Screening for DNA damage response modulators in glioblastoma stem cells

摘要 目前尚无治愈与预后黯淡相关的高级别胶质瘤的方法 和生活质量差。 GBM 患者通常接受手术切除，然后进行放射治疗 疗法（RT）与烷化剂替莫唑胺（TMZ）相结合。然而，几乎所有 GBM 患者要么对这种治疗没有反应，要么最终产生耐药性，这强调了 迫切需要有效的辅助治疗。 RT 和 TMZ 都可以通过引起肿瘤细胞 致命的DNA双链断裂，可以被内在DNA识别和修复 损伤反应（DDR），从而防止 DNA 损伤诱导的细胞死亡。此外，GBM 富含神经胶质瘤干细胞 (GSC) 的子集，其特征是 上调的 DDR 有助于其优异的治疗耐药性。靶向DNA修复 可以增加/恢复脑肿瘤对 TMZ 和 RT 的敏感性，从而增加治疗效果 疗效并最大限度地减少肿瘤复发。我们设计了一种快速、多功能且高度 灵敏的报告基因，可检测和定量微量 DNA 双链断裂 修复，例如同源定向修复以及非同源末端连接，无需 破坏细胞。在此，我们建议使用该报告基因来识别 DNA 调节剂 GSC 中的损伤修复。在目标 1（R61 阶段）中，我们将开发和优化高通量 监测 GSC 中 DDR 的筛选试验。在目标 2（R33 阶段）中，我们将使用此测定来筛选 用于抑制 DNA 损伤修复的化合物。该项目完成后，此屏幕 应鉴定出能够有效提高 GSC 对放化疗敏感性的新型化合物 提高基因毒性治疗的功效。

Abstract There is presently no cure for high grade gliomas which are associated with a bleak prognosis and a poor quality of life. GBM patients typically undergo surgical resection followed by radiation therapy (RT) combined with the alkylating agent temozolomide (TMZ). However, virtually all GBM patients either fail to respond to this treatment, or ultimately develop resistance, underlining the urgency for effective adjuvant therapies. Both RT and TMZ can target tumor cells by causing lethal DNA double-strand breaks, which could be recognized and repaired by the intrinsic DNA damage response (DDR) thus protecting against DNA damage-induced cell death. Further, GBMs are enriched in a subset population of glioma cancer stem cells (GSCs), characterized by an upregulated DDR which contributes to their superior therapeutic resistance. Targeting DNA repair can increase/restore sensitivity of brain tumors to TMZ and RT, thus increasing therapeutic efficacy and minimizing tumor recurrence. We have designed a rapid, versatile and highly sensitive reporter that can detect and quantify minute amounts of DNA double-strand break repairs such as homology-directed repair as well as non-homologous end joining without disrupting cells. Here we propose to use this reporter in order to identify modulators of DNA damage repair in GSCs. In Aim 1 (R61 phase), we will develop and optimize a high-throughput screening assay to monitor DDR in GSCs. In Aim 2 (R33 phase), we will use this assay to screen for compounds which inhibit DNA damage repair. Upon completion of this project, this screen should identify novel compounds which effectively sensitize GSCs to chemoradiotherapy and increase the efficacy of genotoxic therapeutics.