Studies Of The Synergistic Mechanism Of ASP2215 Combined With SAHA On FLT3-ITD Mutant Cell Line

Objective and significance:Acute myeloid leukemia（AML）is a highly aggressive hematologic malignancy that is associated with certain genetic changes.FLT3-ITD mutation is a common mutation in AML,and patients with this mutation have a poor prognosis.In recent years,people have been working on developing FLT3 tyrosine kinase inhibitors,but the effect with single drug application is limited,so the combination of FLT3 inhibitors with other kinds of drugs will be a good choice.This study focused on drug combination therapy in FLT3-ITD mutant AML,and explored the drug synergism and synergistic mechanism of FLT3-ITD mutant cell line treated by FLT3-ITD inhibitor ASP2215 combined with HDAC inhibitor SAHA,in order to provide theoretical basis for subsequent combined treatment of FLT3-ITD mutant AML with similar drugs.Methods:MV4-11 cell line with FLT3-ITD mutation was treated with ASP2215,SAHA along or both at different concentrations,after which cell viability was detected by CCK-8 method,cell morphological changes were observed after cellspin and Wright-Giemsa staining,and flow cytometry was used to detect cell apoptosis and cell cycle.The phosphorylation levels of FLT3 and downstream molecule STAT5 were detected by immunoblotting,and the expression levels of apoptosis-related-proteins caspase-3/9,Mcl-1,Bcl-xl,Bcl-2 and Bax,as well as cell cycle-related-proteins cyclin D1,CDK 6 and P27^Kip1 were also detected by it.Besides,the mRNA levels of cyclin D1,CDK 6 and P27^Kip1 genes were detected by real-time quantitative PCR.Results:（1）.Compared with FLT3 unmutated cell line THP-1,ASP2215 can specifically inhibit the proliferation of FLT3-ITD mutated cell line MV4-11,showing a good effect;（2）.ASP2215 or SAHA alone can inhibit the viability of MV4-11 cells in a dose and time dependent manner.In addition,the combination of these two drugs could synergistically inhibit the viability of MV4-11 cells.（3）.FLT3 inhibitor ASP2215 can reduce the phosphorylation level of FLT3 receptor and downstream molecule STAT5,and SAHA also has a slight inhibitory effect on the phosphorylation of FLT3 and downstream molecule STAT5.The combination of both drugs can further reduce the phosphorylation level of FLT3 and STAT5 compared with single drug alone.（4）.ASP2215 or SAHA alone can induce apoptosis of MV4-11 cells in a dose and time dependent manner,and there is synergistic effect on their combination to induce apoptosis of MV4-11 cells.Apoptosis was accompanied by cleaved activation of caspase-3 and caspase-9.The combination of these two drugs caused more cleaved activation of caspase-3 and caspase-9 than single-drug effect.Morphologically,the changes of apoptosis and necrosis increased with the increase of drug concentration,and the combination of the two drugs coμld lead to more obvious cell apoptosis and necrosis changes.Both ASP2215 and SAHA were able to reduce the level of anti-apoptotic proteins McL-1 and Bcl-xL in a dose dependence manner.While only SAHA could reduce the level of Bcl-2 protein,ASP2215 had little effect on it.The level of pro-apoptotic protein Bax only increased slightly with the concentration increase of ASP2215 or SAHA alone.The combination of the these two drugs further reduced Mcl-1,Bcl-xL expression level and Bcl-2/Bax protein ratio compared with the single-drug effect.（5）.The combination of the these two drugs significantly increased apoptosis and G1 phase arrest compared with the single drug.Both FLT3 inhibitor ASP2215 and HDAC inhibitor SAHA induced up-regμlation of the CDK inhibitor P27^Kip1,and the combination of these two drugs further increased the P27 ^Kip1protein level.In contrast,ASP2215 and SAHA can down-regulate the expression level of cyclin D1,and the combination of the two drugs can further induce the down-regμlation of cyclin D1 at the transcriptional level and translational level.The expression of cyclin dependent kinase CDK6 was only inhibited by ASP2215,and the effect of combination of ASP2215 and SAHA on CDK6 was comparable to that of ASP2215 alone.Conclusion:（1）.The combination of FLT3 inhibitor ASP2215 and HDAC inhibitor SAHA has a synergistic inhibitory effect on the cell viability of FLT3-ITD mutated cell line MV4-11,and the synergistic mechanism involves the inhibition of the FLT3-STAT5signaling pathway by both ASP2215 and SAHA.（2）.The combination of FLT3 inhibitor ASP2215 and HDAC inhibitor SAHA could synergistically induce apoptosis of FLT3-ITD mutated cell line MV4-11,which was accompanied by increased activation of caspase-3and caspase-9.In addition,the co-induction of apoptosis by the two drugs can synergistically reduce the apoptosis-related-proteins McL-1,Bcl-xL protein levels and Bcl-2/Bax protein ratio.（3）.FLT3 inhibitor ASP2215 and HDAC inhibitor SAHA were able to synergistically block MV4-11 cells into G1 phase,and the mechanism involved their combined action on CDK inhibitor P27^Kip1,cyclin-dependent kinase CDK 6 and cyclin D1.Compared with the single drug,the combination of the two drugs can further induce up-regulation of the mRNA level and protein level of P27^Kip1 gene as well as down-regμlation of the mRNA level and protein level of cyclin D1 gene.The expression of CDK 6 gene was only down-regulated by FLT3 inhibitor ASP2215.