Activity And Mechanism Of Action Of Co-targeting Of Mcl-1 And FLT3 In FLT3-mutated Acute Myeloid Leukemia

Acute myeloid leukemia(AML)is a malignant clonal disease of hematopoietic precursor cells with a median age of 68 years at the onset of the disease.The current stardard of care is cytarabine(AraC)and an anthracycline-based chemotherapy.However,the majority of patients relapse post chemotherapy,and relapsed patients usually lose their response to chemotherapy.The 5-year survival rate of AML patients under the age of 60 years is only 47%,while it is less than 10% for patients over 60 years of age due to poor tolerance of chemotherapy.Therefore,new treatment options are urgently needed to improve the prognosis of AML patients.About one-third of AML patients harbor mutations of the FMS-like tyrosine kinase 3(FLT3)gene,which encodes FLT3,a member of the receptor tyrosine kinase family.Mutations in the gene result in constitutive activation of FLT3 downstream RAS/RAF/MEK/ERK,JAK2/STAT5 and PI3K/AKT signaling pathways,which promote the occurrence and development of AML.Patients with FLT3-mutated AML have lower response rate to chemotherapy,are prone to relapse,and have poor prognosis.FLT3 inhibitors,Midostaurin and Gilteritinib,were approved by the US Food and Drug Administration(FDA)in April 2017 and November 2018,respectively,for treating patients with FLT3-mutated AML.In addition,MRX-2843 is another novel promising FLT3 inhibitor currently in clinical trials.However,FLT3 inhibitor monotherapies show limited efficacy due to emergence of drug resistance.Therefore,it is a reasonable strategy to introduce other drugs to combine with FLT3 inhibitors to enhance their antileukemic efficacy.The anti-apoptotic protein Mcl-1 promotes the survival of FLT3-mutant AML cells and causes resistance to FLT3 inhibitors.Mcl-1 selective inhibitor AZD5991 shows good antileukemic activity against preclinical models of AML and has entered clinical trials.Previous studies from our laboratory demonstrate that co-targeting of c-Myc and Mcl-1 results in synergistic anti-AML activity.Given that,inhibition of FLT3 results in down-regulation of c-Myc.Hence,we hypothesized that combing AZD5991 with FLT3 inhibitors would lead to synergistic antileukemic activity against FLT3-mutated AML.To test this hypothesis,we first investigated the antileukemic activity of the combination of AZD5991 and Gilteritinib/MRX-2843 in vitro.The result showed that the combination of AZD5991 and Gilteritinib/MRX-2843 synergistically induced apoptosis in FLT3-mutated AML cells.Although these combinations also resulted in synergistic antileukemic activities against FLT3-wild-type AML cells,higher concentrations of the FLT3 inhibitors were required to induce the same levels of apoptosis.Resistance to the conventional chemotherapy drug AraC and its combination with the Bcl-2 inhibitor Venetoclax is the main cause of treatment failure in AML.In this regard,we developed AraC-and Venetoclax+AraC-resistant AML cell lines,and demonstrated that the combination of Gilteritinib/MRX-2843 and AZD5991 could synergistically induce apoptosis in these drug-resistant cells.In addition,results from our colony formation experiments demonstrated that the combination of Gilteritinib/MRX-2843 and AZD5991 caused significant further suppression of AML progenitor cells compared with single drugs,demonstrating that these combination therapies could effectively target bulk AML cells and AML progenitor cells.Importantly,Gilteritinib,MRX-2843,and AZD5991,alone and in combination,had very limited effects on human peripheral blood mononuclear cells and hematopoietic progenitor cells from healthy donors,suggesting a therapeutic window of these combination therapies.These results demonstrate that the combination of AZD5991 with Gilteritinib/MRX-2843 results in synergistic antileukemic activity against FLT3-mutated bulk AML cells and AML progenitor cells,but largely spares normal hematopoietic cells,indicative of leukemia selectivity.Next,we explored the molecular mechanism underlying the synergistic antileukemic activity between AZD5991 and Gilteritinib/MRX-2843 in FLT3-mutated AML cells.Double knockdown of the apoptotic effectors Bak and Bax largely rescued the cells from the combination treatment.In line with these findings,Gilteritinib/MRX-2843 significantly increased AZD5991-induced release of cytochrome c.These results demonstrate that the combination of AZD5991 and Gilteritinib/MRX-2843-induced apoptosis is at least partially through the intrinsic apoptotic pathway.Further,we found that Gilteritinib/MRX-2843 alone or in combination with AZD5991 substantially down-regulated c-Myc,and knockdown or inhibition of c-Myc significantly enhanced AZD5991-induced apoptosis,demonstrating that down-regulation of c-Myc plays a key role in apoptosis induction by the combination of AZD5991 and Gilteritinib/MRX-2843.We then demonstrated that Gilteritinib/MRX-2843 alone or in combination with AZD5991 could reduce c-Myc m RNA levels,but not c-Myc protein half-life,indicating that the drugs down-regulate c-Myc through transcriptional inhibition.Further studies found that FLT3 inhibitors down-regulated c-Myc through the MEK/ERK and/or JAK2/STAT5 signaling pathways.In summary,FLT3 inhibitors suppress c-Myc transcription through inactivation of the MEK/ERK and/or JAK2/STAT5 pathways,leading to down-regulation of c-Myc,resulting in enhanced cytochrome c release induced by AZD5991,and apoptosis in FLT3-mutated AML cells.Our research provides an important theoretical and experimental basis for the treatment of FLT3-mutated AML with the combination of Mcl-1 and FLT3 inhibitors.