Fig 2: Fes tyrosine kinase inhibitors induce apoptosis in Flt3-ITD+ AML cell lines.The Flt3-ITD+ cell lines MV4-11, MOLM-13, and MOLM-14, along with THP-1 (wild-type FLT3), were incubated with each of kinase inhibitors shown at a final concentration of 1 µM and apoptosis was measured 72 h later using the Apo-ONE Caspase 3/7 Assay (Promega). Cell viability was measured in parallel using the CellTiter-Blue assay, and the resulting data are presented as the ratio of Caspase activity to cell viability as per the manufacturer’s protocol. All assays were performed in triplicate, and data are presented as the mean ratios ± SD. Asterisks indicate statistical significance between each treatment group vs. the corresponding DMSO control group with p < 0.05 (Student’s t-test).

Fig 3: ARQ 531 modulates AML pro-survival kinases. Immunoblot analysis for MOLM-13, MV4-11, and OCI-AML3 AML cell lines (representative of 2-3 independent blots). All cell lines were serum starved overnight followed by 10-min treatment of five to 10 million cells with DMSO, increasing concentrations of ARQ 531 in comparison with 50 nM of quizartinib (Quiz.) or 50 nM gilteritinib (Gilt.), 0.1 µM midostaurin (Mido.), 0.5 µM dasatinib (Das.), 1 µM entospletinib (Ento.), or a combination of 1 µM ARQ 531 and Ento. Twenty to 25 µg of total protein lysate was loaded per lane. GAPDH, HSP90, or ß-Actin were used as loading controls. a, b Modulation of phosphorylated SFK and downstream targets in FLT3-ITD cell lines MOLM-13 and MV-11, respectively. c, d Modulation of phosphorylated SYK and BTK in in all three cell lines

Fig 4: Analysis of Flt3-ITD phosphotyrosine content in TF-1 cells following A-419259 treatment.Each TF-1 cell population from Fig 3 was treated with A-419259 at the concentrations shown or with the DMSO carrier solvent (0.1%) alone as control. Following overnight incubation, Flt3 was immunoprecipitated and analyzed for phosphotyrosine (pTyr) content and Flt3 protein recovery by immunoblotting. Flt3 and pTyr immunoreactivity were quantified using the Odyssey infrared imaging system. Each experiment was performed in triplicate, and the resulting Flt3 pTyr:Flt3 protein ratios were fit by non-linear regression analysis to determine the IC50 value. (A) Individual concentration-response curves for TF-1 cells expressing Flt3-ITD (ITD), as well as the inhibitor-resistant mutant, D835Y, either alone or in the presence of Hck or Fgr. Each replicate is represented by a different color (red, blue, green). (B) Summary of IC50 values for all experiments (Flt3-ITD cells, blue; ITD D835Y cells, red; ITD F691L cells, green). Each point represents a single IC50 determination, with the vertical bar representing the mean value ± SE. Student’s t-tests did not show a significant difference between the IC50 values obtained with TF-1 cells expressing Flt3-ITD alone or in the presence of Hck or Fgr. A-419259 treatment did not inhibit the Flt3 pTyr:Flt3 ratios for cell populations expressing the D835Y mutant alone or the F691L mutant either alone or in the presence of Hck or Fgr (highest concentration tested was 1,000 nM).

Fig 5: Acquired resistance to A-419259 in the Flt3-ITD+ AML cell lines MV4-11, MOLM13 and MOLM14 is a heritable trait.Three independent populations of each AML cell line were passaged in the presence of increasing concentrations of A-419259 until outgrowth was observed with an inhibitor concentration of 1 µM. MV4-11 cells yielded two resistant populations (R2, R3), MOLM13 yielded one (R3), while three were obtained from MOLM14 (R1, R2, R3). P, parent cell line. (A) Three replicates of the parent and resistant cell populations were incubated over a range of A-419259 concentrations the DMSO carrier solvent (0.1%) alone as control. Cell viability was determined 72 hours later using the CellTiter Blue cell viability assay. Results are shown relative to the DMSO control values, and IC50 values were determined by non-linear regression analysis of the resulting concentration-response curves and are plotted for each cell line. The mean IC50 value for each cell population is shown as the black bar, and IC50 values for all resistant populations were significantly higher than those for the corresponding parent cell line (p < 0.05 by Student’s t-test). (B) Resistant cell populations from part A were passaged 8 times over the course of 4 weeks in the absence of A-419259, followed by re-determination of the IC50 values for A-419259. IC50 values from parts A and B are summarized in Table 1.