#METABOLOMICS WORKBENCH Codreags00_20220726_131507 DATATRACK_ID:3366 STUDY_ID:ST002241 ANALYSIS_ID:AN003659 PROJECT_ID:PR001430 VERSION 1 CREATED_ON July 26, 2022, 2:07 pm #PROJECT PR:PROJECT_TITLE ACSS2 Regulates HIF-2α Degradation through the E3-Ubiquitin Ligase MUL1 in PR:PROJECT_TITLE Clear Cell Renal Cell Carcinoma PR:PROJECT_TYPE Untargeted Metabolomics analysis PR:PROJECT_SUMMARY Clear cell renal cell carcinoma (ccRCC) is an aggressive kidney cancer driven by PR:PROJECT_SUMMARY VHL loss and aberrant HIF-2α signaling. Acetate metabolism may contribute to PR:PROJECT_SUMMARY this axis by ACSS2-dependent acetylation of HIF-2α and may provide PR:PROJECT_SUMMARY opportunities to intervention. Here we tested the effects of pharmacological and PR:PROJECT_SUMMARY genetic manipulation of ACSS2 on HIF-2α, ccRCC cells, and tumors. ACSS2 PR:PROJECT_SUMMARY inhibition led to HIF-2α degradation and suppressed ccRCC growth in vitro, in PR:PROJECT_SUMMARY vivo, and in primary cell cultures of ccRCC patient tumors. This treatment PR:PROJECT_SUMMARY resulted in reduced glucose and cholesterol metabolism, mitochondrial biogenesis PR:PROJECT_SUMMARY and altered cristae deformation, that are consistent with loss of HIF-2α. PR:PROJECT_SUMMARY Mechanistically, HIF-2α protein levels are regulated through proteolytic PR:PROJECT_SUMMARY degradation and we found, in parallel to VHL, HIF-2α stability was dependent on PR:PROJECT_SUMMARY ACSS2 activity to prevent direct interaction with the E3 ligase MUL1. These PR:PROJECT_SUMMARY findings highlight ACSS2 as a critical upstream regulator of pathogenically PR:PROJECT_SUMMARY stabilized HIF-2α, and provides a mechanism that may be exploited to overcome PR:PROJECT_SUMMARY resistance to HIF-2α inhibitor therapies. PR:INSTITUTE Vanderbilt University PR:DEPARTMENT Chemistry PR:LABORATORY Center for Innovative Technology PR:LAST_NAME CODREANU PR:FIRST_NAME SIMONA PR:ADDRESS 1234 STEVENSON CENTER LANE PR:EMAIL SIMONA.CODREANU@VANDERBILT.EDU PR:PHONE 16158758422 #STUDY ST:STUDY_TITLE ACSS2 Regulates HIF-2α Degradation through the E3-Ubiquitin Ligase MUL1 in ST:STUDY_TITLE Clear Cell Renal Cell Carcinoma ST:STUDY_TYPE untargeted metabolomics analysis ST:STUDY_SUMMARY Clear cell renal cell carcinoma (ccRCC) is an aggressive kidney cancer driven by ST:STUDY_SUMMARY VHL loss and aberrant HIF-2α signaling. Acetate metabolism may contribute to ST:STUDY_SUMMARY this axis by ACSS2-dependent acetylation of HIF-2α and may provide ST:STUDY_SUMMARY opportunities to intervention. Here we tested the effects of pharmacological and ST:STUDY_SUMMARY genetic manipulation of ACSS2 on HIF-2α, ccRCC cells, and tumors. ACSS2 ST:STUDY_SUMMARY inhibition led to HIF-2α degradation and suppressed ccRCC growth in vitro, in ST:STUDY_SUMMARY vivo, and in primary cell cultures of ccRCC patient tumors. This treatment ST:STUDY_SUMMARY resulted in reduced glucose and cholesterol metabolism, mitochondrial biogenesis ST:STUDY_SUMMARY and altered cristae deformation, that are consistent with loss of HIF-2α. ST:STUDY_SUMMARY Mechanistically, HIF-2α protein levels are regulated through proteolytic ST:STUDY_SUMMARY degradation and we found, in parallel to VHL, HIF-2α stability was dependent on ST:STUDY_SUMMARY ACSS2 activity to prevent direct interaction with the E3 ligase MUL1. These