Sitravatinib as a potent FLT3 inhibitor can overcome gilteritinib resistance in acute myeloid leukemia

Abstract

Background: Gilteritinib is the only drug approved as monotherapy for acute myeloid leukemia (AML) patients harboring FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutation throughout the world. However, drug resistance inevitably develops in clinical. Sitravatinib is a multi-kinase inhibitor under evaluation in clinical trials of various solid tumors. In this study, we explored the antitumor activity of sitravatinib against FLT3-ITD and clinically-relevant drug resistance in FLT3 mutant AML.

Methods: Growth inhibitory assays were performed in AML cell lines and BaF3 cells expressing various FLT3 mutants to evaluate the antitumor activity of sitravatinib in vitro. Immunoblotting was used to examine the activity of FLT3 and its downstream pathways. Molecular docking was performed to predict the binding sites of FLT3 to sitravatinib. The survival benefit of sitravatinib in vivo was assessed in MOLM13 xenograft mouse models and mouse models of transformed BaF3 cells harboring different FLT3 mutants. Primary patient samples and a patient-derived xenograft (PDX) model were also used to determine the efficacy of sitravatinib.

Results: Sitravatinib inhibited cell proliferation, induced cell cycle arrest and apoptosis in FLT3-ITD AML cell lines. In vivo studies showed that sitravatinib exhibited a better therapeutic effect than gilteritinib in MOLM13 xenograft model and BaF3-FLT3-ITD model. Unlike gilteritinib, the predicted binding sites of sitravatinib to FLT3 did not include F691 residue. Sitravatinib displayed a potent inhibitory effect on FLT3-ITD-F691L mutation which conferred resistance to gilteritinib and all other FLT3 inhibitors available, both in vitro and in vivo. Compared with gilteritinib, sitravatinib retained effective activity against FLT3 mutation in the presence of cytokines through the more potent and steady inhibition of p-ERK and p-AKT. Furthermore, patient blasts harboring FLT3-ITD were more sensitive to sitravatinib than to gilteritinib in vitro and in the PDX model.

Conclusions: Our study reveals the potential therapeutic role of sitravatinib in FLT3 mutant AML and provides an alternative inhibitor for the treatment of AML patients who are resistant to current FLT3 inhibitors.

Keywords: AML; Drug resistance; FGF2; FL; FLT3-ITD; FLT3-ITD-F691L; Gilteritinib; Sitravatinib.

Fig. 1

Sitravatinib exerts potent anti-tumor activities against FLT3-ITD AML cell lines. A The IC50 values of each drug for FLT3-WT (THP1, HL60, U937, OCI-AML2, OCI-AML3) or FLT3-ITD (MV4-11 and MOLM13) AML cell lines. Data are mean ± standard error from three independent experiments. B The percentage of cells in different cell cycle phases after 24-h treatment with various concentrations of sitravatinib (S) or gilteritinib (G). Error bars indicate mean ± standard error, n = 3 independent experiments for each cell line. C Apoptotic cell populations of MV4-11 and MOLM13 cell lines after 48-h treatment with sitravatinib (S) or gilteritinib (G). Error bars indicate mean ± standard error, n = 3 independent experiments for each cell line. ***P < 0.001, ****P < 0.0001. D Western blot analysis of PARP1, caspase 8, cleaved-PARP1 (C-PARP1) and cleaved-caspase8 (C-caspase8) expression in MV4–11 and MOLM13 cells after treatment with different doses of sitravatinib for 48 h. E Western blot analysis of p-FLT3, p-STAT5, p-AKT and p-ERK 1/2 in MV4-11 and MOLM13 cells after treatment with sitravatinib at the indicated doses for 4 h

Fig. 2

The interaction of sitravatinib and FLT3. A Overview of the docking results of sitravatinib with FLT3 (Protein Data Bank: 6JQR) from two orthogonal views. B Close-up of the sitravatinib-FLT3 model highlighted the detailed binding sites of sitravatinib with FLT3. The atoms of sitravatinib are colored by the type of element (grey: carbon; blue: nitrogen; red: oxygen; cyan, fluorine; yellow: sulfur). For clarity, the hydrogen atoms are omitted. Molecular interactions are shown as red lines. C BaF3-FLT3-ITD cells were treated with sitravatinib (30 μM/mL) or DMSO for 1 h, temperatures between 38–58.2 °C were defined to perform the test. Quantification was made using western blot to determine the melting curve. Data are mean ± standard error from three independent experiments. D BaF3-FLT3-ITD cells were treated with sitravatinib from 0 to 1000 nM for 30 min and then heated at 51.7 °C for 3 min. Protein quantification was made using western blot and the best fit of the data was monitored. Data are mean ± standard error from three independent experiments

