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

摘要 DNA双链断裂（DSB）是对基因组最具致突变性和细胞毒性的损伤。DSB是 通过同源定向重组（HDR）修复，这在S期和G2期占主导地位， 通过易错的非同源末端连接（NHEJ），其在所有细胞周期阶段中是活跃的。在癌细胞中， 这些修复途径的活性增强与辐射抗性有关。转录抑制子 ZEB 1是一个成熟的驱动器上皮间质转化（EMT）在两个正常的， 发展和肿瘤进展。最近的研究表明，ZEB 1在调节细胞凋亡中发挥着新的作用。 DNA损伤反应（DDR），通过增强检查点激酶CHK 1的稳定性，并在调节 通过聚合酶theta的转录沉默修复DSB，这是微同源性的主要驱动因素， 介导的替代NHEJ（alt-NHEJ）。我们已经发现，ZEB 1被迅速募集到诱导的DSB中， 选择性地在常染色质基因组区域中以DNA-PK依赖的方式，并且对于 招募53 BP 1到这些位点。ZEB 1在这些断裂部位的募集与局部 允许NHEJ修复的染色质修饰。因此，ZEB 1的消耗或缺失抑制了 典型的NHEJ在基于细胞的DSB修复试验中，并显着增加了DSB相关的超 这与ATM非依赖性染色体畸变增加相关， 另一方面，ZEB 1的缺失与对IR的敏感性降低有关。 BRCA缺陷细胞对PARP抑制剂（PARPi）的敏感性，表明这些细胞群中HDR的恢复， 推测是通过抑制53 BP 1募集到DSB和刺激过度切除。重要的是我们 发现ZEB 1依赖的53 BP 1向DSB的募集在缺氧中显著放大。基于这些 新的观察，我们假设ZEB 1通过直接和间接机制促进DSB修复 这对于促进对IR的治疗抗性和赋予BRCA缺陷的敏感性至关重要。 一方面，ZEB 1通过募集抗切除和抗肿瘤细胞直接促进c-NHEJ， c-NHEJ修复蛋白53 BP 1与常染色质结合的DSB。另一方面，ZEB 1促进了 DSB通过增强DDR和通过其染色质间接增强c-NHEJ修复， 修饰以及转录沉默活性。本提案的目标1侧重于了解 ZEB 1调节DSB修复的机制。在目标2中，我们将确定ZEB 1对 在一组乳腺癌细胞系中和在一组乳腺癌细胞系中乳腺癌对IR和PARP抑制剂的治疗反应， 乳腺癌的异种移植小鼠模型。在目标3中，我们将确定ZEB 1如何调节治疗性 低氧乳腺肿瘤对IR和PARPi的反应。了解ZEB 1/53 BP 1轴如何调节DSB 常氧和缺氧中的修复将使我们能够利用该轴作为生物标志物来预测IR/PARPi敏感性 并且靶向轴以降低治疗阻力。

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.