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

靶向 Rad18 依赖性复制应激途径来调节 BRCA1 缺陷癌症的化学反应

• 批准号: 10431909
• 负责人: Emily Cybulla
• 金额: $ 5.18万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-02 至 2024-06-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10431909
• 关键词: Aftercare; BRCA deficient; BRCA1 Mutation; BRCA1 gene; BRCA2 gene; Biological Assay; Breast Cancer Risk Factor; Cancer-Predisposing Gene; Cell Line; Cells; Chemoresistance; Clinical; DNA; DNA Damage; DNA Repair; DNA biosynthesis; DNA replication fork; Data; Development; Electron Microscopy; Event; Fiber; Genetic; Genomic Instability; Goals; Link; Malignant Neoplasms; Malignant neoplasm of ovary; Mediating; Modality; Molecular; Monoubiquitination; Mutation; Nuclear Antigens; Pathway interactions; Patients; Platinum; Play; Poly(ADP-ribose) Polymerases; Polymerase; Positioning Attribute; Proliferating; Proteins; Recovery; Resistance; Resort; Risk; Role; Sampling; Scheme; Testing; Therapeutic; Time; Tissue Microarray; Ubiquitin; Ubiquitination; Work; base; brca gene; cancer cell; cancer therapy; clinically relevant; design; experimental study; genome-wide; homologous recombination; improved; inhibitor; lifetime risk; malignant breast neoplasm; new therapeutic target; novel; novel therapeutics; nuclease; ovarian neoplasm; recruit; replication stress; single molecule; targeted nucleases; therapy development; tumor

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.

项目总结/摘要 乳腺癌易感基因BRCA 1和BRCA 2的突变增加了乳腺癌的终生风险 和卵巢癌临床上，BRCA缺陷型肿瘤对铂类化疗药物敏感， 聚ADP核糖聚合酶抑制剂（PARPi）。然而，对PARPi的耐药性对有效的治疗提出了挑战。 BRCA缺陷癌症治疗。除了在同源重组中的作用外，BRCA 蛋白质在保护复制叉免于广泛的溶核降解中起着新兴的作用。停滞或 受损的复制叉会逆转其进程，以帮助修复DNA损伤，而这些逆转的复制 叉是核酸酶广泛降解的底物。值得注意的是，广泛的溶核降解 反向复制叉不是终末事件，因为BRCA缺陷细胞激活恢复机制 以科普大面积退化。这个项目的总体目标是确定复制的机制 BRCA 1缺陷癌细胞中的分叉恢复，这将有助于开发新的化疗药物。 BRCA缺陷型肿瘤的治疗策略。我的初步数据表明Rad 18蛋白质与叉子的恢复有关 BRCA 1缺陷癌细胞的机制。此外，我发现，在BRCA 1缺陷型癌症中， 细胞加剧了复制叉的降解。Rad 18单泛素化增殖细胞核抗原 增殖细胞核抗原（PCNA），促进转录合成（TLS）聚合酶的招募，以科普DNA损伤。上 根据我的初步数据和Rad 18已建立的泛素化活性，我假设PCNA monoubiquitination调节复制叉恢复，并在BRCA 1- 缺乏癌细胞。该提案的目的1将测试BRCA 1缺陷细胞是否激活Rad 18和PCNA。 复制叉恢复的依赖单泛素化的机制。此外，这一目标将决定 这种机制是否需要特定的TLS聚合酶来恢复停滞的叉。目标2将定义如何 Rad 18和/或PCNA单泛素化介导复制叉保护。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来实现目标1和2。目标3将 测试在BRCA 1 - 1中利用Rad 18和PCNA泛素化途径治疗的临床相关性。 缺陷型癌症我将利用培养的细胞系和组织微阵列来测试Rad 18，TLS 聚合酶或PCNA泛素化可以有效地靶向调节BRCA 1- 背景不足。概述的实验将有助于新疗法的开发，最终 目的是对抗BRCA缺陷肿瘤中日益增长的化疗耐药性。