RAD001 concentrations and inhibition of phosphorylation of S6RP and 4E-BP1 after drug administration. Mice engrafted with ALL-1345 or ALL-0398 cells were treated with RAD001 or vehicle and blood harvested after the indicated time points of continuing treatment and evaluated for RAD001 concentrations. (A) RAD001 (mean ± SD, n = 3) concentration-time profiles in mice engrafted with ALL-1345 () and ALL-0398 (----) up to 48 hours after administration of a single dose of RAD001. (B) RAD001 (mean ± SD, of duplicate determinations from 3 experiments) trough concentrations in mice treated for up to 14 days with RAD001. (C) Ex vivo analysis of the phosphorylation of S6RP and 4E-BP1 in ALL cells from spleens recovered from mice engrafted with ALL-1345 or ALL-1999, 1 or 7 days into the treatment protocol (left and center panels). Cells from xenografts ALL-1196, -2032, and -0398 recovered from untreated mice were treated in vitro for 24 hours with or without 2 μM RAD001 (right panel). The numbers below the blots indicate the intensity of phosphorylation-specific bands for 4E-BP1 and S6RP relative to total 4E-BP1 and S6RP. (D) Representative plots showing the levels of phosphorylated 4E-BP1 assessed by flow cytometry in ALL cells recovered from spleens of mice engrafted with ALL-1345. (E) Time course of the reduction of phosphorylated 4E-BP1 after administration of RAD001 measured by flow cytometry in ALL cells recovered from mice engrafted with ALL-1345.