Molecular characterization of the ETV6-NTRK3 fusion in congenital fibrosarcoma

The t(12;15)(pl3;q25) rearrangement detected in congenital fibrosarcoma splices the ETV6 (TEL) gene on chromosome 12p13 in frame with the NTRK3 (TRKC) neurotrophin-3 receptor gene on chromosome 15q25. Resultant ETV6- NTRK3 fusion transcripts encode the helix-loop-helix (HLH) dimerization domain of ETV6 fused to the protein tyrosine kinase (PTK) domain of NTRK3. We hypothesize that chimeric proteins mediate transformation by dysregulating NTRK3 signal transduction pathways via ligand-independent dimerization and PTK activation. To determine if the fusion protein has transforming activity, NIH3T3 cells were infected with recombinant retroviral vectors carrying the full-length ETV6-NTRK3 cDNA. These cells exhibited a transformed phenotype and formed macroscopic colonies in soft agar. In order to characterize the roles of specific ETV6- NTRK3 domains, we expressed a series of ETV6-NTRK3 mutants in NIH3T3 cells and assessed their transformation activities. Deletion of the ETV6 H L H domain resulted in morphologically non-transformed NIH3T3 cells that failed to grow in soft agar. Mutants of the three PTK activation-loop tyrosines (EN-Y513, EN-Y517, and EN-Y518) had variable PTK activity but had limited to absent transformation activity. The EN-Y513F mutant failed to transform NIH3T3 cells, while the expression of either EN-Y517F or EN-Y518F resulted in a semi-transformed phenotype. The simultaneous mutation of Y517 and Y518 (EN-Yx2F), or of all three tyrosines (EN-Yx3F), resulted in a morphologically untransformed phenotype. The ATP-binding mutant (EN-K380N) failed to autophosphorylate and completely lacked transformation activity. In addition, a series of PTK-active mutants unable to bind phospholipase-Cγ did not show defects in transformation activity. These studies confirm that ETV6-NTRK3 is a transforming protein that requires both an intact dimerization domain and a functional PTK domain for transformation activity.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat