795 278948 293259 2-Hydroxyglutarate/Citramalate 67331 211852 167214 137163 143467 131558 129676 132422 D-Erythrose 4-phosphate 2573 37337 35465 25554 21773 21864 21054 45549 Sedoheptulose 1-phosphate 89065 95597 104036 101856 126856 104901 121285 127339 alpha-D-Ribose 1-phosphate 1010234 1426782 1673058 1661225 1700551 1299537 1541444 1404482 5-Phospho-alpha-D-ribose 1-diphosphate 21197 14139 14849 34510 19668 11538 21400 28178 5-Oxoproline 1090316 2355146 2196939 2275056 2366971 2672286 2379604 2700894 Ascorbate 34534 52948 60850 69034 55377 59955 56189 38435 Dehydroascorbate 25026 79526 103663 122645 117933 92474 120245 112041 gamma-L-Glutamyl-L-cysteine 20888 36101 58605 85415 167338 173456 176561 161140 Cystathionine 104870 202571 283456 331988 286857 317409 299075 304708 N-Acetylneuraminate 71270 197855 300786 412748 575749 686246 545692 752825 UDP-N-acetyl-D-glucosamine 2619636 2345232 2309663 2236198 2327835 1984438 2072730 2462448 CMP-N-acetylneuraminate 121346 124742 94336 96421 120899 107383 105781 118321 Phosphocreatine 200038 356116 200690 339883 63168 52607 82623 56108 trans-4-Hydroxy-L-proline 894874 1037273 817996 651040 801510 716332 699599 728529 N-Succinyl-L-glutamate 5-semialdehyde 13050 19118 26876 27164 16183 25948 8666 21442 Glycerol 3-phosphate 128784 157069 169049 172031 208417 195221 189154 162619 Ethanolamine phosphate 0 21988 36933 72878 70920 57278 53524 73264 Octanoic acid (caprylate) 5036 11264 11671 22025 32202 34758 47236 64928 Nonanoic acid (pelargonate) 12242 18056 24984 38207 44470 45085 48694 59147 Catechin 886829 668846 540986 654552 384468 398758 405799 161786 S-Acylglutathione 235911 258511 284432 247885 255395 248305 290438 321005 Glycerone sulfate 68150 65828 50003 61709 54060 66760 54784 52902 2-Oxo-7-methylthioheptanoic acid 86766 248456 134255 105359 91242 108079 97258 110471 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name CmpdID parent medRt ATP C00002 505.9883 1.07 ADP C00008 426.0221 1.11 Adenosine C00212 266.0889 1.19 Adenine C00147 134.0458 1.20 GTP C00044 521.9832 1.06 GDP C00035 442.0168 1.06 GMP C00144 362.0508 1.16 Guanosine C00387 282.0841 1.19 CTP C00063 481.9767 1.05 CDP C00112 402.0105 1.06 CMP C00055 322.0443 1.12 Cytidine C00475 242.0790 1.13 dTMP C00364 321.0490 1.19 UTP C00075 482.9608 1.05 UDP C00015 402.9952 1.06 UMP C00105 323.0284 1.15 Inosine C00294 267.0732 1.19 Hypoxanthine C00262 135.0298 1.19 Adenylosuccinic acid C03794 462.0665 1.03 UDP-glucose C00029 565.0471 1.07 Diphosphate C00013 176.9349 1.09 D-Glucose C00031 179.0552 1.14 D-Fructose 1-6-bisphosphate C00354 338.9884 1.05 2/3-Phospho-D-glycerate C00631 184.9846 1.05 Phosphoenolpyruvate C00074 166.9738 1.06 Lactate C01432 89.0228 1.13 Mannitol C00392 181.0708 1.18 Citrate C00158 191.0187 1.09 2-Oxoglutarate C00026 145.0135 1.12 Succinate C00042 117.0178 1.13 Fumarate C00122 115.0021 1.12 Malate C00149 133.0128 1.14 Oxaloacetate C00036 130.9994 1.12 2-Hydroxyglutarate/Citramalate C02630 147.0287 1.12 D-Erythrose 4-phosphate C00279 199.0006 1.13 Sedoheptulose 1-phosphate C06222 289.0326 1.09 alpha-D-Ribose 1-phosphate C00620 229.0113 1.11 5-Phospho-alpha-D-ribose 1-diphosphate C00119 388.9444 1.05 5-Oxoproline C01879 128.0338 1.13 Ascorbate C00072 175.0245 1.14 Dehydroascorbate C05422 173.0081 1.08 gamma-L-Glutamyl-L-cysteine C00669 249.0548 1.14 Cystathionine C00542 221.0595 1.12 N-Acetylneuraminate C00270 308.0985 1.13 UDP-N-acetyl-D-glucosamine C00043 606.0740 1.11 CMP-N-acetylneuraminate C00128 613.1394 1.07 Phosphocreatine C02305 210.0277 1.06 trans-4-Hydroxy-L-proline C01157 130.0495 1.12 N-Succinyl-L-glutamate 5-semialdehyde C05932 230.0685 1.11 Glycerol 3-phosphate C00093 171.0057 1.11 Ethanolamine phosphate C00346 140.0105 1.12 Octanoic acid (caprylate) C06423 143.1064 1.21 Nonanoic acid (pelargonate) C01601 157.1221 1.23 Catechin C17590 289.0677 1.19 S-Acylglutathione C02589 333.0598 1.10 Glycerone sulfate C02543 168.9800 1.06 2-Oxo-7-methylthioheptanoic acid C17220 189.0583 1.14 METABOLITES_END #END