Summary of Study ST000055
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 PR000052. The data can be accessed directly via it's Project DOI: 10.21228/M8H01X This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST000055 |
| Study Title | Effect of kinase inhibitors on FLT3-ITD AML cell metabolomes |
| Study Summary | FLT3-ITD AML cells (MR2) obtained from mice were treated with two MEK kinase inhibitors (GSK and AZD) at 10 uM versus media only control. Conditioned media aliquots and cellular fractions comprised of two aliquots (tecnical replicates) for LC-MS metabolomic and one for Western blot analyses were collected at 0, 4, 24, and 48 hours. The experiment was repeated three times. HPLC-MS data were acquired for 24 samples from one biological experiment. |
| Institute | University of North Carolina |
| Department | Systems and Translational Sciences |
| Laboratory | Sumner Lab |
| Last Name | Sumner |
| First Name | Susan |
| Address | Eastern Regional Comprehensive Metabolomics Resource Core, UNC Nutrition Research Institute, 500 Laureate Way, Kannapolis, NC, 28081 |
| susan_sumner @unc.edu | |
| Phone | 704-250-5066 |
| Submit Date | 2014-06-06 |
| Num Groups | 12 |
| Total Subjects | 24 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Waters) |
| Uploaded File Size | 1.2 G |
| Analysis Type Detail | LC-MS |
| Release Date | 2015-06-01 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR000052 |
| Project DOI: | doi: 10.21228/M8H01X |
| Project Title: | Metabonomic profiling of kinase inhibitor response in leukemia |
| Project Type: | Effects of kinase inhibitors in acute myeloid leukemia to understand biohemical mechanisms of disease progression and drug resistance |
| Project Summary: | The goal of this pilot project is to develop the metabonomic parameters necessary to comprehensively profile and analyze large numbers of metabolites and their drug-induced responses in cell and animal models of leukemia. Our long-term objective of these studies is to investigate the effects of select kinase inhibitors in acute myeloid leukemia (AML) in an effort to better understand biochemical mechanisms of disease progression and drug resistance. Our focus will be on FLT3-ITD kinase positive models because this is one of the most aggressive and drug-resistant types of AML. Additionally, because of the central role of the MEK/Erk kinase pathway in regulating cell proliferation and survival in these cells, inhibitors of MEK and other kinases will be tested for their effects on human and mouse metabolic profiles. We have developed a unique model of drug-resistant AML and the metabolic response of these cells to kinase inhibitors will also be evaluated. Cells will be treated with kinase inhibitors for varied times, the cells isolated and the cellular metabolites identified and quantified by mass spectrometry and NMR-based analytical methods. Bioinformatics and statistics will be performed and a comprehensive metabonomic analysis of metabolite profiles will be accomplished. Metabonomic responses will be related to specific changes in cell signaling networks. A second major objective will be to perform metabonomic analyses in response to targeted kinase inhibitors in a mouse model of FLT3-ITD AML. We will measure metabonomic profiles in mouse biofluids before and after exposure to MEK inhibitors. The specific effects of MEK or other kinase inhibitors on individual metabolites will be quantified and analyzed by bioinformatics. We anticipate that these studies will lead to the identification of unique sites of intersection between cell signaling and cell metabolism and provide the foundation for future NIH-funded research projects. |
