Abstract 3247: Novel small molecule inhibitors C9 and C10 specifically disrupt the FAK-VEGFR3 signaling axis in cancer cells.
Authors: Agharkar Priyanka A. , Ethirajan Manivannan , Magis Andrew , Liao Jian , Yemma Michael , Cance William , Pandey Ravindra , Kurenova Elena 
Published: August 2014

Introduction: FAK functions as both, a critical signaling kinase and a scaffolding protein to collectively mediate proliferation, survival, cell adhesion and migration signals. VEGFR3 is a receptor tyrosine kinase that acts as a key modulator of survival signals and lymphangiogenesis. Upregulation of FAK and VEGFR3 in multiple tumor types, with low levels in normal tissues make them attractive anti-cancer targets. Previously we have shown that VEGFR3 binds to FAK and the physical interaction between the two proteins confers a significant survival advantage to tumor cells. Subsequently we discovered small molecule C4 that specifically targets the VEGFR3 binding site on the FAT domain of FAK and exerts anti-tumor effects at moderate concentrations. In this study we designed, synthesized and characterized novel C4 analogs which show enhanced potency and specificity for the target site.

Methods: Modifications were made on the C4 moiety and the resulting analogs were screened for anti-cancer inhibitory effects in different cancer cell lines by MTS assay. Effects of C4 analogs on cancer cell lines were determined by MTS assay (viability), Clonogenecity assay, Western blot (phosphorylation, apoptosis), Immunofluorescence staining (apoptosis) and Flow cytometry (apoptosis). Computer modeling, target specificity (immunoprecipitation) and binding confirmation (Forte-Bio's Octet Red assay) of all analogs were tested. In vitro wound healing and cell invasion assays were also performed. In vivo anti-tumor activity of lead analogs was done using a subcutaneous xenograft mouse model of pancreatic cancer.

Results: Based on the preliminary screening results in several cancer cell lines, we selected analogs C9 and C10 for further analyses. Both analogs inhibit colony formation of pancreatic cancer cells and show target specificity by disrupting FAK-VEGFR3 interaction at ≤10μM. Binding of C9 and C10 to the FAT domain of FAK was confirmed with Octet method. Induction of apoptosis after treatment with C9 and C10 was confirmed via caspase 3/7 activation, caspase-3 and PARP cleavage. Down-regulation of phosphorylated forms of Akt, Erk1/2, Jnk, Grb2 and paxillin was seen in a dose and time dependent manner after 24h treatment with C9 and C10. These drugs also inhibited migration and invasion of pancreatic cancer cells at ≤10μM. In vivo, 30 days treatment with low doses of C9 and C10 led to massive reduction in tumor growth when treated as single agents (P&lt;0.05).

Conclusions: FAK-VEGFR3 targeted therapeutics represents a novel approach towards anti-cancer treatment options. Our promising results thereby form the basis for future clinical applications of FAK-VEGFR3 small molecule inhibitors for cancer therapy.

Citation Format: Priyanka A. Agharkar, Manivannan Ethirajan, Andrew Magis, Jian Liao, Michael Yemma, William Cance, Ravindra Pandey, Elena Kurenova. Novel small molecule inhibitors C9 and C10 specifically disrupt the FAK-VEGFR3 signaling axis in cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3247. doi:10.1158/1538-7445.AM2013-3247