ABT-869 inhibits phosphorylation of FLT3, STAT5, and ERK in the human blood AML model. MV-4-11 cells were spiked into normal human blood, 1 × 107 cells/mL, and treated with varying concentrations of ABT-869 for 2 hours. For FLT3 phosphorylation (A), blood was lysed and the FLT3 was immunoprecipitated from 1 mL of lysate with anti-FLT3 (sc-480; 10 mg/mL) and analyzed by Western blot. The results from at least 3 independent experiments demonstrate similar inhibition of phosphorylation of FLT3 by ABT-869; a representative blot is shown. (B) STAT5 and (C) ERK phosphorylation was analyzed from PBMCs prepared from blood treated as described above. Phospho-STAT5 and -ERK were determined directly in cell lysates. (D) Phospho-STAT5 and Pim-1 expressions were analyzed from PBMCs prepared from blood treated as described in “Materials and methods,” with the exception of spiking with MOLM-13 cells. The results from 2 independent experiments demonstrate similar inhibition of the phosphorylation of STAT5 and ERK and decreased Pim-1 expression by ABT-869; a representative blot is shown. (E) Pharmacokinetic analysis of ABT-869 plasma concentration from orally dosed mice with 3 and 10 mg/kg/day in 2.5% ethanol, 5% Tween-80, and 25% PEG 400 in PBS, and the 40 mg/kg dose in pure refined corn oil. ABT-869 was analyzed from plasma that had been protein precipitated with acidified methanol and concentration determined using LC-MS as described in “Materials and methods.” Data are shown as μM ± SEM.
