Abstract 4733: Activating FGFR2 kinase domain mutations provide resistance to dovitinib (TKI258)

Abstract

Abstract Members of the fibroblast growth factor receptor (FGFR) family are amplified or mutationally activated in a variety of cancers, including breast, endometrial, ovarian, lung, gastric, and bladder cancers, glioblastoma and rhabdomyosarcoma. Consequently FGFRs are attractive therapeutic targets in cancer, with a number of FGFR inhibitors currently progressing through clinical trials. Dovitinib, a lead FGFR kinase inhibitor exhibits activity against FLT3, FGFRs, VEGFRs, and PDGFR, and has demonstrated considerable preclinical activity in cancer models with FGFR activation. Though targeted tyrosine kinase inhibitors (TKIs) have shown dramatic clinical responses, the long-term efficacy of these agents is frequently limited by development of resistance to the targeted agent, often due to mutation of the target kinase. Here we sought to identify the mutational mechanisms of resistance to Dovitinib using a BaF3 cell line screening strategy. The BaF3 cell line is an IL-3 dependent murine pro-B cell line that is commonly employed to model TKI resistant mutations. These cells do not express any FGF ligands or receptors and introduction and activation of FGFRs has been shown to substitute for IL-3 to promote cell proliferation. BaF3 cells transduced with FGFR2 were treated with TKI258 at 5x, 10x, and 15x the cellular IC50 for Dovitinib in these cells. Following clonal selection of Dovitinib resistant cells, the exons encoding the intracellular domain of FGFR2 were sequenced. Mutations in FGFR2 kinase domain were identified in 26 out of 63 (41.2%) resistant clones screened, with an increase in frequency of mutation with increasing selective pressure. Ten distinct Dovitinib-resistant mutations in FGFR2 were identified and subsequently confirmed to result in Dovitinib-resistance and kinase activation. The binding mode of Dovitinib and the mechanisms of action of the resistance mutations were studied using the crystal structures of unphosphorylated and phosphorylated FGFR2Ks. Mutations at N550 and E566 at the kinase hinge/interlobe region are expected to drive the kinase into the active state by disengaging the molecular brake that keeps the kinase in an autoinhibited state. Five additional mutations are also predicted to stabilize the active conformation of the kinase by strengthening a network of hydrophobic interactions between N- and C-terminal lobes of the kinase, termed the hydrophobic spine, that is a hallmark of the active state of the kinase. Hence our biochemical and structural data show that the drug predominantly binds the inactive state of the FGFR2 kinase. Our data have clinical ramifications as they suggest that cancer patients harboring these FGFR2 mutations may not respond to the anti-FGFR activity of Dovitinib. Taken together our study provides the first report of TKI-resistant mutations in FGFR2 and suggests that the active state of the FGFR2 kinase should be targeted for anti-cancer drug discovery. 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 4733. doi:10.1158/1538-7445.AM2011-4733