AZD6244 specifically inhibits ERK phosphorylation and induces cytotoxicity in MM lines resistant to conventional chemotherapy, as well as in primary patient MM cells. (A) Serum-starved MM1S MM cells were pretreated with either AZD6244 (10 nM) or control medium for 1 hour and then stimulated with IGF-1 (100 ng/mL) for indicated time intervals (left panel). Similarly, MM1S cells were pretreated with serial dilutions of AZD6244 (0-10 μM) and stimulated with IL-6 (50 ng/mL, middle panel) or sCD40L (5 μg/mL, right panel). Immunoblotting was performed using anti-pAKT and pERK Abs, as well as anti–β-actin or anti–α-tubulin mAbs as loading controls. (B) Ten MM lines, including drug-sensitive RPMI8226, Dox-resistant RPMI8226 (Dox40), melphalan-resistant RPMI8226 (LR5), Dex-sensitive MM1S and -resistant MM1R, IL-6–dependent INA-6, and MCCAR, 28PE, 28BM, and OPM1 cells, were cultured with AZD6244 for 2 days and then pulsed with [³H]thymidine for the last 8 hours for measurement of DNA synthesis. Data represent mean (+ SE) of quadruplicate cultures; cpm indicates counts per minute. (C) Freshly isolated tumor cells from 3 MM patients (MM 1, MM 2, MM3) were cultured with AZD6244 (0.02-20 μM) for 2 days, and cytotoxicity was assessed by MTT assay.