The Mechanism Of The Combination Of Chidamide And Sorafenib In Anti-FLT3-ID Mutated AML Effect

BackgroundAcute myeloid leukemia(AML)is a type of malignant clonal disease originating from hematopoietic stem/progenitor cells.It is the most common hematological malignancy in adults.About 20%-30%of AML is accompanied with FLT3-ITD mutations.AML patients with FLT3-ITD mutations have a poor prognosis.FLT3 receptor can bind to its ligand to activate downstream signaling pathways,causing abnormal cell proliferation,obstructed apoptosis,and decreased sensitivity to chemotherapy.It is also an important reason for refractory or relapse in FLT3-ITD-positive AML patients.The current treatment for AML is based on traditional chemotherapy,and high-risk patients can submit allogeneic hematopoietic stem cell transplantation(allo-HSCT)as soon as possible after remission and consolidation treatment.In recent years,studies have found that the overall survival(OS)and relapse-free survival(RFS)of patients with FLT3-ITD-positive AML with traditional chemotherapy regimens are short,and the efficacy of combined FLT3 inhibitors is still limited.Allo-HSCT itself cannot benefit FLT3-ITD-positive AML patients for a long time.Maintenance therapy with FLT3 inhibitors after allo-HSCT can significantly reduce the risk of recurrence in FLT3-ITD-positive AML patients.Therefore,the resistant mechanism of FLT3-ITD-positive AML patients,combining with molecular targeted drugs to improve the efficacy is a hot and difficult spot of current research.FMS-like tyrosine kinase 3(FLT3)is a third type of receptor tyrosine kinase,which plays an important role in hematopoietic regulation and lymphocyte proliferation.The abnormal activation of FLT3 is closely related to the occurrence of a variety of tumors.Studies have found that FLT3 signaling is not only involved in the pathogenesis of AML,but can also mediate resistance of AML cells to chemotherapeutic drugs such as cytarabine.Clinical studies have shown that Sorafenib combined with chemotherapy can improve the event-free survival(EFS)and RFS of FLT3-ITD-positive AML,but it does not significantly improve the total OS.Therefore,the combination of targeted drugs in the treatment of FLT3-ITD-positive AML patients has important clinical value in improving the efficacy of patients and enhancing the prognosis.Histone deacetylase(HDAC)inhibitors can inhibit the function of histone deacetylase,increase the level of histone acetylation,regulate a variety of genes and protein levels through epigenetics,and affect cell proliferation and apoptosis.Studies have confirmed that HDAC inhibitors can inhibit tumor cell migration and anti-angiogenesis,thereby inhibiting tumor cell growth.HDAC inhibitors have become a research hotspot in the treatment of solid tumors and lymphomas.Chidamide is a new type of HDAC inhibitor independently developed by our country and approved by the FDA for the treatment of patients with relapsed or refractory peripheral T-cell lymphoma(PTCL).The main targets of Chidamide are.the 1,2,3 and 10 subtypes of the class I HDAC,which are highly related to tumorigenesis and development.By inhibiting specific HDAC subtypes,it exerts chromatin remodeling.With gene transcription regulation,it inhibits the cell cycle,induces apoptosis,and enhances the tumor killing effect mediated by natural killer cells(NK)and antigen-specific cytotoxic T cells(CTL).The role of Chidamide in FLT3-ITD-positive AML and the anti-leukemia effect of combined FLT3 inhibitors are not yet fully understood.Our research studies the effect of Chidamide on increasing sorafenib’s anti-FLT3-ITD-positive AML leukemia effect,and explores its molecular mechanism,laying a theoretical foundation for targeted therapy,and providing new ideas for clinical development of new strategies.Research objective1.To study the anti-FLT3-ITD positive AML leukemia effect of Chidamide combined with sorafenib.2.To study the effect of chidamide combined with sorafenib on