GNF-7, a novel FLT3 inhibitor, overcomes drug resistance for the treatment of FLT3‑ITD acute myeloid leukemia

Abstract

Background: Acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutation accounts for a large proportion of AML patients and diagnosed with poor prognosis. Although the prognosis of FLT3-ITD AML has been greatly improved, the drug resistance frequently occurred in the treatment of FLT3 targeting drugs. GNF-7, a multitargeted kinase inhibitor, which provided a novel therapeutic strategy for overriding leukemia. In this study, we explored the antitumor activity of GNF-7 against FLT3-ITD and clinically-relevant drug resistance in FLT3 mutant AML.

Methods: Growth inhibitory assays were performed in AML cell lines and Ba/F3 cells expressing various FLT3 mutants to evaluate the antitumor activity of GNF-7 in vitro. Western blotting was used to examine the inhibitory effect of GNF-7 on FLT3 and its downstream pathways. Molecular docking and cellular thermal shift assay (CETSA) were performed to demonstrate the binding of FLT3 to GNF-7. The survival benefit of GNF-7 in vivo was assessed in mouse models of transformed Ba/F3 cells harboring FLT3-ITD and FLT3-ITD/F691L mutation. Primary patient samples and a patient-derived xenograft (PDX) model were also used to determine the efficacy of GNF-7.

Results: GNF-7 inhibited the cell proliferation of Ba/F3 cells expressing FLT3-ITD and exhibited potently anti-leukemia activity on primary FLT3-ITD AML samples. Moreover, GNF-7 could bind to FLT3 protein and inhibit the downstream signaling pathway activated by FLT3 including STAT5, PI3K/AKT and MAPK/ERK. In vitro and in vivo studies showed that GNF-7 exhibited potent inhibitory activity against FLT3-ITD/F691L that confers resistant to quizartinib (AC220) or gilteritinib. Importantly, GNF-7 showed potent cytotoxic effect on leukemic stem cells, significantly extend the survival of PDX model and exhibited similar therapy effect compared with gilteritinib.

Conclusions: Our results show that GNF-7 is a potent FLT3-ITD inhibitor and may become a promising lead compound applied for treating some of the clinically drug resistant patients.

Keywords: Acute myeloid leukemia; Drug resistance; FLT3-ITD; GNF-7.

Fig. 1

GNF-7 potently inhibited the proliferation of FLT3-ITD AML cells and targeted FLT3-ITD downstream signaling pathways. A AML cell line MOLM-13, MV4-11, U937 and THP-1 were treated with DMSO or increasing concentrations of GNF-7 for 48 h and the normalized cell proliferation was measured by CellTiter Glo assay. B Mononuclear cells isolated from umbilical cord blood (Normal #1, Normal #2), bone marrow of diagnosed with AML (AML #1, AML #2) and AML harboring FLT3-ITD mutation (AML #3, AML #4, AML #5) were treated with different concentrations of GNF-7 for 48 h, and normalized cell proliferation was detected by CellTiter Glo assay. C Primary bone marrow cells isolated from AML #3 and AML #4 were exposed to GNF-7 for 4 h and then detected by western blotting with antibodies against phosphorylated and total of FLT3, Stat5, AKT and ERK, respectively. D Phosphorylated and total of FLT3, Stat5, AKT and ERK in MOLM-13 and MV4-11 cells treated with GNF-7 for 4 h were detected by western blotting. E Dose response curve of GNF-7 on Ba/F3 FLT3-ITD cells in the presence or absence of IL-3. F Ba/F3 FLT3-ITD cells were treated with the different concentration of GNF-7 for 4 h and subjected to western blotting with the indicated antibodies. All experiments were repeated three times with the same results. Data are presented as mean ± SD, and P values were calculated using Student t test. *p < 0.05, ** p < 0.01, and *** p < 0.001

