A Metabolomics Pilot Study On Desmoid Tumors And Novel .
A Metabolomics Pilot Study onDesmoid Tumors and Novel DrugCandidatesKelly MercierSept 2017RTI International is a registered trademark and a trade name of Research Triangle Institute.www.rti.org
Study DesignStudy aims: To understand the underlying metabolomic differences in paired normalfibroblast and desmoid cells and media from affected patients and normalfibroblast cells from unaffected patients. To investigate the metabolic response to novel desmoid therapies identifiedby the Desmoid Collaboration for a CureCell Growth Conditions All growth conditions previously descripted by the Alman groupPrimary 1 (T41A)Primary 2 (S45F)Unaffected PrimaryFibroblast CellsUntreatedDMSODastinibDesmoid CellsUntreatedDMSODastinibFibroblast CellsUntreatedDMSODastinibDesmoid CellsUntreatedDMSODastinibFibroblast CellsUntreatedDMSODastinibFAK Inhibitor 14FAK Inhibitor 14FAK Inhibitor 14FAK Inhibitor 14FAK Inhibitor 14
Sample Preparation and Data Collection Cells were homogenized in acetonitrile/water (50:50, v/v) with the final cell countapproximately 10x10 6 cells for each sample. An analytical quality control (QC) total pool was created from 6 of the cell samples.Aliquots from these cell lysate samples were combined, divided into three totalpool aliquots, and processed identically to the cell lysate study samples. All study and pool samples were lyophilized to dryness and reconstituted in a 0.2Mphosphate buffer, pH 7.4, in D2O with 10% Chenomx ISTD.
NMR Data Collection and Processing 1H1D NOESY NMR spectra cell samples were acquired on a Bruker 700 MHzNMR spectrometer (David H. Murdock Research Institute in Kannapolis, NC,USA)3 spectra were collected for each study and pool sampleNMR spectra were processed using TopSpin 3.2 software (Bruker-Biospin,Germany) and binned with ACD NMR Processor (ACD Labs Inc, Toronto,Canada).Spectra were:– Zero filled– Fourier transformed after 0.5 Hz EM line broadening– Phase and baseline of the spectra were manually corrected for each spectrum.– Referenced internally to the DSS-d6 signal.Integrals of each of the bins were normalized to total integral of each of thespectrum using intelligent bucket integration of 0.04 ppm bucket width with 50%loosenessNormalized binned NMR data were scaled and centered prior to multivariateanalysis.Spectra were analyzed for resonances associated with the drugs and drugmetabolites.
NMR Data Analysis Multivariate data analysis methods (e.g. principal component analysis [PCA],orthogonal partial least squares discriminant analysis [OPLS-DA]) were usedto enable the visualization of the separation of the study groups.NMR metabolite bins with a VIP 1.0 with a jack-knife confidence intervalthat did not include 0 were included in differentiating the study groups.Chenomx NMR Suite 7.7 Professional software (Edmonton, Alberta,Canada), which has a concentration library of approximately 350compounds, was used to match the signals in the identified bins tometabolites.Concentrations were normalized to the cell count of each sample.Descriptive statistics and two-sided t-tests, using the Satterthwaiteapproximation for unequal variances, were conducted for the binned NMRdata. The exact Wilcoxcon was used when cell counts were small.Metabolites identified by NMR metabolomics were analyzed for pathwayenrichment, using canonical and metabolic pathways in the MetaCoremodule in GeneGo software (Chicago, IL).
Untreated comparisons
Cells: Unsupervised multivariate analysis untreatedTumorNormalUnaffectedN141T141N219T219R2X 0.97 Q2 0.89It is not unexpected that the normal and unaffected fibroblast cells differentiate.
Tumor vs Normal Supervised multivariate analysisT219N141N219R2X 0. R2Y 0.Q2 T141TumorNormalR2X 0.71 R2Y 0.82 Q2 0.62Library-Matched Metabolitesmyo-Inositol Glucose TaurineAlanine LipidsArginine LysineAspartate (3 bins)Choline O-Phosphocholine O-Acetylcholine sn-Glycero-3-phosphocholineCreatine Creatinine Lysine TyrosineGlucose-1-phosphateGlucose-1-phosphate CreatineGlucose-1-phosphate GlutamateGlucose-1-phosphate Glutamate AlanineGlucose-1-phosphate SerineGlucose Glucose-1-phosphate (2 bins)Glucose Glucose-1-phosphate TaurineGlucose myo-Inositol GlycineGlutamateGlutamate GlutamineGlutamate ProlineGlutamineGlutamine PyroglutamateGlutathione Lysine LipidsGlycerol UnknownLactate ThreonineLactate O-Phosphocholine Proline UridineLeucineLeucine IsoleucineLeucine Lysine Lipids (2 bins)Lipids (3 bins)Proline (2 bins)Pyroglutamate Glutamate ProlineSerine Unknown (2 bins)Unknown (6 bins)Uridine (2 bins)ValineValine IsoleucineValine Lipidsmyo-Inositolmyo-Inositol Cholinemyo-Inositol ThreonineVIP1.421.90.7 - 0.9p-value*FC**0.81510.39710.3971.10.017 - 0.071 -1.1 - -1.121.72.72.71.83.32.41.4 - 32.33.50.81.721.70.411.41.70.90.72.31.4 - 2.10.5 - 1.21.1 - 1.21.61.9 - 2.40.1 - 0.51 - 111.1110.815-1 - 2551.10.32 - 0.586 1.1 - 1.10.093 - 0.3971 - 1.10.0931.1 - 1.20.0931.10.071 - 0.586 1.1 - 1.10.053 - 0.317 -19.5 - -2.10.697 - 1-1.1 - Based on the median values (Tumor - Normal)**Wilcoxon Rank Sum Test***A positive fold change indicates median of Tumor median of Normal
Normal vs Unaffected Supervised multivariate analysisNormal vs UnaffectedNormalR2X 0.97 R2Y 1.0 Q2 1.0UnaffectedN141N219Library-Matched MetabolitesVIPp-value*FC**1.50.2 - 0.71.81.70.6 - 0.90.7 - 10.0240.024 - 0.0480.0240.0240.0240.0241.2-3.4 - -1.3-1.4-1.4-1.7 - -1.4-1.4 - -1.31.80.024-1.31.70.50.6 - 12.90.0240.0240.024 - 0.0480.024-1.6-1.11.6 - se Glucose-1-phosphate (2 bins)1.5 - 3.30.0244 - 5.5Glucose Glucose-1-phosphate TaurineGlucose myo-Inositol GlycineGlutamate (2 bins)2.83.10.7 - 0.90.0240.0240.0242.12-1.3 - -1.2Glutamate Glutamine GlutathioneGlutamate ProlineGlutamineGlutathione (3 bins)Glutathione Lysine LipidsGlycerol UnknownLactate Threonine0.921.20.6 - 2-1.21.2-1.6 - -1.3-1.31.3-1.4Lactate O-Phosphocholine Proline UridineLeucine (3 bins)1.10.7 - 0.80.0240.024-1.2-1.3 - -1.3myo-Inositol Glucose Taurine1-Methylnicotinamide (2 bins)Alanine LipidsArginine LysineAsparagine (4 bins)Aspartate (3 bins)Choline O-Phosphocholine O-Acetylcholine