Synergistic Mechanism Of Gilteritinib Combined With ATO On FLT3-ITD Mutant Cells

Objectives: Acute myeloid leukemia(AML)is a heterogeneous malignant clonal disease.Due to repeated genetic changes and transformations,immature myeloid progenitor cells in the bone marrow induce the proliferation and survival of leukemia cells.These cells can self-renew and maintain malignancy and produce subcloning,although the diagnosis and treatment in recent years have improved the prognosis of patients with AML,the overall 5-year survival rate is still less than 30%,and only about 40-45% of young people and 10-20% of elderly patients with acute myeloid leukemia(AML)can achieve remission with current standard chemotherapy.FLT3 protein is a member of the type III tyrosine kinase receptor family and plays an important role in the proliferation and differentiation of early hematopoietic progenitor cells.The FLT3 gene is expressed only in bone marrow CD34 + precursor cells and is located on the 13q12 chromosome encoding the FLT3 protein.Stem cells lacking FLT3 have a reduced ability to rebuild T cells and bone marrow cells.Therefore,FLT3 may play an important role in the development of pluripotent stem cells and B cells.FMS tyrosine kinase 3(FLT3)is one of the most common mutations in AML.It is present in about 40% of patients with normal cytogenetics.Other acute lymphocytic leukemia ALL-T(pre-T)patients also have FLT3-ITD mutations,and the prognosis is not optimistic.FLT3 has two main types of mutations: about 20% to 25% of patients have internal tandem duplications(FLT3-ITDs)in the juxtamembrane domain of FLT3;about 7% to 10% of patients have FLT3 point mutations in the tyrosine-kinase domain(FLT3-TKD).Our research currently involves FLT3-ITD,which is the most common mutation of FLT3.Patients of AML with FLT3-ITD mutation have a high recurrence rate and a poor prognosis.FLT3 tyrosine kinase inhibitors(TKIs)have achieved limited clinical efficacy as a monotherapy.Gilteritinib is a newly applied FLT3 tyrosine kinase inhibitor(TKI)that has been proven to be effective in clinical trials;arsenic trioxide(ATO)is an effective anticancer drug and is currently an acute promyelocytic cell leukemia(APL)standard medication.This study is to explore the potential mechanism of FLT3 tyrosinase inhibitor Gilteritinib combined with ATO on FLT-ITD mutant cell lines,and to provide experimental evidence for the treatment of FLT3-ITD mutant leukemia.Methods: Western blot was used to detect the FLT3 protein in MV4-11 and MOLM13 cell lines with FLT3-ITD mutation,and HL-60 and THP-1 cell lines containing FLT3-WT.MV4-11 and MOLM13 cell lines with FLT3-ITD mutation and HL-60 and THP-1 cell lines with FLT3-WT were treated with Gilteritinib at different concentrations,and MV4-11 was treated with different concentrations of Ara-c,DAC,and ATO,respectively;MV4-11 and MOLM13 cell lines with FLT3-ITD mutation were treated with Gilteritinib,ATO single or two drugs in combination,then CCK-8 was used to detect viability of cells.The MV4-11 and MOLM13 cell lines were treated with 2.5 n M Gilteritinib or 0.5 μM ATO single or two drugs in combination,and the morphological changes of the cells were observed by Wright Staining.Flow cytometry was used to detect cell apoptosis and cell cycle.The levels of FLT3 protein and downstream molecules STAT5,ERK and AKT protein and their protein phosphorylation levels were detected by western blot.Western blot was used to detect the changes of 160 KD FLT3 protein in Gilteritinib-treated MV4-11 and MOLM13 cell lines.Western blot was performed to observe the effect of Gilteritinib on MV4-11 cells on endoplasmic reticulum pathway proteins GRP94,ATF6,PERK and IRE1 a and the effect of ATO combined with Gilteritinib in MV4-11 cells on IRE1a-JNK pathway,and then the small interference RNA targeting IRE1 a gene(si RNA-IRE1a)and negative control interference fragment(si RNA-control)were successfully transfected into MV4-11 and MOLM13 cells to knock down IRE1 a gene.The efficiency of IRE1 a gene knockdown was measured by RT-PCR and western blot.Flow cytometry