| Institute: | University of North Carolina at Chapel Hill |
| Department: | Department of Pharmacology |
| Laboratory: | Graves Laboratory |
| Last Name: | Graves |
| First Name: | Lee |
| Address: | Pharmacology, CB7365, 411 GMB Mason Farm Road, Chapel Hill, NC 27599 |
| Email: | LMG@med.unc.edu |
| Phone: | 919-966-0915 |
Subject:
| Subject ID: | SU000074 |
| Subject Type: | Animal cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | FLT3-ITD AML (MR2) |
| Cell Biosource Or Supplier: | Dr. Tim Pardee (Wake Forest Univ.) |
| Cell Primary Immortalized: | Primary |
| Species Group: | Mammal |
Factors:
Subject type: Animal cells; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Treatment | Collection Time (h) |
|---|---|---|---|
| SA002821 | MR2_AZD_205 | AZD inhibitor | 0 |
| SA002822 | MR2_AZD_206 | AZD inhibitor | 0 |
| SA002823 | MR2_AZD_253 | AZD inhibitor | 24 |
| SA002824 | MR2_AZD_254 | AZD inhibitor | 24 |
| SA002825 | MR2_AZD_230 | AZD inhibitor | 4 |
| SA002826 | MR2_AZD_229 | AZD inhibitor | 4 |
| SA002827 | MR2_AZD_280 | AZD inhibitor | 48 |
| SA002828 | MR2_AZD_279 | AZD inhibitor | 48 |
| SA002829 | MR2_GSK_214 | GSK inhibitor | 0 |
| SA002830 | MR2_GSK_213 | GSK inhibitor | 0 |
| SA002831 | MR2_GSK_262 | GSK inhibitor | 24 |
| SA002832 | MR2_GSK_263 | GSK inhibitor | 24 |
| SA002833 | MR2_GSK_238 | GSK inhibitor | 4 |
| SA002834 | MR2_GSK_237 | GSK inhibitor | 4 |
| SA002835 | MR2_GSK_289 | GSK inhibitor | 48 |
| SA002836 | MR2_GSK_288 | GSK inhibitor | 48 |
| SA002837 | MR2_CTL_198 | Media alone | 0 |
| SA002838 | MR2_CTL_197 | Media alone | 0 |
| SA002839 | MR2_CTL_246 | Media alone | 24 |
| SA002840 | MR2_CTL_245 | Media alone | 24 |
| SA002841 | MR2_CTL_222 | Media alone | 4 |
| SA002842 | MR2_CTL_221 | Media alone | 4 |
| SA002843 | MR2_CTL_270 | Media alone | 48 |
| SA002844 | MR2_CTL_271 | Media alone | 48 |
| Showing results 1 to 24 of 24 |
Collection:
| Collection ID: | CO000057 |
| Collection Summary: | Conditioned Medias:Cellular Fraction_LC-MS:Cellular Fraction_WB |
| Collection Protocol Comments: | Processed simultaneously/time point:Processed simultaneously/time point:Processed simultaneously/time point |
| Sample Type: | conditioned treatment media |
| Collection Method: | Total contents pipette-transferred to tubes & centrifuged at 1800 rpm for 5 minutes. Medias transferred into aliquot tubes.:Cellular fractions pelleted & resuspended in 1X PBS. Aliqotted into collection vials based on cell counts. Re-centrifuged at 8,000 rpm for 3 min at 4 °C. PBS was aspirated off pellets for storage.:Cellular fractions pelleted & resuspended in 1X PBS. Aliqotted into collection vial based on cell counts. Re-centrifuged at 8,000 rpm for 3 min at 4 °C. PBS was aspirated off pellets for storage. |
| Collection Location: | RTI RCMRC Tissue culture suite:RTI RCMRC Wet lab:RTI RCMRC Wet lab |
| Collection Frequency: | 0h, 4h, 24h and 48h:0h, 4h, 24h and 48h:0h, 4h, 24h and 48h |
| Collection Duration: | 10 minutes:10 minutes:5 minutes |
| Collection Time: | 0h, 4h, 24h and 48h:0h, 4h, 24h and 48h:0h, 4h, 24h and 48h |
| Volumeoramount Collected: | 1 mL x 5 aliquots:2x10^7 cells:2x10^6 cells |
