Comparative genomic hybridization to structurally characterize a new-generated dasatinib and imatinib mouse BCR-ABL resistant cell line
Ontology highlight
ABSTRACT:
INSTRUMENT(S): G2505A DNA microarray scanner [Agilent]
ORGANISM(S): Mus musculus
SUBMITTER: bruno quesnel
PROVIDER: E-MEXP-997 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Gene expression profile variation between 4 BCR-Abl transduced cell lines: <br> 1-Mouse DA1-3b cell line as reference, <br> 2-DR1 imatinib resistant clone with E255K BCR-abl mutation<br> 3-DRBMSR 2 dasatinib resistant clone with E255K and T315I BCR-Abl mutation.<br> 4-DRBMSR 7 dasatinib resistant clone with E255K and T315I and V299L BCR-Abl mutation<br> <br> BCR-ABL is an oncogenic tyrosine kinase involved in the development of chronic myelogenous leukaemia (CML).
2007-08-01 | E-MEXP-772 | biostudies-arrayexpress
Project description:Our objective is to decipher a miRNA expression signature associated with Chronic myeloid leukemia (CML), with or without Imatinib or Dasatinib and compared to normal blood leukocytes, and to determine potential target genes and signaling pathways affected by these signature miRNAs. Background/Aims: MicroRNAs (miRNAs) are short non-coding regulatory RNAs that control gene expression and play an important role in cancer development and progression. However, little is known about the role of miRNAs in chronic myeloid leukemia (CML). Our objective is to decipher a miRNA expression signature associated with CML and to determine potential target genes and signaling pathways affected by these signature miRNAs. Results: Using miRNA microarrays we characterized the miRNAs expression profile of K562 cells in reference to healthy blood. We also looked into the expression profile of K562 cells treated with imatinib or dasatinib. We identified a miRNA signature that distinguishes K562 cells from healthy blood that included downregulation of miRNAs such as miR-31, miR-34a, miR-143, miR-145, miR-155, miR-196b and miR-564 and upregulation of miR-128. We also identified a miRNA signature that distinguishes untreated K562 cells from the treated ones that included downregulation of miRNAs such as miR-128 and upregulation of miR-564. Results: Using miRNA microarrays we characterized the miRNAs expression profile of K562 cells in reference to healthy blood. We also looked into the expression profile of K562 cells treated with imatinib or dasatinib. We identified a miRNA signature that distinguishes K562 cells from healthy blood that included downregulation of miRNAs such as miR-31, miR-34a, miR-143, miR-145, miR-155, miR-196b and miR-564 and upregulation of miR-128. We also identified a miRNA signature that distinguishes untreated K562 cells from the treated ones that included downregulation of miRNAs such as miR-128 and upregulation of miR-564. We next analyzed predicted targets and affected pathways of the deregulated miRNAs. Reassuringly, the analysis identified CML as the main disease associated with these miRNAs. MAPK, TGF-beta and Wnt were the main molecular pathways related with these expression patterns. Utilizing Venn diagrams we found appreciable overlap between the CML-related miRNAs and the MAPK and TGF-beta signaling pathways and focal adhesion-related miRNAs. Conclusions: The miRNAs identified in this study might offer a pivotal role in CML. Nevertheless, while these data point to a central disease, the precise molecular pathway/s targeted by these miRNAs is variable implying a high level of complexity of miRNA target selection and regulation. These deregulated miRNAs highlight new candidate gene targets allowing for a better understanding of the molecular mechanism underlying the development of CML, and propose possible new avenues for therapeutic treatment. MiRNA profiling of CML: With the objective of deciphering a potential miRNA expression signature associated with CML, we analyzed the miRNAs expression profile of K562 in reference to a pool of 3 healthy blood samples using an miRNA-based microarray chip assay. In addition, to search for Bcr-Abl-dependent or Src-dependant miRNA expression patterns, K562 cells were treated or not with imatinib or with dasatinib to inhibit Bcr-Abl tyrosine kinase activity or Bcr-Abl and Src tyrosine kinase activity, respectively. In order to validate the microarray data and to ensure that the variability observed among samples was not technical, we carried out Taqman miRNA quantitative real-time PCR analysis (Applied Biosystems, USA) on miRNAs that we find to be most relevant, according to bioinformatics analysis (see below) and published data.
