Differential transactivation by alternative EWS-FLI1 fusion proteins correlates with clinical heterogeneity in Ewing's sarcoma

The t(11;22)(q24;q12) translocation is present in up to 95% of cases of Ewi ng's sarcoma and results in the formation of an EWS-FLI1 fusion gene which encodes a chimeric transcription factor. The proximate role of EWS-FLI1 in the pathogenesis of Ewing's sarcoma is thought to involve the activation of as yet largely unknown target genes. Many alternative forms of EWS-FLI1 ex ist because of variations in the locations of the EW'S and FLI1 genomic bre akpoints. The most common form, designated "type 1," consists of the first seven exons of EWS joined to exons 6-9 of FLI1 and accounts For approximate ly 60% of cases. The "type 2" EWS-FLI1 fusion also includes FLI1 exon 5 and is present in another 25%. We and others have observed previously that the type I fusion is associated with a significantly better prognosis than the other fusion types. Because EWS-FL1 is an aberrant transcription factor, w e investigated whether these differences in clinical behavior may he correl ated to functional differences by comparing transactivation by the type 1 E WS-FLI with other types in both heterologous cells (HeLa, NIH3T3) and homol ogous cells (Ewing's sarcoma cell lines). In a panel of seven Ewing's sarco ma cell lines, we found transactivation of a transiently transfected FLI1-r esponsive reporter construct to be significantly lower in cell lines with t he type 1 fusion than in cell lines with the type 2 fusion (P = 0.003). Cot ransfection of the same reporter construct with each of a series of seven E WS-FLI1 expression constructs (corresponding to the two major fusion types and five less common types) also showed that type 1 EWS-FLI1 was a signific antly weaker transactivator than the type 2 product in both HeLa and NIH3T3 cells (P = 0.003, and P = 0.033, respectively). Electromobility shift assa ys showed equivalent binding of the type 1 and type 2 EWS-FLI1 to the conse nsus FLI1-responsive binding site, indicating that differences in transacti vation were not due simply to differences in DNA binding affinity. The find ing that the type 1 EWS-FLI1 fusion, associated with less aggressive clinic al behavior, encodes a less active chimeric transcription factor may provid e the basis for a molecular explanation of clinical heterogeneity in Ewing' s sarcoma.