snGlycero-3-phosphocholineCreatine Creatinine Lysine TyrosineDimethylamineGlucose (4 bins)Glucose-1-phosphateGlucose-1-phosphate CreatineGlucose-1-phosphate GlutamateGlucose-1-phosphate Glutamate AlanineGlucose-1-phosphate SerineLeucine IsoleucineLeucine Lysine Lipids (2 bins)Lipids (30 bins)Lipids GlycocholateLysine LipidsLysine ProlineMethanol ProlineMethionine2.21.4 - 1.90.3 - 1.20.70.70.80.90.50.0240.0240.024 - 0.0950.0240.0240.0240.0240.024-1.4-1.4 - -1.4-2 - -1.1-1.2-1.4-1.4-1.31.1O-Phosphocholine PyroglutamatePhenylalanine (5 bins)Proline (3 bins)0.60.3 - 0.90.6 - 1.10.0240.0240.024-1.3-1.5 - -1.3-1.4 - -1.4Pyroglutamate Glutamate ProlinePyroglutamate GlutamineRibose LactateSerine Unknown (2 bins)ThreonineTyramineTyrosine (3 bins)Unknown (14 bins)Uracil (2 bins)Uridine (2 bins)ValineValine IsoleucineValine Lipidsmyo-Inositolmyo-Inositol Cholinemyo-Inositol Threoninep-Methylhistidine (2 bins)1.70.80.81.7 - 1.90.90.60.6 - 0.70.2 - 0.80.5 - 0.60.7 - 0.91.31.41.211.62.30.3 - 0.40.0240.0240.0240.0240.0240.0240.0240.024 - 10.0240.0240.0240.0240.0240.0240.0240.0240.024 - 0.095-1.2-1.3-1.2-1.6 - -1.5-1.2-1.7-1.4 - -1.3-3.3 - 2.3-8.7 - -3.83.4 - 22-1.4-1.3-1.31.91.41.8-1.4 - -1.2*Exact Wilcoxon Rank Sum Test**A positive fold change indicates median of Normal median of UnaffectedVIP Variable Influence on Projection
Tumor and Unaffected Supervised multivariate analysisTumorUnaffectedT141R2X 0.89 R2Y 0.99 Q2 0.99T219Library-Matched Metabolitesmyo-Inositol Glucose Taurine1-Methylnicotinamide (3 bins)Alanine LipidsArginine LysineAsparagine (3 bins)Aspartate (3 bins)Choline O-Phosphocholine OAcetylcholine sn-Glycero-3phosphocholineCreatine Creatinine Lysine cose-1-phosphate CreatineGlucose-1-phosphate GlutamateGlucose-1-phosphate Glutamate AlanineGlucose-1-phosphate SerineGlucose Glucose-1-phosphate (2 bins)Glucose Glucose-1-phosphate TaurineGlucose myo-Inositol GlycineGlutamate (2 bins)Glutamate Glutamine GlutathioneGlutamate ProlineGlutamine PyroglutamateGlutathione (2 bins)Glutathione Lysine LipidsGlycerol UnknownIsoleucineIsoleucine 3-HydroxyisovalerateLactate ThreonineVIP1.50.3 - 0.61.81.80.6 - 0.70.9 - 1.3p-value*Fold Change**0.3811.20.024 - 0.095-6.1 - -1.20.024-1.30.024-1.40.024 - 0.095-1.4 - -1.20.024-1.6 - -1.40.91.70.70.63.33.32.243.21.6 - 3.73.51.70.5 - 0.70.91.50.60.7 - .0240.0240.0240.02410.024 - 0.0480.0240.3810.0240.048 - 12.61.51.84.3 - 5.32.51.2-1.2 - -1.1-1.1-1.1-1.2-1.2 - -1.2-1.2-1-1.3-1.3-1.2Lactate