MZH29 Is A Novel Potent Inhibitor That Overcomes Drug Resistance FLT3 Mutations In Acute Myeloid Leukemia

More than one-third of patients with acute myeloid leukemia(AML)harbor aberrant mutations in FMS-like Tyrosine Kinase 3(FLT3).Among them,the internal tandem duplication(ITD)mutation predicts poor prognosis.Targeting FLT3,no clinical drugs were approved for the treatment of FLT3 mutant AML.AC220(Quizartinib)was a well-known and potent FLT3 inhibitor,and was studied for the clinical trial at the phase III.One of the main issues facing AC220,as well as other type Ⅱ FLT3 inhibitors,was the drug induced resistance mutation.This study was thus to find out a new compound with strong FLT3 inhibitory activity,to be effective for drug resistance mutations,and to elaborate its underling mechanism.An in vitro screening experiment targeting 46 kinds of tyrosine kinase was previously performed on LS177,which was a c-MET tyrosine kinase inhibitor.It has been found that LS177 also had FLT3 inhibitory effect.However,the inhibitory effect of LS177 was lower than that of AC220 in cell line level.The inhibitory effects of cell proliferation on a series of compounds were tested on MV4-11 cell line by MTT assay.Based on the screening results,4-phenoxyquinoline derivatives were designed and synthesized.Then MZH29 was identified to have a strong inhibitory activity against FLT3-WT and FLT3-ITD kinases,which was expressed in vitro.The half effective inhibition concentration(IC50)were 9 nM and 7 nM respectively.The inhibitory effects were equal to that of AC220.MZH29 also had a pro-apoptotic effect on FLT3-WT-dependent AML cell lines and FLT3 mutant cell lines by the up-regulation of Cleaved Caspase 3 and Cleaved PARP.MZH29 also inhibits FLT3-WT-dependent AML cell lines and FLT3 mutant cell lines.The results showed that MZH29 had a strong inhibitory effect on drug-resistance mutant cell lines,including mutant cell lines such as FLT3-ITD-F691L,FLT3-D835H/Y/V and FLT3 K663Q.The inhibitory effects were significantly better than that of AC220.The inhibitory effect of MZH29 on cell proliferation was driven by the inhibition of the phosphorylation of FLT3 and its downstream STAT5 and ERK.MZH29 has retained its inhibitory effects on the phophoralytion of FLT3 and its downstream proteins such as STAT5 and ERK on the constructed drug resistant mutantion cell line.The results indicated that MZH29 can be used as an inhibitor for drug resistant mutation of FLT3.Docking results demonstrated that MZH29 can form a stable binding conformation to both the FLT3 and the drug resistance mutation of FLT3-F691L,indicating that the mechanism of MZH29 is effective on drug resistance mutations.Oral administration of 10 mg/kg MZH29 causes complete tumor regression and extends survival in a mouse model of AML with less toxicity.Subsequent proteomics study revealed less proteome perturbation in the MZH29-treated group than in the AC220-treated group.MZH29 demonstrated potential and potent novel FLT3 inhibitory effects for the treatment of AML.