2011-10-20 | E-GEOD-28825 | biostudies-arrayexpress
Project description:Using a three-pronged MS-based proteomic approach, we assessed the mode-of-action of four clinically-relevant tyrosine kinase inhibitors in an epithelial cancer cell line. Here we defined the: (I) semi-quantitative tyrosine kinome; (II) drug interactome; and (III) drug-induced effects on the tyrosine signalome. We first examined the entire proteome and kinome profile expressed in the A431 cell line, and determined the relative abundance of all possible protein targets of the tyrosine kinase inhibitors. We therefore utilized a ‘shot-gun’ approach that involved a multidimensional separation-based proteomic strategy. To ensure maximal protein identification, we reduced sample complexity by strong cation exchange (SCX) chromatography, and analyzed all fractions by LC-MS/MS. To determine the direct targets of the four selected TKIs, we subjected the proteins extracted from the A431 cancer cell line to drug-coupled affinity matrices. By comparing the pull-down results from a lysate with a second identical lysate to which 20 uM of the inhibitor had been added to block binding to the beads, we were able to identify selective binding interactors using LC-MS/MS. This differential affinity pull-downs approach allowed us to also quantitatively evaluate the binding affinity of the tyrosine kinases to each of the drugs. To evaluate the global tyrosine phosphorylation dynamics that occurs in A431 cells upon treatment with each of the four inhibitors, phosphotyrosine peptides were enriched and identified by quantitative mass spectrometry. For this purpose, we treated the cells with each inhibitor for 2 h at appropriate concentrations adopted from reported studies. Cells were lysed, and the proteins digested with the proteases Lys-C and trypsin. To allow quantitation of the phosphorylated peptides originating from the three different peptide pools (control/imatinib/dasatinib or control/bosutinib/nilotinib), we used stable isotope dimethyl labeling. Subsequently, tyrosine-phosphorylated peptides were enriched by immunoprecipitation and analyzed by LC-MS/MS.
2014-05-12 | PXD000681 | Pride
Project description:Chronic myeloid leukemia (CML) epitomizes successful targeted therapy, with 86% of patients in the chronic phase treated with tyrosine kinase inhibitors (TKIs) attaining remission. However, resistance to TKIs occurs during treatment, and patients with resistance to TKIs progress to the acute phase called Blast Crisis (BC), wherein the survival is restricted to 7-11 months. About 80 % of patients in BC are unresponsive to TKIs. This issue can be addressed by identifying a molecular signature which can predict resistance in CML-CP prior to treatment as well as by delineating the molecular mechanism underlying resistance. Herein, we report genomic analysis of CML patients and imatinib-resistant K562 cell line to achieve the same. WGS was performed on imatinib-sensitive and -resistant K562 cells. Library preparation was done by 30x WGS KAPA PCR-Free v2.1 kit, and Illumina HiSeq X sequencer was used for 2 x 150 bp paired-end sequencing. Our study identified accumulation of aberrations on chromosomes 1, 3, 7, 16 and 22 as predictive of occurrence of resistance. Further, recurrent amplification in chromosomal region 8q11.2-12.1 was detected in highly resistant K562 cells as well as CML patients. The genes present in this region were analyzed to understand molecular mechanism of imatinib resistance.
2024-01-01 | E-MTAB-11896 | biostudies-arrayexpress
Project description:KCL-22 is a chronic myeloid leukemia (CML) cell line derived from a patient in blast crisis phase and harbors the BCR-ABL translocation. The catalytic (ATP-competitive) BCR-ABL inhibitors imatinib and nilotinib have dramatically improved CML patient outcome, but the development of resistance remains a clinical challenge. The recent identification of allosteric BCR-ABL inhibitors, such as GNF-2, which target the enzyme by binding to the myristoyl pocket rather than catalytic site of ABL1, may provide a strategy to broadly overcome resistance to the class of ABL1 ATP competitive inhibitors. We therefore wanted to use the ClonTracer barcoding system to compare the clonal responses of KCL-22 to imatinib, nilotinib and GNF-2. RNA-seq was employed to characterize genetic alterations and gene expression signatures in the pooled cell populations resistant to BCR-ABL inhibitors as well as single clones showing differential response to the three inhibitors. mRNA profiling of the subpopulations and single clones of human CML cell line KCL-22 that contribute to BCR-ABL inhibitor resistance
2014-10-07 | E-GEOD-62121 | biostudies-arrayexpress
Project description:Genomic profiling of K562 cells resistant to imatinib as well as imatinib resistant CML patients harboured a recurrent chromosomal amplification in 8q11.2-12.1. Gene encoding PLAG1, a transcription factor associated with cancers and chemo resistance, resides on this locus and was found to be amplified in resistant cells. Upon PLAG1 knockdown we observed a decrease in imatinib resistance, and to understand the molecular events underlying PLAG1-mediated imatinib resistance, RNA sequencing was carried out. Imatinib-resistant K562 cells were transfected with lentiviral particles containing shRNA against PLAG1 and lentiviral particles containing pLKO.1-empty vectors. Monoclonal populations were established. Total RNA sequencing was performed. For QC, RNA samples were quantified using the Qubit RNA BR Assay kit (Invitrogen, Cat# Q10211). RNA purity was checked using QIAxpert, and RNA integrity was assessed on TapeStation using High Sensitivity RNA ScreenTape® (Agilent, Cat# 5067-5579). QC passed RNA samples were subjected to the library preparation, and NEB Ultra RNA-Seq Library Prep kit protocol (Cat# E7530L) was used and sequenced on Illumina NovaSeq 6000 instrument. Transcriptomic analysis identified several genes down-regulated upon PLAG1 knockdown which were further investigated.