| Storage Conditions: | -80C:-80C:-80C |
| Collection Vials: | 15 mL conical tubes:15 mL conical tubes:15 mL conical tubes |
| Storage Vials: | 1.5 mL Eppendorf Protein Lo-Bind tubes:1.5 mL Eppendorf Protein Lo-Bind tubes:1.5 mL Eppendorf Protein Lo-Bind tubes |
| Collection Tube Temp: | chilled in ice:chilled in ice:chilled in ice |
Treatment:
| Treatment ID: | TR000075 |
| Treatment Summary: | MEK kinase inhibitors |
| Treatment Compound: | GSK | AZD |
| Treatment Route: | added to media |
| Treatment Dose: | 10nM | 10nM |
| Treatment Vehicle: | dilutions made in 1X sterile PBS (orginally dissolved in DMSO) |
| Cell Storage: | liquid nitrogen, live maintenance in 5% CO2, humidified incubator |
| Cell Growth Container: | T-75 culture flask |
| Cell Growth Config: | suspension |
| Cell Growth Rate: | double ~every 20 hours |
| Cell Media: | 50:50 DMEM:IMDM + 10% FBS, 1% P/S |
| Cell Harvesting: | Transfer 1 mL to new flask containing 10 mL of fresh culture media |
| Cell Pct Confluence: | maintained at ~ 2 x10^6/mL |
Sample Preparation:
| Sampleprep ID: | SP000070 |
| Sampleprep Summary: | Samples were extracted using 90:10 methanol:chloroform. For reversed phase, samples were dried down and reconstituted in 95:5 water:methanol. For HILIC, samples were directly transferred to autosampler vials. |
| Sampleprep Protocol Filename: | RTI_GRAVES_Metabolomics_Procedure.docx |
| Extraction Method: | 90:10 methanol:chloroform |
| Extract Storage: | -80 C |
| Sample Resuspension: | 95:5 Water:Methanol |
| Sample Spiking: | L-Tryptophan-d5 |
| Cell Type: | FLT3-ITD AML (myeloid) |
Combined analysis:
| Analysis ID | AN000093 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Waters Acquity |
| Column | Waters Acquity HSS T3 (100 x 2.1mm,1.8um) |
| MS Type | ESI |
| MS instrument type | QTOF |
| MS instrument name | Waters Synapt G2 |
| Ion Mode | POSITIVE |
| Units | Normalized abundance |
Chromatography:
| Chromatography ID: | CH000061 |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters Acquity HSS T3 (100 x 2.1mm,1.8um) |
| Column Pressure: | 6000-10000 |
| Column Temperature: | 50 C |
| Flow Gradient: | Time(min) Flow Rate %A %B Curve ; 1. Initial 0.400 100.0 0.0 ; 2. 1.00 0.400 100.0 0.0 6 ; 3. 16.00 0.400 0.0 100.0 6 ; 4. 20.00 0.400 0.0 100.0 6 ; 5. 22.00 0.400 100.0 0.0 6 |
| Flow Rate: | 0.4 mL/min |
| Injection Temperature: | 8 C |
| Internal Standard: | L-Tryptophan-d5 |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% methanol; 0.1% formic acid |
| Analytical Time: | 22 min |
| Weak Wash Solvent Name: | 5%MeOH |
| Weak Wash Volume: | 1000 uL |
| Strong Wash Solvent Name: | 80%MeOH |
| Strong Wash Volume: | 1000 uL |
| Target Sample Temperature: | 8 C |
| Sample Loop Size: | 10 uL |
| Sample Syringe Size: | 100 uL |
| Randomization Order: | Yes |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS000070 |
| Analysis ID: | AN000093 |
| Instrument Name: | Waters Synapt G2 |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 110 C |
| Capillary Voltage: | 3.2 kV |
| Collision Energy: | 4 |
| Helium Flow: | 180 |
| Ionization: | ES+ |
| Mass Accuracy: | 10 ppm |
| Source Temperature: | 110 C |
| Dataformat: | Continuum |
| Desolvation Gas Flow: | 400 L/Hr |
| Desolvation Temperature: | 400 C |
| Resolution Setting: | 18000 |
| Scan Range Moverz: | 50-1000 m/s |
| Scanning Cycle: | 1 s |
| Tube Lens Voltage: | 75 |
