Cell lines and compounds

MV4-11, MOLM-13, U937 and THP-1 cells were cultured with RPMI-1640 (Gibco) containing 10% FBS (Corning). Ba/F3 cells expressing FLT3-ITD, FLT3-ITD/F691L, FLT3-ITD/D835Y, FLT3-ITD/D835V, FLT3-ITD/D835F, FLT3-ITD/Y842C, BCR::ABL1/P190 (P190) and BCR::ABL1/T315I (T315I) were generated by retroviral infection as previously described11 and cultured with RPMI-1640 containing 10% FBS. AC220, imatinib and dasatinib were purchased from Selleck (Shanghai, China), GNF-7 was purchased from CSNpharm (Shanghai, China), gilteritinib was purchase from AbMole (Shanghai, China).

Mononuclear cells isolated from bone marrow

Bone marrow samples were collected from three patients with diagnosed FLT3-ITD-AML (detailed information for these patients are provided in Additional file 1: Table S1). Mononuclear cells (MNCs) were then separated from umbilical cord blood or bone marrow samples as previously reported [14] and supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma Aldrich). This study was approved by the Institutional Review Board of the Guangzhou Women and Children’s Medical Center affiliated to Guangzhou Medical University and Guangzhou First People's Hospital. Informed consent for the in vitro drug testing studies was obtained in accordance with the declaration of Guangzhou Women and Children’s Medical Center affiliated to Guangzhou Medical University and Guangzhou First People's Hospital.

Cell growth inhibition assay

Normalized cell proliferation detected in the study were carried out using the CellTiter-Glo® Luminescent Cell Viability Assay as described previously [14]. Leukemia cell lines and primary cells were seeded into 96-well cell culture plates at a density of 104 and 2 × 104 cells per well, and then added with indicated drugs at various concentrations. After 48 h incubation, cells were lysed by CellTiter Glo reagent (Promega, #G7572) and the luminescence signals were detected through a multimode microplate reader (VICTOR Nivo).

Cell apoptosis assay

After treated with different concentrations of GNF-7, gilteritinib or AC220 for 48 h, leukemia cells were harvested and incubated with Annexin V-FITC and PI (KeyGEN BioTECH, China). The portion of Annexin V+ cells were detected by flow cytometry (BD Bioscience, San Jose, CA, USA).

Western blotting

Western blotting was performed as previously described [15]. Briefly, cell samples were harvested and lysed by 1 × SDS lysis buffer. Equal amount of protein samples was loaded on polyacrylamide gel and then transferred to nitrocellulose membrane. The membrane was then blotted with specific primary antibodies against p-FLT3 (Tyr591, #3466, CST), p-AKT (Ser473, #4060S, CST), ERK (#4695S, CST), p-ERK (T202/Y204, #4370S, CST), p-Stat5 (Y694, #AP0887, Abclonal), FLT3 (#ab245116, Abcam), and Stat5 (#13179-1-AP, Proteintech), AKT (#10176-2-AP, Proteintech), actin-HRP (#HRP-60008, Proteintech). After overnight incubation at 4℃, HRP-conjugated secondary antibodies were applied and luminescence signals on membrane were detected with electrochemical luminescence (BIO-RAD).

Cellular thermal shift assay (CETSA)

CETSA assay was performed as previously described [16]. Ba/F3 FLT3-ITD cells were treated with GNF-7 (1 μM) or DMSO for one hour, then harvested and lysed by liquid nitrogen. The lysates were divided into equal volume and heated at different temperatures for three minutes, after cooled to room temperature, lysates were centrifuged and the supernatants were collected and subjected to SDS-PAGE and western blotting analysis.

The dose effect of GNF-7 on the thermal stability of FLT3 was evaluated as follows. Same number of Ba/F3 FLT3-ITD cells were exposed to various concentrations of GNF-7 for one hour and then lysed by liquid nitrogen. Subsequently, the lysate solutions were heated at 50 °C for three min and then centrifuged, and the supernatants were subjected to SDS-PAGE and western blotting analysis.

Animal models

0.8 × 106 Ba/F3 FLT3-ITD cells or 0.5 × 106 Ba/F3 FLT3-ITD-F691L cells were injected intravenously into BALB/c mice (6–8 weeks old, female, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), respectively. The mice were randomly divided into four groups. Three days after the cell injection, the treatment group received GNF-7, gilteritinib, AC220 or the same volume of solvent. Oral dosing with 15 mg/kg GNF-7 was administered to the mice once a day, 10 mg/kg AC220 and 30 mg/kg gilteritinib were used as the control. In order to assess the effect of the therapy, leukemia cells infiltrating peripheral blood or bone marrow was collected from each group of mice and analyzed by flow cytometry. Moreover, the leukemia burden of mice model was also measured by the spleen weight. The survival time of the mice was determined.

In the patient-derived xenograft (PDX) model, five weeks old female NOG mice (Charles River) were sub-lethally administrated with busulfan (30 mg/kg) before tail vein injection of AML #3 or AML #4 primary cells. After engraftment was successfully established, the primary blasts (2 × 106 human AML #3 cells or 5 × 105 human AML #4 cells) were collected, and then reinjected into busulfan treated NOG mice for the secondary transplantation. Soon afterwards, the transplanted mice were randomly divided into three groups. 15 days or 44 days after the engraftment, AML #3 or AML #4 cells transplanted mice was oral administrated with vehicle, 30 mg/kg gilteritinib and 15 mg/kg GNF-7, respectively. Residual leukemia cells were identified with hCD45 antibody (Biolegend) and leukemia stem and progenitor cells were identified with hCD45 plus with hCD34 antibody (BD Bioscience) through flow cytometry.

Animal experiments were conducted in accordance with established guidelines and were approved by the Institutional Animal Care and Welfare Committee of Guangzhou Women and Children’s Medical Center, Guangzhou Medical University.

Model of GNF-7 bound to FLT3 protein

AutoDock Vina 1.1.2 software was used for molecular docking work, and the structure of the small molecule GNF-7 was energy minimized using AVOGADR 1.2.0 under the MMFF94 force field before the docking began. The FLT3 protein (5X02) was hydrotreated using PyMol software. Then ADFRsuite 1.0 was used to convert small molecules and receptor proteins into PDBQT format necessary for AutoDock Vina 1.1.2 docking. Before docking, center the box with the ATP site of the FLT3 protein was required. The detail of the global search for docking was set to 32, and the rest of the parameters remained the default settings. Finally, the highest-scoring docked conformation output was regarded as binding conformation and the docking results were visualized using PyMol and Ligplot 2.2.4 software.

Statistical analysis

GraphPad Prism 8.0 software was used for statistical data analysis. Two-tailed paired Student’s t test was used for mean comparison between two groups, whereas the Kaplan–Meier survival curve and log-rank test were used for survival analysis. P values < 0.05 were considered statistically significant, and different levels were denoted as *, P < 0.05, **, P < 0.01, and ***, P < 0.001, respectively.