{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002713","ANALYSIS_ID":"AN004397","VERSION":"1","CREATED_ON":"May 25, 2023, 1:01 pm"},

"PROJECT":{"PROJECT_TITLE":"Ranolazine induced metabolic rewiring improves melanoma responses to targeted therapy and immunotherapy","PROJECT_SUMMARY":"Metabolic rewiring affects resistance of melanoma to targeted- and immuno-therapy. We have found that increased fatty acid oxidation (FAO) during late stages of BRAF inhibitor (BRAFi) treatment enables the establishment of acquired resistance. Targeting FAO with ranolazine in vivo once acquired BRAFi-resistance emerges delays tumour recurrence. Single cell RNAseq analysis revealed that ranolazine diminishes the transcriptional NGFRhigh neural crest stem cell subpopulation, which is refractory against BRAFi and immunotherapy. Moreover, by rewiring the methionine salvage pathway, ranolazine enhanced melanoma immunogenicity through increased antigen presentation and interferon signalling. Combination of ranolazine with anti-PD-L1 antibodies strongly improved survival in mice, where it increased lymphocyte infiltration and enhanced anti-tumour responses. Altogether, we show that ranolazine increases the efficacy of targeted melanoma therapy through fatty acid and methionine salvage metabolic rewiring. Importantly, our study suggests that ranolazine could sensitize BRAFi-resistant tumours to immunotherapy, by modulating melanoma cell recognition and immune infiltration. Ranolazine is an FDA and EMA-approved anti-anginal drug with very mild side effects, and our preclinical data encourage its use as a therapeutic option to improve the two main therapeutic strategies currently used to treat metastatic melanoma.","INSTITUTE":"University of Colorado Denver","LABORATORY":"Lab of Angelo D'Alessandro in collaboration with lab of Imanol Arozarena","LAST_NAME":"Haines","FIRST_NAME":"Julie","ADDRESS":"12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA","EMAIL":"julie.haines@cuanschutz.edu","PHONE":"3037243339"},

"STUDY":{"STUDY_TITLE":"Ranolazine induced metabolic rewiring improves melanoma responses to targeted therapy and immunotherapy - lipidomics","STUDY_SUMMARY":"Lipidomics analysis was performed on A375 melanoma cells resistant to BRAF inhibitor vemurafenib (VR) and cells resistant to VR and treated with fatty acid oxidation inhibitor ranolazine.","INSTITUTE":"University of Colorado Denver","LAST_NAME":"Haines","FIRST_NAME":"Julie","ADDRESS":"12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA","EMAIL":"julie.haines@cuanschutz.edu","PHONE":"3037243339"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","GENDER":"Female","CELL_BIOSOURCE_OR_SUPPLIER":"ATCC","CELL_STRAIN_DETAILS":"A375"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"A375P 1",
"Factors":{"Treatment":"VEHICLE"},
"Additional sample data":{"RAW_FILE_NAME":"AG-L-1"}
},
{
"Subject ID":"-",
"Sample ID":"A375P 2",
"Factors":{"Treatment":"VEHICLE"},
"Additional sample data":{"RAW_FILE_NAME":"AG-L-2"}
},
{
"Subject ID":"-",
"Sample ID":"A375P 3",
"Factors":{"Treatment":"VEHICLE"},
"Additional sample data":{"RAW_FILE_NAME":"AG-L-3"}
},
{
"Subject ID":"-",
"Sample ID":"A375P 4",
"Factors":{"Treatment":"VEHICLE"},
"Additional sample data":{"RAW_FILE_NAME":"AG-L-4"}
},
{
"Subject ID":"-",
"Sample ID":"A375 VR 1",
"Factors":{"Treatment":"VEMURA"},
"Additional sample data":{"RAW_FILE_NAME":"AG-L-5"}
},
{
"Subject ID":"-",
"Sample ID":"A375 VR 2",
"Factors":{"Treatment":"VEMURA"},
