The Effects Of Inhibiting BCR/ABL Protein Through ATP-competitive Independent Pathway In CML

Part Ⅰ:The mechanisms of a novel anticancer agent JNJ-165 inhibits T315I mutant BCR/ABL and the value of clinical applicationObjective:In previous studies,we found that the novel anticancer agent JNJ-26854165 was active in CML cells with unmutated BCR/ABL through p53-independent pathway.In this study,we focused on the effects of growth-inhibition mediated by JNJ-165 on TKIs-resistant CML cells with mutated BCR/ABL,including T315I mutation,and then investigated the ATP-competitive independent mechanisms from the aspects of transcription,translation and protein degradation of BCR/ABL.Methods:Cell growth was measured by MTT assay and colony culture.Western blot method analyzed the expression of BCR/ABL and BCR/ABL downstream proteins.Real-time PCR assay for detection of BCR/ABL mRNA expression level,and BCR promoter luciferase reporter assay was performed to study the effect of JNJ-165 on the activity of the BCR promoter.Western blot method measured the regulation of BCR/ABL and BCR/ABL downstream proteins expression mediated by JNJ-165 with or without proteasome inhibitor MG 132,caspase-3 inhibitor z-DEVD-fink and protein synthesis inhibitor cycloheximide(CHX).Immunoprecipitation and immunoblot analysis examined the ubiquitin level of BCR/ABL.Colony-forming cell assay was used to observe the cells cloning ability.Furthermore,we investigated the effect of JNJ-165 with or without TKIs on the tumor growth-inhibition of K562 and 32D-p210-T315 xenograft-bearing SCID mice.SPSS 16.0 statistic analysis software was used for analysis.Statistical significance was defined as p<0.05.Results:1.JNN-165 could inhibit the proliferation in CML cell lines(32D-p210,32D-p210-T315I)and primary cells in a dose-dependent manner,while non-toxic side effects on normal stem(progenitor)cells from bone marrow.2.JNJ-165 could inhibit the expression of BCR/ABL mRNA,but did not induce significantly decreased transactivation of BCR promoter.3.The down-regulation of BCR/ABL mediated by JNJ-165 could be reversed by proteasome inhibitor MG132.JNJ-165 treatment led to a marked accumulation of polyubiquitinated c-Abl,indicating that JNJ-165 mediated degradation of T315I mutant BCR/ABL proteins may through promoting proteosomal degradation pathway.4.JNJ-165 induced over-expression of PP2A in K562 and 32D-p210-T315I cells in a dose-dependent fashion,which coincided with the up-regulation of SHP-1,indicating that JNJ-165 may trigger BCR/ABL degradation by the proteasome via PP2A-dependent mechanisms.5.The growth of xenograft tumors established from K562 cells was significantly suppressed in the mice treated with either JNJ-165 or TKI as compared with the control mice,and the combination treatment with JNJ-165 and TKI showed an impressive antitumor activity.Neither PD180970 nor Imatinib was growth inhibitory to the 32D-p210-T315I tumors and prolonged the survival of mice.In contrast,JNJ-165 alone caused an almost complete remission of tumors established from 32D-p210-T315I cells and significantly prolonged the survival of mice.6.As expected,significant down-regulation of BCR/ABL and downstream molecules were observed in the tumors from JNJ-165-treated mice.Conclusion:We demonstrated that JNJ-165 could trigger BCR/ABL degradation by promoting the proteasome pathway via PP2A-dependent mechanisms,inducing growth-inhibition in a variety of CML cell lines,including those resistance to Imatinib or harboring T3151 mutation.Our results suggested that JNJ-165 down-regulated BCR/ABL protein through ATP-competitive independent pathway,which is different from the mechanism of TKIs.The novel anticancer agent JNJ-165,used either alone or in combination with TKIs,represents a promising novel targeted approach to overcome TKIs resistance and improve patient outcome in CML.Part Ⅱ:The study of antitumor activity of oncolytic adenovirus expressing beclin-1 via inhibiting BCR/ABL protein in TKIs-resistant CML with MDR over-expressingObjective:In our previous studies,we demonstrated that the potent antitumor activity of oncolytic adenovirus expressing beclin-1(SG511-BECN)via targeting the autophagic pathway in AML and TKIs-sensitive CML.In this study,we will further explore the antitumor effects of SG511-BECN in multidrug resistant CML cells K562-A02,and compare its antitumor activity with SG511 and chemotherapy agent Dox,and then focus on the mechanisms associated with autophagy gene beclin-1 over-expression and its induced classical autophagic pathway.The interaction between autophagy and ROS production induced by SG511-BECN in CML