Abstract 1379: Histone demethylase KDM6 as a target for glioblastoma radiosensitization

Abstract Radiotherapy is a primary treatment modality for glioblastomas (GBMs). 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 the effect of radiation on histone methylation in glioblastoma stem-like cells (GSCs) and the established glioma cell line (U251). For these studies we focused on histone H3K27 tri-methylation (H3K27me3), whose opposing site H3K36me2 has already been linked to DNA repair. Exposure of GSCs and U251 to 10Gy decreased H3K27me3 by 30min, reaching a cell line depended maximum between 1h and 3h, followed by a return to control levels at 6h. According to immunoblot analysis, GSCs and U251 had significant higher levels of KDM6A compared to KDM6B, suggesting a more prominent role for KDM6A in the demethylation of H3K27me3 after irradiation. To determine whether KDM6 could serve as a target for GBM radiosensitization, we focused on GSKJ4, which is an inhibitor for KDM6A and KDM6B. GSKJ4 treatment (4uM) of GSCs and U251 increased the amount of H3K27me3 within 1h of exposure and was found to block the radiation-induced decrease of H3K27me3. Based on clonogenic survival analysis addition of GSKJ4 immediately prior to irradiation significantly enhanced the radiosensitivity of GSCs and U251. To begin to investigate the mechanism responsible for this radiosensitization, GSCs and U251 were irradiated (10Gy), treated with GSKJ4 and collected at 0.5-24h later for neutral comet assay, a measure of DNA double strand breaks. GSKJ4 had no effect on the initial comet-tail moment, yet significantly increased the comet tail-moment up to 24h after radiation, suggestive of an inhibition of DSB repair. To further investigate the potential role of histone demethylase as a potential target to radiosensitize GBM, siRNA was used to knock down KDM6A expression. Cells treated with siKDM6A showed a significant increase in H3K27me3 and the radiation-induced decrease of H3K27me3 was abolished. Analysis of γH2AX nuclear foci after irradiation (2Gy) of siKDM6A treated cells showed a significant delay in foci dispersal, consistent with an inhibition of DSB repair. To investigate whether similar changes in histone methylation occurred in an in vivo setting, H3K27me3 levels were determined in GSC orthotopic xenografts as a function of time after brain irradiation. Similar to in vitro conditions, radiation reduced H3K27me3 levels at 1h, with a decrease still present at 6h. Taken together, these results suggest that the histone demethylase KDM6A is a target for GBM radiosensitization. Citation Format: Barbara Helen Rath, Marzia F. Shah, Kevin Camphausen, Philip J. Tofilon. Histone demethylase KDM6 as a target for glioblastoma radiosensitization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1379. doi:10.1158/1538-7445.AM2017-1379