In vivo antileukemic activity of bortezomib in FLT3-ITD driven leukemia. (A-D) Bortezomib (Bort) inhibited tumor growth and targeted FLT3-ITD protein expression in NOD/SCID mice subcutaneously engrafted with MV4-11 cells. Mice were treated twice a week with 1 mg/mL bortezomib IP. (A-B) Tumor growth was evaluated by measuring tumors with a caliper. (C) Tumor cells were lysed and analyzed by western blot using indicated antibodies. (D) Western blot quantification of FLT3-ITD protein expression. (E-J) Antileukemic activity of bortezomib in NSG mice engrafted by IV injection of MV4-11 cells. After engraftment mice were treated twice a week with IV injections of 0.75 mg/kg bortezomib. After 3 daily doses of bortezomib, vehicle mice (n = 5) and treated mice (n = 5) were sacrificed and analyzed. (F) Percentage of human cells in the murine bone marrow was assessed by hCD45⁺/hCD33⁺/hCD44⁺ labeling and flow cytometry. (G) In vivo bortezomib-induced cell death was evaluated onto the hCD45⁺/hCD33⁺/hCD44⁺ compartment after Annexin-V staining via flow cytometry. (H) Viable human cells hCD33⁺/hCD45⁺/Annexin-V− were cell sorted by flow cytometry, lysed, resolved by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, and immunoblotted with indicated antibodies. (I-J) Ratio FLT3-ITD/actin (I) or LC3-II/actin (J) using GeneTool software (western blot quantification). (K) Kaplan Meier survival curves of mice treated by bortezomib or vehicle (Veh). *P < .05; **P < .01; ***P < .001.