To the editor

FLT3 mutations occur in more than 30% of patients with acute myeloid leukemia (AML) and are associated with short relapse-free and overall survival, including internal tandem duplication (ITD) and point mutations within the tyrosine kinase domain (TKD) [1, 2]. To date, multiple FLT3 kinase inhibitors have been developed and some are approved for clinical use including sorafenib, Quizartinib (AC220) and Gilteritinib [3]. However, clinical responses to these drugs are transient because of high rates of relapse and drug resistance after treatment, which contributes to disease progression and poor overall survival [4, 5]. One particular mechanism for resistance involves acquired additional mutations in the TKD and a “gatekeeper” mutation (F691L) is resistant to most currently available FLT3 inhibitors [6, 7]. Therefore, finding effective compounds to overcome the drug resistance caused by F691L and other mutations is an urgent problem. Herein, we identified KX2-391 as a FLT3 inhibitor and evaluated its activity against FLT3-ITD-TKD mutations using in vitro and in vivo models.

We used molecular docking simulations to computationally screen 1487 small molecule ligands from the L3400 Clinical Compound Library and identified KX2-391 as a candidate FLT3 inhibitor (Additional file 1: Table S1). Previous studies reported KX2-391 was an SRC/tubulin dual inhibitor and exhibited anticancer activities in some tumors [8]. Our modeling predicted the interaction of KX2-391 with FLT3’s L616, V624 and E661 residues (Additional file 2: Fig. S1). We further confirmed the interactions between FLT3 and KX2-391 by cellular thermal shift assay. Compared with DMSO, an obvious thermal shift of the melting curve was detected in the KX2-391-treated sample. The thermal stability of FLT3 protein was increased by KX2-391 in a dose-dependent manner (Fig. 1a). We evaluated the inhibitory activity of KX2-391 against different forms of FLT3 in Ba/F3 cells and found it potently inhibited the growth of FLT3-ITD-expressing Ba/F3 cells and all tested cells expressing FLT3-ITD-TKD mutations previously linked with drug resistance to FLT3 inhibitors, such as AC220 (Fig. 1b, c, Additional file 1: Table S2). Notably, Ba/F3-ITD-F691L cells were tenfold more sensitive to KX2-391 than parental Ba/F3 cells (0.032 μM vs 0.372 μM). KX2-391 also displayed higher inhibitory efficacy on human leukemia cell lines harboring FLT3-ITD (MV4-11 and MOLM13) than that on FLT3 nonmutated leukemia cell lines (Fig. 1d). We observed dose-dependent induction of apoptosis in Ba/F3 cells expressing FLT3-ITD, FLT3-ITD-D835Y and FLT3-ITD-F691L as well as in two FLT3-ITD positive AML cell lines (Fig. 1e, f). KX2-391 prominently inhibited the phosphorylation of FLT3 and downstream targets STAT5, ERK and AKT in FLT3-ITD, FLT3-ITD-F691L-expresssing Ba/F3 cells and other cells of our assay panel (Fig. 1g, h). Recalling that KX2-391 has to date understood as an SRC/tubulin inhibitor [9], we monitored SRC phosphorylation and assessed KX2-391’s effects on microtubule morphology. KX2-391 treatment did not alter phosphorylation of SRC in FLT3 mutant cells (Fig. 1g, h). We did detect disrupted tubulin polymerization in MOLM13 cells treated with KX2-391 and with various known tubulin inhibitors (Additional file 2: Fig. S2). Excluding a direct impact of tubulin modulation, assays showed that the known tubulin inhibitor Vincristine did not affect phosphorylation of FLT3 or its downstream targets (Additional file 2: Fig. S3).

Fig. 1
figure 1

KX2-391 is active against ITD-TKD resistance-causing FLT3 mutations and blocks FLT3 signaling in FLT3-ITD and FLT3-ITD-TKD cells. a Quantification of cellular thermal shift assay was made using western blot Ba/F3 cells expressing FLT3-ITD cells were treated with KX2-391 (1 µM) for 1 h, and temperatures between 43 and 55℃ were defined to perform the test. KX2-391were treated based on 10 different concentrations for 1 h at 51℃. Data were normalized by setting the highest and lowest value in each set to 100% and 0%, respectively. Data were obtained in the presence of the KX2-391 (blue circle) as the positive control and DMSO (red square) as the negative control. b Normalized cell viability of Ba/F3 cells expressing FLT3-ITD TKD mutations after a 48 h exposure to various concentrations of KX2-391 and AC220 (c), measured using CellTiter Glo assays (error bars represent the SD of 3 or more independent experiments). d Viability of FLT3-ITD mutated cells (MV4-11, MOLM-13), and FLT3 nonmutated cells (NB4, SKNO1, TF1) treated with various concentrations of KX2-391 for 48 h, measured by CellTiter Glo assays. e Ba/F3 cells expressing FLT3-ITD TKD mutations were treated with different concentrations of KX2-391 for 24 h and then parental Ba/F + IL3, FLT3-ITD, FLT3-ITD-D835Y, and FLT3-ITD-F691L cells, as well as f MOLM13 and MV4-11 cells were examined by flow cytometry (Annexin V). g Ba/F3 cells expressing FLT3-ITD, FLT3-ITD-D835Y, FLT3-ITD/F691L, FLT3-D835V, and FLT3-D835F, as well as FLT3-ITD positive human leukemia cell lines h MOLM13 and MV4-11 were incubated for 12 h with the indicated concentrations of KX2-391 (based on the IC50 values) and subsequently examined by western blotting using antibodies against FLT3/P-FLT3, STAT5/P-STAT5, ERK/P-ERK, AKT/P-AKT and SRC/P-SRC. GAPDH was used as a loading control. All values represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001versus the control

