Pb1707: combination of azacitidine and flumatinib induces apoptosis in sup-b15 cells by blocking the cell cycle through the mdm2-p53 signaling pathway

Topic: 1. Acute lymphoblastic leukemia - Biology & Translational Research Background: Acute lymphoblastic leukemia (ALL) in adults is an aggressive clonal hematologic cancer of lymphoid progenitor cells with marrow origin. Philadelphia (Ph) chromosome is the long arms of chromosomes 9 and 22 that translocates with each other, resulting in the production of BCR-ABL fusion protein. It is the most common cytogenetic change in adult ALL. Flumatinib (FLU), a second-generation, small molecule protein tyrosine kinase (TKIs) inhibitor, induces cell proliferation arrest and apoptosis in Ph+ALL. On the other hand, aberrant methylation of tumor suppressor gene promoters is frequently observed in ALL. Azacitidine (AZA) is a methyltransferase inhibitor (hypomethylating agents, HMA) that partially reverses aberrant DNA methylation. However, the anti-leukemic activity of AZA in combination with TKI has not been reported. These drugs are expected to provide additional treatment options for patients with Ph+ ALL. Aims: In this study, we investigate the synergistic effects of TKI Flumatinib combined with the methyltransferase inhibitor AZA in Ph+ALL cells and the potential underlying mechanisms. Methods: Logarithmic growth phase cells Ph+ ALL cell line, SUP-B15 were used, and the cell proliferation arrest was detected by CCK-8 in the cells treated with DMSO control, FLU only, AZA only, and FLU+AZ, and the Synergy effects were determined by combination indices using CompuSyn software. The cell cycle was detected by PI staining following flow cytometry analysis. The mortality of apoptotic cells was examined by double staining with Annexin V-FITC and 7-Amino-Actinomycin D (7-AAD). The expression of apoptosis, cell cycle, and signaling pathway-related mRNA and protein were detected by Real-Time Quantitative PCR (qPCR) and Western blot (WB). The GEO database was utilized to investigate the expression of MDM2. Results: A dose-dependent and time-dependent effect on cell proliferation arrest was observed in the SUP-B15 cells treated with single AZA or FLU drugs in different days, respectively. The 50% inhibitory concentrations (IC50) values for AZA and FLU were 5.6 μM and 0.9 μM, respectively (Fig.1A). Compared to the FLU group, the combination group dramatically increased the cell proliferation arrest (Fig.1B). CompuSyn analysis showed a synergistic effect of AZA+FLU (Fig.1C). The combination of AZA and FLU induces G1 phase arrest (Fig.1D) and apoptosis (Fig. 1E) compared to single drug controls (Fig.1D). The qPCR data showed that BAX and P21 were up-regulated at the mRNA level, while BCL2 and CyclinE were down-regulated upon the combination treatment compared to the single drug controls (Fig.1F). Additionally, the tumor suppressor TP53 was up-regulated whereas the oncogenes MDM2 was down-regulated upon the combination treatment in mRNA level (Fig.1F). and protein level (Fig.1H) compared to the controls. MDM2 was significantly over-expressed in Ph+ ALL patients cohorts from the GEO database (P<0.05) (Fig.1G). The mechanism model of the synergy is summarized in (Fig.1I). Summary/Conclusion: The combination of AZA and FLU has a synergistic anti-leukemia effect on cell proliferation arrest and apoptosis in Ph+ALL cells. Our data provide experimental evidence for a new potential combination of AZA with FLU in the therapy of Ph+ALL and highlight the likelihood of further in vivo pre-clinical study for the potential clinical trial of the novel combination in ALL patients.Keywords: Azacitidine, Apoptosis, Ph+ ALL, Cell cycle