The processes mediating the repair of DNA double strand breaks (DSBs) are critical determinants of radiosensitivity and provide a source of potential targets for tumor radiosensitization. Among the events required for efficient DSB repair are a variety of post-translational histone modifications including methylation. Because trimethylation of histone H3 on lysine 27 (H3K27me3) has been associated with chromatin condensation, which can influence DSB repair, we determined the effects of radiation on H3K27me3 levels in three in three human tumor cell lines: U251; MDA-MB-231 and A549, and two normal cell lines. Irradiation (10Gy) of tumor cells resulted in a rapid loss of H3K27me3. According to immunoblot analysis, tumor cells had significant higher levels of the histone demethylase UTX compared to JMJD3, suggesting a more prominent role for UTX in the demethylation of H3K27me3 after irradiation. The rapid loss of H3K27me3 was prevented by the siRNA-mediated knockdown UTX and enhanced the radiosensitivity of each tumor cell line, whereas the knockdown of JMJD3 had no effect. To investigate whether inhibition of UTX could serve as a potential target to target to radiosensitize tumor cells, we focused on GSKJ4 a H3K27 demethylase inhibitor. GSKJ4 treatment (4 μM) of tumor cells was found to block the radiation-induced decrease of H3K27me3. Further, based on clonogenic survival analysis addition of GSKJ4 immediately prior to irradiation significantly enhanced the radiosensitivity of tumor cells but not that of normal cells. To begin to investigate the mechanism responsible for this radiosensitization tumor cells were irradiated (10Gy), treated with GSKJ4 and collected at 0.5-24h later for neutral comet assay, a measure of DNA double strand breaks (DSBs). 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. Analysis of γH2AX nuclear foci, a surrogate marker for DNA DSBs 16h after irradiation (6Gy) and treatment of tumor cells with GSKJ4 (4μM) increased the percentage of cells expressing γH2AX as compared to radiation alone suggesting that GSKJ4 inhibits DSB repair. Thus, data generated from the neutral comet and γH2AX assays suggest that the inhibition of H3K27m3 demethylation mediated by GSKJ4 inhibits the repair of radiation-induced DSBs, which could then account for the observed radiosensitization. Consistent with in vitro results radiation reduced the H3K27me3 methylation in mice bearing leg tumor xenografts. Treatment of mice bearing leg tumor xenografts with GSKJ4 and radiation significantly enhanced radiation-induce tumor growth delay. These data suggest that H3K27me3 demethylation contributes to DSB repair in tumor cells and that UTX, the demethylase responsible, provides a target for selective tumor cell radiosensitization in vivo.
Citation Format: Barbara H. Rath, Isabella Waung, Kevin Camphausen, Philip Tofilon. Inhibition of the histone H3K27 demethylase UTX enhances tumor cell radiosensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1389.