Functional investigation of the EWS /ATF1 fusion protein: Implications for molecular therapy in clear cell sarcoma

Soft tissue sarcomas are characterized by the presence of consistent chromosomal translocations that frequently involve genes encoding transcription factors. In clear cell sarcoma (CCS) of tendon sheath and aponeuroses, the translocation t(12;22)(q13;q12) juxtaposes the genes for Ewing's sarcoma protein (EWS) and activating transcription factor 1 (ATF1) to generate the EWS/ATF1 fusion protein. The activity of EWS/ATF1 was disrupted by intracellular expression of an inhibitory single chain antibody fragment (scFv41.4). In studies investigating the role of EWS/ATF1 in maintenance of the clear cell sarcoma tumor phenotype, inhibition of EWS/ATF1 DNA binding and transcriptional activity in a human CCS cell line (SU-CCS-1) resulted in a loss of tumor cell viability through an apoptotic mechanism. Phosphorylation of the EWS IQ domain was shown to regulate EWS/ATF1 activity in tumor cells and potentiate their neoplastic activity. Molecular modeling studies were performed to identify the structural determinants responsible for scFv41.4 binding to EWS/ATF1. ScFv41.4 was demonstrated to be a disulfide-deficient single domain antibody fragment, with the heavy chain (VH) CDR1 and CDR3 being necessary for retention of inhibitory activity. Overall, our studies suggest that sarcoma associated fusion proteins may serve as molecular targets for therapeutic development. The unique ability of scFv41.4 to disrupt EWS/ATF1 function in cells provides a suitable model for the design of pharmacophore models or peptidomimetic derivatives having clinical relevance to human cancer.