Antileukemic activity of bortezomib is linked to FLT3-ITD expression. (A) FLT3-ITD AML cell lines are more sensitive than FLT3-WT to bortezomib (Bort)-induced cell death. Apoptosis was evaluated by treating MV4-11, MOLM-14, U-937, HL-60, or Kasumi-1 AML cell lines 24 hours with bortezomib at 1, 3, or 10 nM before Annexin-V/7-AAD staining and flow cytometry analysis. (B-C) Bortezomib targets preferentially FLT3-ITD primary AML samples. Frozen primary AML samples were thawed and treated with bortezomib at 3, 10, 30, or 100 nM for 24 hours. Bortezomib-induced apoptosis was evaluated by labeling cells with Annexin-V/7-AAD via fluorescence-activated cell sorter (FACS) analysis. (D) Viability was assessed by Annexin-V/7-AAD via FACS analysis, and IC50 values were calculated using GraphPad Prism software. (E) Correlation between bortezomib sensitivity and FLT3-ITD mutation load estimated by the level of FLT3-ITD to FLT3 wild-type (WT) allele ratio. (F) Bortezomib activity and FLT3-ITD mutational load in a patient sample assessed at diagnosis and relapse. (G-H). Antileukemic activity of bortezomib, according to NPM1 mutational status. *P < .05; **P < .01; ***P < .001.