Fig. 6.

Synergy of inhibitor 16 and kinase inhibitor midostaurin (PKC412). Activity tested in a murine cancer model. a MV-411 cells were treated with PKC412 (10 nm) or compound 16 (10 µm) alone or in combination and incubated for 24 h followed by annexin-V/ propidium iodide staining and flow cytometry. Apoptosis was quantitated for three independent experiments (n = 3) and error bars denote mean ± S.D. b Cell viability assays were carried out by treating MV-411 cells with compound 16 (10 µm) and PKC412 (10 nm) alone or in combination. The number of viable cells was distinguished using an ATP-dependent bioluminescence assay (CellTiter-Glo, Promega) (n = 3, error bars denote mean ± S.D.). c Combination index (CI) plot showing the synergistic effect of compound 16 and PKC412 in MV-411 cells. CI values were generated using CalcuSyn software (Conservion, Ferguson, MO) and plotted as a function of fractional growth inhibition (n = 3) (Fa) where Fa = (A₅₇₀ control−A₅₇₀ treated)/A₅₇₀control. CI values of < 1, = 1, and > 1 indicate synergism, additivity, and antagonism, respectively. Error bars denote mean ± S.D. d Relative STAT5 phosphorylation levels were plotted based on the raw data from western blotting chemiluminescence readings to study the synergistic effect of both compounds on STAT5 phosphorylation reduction. MV-411 cells were treated with compound 16 or PKC412 alone or in combination and incubated for 6 h (n = 3, error bars denote mean ± S.D.). e The corresponding body weight changes in non-xenografted mice during compound 16 treatments (n = 6, error bars denote mean ± S.D.). f Compound 16 significantly inhibits tumor growth in BaF3/FLT3-ITD xenograft tumor model. Time course of tumor growth suppressed by compound 16 (200 mg kg⁻¹) in mice bearing BaF3/FLT3-ITD tumor (n = 6, error bars denote mean ± S.D.)