Midostaurin synergizes with bosutinib in producing apoptosis in HMC-1 cells. (A) HMC-1.1 and HMC-1.2 cells were kept in control medium or in the presence of bosutinib (0.01-5μM). After 4 hours, Western blot analysis was performed using Abs directed against p-Lyn^Tyr507, p-Src^Tyr416, Lyn, p-Btk^Tyr223, Btk, KIT, and β-actin. To detect expression of p-KIT, an immunoprecipitation was conducted using an anti-KIT Ab followed by immunoblotting using the anti-phosphotyrosine Ab 4G10. (B) HMC-1.1 cells and HMC-1.2 cells were kept in control medium (Co) or in various concentrations of bosutinib or dasatinib (as indicated) for 48 hours. BM cells of 4 patients with SM (nos. 13, 17, 19, and 25 in Table 1; all KIT D816V⁺) were incubated in control medium or bosutinib (0.1-10μM) for 48 hours. After incubation, proliferation was determined by ³H uptake. Results are expressed as percent of control and represent the mean ± SD of 3 independent experiments (cell lines), or the mean ± SD of 4 experiments performed with primary neoplastic mast cells (4 patients). (C) In HMC-1 cells, the percentage of apoptotic cells was determined by light microscopy after 48 hours of incubation with 500-1000nM bosutinib. Results represent the mean ± SD of 3 independent experiments. *P < .05. (D,E) HMC-1.1 cells and HMC-1.2 cells were incubated with midostaurin and bosutinib as single drugs or in combination. (D) HMC-1.2 cells were incubated in control medium (0/0) or in medium containing midostaurin and bosutinib alone or in combination (in fixed ratio: 1:1 concentration) as indicated. Three different drug concentrations are shown (100, 200, and 300nM). After 48 hours of incubation, the percentages of apoptotic cells were determined by light microscopy. Results represent the mean ± SD of 3 independent experiments. *P < .05 compared with control (0). The bottom right panel shows combination index (CI) values obtained in these experiments using Calcusyn Software. As visible, the CI values are all below 1.0 indicating synergistic drug interactions. (E) After 4 hours of incubation in control medium, midostaurin (1μM), bosutinib (1μM), or a combination of both drugs (1μM each), Western blot analysis was performed using Abs directed against p-Lyn^Tyr507, Lyn, p-Btk^Tyr223, Btk, KIT, and β-actin. To detect expression of p-KIT, an immunoprecipitation was conducted using an anti-KIT Ab followed by immunoblotting with anti-phosphotyrosine Ab 4G10.