Targeting the SLC1A5 glutamine transporter has proapoptotic effects and in vivo antileukemic activity. (A) SLC1A5 expression was studied by western blotting in the 4 indicated AML cell lines. (B-F) MOLM-14 cells were infected with a lentiviral vector expressing either an inducible SLC1A5 or a control (CTR) shRNA. Stably infected cell lines were established after selection in puromycin. (B) MOLM-14 cells infected with SLC1A5 or CTR shRNA were cultured with or without doxycycline for the indicated times. The inhibition of SLC1A5 expression with doxycycline was assessed by western blotting. (C) MOLM-14/SLC1A5 shRNA cells and MOLM-14/CTR shRNA cells were starved or not for 6 hours of glutamine and leucine to inhibit mTORC1, as detected by the inhibition of p70S6K T389 phosphorylation. Previously starved cells were then stimulated for 15 or 30 minutes with leucine + glutamine in the presence or absence of doxycycline. The reactivation of mTORC1 was then determined. (D) Apoptosis was evaluated in MOLM-14/SLC1A5 shRNA cells and MOLM-14/CTR shRNA cells in the presence of doxycycline by flow cytometry analysis of Annexin-V fixation or by western blotting analysis of PARP and caspase-3 cleaved fragments. Analysis was performed every day from day 2 to day 6. (E) MOLM-14/SLC1A5shRNA cells (red) or MOLM-14/CTR shRNA cells (black) were injected into nude mice. The mice were then treated with doxycycline by oral gavage, and the tumor volumes were determined at the indicated times. The means of the individual tumor sizes were then plotted (n = 8 in each group). (F) Kaplan-Meier survival curves of nude mice treated with doxycycline. (G) SLC1A5 expression was detected by western blotting analysis of tumor extracts from MOLM-14/SLC1A5 shRNA or MOLM-14/CTR shRNA mice treated with doxycycline (day 22).