FL impairs inhibition of FLT3 autophosphorylation in vivo. (A) Plasma samples from 2 individual patients treated on the MRC AML15 trial were collected at different time points and then assessed for FLT3 inhibitory activity (PIA assay). Cells were exposed to the plasma for 3 hours and then lysed. FLT3 was immunoprecipitated, subjected to electrophoresis, and transferred to a membrane. The blot was probed with antiphosphotyrosine (top row), then stripped and reprobed with anti-FLT3 (bottom row). FL and lestaurtinib levels were determined from the same plasma samples as described in “FL ELISA” and “Pharmacokinetics.” (B) Plasma was collected from a single newly diagnosed AML patient at different time points after diagnosis and treatment with induction chemotherapy (cytarabine, daunorubicin, and etoposide). The plasma was assayed for FL levels by enzyme-linked immunosorbent assay and plotted (○). In parallel, AC220 was added to a concentration of 2μM for each time point and used to incubate Molm14 cells for 2 hours. Each sample was then assayed for FLT3 inhibitory activity as in panel A. The densitometric analysis of the phospho-FLT3 blot (top blot; ●).