Mithramycin A Radiosensitizes EWS:Fli1⁺ Ewing Sarcoma Cells by Inhibiting Double Strand Break Repair.

Abstract

The oncogenic EWS:Fli1 fusion protein is a key transcriptional mediator of Ewing sarcoma initiation, progression, and therapeutic resistance. Mithramycin A (MithA) is a potent and specific inhibitor of transcription mediated by the EWS:Fli1. We tested the hypothesis that pretreatment with MithA could selectively radiosensitize EWS:Fli1⁺ tumor cells by altering the transcriptional response to radiation injury. A panel of 4 EWS:Fli1⁺ and 3 EWS:Fli1^- Ewing sarcoma cell lines and 1 nontumor cell line were subjected to MithA dose-response viability assays to determine the relative potency of MithA in cells possessing or lacking the EWS:Fli1 fusion. Radiosensitization by MithA was evaluated by clonogenic survival assays in vitro and in a murine xenograft model. DNA damage was evaluated by comet assay and γ-H2Ax flow cytometry. Immunoblotting, flow cytometry, and reverse-transcription, polymerase chain reaction were used to evaluate DNA damage-induced signaling and repair processes and apoptosis. We found that MithA alone could potently and selectively inhibit the growth of EWS:Fli1⁺ tumor cells, but not cells lacking this fusion. Pretreatment with MithA for 24 hours before irradiation significantly reduced clonogenic survival in vitro and delayed tumor regrowth in vivo, prolonging survival of EWS:Fli1⁺ tumor-bearing mice. Although MithA did not increase the level of DNA double-strand breaks, mechanistic studies revealed that MithA pretreatment selectively inhibited DNA double-strand break repair through downregulation of EWS:Fli1-mediated transcription, leading to tumor cell death by apoptosis. Our data indicate that MithA is an effective radiosensitizer of EWS:Fli1⁺ tumors and may achieve better local control at lower doses of radiation.