Targeting Y397 FAK scaffolding compared with FAK kinase inhibition: Efficacy and specificity for tumor cells.

Abstract

e13547 Background: Focal Adhesion Kinase is a non-receptor kinase which is highly overexpressed and activated or autophosphorylated in many types of tumors and plays a major role in survival signaling and metastasis. Recently, our group developed a highly specific allosteric scaffolding FAK autophosphorylation inhibitor, Y15, that blocked FAK Y397 autophosphorylation, decreased cancer cell viability, and significantly decreased breast, neuroblastoma, pancreatic and glioblastoma tumor growth. In addition, Y15 was not toxic in mice. In this study, we compared side by side efficacy of Y15 with two other FAK inhibitors in clinical trials, Pfizer PF-04554878 and Glaxo GSK-2256098. Methods: MTT, clonogenicity and Western blotting assays were used to detect the effect of FAK inhibitors on different cancer and normal cells. Student’s t-test was used for statistical analysis and p<0.05 was considered significant. Results: We tested different cancer cell lines: kidney 293T, colon SW620 and LoVo; lung A549; glioblastoma U251 and U87; breast: BT474, MDA-231, MDA-361, MDA-453, MDA-468 and T47D and pancreatic PANC-1 cancer cells with different doses of Y15, PF and GSK inhibitors by MTT assay and found that Y15 was the most effective at decreasing cancer cell viability in most cancer cell lines, with higher efficacy than PF and much higher efficacy than GSK inhibitor. The same was observed for clonogenicity assay. In addition, we found that Y15 did not significantly affect viability of normal colon (CCD-112) cells, while viability of these cells with PF and GSK decreased to 54% and 57% at a 2 microM dose, respectively, from 100% in untreated cells. In addition, Y15 significantly decreased Y397-FAK, Y418-Src, phospho- Ser 473-AKT and phospho-ERK1/2, while PF had less effect and GSK only a minimal effect in four different cancer cell lines: U251, A549, 293T and Lovo at 10 microM and 100 microM doses, showing high potency of the FAK Y397 scaffolding inhibitor. Conclusions: These data show that Y15, which inhibits the Y397 site of FAK, is more potent against cancer cells and less toxic to normal cells than FAK inhibitors that target the conserved ATP-binding site in the kinase domain.