| Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder that is characterized by the Ph chromosome. The reciprocal translocation between the chromosomes 9 and 22 results in the Ph chromosome, and produces a fusion gene known as Bcr-Abl.This fusion gene encodes a chimeric protein which turns on a dysregulated tyrosine kinase activity and drives CML. In CML, a p210 Bcr-Abl isoform is initially expressed in haematopoietic stem cells (HSCs) capable of giving rise to both differentiated myeloid and lymphoid progeny.The traditional therapy of CML includes Hydroxycarbamide, Myleran, Meisoindigo, interferon with high side effects. The biology of CML has enabled preclinical and clinical oncology researches with targeted therapies. Imatinib is a small molecule inhibitor of the Bcr-Abl tyrosine kinase that produces clinical remissions in CML patients with minimal toxicity. Imatinib is now frontline therapy for CML. However, despite the stunning efficacy of this agent, resistance or intolerance to imatinib may become increasingly important. Moreover, imatinib does not completely eradicate residual leukemic stem cells and progenitors, which present a persistent risk of desease relapse. Therefore, there is a clear need for CML research to focus on novel targets and targeted drugs.Various mechanisms may contribute to imatinib resistance, and it can be categorized into two broad groups:Bcr-Abl-dependent and Bcr-Abl-independent. The main cause in Bcr-Abl-dependent imatinib resistance involves point mutations in the Abl kinase domain of the fusion protein and over-expression of Bcr-Abl kinase through gene amplification. In addition, the Src family of kinase (SFKs) members Hck and Lyn are over-expressed in some imatinib-resistant patient isolated and cell lines, suggesting that SFKs may be involved in Bcr-Abl-independent imatinib resistance. Abl shares significant sequence homology and remarkable structural resemblance in its active state with Src family members. Several Src inhibitors from various chemical classes, including bosutinib, dasatinib and INNO-406 have been developed. These agents are more effective than imatinib in blocking Bcr-Abl tyrosine kinase autophosphorylation, and these effects extend to point mutantions of Bcr-Abl.FB2 is a novel N-(thiazol-2-yl)pyrimidin-4-amine derivative, and it had shown that FB2 inhibited imatinib-sensitive and resistance CML cell lines with the wild-type Bcr-Abl fusion gene. In this report, it was sought to identify this novel compound for treating Ph+ chronic myeloid leukemia that is potent in blocking Bcr-Abl kinase activity, including point mutations in the kinase domain, and inhibits src kinase activity. To assess its potential as a therapeutic agent, it was investigated the effect of FB2 on survival span of mice inoculated with K562 cells, K562/G7.0 cells and Ba/F3 cells expressing different isoforms of Bcr-Abl (wild-type, Y253F).On the basis of prior structural insights from dual Abl and Src inhibitor Dasatinib, we reasoned that FB2 may impose less stringent conformational requirements on Abl for kinase inhibition, and we therefore purified enzymes to assesse its activity against imatinib-resistant Bcr/Abl mutants. As expected, FB2 was potent at inhibiting nonmutanted Bcr/Abl (Bcr/Abl WT) and mutanted Bcr-Abl (Y253F) kinase activity, while not on mutanted Bcr-Abl with T315I. Furthermore, Src and Lyn protein kinase assays were done and the results exhibited FB2 was a nanomolar inhibitor of two Src kinases (Src and Lyn with IC50 of 1.37 nmol/L,2.83nmol/L, respectively).Ba/F3 p210 cells were obtained by transfecting the IL-3-dependent murine hematopoietic Ba/F3 cell lines with pSRa-p210Bcr-Abl plasmid. The mutations (Y253F and T315I) were introduced into full-length p210Bcr-Abl. MTT assay were performed to investigate the inhibition of FB2 on Ba/F3 p210 (WT, Y253F and T315I) cells. And the mean IC50 values for FB2 were 1.30 and 2.56 nmol/L in Ba/F3 p210 WT and Ba/F3 p210 Y253F cells respectively. However, it had no effects on proliferation of Ba/F3 p210 T315I cells. FB2 inhibited the activities of Bcr-Abl and Src kinases as assayed by reduction of the phosphorylated forms of Bcr-Abl,c-Src and Lyn, respectively. Ba/F3 p210 WT and Y253F cells presented the marked dose-dependent reduction in Bcr-Abl, c-Src and Lyn phosphorylation when treated with FB2 from 0.2 to 5 nmol/L, and its potency of inhibition in c-src phosphorylation was stronger than dasatinib on it. FB2 reduced the level of p-c-Src and p-Lyn in Ba/F3-T315I cells while not the level of p-Bcr-Abl. To determine the antiproliferative effects of FB2 involved growth arrest at specific phases of the cell cycle, flow cytometric studies were performed.Our data showed that, in the low nanomolar range, FB2 induced the inhibition of cell growth and cell cycle progression of Ba/F3 p210 (WT, Y253F) cell lines mainly by inducing the G0/G1 phase arrest, and exhibited the dose-dependent relationship. Increasing the concentration of agent to micromolar range, FB2 could induce Ba/F3 p210 T315I cells arrested in G0/G1 phase.To assess its potential as a therapeutic agent, we studied FB2 in a mouse model of imatinib-resistant, Bcr/Abl dependent disease. Nod/Scid mice were injected intravenously with K562 and K562/G7.0 cells, and Balb/c mice were harbored Ba/F3 cells expressing different Bcr/Abl isoforms. Untreated mice harboring cells expressing nonmutant or imatinib-resistant mutant Bcr/Abl developed aggressive disease, typically resulting in death in 27-75 days. All the three doses tested groups showed significantly prolonged survival span and the increases in survival times were in dose dependent manner. Mice bearing K562 and Ba/F3 p210 cells tolerated administrations of FB2 well, and obvious evidence of toxicity did not occurred in three months.In summary, our current studies demonstrated an effective inhibition of FB2 on Bcr-Abl and Src kinases. These data provide the framework for clinical trials with FB2 in Ph+CML and imtinib-resisitant CML。 