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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

基本信息

批准号：
10667500
负责人：
Emily Cybulla
金额：
$ 5.27万
依托单位：
SAINT LOUIS UNIVERSITY
依托单位国家：
美国
项目类别：
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/10667500
关键词：
Aftercare BRCA deficient BRCA1 Mutation BRCA1 gene BRCA2 gene Biological Assay Breast Cancer Risk Factor Cancer-Predisposing Gene Cell Line Cell Proliferation 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) Polymerase Inhibitor Polymerase Positioning Attribute Proteins Recovery Resistance Risk Role Sampling Scheme Testing Therapeutic Time Tissue Microarray Ubiquitin Ubiquitination Work brca gene cancer cell cancer therapy clinically relevant coping design experimental study genome-wide homologous recombination improved lifetime risk malignant breast neoplasm new therapeutic target novel novel therapeutics nuclease ovarian neoplasm recruit replication stress response single molecule targeted nucleases therapeutic development therapy development tumor

项目摘要

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。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Leveraging the replication stress response to optimize cancer therapy.

DOI：
10.1038/s41568-022-00518-6
发表时间：
2023-01
期刊：
Nature reviews. Cancer
影响因子：
0
作者：
通讯作者：

Temporally distinct post-replicative repair mechanisms fill PRIMPOL-dependent ssDNA gaps in human cells.

DOI：
10.1016/j.molcel.2021.09.013
发表时间：
2021-10-07
期刊：
Molecular cell
影响因子：
16
作者：
Tirman S;Quinet A;Wood M;Meroni A;Cybulla E;Jackson J;Pegoraro S;Simoneau A;Zou L;Vindigni A
通讯作者：
Vindigni A

PRIMPOL ready, set, reprime!