[CANCER RESEARCH 59, 1428 –1432, April 1, 1999] Advances in Brief Differential Transactivation by Alternative EWS-FLI1 Fusion Proteins Correlates with Clinical Heterogeneity in Ewing’s Sarcoma 1 Patrick P. Lin, Rachel I. Brody, Aime ´e C. Hamelin, James E. Bradner, John H. Healey, and Marc Ladanyi 2 Departments of Surgery [P. P. L., J. H. H.], Pathology [R. I. B., A. C. H., J. E. B., M. L.], and Human Genetics [M. L.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021 Abstract The t(11;22)(q24;q12) translocation is present in up to 95% of cases of Ewing’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 exist because of variations in the locations of the EWS and FLI1 genomic breakpoints. 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 approximately 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 1 fusion is associated with a significantly better prognosis than the other fusion types. Because EWS- FLI1 is an aberrant transcription factor, we investigated whether these differences in clinical behavior may be correlated to functional differences by comparing transactivation by the type 1 EWS-FLI1 with other types in both heterologous cells (HeLa, NIH3T3) and homologous cells (Ewing’s sarcoma cell lines). In a panel of seven Ewing’s sarcoma cell lines, we found transactivation of a transiently transfected FLI1-responsive re- porter construct to be significantly lower in cell lines with the type 1 fusion than in cell lines with the type 2 fusion (P  0.003). Cotransfection of the same reporter construct with each of a series of seven EWS-FLI1 expres- sion constructs (corresponding to the two major fusion types and five less common types) also showed that type 1 EWS-FLI1 was a significantly weaker transactivator than the type 2 product in both HeLa and NIH3T3 cells (P  0.003, and P  0.033, respectively). Electromobility shift assays showed equivalent binding of the type 1 and type 2 EWS-FLI1 to the consensus FLI1-responsive binding site, indicating that differences in transactivation were not due simply to differences in DNA binding affin- ity. The finding that the type 1 EWS-FLI1 fusion, associated with less aggressive clinical behavior, encodes a less active chimeric transcription factor may provide the basis for a molecular explanation of clinical heterogeneity in Ewing’s sarcoma. Introduction Ewing’s sarcoma is a primitive neuroectodermal tumor arising in bone or soft tissue, which strikes over 300 children and young adults in the United States annually. The t(11;22)(q24;q12) translocation is present in up to 95% of cases of Ewing’s sarcoma and results in the formation of the EWS-FLI1 fusion gene that encodes an oncogenic chimeric transcription factor (reviewed in Ref. 1). EWS is a functional RNA-binding protein that, along with TLS and a novel TATA-binding protein-associated factor, TAF II 68 (also known as RBP56), forms a new family of related proteins. Both TLS and EWS, but not EWS- FLI1, can function as TATA-binding protein-associated factors (2). Thus, EWS may mediate direct positive interactions of nascent tran- scripts with the basal transcription apparatus. However, the EWS portion of EWS-FLI1 may be redirected to other protein targets because of possible conformational changes induced by the gene fusion, as suggested by the recent demonstration of specific binding in vitro and in vivo of the EWS portion of EWS-FLI1— but not of native EWS—to hsRPB7, a subunit of RNA polymerase II (2, 3). FLI1 encodes a member of the ETS family of transcription factors and seems to be involved in early hematopoietic, vascular, and neuroec- todermal development in model organisms (4). The highly restricted tissue expression of FLI1 contrasts with that of native EWS, which is high and ubiquitous. Because of the genomic structure of the fusion, the EWS promoter drives the expression of EWS-FLI1. In standard transactivation assays, the EWS-FLI1 chimeric tran- scription factor functions as a 5- to 10-fold stronger transactivator than native FLI1 at promoters containing binding sites for FLI1 (5). The sequences recognized by its DNA binding domain are indistin- guishable from those recognized by native FLI1 (5). The oncogenicity of EWS-FLI1 may depend on a number of features, including consti- tutive, tissue-inappropriate, differentiation stage-inappropriate expres- sion driven by the EWS promoter, the stronger transactivating poten- tial of the EWS NH 2 -terminal domain, and its possible interaction with a different subset of coactivators. It is thus likely that the proximate effect of EWS-FLI1 is the deregulated activation of both FLI1 target genes as well as some genes whose expression is not normally regulated by FLI1. Many alternative forms of EWS-FLI1 exist because of variations in the locations of the EWS and FLI1 genomic breakpoints (6). At least 12 types of in-frame EWS-FLI1 chimeric transcripts have been ob- served clinically. They contain different combinations of exons from EWS and FLI1, reflecting different combinations of EWS and FLI1 genomic breakpoints (6) (Fig. 1). The two main types, fusion of EWS exon 7 to FLI1 exon 6 (type 1) and fusion of EWS exon 7 to FLI1 exon 5 (type 2), account for about 60 and 25%, respectively, of EWS-FLI1 fusions (6 – 8). Biologically significant alternative splicing of the EWS-FLI1 transcript is rarely detected (8); therefore, each tumor usually expresses only a single type of fusion transcript. The minimal components of each EWS-FLI1 fusion protein are the NH 2 -terminal domain of EWS (encoded by exons 1–7), which functions in vitro as a strong transactivation domain, and the intact DNA-binding domain of FLI1 (encoded by exon 9). The portion of the chimeric transcript between these two domains is variable in size and composition, and this heterogeneity is clinically significant. Specifically, we and others have recently shown (7, 8) that the survival of patients whose Ewing’s sarcomas bear the type 1 EWS- FLI1 fusion is markedly better than those with tumors containing other fusion types, regardless of tumor site, stage, or size. The clinical impact may be substantial, and it is possible that patients with non- type 1 fusions may be candidates for more aggressive therapy such as bone marrow ablation and total body irradiation. Before such meas- Received 11/19/98; accepted 2/15/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the Byrne Fund (to M. L.). In addition, fellowship support was provided by NIH T32-CA60376 (for R. I. B.), and J. E. B. was a visiting medical student from the University of Chicago-Pritzker School of Medicine sponsored by the NIH Cancer Education Program. 2 To whom requests for reprints should be addressed, at at Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. 1428 Research. on October 21, 2021. © 1999 American Association for Cancer cancerres.aacrjournals.org Downloaded from