Fig. 2

GNF-7 interacts with FLT3 protein. A Docking model of FLT3 bound to GNF-7: the yellow dotted line represents the hydrogen bond interaction, the green line represents the amino acid that forms hydrogen bonds with GNF-7, the cartoon represents the FLT3 protein, and the purple stick represents the GNF-7 molecule. B 2D interaction diagram of the FLT3/GNF-7 complex: GNF-7 is bound to the FLT3 protein in a pocket surrounded by LYS706, TYR696, GLY697, CYS694, ALA642, LEU818, PHE830, VAL624, LEU616, CYS695, and SER705 amino acids, GNF-7 forms hydrogen bonds with SER705 and forms hydrophobic interaction with LYS706, TYR696, GLY697, CYS694, ALA642, LEU818, PHE830, VAL624, LEU616, CYS695. C, E Thermal stabilization of FLT3 in Ba/F3 FLT3-ITD cells treated with GNF-7 (1 μM) or DMSO in various temperatures (C) and treated with various concentrations of GNF-7 (E) was analyzed through CETSA assay. D–F The density of the FLT3 bands were quantified by quantity one software. All experiments were repeated three times with the same results. Data are presented as mean ± SD, and P values were calculated using Student t test. * p < 0.05, ** p < 0.01, and *** p < 0.001

Fig. 3

GNF-7 showed significant therapy effect on the mice model engrafted with Ba/F3 FLT3-ITD cells. A The mice engrafted with Ba/F3 FLT3-ITD cells were treated with vehicle, AC220 (10 mg/kg), gilteritinib (30 mg/kg) and GNF-7 (15 mg/kg) for 8 days and the percentages of leukemia cells infiltrated in peripheral blood were then analyzed by flow cytometry. B The mice engrafted with Ba/F3 FLT3-ITD cells were treated with vehicle, AC220 (10 mg/kg), gilteritinib (30 mg/kg) and GNF-7 (15 mg/kg) for 9 days and the percentages of leukemia cells infiltrated in bone marrow were then analyzed by flow cytometry. C The spleen weight in each group were measured. D The survival curve of mice was calculated. Data are presented as mean ± SD, and P values were calculated using Student t test. * p < 0.05, ** p < 0.01, and *** p < 0.001

Fig. 4

Activity of GNF-7 against drug resistant Ba/F3 FLT3-ITD/F691L cells. A Relative proliferation of Ba/F3 populations stably expressing FLT3-ITD mutant isoforms after 48 h in various concentrations of GNF-7 were measured by CellTiter Glo assay. B IC₅₀ values of Ba/F3 stably expressing FLT3-ITD and FLT3-ITD/F691L cells treated with various concentrations of AC220, gilteritinib and GNF-7 were analyzed by CellTiter Glo assay. C After treated with vehicle, AC220 (10 mg/kg), gilteritinib (30 mg/kg) and GNF-7 (15 mg/kg) for 8 days, the percentages of leukemia cells infiltrated in peripheral blood of mice (n = 6) engrafted with Ba/F3 FLT3-ITD/F691L cells were evaluated by flow cytometry. D After treated with vehicle, AC220 (10 mg/kg), gilteritinib (30 mg/kg) and GNF-7 (15 mg/kg) for 9 days, the burden of leukemia cells in bone marrow of mice (n = 3) which was randomly selected from each group were detected. E The spleen weights were analyzed. F The Kaplan–Meier survival curves of animal survival of mice treated with vehicle, AC220 (10 mg/kg), gilteritinib (30 mg/kg) and GNF-7 (15 mg/kg). All cell line experiments were repeated three times with the same results. P values were calculated by log-rank test and shown. Data are presented as mean ± SD, and P values were calculated using Student t test. * p < 0.05, ** p < 0.01, and *** p < 0.001

Fig. 5

GNF-7 exerts potent therapy effect on AML PDX model. A, B Primary patient cells from AML #3 or AML #4 carrying with FLT3-ITD were transplanted into busulfan pretreated NOG mice and then randomly divided into three groups. Peripheral blood of 5 mice administrated with vehicle, 15 mg/kg GNF-7 and 30 mg/kg Gilteritinib were collected at the indicated time, and the leukemia cells content was detected by flow cytometry using human CD45 antibody. C, D The percentages of leukemia stem and progenitor cells in peripheral blood were detected by flow cytometry using human CD45 and CD34 antibodies. E, F The Kaplan–Meier survival curves of animal survival of mice treated with vehicle, AC220 (10 mg/kg), gilteritinib (30 mg/kg) and GNF-7 (15 mg/kg)