BIBF 1000–induced apoptosis in t(4;14)–positive myeloma cells and related inhibition of MAPK (p44/42) phosphorylation. (A) Dose-dependent induction of apoptosis in t(4;14)–positive, FGFR3-overexpressing OPM-2 cells by BIBF 1000 (125-500 nM, top). Induction of apoptosis was completely reverted by the pan-caspase-inhibitor z-VAD-FMK (100 μM). No significant BIBF 1000-induced apoptosis occurred in U-266 cells serving as a t(4;14)–negative control (bottom). (B) FGFR3 protein expression in t(4;14)–positive OPM-2 and KMS-11 versus t(4;14)–negative RPMI-8226 and U-266 cells. Representative immunoblots are shown. (C) Inhibition of MAPK phosphorylation in t(4;14) OPM-2 cells. Constitutive and IL-6–induced MAPK phosphorylation were almost completely or partially inhibited by BIBF 1000, whereas STAT3 phosphorylation was not affected. (D) Inhibition of MAPK phosphorylation in t(4;14) KMS-11 cells. Results were similar to those obtained with OPM-2 cells. (E-F) Neither MAPK nor STAT3 phosphorylation were altered by BIBF 1000 in N-Ras mutated t(4;14)–positive NCI-H929 cells (E) and in t(4;14)–negative U-266 cells (F). In panels C through F, myeloma cell lines were starved for 2 hours and exposed to IL-6 (10 ng/mL) and/or BIBF 1000 (500 nM) for 5 minutes prior to extraction of proteins by RIPA-buffer containing protease and phosphatase inhibitors.