O-Phosphocholine Proline UridineLeucine (3 bins)Leucine IsoleucineLeucine Lysine Lipids (2 bins)Lipids (29 bins)Lysine LipidsLysine ProlineMethanol ProlineO-Phosphocholine PyroglutamatePhenylalanine (3 bins)Proline (3 bins)Pyroglutamate Glutamate ProlinePyroglutamate GlutaminePyroglutamate GlutathioneRibose LactateSerine Unknown (2 bins)ThreonineTyrosine (2 bins)Unknown (14 bins)Uracil (2 bins)Uridine (2 bins)ValineValine IsoleucineValine Lipidsmyo-Inositol Cholinemyo-Inositol Threoninep-Methylhistidine (2 bins)0.60.6 - 0.72.11.4 - 1.90.4 - 1.20.70.70.90.60.6 - 0.90.8 - 11.30.80.70.81.5 - 1.80.80.6 - 0.60.3 - 0.90.4 - 0.70.7 - 11.51.31.211.20.3 - 0.40.0950.0240.0240.0240.024 - 0.5480.0240.0240.0240.0240.024 - 0.0950.0240.5480.0240.0240.0240.024 - 0.1670.0480.0240.024 - 0.1550.012 - 0.1550.0240.0240.0240.024110.095-1.1-1.2 - -1.2-1.3-1.4 - -1.3-2.6 - -1.1-1.2-1.2-1.2-1.4-1.4 - -1.2-1.6 - -1.2-1.1-1.4-1.3-1.2-1.4 - -1.3-1.2-1.2 - -1.1-20.2 - -1.1-2.5 - 03.2 - 22.4-1.4-1.3-1.2-1.11-1.4 - -1.1*Exact Wilcoxon Rank Sum Test**A positive fold change indicates median of Tumor median of UnaffectedVIP Variable Influence on Projection
Semi-Quantified MetabolitesTumor vs utaminePyroglutamateNormal vs lutamate*Wilcoxon Rank Sum Test**A positive fold change indicates median ofTumor median of Normalp-value*0.0240.0240.095FC**55.51.4-1.3*Exact Wilcoxon Rank Sum Test**A positive fold change indicates medianof Normal median of UnaffectedTumor vs 24-1.9Tyrosine0.095-1.4*Exact Wilcoxon Rank Sum Test**A positive fold change indicates median of Tumor median of UnaffectedCell line 141 (T41A)Tumor vs NormalCell line 219 (S45F)Tumor vs NormalSemi-Quantified Metabolites p-value*FC**Semi-Quantified Tyrosine0.1-1.5Valine0.1-1.40.11.6Uridine*Exact Wilcoxon Rank Sum Test**A positive fold change indicates median of Das. median of DMSO
Pathway AnalysisOrange: Tumor vs NormalBlue: Tumor vs UnaffectedRed: Normal vs Unaffected
Aminoacyl-tRNA biosynthesis in mitochondrion1. Tumor vs Normal2. Tumor vs Unaffected3. Normal vs UnaffectedNature Reviews Cancer 11, 708-718(October 2011) doi:10.1038/nrc3124Aminoacyl-tRNA synthetases andtumorigenesis: more thanhousekeepingEMBO Mol Med. 2013Mar;5(3):332-43. doi:10.1002/emmm.201100626. Epub2013 Feb 21.Aminoacyl-tRNA synthetases inmedicine and disease.Proc Natl Acad Sci U S A. 2008 Aug12;105(32):11043-9. doi:10.1073/pnas.0802862105. Epub 2008Aug 5.Aminoacyl tRNA synthetases andtheir connections to disease.
