4E-BP1 phosphorylation escapes mTORC1 in AML. (A) Samples from AML patients were starved for 4 hours in cytokine- and serum-free medium, except for sample 1, starved for 1, 4, and 12 hours, and then lysed in Laemmli sampler buffer. Western blot was performed with anti–phospho-P70S6K T³⁸⁹, anti–phospho-4E-BP1 S⁶⁵, anti-P70S6K, anti–4E-BP1, and antiactin antibodies. Five representative samples of AML are depicted in panel A. (B) Bone marrow blast cells from 8 AML patients, cells from the MOLM-14 leukemic cell line, and normal CD34⁺ immature hematopoietic cells were starved for 4 hours in cytokine- and serum-free medium. A total of 10 nM RAD001 or 25 μM LY294002 was added during the last hour of starvation. Blast cells from 8 other AML patients were cultured for 24 hours in αMEM containing 10% FCS without or with 10 nM RAD001 or 25 μM LY294002. Proteins extracts from 10⁶ cells were resolved by SDS-PAGE electrophoresis, transferred to nitrocellulose, and probed with anti–phospho-P70S6K T³⁸⁹, anti–phospho-4E-BP1 S⁶⁵, anti-P70S6K, anti–4E-BP1, and antiactin antibodies. (C) Western blot signals from experiments done in primary blast cells (B) were quantified using the Multi Gauge, Version 3.0 software from Fuji. Ratios of phospho-4E-BP1 S⁶⁵/actin signal intensity were first calculated to correct for loading variations. Results were expressed relative to the control condition (without inhibitor) in each experiment. Each histogram represents the mean of 8 independent experiments. Vertical bars represent SD. (D) Primary AML cells were cultured without or with RAD001 during 1 hour and lysed in 0.3% CHAPS buffer. Then mTOR was immunoprecipitated (with goat anti-mTOR antibody from Santa Cruz Biotechnology), and Western blot was performed using anti-mTOR and antiraptor antibodies. Whole-cell extracts collected before mTOR immunoprecipitation were submitted to Western blots using anti–phospho-P70S6K T³⁸⁹, anti–phospho-4E-BP1 S⁶⁵, anti-P70S6K, anti–4E-BP1, and antiactin antibodies. (E) Bone marrow blast cells from 6 AML patients were transfected with 1 μM raptor siRNA or 1μM nontargeted control siRNA. Cell extracts were analyzed by Western blot 24 hours after electroporation to assess the expression of raptor and the phosphorylation level of P70S6K on T³⁸⁹ and of 4E-BP1 on S⁶⁵. The intensities of phospho-4E-BP1 S⁶⁵, phospho-P70S6K T³⁸⁹, and actin signals were quantified using the Multi Gauge, Version 3.0 software from Fuji, and ratios of phospho-4E-BP1 S⁶⁵ or phospho-P70S6K T³⁸⁹ to actin were calculated and set to 1 for control cultures. Each histogram represents the mean of 6 independent experiments. ***P < .001. ns indicates not significant.