Stalled replication fork repair in cancer predisposition and cancertherapy

癌症易感性和癌症治疗中停滞的复制叉修复

• 批准号: 10517824
• 负责人: Ralph Scully
• 金额: $ 102.2万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-10 至 2029-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10517824
• 关键词: ATP phosphohydrolase; Ablation; BRCA1 gene; BRCA2 gene; Breast Cancer therapy; Cell Cycle; Cells; Chromatin; Clinical; Code; DNA; DNA Damage; DNA Repair; DNA analysis; DNA biosynthesis; DNA replication fork; Defect; Disease; Fanconi' s Anemia; Funding; Genes; Genetic; Genomic Instability; Goals; Hereditary Breast and Ovarian Cancer Syndrome; Human; Light; Link; Malignant Neoplasms; Malignant neoplasm of ovary; Motor; Mutation; Oncogenes; Outcome; Prevention; Proteins; Regulation; Replication-Associated Process; Reporter; Rest; Role; Site; Techniques; Therapeutic; Time; Variant; Work; Yeasts; cancer genome; cancer predisposition; druggable target; homologous recombination; human disease; innovation; insight; loss of function mutation; malignant breast neoplasm; molecular targeted therapies; mutant; novel therapeutics; prevent; repaired; response; success; targeted cancer therapy; targeted treatment; tool

PROJECT SUMMARY Error-free DNA repair initiated at the sites of replication fork stalling is critical for the prevention of genomic instability in cycling cells. Defects in stalled fork repair have been directly implicated in cancer predisposition and other human diseases. The clinical burden associated with failed stalled fork repair may include hereditary breast and ovarian cancer (HBOC) predisposition, in light of the involvement of BRCA1 and BRCA2 in repair of stalled replication forks, and Fanconi Anemia (FA)—a rare, autosomal recessive (or X-linked) disease caused by inactivation of any one of several FA genes. Our work previously established roles for BRCA1 and BRCA2 in regulating HR at both double strand breaks (DSBs) and in stalled fork repair. We developed innovative tools for quantifying homologous recombination (HR) and other repair outcomes at stalled mammalian replication forks and, more recently, at broken replication forks. A major goal of this proposal is to define the fundamental mechanisms of repair of stalled forks. We have developed an array of cutting-edge tools to support this study, including unique, sophisticated HR reporters that can distinguish between error-free “short tract” HR and error- prone “long tract” HR—a replicative response analogous to break-induced replication in yeast. One unusual aberrant replicative response that we observe at stalled forks specifically in BRCA1 mutant cells is the formation of <10 kb non-homologous tandem duplications (TDs). In a paradigm-shifting discovery, we found that these highly specific forms of structural variation are also abundant in the human BRCA1-linked breast and ovarian cancer genome. A major goal of this proposal is to define the genetic regulation and full mechanism of TD formation at stalled forks in BRCA1 mutant cells. Success in this project will reveal in unprecedented detail the mechanisms that regulate mammalian stalled (or broken) fork repair and their relationship to cancer predisposition. In support of this, we will develop new techniques for analyzing DNA structural intermediates, chromatin responses to fork stalling and protein composition of the stalled mammalian replication fork. These analytical studies may also identify new molecular targets for therapy of breast and ovarian cancer. Indeed, our recent work on the mechanisms underlying formation of BRCA1-linked TDs led us to discover a synthetic lethal interaction between BRCA1 and FANCM loss-of-function mutations. FANCM is a motor protein and, hence, an ATPase. We find that ablation of FANCM ATPase activity alone (leaving the rest of the protein intact and stable within the cell) is sufficient to confer lethality on BRCA1 mutant cells. Thus, FANCM may be a “druggable” target for therapy in BRCA1-linked cancer. In work proposed herein, we will define the therapeutic potential of this discovery. During the funding period, we expect to make important discoveries in this field and to open the door to new therapies in HBOC and perhaps other forms of cancer.

项目概要 在复制叉停滞位点启动的无错误 DNA 修复对于预防基因组修复至关重要 循环细胞的不稳定性。停滞叉修复缺陷与癌症易感性直接相关 和其他人类疾病。与失速前叉修复失败相关的临床负担可能包括遗传性 鉴于 BRCA1 和 BRCA2 参与修复乳腺癌和卵巢癌 (HBOC) 复制叉停滞和范可尼贫血 (FA)——一种罕见的常染色体隐性遗传（或 X 连锁）疾病 通过使几个 FA 基因中的任何一个失活。我们之前的工作确定了 BRCA1 和 BRCA2 的角色 调节双链断裂 (DSB) 和失速叉修复时的 HR。我们开发了创新工具 用于量化哺乳动物复制停滞时的同源重组 (HR) 和其他修复结果 分叉，以及最近出现的损坏的复制分叉。该提案的一个主要目标是定义基本的 修复失速货叉的机制。我们开发了一系列尖端工具来支持这项研究， 包括独特、先进的人力资源报告员，可以区分无错误的“简短”人力资源和错误的人力资源 倾向于“长链”HR——一种类似于酵母中断裂诱导复制的复制反应。一个不寻常的 我们在 BRCA1 突变细胞中特别是在停滞叉处观察到的异常复制反应是 形成 <10 kb 非同源串联重复 (TD)。在一个范式转变的发现中，我们发现 这些高度特异性的结构变异形式在人类 BRCA1 相关乳房中也很丰富 和卵巢癌基因组。该提案的一个主要目标是定义遗传调控和完整的 BRCA1 突变细胞中停滞叉处 TD 形成机制。该项目的成功将在 前所未有的详细描述了调节哺乳动物停滞（或损坏）叉修复的机制及其 与癌症易感性的关系。为了支持这一点，我们将开发分析 DNA 的新技术 结构中间体、染色质对叉失速的反应以及失速哺乳动物的蛋白质组成 复制叉。这些分析研究还可能确定治疗乳腺癌和乳腺癌的新分子靶点。 卵巢癌。事实上，我们最近对 BRCA1 相关 TD 形成机制的研究引导我们 发现 BRCA1 和 FANCM 功能丧失突变之间的合成致死相互作用。 FANCM 是一个 运动蛋白，因此也称为 ATP 酶。我们发现单独消除 FANCM ATP 酶活性（留下其余部分） 细胞内完整且稳定的蛋白质）足以赋予 BRCA1 突变细胞致死性。因此， FANCM 可能是治疗 BRCA1 相关癌症的“可成药”靶点。在本文提出的工作中，我们将 定义这一发现的治疗潜力。在资助期间，我们希望做出重要的 该领域的发现为 HBOC 以及其他形式的癌症的新疗法打开了大门。