mTORC1 signal transduction1. Tumor vs Normal2. Tumor vs Unaffected3. Normal vs UnaffectedCancer Cell. 2016 Apr 11;29(4):548-62. doi:10.1016/j.ccell.2016.02.018. Epub 2016 Mar 24.mTORC1-Dependent MetabolicReprogramming Underlies Escape fromGlycolysis Addiction in Cancer Cells.Sci Rep. 2015 Nov 24;5:17046. doi:10.1038/srep17046.PRL-3 activates mTORC1 in CancerProgression.Int J Mol Sci. 2012; 13(2): 1886–1918.Published online 2012 Feb 10.doi: 10.3390/ijms13021886PMCID: PMC3291999The mTOR Signalling Pathway in HumanCancer
T41A vs S45FS45FT41A*Based on the median values (219 - 141)**Exact Wilcoxon Rank Sum Test***A positive fold change indicatesmedian of 219 median of 141R2X 0.94 R2Y 0.98 Q2 0.96Clin Cancer Res. 2005 Sep 15;11(18):6550-7.Identification of nicotinamide Nmethyltransferase as a novel serum tumormarker for colorectal cancer.Crit Rev Clin Lab Sci. 1993;30(3):223-72.Protein and amino acid metabolism incancer cachexia: investigative techniquesand therapeutic interventions.Semi-Quantified d Metabolitesmyo-Inositol Glucose Taurine1-Methylnicotinamide (4 bins)Alanine LipidsArginine LysineAsparagine (4 bins)Aspartate (3 bins)Choline O-Phosphocholine O-Acetylcholine sn-Glycero-3-phosphocholineCreatine Creatinine Lysine TyrosineGlucose (3 bins)Glucose-1-phosphateGlucose-1-phosphate CreatineGlucose-1-phosphate GlutamateGlucose-1-phosphate Glutamate AlanineGlucose-1-phosphate SerineGlucose Glucose-1-phosphate (2 bins)Glucose Glucose-1-phosphate TaurineGlucose myo-Inositol GlycineGlutamate GlutamineGlutamate ProlineGlutamineGlutathione (3 bins)Leucine Lysine Lipids (2 bins)Lipids (16 bins)Lipids GlycocholateMethanol ProlinePhenylalanine (2 bins)Proline (2 bins)Pyroglutamate Glutamate ProlineSerine UnknownTyramineUnknown (11 bins)ValineValine Lipidsmyo-Inositolmyo-Inositol Cholinemyo-Inositol Threoninep-MethylhistidineVIP2.40.2 - 0.51.51.60.8 - 10.9 - 1.2p-value*0.10.1 - 0.20.10.10.10.1FC**1.42.1 - 4.91.21.21.2 - 1.61.2 - 1.31.31.10.6 - 1.132.91.84.32.61.4 - 2.51.93.22.62.51.61 - 1.31.1 - 1.20.5 - 1.70.91.11.1 - 1.20.7 - 0.92.22.30.80.2 - 0.10.10.10.10.10.1 - 0.40.10.10.10.10.10.10.10.1 - 0.70.10.10.10.10.10.71.11.11.1 - 1.9-1.9-1.4-1.4-1.3-1.3-1.6 - -1.3-1.21.8-1.2-1.1-1.31.3 - 3.31.1 - 1.1-1.3 - 3.1-1.21.31.3 - 1.41.1 - 1.7-1.11.41.61.1 - 9.91.31.121.41.74.5
Treated comparisonsDas (Dasatinib)FAK Inhibitor 14
Tumor: Das vs line
Response to Das Venn hanolRiboseAlanineLipids
Response to FAK Venn holateMethionine
To Be Continued Progress–Evaluated a small number of cells and exposures to novel desmoid tumortherapies– Preliminary data provides clues as to mechanisms important todifferentiating groups Future––––Many, many more cell lines and replicates.Evaluate cell growth media for secreted metabolites and proteinsInvestigate Leucyl-tRNA synthetase (LARS) as a sensor for mTORC1 withthe leucine supplementationFollow glutamine or glucose metabolism with 13C media labeling
ContributionsRTI Systems and Translational SciencesKelly Mercier, PhDZach Acuff, BSAcknowledgments:Mushriq Al-JazraweRaymond PoonEastern Regional NIH Common Fund MetabolomicsCoreThe metabolomics study was performed under the New InvestigatorAward (Desmoid Tumor Research Foundation).The Desmoid Collaboration for a Cure (Alman) provided the treatedand untreated cell lysates (Desmoid Tumor Research Foundation).
Aug 16, 2015 · NMR Data Collection and Processing 1H 1D NOESY NMR spectra cell samples were acquired on a Bruker 700 MHz NMR spectrometer (David H. Murdock Research Institute in Kannapolis, NC, USA) 3 spectra were collected for each study and pool sample NMR spectra were processed using TopSpin 3.2 software (Bruker-Biospin, Germany) and binned with
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