#METABOLOMICS WORKBENCH ducker_lab_20241202_172418 DATATRACK_ID:5418 STUDY_ID:ST003613 ANALYSIS_ID:AN005938 PROJECT_ID:PR002233 VERSION 1 CREATED_ON December 3, 2024, 1:29 pm #PROJECT PR:PROJECT_TITLE SLC7A5 is required for cancer cell growth in arginine-limited conditions PR:PROJECT_TYPE LCMS quantiative analysis PR:PROJECT_SUMMARY Tumor cells must optimize metabolite acquisition between synthesis and uptake PR:PROJECT_SUMMARY from a microenvironment characterized by hypoxia, lactate accumulation, and PR:PROJECT_SUMMARY depletion of many amino acids including arginine. We performed a PR:PROJECT_SUMMARY metabolism-focused functional screen using CRISPR/Cas9 to identify pathways and PR:PROJECT_SUMMARY factors that enable tumor growth in an arginine-depleted environment. Our screen PR:PROJECT_SUMMARY identified the SLC-family transporter SLC7A5 as required for growth, and we PR:PROJECT_SUMMARY hypothesized that this protein functions as a high-affinity citrulline PR:PROJECT_SUMMARY transporter. Using metabolomics, we show that metabolism of citrulline into PR:PROJECT_SUMMARY arginine are dependent upon expression of SLC7A5. Pharmacological inhibition of PR:PROJECT_SUMMARY SLC7A5 blocks growth in low arginine conditions across a diverse group of cancer PR:PROJECT_SUMMARY cell lines. Loss of SLC7A5 reduces tumor growth and citrulline import in a mouse PR:PROJECT_SUMMARY tumor model. We identify a conditionally essential role for SLC7A5 in arginine PR:PROJECT_SUMMARY metabolism, and we propose that SLC7A5-targeting therapeutic strategies in PR:PROJECT_SUMMARY cancer may be effective in the context of arginine limitation. PR:INSTITUTE University of Utah PR:DEPARTMENT Biochemistry PR:LABORATORY Ducker Lab PR:LAST_NAME Dunlap PR:FIRST_NAME Kyle PR:ADDRESS 15 N Medical E Drive, Salt Lake City UT 84113 PR:EMAIL kyle.dunlap@biochem.utah.edu PR:PHONE 2489310065 #STUDY ST:STUDY_TITLE SLC7A5 is required for cancer cell growth in arginine-limited conditions ST:STUDY_TYPE LCMS quantitative analysis ST:STUDY_SUMMARY Polar metabolite abundance studies from A375m human melanoma cells containing ST:STUDY_SUMMARY native levels of SLC7A5 (WT), CRISPR-Cas9 induced knockout of SLC7A5 (KO) or ST:STUDY_SUMMARY lentiviral induced overexpression of SLC7A5 (OE). Studies used RPMI media with ST:STUDY_SUMMARY no arginine. In place of arginine was 110uM citrulline. ST:INSTITUTE University of Utah ST:DEPARTMENT Biochemistry ST:LABORATORY Ducker Lab ST:LAST_NAME Dunlap ST:FIRST_NAME Kyle ST:ADDRESS 15 N Medical Drive E, Salt Lake City UT 84132 ST:EMAIL kyle.dunlap@biochem.utah.edu ST:PHONE 2489310065 ST:NUM_GROUPS 3 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_STRAIN_DETAILS A375m #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 WT_1 WT_1 Genotype:WT | Sample source:A375m Cells RAW_FILE_NAME=WT_1.mzML SUBJECT_SAMPLE_FACTORS WT_2 WT_2 Genotype:WT | Sample source:A375m Cells RAW_FILE_NAME=WT_2.mzML SUBJECT_SAMPLE_FACTORS WT_3 WT_3 Genotype:WT | Sample source:A375m Cells RAW_FILE_NAME=WT_3.mzML SUBJECT_SAMPLE_FACTORS WT_4 WT_4 Genotype:WT | Sample source:A375m Cells RAW_FILE_NAME=WT_4.mzML SUBJECT_SAMPLE_FACTORS KO_1 KO_1 Genotype:KO | Sample source:A375m