was used to detect the effect of IRE1 a gene knockdown on the combined treatment of Gilteritinib and ATO on the apoptosis of MV4-11 and MOLM13 cells and the effects of IRE1 a gene knockdown on the combined treatment of Gilteritinib and ATO on the BAX protein and BCL-2 protein of MV4-11 and MOLM13 cells were examined by western blot.Endoplasmic reticulum activator tunicamycin and Gilteritinib were used to treat MV4-11 and MOLM13 cells.Apoptosis was detected by flow cytometry and apoptosis-related proteins were detected by western blot.Mouse xenograft models of FLT3-ITD AML were established and treated with Gilteritinib,ATO single or two drugs in combination,then the growth of the tumor was observed.Western blot was used to detect the levels of FLT3 protein and downstream STAT5,ERK and AKT protein,as well as their protein phosphorylation level and IRE1a-JNK signaling pathway.Immunohistochemistry was used to detect tumor proliferation and Tunel kit was used to detect tumor apoptosis.Results:(1)FLT3 protein expression of FLT3-ITD mutant MV4-11 and MOLM13 cell lines is higher than that of FLT3 wild-type THP-1 and HL-60 cell lines.Gilteritinib can specifically inhibit the proliferation of MV4-11 and MOLM13.ATO inhibited MV4-11 cells more obviously than DAC and Ara-c.The combination of Gilteritinib and ATO can synergistically inhibit the proliferation,increase apoptosis and inhibit invasion of MV4-11 and MOLM13 cell lines;morphologically,the combination of two drugs can cause more pronounced apoptosis and necrotic changes in cells.(2)Gilteritinib monotherapy can significantly reduce the phosphorylation level of FLT3 protein and downstream molecules STAT5,ERK and AKT protein in MV4-11 and MOLM13 cell lines,and increase the expression of 160 KD FLT3 protein on the surface of MV4-11 and MOLM13 cell membranes.After Gilteritinib treatment of MV4-11 cells,endoplasmic reticulum marker protein GRP94 was down-regulated,ATF6 and PERK were not changed significantly,and IRE1 a and its downstream signal phosphorylated JNK protein were inhibited.Gilteritinib combined with ATO can significantly up-regulate IRE1 a and its downstream phosphorylated JNK protein compared to single drug of Gilteritinib.(3)Both RT-PCR and western blot results showed that the IRE1 a gene was successfully knocked down.IRE1 a gene knockdown reduced the combined effect of Gilteritinib and ATO,decreased apoptosis and inhibited apoptotic pathways.Tunicamycin(endoplasmic reticulum pathway activator)and Gilteritinib combined treatment of MV4-11 and MOLM13 cell lines can also cause increased apoptosis and activation of apoptotic pathways.(4)The combination of Gilteritinib and ATO can significantly inhibit the growth of MV4-11 tumors in nude mice.Gilteritinib monotherapy can significantly reduce the phosphorylation levels of FLT3 protein and downstream molecules STAT5,ERK and AKT protein in tumor cells of nude mice.Gilteritinib combined with ATO can upregulate the IRE1a-JNK pathway.Gilteritinib combined with ATO can inhibit proliferation and up-regulate apoptosis of tumor cells in nude mice.Conclusion: The combined application of Gilteritinib and ATO has synergistic effects on FLT3-ITD mutant MV4-11 and MOLM13 cell lines in inhibiting proliferation,increasing apoptosis,weakening invasion ability and reducing tumor growth in nude mice.Gilteritinib single drug can significantly reduce phosphorylation levels of FLT3 protein and downstream molecules STAT5,ERK and AKT and western blot results showed that Gilteritinib increased the FLT3 protein at 160 KD on the cell membrane surface of the FLT3-ITD mutant MV4-11 and MOLM13 cell lines.Gilteritinib-treated MV4-11 cells inhibited IRE1 a and its downstream phosphorylation of JNK signals.Adding ATO reverses Gilteritinib’s down-regulation of IRE1 a.IRE1a gene knockdown reduced the combined effect of Gilteritinib and ATO.Apoptosis was decreased and apoptotic pathway was inhibited.The combination of tunicamycin(endoplasmic reticulum pathway activator)and Gilteritinib in the treatment of MV4-11 and MOLM13 cell lines could cause further apoptosis.