2024-01-01 | E-MTAB-11893 | biostudies-arrayexpress
Project description:Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, with at least one-third of its patients not responding to the current chemotherapy regimen, R-CHOP. By gene expression profiling, patients with DLBCL can be categorized into two clinically relevant subtypes: activated B-cell (ABC) DLBCL and germinal center B-cell (GCB). Patients with ABC DLBCL have a worse prognosis, and are defined by chronic, overactive signaling through the B-cell receptor and NF-κB pathways. We examined the effects of the Src family kinase (SFK) inhibitor dasatinib in a panel of ABC and GCB DLBCL cell lines, and found that the ABC DLBCL cell lines are much more sensitive to dasatinib than the GCB DLBCL cell lines. However, using multiplexed inhibitor bead coupled to mass spectrometry (MIB/MS) kinome profiling competition and western blot analysis, both subtypes display inhibition of the SFKs in response to dasatinib after both short- and long-term treatment. MIB/MS analyses revealed several cell cycle kinases, including CDK4, CDK6, and the Aurora kinases, are inhibited by dasatinib treatment in the ABC DLBCL subtype, but not in the GCB DLBCL subtype. The present findings have important implications for the clinical use of dasatinib for the treatment of ABC DLBCL, either alone or in combination with other agents.
2018-12-14 | PXD009380 | Pride
Project description:Total RNA from 1e7 K562 cells either mock-treated with DMSO or treated with 100nM dasatinib in two biological repeats for 3 hours
2009-04-02 | E-MEXP-1561 | biostudies-arrayexpress
Project description:Chronic myeloid leukemia (CML) epitomizes successful targeted therapy, with 86% of patients in the chronic phase treated with tyrosine kinase inhibitors (TKIs) attaining remission. However, resistance to TKIs occurs during treatment, and patients with resistance to TKIs progress to the acute phase called Blast Crisis (BC), wherein the survival is restricted to 7-11 months. About 80 % of patients in BC are unresponsive to TKIs. This issue can be addressed by identifying a molecular signature which can predict resistance in CML-CP prior to treatment as well as by delineating the molecular mechanism underlying resistance. Herein, we report genomic analysis of CML patients and imatinib-resistant K562 cell line to achieve the same. Thirteen CML patients (sensitive and resistant to TKIs) and 2 BMT donors (as control) were recruited for the study. DNA was isolated from an enriched CD34+ fraction for each sample as well as from K562 cells made resistant to imatinib which provided a model system for further molecular investigations. DNA was subjected to Cytoscan HD array (Affymatrix) analysis from patient samples and cell lines. Affymetrix CytoScan™ HD array (Applied Biosystems™, Cat# 901835) chip consists of 2.6 M oligonucleotide probes across the genome, including 1953K unique non-polymorphic probes and 750K bi-allelic SNP (single nucleotide polymorphism) probes. Our study identified accumulation of aberrations on chromosomes 1, 3, 7, 16 and 22 as predictive of occurrence of resistance. Further, recurrent amplification in chromosomal region 8q11.2-12.1 was detected in highly resistant K562 cells as well as CML patients. The genes present in this region were analyzed to understand molecular mechanism of imatinib resistance.
2024-01-01 | E-MTAB-11899 | biostudies-arrayexpress
Project description:K562 cells were treated with the BCR-ABL kinase inhibitor dasatinib over an extended period of time to determine how BCR-ABL inhibition affects BCR-ABL-dependent negative feedback and erythropoietin receptor (EPO-R) signaling. Specifically, what types of changes (upregulation versus downregulation) occur in both the negative and positive regulators of growth-factor receptor signaling. Total RNA was extracted from K562 cells treated with 0.2% DMSO for 24hrs or 100nM dasatinib for 4hrs, 8hrs, and 24hrs.
2013-09-24 | E-GEOD-51083 | biostudies-arrayexpress