PB1749: APOPTOTIC EFFECT OF FLUMATINIB COMBINED WITH BCL2 INHIBITOR ON PH+ B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

Abstract

Background: Philadelphia chromosome (Ph+) is the most common genetic abnormality of B-cell acute lymphoblastic leukemia (B-ALL), and also an independent prognostic high-risk marker. In addition to allogeneic hematopoietic stem cell transplantation, now tyrosine kinase inhibitor (TKI) has greatly improved the prognosis of patients with Ph+ B-cell ALL. Flumatinib is a kind of TKI originally developed in China and has been approved for chronic myeloid leukemia (CML). ABT-199, a selective Bcl-2 inhibitor, was effective in reducing leukemic burden in vitro and in vivo in B-ALL. However, there is no report about the anti-tumor effect of flumatinib in combination with Bcl-2 inhibitor in B-ALL. Aims: We explore the effect of flumatinib combined with Bcl-2 inhibitor on cell proliferation arrest and apoptosis in SUP-B15 human Ph+ B-cell ALL cells. Methods: CCK-8 assay was used to analyze the proliferation of SUP-B15 cells treated with flumatinib only, ABT-199 only, combination group, and vehicle control. All groups were treated for 48h at the same time. Apoptosis assay was performed with Annexin V staining following flow cytometry analysis. Expression of the apoptosis-associated gene and protein level was detected by real-time PCR and western blot, respectively. Results: Flumatinib and ABT-199 were sensitive to SUP-B15 in both single or combined treatment in a dose-dependent manner (Fig. 1A). The IC50 of flumatinib was 2.4uM, and ABT-199 5.5nM in the cells treated for 48 hours. Combination of various doses of flumatinib with 5.5nM (IC50) ABT-199 showed significantly higher proliferation arrest than that of either single drug, and CalcuSyn analysis showed a synergistic effect of the two drugs on the cell proliferation arrest (Fig. 1B). The combination of flumatinib with ABT-199 also significantly increased the apoptosis rate of SUP-B15 (39.2±1.2%) compared to either flumatinib (25.3±0.3%) or ABT-199 (10.6±0.7%) (p<0.05) (Fig. 1C). Furthermore, the combination treatment showed significantly higher expression of apoptosis-related proteins, Bax, Bad, Caspase-3 and Caspase-9 compared to either single drug in mRNA level by qPCR (p<0.05) (Fig. 1D) and protein level by western blot (Fig. 1E). Image:Summary/Conclusion: Both single drugs of flumatinib and ABT-199 are sensitive to SUP-B15 human Ph+ B-cell ALL cells. The combination of flumatinib with ABT-199 has a synergistic effect on cell proliferation arrest and apoptosis of the cells, revealing the potential of the novel combination for therapy of Ph+ B-cell ALL.