New Bcr-abl And Src / Abl Inhibitors Against Chronic Myelogenous Leukemia In Experimental Research

Chronic myelogenous leukemia(CML) is a myeloproliferative disorder of hematopoietic stem cells.The pathologic hallmark of this disease is the Philadelphia chromosome(Ph),which is seen in＞90%of CML patients.The Ph produces Bcr-Abl fusion protein,which displays constitutive tyrosine kinase activity and activates several signaling transduction pathways.The 2-phenylamino-pyrimidine derivative Imatinib(Gleevec,STI571) was developed as the first molecularly targeted therapy that specifically inhibits the Bcr-Abl tyrosine kinase activity in patients with Ph positive(Ph⁺) CML.Imatinib acts by revoking the effects of the Bcr-Abl oncoprotein through inhibition of Bcr-Abl autophosphorylation,inhibition of proliferation,and induction of apoptosis.Due to its excellent hematologic and cytogenetic responses,particularly in patients with chronic phase CML,Imatinib has moved towards first-line treatment for newly diagnosed CML. However,reponses to Imatinib in patients with more advanced disease are generally transient.Nevertheless,resistance to the drug has been frequently reported.As a result,a variety of strategies derived from structural studies of the Abl-Imatinib complex have been developed,resulting in the design of novel Abl inhibitors.Several preclinical studies have shown promising results for second generation Abl inhibitor AMN107.Using numerous Bcr-Abl tranformed hematopoietic cell lines, AMN107 was found to be 20-50 fold more potent compared with Imatinib in reduction of both autophosphorylation and proliferation.Nevertheless,cell lines expressing the Imatinib-resistant Bcr-Abl mutants can be inhibited by AMN107.AMN107 has made its way into clinical trial.Unfortunately,resistance to AMN107 reduced inhibition of cell growth.The molecular mechanisms underlying resistance still need to be determined.In this study,we investigated the activity of a novel phenylamino pyrimidine derivative FAB107,compared with Imatinib and 368485,in both K562 and K562/G3.0 cells.Effects of FAB107 on the growth of K562 and K562/G3.0 cells were analyzed by MTT assay.The growth of K562 and K562/G3.0 cells was inhibited by FAB 107 with the IC₅₀ value of 0.03μmol/L and 0.07μmol/L,respectively.The inhibition was stronger than that of Imatinib(IC₅₀:0.31μmol/L and 6.32μmol/L) and equal to 368485(IC₅₀: 0.02μmol/L and 0.082μmol/L).In vivo result from nude mice bearing hollow fibers with leukemia cells showed that FAB107 inhibited growth of K562 and K562/G3.0 cells in nude mice.The inhibitory effects of FAB107 were in a dose-dependent manner and 15μmol/kg,30μmol/kg and 45μmol/kg on FAB107 had P values less than 0.01,compared with control group.The inhibitory activity was stronger than that of Imatinib and equal to 368485.Flow cytometric assay was perfomed to evaluate the cell cycle and apoptosis. Exposure of K562 and K562/G3.0 cells to FAB107,Imatinib and 368485 resulted in an increase of percentage of apoptotic cells.The potency of inhibition was stronger than that of Imatinib and equral to 368485.To investigate the mechanism of FAB107 on the growth inhibition of K562 and K562/G3.0 cells,we tested the effect of FAB107 on the autophosphorylation to Bcr-Abl in K562 and K562/G3.0 cells.FAB107 inhibited autophorylation of Bcr-Abl kinase more effectively than that of Imatinib in both cell lines,and its potency of inhibition was equal to 368485.In conclusion,FAB107,a novel phenylamino pyrimidine class derivative,inhibited growth of K562 and K562/G3.0 cells in vitro and in vivo,and the probAble mechanisms was inhibition on autophosphorylation to Bcr-Abl kinase.These results suggest that FAB107 is a promising candidate as a novel therapeutic agent for Imatinib-sensitive and Imatinib-resistance CML. Chronic myelogenous leukemia(CML) is a hematopoietic disorder characterizd by the malignant expansion of bone marrow stem cells.Its malignant clonal marker is Philadelphia chromosome(Ph) which harbors the Bcr-Abl fusion gene.The latter encdes a chimeric Bcr-Abl protein,P210^Bcr-Abl,with a deregulated tyrosine kinase activity and identified as having a central role in the pathogenesis of CML.Imatinib(Gleevec,STI-571;Novartis,Inc.) is a tyrosine kinase inhibitor that competitively inhibitis Bcr-Abl kinases.It inhibits proliferation and induces apoptosis in Bcr-Abl positive cells and has shown remarkAble clinical activity in patients with CML. However,a significant proportion of patients treated with Imatinib develop resistance and some of them have primitive resistance,which