Silencing of Suppressor of Cytokine Signaling-3 due to Methylation Results in Phosphorylation of STAT3 in Imatinib Resistant BCR-ABL Positive Chronic Myeloid Leukemia Cells

Abstract

<b>Background:</b> Silencing due to methylation of suppressor of cytokine signaling-3 (SOCS-3), a negative regulatorgene for the JAK/STAT signaling pathway has been reported to play important roles in leukemogenesis. Imatinibmesylate is a tyrosine kinase inhibitor that specifically targets the BCR-ABL protein and induces hematologicalremission in patients with chronic myeloid leukemia (CML). Unfortunately, the majority of CML patientstreated with imatinib develop resistance under prolonged therapy. We here investigated the methylation profileof SOCS-3 gene and its downstream effects in a BCR-ABL positive CML cells resistant to imatinib. Materials and<br/><b>Methods</b>: BCR-ABL positive CML cells resistant to imatinib (K562-R) were developed by overexposure of K562cell lines to the drug. Cytotoxicity was determined by MTS assays and IC50 values calculated. Apoptosis assayswere performed using annexin V-FITC binding assays and analyzed by flow cytometry. Methylation profileswere investigated using methylation specific PCR and sequencing analysis of SOCS-1 and SOCS-3 genes. Geneexpression was assessed by quantitative real-time PCR, and protein expression and phosphorylation of STAT1,2 and 3 were examined by Western blotting. <br/><b>Results</b>: The IC50 for imatinib on K562 was 362nM compared to3,952nM for K562-R (p=0.001). Percentage of apoptotic cells in K562 increased upto 50% by increasing theconcentration of imatinib, in contrast to only 20% in K562-R (p<0.001). A change from non-methylation ofthe SOCS-3 gene in K562 to complete methylation in K562-R was observed. Gene expression revealed downregulationof both SOCS-1 and SOCS-3 genes in resistant cells. STAT3 was phosphorylated in K562-R but notK562. <br/><b>Conclusions</b>: Development of cells resistant to imatinib is feasible by overexposure of the drug to the cells.Activation of STAT3 protein leads to uncontrolled cell proliferation in imatinib resistant BCR-ABL due to DNAmethylation of the SOCS-3 gene. Thus SOCS-3 provides a suitable candidate for mechanisms underlying thedevelopment of imatinib resistant in CML patients.