Biological Functional Analysis of Novel FGFR2 Mutations
Hyder, Pilar1,2; Gartside, Mike2; Byron, Sara2; Wellens, Candice2; and Pollock, Pamela2
1School of Life Sciences, Arizona State University, Tempe, AZ; 2Translational Genomics Research Institute, Phoenix, AZ
Cutaneous malignant melanoma is the most aggressive form of skin cancer. Presently, metastatic melanoma remains an incurable disease. The development of molecularly targeted therapies relies on the identification of the genetic events underlying melanoma progression. Our lab has identified mutations in FGFR2 in 14/113 melanoma cells lines, 9/100 primary tumors. The FGFRs are members of the immunoglobulin family of receptor tyrosine kinases (RTKs). Crystal structure analysis of a number of the identified mutations suggested that these would result in receptor loss-of-function. Biochemical analysis of the FGFR2 mutations revealed that the majority of the mutations result in receptor inactivation through either loss of ligand binding, misfolding and retention of the receptor in the ER, or complete or partial loss in kinase activity. In contrast to the majority of data, two of the mutations, G271E and E475K, were found to result in constitutive receptor activation. To determine whether these biochemically activating mutations were indeed biologically loss-of-function, we performed proliferation studies in BaF3 cells. The BaF3 murine cell line is often used to test kinase function as it is IL3 dependent and doesn’t express any RTKs. Stable cell lines can be engineered to express RTK mutants and proliferation in the absence of IL3 and the presence of the appropriate ligand then serves as a surrogate measurement of receptor function. These studies revealed that G271E and E475K mutations resulted in decreased BaF3 proliferation compared to wildtype FGFR2, conclusively demonstrating for the first time that all mutations result in receptor loss of function.