The ETV6-NTRK3 gene fusion encodes a chimeric protein tyrosine kinase thattransforms NIH3T3 cells

The congenital fibrosarcoma t(12;15)(p13;q25) rearrangement splices the ETV 6 (TEL) gene on chromosome 12p13 in frame with the NTRK3 (TRKC) neurotrophi n-3 receptor gene on chromosome 15q25, Resultant ETV6-NTRK3 fusion transcri pts encode the helix-loop-helix (HLH) dimerization domain of ETV6 fused to the protein tyrosine kinase (PTK) domain of NTRK3, We show here that ETV6-N TRK3 homodimerizes and is capable of forming heterodimers with wild-type ET V6, Moreover, ETV6-NTRK3 has PTK activity and is autophosphorylated on tyro sine residues. To determine if the fusion protein has transforming activity , NIH3T3 cells were infected with recombinant retroviral vectors carrying t he full-length ETV6-NTRK3 cDNA, These cells exhibited a transformed phenoty pe, grew macroscopic colonies in soft agar, and formed tumors in severe com bined immunodeficient (SCID) mice, We hypothesize that chimeric proteins me diate transformation by dysregulating NTRK3 signal transduction pathways vi a ligand-independent dimerization and PTK activation. To test this hypothes is, we expressed a series of ETY6-NTRK3 mutants in NIH3T3 cells and assesse d their transformation activities, Deletion of the ETV6 HLH domain abolishe d dimer formation with either ETV6 or ETV6-NTRK3, and cells expressing this mutant protein were morphologically non-transformed and failed to grow in soft agar, An ATP-binding mutant failed to autophosphorylate and completely lacked transformation activity. Mutants of the three NTRK3 PTK activation- loop tyrosines had variable PTK activity but had limited to absent transfor mation activity. Of a series of signaling molecules well known to bind to w ild-type NTRK3, only phospholipase-C gamma (PLC gamma) associated with ETV6 -TRK3, However, a PTK active mutant unable to bind PLC gamma did not show d efects in transformation activity, Our studies confirm that ETV6-NTRK3 is a transforming protein that requires both an intact dimerization domain and a functional PTK domain for transformation activity.