LKB1/AMPK activation using metformin strongly decreases AML cell survival, but does not affect normal hematopoietic cells ex vivo. (A) Top panel, Western blot analysis of CDK2, SCF^SKP2, and p21^CIP1 in primary AML cells treated or not with metformin. Bottom panel, proliferation was assessed in primary AML cells cultured without or with 5, 10, or 15mM metformin and then pulsed for 6 hours with [³H]thymidine. Ratios to the controls without inhibitor were calculated, and the results are presented as a mean of 5 independent experiments. The vertical bars indicate the SEM (B) clonogenic assays of primary AML cells (CFU-L) and normal CD34⁺ hematopoietic progenitors (erythroid, BFU-E; and granulo-macrophagic, CFU-GM). Cells were cultured for 24 hours without or with 5, 10, or 15mM metformin, washed twice in PBS buffer, and then plated in methylcellulose medium. We used 25μM LY294002 as a control for CD34⁺ clonogenic growth inhibition.^9,49  Each histogram represents the mean of 5 independent experiments, performed in duplicate. The results are expressed as a ratio to the control incubation without inhibitor and presented as a mean of 5 independent experiments. Vertical bars indicate the SEM. (C) Top panel, Western blot analysis of caspase-3 and Mcl-1 expression in primary AML cells cultured for 24 hours without or with 5, 10, or 15mM metformin (sample 4 is representative of 5 independent experiments). Bottom panel, Annexin V binding assays of cells cultured without or with 5, 10, or 15mM metformin for 24 hours. Results are expressed separately as the mean of 5 primary AML samples and 5 CD34⁺ samples, respectively. The vertical bars indicate the SEM.