"Additional sample data":{"RAW_FILE_NAME":"AG-L-6"}
},
{
"Subject ID":"-",
"Sample ID":"A375 VR 3",
"Factors":{"Treatment":"VEMURA"},
"Additional sample data":{"RAW_FILE_NAME":"AG-L-7"}
},
{
"Subject ID":"-",
"Sample ID":"A375 VR 4",
"Factors":{"Treatment":"VEMURA"},
"Additional sample data":{"RAW_FILE_NAME":"AG-L-8"}
},
{
"Subject ID":"-",
"Sample ID":"A375 VR-Rano 1",
"Factors":{"Treatment":"VEMURA+RANO"},
"Additional sample data":{"RAW_FILE_NAME":"AG-L-9"}
},
{
"Subject ID":"-",
"Sample ID":"A375 VR-Rano 2",
"Factors":{"Treatment":"VEMURA+RANO"},
"Additional sample data":{"RAW_FILE_NAME":"AG-L-10"}
},
{
"Subject ID":"-",
"Sample ID":"A375 VR-Rano 3",
"Factors":{"Treatment":"VEMURA+RANO"},
"Additional sample data":{"RAW_FILE_NAME":"AG-L-11"}
},
{
"Subject ID":"-",
"Sample ID":"A375 VR-Rano 4",
"Factors":{"Treatment":"VEMURA+RANO"},
"Additional sample data":{"RAW_FILE_NAME":"AG-L-12"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"All cell lines had been authenticated in 2021. Cells were expanded to generate enough vials from a single batch before the start of the study. Cell lines were cultured in Dulbecco’s Eagle’s Medium (DMEM) (cat#61965026, Gibco) supplemented with 10% fetal bovine serum (cat#10500064, Gibco) plus 1% penicillin/streptomycin (cat#15140122, Gibco). Cells were grown at 37°C in a 5% CO2 environment.","SAMPLE_TYPE":"Melanoma cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"Melanoma cells seeded in 6-well plates were treated for 7 days with a high dose (5 or 10μM as indicated) of vemurafenib before switching to a lower concentration of the drug (0,5 or 1μM). Fresh medium and vemurafenib was added once weekly for further 3-4 weeks until arising colonies grew to confluence. Fatty Acid oxidation inhibitors ranolazine, etomoxir or thiroridazine were added once a week."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Lipids from frozen cell pellets were extracted with cold methanol at a concentration of 2 million cells/mL. Samples were vortexed 30 min at 4 degrees C then supernatants clarified by centrifugation (10 min, 10,000 g, 4 degrees C) and transferred to autosampler vials.","PROCESSING_STORAGE_CONDITIONS":"4℃","EXTRACT_STORAGE":"-80℃"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Negative C18","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Phenomenex Kinetex C18 (30 x 2.1mm,1.7 um)","SOLVENT_A":"75% water 25% acetonitrile 5 mM ammonium acetate","SOLVENT_B":"90% isopropanol 10% acetonitrile 5 mM ammonium acetate","FLOW_GRADIENT":"0-3 min 10-95% B at 0.3 mL/min, 3-4.2 min hold at 95% B at 0.3 mL/min, 4.2-4.3 min drop to 10% B at 0.45 mL/min, 4.3-4.9 min hold at 10%B while lowering flow rate to 0.4 mL/min, 4.9-5 min hold at 10%B while lowering flow rate to 0.3 mL/min.","FLOW_RATE":"see gradient","COLUMN_TEMPERATURE":"45","SAMPLE_INJECTION":"10 uL"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"The Q Exactive was run independently in positive and negative ion mode, scanning using full MS from 125-1500 m/z at 70,000 resolution and top 10 data-dependent MS2 at 17,500 resolution. Electrospray ionization was achieved with 45 Arb sheath gas, 25 Arb auxiliary gas, and 4 kV spray voltage. Calibration was performed prior to the run using the PierceTM Positive and Negative Ion Calibration Solutions (Thermo Fisher Scientific). Run order of samples was randomized and technical replicates were injected after every 4 samples to assess quality control."},

"MS_METABOLITE_DATA":{
"Units":"peak area",

