Combined exposure to sorafenib and obatoclax results in enhanced lethality in primary AML cells. (A) Leukemic blasts were isolated from the BM of 4 patients with AML (FAB classification M2; AML#1, AML#2, and AML#4 with wild-type FLT3, and AML#3 with a FLT3-ITD mutation), exposed to sorafenib (7.5μM) and obatoclax (0.5μM) for 48 hours, after which the extent of cell death was assessed using the 7-AAD analysis. Results are presented as percentage of dead cells specific for each treatment using the formula ([treatment − control]/[100 − control]) × 100. Cell death for untreated control samples ranged from 10% to 25%. (B) Alternatively, protein lysates were prepared from AML patient #1 and subjected to Western blot analysis. Densitometric analysis of cleaved PARP and caspase-3 bands was performed using Adobe Photoshop. Values shown were normalized to ERK1/2 and represent relative changes compared with control. (C) Normal CD34⁺ cells were isolated as described in “Methods” from the BM of normal subjects (nonleukemic; N#1, N#2, and N#3) and exposed to increasing concentrations of sorafenib and obatoclax alone or in combination for 48 hours, after which the extent of cell death was determined using the 7-AAD analysis. (D) Two primary AML specimens were plated in methylcellulose in the presence of 5μM sorafenib and 75nM obatoclax alone or in combination for 14 days, after which CFUs were enumerated and expressed as a percentage relative to untreated cells. (E) Normal CD34⁺ cells from 2 subjects were plated in methylcellulose in the presence of increasing concentrations of sorafenib and obatoclax alone or in combination for 8 days, after which CFUs were enumerated and expressed as in panel D. For panels A, C, D, and E, data for each patient were obtained from a single experiment performed in triplicate; values represent the means ± SD.