Ewing's sarcoma is an aggressive pediatric cancer of the bone and soft tissue, in which patients' whose tumors have a poor histological response to initial chemotherapy have a poor overall prognosis. Importantly, childhood cancer survivors are faced with long-term chemotherapy-associated toxicities many years after they have been cured of their primary disease. Therefore, targeting molecules involved in resistance to chemotherapy should benefit patients with resistant disease and allow lower doses of toxic chemotherapies to be used in treatment regimens, thus improving outcomes for children suffering from Ewing's sarcoma on multiple fronts. The vast majority of Ewing's sarcomas harbor a characteristic translocation, t(11;22), encoding the EWS/Fli1 fusion protein. We have identified the DNA-repair protein and transcriptional cofactor, Eya3, as a downstream target of EWS/Fli1. Eya3 is highly expressed in Ewing's sarcoma tumor samples and cell lines compared with mesenchymal stem cells, the presumed cell of origin of Ewing's sarcoma. We demonstrate that EWS/Fli1 mediates upregulation of Eya3 via repression of miR-145 and miR-708, microRNAs that target the Eya3 3'UTR. Furthermore, replacement of miR-145 and miR-708 in a Ewing's sarcoma cell line decreases Eya3 levels. It was recently shown that Eya3 promotes DNA repair by dephosphorylating a critical tyrosine residue on H2AX in response to DNA damage, resulting in the recruitment of DNA repair factors to the sites of DNA damage. Thus we tested whether knockdown of Eya3 in Ewing's sarcoma cells would decrease cell survival and sensitize cells to DNA-damaging chemotherapeutics used in the treatment of Ewing's sarcoma: etoposide and doxorubicin. Indeed, Eya3 knockdown led to increased cell death and sensitization to chemotherapy, and, as expected, Eya3 knockdown cells contained more DNA damage following treatment with etoposide than their control counterparts. These data strongly suggest that Eya3 mediates chemoresistance in Ewing's sarcoma through its ability to enhance DNA repair. Because Eya3 mediates DNA repair and survival via its intrinsic phosphatase activity, and because the phosphatase activity of Eya is also known to play a role in metastasis of other cancers, we have begun to develop inhibitors targeting the Eya phosphatase activity and to demonstrate their efficacy both in vitro and in cell culture. In closing, Eya phosphatase inhibitors or synthetic mimics of the microRNAs targeting Eya3 have the potential to improve outcomes for Ewing's sarcoma patients by improving therapeutic efficacy and decreasing associated toxicities of conventional chemotherapeutics.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B78.