The t(11;22)(q24;q12), present in 85% of Ewing's sarcoma and related tumours, fuses the EWS gene from chromosome 22q12 and the ETS family member, FLI-1. This results in the expression of a chimaeric protein containing the amino-terminal portion of EWS fused to the ETS DNA-binding domain of FLI-1. We have identified a second Ewing's sarcoma translocation, t(21;22)(q22;q12), that fuses EWS to a different ETS family member, the ERG gene located on band 21q22. Identical EWS nucleotide sequences found in the EWS/FLI-1 fusion transcripts are fused to portions of ERG encoding an ETS DNA-binding domain resulting in expression of a hybrid EWS/ERG protein. These findings suggest that fusion of EWS to different members of the ETS family of transcription factor genes may result in the expression of similar disease phenotypes.

The 11;22 chromosomal translocation specificaOly linked to Ewing sarcoma and primitive neuroectodermal tumor results in a chimeric molecule fusing the aminoterminal-encoding portion of the EWS gene to the carboxylterminal DNA-binding domain encoded by the FLII gene. We have isolated a fourth EWS-FLII fusion cDNA that is structurally distinct from the three forms previously described. To determine the transforming activity of this gene, alternative forms of the EWS-FLI1 fusion were transduced into NIH 3T3 Structural alteration or aberrant expression of transcription factors is also common in human malignancies but usually results through somatic genomic mutation (for reviews, see refs. 2 and 3). Karyotypic analyses have revealed a tumor-specific t(11;22)(q24;ql2) chromosomal translocation in 86% of both Ewing sarcoma and primitive neuroectodermal tumor (PNET), suggesting that the product of this rearrangement is necessary for the formation of both these malignancies (4,5 MATERIALS AND METHODSPNET cDNA Library Construction and Isolation of EWS-FL!) Chimeras. TC-32, a PNET tumor cell line containing the 11;22 translocation, was grown in RPMI medium/10% fetal calf serum, as described (5). Total RNA was harvested by lysis with guanidine isothiocyanate and purified over cesium chloride (9). Poly(A)+ RNA was obtained by using columns packed with oligo(dT)-cellulose (Collaborative Research) and used for construction of cDNA libraries.A TC-32 cDNA library was made according to previously published procedures (10). Briefly, first-strand synthesis was accomplished using methyl mercury-denatured poly(A)+ RNA primed with oligo(dT) and murine leukemia virus reverse transcriptase (GIBCO/BRL). Second-strand synthesis was done by using RNase H and polymerase I (GIBCO/ BRL), and synthesized products were purified over a Sephadex G100 column (Pharmacia). cDNAs were blunted by using T4 polymerase (GIBCO/BRL) and ligated to a molar excess of EcoRI adaptors (Invitrogen, San Diego). The adaptor ends were phosphorylated with T4 polynucleotide kinase (United States Biochemical), and cDNAs were fractionated over a 6% acrylamide gel. DNA species >600 bp were recovered from gel slices by electroelution, purified over

EWS/FLI-1 is a chimeric protein formed by a tumor-specific 11;22 translocation found in both Ewing's sarcoma and primitive neuroectodermal tumor of childhood. EWS/FLI-I has been shown to be a potent transforming gene Aberrant expression and structural alteration of transcription factors are frequent, primary molecular mechanisms in oncogenesis (11). In lower mammals and avian species, these alterations are often mediated by retroviral insertion. In humans, deregulation or structural alteration of transcription factors is frequently the result of somatic genomic rearrangement.The 11;22 chromosomal translocation found in Ewing's sarcoma and primitive neuroectodermal tumor of childhood (PNET) juxtaposes the 5' sequences from a newly described gene, termed EWS, with the 3' sequences from FLI-1, which encodes a member of the Ets transcription factor family (6). Like most Ets family members (for a review, see reference 27), the carboxyl domain of FLI-1 mediates sequencespecific DNA binding. The FLI-1 amino terminus contains a putative transcription activation domain that may interact on a protein-protein level with other transcription factors. As with other Ets proteins, it is probably the combination of protein-DNA and protein-protein binding specificities that determines which genes are transcriptionally modulated by FLI-1. As a result of the 11;22 rearrangement, the aminoterminal domain of FLI-1 is replaced by a portion of EWS containing a series of degenerate, glutamine-rich repeats. The carboxyl terminus of EWS has amino acid similarity to proteins involved in RNA synthesis and processing (6). However, the function in tumor cells of the EWS aminoterminal domain that is fused to FLI-1 is unknown.We have recently demonstrated that the EWS/FLI-1 chi-

Tumor-associated chromosomal translocations lead to the formation of chimeric fusions between the EWS gene and one of ®ve dierent ETS transcription factors in Ewing's family tumors (EFTs). The resultant EWS/ETS proteins promote oncogenesis in a dominant fashion in model systems and are necessary for continued growth of EFT cell lines. EWS belongs to a family of genes that encode proteins that may serve as adapters between the RNA polymerase II complex and RNA splicing factors. EWS/ETS fusions have biochemical characteristics of aberrant transcription factors and appear to promote abnormal cellular growth by transcriptionally modulating a network of target genes. Early evidence suggests that EWS/ETS proteins may also impact gene expression through alteration in RNA processing. Elucidation of EWS/ETS target gene networks in the context of other signaling pathways will hopefully lead to biology based therapeutic strategies for EFT. Oncogene (2001) 20, 5747 ± 5754.

Clear cell sarcoma (CCS) harbors a pathognomonic chromosomal translocation fusing the Ewing's sarcoma gene (EWS) to the CREB family transcription factor ATF1 and exhibits melanocytic features. We show that EWS-ATF1 occupies the MITF promoter, mimicking melanocyte-stimulating hormone (MSH) signaling to induce expression of MITF, the melanocytic master transcription factor and an amplified oncogene in melanoma. Knockdown/rescue studies revealed that MITF mediates the requirement of EWS-ATF1 for CCS survival in vitro and in vivo as well as for melanocytic differentiation. Moreover, MITF and TFE3 reciprocally rescue one another in lines derived from CCS or pediatric renal carcinoma. Seemingly unrelated tumors thus employ distinct strategies to oncogenically dysregulate the MiT family, collectively broadening the definition of MiT-associated human cancers.