Abstract AZD6244 is a small-molecule-based inhibitor targeting the MEK pathway that is currently in clinical trials. However, the mechanisms mediating intrinsic resistance to MEK inhibition remain to be completely characterized. To identify the molecular mechanism of MEK inhibitor resistance, we analyzed responses of 38 lung cancer cell lines following AZD6244 treatment and their genome-wide gene expression profiles and identified a panel of genes correlated with sensitivity or resistance to AZD6244 treatment. Ingenuity pathway analysis revealed that activation of the signal transducer and activator of transcription 3 (STAT3) pathway was associated with MEK inhibitor resistance. Inhibition of the STAT3 pathway using JSI-124, a STAT3-specific inhibitor, sensitized lung cancer cells to AZD6244 treatment and induced apoptosis in vitro and in vivo. Moreover, combining a STAT3 inhibitor with AZD6244 induced the expression of BIM and poly (ADP-ribose) polymerase (PARP) cleavage, whereas activation of the STAT3 pathway inhibited BIM expression and thus induced MEK inhibitor resistance. In addition, we found that miR-17, which is regulated by STAT3, played an important role in STAT3-mediated MEK inhibitor resistance by targeting BIM. Inhibition of miR-17 with anti-miR-17 sensitized resistant cells to AZD6244 by inducing BIM and PARP cleavage. We conclude that STAT3-mediated overexpression of miR-17 blocked BIM expression, which resulted in resistance to AZD6244. These results implicate novel alternative approaches for overcoming the MEK inhibitor resistance by combining AZD6244 with STAT3 inhibitors or miR-17 antagomirs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-400. doi:10.1158/1538-7445.AM2011-LB-400