BRD4拮抗剂GSK525762A抗普通型急性B淋巴细胞白血病的体外研究

OBJECTIVE: To investigate the effects of bromodomain-containing protein 4 (BRD4) inhibitor GSK525762A on the proliferation and apoptosis of primary common B-cell acute lymphoblastic leukemia (common B-ALL) cells from adult patients, then to further explore the possible mechanisms. METHODS: Purified leukemia cells from 14 common B-ALL adult patients (4 Ph(+) and 10 Ph(−)cases) were obtained by flow cytometry sorting, and maintained in a mimic bone marrow microenvironment culture system for short-term culture. Leukemia cells were treated with various concentrations of GSK525762A. The inhibitory effects of BRD4 inhibitor on common B-ALL leukemia cells were measured by CCK-8 assay and the apoptosis of those cells was determined by AnnexinⅤ/7-AAD staining using flow cytometry. The transcripts of c-MYC, CDK6 and Bcl-2 were detected by quantitative RT-PCR, and the expression of c-MYC, CDK6 and Bcl-2 proteins were detected via Western blot. RESULTS: GSK525762A could inhibit the proliferation of leukemia cells from all 14 common B-ALL patients in a dose-dependent manner, the median value of IC(50) was 256.25 (90.64–1 378.39)nmol/L. GSK525762A could promote cells apoptosis of B-ALL leukemia cells in a dose-dependent manner, the median apoptosis rates respectively were 45.17%(9.38%–70.91%), 66.02% (24.36%–96.34%) and 89.29% (39.29%–99.37%) after treated by 500, 1 000 and 2 500 nmol/L GSK525762A. GSK525762A has a similar effect on Ph(+) ALL and Ph(−)B-ALL, but the effect of proliferation inhibition and apoptosis enhancement on Ph(+) B-ALL is weaker than that on Ph(−)B-ALL. Compared with vehicle control group, the levels of c-MYC, Bcl-2 and CDK6 transcripts in leukemic cells were reduced after treatment for 24 h and 48 h by 1 000 nmol/L GSK525762A, and there are no significant differences in the downregulation of c-MYC and CDK6 mRNA between Ph(+) and Ph(−)B-ALL; however, the inhibitory effect on Bcl-2 transcription was weaker in Ph(+) B-ALL cells than that in Ph(−)B-ALL cells. Moreover, c-MYC, Bcl-2 and CDK6 protein levels decreased in GSK525762A treated group. CONCLUSION: GSK525762A could strongly inhibit the proliferation of common B-ALL and trigger apoptosis; meanwhile it has certain effects against Ph(+) ALL in vitro. The effect may be achieved by down-regulation of c-MYC, CDK6 and Bcl-2 expression.