Imatinib is the first small molecular drug for targeted therapies, and it is a selective inhibitor of the tyrosine kinase activity of Bcr-Abl fusion oncoprotein. Imatinib is indicated for the treatment of patients with Ph+CML in chronic phase,blast crisis and accelerated phase, and Ph+ALL. Most patients with chronic phase (CP) CML treated with imatinib have well-controlled disease. Imatinib at an oral dose of 400mg daily has now become standard initial treatment for all CML patients who present in CP. It is highly effective, too, in the short term for patients who present in advanced phases, but the duration of response is usually much shorter and the probability of relapse to blastic phase is high. Even for CP, however, the drug is not perfect. Only approximately 60% of patients are still taking imatinib at standard dosage after 6 years, which means that approximately 40% have needed higher doses of imatinib or alternative therapy. Therefore the mechanisms of resistance emerged as a major question.Multiple studies have addressed the problem of imatinib resistance and helped to define the major elements contributing to this occurrence. In simple terms, the mechanisms of imatinib resistance include treatment compliance excessive binding of imatinib to the plasma protein al-acid glycoprotein-1(AGP-1), increase of metabolization by the cytochrome p450 isoenzymes, changes on intracellular uptake of imatinib, Bcr-Abl overexpression and point mutations in the kinase domain of Bcr-Abl. There are many methods to research imatinib resistance. Some imatinib resistant cells were derived from CML patients, while it is useful to establish the resistant cells by adding imatinib to the culture. On the basis of the resistant cell line K562/G5.0 which was established by our laboratory before, K562/7.0 cells were induced and could be maintained in liquid culture with 7μmol/L imatinib. Then the biological characteristics of K562/G cell lines including K562/G01, K562/G3.0, K562/G5.0, K562/G7.0, and the changes of resistant mechanisms, were investigated in order to find some new targets to overcome the imatinib resistance.First, cell proliferation assay was performed using MTT, and IC50 for K562, K562/G01, K562/G3.0, K562/G5.0, K562/G7.0 was,0.36,4.84,14.06,36.65 and 51.26μmol/L, respectively. And there was no obviously change of doubling generation time between these cell lines. Furthermore, flow cytometric studies were performed to investigate the distribution of cell cycle, our data showed that cells in G2/M phase were increased gradually accompanied by the increase of resistant concentrations. All these results showed that the biological characteristics were changed between K562/G cell lines and K562 cells.To address whether there were any mutations in the ATP binding site of the Abl kinase domain, thought to be the target of imatinib, the ATP binding domains of K562 sensivive and resistant cells were sequenced and compared these to the the pubished sequences. No mutation was found in the fragment in any of the cell lines. The expression levels of Bcr-Abl, Lyn and p-gp were studied by immunoblotting. Compared with the sensitive parental line, the level of Lyn overexpressed increasing with the degree of resistance. However, the expression of p-gp was increased at lower level in K562/G01 cells and decreased at higher resistance fold in K562/G3.0, K562/G5.0 and K562/G7.0. The Bcr-Abl expression in K562/G3.0 cells was higher than that in K562, while it was reduced in K562/G5.0 and K562/G7.0 which was lower than in K562. The different changes of resistant proteins probably explained that the different results were gotten by different researchers about the resistant mechanisms.Proteomics can provide a global, systemic, and comprehensive approach to the identification and description of the biochemical processes, pathways, and networks involved in both normal and abnormal physiological states at the protein level. As a typical proteomic analysis, two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS) has been used widely in research. Differences in protein expression levels were detected in cell states between K562 and K562/G cell lines by 2-DE, and 14 protein spots showing significant changes were gotten. There were 9 proteins identified via mass spectrometry correctly. Among those protein, Vimentin, Moesin, Ezrin and ANXA1 were correlated with cancer closely.Currently, resistance to imatinib is believed to be a consequence of the interaction of multiple factors. Different research methods may exhibit different resistant mechanisms. Varies of imatinib resistant K562/G cell lines were used to explore the changes of mechanisms, and it was found the higher expression proteins in K562/G cells by 2-DE and MS. All these results were provided experimental basis for further exploration of imatinib resistance. |
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