Summary of Study ST003148

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 PR001941. The data can be accessed directly via it's Project DOI: 10.21228/M8TM76 This work is supported by NIH grant, U2C- DK119886.

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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 IDST003148
Study TitleBT-474 cells fed with [U-13C6] D-glucose or [U-13C5] L-glutamine and treated with Fasnall and GSK2194069 for 24 h
Study TypeIntracellular metabolomics, [U-13C6] glucose and [U-13C5] L-glutamine tracing
Study SummaryBT-474 cells fed with [U-13C6] D-glucose or [U-13C5] L-glutamine and treated with Fasnall and GSK2194069 for 24 h.
Institute
Wistar Institute
DepartmentMolecular and Cellular Oncogenesis Program, Ellen and Ronald Caplan Cancer Center
LaboratorySchug's Lab
Last NameMukha
First NameDzmitry
Address3601 Spruce St, Philadelphia, PA 19104, USA
Emaildmukha@wistar.org
Phone2154956903
Submit Date2024-03-09
Num Groups6
Total Subjects18
PublicationsSubmission Pending
Raw Data AvailableYes
Raw Data File Type(s)mzXML, raw(Thermo)
Analysis Type DetailLC-MS
Release Date2024-04-02
Release Version1
Dzmitry Mukha Dzmitry Mukha
https://dx.doi.org/10.21228/M8TM76
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001941
Project DOI:doi: 10.21228/M8TM76
Project Title:The shutdown of NADH oxidation via Respiratory Complex I mimics fatty acid biosynthesis inhibition
Project Type:LC-MS Quantitative Analysis
Project Summary:Proliferating cancer cells actively utilize anabolic processes for biomass production, including de novo biosynthesis of amino acids, nucleotides, and fatty acids. The key enzyme of the fatty acid biosynthesis pathway, fatty acid synthase (FASN), is widely recognized as a promising therapeutic target in cancer and other health conditions. Here, we establish a metabolic signature of FASN inhibition using a panel of pharmacological inhibitors (GSK2194069, TVB-2640, TVB-3166, C75, cerulenin, and Fasnall). We find that the activity of some commonly used FASN inhibitors is inconsistent with the metabolic signature of FASN inhibition (accumulation of malonate, succinate, malonyl coenzyme A, succinyl coenzyme A, and other metabolic perturbations). Moreover, we show that one of these putative FASN inhibitors, Fasnall, is a respiratory Complex I inhibitor that mimics FASN inhibition through NADH accumulation and consequent depletion of the tricarboxylic acid cycle metabolites. We demonstrate that Fasnall impairs tumor growth in several oxidative phosphorylation-dependent cancer models, including combination therapy-resistant melanoma patient-derived xenografts. Fasnall administration does not reproduce neurological side effects in mice reported for other Complex I inhibitors. Our results have significant implications for understanding the FASN role in human health and disease and provide evidence of therapeutic potential for Complex I inhibitors with fast systemic clearance. Our findings also highlight the continuing need for validation of small molecule inhibitors to distinguish high-quality chemical probes and to expand the understanding of their application.
Institute:Wistar Institute
Department:Molecular and Cellular Oncogenesis Program, Ellen and Ronald Caplan Cancer Center
Laboratory:Schug's Lab
Last Name:Mukha
First Name:Dzmitry
Address:3601 Spruce St., Philadelphia, Pennsylvania 19104, USA
Email:dmukha@wistar.org
Phone:+12154956903
Funding Source:This work was supported by grants from the National Institutes of Health (NIH) National Cancer Institute (NCI) DP2 CA249950-01 (Z.T.S.), NIH NCI P01 CA114046 (Z.T.S.), Melanoma Research Foundation 717173 (Z.T.S.), and Susan G. Komen CCR19608782 (Z.T.S.).
Publications:Submission Pending
Contributors:Dzmitry Mukha, Jena Dessain, Seamus O’Connor, Katherine Pniewski, Fabrizio Bertolazzi, Jeet Patel, Mary Mullins, Zachary T. Schug

Subject:

Subject ID:SU003265
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Age Or Age Range:60
Gender:Female
Cell Strain Details:BT-474, breast cancer cell line
Species Group:Mammals

Factors:

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

mb_sample_id local_sample_id Sample source Isotopic Tracer Drug Treatment
SA34075719_BT474_Gln_fasnallBT-474 [13C5] L-glutamine 1 uM Fasnall
SA34075816_BT474_Gln_fasnallBT-474 [13C5] L-glutamine 1 uM Fasnall
SA34075913_BT474_Gln_fasnallBT-474 [13C5] L-glutamine 1 uM Fasnall
SA34076017_BT474_Gln_GSKBT-474 [13C5] L-glutamine 1 uM GSK2194069
SA34076120_BT474_Gln_GSKBT-474 [13C5] L-glutamine 1 uM GSK2194069
SA34076214_BT474_Gln_GSKBT-474 [13C5] L-glutamine 1 uM GSK2194069
SA34076318_BT474_Gln_contrBT-474 [13C5] L-glutamine Vehicle
SA34076415_BT474_Gln_contrBT-474 [13C5] L-glutamine Vehicle
SA34076512_BT474_Gln_contrBT-474 [13C5] L-glutamine Vehicle
SA34076609_BT474_Gluc_fasnallBT-474 [13C6] D-glucose 1 uM Fasnall
SA34076706_BT474_Gluc_fasnallBT-474 [13C6] D-glucose 1 uM Fasnall
SA34076803_BT474_Gluc_fasnallBT-474 [13C6] D-glucose 1 uM Fasnall
SA34076904_BT474_Gluc_GSKBT-474 [13C6] D-glucose 1 uM GSK2194069
SA34077007_BT474_Gluc_GSKBT-474 [13C6] D-glucose 1 uM GSK2194069
SA34077110_BT474_Gluc_GSKBT-474 [13C6] D-glucose 1 uM GSK2194069
SA34077205_BT474_Gluc_contrBT-474 [13C6] D-glucose Vehicle
SA34077308_BT474_Gluc_contrBT-474 [13C6] D-glucose Vehicle
SA34077402_BT474_Gluc_contrBT-474 [13C6] D-glucose Vehicle
SA34075321_BlankBT-474 NA NA
SA34075401_BlankBT-474 NA NA
SA34075511_BlankBT-474 NA NA
SA34075601_Blank_20220515015111BT-474 NA NA
Showing results 1 to 22 of 22

Collection:

Collection ID:CO003258
Collection Summary:One PBS washing, 80% methanol extraction.
Collection Protocol Filename:DM_metabolomics_samples.txt
Sample Type:Cultured cells
Collection Method:80% methanol extraction
Volumeoramount Collected:500 ul
Storage Conditions:-80℃
Collection Vials:1.5 ml plastic centrifuge tubes
Storage Vials:1.5 ml plastic centrifuge tubes

Treatment:

Treatment ID:TR003274
Treatment Summary:Cells were grown in RPMI-1640 supplemented with [U-13C6] D-glucose or [U-13C5] L-glutamine and treated with 1 uM Fasnall or 1 uM GSK2194069.
Treatment Compound:Fasnall, GSK2194069
Treatment Dose:1 uM
Treatment Doseduration:24 h
Treatment Vehicle:DMSO
Cell Growth Container:6-cm Petri dishes
Cell Media:RPMI-1640
Cell Envir Cond:37C, 5% CO2
Cell Pct Confluence:~70%

Sample Preparation:

Sampleprep ID:SP003272
Sampleprep Summary:Metabolites were extracted with ice-cold 80% methanol.
Sampleprep Protocol Filename:DM_metabolomics_samples.txt
Extraction Method:80% methanol
Extract Enrichment:None
Extract Cleanup:None
Extract Storage:-80℃
Sample Resuspension:None
Sample Derivatization:None
Sample Spiking:None
Subcellular Location:Intracellular metabolites

Combined analysis:

Analysis ID AN005165 AN005166
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Thermo Dionex Ultimate 3000 RS Thermo Dionex Ultimate 3000 RS
Column Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um) Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive HF-X Orbitrap Thermo Q Exactive HF-X Orbitrap
Ion Mode NEGATIVE POSITIVE
Units AU AU

Chromatography:

Chromatography ID:CH003909
Instrument Name:Thermo Dionex Ultimate 3000 RS
Column Name:Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um)
Column Temperature:40
Flow Gradient:A linear solvent gradient of a total duration 22.5 min was starting with 0.2 ml/min flow rate of 80% solvent B, 12.5 min – 30%, 15 min – 30%, 15.2 min – 80%, 20 min – 80%, 21 min – flow rate 0.3 ml/min, 22 min – flow rate 0.3 ml/min, 22.1 min – flow rate 0.2 ml/min.
Flow Rate:0.2 ml/min
Solvent A:100% Water; 0.01% ammonium hydroxide; 20 mM ammonium bicarbonate
Solvent B:100% Acetonitrile
Chromatography Type:HILIC

MS:

MS ID:MS004901
Analysis ID:AN005165
Instrument Name:Thermo Q Exactive HF-X Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The HESI ion source voltage was set to the following parameters: 3,600 V in both polarity modes, sheath gas 30, auxiliary gas 5, spare gas 0, probe heater 200 °C, capillary temperature 325 °C, S-Lens RF level 65. The mass spectrometer was set to acquire data in the polarity-switching mode averaging two microscans with 60,000 resolution, automatic gain control (AGC) target 5e6, scan range 72-1080 m/z, and maximum orbitrap injection time (IT) 200 ms. Data were converted into the mzXML format by ProteoWizard and analyzed in MAVEN.
Ion Mode:NEGATIVE
Ion Source Temperature:200
Ion Spray Voltage:3600
Ionization:Both
Source Temperature:200
Automatic Gain Control:5e6
Dataformat:RAW
  
MS ID:MS004902
Analysis ID:AN005166
Instrument Name:Thermo Q Exactive HF-X Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The HESI ion source voltage was set to the following parameters: 3,600 V in both polarity modes, sheath gas 30, auxiliary gas 5, spare gas 0, probe heater 200 °C, capillary temperature 325 °C, S-Lens RF level 65. The mass spectrometer was set to acquire data in the polarity-switching mode averaging two microscans with 60,000 resolution, automatic gain control (AGC) target 5e6, scan range 72-1080 m/z, and maximum orbitrap injection time (IT) 200 ms. Data were converted into the mzXML format by ProteoWizard and analyzed in MAVEN.
Ion Mode:POSITIVE
Ion Source Temperature:200
Ion Spray Voltage:3600
Ionization:Both
Source Temperature:200
Automatic Gain Control:5e6
Dataformat:RAW
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