Abstract 2406: Targeting prmt5 inhibits DNA repair and enhances the radiosensitivity of GBM cells

Abstract Glioblastoma (GBM) is considered highly radioresistant. To improve the efficacy of GBM radiotherapy, one approach is the addition of a targeted radiosensitizer, which requires an understanding of the molecules that determine GBM radioresponse. Along these lines, PRMT5 is a type II arginine methyltransferase that symmetrically dimethylates histone and non-histone substrates to regulate chromatin structure and mRNA splicing, two parameters that can influence radiosensitivity. As an initial test of PRMT5 as a target for GBM radiosensitization, PRMT5 knockdown using siRNA was shown to increase the in vitro radiosensitivity of U251 GBM cells. PRMT5 was then targeted using the small molecule inhibitor LLY-283, which penetrates the blood brain barrier. Exposure of U251 cells to LLY-283 (24h) reduced the levels of symmetric dimethylated arginine as determined by western blot, indicating an inhibition of PRMT5 activity. Based on clonogenic analysis, LLY-283 exposure beginning 1h prior to irradiation significantly enhanced the radiosensitivity of U251 cells. Whereas treatment of the normal fibroblast cell line MRC9 with LLY-283 reduced PRMT5 activity, no effect was detected on radiosensitivity, suggesting tumor cell selective radiosensitization. As an initial investigation of the radiosensitizing mechanism, U251 were treated with LLY-283 1h prior irradiation (2Gy) and the number of γH2AX foci, marker for DNA double strand breaks (DSBs), was determined. Whereas LLY-283 had no effect on the initial level of radiation-induced γH2AX foci, the dispersal of foci was significantly delayed in LLY-283 treated cells. These results suggest that LLY-283 inhibits the repair of radiation-induced DSBs. The neutral comet assay, an alternative measure of radiation-induced DSBs showed that LLY-283 delivered 1h prior irradiation (10Gy) significantly increased the comet tail moment detected at 6 and 24h as compared to 10Gy only, consistent with an inhibition of DSB repair. Based on these two approaches LLY-283-induced radiosensitization appears to be mediated by an inhibition of DSB repair. Extending these studies to GBM stem-like cell lines (GSCs) showed that LLY-283 inhibited PRMT5 activity and when delivered 1h prior to irradiation enhanced radiosensitivity. Analysis of γH2AX foci showed a delay in foci dispersal in LLY-283 treated cells consistent with an inhibition of DSB repair. Because a 1h LLY-283 pretreatment was sufficient for the inhibition of DSB repair and enhanced radiosensitivity, it appears that the radiosensitization was primarily the result of altered chromatin function rather than changes in gene expression due to alternative mRNA splicing. Altogether, these results suggest targeting PRMT5 as a potential strategy for enhancing the radiosensitivity of GBMs. Citation Format: Charlotte M. Degorre, Steven Lohard, Philip J. Tofilon. Targeting prmt5 inhibits DNA repair and enhances the radiosensitivity of GBM cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2406.