"Data":[{"Metabolite":"Cer(d18:1_16:0)+CH3COO","A375P 1":"15560054","A375P 2":"16302888","A375P 3":"17918347","A375P 4":"17030173","A375 VR 1":"8054552","A375 VR 2":"12852109","A375 VR 3":"8657413","A375 VR 4":"9199501","A375 VR-Rano 1":"11838956","A375 VR-Rano 2":"11007987","A375 VR-Rano 3":"12084895","A375 VR-Rano 4":"10712601"},{"Metabolite":"Cer(d18:1_16:0)-H","A375P 1":"2773714","A375P 2":"3161415","A375P 3":"5241280","A375P 4":"3165333","A375 VR 1":"1572443","A375 VR 2":"2597391","A375 VR 3":"2572183","A375 VR 4":"1902247","A375 VR-Rano 1":"2465997","A375 VR-Rano 2":"2243929","A375 VR-Rano 3":"3263568","A375 VR-Rano 4":"3495964"},{"Metabolite":"Cer(d18:2_16:0)+CH3COO","A375P 1":"1451731","A375P 2":"1605488","A375P 3":"1838127","A375P 4":"1555265","A375 VR 1":"878632","A375 VR 2":"883022","A375 VR 3":"1174251","A375 VR 4":"740016","A375 VR-Rano 1":"1854345","A375 VR-Rano 2":"1958576","A375 VR-Rano 3":"1567836","A375 VR-Rano 4":"1385314"},{"Metabolite":"Cer(d18:1_18:0)+CH3COO","A375P 1":"7874940","A375P 2":"6787285","A375P 3":"9522533","A375P 4":"6111380","A375 VR 1":"2389459","A375 VR 2":"3459152","A375 VR 3":"4272484","A375 VR 4":"2493256","A375 VR-Rano 1":"752470","A375 VR-Rano 2":"699787","A375 VR-Rano 3":"893881","A375 VR-Rano 4":"808043"},{"Metabolite":"Cer(d36:1)-H","A375P 1":"1527751","A375P 2":"1676891","A375P 3":"1827123","A375P 4":"1172814","A375 VR 1":"527963","A375 VR 2":"441432","A375 VR 3":"716325","A375 VR 4":"755147","A375 VR-Rano 1":"168225","A375 VR-Rano 2":"161882","A375 VR-Rano 3":"184461","A375 VR-Rano 4":"248139"},{"Metabolite":"Cer(d18:2_18:0)+CH3COO","A375P 1":"2711947","A375P 2":"3364419","A375P 3":"3722503","A375P 4":"2499509","A375 VR 1":"1214147","A375 VR 2":"1051358","A375 VR 3":"914191","A375 VR 4":"893128","A375 VR-Rano 1":"354999","A375 VR-Rano 2":"387726","A375 VR-Rano 3":"584626","A375 VR-Rano 4":"639246"},{"Metabolite":"Cer(d18:1_22:0)+CH3COO","A375P 1":"10836840","A375P 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1":"3623740","A375 VR 2":"3337588","A375 VR 3":"3676527","A375 VR 4":"3521614","A375 VR-Rano 1":"2536405","A375 VR-Rano 2":"2756470","A375 VR-Rano 3":"2365683","A375 VR-Rano 4":"2415126"},{"Metabolite":"Cer(d15:0_26:2)+CH3COO","A375P 1":"3762596","A375P 2":"4113142","A375P 3":"4304920","A375P 4":"4500796","A375 VR 1":"2715496","A375 VR 2":"2879401","A375 VR 3":"2905846","A375 VR 4":"2822671","A375 VR-Rano 1":"2044724","A375 VR-Rano 2":"2059041","A375 VR-Rano 3":"1978409","A375 VR-Rano 4":"1811386"},{"Metabolite":"Cer(d18:1_24:0)+CH3COO","A375P 1":"26765281","A375P 2":"28017967","A375P 3":"21622197","A375P 4":"27193204","A375 VR 1":"17124577","A375 VR 2":"15434939","A375 VR 3":"17895176","A375 VR 4":"16684220","A375 VR-Rano 1":"13322039","A375 VR-Rano 2":"13815189","A375 VR-Rano 3":"12230694","A375 VR-Rano 4":"12666560"},{"Metabolite":"Cer(d18:1_24:0)-H","A375P 1":"3569747","A375P 2":"3802321","A375P 3":"4390903","A375P 4":"3719579","A375 VR 1":"2130437","A375 VR 2":"2032161","A375 VR 3":"2273949","A375 VR 4":"2134161","A375 VR-Rano 1":"1351413","A375 VR-Rano 2":"1724904","A375 VR-Rano 3":"1555448","A375 VR-Rano 4":"1334748"},{"Metabolite":"Cer(d18:1_24:1)+CH3COO","A375P 1":"25692610","A375P 2":"27426660","A375P 3":"29721358","A375P 4":"20514421","A375 VR 1":"19933542","A375 VR 2":"18393410","A375 VR 3":"21020708","A375 VR 4":"19776052","A375 VR-Rano 1":"15855358","A375 VR-Rano 2":"15982432","A375 VR-Rano 3":"14695620","A375 VR-Rano 4":"14929321"},{"Metabolite":"Cer(d18:1_24:1)-H","A375P 