Abstract 1613: High-throughput tyrosine kinase activity profiling identifies focal adhesion kinase (FAK) as a new target in Ewing sarcoma

Abstract

Abstract Aberrant activation of tyrosine kinases is a frequent driver event in cancer. Nevertheless, for many tumors, the critical kinase abnormalities have yet to be discovered. Emerging technologies are enabling the rapid profiling in high-throughput format of not only kinase expression, but kinase activation. We leveraged a bead-based approach for profiling tyrosine kinase phosphorylation in Ewing sarcoma and identified focal adhesion kinase (FAK) as a top candidate. Ewing sarcoma is the second most common bone tumor diagnosed in children. Current treatment for de novo disease employs intensive cytotoxic chemotherapy in combination with radiation or surgical resection. Outcomes for metastatic and recurrent disease remain poor. One potential target in Ewing sarcoma is the EWS/FLI rearrangement. However, development of small molecule modulators of aberrant transcription factors poses a challenge. IGF1R has emerged as a tractable target with modest success in a small subset of relapsed patients treated with IGF1R antibody therapy. However, single agent IGF1R-directed therapy is unlikely to be curative. New targeted approaches are needed for treating this disease. Utilizing a fluorescent, bead-based technology, we screened six human Ewing sarcoma cell lines. This approach is based on the multiplexed coupling of 62 kinase-specific antibodies to microspheres. Protein lysates from cell lines are incubated with antibody-coupled beads and a biotinylated antiphosphotyrosine antibody and the bead complexes detected with a Luminex flow cytometer. FAK phosphorylation was detected at higher levels in Ewing sarcoma cell lines compared to a control cell line and a no lysate BSA control. Phosphorylation at the FAK Y397 activation site was confirmed in all six lines by western immunoblotting. We next addressed the functional consequences of loss of FAK in Ewing sarcoma cell lines with both genetic and pharmacological inhibition. Multiple shRNAs directed against FAK inhibited cell viability and profoundly decreased colony formation and size in methylcellulose in three Ewing sarcoma cell lines. Similarly, chemical inhibition of FAK at doses that decrease phosphorylation at the kinase activity site decreased growth of Ewing sarcoma cell lines and inhibited anchorage independent growth. Based on prior reports of a synergistic interaction of inhibiting FAK and IGF1R in epithelial tumors, we hypothesized that a similar interaction would be observed in Ewing sarcoma. Indeed, we observed enhanced effects of small molecule inhibition of FAK on shRNA-directed knockdown of IGF1R on both cell viability and colony formation. In vivo studies of FAK inhibition as a single perturbation, as well as in combination with IGF1R inhibitors, are currently underway. With FAK small molecule inhibitors already in clinical trials, these studies may have immediate translational relevance to Ewing sarcoma. 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 1613. doi:10.1158/1538-7445.AM2011-1613