Mutations in the SAM Domain of the ETV6-NTRK3 Chimeric Tyrosine Kinase Block Polymerization and Transformation Activity

Abstract

The 12p13 ETV6 (TEL) gene is frequently targeted by chromosomal translocations in human malignancies, resulting in the formation of oncogenic ETV6 gene fusions. Many of the known partner genes encode protein tyrosine kinases (PTKs), generating fusion proteins that function as chimeric PTKs. ETV6-NTRK3 (EN), comprised of the ETV6 SAM domain fused to the NTRK3 PTK, is unique among ETV6 chimeric oncoproteins, as it is expressed in cancers of multiple lineages. We initially hypothesized that, similar to other ETV6-PTK chimeras, SAM-mediated dimerization of EN leads to constitutive activation of the PTK and downstream signaling cascades. However, when the EN SAM domain was replaced with an inducible FK506 binding protein (FKBP) dimerization system, resulting FKBP-NTRK3 chimeras failed to transform NIH 3T3 cells even though PTK activation was preserved. It was recently shown that the ETV6 SAM domain has two potential interacting surfaces, raising the possibility that this domain can mediate protein polymerization. We therefore mutated each EN SAM binding interface in a manner shown previously to abolish self-association of wild-type ETV6. Each mutation completely blocked the ability of EN to polymerize, to activate its PTK, and to transform NIH 3T3 cells. Furthermore, EN itself formed large polymeric structures within cells while mutant EN proteins were present only as monomers. Finally, we observed a dominant negative effect on the transformation of isolated SAM domains coexpressed in EN-transformed cells. Taken together, our results suggest that higher-order polymerization may be a critical requirement for the transformation activity of EN and possibly other ETV6-PTK fusion proteins.

We thank Martin Gleave and Mary Bowden for assistance with the mouse studies, Wayne Vogl for technical assistance, and Philip Webster for help with protein expression.

This work was supported by funds from the Canadian Institutes for Health Research (CIHR) (to P.H.B.S.), the CIHR/Candlelighter's Fund (to C.E.T.), the National Cancer Institute of Canada with funds from the Canadian Cancer Society (to L.P.M.), the Natural Sciences and Engineering Research Council of Canada (to C.D.M.), the NCIC and Michael Smith Foundation (to A.M.S.), and the Johal Program in Pediatric Oncology Basic and Translational Research at the BC Research Institute for Children's and Women's Health. L.P.M. is a CIHR Scientist.