#METABOLOMICS WORKBENCH DavideS_20240228_063403 DATATRACK_ID:4679 STUDY_ID:ST003113 ANALYSIS_ID:AN005101 PROJECT_ID:PR001933 VERSION 1 CREATED_ON February 29, 2024, 3:20 pm #PROJECT PR:PROJECT_TITLE Inhibition of Asparagine Synthetase Effectively Retards Polycystic Kidney PR:PROJECT_TITLE Disease Progression. PR:PROJECT_SUMMARY Polycystic Kidney Disease (PKD) is a genetic disorder characterized by bilateral PR:PROJECT_SUMMARY cyst formation. We showed that PKD cells and kidneys display metabolic PR:PROJECT_SUMMARY alterations, including the Warburg effect and glutaminolysis, sustained in vitro PR:PROJECT_SUMMARY by the enzyme asparagine synthetase (ASNS). Here, we used antisense PR:PROJECT_SUMMARY oligonucleotides (ASO) against ASNS in orthologous and slowly progressive PKD PR:PROJECT_SUMMARY murine models and show that treatment leads to a drastic reduction of total PR:PROJECT_SUMMARY kidney volume (measured by MRI) and a prominent rescue of renal function in the PR:PROJECT_SUMMARY mouse. Mechanistically, the upregulation of an ATF4-ASNS axis in PKD is driven PR:PROJECT_SUMMARY by the amino acid response (AAR) branch of the integrated stress response (ISR). PR:PROJECT_SUMMARY Metabolic profiling of PKD control kidneys treated with ASNS-ASO or Scr-ASO PR:PROJECT_SUMMARY revealed major changes in the mutants, several of which are rescued by ASNS PR:PROJECT_SUMMARY silencing in vivo. Indeed, ASNS drives glutamine-dependent de novo pyrimidine PR:PROJECT_SUMMARY synthesis and proliferation in cystic epithelia. Notably, while several PR:PROJECT_SUMMARY metabolic pathways were completely corrected by ASNS-ASO, glycolysis was only PR:PROJECT_SUMMARY partially restored. Accordingly, combining the glycolytic inhibitor 2DG with PR:PROJECT_SUMMARY ASNS-ASO further improved efficacy. Our studies identify a new therapeutic PR:PROJECT_SUMMARY target and novel metabolic vulnerabilities in PKD. Of interest, in these tracing PR:PROJECT_SUMMARY studies we could confirm that the pyrimidine biosynthesis pathway is increased PR:PROJECT_SUMMARY and rescued by silencing of Asns. PR:INSTITUTE San Raffaele University PR:LAST_NAME Stefanoni PR:FIRST_NAME Davide PR:ADDRESS Via Olgettina 58, Milan, Milan, 20132, Italy PR:EMAIL stefanoni.davide@hsr.it PR:PHONE +393337686005 #STUDY ST:STUDY_TITLE Inhibition of Asparagine Synthetase Effectively Retards Polycystic Kidney ST:STUDY_TITLE Disease Progression, investigated with targeted tracing metabolomics analysis in ST:STUDY_TITLE MEF cells using 15N2-glutamine. ST:STUDY_SUMMARY Polycystic Kidney Disease (PKD) is a genetic disorder characterized by bilateral ST:STUDY_SUMMARY cyst formation. We showed that PKD cells and kidneys display metabolic ST:STUDY_SUMMARY alterations, including the Warburg effect and glutaminolysis, sustained in vitro ST:STUDY_SUMMARY by the enzyme asparagine synthetase (ASNS). Here, we used antisense ST:STUDY_SUMMARY oligonucleotides (ASO) against Asns in orthologous and slowly progressive PKD ST:STUDY_SUMMARY murine models and show that treatment leads to a drastic reduction of total ST:STUDY_SUMMARY kidney volume (measured by MRI) and a prominent rescue of renal function in the ST:STUDY_SUMMARY mouse. Mechanistically, the upregulation of an ATF4-ASNS axis in PKD is driven ST:STUDY_SUMMARY