Abstract 1663: Histone methylation as a target for the radiosensitization of glioblastoma-stem like cells

Radiotherapy is a primary treatment modality for glioblastomas (GBMs). However, while many GBMs initially respond to radiation, even in combination with surgery and chemotherapy, median survival continues to be less than 2 years after diagnosis. Because post-translational histone modifications have been implicated in the regulation of radiosensitivity, as a potential strategy for enhancing the response of GBMs to radiotherapy we have evaluated modifiers of histone methylation on the radiosensitivity of glioblastoma stem-like cells (GSCs). For these studies we initially focused on DZNep (3-deazaneplanocin A), which induces degradation of the methyltransferase EZH2. DZNep treatment (0.5μM) of GSCs resulted in a decrease in the amount of H3K27me3 within 6hrs of exposure. Using clonogenic survival analysis, exposure of GSCs to DZNep was then found to enhance their radiosensitivity in a schedule dependent manner: addition of DZNep immediately after irradiation significantly enhanced radiosensitivity, whereas treatment with DZNep for 24h pre-irradiation had minimal to no effect on radiation-induced cell killing. To begin to investigate the mechanism responsible for this radiosensitization, GSCs were irradiated (2Gy), treated with DZNep and collected at 1-24h later for analysis of γH2AX nuclear foci, a surrogate marker of DNA double strand breaks (DSBs). DZNep had no effect on the initial level of radiation-induced γH2AX foci, yet significantly delayed foci dispersal, suggestive of an inhibition of DSB repair as the mechanism of DZNep mediated radiosensitization. To further investigate the potential role of histone methylation as a determinant of radiosensitivity, GSCs were exposed to GSKJ4, an inhibitor of the histone demethylase JMJD3, which results in an increase histone methylation. Given the results from the DZNep experiments, it was anticipated that GSKJ4 would have no effect on or possibly reduce GSC radiosensitivity. However, based on clonogenic survival analysis, addition of GSKJ4 (4μM) 24h prior to or immediately after irradiation significantly enhanced GSC radiosensitivity. With respect to DSBs, GSKJ4 had no effect on the initial level of radiation-induced γH2AX foci or, in contrast to DZNep, on foci dispersal. The γH2AX data suggest that the two modifiers of histone methylation induced radiosensitization through different mechanisms. The radiosensitization induced by an increase or decrease in histone methylation may be accounted for by the biphasic changes in methylation pattern observed after irradiation. Exposure of GSCs to 10Gy resulted in an initial decrease in H3K27me3 levels at 30min followed by an increase above control at 60min. Taken together, these results suggest that disrupting histone methylation status induces GSC radiosensitization. Citation Format: Barbara Helen Rath, Kevin Camphausen, Philip J. Tofilon. Histone methylation as a target for the radiosensitization of glioblastoma-stem like cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1663.