Abstract Background However potency of Alectinib, a selective ALK inhibitor, for ALK rearranged NSCLC and for that with secondary mutation have been reported, intrinsic and acquired resistance are likely to emerge. Recent study showed tumor microenvironment triggered resistance to Crizotinib and TAE684. In this study, we examine whether emerging of intrinsic and acquired resistance are affected by tumor microenvironment in H3122, EML4-ALK positive lung cancer cell line, and in Alectinib-resistant clone of H3122 (AFR) which is generated in vitro. Elucidating intrinsic resistant mechanisms and preceding development of acquired resistance to understand its mechanisms will provide beneficial information for the development of novel therapeutic strategies in clinical practice. Methods We examined the effect of EGF produced by endothelial cells on Alectinib sensitivity of H3122 cell line, and EGF and HGF produced by fibroblasts on Alectinib sensitivity of H3122AFR by clonogenic assay. Downstream pathways of ALK in both cells were analyzed by western blotting. Combination treatments of Alectinib and other TKI were tested both in vitro and in vivo. Results H3122 cells which had been cultured with EGF acquired resistance to Alectinib treatment. Combination treatment of Alectinib and Erlotinib overcome EGF induced resistance and showed down-regulation in phosphorylation of AKT and ERK1/2. H3122AFR cells were also reduced their sensitivity to Alectinib at 100 uM or higher concentration when the media contained EGF or HGF. Erlotinib or Crizotinib combined with Alectinib canceled the EGF-induced or HGF-induced resistance by down-regulating phosphorylation of AKT and ERK1/2. In vivo, tumor xenografts of nude mice which had been orally administered Alectinib and Erlotinib showed tumor regression in both H3122 and H3122AFR, and those received Alectinib and Crizotinib showed regression in H3122AFR. Conclusion In our study, we showed ligands produced by tumor microenvironment, such as EGF and HGF, contributed the emerging of intrinsic resistance and acquired resistance by activating survival pathway. Further, the efficacy of combination treatment of Alectinib with Erlotinib or that with Crizotinib was shown. Collectively, Alectinib combined with EGFR-TKI would be an effective strategy for Alectinib-resistant lung cancer, and Alectinib with crizotinib is another considerable strategy. Citation Format: Yukiko Hibino, Sasaki Takaaki, Yoshinori Minami, Pasi A. Janne, Yoshinobu Ohsaki. Intrinsic and acquired resistance mechanisms of alectinib in ALK rearranged cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3720. doi:10.1158/1538-7445.AM2014-3720