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.

Study ID	ST001339
Study Title	Disruption of Redox Balance Enhances the Effects of BRAF-inhibitors in Melanoma
Study Summary	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
Submit Date	2020-03-31
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2021-03-31
Release Version	1

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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
SA097565	SD5a	resistant	DMSO
SA097566	SD5b	resistant	DMSO
SA097567	SD4a	resistant	DMSO
SA097568	SD1a	resistant	DMSO
SA097569	SD4b	resistant	DMSO
SA097570	SD3b	resistant	DMSO
SA097571	SD1b	resistant	DMSO
SA097572	SD2b	resistant	DMSO
SA097573	SD2a	resistant	DMSO
SA097574	SD3a	resistant	DMSO
SA097575	SP4b	resistant	PLX4720
SA097576	SP5a	resistant	PLX4720
SA097577	SP5b	resistant	PLX4720
SA097578	SP4a	resistant	PLX4720
SA097579	SP2a	resistant	PLX4720
SA097580	SP1a	resistant	PLX4720
SA097581	SP3b	resistant	PLX4720
SA097582	SP1b	resistant	PLX4720
SA097583	SP2b	resistant	PLX4720
SA097584	SP3a	resistant	PLX4720
SA097585	WD4b	sensitive	DMSO
SA097586	WD5a	sensitive	DMSO
SA097587	WD5b	sensitive	DMSO
SA097588	WD4a	sensitive	DMSO
SA097589	WD1a	sensitive	DMSO
SA097590	WD3b	sensitive	DMSO
SA097591	WD2a	sensitive	DMSO
SA097592	WD1b	sensitive	DMSO
SA097593	WD3a	sensitive	DMSO
SA097594	WD2b	sensitive	DMSO
SA097595	WP4b	sensitive	PLX4720
SA097596	WP5b	sensitive	PLX4720
SA097597	WP4a	sensitive	PLX4720
SA097598	WP5a	sensitive	PLX4720
SA097599	WP2b	sensitive	PLX4720
SA097600	WP1a	sensitive	PLX4720
SA097601	WP1b	sensitive	PLX4720
SA097602	WP2a	sensitive	PLX4720
SA097603	WP3a	sensitive	PLX4720
SA097604	WP3b	sensitive	PLX4720

Showing results 1 to 40 of 40

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.

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.

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