The Role of ZEB1 in promoting therapeutic resistance through its interaction with 53BP1

摘要 DNA双链断裂(DSB)是对基因组最具诱变性和细胞毒性的侮辱。DSB是 通过同源定向重组修复，在S和G2期占主导地位，也 通过容易出错的非同源末端连接(NHEJ)，它在所有细胞周期阶段都是活跃的。在癌细胞中， 这些修复途径的高活性与辐射抗性有关。转录抑制因子 ZEB1是一种公认的驱动上皮向间充质转化(EMT)的因素 发展和肿瘤进展。最近的研究表明，ZEB1在调节细胞周期中发挥着新的作用。 DNA损伤反应(DDR)，通过增强检查点激酶CHK1的稳定性，并在调节 通过转录沉默聚合酶theta修复DSB，聚合酶theta是微同源性的主要驱动力- 中介替代NHEJ(ALT-NHEJ)。我们发现ZEB1被迅速招募到诱导的DSB中 以DNA-PK依赖的方式选择性地在常着色质基因组区域中，并且对于 向这些网站招聘53BP1。ZEB1在这些休息地点的招募与当地 染色质修饰可用于NHEJ修复。因此，ZEB1的耗尽或缺失被抑制 经典的NHEJ在基于细胞的DSB修复分析中，并显著增加DSB相关的两个高 切除和HDR，这与ATM非依赖的染色体畸变率增加和 增强了对IR的敏感度。另一方面，ZEB1基因的缺失与降低的敏感性有关 BRCA缺陷细胞对PARP抑制剂(PARPI)的作用，表明这些细胞群体中HDR的恢复， 推测是通过抑制53BP1向DSB募集和刺激超切除来实现的。重要的是，我们 发现在低氧条件下，53BP1对DSB的ZEB1依赖的募集显著放大。基于这些 新的观察结果，我们假设ZEB1通过直接和间接机制促进DSB修复 这对于促进对IR的治疗抵抗和赋予BRCA缺陷的敏感性至关重要 一方面，ZEB1通过对c-NHEJ的募集，直接促进c-NHEJ的抗切除和 C-NHEJ修复蛋白53BP1与常染色质结合的DSB。另一方面，ZEB1促进了对 DSB通过增加DDR和通过其染色质间接增强c-NHEJ修复- 修饰和转录沉默活性。本提案的目标1侧重于了解 ZEB1调控双链断裂修复的机制(S)在目标2中，我们将确定ZEB1对 乳腺癌对IR和PARP抑制剂的治疗反应 异种移植小鼠乳腺癌模型。在目标3中，我们将确定ZEB1如何调节治疗 缺氧性乳腺肿瘤对IR和PARPI的反应了解ZEB1/53BP1轴如何调节DSB 在常氧和低氧条件下的修复将使我们能够利用这个轴作为生物标志物来预测IR/PARPI的敏感性 以及靶向轴线以降低治疗阻力。

ABSTRACT DNA double strand breaks (DSBs) are the most mutagenic and cytotoxic insults to the genome. DSBs are repaired through homology directed recombination (HDR), which is predominant in S and G2 phases, and also by error prone non-homologous end joining (NHEJ), which is active in all cell cycle phases. In cancer cells, heightened activity of these repair pathways has been linked to radioresistance. The transcriptional repressor ZEB1 is a well-established driver of the epithelial-to-mesenchymal transition (EMT) in both normal development and tumor progression. Recent studies suggest that ZEB1 plays new roles in the regulation of the DNA damage response (DDR), via enhancing the stability of the checkpoint kinase CHK1, and in regulating the repair of DSBs through the transcriptional silencing of polymerase theta, a major driver of microhomology- mediated alternative NHEJ (alt-NHEJ). We have discovered that ZEB1 is rapidly recruited to DSBs induced selectively in euchromatic genomic regions in a DNA-PK-dependent manner, and is essential for the recruitment 53BP1 to these sites. The recruitment of ZEB1 to these break sites was associated with local chromatin modifications permissive for NHEJ repair. Consequently, depletion or deletion of ZEB1 suppressed canonical NHEJ in cell-based DSB repair assays, and significantly increased both DSB-associated hyper- resection and HDR, This correlated with ATM-independent increases in chromosomal aberrations and enhanced sensitivity to IR. On the other hand, loss of ZEB1 was associated with decreased sensitivity of BRCA-deficient cells to PARP inhibitors (PARPi), indicative of restoration of HDR in these cell population, presumably through inhibiting 53BP1 recruitment to DSBs and stimulation of hyper-resection. Importantly, we found that ZEB1-dependent recruitment of 53BP1 to DSB is significantly amplified in hypoxia. Based on these novel observations, we hypothesize that ZEB1 promotes DSB repair via both direct and indirect mechanisms and this is critical for promoting therapeutic resistance to IR and for conferring sensitivity of BRCA-deficient cells to PARPi; on one hand, ZEB1 directly promotes c-NHEJ through the recruitment of the anti-resection and c-NHEJ repair protein 53BP1 to euchromatin-bound DSBs. On the other hand, ZEB1 facilitates the repair of DSBs through augmenting the DDR and through indirectly enhancing c-NHEJ repair through its chromatin- modifying as well as transcriptional silencing activity. Aim 1 of this proposal focuses on understanding the mechanism(s) by which ZEB1 regulates DSB repair. In Aim 2, we will determine the impact of ZEB1 on the therapeutic response of breast cancer to IR and PARP inhibitors in a panel of breast cancer cell lines and in xenograft mouse models of breast cancer. In Aim 3, we will determine how ZEB1 modulates the therapeutic response of hypoxic breast tumors to IR and PARPi. Understanding how the ZEB1/53BP1 axis regulates DSB repair in normoxia and hypoxia will allow us to exploit this axis as a biomarker to predict IR/PARPi sensitivity and well as to target the axis to decrease therapeutic resistance.