usually are found in patients treated with other chemotherapeutic drugs.So the potential mechanisms of Imatinib resistance become a new focal point.There are 5 main mechanisms currently known that may result in Imatinib resistance.The first is plasma protein binding.The second mechanism is drug efflux.The third mechanism is the mutation of the Bcr-Abl kinase.The fourth mechanism is independent of Bcr-Abl.The fifth mechanism is gene amplification.Src family kinase activation is involved in leukemia mediated by Bcr-Abl,and may function in cases of resistance to Imatinib.Dasatinib(Bristol-Myers Squibb Inc.) is a thiazole-based dual Src/Abl kinase inhibitor.Dasatinib has recently been approved by U.S.Food and Drug Administration in 2006 for use in patients with CML or Philadelphia chromosome-positive acute lympho-blastic leukemia(ALL) who are unable to tolerate or have not responded to other treatments.To investigate possible mediators of acquired Imatinib resistance,K562 cells of resistant to 5μM Imatinib(K562/G5.0) were cloned and compared with the parental cell population.In this study,We also investigated the activity of a novel thiazole-based derivative FB2,relative to Dasatinib,in both K562 and K562/G5.0 cells.1.The induction of Imatinib resistance cell line(K562/G5.0) and analysis of the induced-resistane mechanisms The wild-type K562 cells were cultured in gradually increased concentrations of Imatinib.The cytotoxic effects of K562 and K562/G5.0 treated with different concentration of Imatinib were analyed by MTT assay and the cell cycle was evaluated by flow cytometric assay.Effects of Imatinib,doxorubicin and Dasatinib on the growth of K562 and K562/G5.0 cells were analyzed by MTT assay.C-Abl,P-C-Abl,Lyn and P-Lyn protein expression,sequence analysis were used to study the potential mechanisms of acquired resistance of K562/G5.0.IC₅₀s(50%inhibiting concentration) of K562 and K562/G5.0 were 0.37μmol/L and 44.40μmol/L respectively by MTT assay and the resistance to Imatinib increased to 122.5 fold.The analysis to the cell cycle in K562/G5.0 cells indicated that the cells of G0/G1 and G2/M phase decreased and the cells of S phase increased.In contrast with K562 and KB/V,the MDR-1 and BCRP mRNA expression in K562/G5.0 cells decreased. It was testified that there was no statistic difference after comparing the cytotoxic effects of K562 and K562/G5.0 treated with different concentration of doxorubicin by MTT assay.No point mutant in the Bcr-Abl ATP-binding site was detected.By comparison with K562,expression of Lyn mRNA in K562/G5.0 cells increased.Interesting,the expression of Lyn and p-Lyn increasd following the increase of the Imatinib resistance fold in K562 resistance cell lines.However,the expression of c-Abl and p-c-Abl increased at lower level in K562/G3.0 cells and decreased at higer resistance fold in K562/G5.0 cells.Furthermore,Dasatinib,the dual Src/Abl tyrosine kinase inhibitor,was still effective in K562/G5.0 cells,with resistance presumed due to expressing a high number of Lyn gene copies and transcripts.2.The inhibition of FB2 on Imatinib-resistant chronic myeloid leukemia in vitro and in vivoThe cytotoxic effects in K562,K562/G5.0,MDA-MB-231 and DU145 treated with different concentrations of FB2 were analyed by MTT assay.Western blot analysis was used to evaluate the effects of FB2 on the inhibitions of c-Abl,c-Src,and Lyn protein autophosphorylation in K562 and K562/G5.0 cells.The efficacy of FB2 was assessed in a nonobese diabetic/severe combined immunodeficient(NOD/SCID) mouse model bearing of Imatinib-resistant CML cells.IC₅₀s of K562,K562/G01 and K562/G5.0 were 0.03nmol/L,0.10nmol/L and 0.38nmol/L respectively by MTT assay.Besides Imatinib-sensitive cell line(K562),FB2 significantly inhibited the growth of Imatinib-resistant cell lines with different resistance mechanisms(K562/G5.0 and K562/G01),and decreased the expressions of autophosphorylation of Bcr-Abl,c-Src and Lyn kinases in them.It also inhibited the proliferations of Src overactivated cells DU145 and MDA-MB-231.Furthermore,FB2 potently prolonged the survival time of NOD/SCID mice harbored K562/G5.0 cells.In vitro,we successfully established a resistant cell line(K562/G5.0) with resistance to 5μmol/L and 122.5 fold resistance as compared with that of K562.The potential mechanism of the resistant cell line above established was the Bcr-Abl-independent and Lyn-activated phenotype.Our results also indicated that FB2,an Abl/Src dual tyrosine kinase inhibitor,is a promising candidate for Imatinib-resistant CML and Src overactivated cancer.