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Next, we used the previously described Ba/F3-ITD-F691L leukemia model to measure the efficacy of KX2-391 in vivo [10]. KX2-391 showed a significant benefit on median survival, prolonging the survival period from 12 days in the control group to 23.5 days (Fig. 2a, P < 0.0001). No significant weight loss (Fig. 2b) or other signs of toxicity were observed during treatment. Compared to the vehicle control, AC220 and Gilteritinib groups, KX2-391-treated mice had fewer leukemia cells (in peripheral blood, spleen, and bone marrow) (Fig. 2c, d) and had smaller spleens (Fig. 2d). H&E staining demonstrated that KX2-391 treatment significantly reduced AML cell infiltration into the spleen and liver (Fig. 2e).

Fig. 2
figure 2

In vivo effects of KX2-391 in mice bearing FLT3-ITD-F691L leukemia and against patient leukemic blast cells harboring FLT3-ITD or FLT3-ITD-D835Y mutations. a Kaplan–Meier survival curves of FLT3-ITD-F691L leukemia mice administered vehicle, Gilteritinib (30 mg/kg), AC220 (10 mg/kg), or KX2-391 (10 mg/kg) once daily for 10 days (orally). **** P < 0.0001. b No change in body weight was detected between the KX2-391 treatment and vehicle control groups. c The percentage of GFP-positive Ba/F3 FLT3-ITD-F691L leukemia cells in peripheral blood (PB) samples from mice treated with vehicle, Gilteritinib (30 mg/kg/d), AC220 (10 mg/kg/d), or KX2-391 (10 mg/kg/d) for 4 days and for 8 days. d Representative weight of spleens at 10 days after injection with Ba/F3 FLT3-ITD-F691L leukemia cells, and Flow cytometry analysis of bone marrow cells and spleens from mice as described in (c). e Hematoxylin and eosin staining of spleens and liver from mice treated as described in (a). Scale bars in the panel are 50 μm. f Patient-derived AML leukemic blast cells expressing FLT3-ITD/D835Y (patients 1 and 2), FLT3-ITD (patient 3) and peripheral blood mononuclear cells (PBMCs) from healthy donors were incubated for 48 h with the indicated concentrations of KX2-391 (g), and the cell viability was then determined. For each FLT3 inhibitor, the percentage over DMSO control was presented as a mean value, with error bars representing ± SD. h KX2-391 suppresses FLT3 phosphorylation in primary AML cells incubated for 12 h with the indicated concentrations (based on the IC50 values), as determined by western blotting using the indicated antibodies. GAPDH was used as a loading control. ****P < 0.0001

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We also evaluated the anti-leukemia effects of KX2-391 on primary AML cells isolated from 6 newly diagnosed FLT3-ITD AML patients (Additional file 1: Table S3). KX2-391 effectively reduced cell viability in 4 primary blasts with FLT3-ITD mutations (comparable with AC220’s effect). It also significantly inhibited the growth of 2 primary AML cells expressing FLT3-ITD-D835Y—both of which were resistant to AC220 (Fig. 2f, Additional file 2: Fig. S4). KX2-391 did not affect the growth of healthy peripheral blood mononuclear cells at the same concentrations (Fig. 2g). We confirmed that KX2-391 inhibited FLT3 phosphorylation in primary AML cells (Fig. 2h).

Collectively, we show that KX2-391 is an FLT3/tubulin inhibitor that exerts strong therapeutic effects both in vitro and in vivo against FLT3-ITD and drug resistant TKD mutations including FLT3-ITD-F691L, which is understood as the most difficult mutation to overcome clinically. Considering the detected therapeutic effects and its apparently minimal toxicity [11, 12], KX2-391 may become a useful second-line drug suitable for treating some of the most clinically challenging AML cases.