Summary of Study ST000055

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench,, 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.


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Study IDST000055
Study TitleEffect of kinase inhibitors on FLT3-ITD AML cell metabolomes
Study SummaryFLT3-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.
University of North Carolina
DepartmentSystems and Translational Sciences
LaboratorySumner Lab
Last NameSumner
First NameSusan
AddressEastern Regional Comprehensive Metabolomics Resource Core, UNC Nutrition Research Institute, 500 Laureate Way, Kannapolis, NC, 28081
Submit Date2014-06-06
Num Groups12
Total Subjects24
Raw Data AvailableYes
Raw Data File Type(s)raw(Waters)
Uploaded File Size1.2 G
Analysis Type DetailLC-MS
Release Date2015-06-01
Release Version1
Susan Sumner Susan Sumner application/zip

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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


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


Subject type: Animal cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Treatment Collection Time (h)
SA002821MR2_AZD_205 AZD inhibitor 0
SA002822MR2_AZD_206 AZD inhibitor 0
SA002823MR2_AZD_253 AZD inhibitor 24
SA002824MR2_AZD_254 AZD inhibitor 24
SA002825MR2_AZD_230 AZD inhibitor 4
SA002826MR2_AZD_229 AZD inhibitor 4
SA002827MR2_AZD_280 AZD inhibitor 48
SA002828MR2_AZD_279 AZD inhibitor 48
SA002829MR2_GSK_214 GSK inhibitor 0
SA002830MR2_GSK_213 GSK inhibitor 0
SA002831MR2_GSK_262 GSK inhibitor 24
SA002832MR2_GSK_263 GSK inhibitor 24
SA002833MR2_GSK_238 GSK inhibitor 4
SA002834MR2_GSK_237 GSK inhibitor 4
SA002835MR2_GSK_289 GSK inhibitor 48
SA002836MR2_GSK_288 GSK inhibitor 48
SA002837MR2_CTL_198 Media alone 0
SA002838MR2_CTL_197 Media alone 0
SA002839MR2_CTL_246 Media alone 24
SA002840MR2_CTL_245 Media alone 24
SA002841MR2_CTL_222 Media alone 4
SA002842MR2_CTL_221 Media alone 4
SA002843MR2_CTL_270 Media alone 48
SA002844MR2_CTL_271 Media alone 48
Showing results 1 to 24 of 24


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 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 instrument type QTOF
MS instrument name Waters Synapt G2 QTOF
Units Normalized abundance


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 ID:MS000070
Analysis ID:AN000093
Instrument Name:Waters Synapt G2 QTOF
Instrument Type:QTOF
Capillary Temperature:110 C
Capillary Voltage:3.2 kV
Collision Energy:4
Helium Flow:180
Mass Accuracy:10 ppm
Source Temperature:110 C
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