Summary of study ST001339

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000915. The data can be accessed directly via it's Project DOI: 10.21228/M8DQ2M This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST001339
Study TitleDisruption of Redox Balance Enhances the Effects of BRAF-inhibitors in Melanoma
Study SummarySpecifically, we report that drug-insensitive melanoma cells can maintain higher levels of antioxidant metabolites to withstand the lethal effects of drugs. By extending our analysis to other melanoma subtypes in the TCGA, we show that elevated redox capacity could indeed be a general feature of melanoma. Our results suggest that redox vulnerabilities could be exploited for therapeutic benefits and identify unsuspected combination targets to enhance the effects of BRAFi in pan-melanoma.
Institute
Vanderbilt University
Last NameCodreanu
First NameSimona
Address1234 Stevenson Center Lane
Emailsimona.codreanu@vanderbilt.edu
Phone6158758422
Submit Date2020-03-31
Raw Data AvailableYes
Raw Data File Type(s).raw
Analysis Type DetailLC-MS
Release Date2021-03-31
Release Version1
Simona Codreanu Simona Codreanu
https://dx.doi.org/10.21228/M8DQ2M
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:


Project:

Project ID:PR000915
Project DOI:doi: 10.21228/M8DQ2M
Project Title:Disruption of Redox Balance Enhances the Effects of BRAF-inhibitors in Melanoma
Project Type:Untargeted-Targeted Metabolomics analysis
Project Summary:Melanomas harboring BRAF mutations can be treated with BRAF inhibitors, but tumor recurrence is inevitable. In spite of large-scale attempts, there remains an unmet need to uncover molecular determinants of BRAFi insensitivity and devise actionable combination targets to overcome resistance. Here, using an integrative approach of experimentation, and mathematical flux balance analyses in a panel of BRAF-mutated melanoma cells, we show that elevated antioxidant capacity of melanoma cells is linked to their drug sensitivity. Specifically, we report that drug-insensitive melanoma cells can maintain higher levels of antioxidant metabolites to withstand the lethal effects of drugs. By extending our analysis to other melanoma subtypes in the TCGA, we show that elevated redox capacity could indeed be a general feature of melanoma. Our results suggest that redox vulnerabilities could be exploited for therapeutic benefits and identify unsuspected combination targets to enhance the effects of BRAFi in pan-melanoma.
Institute:Vanderbilt University
Last Name:Codreanu
First Name:Simona
Address:1234 Stevenson Center Lane
Email:simona.codreanu@vanderbilt.edu
Phone:6158758422

Subject:

Subject ID:SU001413
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id genotype treatment
SA097565SD5aresistant DMSO
SA097566SD5bresistant DMSO
SA097567SD4aresistant DMSO
SA097568SD1aresistant DMSO
SA097569SD4bresistant DMSO
SA097570SD3bresistant DMSO
SA097571SD1bresistant DMSO
SA097572SD2bresistant DMSO
SA097573SD2aresistant DMSO
SA097574SD3aresistant DMSO
SA097575SP4bresistant PLX4720
SA097576SP5aresistant PLX4720
SA097577SP5bresistant PLX4720
SA097578SP4aresistant PLX4720
SA097579SP2aresistant PLX4720
SA097580SP1aresistant PLX4720
SA097581SP3bresistant PLX4720
SA097582SP1bresistant PLX4720
SA097583SP2bresistant PLX4720
SA097584SP3aresistant PLX4720
SA097585WD4bsensitive DMSO
SA097586WD5asensitive DMSO
SA097587WD5bsensitive DMSO
SA097588WD4asensitive DMSO
SA097589WD1asensitive DMSO
SA097590WD3bsensitive DMSO
SA097591WD2asensitive DMSO
SA097592WD1bsensitive DMSO
SA097593WD3asensitive DMSO
SA097594WD2bsensitive DMSO
SA097595WP4bsensitive PLX4720
SA097596WP5bsensitive PLX4720
SA097597WP4asensitive PLX4720
SA097598WP5asensitive PLX4720
SA097599WP2bsensitive PLX4720
SA097600WP1asensitive PLX4720
SA097601WP1bsensitive PLX4720
SA097602WP2asensitive PLX4720
SA097603WP3asensitive PLX4720
SA097604WP3bsensitive PLX4720
Showing results 1 to 40 of 40

Collection:

