Gilteritinib (ASP2215), a Novel FLT3/AXL Inhibitor: Preclinical Evaluation in Combination with Azacitidine in Acute Myeloid Leukemia
Authors: Ueno Yoko , Mori Masamichi , Kamiyama Yoshiteru , Kaneko Naoki , Isshiki Eriko , Takeuchi Masahiro 
Published: October 2019

Abstract
Background FMS-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in acute myeloid leukemia (AML). Activating mutations in FLT3 such as internal tandem duplications (ITD) at the juxtamembrane domain are present in approximately 25-30% of newly diagnosed AML cases. Patients with AML harboring the FLT3-ITD mutation have poorer prognosis following the current induction chemotherapy treatment of cytarabine (AraC) and an anthracycline (daunorubicin [DNR] or idarubicin [IDR]). Azacitidine (Aza) is a treatment option for AML patients who are not eligible for intensive chemotherapy. Gilteritinib, a novel, small molecule inhibitor of the tyrosine kinases FLT3/AXL, is in Phase 3 development for treatment of FLT3 mutation-positive AML, including patients with FLT3-ITD mutations. In previous preclinical studies, gilteritinib has demonstrated superior antitumor effects when given in combination with AraC and either DNR or IDR compared with combination chemotherapy. Here, we report preclinical antileukemic activity of gilteritinib in combination with Aza against FLT3-ITD mutation-positive AML.
Methods: The antitumor effects of gilteritinib alone and in combination with Aza were investigated in MV4-11 cells harboring the FLT3-ITD mutation. Apoptosis was detected using annexin V staining via flow cytometry; PARP cleavage was evaluated via Western blot analysis. Anti-apoptotic protein expression was evaluated by Western blot. Furthermore, the combination antitumor effects were evaluated in MV4-11-xenografted nude mice administered once-daily, oral gilteritinib at 3 mg/kg for 21 days alone and concomitantly with once-daily intravenous Aza at 3 mg/kg for 5 days. Pharmacokinetic parameters were also investigated in MV4-11 xenografted nude mice. Statistical differences in antitumor effects of combination therapy versus gilteritinib alone and combination therapy versus Aza alone were assessed on Day 21 using the Student's t-test.
Results: Gilteritinib treatment for 48h resulted in an induction of apoptosis in MV4-11 cells as determined by an increase in annexin V-positive cells. When used in combination, gilteritinib augmented the Aza-induced increase in annexin V-positive cells. Gilteritinib also decreased the expression of anti-apoptotic proteins such as MCL-1, BCL2L10, and survivin, which are reported to be important in chemotherapy sensitivity, following 24h treatment. In combination with annexin results, an increase in PARP cleavage was also observed in MV4-11 cells following gilteritinib treatment; cells co-treated with gilteritinib and Aza showed a further increase in PARP cleavage. In mice xenografted with MV4-11 cells, oral gilteritinib (3 mg/kg/day) inhibited the tumor growth by 71%; Aza alone did not suppress tumor growth. The tumor volume in the doublet combination group (94% growth inhibition) was significantly smaller than in each single treatment group on Day 21 (Figure). No obvious influences on body weight, behavior, or diarrhea were noted in the combination group. Plasma concentrations for either gilteritinib or Aza were not considerably increased by their use in combination therapy when compared with the concentrations observed with single treatment.
Conclusions: Gilteritinib treatment augmented Aza-induced apoptosis in part by reductions of anti-apoptotic protein expressions in vitro. In the non-clinical models used, gilteritinib, in combination with Aza, showed superior antitumor efficacy compared with each single agent alone. These findings support the development of gilteritinib in combination with Aza as a potential treatment of FLT3-ITD mutation-positive AML.
Figure Antitumor Effect of Gilteritinib in Combination with Azacitidine in Mice Xenografted with MV4-11 Cells ***P<.001 vs gilteritinib alone; +++P<.001 vs azacitidine alone (Student's t-test). Figure. Antitumor Effect of Gilteritinib in Combination with Azacitidine in Mice Xenografted with MV4-11 Cells. / ***P<.001 vs gilteritinib alone; +++ P<.001 vs azacitidine alone (Student's t-test).

Disclosures
Ueno: Astellas Pharma Inc: Employment. Mori:Astellas Pharma Inc: Employment. Kamiyama:Astellas Pharma Inc: Employment. Kaneko:Astellas Pharma Inc: Employment. Isshiki:Astellas Pharma Inc: Employment. Takeuchi:Astellas Pharma Inc: Employment.