1":"3361883","A375P 2":"3551647","A375P 3":"3690398","A375P 4":"3401824","A375 VR 1":"2471296","A375 VR 2":"2282008","A375 VR 3":"2524511","A375 VR 4":"2489519","A375 VR-Rano 1":"1943458","A375 VR-Rano 2":"1941767","A375 VR-Rano 3":"1845749","A375 VR-Rano 4":"1754073"},{"Metabolite":"Cer(d18:2_24:1)+CH3COO","A375P 1":"5575859","A375P 2":"6020921","A375P 3":"6614258","A375P 4":"5867111","A375 VR 1":"5446704","A375 VR 2":"5288754","A375 VR 3":"5815206","A375 VR 4":"5300245","A375 VR-Rano 1":"4229842","A375 VR-Rano 2":"4006738","A375 VR-Rano 3":"3840607","A375 VR-Rano 4":"3785777"},{"Metabolite":"CL(66:1)-2H","A375P 1":"11373395","A375P 2":"7402578","A375P 3":"8555505","A375P 4":"7677290","A375 VR 1":"6442229","A375 VR 2":"4356280","A375 VR 3":"6896621","A375 VR 4":"4077033","A375 VR-Rano 1":"3370390","A375 VR-Rano 2":"2808584","A375 VR-Rano 3":"3283443","A375 VR-Rano 4":"4868708"},{"Metabolite":"CL(14:0_18:1_18:1_16:0)-H","A375P 1":"1627731","A375P 2":"1503423","A375P 3":"1300507","A375P 4":"1512016","A375 VR 1":"8010577","A375 VR 2":"6980924","A375 VR 3":"7830099","A375 VR 4":"7677122","A375 VR-Rano 1":"5453550","A375 VR-Rano 2":"5857166","A375 VR-Rano 3":"5173623","A375 VR-Rano 4":"5486538"},{"Metabolite":"CL(15:0_18:1_16:0_18:1)-H","A375P 1":"482567","A375P 2":"460268","A375P 3":"484855","A375P 4":"463047","A375 VR 1":"3927684","A375 VR 2":"3335167","A375 VR 3":"3767080","A375 VR 4":"3941631","A375 VR-Rano 1":"2181201","A375 VR-Rano 2":"2276015","A375 VR-Rano 3":"2136823","A375 VR-Rano 4":"2276463"},{"Metabolite":"CL(68:1)-2H","A375P 1":"4063635","A375P 2":"4154850","A375P 3":"4516778","A375P 4":"4187736","A375 VR 1":"4003798","A375 VR 2":"3634119","A375 VR 3":"4107421","A375 VR 4":"2525634","A375 VR-Rano 1":"2922178","A375 VR-Rano 2":"3033163","A375 VR-Rano 3":"2946411","A375 VR-Rano 4":"1796255"},{"Metabolite":"CL(18:1_16:0_16:0_18:1)-H","A375P 1":"7279577","A375P 2":"4769237","A375P 3":"7622664","A375P 4":"5473317","A375 VR 1":"37037591","A375 VR 2":"28484088","A375 VR 3":"34208122","A375 VR 4":"45290767","A375 VR-Rano 1":"31464124","A375 VR-Rano 2":"32654137","A375 VR-Rano 3":"29286800","A375 VR-Rano 4":"32668760"},{"Metabolite":"CL(68:2)-2H","A375P 1":"311754","A375P 2":"272015","A375P 3":"283809","A375P 4":"271752","A375 VR 1":"2006216","A375 VR 2":"1730088","A375 VR 3":"1888246","A375 VR 4":"2175538","A375 VR-Rano 1":"1374721","A375 VR-Rano 2":"1474395","A375 VR-Rano 3":"1358873","A375 VR-Rano 4":"1379961"},{"Metabolite":"CL(18:1_16:0_18:1_16:1)-H","A375P 1":"4586600","A375P 2":"5460006","A375P 3":"6280247","A375P 4":"4472793","A375 VR 1":"8350270","A375 VR 2":"9789969","A375 VR 3":"8152614","A375 VR 4":"8273078","A375 VR-Rano 1":"5594646","A375 VR-Rano 2":"8617260","A375 VR-Rano 3":"6173734","A375 VR-Rano 4":"8174417"},{"Metabolite":"CL(68:4)-H","A375P 1":"6364609","A375P 2":"6112595","A375P 3":"6751005","A375P 4":"4631612","A375 VR 1":"4265264","A375 VR 2":"4019334","A375 VR 3":"3458618","A375 VR 4":"3060151","A375 VR-Rano 1":"4086647","A375 VR-Rano 2":"4283825","A375 VR-Rano 3":"3008903","A375 VR-Rano 4":"3970174"},{"Metabolite":"CL(18:2_16:1_16:1_18:1)-H","A375P 1":"3440775","A375P 2":"3452418","A375P 3":"3647496","A375P 4":"3453514","A375 VR 1":"3267784","A375 VR 2":"3147077","A375 VR 3":"3352066","A375 VR 4":"3395980","A375 VR-Rano 1":"2246344","A375 VR-Rano 2":"2447889","A375 VR-Rano 3":"2288125","A375 VR-Rano 4":"2289192"},{"Metabolite":"CL(69:3)-H","A375P 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}