Targeting Rad18-dependent replication stress pathways to modulate chemoresponse in BRCA1-deficient cancers

项目摘要/摘要 乳腺癌易感基因BRCA1和BRCA2的突变增加了乳腺癌的终生风险 和卵巢癌。临床上，BRCA缺乏的肿瘤对铂类化疗药物和 多聚ADP核糖聚合酶抑制剂(PARPI)。然而，对PARPI的抗药性对有效 BRCA缺乏的癌症治疗。除了它们在同源重组中的既定作用外，BRCA 蛋白质在保护复制叉子免受广泛的核溶解降解方面发挥着新的作用。停滞不前或 受损的复制叉会逆转它们的路线，以帮助修复DNA损伤，这些复制叉会逆转复制 叉子是核酸酶广泛降解的底物。值得注意的是，广泛的核溶解降解 反向复制分叉不是终极事件，因为BRCA缺陷细胞激活了恢复机制 以应对广泛的退化。这个项目的总体目标是确定复制的机制 BRCA1缺陷癌细胞中的叉状恢复，这将有助于开发新的化疗药物 BRCA缺陷肿瘤的治疗策略。我的初步数据显示RAD18蛋白与叉子的恢复有关 BRCA1缺陷癌细胞的机制。此外，我发现在BRCA1缺陷型癌症中RAD18的缺失 细胞会加剧复制分叉的退化。RAD18单胞素化增殖细胞核抗原 (增殖细胞核抗原)，促进招募跨损伤合成(TLS)聚合酶，以应对DNA损伤。论 根据我的初步数据和RAD18‘S建立的泛素化活性，我假设增殖细胞核抗原 单素化调控复制分叉恢复，并在BRCA1-1的分叉保护中发挥新的作用 缺乏癌细胞。这项提案的目标1将测试BRCA1缺陷细胞是否激活RAD18-和增殖细胞核抗原 复制分叉恢复的单素化依赖机制。此外，这一目标将决定 这一机制是否需要特定的TLS聚合酶来恢复停滞的叉子。目标2将定义如何 RAD18和/或增殖细胞核抗原单素化介导复制分叉保护。我将使用一种独特的组合 单分子DNA纤维分析和电子显微镜方法实现目标1和2。目标3将 检测在BRCA1中利用Rad18和增殖细胞核抗原泛素化途径治疗的临床意义 有缺陷的癌症。我将利用培养的细胞系和组织芯片来测试Rad18，TLS 聚合酶，或增殖细胞核抗原泛素化，可以有效地靶向调节BRCA1- 背景不足。概述的实验将有助于新的治疗方法的开发，最终 对抗BRCA缺陷性肿瘤中日益增长的化疗耐药性的目标。

Project Summary/Abstract Mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 confer an increased lifetime risk of breast and ovarian cancers. Clinically, BRCA-deficient tumors are sensitive to platinum-based chemotherapeutics and poly-ADP ribose polymerase inhibitors (PARPi). However, resistance to PARPi presents a challenge for effective BRCA-deficient cancer treatment. In addition to their established roles in homologous recombination, BRCA proteins play an emerging role in protecting replication forks from extensive nucleolytic degradation. Stalled or damaged replication forks reverse their course to aid in the repair of DNA damage, and these reversed replication forks are the substrates for extensive degradation by nucleases. Notably, extensive nucleolytic degradation of reversed replication forks is not a terminal event because BRCA-deficient cells activate recovery mechanisms to cope with extensive degradation. The overall goal of this project is to determine the mechanism of replication fork recovery in BRCA1-deficient cancer cells, which will contribute to development of novel chemotherapeutic strategies for BRCA-deficient tumors. My preliminary data implicate the Rad18 protein in the fork recovery mechanism of BRCA1-deficient cancer cells. Moreover, I found that loss of Rad18 in BRCA1-deficient cancer cells exacerbates replication fork degradation. Rad18 monoubiquitinates proliferating cellular nuclear antigen (PCNA), promoting recruitment of Translesion Synthesis (TLS) polymerases to cope with DNA damage. On the basis of my preliminary data and Rad18’s established ubiquitination activity, I hypothesize that PCNA monoubiquitination modulates replication fork recovery and plays a novel role in fork protection in BRCA1- deficient cancer cells. Aim 1 of this proposal will test whether BRCA1-deficient cells activate a Rad18- and PCNA monoubiquitination-dependent mechanism of replication fork recovery. Furthermore, this aim will determine whether this mechanism requires specific TLS polymerases to recover the stalled forks. Aim 2 will define how Rad18 and/or PCNA monoubiquitination mediate replication fork protection. I will use a unique combination of single-molecule DNA fiber assay and electron microscopy approaches to accomplish Aims 1 and 2. Aim 3 will test the clinical relevance of exploiting the Rad18 and PCNA ubiquitination pathways therapeutically in BRCA1- deficient cancers. I will utilize both cultured cell lines and tissue microarrays to test whether Rad18, TLS polymerases, or PCNA ubiquitination can be effectively targeted to modulate chemoresponse in a BRCA1- deficient background. The outlined experiments will contribute to novel therapy development, with the ultimate goal of combating growing chemoresistance in BRCA-deficient tumors.