Cells RAW_FILE_NAME=KO_1.mzML SUBJECT_SAMPLE_FACTORS KO_2 KO_2 Genotype:KO | Sample source:A375m Cells RAW_FILE_NAME=KO_2.mzML SUBJECT_SAMPLE_FACTORS KO_3 KO_3 Genotype:KO | Sample source:A375m Cells RAW_FILE_NAME=KO_3.mzML SUBJECT_SAMPLE_FACTORS KO_4 KO_4 Genotype:KO | Sample source:A375m Cells RAW_FILE_NAME=KO_4.mzML SUBJECT_SAMPLE_FACTORS OE_1 OE_1 Genotype:OE | Sample source:A375m Cells RAW_FILE_NAME=OE_1.mzML SUBJECT_SAMPLE_FACTORS OE_2 OE_2 Genotype:OE | Sample source:A375m Cells RAW_FILE_NAME=OE_2.mzML SUBJECT_SAMPLE_FACTORS OE_3 OE_3 Genotype:OE | Sample source:A375m Cells RAW_FILE_NAME=OE_3.mzML SUBJECT_SAMPLE_FACTORS OE_4 OE_4 Genotype:OE | Sample source:A375m Cells RAW_FILE_NAME=OE_4.mzML #COLLECTION CO:COLLECTION_SUMMARY Cells were seeded and allowed to grow overnight. Then, 60x of the packed cell CO:COLLECTION_SUMMARY volume (PCV) of 80:20 methanol:water was added to the cells after 2 washes with CO:COLLECTION_SUMMARY PBS. Cells were then scraped, allowed to sit on dry ice for ten minutes, CO:COLLECTION_SUMMARY centrifuged twice to remove impurities, and then placed in an MS tube for LC-MS CO:COLLECTION_SUMMARY analysis. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY No treatment was performed. The difference in groups has to do with genotype. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples were processed as indicated in the "collection summary" section. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME BEH Z-HILIC Column, (1.7 µm, 2.1 mm X 50 mm) CH:SOLVENT_A 100% water, 20 mM Ammonium Formate, pH 3 (with formic acid) CH:SOLVENT_B 90% acetonitrile/10% water, 20 mM Ammonium Formate, pH 3 (with formic acid) CH:FLOW_GRADIENT 0.25 ml/min; 0-0.1 min: 0% B, 2 min: 85% B, 8 min: 25% B, 9 min: 25% B, 9.5 min: CH:FLOW_GRADIENT 100% B CH:FLOW_RATE 0.25 ml/min CH:COLUMN_TEMPERATURE 30 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE Other MS:ION_MODE POSITIVE MS:MS_COMMENTS Extracted aqueous and polar metabolites were analyzed by LC-MS using a Vanquish MS:MS_COMMENTS HPLC system (Thermo Fisher Scientific) and a QExactive Plus Orbitrap mass MS:MS_COMMENTS spectrometer (Thermo Fisher Scientific). For aqueous phase polar metabolites, MS:MS_COMMENTS separation was achieved by hydrophilic interaction liquid chromatography (HILIC) MS:MS_COMMENTS performed on an Atlantis Premier BEH Z-HILIC column. The autosampler temperature MS:MS_COMMENTS was 4°C, column temperature was 30°C, and injection volume was 3 µL. Samples MS:MS_COMMENTS were injected into the mass spectrometer by electrospray ionization operating in MS:MS_COMMENTS either negative or positive ion mode. Samples were analyzed using a full scan MS:MS_COMMENTS method with a resolving power of 70,000 at m/z of 200 and range of 74 – 1110 MS:MS_COMMENTS m/z. Full scan data were analyzed using the Maven software package with specific MS:MS_COMMENTS peaks assigned based on exact mass and comparison with known standards. MS:MS_COMMENTS Extracted peak intensities were corrected for natural isotopic abundance using MS:MS_COMMENTS the R package AccuCor. MS:MS_RESULTS_FILE ST003613_AN005938_Results.txt UNITS:Peak Area Has m/z:Yes Has RT:Yes RT units:Minutes #END