also was preliminarily investigated.The synergistic effects of SG511-BECN and chemotherapeutic drugs Dox on K562-A02 and its potential underlying mechanism.Of particular note,we found that the inhibition of BCR/ABL expression induced by SG511-BECN in CML cells was closely related with the over-expression of beclin-1 protein.Interestingly,autophagy associated immunity and the crosstalk between autophagy-lysosomal and endocytic-exosomal pathways in antigen processing for MHC presentation in anticancer have become the focus of current research.In order to further study,we have covered the relevant literature and completed a review.Methods:Cell growth inhibition was measured by MTT assay.We adopted methods including,transmission electron microscopy(EM)and LC3-GFP-K562 cell fluorescence to detect autophagy induced by SG511-BECN in CML cells,including K562-A02.AV/PI flow cytometry was used to detect apoptosis.Western blot method was used to analyze the expression of caspase family proteins and autophagy associated proteins.We used siRNA to knockdown ATG5/ATG7 genes in K562 cell,and then detected the suppressive effects of SG511-BECN by MTT assay.We tested the generation of ROS in CML cells treated with SG511-BECN and/or ROS inhibitors(Tiron or NAC)by ROS-Kit,and further measured the cells viability by MTT assay.Flow cytometry analysis was used for detecting the changes of infection rate of SG511-EGFP in CML cells when combined with chemotherapy agent Dox.Real-time PCR assay for detection of BCR/ABL mRNA expression level and western blot to measure the regulation of BCR/ABL and BCR/ABL downstream proteins expression mediated by SG511-BECN in CML cells.Subcellular localization of beclin-1 and BCR/ABL proteins in K562 cell treated with SG511-BECN were observed by confocal microscope.SPSS 16.0 statistic analysis software was used for analysis.Statistical significance was defined as p<0.05.Results:1.Compared with SG511,SG511-BECN is a more potential oncolytic virus to kill multidrug resistant CML cells in a apoptotic-independent pathway.2.The infection of CML cells with SG511-BECN can up-regulate autophagy associated proteins beclin-1 and LC3-Ⅱ,but not caspase family proteins.Furthermore,SG511-BECN also resulted in a significant formation of GFP-LC3-labled vacuoles,whereas the SG511 vector did not.In agreement with those results,EM showed many autophagic vacuoles in the CML cells treated with SG511-BECN.3.siRNA-mediated ATG5,or ATG7 knockdown reverses the suppressive effects of SG511-BECN.4.Autophagy up-regulation induced by SG511-BECN in CML was accompanied with the up-expression of ROS,and inhibition of ROS could inhibit the level of autophagy.5.Chemotherapy agent Dox functions synergistically with SG511-BECN to inhibit multidrug resistant CML cells growth via enhancing infectious efficiency.6.SG511-BECN can inhibit the expression of mRNA BCR/ABL and BCR/ABL protein.7.Confocal microscope test showed that BCR/ABL and beclin-1 proteins colocated in the CML cells.8.Compared with SG511,SG511-BECN treated CML cells were observed more virus aggregation in cytoplasm and nucleus under electron microscope.Conclusion:In summary,we demonstrated for the first time that,by introducing the Beclin-1 gene into the oncolytic adenoviral backbone,antileukemia activity of the virus on multidrug resistant cell lines(including K562-A02)can be significantly improved via targeting autophagy pathway and inhibiting BCR/ABL expression.Furthermore,chemotherapy agent Dox could synergize with SG511-BECN to kill CML cells through improving the infectious efficiency of oncolytic adenovirus.We also found that SG511-BECN could specific down-regulate BCR/ABL expression,which may be related to the consequence of direct interaction between beclin-1 and BCR/ABL proteins in CML cells.Interestingly,compared with SG511,SG511-BECN treated CML cells were observed more virus aggregation in cytoplasm and nucleus under electron microscope.The present study has showed that autophagy-lysosomal pathway and endo-/exosomal pathway are closely interrelated and interacted on each other,under the circumstance of up-regualtion of autophagy,adenovirus infected cells were observed more easily to be dissoluted and thus,adenovirus easier amplificated in intracellular and released to extracellular.Therefore,in our study,could the SG511-BECN induced autophagy affect the endo-/exosomal pathway,leading to further amplification of oncolytic adenovirus in the CML cells?And what is the antitumor immunity changes in this process?In order to further study,we have covered the relevant literature and completed a review(see attachment part).