by the amino acid response (AAR) branch of the integrated stress response (ISR). ST:STUDY_SUMMARY Metabolic profiling of PKD or control kidneys treated with Asns-ASO or Scr-ASO ST:STUDY_SUMMARY revealed major changes in the mutants, several of which are rescued by Asns ST:STUDY_SUMMARY silencing in vivo. Indeed, ASNS drives glutamine-dependent de novo pyrimidine ST:STUDY_SUMMARY synthesis and proliferation in cystic epithelia. Notably, while several ST:STUDY_SUMMARY metabolic pathways were completely corrected by Asns-ASO, glycolysis was only ST:STUDY_SUMMARY partially restored. Accordingly, combining the glycolytic inhibitor 2DG with ST:STUDY_SUMMARY Asns-ASO further improved efficacy. Our studies identify a new therapeutic ST:STUDY_SUMMARY target and novel metabolic vulnerabilities in PKD. Of interest, in these tracing ST:STUDY_SUMMARY studies we could confirm that the pyrimidine biosynthesis pathway is increased ST:STUDY_SUMMARY and rescued by silencing of Asns. ST:INSTITUTE San Raffaele University ST:LAST_NAME Stefanoni ST:FIRST_NAME Davide ST:ADDRESS Via Olgettina 58, Milan, Milan, 20132, Italy ST:EMAIL stefanoni.davide@hsr.it ST:PHONE +393337686005 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - 1 Sample source:MEF cells | Factor:Unlabeled_Untreated_11_1 RAW_FILE_NAME(compound)=CA24_06-1 SUBJECT_SAMPLE_FACTORS - 2 Sample source:MEF cells | Factor:Unlabeled_Untreated_11_2 RAW_FILE_NAME(compound)=CA24_06-2 SUBJECT_SAMPLE_FACTORS - 3 Sample source:MEF cells | Factor:Unlabeled_Untreated_11_3 RAW_FILE_NAME(compound)=CA24_06-3 SUBJECT_SAMPLE_FACTORS - 4 Sample source:MEF cells | Factor:Unlabeled_Untreated_11_4 RAW_FILE_NAME(compound)=CA24_06-4 SUBJECT_SAMPLE_FACTORS - 5 Sample source:MEF cells | Factor:Unlabeled_Untreated_11_5 RAW_FILE_NAME(compound)=CA24_06-5 SUBJECT_SAMPLE_FACTORS - 6 Sample source:MEF cells | Factor:Unlabeled_Untreated_11_6 RAW_FILE_NAME(compound)=CA24_06-6 SUBJECT_SAMPLE_FACTORS - 7 Sample source:MEF cells | Factor:Unlabeled_Untreated_14_1 RAW_FILE_NAME(compound)=CA24_06-7 SUBJECT_SAMPLE_FACTORS - 8 Sample source:MEF cells | Factor:Unlabeled_Untreated_14_2 RAW_FILE_NAME(compound)=CA24_06-8 SUBJECT_SAMPLE_FACTORS - 9 Sample source:MEF cells | Factor:Unlabeled_Untreated_14_3 RAW_FILE_NAME(compound)=CA24_06-9 SUBJECT_SAMPLE_FACTORS - 10 Sample source:MEF cells | Factor:Unlabeled_Untreated_14_4 RAW_FILE_NAME(compound)=CA24_06-10 SUBJECT_SAMPLE_FACTORS - 11 Sample source:MEF cells | Factor:Unlabeled_Untreated_14_5 RAW_FILE_NAME(compound)=CA24_06-11 SUBJECT_SAMPLE_FACTORS - 12 Sample source:MEF cells | Factor:Unlabeled_Untreated_14_6 RAW_FILE_NAME(compound)=CA24_06-12 SUBJECT_SAMPLE_FACTORS - 13 Sample source:MEF cells | Factor:Labeled_Untreated_11_1 RAW_FILE_NAME(compound)=CA24_06-13 SUBJECT_SAMPLE_FACTORS - 14 Sample source:MEF cells | Factor:Labeled_Untreated_11_2 RAW_FILE_NAME(compound)=CA24_06-14 SUBJECT_SAMPLE_FACTORS - 15 Sample source:MEF cells | Factor:Labeled_Untreated_11_3 RAW_FILE_NAME(compound)=CA24_06-15 SUBJECT_SAMPLE_FACTORS - 16 Sample source:MEF cells | Factor:Labeled_Untreated_11_4 RAW_FILE_NAME(compound)=CA24_06-16 SUBJECT_SAMPLE_FACTORS - 17 Sample source:MEF cells | Factor:Labeled_Untreated_11_5 RAW_FILE_NAME(compound)=CA24_06-17 SUBJECT_SAMPLE_FACTORS - 18 Sample source:MEF cells | Factor:Labeled_Untreated_11_6 RAW_FILE_NAME(compound)=CA24_06-18 SUBJECT_SAMPLE_FACTORS - 19 Sample source:MEF cells | Factor:Labeled_Untreated_14_1 RAW_FILE_NAME(compound)=CA24_06-19 SUBJECT_SAMPLE_FACTORS - 20 Sample source:MEF cells | Factor:Labeled_Untreated_14_2 RAW_FILE_NAME(compound)=CA24_06-20 SUBJECT_SAMPLE_FACTORS - 21 Sample source:MEF cells | Factor:Labeled_Untreated_14_3 RAW_FILE_NAME(compound)=CA24_06-21 SUBJECT_SAMPLE_FACTORS - 22 Sample source:MEF cells | Factor:Labeled_Untreated_14_4 RAW_FILE_NAME(compound)=CA24_06-22 SUBJECT_SAMPLE_FACTORS - 23 Sample source:MEF cells | Factor:Labeled_Untreated_14_5 RAW_FILE_NAME(compound)=CA24_06-23 SUBJECT_SAMPLE_FACTORS - 24 Sample source:MEF cells | Factor:Labeled_Untreated_14_6 RAW_FILE_NAME(compound)=CA24_06-24 SUBJECT_SAMPLE_FACTORS - 37 Sample source:MEF cells | Factor:Labeled_siASNS_11_1 RAW_FILE_NAME(compound)=CA24_06-37 SUBJECT_SAMPLE_FACTORS - 38 Sample source:MEF cells | Factor:Labeled_siASNS_11_2 RAW_FILE_NAME(compound)=CA24_06-38 SUBJECT_SAMPLE_FACTORS - 39 Sample source:MEF cells | Factor:Labeled_siASNS_11_3 RAW_FILE_NAME(compound)=CA24_06-39 SUBJECT_SAMPLE_FACTORS - 40 Sample source:MEF cells | Factor:Labeled_siASNS_11_4 RAW_FILE_NAME(compound)=CA24_06-40 SUBJECT_SAMPLE_FACTORS - 41 Sample source:MEF cells | Factor:Labeled_siASNS_11_5 RAW_FILE_NAME(compound)=CA24_06-41 SUBJECT_SAMPLE_FACTORS - 42 Sample source:MEF cells | Factor:Labeled_siASNS_11_6 RAW_FILE_NAME(compound)=CA24_06-42 SUBJECT_SAMPLE_FACTORS - 43 Sample source:MEF cells | Factor:Labeled_siASNS_14_1 RAW_FILE_NAME(compound)=CA24_06-43 SUBJECT_SAMPLE_FACTORS - 44 Sample source:MEF cells | Factor:Labeled_siASNS_14_2 RAW_FILE_NAME(compound)=CA24_06-44 SUBJECT_SAMPLE_FACTORS - 45 Sample source:MEF cells | Factor:Labeled_siASNS_14_3 RAW_FILE_NAME(compound)=CA24_06-45 SUBJECT_SAMPLE_FACTORS - 46 Sample source:MEF cells | Factor:Labeled_siASNS_14_4 RAW_FILE_NAME(compound)=CA24_06-46 SUBJECT_SAMPLE_FACTORS - 47 Sample source:MEF cells | Factor:Labeled_siASNS_14_5 RAW_FILE_NAME(compound)=CA24_06-47 SUBJECT_SAMPLE_FACTORS - 48 Sample source:MEF cells | Factor:Labeled_siASNS_14_6 RAW_FILE_NAME(compound)=CA24_06-48 #COLLECTION CO:COLLECTION_SUMMARY Immortalized Pkd1+/+ and Pkd1−/− MEFs were cultured in either culture media CO:COLLECTION_SUMMARY enriched with and without 15N2-glutamine. Following 24h were collected, pellet CO:COLLECTION_SUMMARY and instantly extracted with metabolomics lysis buffer. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY Immortalized Pkd1+/+ and Pkd1−/− MEFs were cultured in either culture media TR:TREATMENT_SUMMARY enriched or not with 15N2-glutamine. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY MEF cells samples were extracted in a ratio of 1milion/1mL of ice cold SP:SAMPLEPREP_SUMMARY extraction solution (methanol:acetonitrile:water 5:3:2 v/v/v). Suspensions were SP:SAMPLEPREP_SUMMARY vortexed continuously for 30 min at 4°C. Insoluble material was removed by SP:SAMPLEPREP_SUMMARY centrifugation at 18,000 g for 10 min at 4°C and supernatants were isolated for SP:SAMPLEPREP_SUMMARY metabolomics analysis by UHPLC-MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY 5MM_NEG_ESI CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) CH:SOLVENT_A 5% acetonitrile/95% water; 1mM