The AZD7762/AZD6244 regimen induces apoptosis in IL-6–dependent MM cells and selectively targets primary CD138⁺ MM cells while sparing their normal hematopoietic counterparts. (A) IL-6–dependent ANBL-6 cells were exposed to 2.5μM AZD6244 or 2.5μM PD184352 for 24 hours, followed by 50nM AZD7762 for another 48 hours, after which time cells were immunofluorescently stained with AF 488–conjugated Ab directed against cleaved (activated) caspase-3 to monitor caspase-3 activation. Images were captured microscopically at 40×/0.65. (B) Normal cord blood CD34⁺ cells were exposed to 5μM AZD6244 ± 300nM AZD7762 or 4μM VP-16 for 24 hours, after which time the percentage of apoptotic (annexin V⁺) cells was determined by flow cytometry. (C-D) Primary CD138⁺ MM cells (C) and their CD138⁻ counterparts (D) were isolated from the BM samples of 9 patients with MM. Cell death responses of cells exposed for 24 hours to 250nM AZD7762 ± 5μM AZD6244 were examined by trypan blue exclusion. (E) Once the number of CD138⁺ cells was suitable, followed by drug treatment as described in panels C and D, primary cells were immunofluorescently stained with AF 488–conjugated cleaved (activated) caspase-3 Ab to confirm apoptosis. The representative images shown were captured microscopically at 40×/0.65. (F) Primary CD138⁺ MM cells from a MM patient were treated as described in panel C, and then subjected to Western blot analysis to monitor ERK1/2 phosphorylation and Bim expression. Lanes were loaded with 10 μg of protein; blots were reprobed with Abs to β-actin to ensure equivalent loading and transfer.