Objective: Somatic mutations in FGFR2 contribute to development of malignant tumors. It has been reported that 10-12% of endometrial carcinomas have mutations in the FGFR2 gene and FGFR2 is thought to be a target for novel anti-cancer agents. Lenvatinib is an inhibitor of multiple receptor tyrosine kinases, such as VEGFR, FGFR, PDGFR, RET and Kit. Lenvatinib inhibited angiogenesis driven by both VEGF and FGF in preclinical models. The purpose of this study is to determine the effect of lenvatinib on site specific phosphorylation of FGFR2 expressed in tumor cells, which are addicted to FGF signaling by either amplification or active mutations of FGFR2. Methods: The gastric cancer cell line, SNU-16, and endometrial cancer cell lines, AN3CA and MFE-296, were examined for phosphorylation status of FGFR2. Phosphorylation of FGFR2 was detected by western blotting after immunoprecipitation (IP-WB) with anti-FGFR2 antibody and the site specific phosphorylation status was assessed by liquid chromatography mass spectrometry (LC-MS). Inhibitory activity of lenvatinib and sorafenib against the phosphorylation of FGFR2 was examined followed by FGF-2 stimulation. Results: Phosphorylation of FGFR2 was promoted in SNU-16 because of gene amplification. Inhibitory activity of lenvatinib and sorafenib (0.001-10 uM) was examined against the phosphorylation of FGFR2. IP-WB analysis showed that lenvatinib inhibited the phosphorylation of FGFR2 at 1 uM and 10 uM, while sorafenib inhibited only at 10 uM. Next, we performed LC-MS analysis to determine whether lenvatinib and sorafenib selectively affect site specific phosphorylation of FGFR2. LC-MS analysis showed three different statuses of phosphorylation in FGFR2, single phosphorylation of either Y656 or Y657 and dual phosphorylation of Y656 and Y657, and both agents inhibited phosphorylation at 10 uM despite the phosphorylation status. IP-WB analysis also showed a constitutive activation of FGFR2 in AN3CA and MFE-296 (mutation in FGFR2 kinase domain) compared to other endometrial cancer cells without mutation. Lenvatinib, but not by sorafenib, decreased the phosphorylation of FGFR2 in AN3CA and MFE-296 at 1 uM, consistent with the observation in SNU-16. LC-MS analysis in AN3CA demonstrated the phosphorylation on Y657 was clearly inhibited by both agents, although the level of phosphorylation on Y656 was increased by sorafenib, but not by lenvatinib. Conclusions: IP-WB analysis showed that lenvatinib inhibited the phosphorylation of FGFR2 at lower concentration in comparison with sorafenib. LC-MS analysis indicated that inhibitory activity of both agents was different among phosphorylation sites of FGFR2 and also types of FGFR2-activated cancer cells. These results suggested that lenvatinib may exert an unique anti-tumor activity through the inhibition of FGFR2 signaling, in addition to the anti-angiogenic activity via inhibition of VEGFR2 kinase.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1089. doi:1538-7445.AM2012-1089