ST:STUDY_SUMMARY findings highlight ACSS2 as a critical upstream regulator of pathogenically ST:STUDY_SUMMARY stabilized HIF-2α, and provides a mechanism that may be exploited to overcome ST:STUDY_SUMMARY resistance to HIF-2α inhibitor therapies. ST:INSTITUTE Vanderbilt University ST:DEPARTMENT Chemistry ST:LABORATORY Center for Innovative Technology ST:LAST_NAME CODREANU ST:FIRST_NAME SIMONA ST:ADDRESS 1234 STEVENSON CENTER LANE ST:EMAIL SIMONA.CODREANU@VANDERBILT.EDU ST:PHONE 6158758422 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 10 ST:NUM_MALES 10 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN NOD-scid IL2Rgnull (NSG) SU:AGE_OR_AGE_RANGE 6 weeks SU:GENDER Male SU:ANIMAL_ANIMAL_SUPPLIER Jackson Laboratory (catalog number 005557) SU:ANIMAL_HOUSING Mice were housed five to a cage in a temperature- and humidity-controlled space SU:ANIMAL_HOUSING (20-25°C, 45%–64% humidity) with regulated water and lighting (12 h SU:ANIMAL_HOUSING light/dark) within the animal facility. #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 Mouse_1 RCC_C01 Genotype:Wild-type RAW_FILE_NAME=SC_20200921_RPLCp_FMS_RCC_C01 SUBJECT_SAMPLE_FACTORS Mouse_2 RCC_C02 Genotype:Wild-type RAW_FILE_NAME=SC_20200921_RPLCp_FMS_RCC_C02 SUBJECT_SAMPLE_FACTORS Mouse_3 RCC_C03 Genotype:Wild-type RAW_FILE_NAME=SC_20200921_RPLCp_FMS_RCC_C03 SUBJECT_SAMPLE_FACTORS Mouse_4 RCC_C04 Genotype:Wild-type RAW_FILE_NAME=SC_20200921_RPLCp_FMS_RCC_C04 SUBJECT_SAMPLE_FACTORS Mouse_5 RCC_C05 Genotype:Wild-type RAW_FILE_NAME=SC_20200921_RPLCp_FMS_RCC_C05 SUBJECT_SAMPLE_FACTORS Mouse_6 RCC_KD01 Genotype:doxycycline-inducible shRNA RAW_FILE_NAME=SC_20200921_RPLCp_FMS_RCC_KD01 SUBJECT_SAMPLE_FACTORS Mouse_7 RCC_KD02 Genotype:doxycycline-inducible shRNA RAW_FILE_NAME=SC_20200921_RPLCp_FMS_RCC_KD02 SUBJECT_SAMPLE_FACTORS Mouse_8 RCC_KD03 Genotype:doxycycline-inducible shRNA RAW_FILE_NAME=SC_20200921_RPLCp_FMS_RCC_KD03 SUBJECT_SAMPLE_FACTORS Mouse_9 RCC_KD04 Genotype:doxycycline-inducible shRNA RAW_FILE_NAME=SC_20200921_RPLCp_FMS_RCC_KD04 SUBJECT_SAMPLE_FACTORS Mouse_10 RCC_KD05 Genotype:doxycycline-inducible shRNA RAW_FILE_NAME=SC_20200921_RPLCp_FMS_RCC_KD05 #COLLECTION CO:COLLECTION_SUMMARY Mice which were inoculated with 786-O cells transduced to express the pTRIPZ CO:COLLECTION_SUMMARY doxycycline-inducible shRNA system were provided a 200 mg/kg doxycycline rodent CO:COLLECTION_SUMMARY diet (Bio-Serv) and allowed to feed ad libitum. Tumor burden was monitored via CO:COLLECTION_SUMMARY weekly manual, digital caliper measurement with intermittent measurements taken CO:COLLECTION_SUMMARY as needed. Mice were euthanized once the first tumor reached size endpoint CO:COLLECTION_SUMMARY (1,000 mm3) or if discomfort was observed as outlined in the IACUC approved CO:COLLECTION_SUMMARY protocol. CO:SAMPLE_TYPE Tumor cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Mice which were inoculated with 786-O cells transduced to express the pTRIPZ TR:TREATMENT_SUMMARY doxycycline-inducible shRNA system were provided a 200 mg/kg doxycycline rodent TR:TREATMENT_SUMMARY diet (Bio-Serv) and allowed to feed ad libitum. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Fresh tumor sample materials were flash frozen and stored at -80°C until SP:SAMPLEPREP_SUMMARY analyzed via Liquid Chromatography-Mass Spectrometry (LC-MS and LC-MS/MS)- SP:SAMPLEPREP_SUMMARY untargeted metabolomics in the Vanderbilt Center for Innovative Technology SP:SAMPLEPREP_SUMMARY (CIT). Individual tissue samples were thawed on ice and lysed in 400 µl SP:SAMPLEPREP_SUMMARY ice-cold lysis buffer (1:1:2, acetonitrile: methanol: 0.1M ammonium bicarbonate, SP:SAMPLEPREP_SUMMARY pH 8.0) and sonicated 2 x 10 pulses using a probe sonicator at 50% power with SP:SAMPLEPREP_SUMMARY cooling in ice in-between. Homogenized samples were normalized by protein amount SP:SAMPLEPREP_SUMMARY (200 µg total protein per tissue sample) based on BCA assay (Thermo Fisher SP:SAMPLEPREP_SUMMARY Scientific, Fair Lawn, NJ). Isotopically labeled phenylalanine-D8 and biotin-D2 SP:SAMPLEPREP_SUMMARY were added to individual samples, and proteins wwere precipitated by addition of SP:SAMPLEPREP_SUMMARY 800 µL of ice-cold methanol followed by overnight incubation at -80°C. SP:SAMPLEPREP_SUMMARY Precipitated proteins were pelleted by centrifugation (15,000 rpm, 15 min), and SP:SAMPLEPREP_SUMMARY metabolite extracts were dried down in vacuo. Individual samples were SP:SAMPLEPREP_SUMMARY reconstituted in 100 µl of reconstitution buffer (acetonitrile: water, 90:10, SP:SAMPLEPREP_SUMMARY v:v) containing tryptophan-D3, valine-D8, and inosine-4N15 and cleared by SP:SAMPLEPREP_SUMMARY centrifugation. A quality control (QC) sample was prepared by pooling equal SP:SAMPLEPREP_SUMMARY volumes from individual samples following reconstitution. Quality control SP:SAMPLEPREP_SUMMARY samples were used for column conditioning, retention time alignment and to SP:SAMPLEPREP_SUMMARY assess mass spectrometry instrument reproducibility throughout the sample set SP:SAMPLEPREP_SUMMARY and allows for sample batch acceptance. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Following lysis and standard addition, protein precipitation was performed by SP:EXTRACTION_METHOD adding 800µL of ice-cold methanol (4x by volume). Samples were incubated at SP:EXTRACTION_METHOD -80°C overnight. Following incubation, samples were centrifuged at 10,000 rpm SP:EXTRACTION_METHOD for 10 min to eliminate proteins. The supernatants containing metabolites were SP:EXTRACTION_METHOD dried via speed-vacuum. SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The LC-MS and LC-MS/MS analyses were performed on a high-resolution Q-Exactive CH:CHROMATOGRAPHY_SUMMARY HF hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, CH:CHROMATOGRAPHY_SUMMARY Bremen, Germany) equipped with a Vanquish UHPLC binary system and autosampler CH:CHROMATOGRAPHY_SUMMARY (Thermo Fisher Scientific, Germany) using previously described methods. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME SeQuant ZIC-HILIC (100 x 2.1mm, 3.5um) CH:FLOW_GRADIENT 45 min CH:FLOW_RATE 0.20mL/min CH:COLUMN_TEMPERATURE 40 CH:SOLVENT_A 90% water, 10% acetonitrile, 5mM Ammonium Formate CH:SOLVENT_B 10% water, 90% acetonitrile, 5mM Ammonium Formate #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The acquired raw data were imported, processed, normalized and reviewed using MS:MS_COMMENTS Progenesis QI v.3.0 (Non-linear Dynamics, Newcastle, UK). All MS and MS/MS MS:MS_COMMENTS sample runs were aligned against a QC sample MS:MS_RESULTS_FILE ST002241_AN003659_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END