Bortezomib regulates wild-type FLT3 expression. (A) Downregulation of FLT3-wild-type by bortezomib (Bort). OCI-AML3 cells were treated for 24 hours with increasing concentrations of bortezomib. Total cell lysates were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immunoblotted with the indicated antibodies. (B) Bortezomib induces an active autophagic flux in FLT3-WT cells. OCI-AML3 cells were incubated for 24 hours with 10 nM bortezomib alone or in combination with 10 nM Bafilomycin A for the last 2 hours. Cells were lysed and LC3-I to LC3-II conversion was assessed by western blot analysis. (C) Inhibition of autophagy rescues bortezomib-induced downregulation of FLT3-WT. OCI-AML3 were treated for 24 hours with 10 nM bortezomib alone or in combination with 5 mM 3-MA, before cell lysis and immunoblotting analysis. (D) Bortezomib effects in primary FLT3-WT AML samples. Patient samples were treated 24 hours with increasing concentrations of bortezomib and then analyzed by western blot. (E) Autophagy inhibition by 3-MA affects bortezomib-induced cell death in OCI-AML3 cells. OCI-AML3 cells were treated 24 hours with 10 nM bortezomib alone or in combination of 5 mM 3-MA before Annexin-V/7-AAD staining and flow cytometry analysis. (F-I) Activity of bortezomib in NSG mice engrafted by IV injection of OCI-AML3 cells. After engraftment, mice were treated twice a week with intraperitoneal injections of 0.75 mg/kg bortezomib. (F) Viable human cells hCD33⁺/hCD45⁺/Annexin-V⁻ were cell sorted by flow cytometry, lysed, resolved by SDS-PAGE, and immunoblotted with indicated antibodies. (G-H) Ratio FLT3-ITD/actin (G) or LC3-II/actin (H) using GeneTool software. (I) Kaplan-Meier survival curves of mice treated by bortezomib or vehicle.