Fig 2: ARQ 531 demonstrates in vitro anti-leukemic effect in primary AML. MTS proliferation assay using a human stroma co-culture assay. Primary patient blasts (n = 11–12 pts) were co-cultured with HS5-GFP stroma cells and treated with increasing concentrations of ARQ 531 (a) or gilteritinib (b) for 96 h. FLT3 wild-type (WT) samples (red) and FLT3-ITD (black). Colony-Forming Unit-Granulocyte and Macrophage (CFU-GM) assay. Four primary patient blasts were treated with DMSO or three µM ARQ 531 or 0.1 µM gilteritinib (Gilt.) and then cultured in methylcellulose in duplicate dishes. Number of colonies were counted after 14 days (c)

Fig 3: AXL-targeted agents inhibit the proliferation of FLT3-mutant AML cell lines and FLT3-ITD+ AML blast cells in a dose-dependent manner. (A) DAXL-88 inhibited the proliferation of FLT3-WT+ THP-1, FLT3-ITD+ MV4-11 and MV4-11/AC220 cells. (B) DAXL-88 inhibited the proliferation of FLT3-ITD+ AML blast cells. (C) DAXL-88-MMAE inhibited the proliferation of AXL-expressing AML cells. (D) DAXL-88-MMAE inhibited the proliferation of AXL-expressing AML blast cells. (E) R428 inhibited the proliferation of AXL-expressing AML cells. (F) R428 inhibited the proliferation of AXL-expressing AML blast cells. Cells were treated with DAXL-88, DAXL-88-MMAE and R428 at the indicated concentrations for 48 h. Cell viability was measured using a Cell Counting Kit-8 assay. #P<0.05, *P<0.01, and **P<0.001 (ANOVA and Dunnett test; AML cells vs. MV4-11/AC220 cell line; AML blast cells vs. patient's sample 076). Data are presented as the mean ± SEM. AXL, AXL receptor tyrosine kinase; AML, acute myeloid leukemia; FLT3, FMS-like tyrosine kinase 3; ITD, internal tandem duplication; WT, wild-type.

Fig 4: Effect of A674563 on FLT3- and AKT-mediated signaling and overriding of FLT3 ligand-induced drug resistance(A) Combination study with FLT3 inhibitor TCS359, and AKT inhibitor MK2206. (B) Anti-proliferation effects of A674563 and FLT3 inhibitors, TCS359 and MLN518, against FLT3-ITD-positive AML cells in the presence of 10 ng/mL FLT3 ligand. (C) Effect of A674563 and MLN518 on FLT3-mediated signaling in MOLM14 (FLT3-ITD/wt) in the presence/absence of FLT3 ligand. (D) Effect of A674563 and MLN518 on AKT-mediated signaling in MOLM14 (FLT3-ITD/wt) in the presence/absence of FLT3 ligand.

Fig 5: The chemical structure of SKLB-677 and its in vitro bioactivities.(a) Chemical structures of compound 1 and SKLB-677. (b) Binding affinities of SKLB-677 against human FLT3-wt and the FLT3-ITD mutant measured by KINOMEscan kinase binding assays (Ambit Biosciences). The y-axis indicates the concentrations of the tested kinases, which are represented by the signal of qPCR (the tested kinases were tagged with DNA and detected using qPCR assays). All data points are means of triplicates ± SD. IC50 values are presented. (c) SKLB-677 was screened against a KINOMEscan ( http://www.kinomescan.com) panel of 456 kinase assays. The red circles indicate bound kinases, and the circle size indicates the degree of binding affinity. The complete dataset is shown in Supplementary Table S3. (d) MV4-11 or Molm-13 cells were incubated in the presence of various concentrations of SKLB-677. The viabilities of the cultured cells after 72 h were measured by MTT assays. All data points are means of triplicates ± SD. IC50 values are presented. (e), Primary AML PBSCs were incubated in the presence of various concentrations of SKLB-677 and/or AC220. The viabilities of the cultured cells after 72 h were measured by the CellTiterGlo assays. The primary AML cells were obtained from 6 patients (sample #1, #2, #3: FLT3-ITD positive, sample #4, #5, #6: FLT3-ITD negative). All data points are means of triplicates ± SEM. IC50 values are presented in the right-hand column. (f) Normal PBMCs were incubated in the presence of various concentrations of SKLB-677 for 72 h, followed by the measurement using CellTiterGlo assays. All data points are means of triplicates ± SEM. All the IC50 values are >10 µM.