Activity of AC220 in primary AML cells. (A) Inhibition of FLT3 phosphorylation. Blasts were incubated in increasing concentrations of AC220 with 10% FBS for 1 hour at 37°C. For the Western blot, cell lysates were subjected to immunoprecipitation, sodium dodecylsulfate polyacrylamide electrophoresis, and immunoblotting for phosphorylated and total FLT3. As is frequently observed in patient samples for which limited numbers of cells are available, phospho-FLT3 (pFLT3) levels are relatively low and only weakly detected by Western blot. Robust quantitation of phospho-FLT3 was achieved by a more sensitive ELISA (“Cellular assays”). The graph shows phospho-FLT3 levels, normalized for total FLT3, obtained by ELISA. The line represents a fit of the data to the Hill equation. (B) Effect of AC220 on cell viability. In parallel to the phosphorylation assay, using the same AC220 medium preparation, blasts from the same patient and the same thawing were incubated at 37°C in 5% CO₂. After 72 hours, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay was performed, and the results were plotted as a fraction of untreated control. The line represents a fit of the data to the Hill equation. (C) Effect of AC220 on cell viability in primary cell samples from additional patients. The experiments were performed and the data are plotted as in panel B. The primary cells were obtained from a 65-year-old male (■; IC₅₀ = 0.8 nM), a 53-year-old male (▲; IC₅₀ = 1 nM), a 56-year-old female (●; IC₅₀ = 1 nM), and a 34-year-old male (▼; IC₅₀ = 2 nM), all with relapsed AML harboring FLT3-ITD mutations.