ammonium acetate CH:SOLVENT_B 95% acetonitrile/5% water; 1mM ammonium acetate CH:FLOW_GRADIENT 0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min CH:FLOW_GRADIENT 100-0% B, 3-5 min hold at 0% B. CH:FLOW_RATE 0.450ml/min CH:COLUMN_TEMPERATURE 45°C #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Angelo D'Alessandro Lab #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The Q Exactive was run independently in positive and negative ion mode, scanning MS:MS_COMMENTS using full MS from 125-1500 m/z at 70,000 resolution and top 10 data-dependent MS:MS_COMMENTS MS2 at 17,500 resolution. Electrospray ionization was achieved with 45 Arb MS:MS_COMMENTS sheath gas, 25 Arb auxiliary gas, and 4 kV spray voltage. Calibration was MS:MS_COMMENTS performed prior to the run using the PierceTM Positive and Negative Ion MS:MS_COMMENTS Calibration Solutions (Thermo Fisher Scientific). Run order of samples was MS:MS_COMMENTS randomized and technical replicates were injected after every 4 samples to MS:MS_COMMENTS assess quality control. Metabolite assignments and correction for expected MS:MS_COMMENTS natural abundances of 13C isotopes were performed using MAVEN. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak Area MS_METABOLITE_DATA_START Samples 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 37 38 39 40 41 42 43 44 45 46 47 48 Factors Sample source:MEF cells | Factor:Unlabeled_Untreated_11_1 Sample source:MEF cells | Factor:Unlabeled_Untreated_11_2 Sample source:MEF cells | Factor:Unlabeled_Untreated_11_3 Sample source:MEF cells | Factor:Unlabeled_Untreated_11_4 Sample source:MEF cells | Factor:Unlabeled_Untreated_11_5 Sample source:MEF cells | Factor:Unlabeled_Untreated_11_6 Sample source:MEF cells | Factor:Unlabeled_Untreated_14_1 Sample source:MEF cells | Factor:Unlabeled_Untreated_14_2 Sample source:MEF cells | Factor:Unlabeled_Untreated_14_3 Sample source:MEF cells | Factor:Unlabeled_Untreated_14_4 Sample source:MEF cells | Factor:Unlabeled_Untreated_14_5 Sample source:MEF cells | Factor:Unlabeled_Untreated_14_6 Sample source:MEF cells | Factor:Labeled_Untreated_11_1 Sample source:MEF cells | Factor:Labeled_Untreated_11_2 Sample source:MEF cells | Factor:Labeled_Untreated_11_3 Sample source:MEF cells | Factor:Labeled_Untreated_11_4 Sample source:MEF cells | Factor:Labeled_Untreated_11_5 Sample source:MEF cells | Factor:Labeled_Untreated_11_6 Sample source:MEF cells | Factor:Labeled_Untreated_14_1 Sample source:MEF cells | Factor:Labeled_Untreated_14_2 Sample source:MEF cells | Factor:Labeled_Untreated_14_3 Sample source:MEF cells | Factor:Labeled_Untreated_14_4 Sample source:MEF cells | Factor:Labeled_Untreated_14_5 Sample source:MEF cells | Factor:Labeled_Untreated_14_6 Sample source:MEF cells | Factor:Labeled_siASNS_11_1 Sample source:MEF cells | Factor:Labeled_siASNS_11_2 Sample source:MEF cells | Factor:Labeled_siASNS_11_3 Sample source:MEF cells | Factor:Labeled_siASNS_11_4 Sample source:MEF cells | Factor:Labeled_siASNS_11_5 Sample source:MEF cells | Factor:Labeled_siASNS_11_6 Sample source:MEF cells | Factor:Labeled_siASNS_14_1 Sample source:MEF cells | Factor:Labeled_siASNS_14_2 Sample source:MEF cells | Factor:Labeled_siASNS_14_3 Sample source:MEF cells | Factor:Labeled_siASNS_14_4 Sample source:MEF cells | Factor:Labeled_siASNS_14_5 Sample source:MEF cells | Factor:Labeled_siASNS_14_6 L-Glutamine_C12 PARENT 17107280 17701536 17986014 17905858 16950336 17565010 20431292 20398654 17494178 19886606 18162178 21432094 4049562.75 4118810.75 4466336 4130551.25 4317054.5 4253345 41780.91 73915.65 60679.61 85437.26 76366.12 91239.16 0 36328.04 28211.39 34158.32 75080.98 55641.39 0 0 0 25996.42 34259.81 42084.15 L-Glutamine_N15-label-2 0 0 0 0 0 0 0 0 0 0 0 0 1799715.392 2087673.604 1607745.111 2111668.469 1595747.775 2027674.218 6276597.299 6350877.27 6796551.847 2640625.167 5385243.666 4753872.46 27195032.05 29935613.06 26984218.07 28670728.7 26984216.68 27616659.94 20451923.69 23032543.61 27494335.66 26529625.13 23418420.81 23032532.53 L-Glutamate_C12 PARENT 120883840 124247528 119585376 130986280 119668264 125629184 130192232 138345472 50399744 156129456 66368468 155644272 61637364 70240312 72688928 64167884 72532472 70753944 67719168 68320400 55399188 8383101.5 61672460 63421152 51387936 50858032 50358724 48552848 54732492 51460896 63540096 60866240 65912148 63972720 63425360 56930324 L-Glutamate_N15-label-1 0 0 0 0 0 0 0 0 0 0 0 0 774909.3022 925703.818 727435.5262 1027759.381 971986.3978 1016947.228 2076825.67 2095207.4 2256942.377 937346.9958 1970478.346 1874882.574 919165.9363 1023620.086 961008.7644 1015172.002 1040340.396 1265900.192 410446.7088 405649.2548 415235.4156 463008.7683 436627.1448 370022.6147 (S)-Dihydroorotate_C12 PARENT 222754.89 208810.25 243374.7 201283.92 196704.7 276908.53 333957.53 331781.81 1806169.62 320445.47 1127557.88 319203.25 154655 152600.89 133662.86 219710.95 125412.24 211940.67 190044.08 261505.83 313167.91 277438.84 212239.56 235470.17 49019.02 35833.54 65556.57 101045.52 71427.6 112616 32544.57 33936.9 73806.07 80489.3 50129.72 111161.23 (S)-Dihydroorotate_N15-label-1 0 0 0 0 0 0 0 0 0 0 0 0 1229.389319 855.4915239 475.9075043 1618.10279 948.7656633 1630.752571 7014.243178 9399.305256 11642.07282 787.0581034 9005.900571 6891.102037 2113.279556 1012.910089 2089.73471 2252.287419 1036.845355 2481.424253 1094.521246 0 1942.469446 1789.952345 728.6938559 569.0656415 Carbamoyl phosphate_C12 PARENT 5327.67 7864.16 5279.66 6639.37 8095.85 8316.54 16306.52 11757.15 91486.37 11313.9 61750.87 11654.12 5939.09 2085.99 9973.32 8260.23 26434.39 5718.53 3937.41 1804.61 6445.79 5748.72 48500.61 10822.47 19019.5 4041.68 7104.59 14075.37 3341.08 20716 16508.08 26347.37 19185.34 4968.68 6816.3 19376.91 Carbamoyl phosphate_N15-label-1 0 0 0 0 0 0 0 0 0 0 0 0 107.7793564 13.17322566 84.29084691 52.92570835 52.73970884 76.92356526 33.35313385 172.794885 876.6884453 583.0920593 174.188694 922.1252777 72.91981117 70.00054491 84.2371083 90.35147796 70.65445365 104.9308361 176.2327878 450.4535896 262.6321892 25.3178562 32.51543327 23.18742771 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID m/z r.t. L-Glutamine_C12 PARENT C00064 145.061859 0.671 L-Glutamine_N15-label-2 C00064 147.056 0.673 L-Glutamate_C12 PARENT C00025 146.045883 0.6184 L-Glutamate_N15-label-1 C00025 147.042755 0.6139 (S)-Dihydroorotate_C12 PARENT C00337 157.02792 0.652 (S)-Dihydroorotate_N15-label-1 C00337 158.022009 0.669 Carbamoyl phosphate_C12 PARENT C00169 139.980881 0.575 Carbamoyl phosphate_N15-label-1 C00169 140.97905 0.585 METABOLITES_END #END