Abstract
EWSR1/FLI1, the most common fusion gene in Ewing sarcoma, upregulates expression of the Eyes Absent 3 (EYA3) transactivator-phosphatase protein. The purpose of this study was to investigate molecular and cellular mechanisms through which EYA3 might promote Ewing sarcoma tumor growth and to determine whether the EYA3 tyrosine phosphatase activity represents a viable therapeutic target. We used genetic and pharmacologic modulation of EYA3 in cell line-based xenografts to examine how loss of EYA3 tyrosine phosphatase activity affects tumor growth and angiogenesis. Molecular mechanisms were evaluated in vivo and in vitro through analyses of tumor tissue and multicellular tumor spheroids. Our results show that both loss of EYA3 in Ewing sarcoma cells and pharmacologic inhibition of the EYA3 tyrosine phosphatase activity inhibit tumor growth and tumor angiogenesis. EYA3 regulates levels of VEGFA in Ewing tumors, as well as promoting DNA damage repair and survival of Ewing sarcoma tumor cells. Target engagement is demonstrated in tumor tissue through elevated levels of the EYA3 substrate H2AX-pY142 upon loss of EYA3 or with Benzarone treatment. The efficacy of EYA3 tyrosine phosphatase inhibition in attenuating tumor growth and angiogenesis is corroborated in an Ewing sarcoma patient-derived tumor xenograft. Together, the results presented here validate EYA3 as a target for the development of novel Ewing sarcoma therapeutic strategies, and set the stage for evaluating the efficacy of combining the antiangiogenic and anti-cell survival effects of EYA3 inhibition with cytotoxic chemotherapy.