Bortezomib overcomes resistance to quizartinib induced by tyrosine kinase domain mutations of FLT3-ITD. (A-B) MOLM-14/TKD-D835Y cells were as sensitive to bortezomib (Bort) as parental MOLM-14. (A) Cells were treated with bortezomib before Annexin-V/7-AAD labeling and analysis by flow cytometry. (B) MOLM-14/TKD cell proliferation upon bortezomib treatment was assessed by trypan blue counting. (C-J) Activation of autophagy by bortezomib in MOLM-14/TKD cells. (C) MOLM-14/TKD cells were incubated 24 hours with 10 nM bortezomib before immunofluorescence analysis using LC3-B antibody and an anti-mouse-Alexa 488 antibody, and labeling with DAPI. Analyses were performed on a Zeiss LSM710 confocal microscope. (D) Quantification of LC3-positive dots. At least 100 cells per condition of three independent experiments were counted. (E) Cells were treated 24 hours with 10 nM bortezomib before immunoblotting analyses using appropriate antibodies. (F-G) Downregulation of FLT3-ITD after bortezomib treatment. MOLM-14, MOLM-14/TKD cells (F) or a FLT3-TKD primary AML sample (G) were treated 24 hours with 10 nM or 3 nM bortezomib, respectively, and then analyzed by western-blotting. (H-I) MOLM-14/TKD cells were infected with TRIPZ lentiviral shRNA targeting ATG12 or VPS34. ShRNA expressions were induced by 72 hours 1 µg/mL doxycycline treatment and cells were incubated 24 hours with 10 nM bortezomib. Cells were then lysed and analyzed by immunoblotting. (J) Cells were treated 24 hours with 10 nM bortezomib in the presence or not of doxycycline and apoptosis was assessed by flow cytometry after Annexin-V/7-AAD staining. (K) Kaplan Meier survival curves of MOLM-14 and MOLM-14/TKD mice treated by bortezomib or vehicle. Dapi, 4,6 diamidino-2-phenylindole. *P < .05; **P < .01; ***P < .001.