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

Abstract

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 + ES 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 24 h later by exposure to 2 Gy 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 pretreatment with 1 mg/kg MithA, followed 24 h later by a single 4 Gy fraction of x-radiation. Results MithA-treated cells showed reduced levels of ROS, and were associated with increased expression of antioxidant genes SOD1, SOD2, and CAT. It nonetheless induced persistent G0/G1 arrest and a 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 h after MithA exposure, reducing clonogenic survival. Tumors from xenograft mice treated with either radiation alone, or in combination with MithA showed a significant reduction of tumor cell proliferation, while apoptosis was significantly increased in the group receiving the 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+ ES, rather than the result of acutely enhanced ROS levels.