Summary of Study ST003147
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.
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 | ST003147 |
| Study Title | BT-474 cells fed with [13C2] acetate and treated with 1 uM Fasnall or 1 uM GSK2194069 for 24 h |
| Study Type | Intracellular metabolomics, [13C2] acetate |
| Study Summary | BT-474 breast cancer cells were fed with [13C2] acetate and treated with 1 uM Fasnall or 1 uM GSK2194069 for 24 h. Cells were grown in RPMI-1640 with 10% dialyzed FBS. |
| 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, PA 19104, USA |
| dmukha@wistar.org | |
| Phone | 2154956903 |
| Submit Date | 2024-03-09 |
| Num Groups | 3 |
| Total Subjects | 9 |
| Publications | Submission Pending |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzXML, raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-04-02 |
| Release Version | 1 |
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: | SU003264 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Age Or Age Range: | 60 |
| Gender: | Female |
| Cell Biosource Or Supplier: | ATCC |
| Cell Strain Details: | BT-474, breast cancer cell line |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | [13C2] Acetate | Drug Treatment |
|---|---|---|---|---|
| SA340740 | m22-L025_SO_04 | BT-474 | 200 uM | 1 uM Fasnall |
| SA340741 | m22-L025_SO_05 | BT-474 | 200 uM | 1 uM Fasnall |
| SA340742 | m22-L025_SO_06 | BT-474 | 200 uM | 1 uM Fasnall |
| SA340743 | m22-L025_SO_08 | BT-474 | 200 uM | 1 uM GSK2194069 |
| SA340744 | m22-L025_SO_09 | BT-474 | 200 uM | 1 uM GSK2194069 |
| SA340745 | m22-L025_SO_07 | BT-474 | 200 uM | 1 uM GSK2194069 |
| SA340746 | m22-L025_SO_03 | BT-474 | 200 uM | NA |
| SA340747 | m22-L025_SO_02 | BT-474 | 200 uM | NA |
| SA340748 | Blank-80MeOH_20220211171023 | BT-474 | NA | NA |
| SA340749 | Blank-80MeOH_20220211180427 | BT-474 | NA | NA |
| SA340750 | m22-L025_SO_01 | BT-474 | NA | NA |
| SA340751 | Blank-80MeOH | BT-474 | NA | NA |
| SA340752 | Blank_NoFlow | BT-474 | NA | NA |
| Showing results 1 to 13 of 13 |
Collection:
| Collection ID: | CO003257 |
| 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: | TR003273 |
| Treatment Summary: | Cells were grown in RPMI-1640 supplemented with 200 uM [13C2] acetate and treated with 1 uM Fasnall or 1 uM GSK2194069. |
| Treatment Compound: | Fasnall, GSK2194069 |
| Treatment Dose: | 0 - 1 uM |
| Treatment Doseduration: | 24 h |
| Treatment Vehicle: | DMSO |
| Cell Growth Container: | 6-well plates |
| Cell Media: | RPMI-1640 |
| Cell Envir Cond: | 37C, 5% CO2 |
| Cell Pct Confluence: | ~70% |
Sample Preparation:
| Sampleprep ID: | SP003271 |
| 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 | AN005163 | AN005164 |
|---|---|---|
| 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: | CH003908 |
| 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: | MS004899 |
| Analysis ID: | AN005163 |
| 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: | MS004900 |
| Analysis ID: | AN005164 |
| 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 |
