Synergistic Anti-leukemic Activity And Underlying Molecular Mechanism Of FLT3 Inhibitors Combined With Venetoclax In FLT3-mutated Acute Myeloid Leukemia

Acute myeloid leukemia(AML)is clonal proliferation of immature myeloid precursor cells at an arrest stage of development.The prognosis of AML remains poor,with 5-year overall survival rates approximating 65% and 25%,respectively,in the pediatric and adult populations.The 7+3 regimen(7 days on standard-dose cytarabine and 3 days on an anthracycline)is the first-line induction therapy for AML.However,the majority of older patients cannot tolerate high intensity chemotherapy.Therefore,there is an urgent needed for new therapies for the treatment AML patients,especially the older ones.FMS-like tyrosine kinase 3(FLT3)is a receptor tyrosine kinase in which is mutated in approximately one third of AML patients.FLT3-internal tandem duplications(ITD)mutations occur in approximately 25% of AML patients and FLT3-tyrosine kinase domain(TKD)mutations occur in approximately 5%-10% of AML patients.FLT3 mutations result in constitutive activation of downstream signaling pathways including the PI3K/AKT,RAS/RAFMEK/ERK,and JAK/STAT5 pathways.Activation of these pathways promotes cell survival,proliferation and inhibits apoptosis.FLT3-ITD is an independent prognostic marker and AML patients with FLT3-ITD have extremely poor prognosis.Therefore,FLT3 represents a promsing therapeutic target for this patient population.Midostaurin,a first generation multi-kinase inhibitor was approved by the US Food and Drug Administration(FDA)in April 2017 for use in newly diagnosed FLT3-mutated AML patients,in combination with standard 7+3 chemotherapy.However,7+3 chemotherapy cannot be tolerated in the majority of patients over the age of 60 and those with other comorbidities due to extensive toxicities and severe side effects,limiting a wider use of midostaurin in AML population.Gilteritinib was approved by the US FDA in November 2018 for treating adult patients with relapsed or refractory FLT3-mutated AML,however its clinical activity is incremental rather than curative.Therefore,new therapies are still needed for the treatment of FLT3-mutated AML.Venetoclax(ABT-199)is a selective Bcl-2 inhibitor,and was granted approval by the US FDA in November 2018 for first-line use in combination with low-dose cytarabine or the hypomethylating agents azacitidine or decitabine in newly diagnosed AML in adults over 75 or those with comorbidities precluding use of intensive induction chemotherapy.Our previous work demonstrated that Venetoclax releases proapoptotic Bim from antiapoptotic protein Bcl-2.However,another antiapoptotic Bcl-2 family protein Mcl-1 sequesters released Bim preventing it inducing apoptosis in AML cells.Therefore,combining agents which can downregulate Mcl-1 with Venetoclax would result in synergistic antileukemic activity against AML.It has been Reported that activation of FLT3 downstream pathways including PI3K/AKT and RAS/ERK signaling leads to phosphorylation of the anti-apoptotic protein Mcl-1 on T163,which increases Mcl-1 protein stability.Activation of JAK/STAT5 signaling leads to increased Mcl-1 transcript levels via transcription factor NF-?B.We therefore hypothesized that combining FLT3 inhibitors Gilteritinib or Midostaurin with Venetoclax would result in synergistic induction of apoptosis in FLT3-mutated AML.To test this hypothesis,we first investigated the antileukemic activity of the combination of Midostaurin or Gilteritinib with Venetoclax in vitro.Consistent with our hypothesis,the combinations synergistically induced apoptosis in both AML cell lines and primary patient samples.In contrast,the levels of apoptosis induced by these combinations in normal peripheral blood mononuclear cells was minimal,suggesting good tumor selectivity and safety.In an early stage MV4-11-derived xenograft mouse mode,the combination of Gilteritinib and Venetoclax showed striking in vivo efficacy against AML.Next,we investigate the molecular mechansims underlying the synergistic antileukemic activity between Venetoclax and Midostaurin or Gilteritinib in FLT3-mutated AML cell lines and a primary patient sample.We found that Midostaurin and Gilteritinib inactivates the ERK and JAK2/STAT5 pathways,leading to decreased levels of Mcl-1 transcripts and protein.This allows Bim released from Bcl-2 induced by Venetoclax to activate Bax/Bak,resulting in intrinsic apoptosis.Surprisingly,we also found potent induction of p-ERK by Venetoclax monotherapy and a rebound of p-ERK with prolonged Midostaurin treatment,resulting in resistance to both Midostaurin and Venetoclax.However,the combination of Midostaurin and Venetoclax completely abolish the induction of p-ERK by individual treatments,leading to synergistic antileukemic activity against FLT3-mutated AML.Essentially the same results were also obtained with Gilteritinib in combination with Venetoclax.In summary,FLT3 inhibitors and Venetoclax synergistically induce apoptosis in FLT3-mutated AML both in virto and in vivo.Simultaneous downregulation of Mcl-1,inhibition of Bcl-2,and abrogation of ERK activation represent the underlying molecular mechanisms.Our study strongly supports the clinical translation of the combination of Venetoclax with Midostaurin or Gilteritinib for the treatment of FLT3-mutated AML patients,especially the elderly.