Abstract 3683: Small molecule inhibitors of the Fes non-receptor tyrosine kinase
Authors: Hellwig Sabine , Zhang Jianming , Filippakopoulos Panagis , Salah Eidarus , Knapp Stefan , Gray Nathanael S. , Smithgall Thomas E. 
Published: February 2011

The c-fes/fps proto-oncogene encodes a unique 93 kDa protein-tyrosine kinase (Fes) expressed in myeloid, vascular endothelial, neuronal and epithelial cell types. Basal Fes catalytic activity is tightly regulated in vivo, with Fes adopting an inactive conformation in the absence of activating signals. The role of Fes in cancer is paradoxical, with some observations pointing to a tumor-suppressor role. For example, while Fes is strongly expressed in normal colonic epithelium, promoter methylation represses Fes expression in primary tumor tissue and many colorectal cancer cell lines. Restoration of Fes expression from a viral vector suppresses anchorage-independent growth of colorectal cancer cells. In a mouse model of breast cancer, disruption of the c-fes/fps locus correlates with more rapid tumor onset, and this effect can be rescued with a c-fes/fps transgene. In contrast to these observations, other lines of evidence support a pro-oncogenic role for Fes. Constitutively active mutants of Fes are capable of transforming rodent fibroblasts. In the mouse, expression of a gain-of-function human c-fes/fps allele results in hypervascularization and hemangioma formation. Fes has been identified as a phosphorylation target for a Kit mutant (D816V) implicated in mastocytosis, hematologic neoplasms, germ-cell tumors and melanomas. Targeting of Fes by siRNA knockdown inhibited proliferation of cells carrying the KITD816V mutation, identifying Fes as a potential therapeutic target in this cellular context. Another study demonstrated that siRNA knockdown of Fes reduces proliferation of two human renal carcinoma cell lines. One caveat of siRNA-mediated knockdown of Fes is that the resulting growth-inhibitory effect can not be attributed to loss of Fes activity as opposed to loss of the Fes protein. Delineation of kinase-dependent vs. kinase-independent cellular functions of Fes calls for Fes-specific kinase inhibitors, which have not been reported to date. In this study we aimed to identify inhibitors of the Fes tyrosine kinase. Using the recombinant Fes SH2-kinase region and an in vitro kinase assay (Z’-Lyte; Invitrogen), we screened a kinase-biased library of 586 compounds for inhibition of Tyr2 peptide phosphorylation by Fes. Lead compounds identified in vitro were then tested for their ability to inhibit full-length Fes autophosphorylation and microtubule association in transfected COS-7 cells and for their effect on rodent fibroblast transformation driven by constitutively active Fes mutants. A subset of active compounds was further evaluated for inhibition of tubulin phosphorylation by wild-type and active Fes in vitro. We will present a panel of type I and type II small molecule kinase inhibitors from various chemical classes with activity against Fes that are also non-toxic at inhibitory concentrations in cell lines. Our findings represent the first step towards developing a potent and selective inhibitor of Fes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3683.