the apoptosis of FLT3-ITD positive AML cells under the condition of MSC co-culture.3.To study the anti-FLT3-ITD-positive AML leukemia mechanism of Chidamide combined with sorafenib.Contents and Methods1.CCK8 detects the inhibition rate of chidamide combined with sorafenib on the proliferation of FLT3-ITD positive AML cells,and the flow cytometric detection of chidamide combined with sorafenib induces the apoptosis rate of FLT3-ITD positive AML cells.2.The anti-FLT3-ITD positive AML leukemia effect of chidamide combined with sorafenib under the condition of MSC co-cultured.In vitro,AML patient-derived bone marrow MSCs were co-cultured with Molm-13 and MV4-11 cell lines.CCK8 and flow cytometry were used to inhibit the proliferation of FLT3-ITD positive AML cells and induce apoptosis.3.Use Western blot technology to detect GADPH,H3,Acety-H3,FLT3,P-FLT3,AKT,P-AKT,ERK,P-ERK,Bax/Bcl2,Bim and other protein expression levels,and explore the mechanism of the combination of chidamide’s and sorafenib’s anti-FLT3-ITD positive AML leukemia effect.4.Statistical analysis:SPSS 23.0 and GraphPad Prism 8.0 software were used for data analy sis.The measurement data of the two groups were compared by t test,and the means of multiple groups were compared by single-factor analysis of variance.P value<0.05 was considered to be statistically significant.Results:1.The results of CCK8 showed that treatment of FLT3-ITD-positive AML cells with chidamide and sorafenib as a single agent,the proliferation inhibition of FLT3-ITD-positive AML cells was significantly enhanced with the increase of drug concentration,showing a significant dose-dependent relationship.The cell proliferation inhibition rate of the combination group was significantly higher than that of the single drug group after 24,48,and 72 hours of drug treatment(P=0.00028,0.00039,0.00036).Calcusyn 2.0 software calculated the cell proliferation inhibition rate of chidamide combined with sorafenib The CI<1(24h,48h,72h),and P<0.05,indicating that Chidamide combined with sorafenib had a synergistic inhibitory effect on the proliferation of FLT3-ITD-positive AML cells.2.The results of flow cytometry showed that the apoptosis rate of FLT3-ITD-positive AML cells in the combination group was significantly higher than that of the single-drug group(P=0.00037).Under the condition of MSC co-culture,the apoptosis rate of FLT3-ITD-positive AML cells in the combination group was significantly higher.The mortality rate is also higher than that of the single-drug group(P=0.00045).However,the apoptotic rate of non-coculture was low when MSC co-cultured(P=0.0008),suggesting that MSC co-culture may induce drug resistance in FLT3-ITD-positive AML cells.Calcusyn 2.0 software calculates the CI value of the apoptosis rate of FLT3-ITD-positive AML cells induced by chidamide combined with sorafenib.The CI<1,and the P<0.05,indicating that the combination of chidamide and sorafenib is synergistically induced FLT3-ITD positive AML cell apoptosis.3.Western blot detection of cell signaling pathway protein expression,the results showed that compared with the control group,after the treatment of Sorafenib or Chidamide in FLT3-ITD positive AML cells about24h,Bax/Bcl2 and Bim did not change significantly,but the P-ERK and P-AKT activities were inhibited.Chidamide combined with sorafenib treated FLT3-ITD-positive AML cells for 48h,the P-ERK and P-AKT activities were significantly inhibited,and the expression of Bax/Bcl2 and Bim was significantly increased.Conclusion1.Chidamide combined with sorafenib has a synergistic effect on anti-FLT3-ITD positive AML leukemia.2.Under the condition of MSC co-culture,Chidamide can increase the apoptotic effect of FLT3-ITD-positive AML cells when combined with sorafenib.3.Chidamide can increase the anti-FLT3-ITD-positive AML leukemia effect when combined with sorafenib by inhibiting the activities of P-AKT and P-ERK in FLT3-ITD-positive AML cells,and up-regulating the Bax/Bcl2 and Bim pathways.