Cell cycle arrest and apoptosis are early events in radiosensitization of EWS::FLI1+ Ewing sarcoma cells by Mithramycin A.

PURPOSE The oncogenic fusion protein EWS::FLI1 is an attractive therapeutic target in Ewing sarcoma (ES). Mithramycin A (MithA) is a potent and specific inhibitor of EWS::FLI1 that can selectively radiosensitize ES cells through transcriptional inhibition of DNA double-strand break (DSB) repair. Here, we evaluate temporal changes in cell cycle progression and apoptosis in ES cells treated with MithA and/or ionizing radiation (RTx), testing the hypothesis that combining MithA with ionizing radiation would synergistically impair cell cycle progression and enhance apoptotic elimination to a greater extent than either agent alone. MATERIALS AND METHODS Four EWS::FLI1+ Ewing sarcoma cell lines TC-71, RD-ES, SK-ES-1, and A673, and one EWS::Erg cell line (CHLA-25) were exposed to 10nM MithA or vehicle and followed twenty-four hours later by exposure to 2Gy x-radiation or sham irradiation. Reactive oxygen species (ROS) activity was evaluated by cytometric assay, and assay of antioxidant gene expression by RT-qPCR. Cell cycle changes were evaluated by flow cytometry of nuclei stained with propidium iodide. Apoptosis was assessed by cytometric assessment of Caspase-3/7 activity and by immunoblotting of PARP-1 cleavage. Radiosensitization was evaluated by clonogenic survival assay. Proliferation (EdU) and apoptosis (TUNEL) were evaluated in SK-ES-1 xenograft tumors following pre-treatment with 1mg/kg MithA, followed 24 hours later by a single 4Gy fraction of x-radiation. RESULTS MithA treated cells showed reduced levels of ROS, and was associated with increased expression of antioxidant genes SOD1, SOD2 and CAT. It nonetheless induced persistent G0/G1 arrest and progressive increase of the sub-G1 fraction, suggesting apoptotic degeneration. In vitro assays of Caspase-3/7 activity and immunoblotting of Caspase-3/7 dependent cleavage of PARP-1 indicated that apoptosis began as early as 24-hours after MithA exposure, reducing clonogenic survival. Tumors from xenograft mice treated with either radiation alone, or in combination with MithA showed significant reduction of tumor cell proliferation, while apoptosis was significantly increased in the group receiving combination of MithA and RTx. CONCLUSIONS Taken together, our data show that the anti-proliferative and cytotoxic effects of MithA are the prominent components of radiosensitization of EWS::FLI1+ Ewing sarcoma, rather than the result of acutely enhanced ROS levels.