Fig. 3

Sitravatinib suppresses leukemic progression in FLT3-ITD engrafted models. A Left: Schematic representation of xenograft experiments using human MOLM13 cells; Right: Schematic representation of transplant experiments using mouse BaF3-FLT3-ITD cells. B The percentage of human CD45 positive cells in BM and SP of MOLM13-diseased NSG mice detected by flow cytometry on day 24 (n = 3 mice per group). C The survival curves of MOLM13-diseased NSG mice treated with vehicle, sitravatinib (20 mg/kg/day), gilteritinib (30 mg/kg/day) or quizartinib (10 mg/kg/day) (n = 7 mice per group). D The percentage of GFP positive cells in BM and SP of BaF3-FLT3-ITD-diseased BALB/c mice on day 11 (n = 3 mice per group). E The survival curves of BaF3-FLT3-ITD-diseased mice treated with vehicle (n = 11), sitravatinib (20 mg/kg/day, n = 9), gilteritinib (30 mg/kg/day, n = 10) or quizartinib (10 mg/kg/day, n = 10). Error bars indicate mean ± standard error, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

Sitravatinib is efficient to eliminate FLT3-ITD-F691L cells in vitro and in vivo. A IC50 values of BaF3-FLT3-ITD and BaF3-FLT3-ITD-F691L cells treated with each drug for 48 h. Error bars indicate mean ± standard error, n = 3 independent experiments. B Fold changes of IC50 values of FLT3 inhibitors for BaF3-FLT3-ITD cells with or without secondary TKD mutations. Error bars indicate mean ± standard error, n = 3 independent experiments. C Western blot analysis of p-FLT3, p-STAT5, p-AKT and p-ERK 1/2 in BaF3-FLT3-ITD-F691L cells after treatment with sitravatinib at the indicated concentrations for 4 h. D BALB/c mice were intravenously inoculated with BaF3-FLT3-ITD-F691L cells. From 2 days after the injection, mice were administrated with vehicle, sitravatinib (20 mg/kg/day), gilteritinib (30 mg/kg/day) or quizartinib (10 mg/kg/day) until the first mouse died in the vehicle group. The percentage of leukemia cells in PB of BaF3-FLT3-ITD-F691L-diseased mice on day 10 was detected by flow cytometry (n = 6 mice per group). E The percentage of leukemia cells in BM and SP of BaF3-FLT3-ITD-F691L-diseased mice on day 10 (n = 3 mice per group). F Top: The spleen image and weight of mice in (E); Bottom: One mouse of each group in (E) was randomly selected for the H&E staining of spleens. A normal mouse used as control. Scale bars: 1500 μm. Green arrows: typical spleen lymph nodules; red arrows: fuzzy lymph nodules. G Body weight measurements of the mice during drug administration. H The survival curves of BaF3-FLT3-ITD-F691L-diseased mice treated with vehicle (n = 10), sitravatinib (n = 10), gilteritinib (n = 12) or quizartinib (n = 9). Error bars indicate mean ± standard error, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 5

Sitravatinib efficiently suppresses the resistance mediated by FGF2 and FL. A-B The IC50 values of sitravatinib (S) and gilteritinib (G) for MV4-11 or MOLM13 cells in the absence/presence of FGF2 or FL. Error bars indicate mean ± standard error, n = 3 independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001. C-F MV4-11 and MOLM13 cells were treated with sitravatinib or gilteritinib at indicated concentrations for 3 h, then FGF2 or FL (10 ng/mL) was added to the culture for incubation of 3 h. Cell lysates were immunoblotted to evaluate p-FLT3, p-STAT5, p-AKT and p-ERK 1/2. The densitometry ratio of p-AKT to AKT and p-ERK to ERK was assessed via ImageJ, and expressed as fold change compared with vehicle control

Fig. 6

Anti-leukemia activity of sitravatinib against AML primary blasts. A-D Dose–response curves of primary AML patient samples diagnosed as FLT3-ITD treated with sitravatinib, gilteritinib or quizartinib at indicated concentrations for 48 h. E–G Western blot analysis of p-FLT3, p-STAT5, p-AKT, p-ERK 1/2 in AML blasts harboring FLT3-ITD after 6-h sitravatinib treatment. H Schematic representation of the PDX model experiments. I The percentage of human CD45 positive cells in PB of PDX mice detected by flow cytometry on day 39 (n = 3 mice per group) and day 82 (n = 5 or 6 mice per group). J The percentage of human CD45 positive cells in BM and SP of PDX mice detected by flow cytometry on day 120 (n = 3 mice per group). Error bars indicate mean ± standard error, *P < 0.05, **P < 0.01, ***P < 0.001