Targeting the hydrophobic pockets of FAK/PYK2 FAT domain: a highly effective inhibitory strategy suppressing tumor growth and eliminating metastasis.

backgroundFAK is a non-receptor tyrosine kinase and an adaptor protein commonly overexpressed in cancer. It regulates multiple tumorigenic pathways through both kinase-dependent and kinase-independent scaffolding functions and thus represents a promising therapeutic target for various cancers. Several FAK kinase inhibitors shown to be effective in preclinical studies advanced to clinical trials, however none produced objective clinical responses. These results are in part attributed to drug resistance and the inability to simultaneously target kinase-dependent and kinase-independent functions of the protein, both of which have been shown to promote tumorigenesis. This has led to the development of scaffold inhibitors that could be used as adjuvants, none of which have so far reached the clinical stage. Importantly, FAK's closely related paralogue, PYK2, compensates for the loss of FAK thus it is also important to target both kinases. In the present study, we evaluate a novel strategy for the inhibition of kinase-dependent and kinase-independent functions of FAK and PYK2 through the expression of the FAT HP-site-specific LD2-LD4 peptide that leads to their displacement from focal adhesions.

methodsThe impact of LD2-LD4 expression on FAK and PYK2 was assessed through co-immunoprecipitation experiments, Western Blot analysis and quantitative immunofluorescence. In vitro investigation of the effects of LD2-LD4 expression on tumor cell migration and proliferation was carried out using 2D migration, 3D invasion and proliferation assays. The preclinical experiments of this study were carried out using an orthotopic xenograft model, followed by immunohistochemical analysis.

resultsWe show that LD2-LD4 expression leads to the displacement of FAK and PYK2 from focal adhesions, blocking both enzymatic and non-enzymatic activities. It also dramatically inhibits 2D cell migration, as well as invasion in vitro. Importantly, LD2-LD4 exerts promising anti-tumor effects and nearly abolishes the appearance of metastatic foci. Finally, we show that an LD monomer can also displace both FAK and PYK2 from FAs suggesting that organic molecules with high affinity for the FAT HPs could mimic the LD2-LD4 activity.

conclusionsTargeting the FAT domain hydrophobic patches of FAK/PYK2 is a highly effective inhibitory strategy that can overcome the limitations of existing ATP competitive inhibitors and lead to the development of novel inhibitors with strong antitumor and antimetastatic activity.