Collection ID:CO001408
Collection Summary:Single-cell derived BRAF-mutated SKMEL5 subclones were derived as previously described (Paudel et al., 2018). BRAF-mutated melanoma cells (SKMEL5, WM88), including the SKMEL5 subclones, NRAS-mutated melanoma cells (SKMEL2), and NF1-mutated melanoma cells (MeWO) were grown and cultured in Dulbecco’s modified Eagle’s medium and Ham’s F-12 media (DMEM:F12, 1:1, Cat. No. 11330-032). Media were obtained from Gibco (Grand Island, NY), and supplemented with 10% fetal bovine serum. All cells were cultured in humidified incubators that were CO2 and temperature (37oC) controlled. Cells were passaged 1–2 times per week and were maintained as exponentially growing cultures for a maximum of less than 20 passages. All cells were tested for mycoplasma, and tested negative.
Sample Type:Tumor cells

Treatment:

Treatment ID:TR001428
Treatment Summary:PLX4720 (Cat. No. S1152) and vemurafenib (Cat. No. S1267) were obtained from Selleckchem (Houston, TX). Dabrafenib (Cat No. HY-14660), (1S,3R)-RSL3 (Cat No. HY-100218A), Ferrostatin-1 (FER1, Cat No. HY-100579), Erastin (Cat No. HY-15763), (E)-Daporinad (FK866) (Cat No. HY-50876) were obtained from MedChem Express (Monmouth Junction, NJ) and solubilized in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM. Powdered L-Buthionine-sulfoximine (BSO) (Product No. B2515), powdered Diphenyleneiodonium chloride (DPI) (Product No. D2926), and powdered L-Glutathione reduced (GSH) were obtained from Sigma-Aldrich. BSO, and GSH was freshly made at a stock concentration of 100 mM in H2O, while DPI was solubilized in DMSO at a stock concentration of 10mM. CellRoxTM DeepRed Reagent (Cat No. C10422) for oxidative stress detection was obtained from ThermoFisher Scientific. Acetonitrile (AcCN) (Cat No. A955-1), Methanol (MeOH) (Cat No. A456-1) and water (Cat No. W6-1), Optima LC-MS grade, for the mass spectrometry analysis were obtained from ThermoFisher Scientific.

Sample Preparation:

Sampleprep ID:SP001421
Sampleprep Summary:The combined global untargeted-targeted metabolomic analysis used BRAF-mutated melanoma cells (WM88, SKMEL5-SC10) treated with either DMSO or 8μM PLX4720 for 24 hrs. Cell pellet samples were lysed using 400µL ice cold lysis buffer (1:1:2, ACN:MeOH:Ammonium Bicarbonate 0.1M, pH 8.0, LC-MS grade) and vortexed well until the cells mixed well with the solvent. Each sample was sonicated using a probe tip sonicator, 10 pulses at 30% power, cooling down in ice between samples. A BCA protein assay was used to determine the protein concentration for each individual sample, and adjusted to a total amount of protein of 200µg total protein in 200 µL of lysis buffer. Heavy labeled standard molecules, Phenylalanine-D8 (CDN Isotopes, Quebec, CA), and Biotin-D2(Cambridge Isotope Laboratories, Inc., MA, USA), were added to each sample to assess sample handling steps. Samples were subjected to protein precipitation by addition of 800µL of ice cold methanol (4x by volume), then incubated at -80°C overnight. Samples were centrifuged at 10,000 rpm for 10 min to eliminate precipitated proteins and the metabolite containing supernatant was dried in vacuo and stored at -80°C until LC-MS analysis.

Combined analysis:

Analysis ID AN002233
Analysis type MS
Chromatography type HILIC
Chromatography system Thermo Vanquish UHPLC
Column SeQuant ZIC-HILIC (100 x 2.1mm, 3.5um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive HF hybrid Orbitrap
Ion Mode NEGATIVE
Units peak area

Chromatography:

Chromatography ID:CH001639
Chromatography Summary:Vanquish UHPLC binary system and autosampler
Instrument Name:Thermo Vanquish UHPLC
Column Name:SeQuant ZIC-HILIC (100 x 2.1mm, 3.5um)
Chromatography Type:HILIC

MS:

MS ID:MS002079
Analysis ID:AN002233
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:LC-MS/MS raw data were imported, processed, normalized and reviewed using Progenesis QI v.2.1 (Non-linear Dynamics, Newcastle, UK). All FMS, DDA and PRM sample runs were chromatographically aligned against a QC reference run. Following peak picking, unique spectral features (retention time and m/z pairs) were grouped based on adducts and isotopes, and individual features or metabolites were normalized to all features. Further filtering was carried out by removing features or metabolites that had >30% coefficient of variance.
Ion Mode:NEGATIVE
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