Combinatorial Inhibition Of Notch And FLT3 Produces Synergistic Cytotoxic Effects In FLT3-ITD+Acute Myeloid Leukemia

Objective Internal tandem duplication(ITD)mutations of FLT3 are the most frequent genetic alterations in acute myeloid leukemia(AML)and portend a poor prognosis.FLT3 tyrosine kinase inhibitors(TKIs)provide short-term clinical responses,but the long-term prognosis remains poor in FLT3-ITD+ AML patients.Notch signaling is important in numerous types of tumors.However,the role of Notch signaling in FLT3-ITD+ AML remains to be elucidated.Patients with AML who have FLT3-ITD mutations also need effective treatment.Methods We first explored the role of the Notch pathway in primary samples and cell lines with FLT3-ITD mutations by q PCR and western blot.The role of FLT3 inhibitors in combination with Notch inhibitors in cell proliferation,apoptosis,colony formation,and the like was then examined in AML cell lines,either with ITD-mutanted FLT3 or wild-type FLT3.To exclude the influence of cytogenetic background and confirme that the synergy between FLT3 inhibitor and Notch inhibitor exists only in FLT3-ITD mutant AML cells,we constructed the FLT3-ITD knock-in cell line in SKM-1 cell line using CRISPR/CAS9 system.The effects of FLT3 inhibitors and Notch inhibitors were examined in SKM-1wild-type and FLT3-ITD knock-in cell lines.To further validate the relevance of Notch signaling inhibition to the observed synergistic effects,small interfering RNA(si RNA)targeting Hes1 and lentivirus expressing dominant-negative mastermindlike-1(DNMAML)were used.In AML primary samples,the results of cell line proliferation and apoptosis were verified in vitro,and the proportion of CD34+ cells in the AML and healthy donor samples after treatment with FLT3 inhibitors and/or Notch inhibitors was examined.In vivo,we used a PDX mouse model induced by AML patients with FLT3-ITD mutations to detect the effficiacy of the FLT3 inhibitor sorafenib and the Notch inhibitor DAPT alone or in combination in NOD/SCID mice,determined by tumor growth,organ infiltration,andsurvival.The expression profile of RNA-seq was used to analyze the possible mechanism underlying the combined effect of FLT3 inhibitor and Notch inhibitor,and the potential target genes were screened.Finally,in the FLT3-ITD mutant cell lines,the sequencing results were verified by simulation and rescue experiments.Results In current study,we found that Notch signaling was activated upon FLT3-TKIs treatment in FLT3-ITD+ cell lines and primary cells.In the FLT3-ITD+ AML cell line and primary AML cells,we observed synergistic cytotoxicity of FLT3 inhibitors with Notch inhibitors,mainly presented in inhibiting cell proliferation,inducing apoptosis and inhibiting cell clone formation.In addition,in a FLT3-ITD+ patient-derived xenograft(PDX)AML mouse model,the combination of a FLT3 inhibitor and a Notch inhibitor significantly cleared leukemia cells and prolonged survival.In terms of mechanism,RNA sequencing showed that decreased expression of CXCR3 may be partly responsible for the synergistic effect of FLT3 inhibitors and Notch inhibitors.Protein level verification revealed that down-regulation of ERK signaling activity may be one of the underlying mechanisms.When FLT3-ITD+ AML cells treated with CXCR3 inhibitor combined with the FLT3 inhibitor,we observed a similar proliferative and pro-apoptotic effect as the Notch inhibitor,and the CXCR3 ligand CXCL10 can partially rescue the apoptosis induced by the FLT3 inhibitor or the FLT3 inhibitor in combination with the Notch inhibitor.These results support that CXCR3 is the result of sequencing of important genes involved in the synergy of FLT3 inhibitors and Notch inhibitors.Conclusion Our findings suggest that Notch pathway plays an indispensable role in FLT3-ITD mutant AML,combined therapies of FLT3-TKIs with GSI may be exploited as a potential therapeutic strategy to treat FLT3-ITD+ AML.