FEN1 Endonuclease as a Synthetic Lethal Target for Cancer Therapy

项目总结 散发性癌症是恶性进展的重要驱动力。例如，遗传性乳房和 BRCA1和BRCA2胚系突变导致的卵巢癌发生同源重组 修复(HRR)增加模型系统中GCR的缺陷。我们之前的研究已经建立了一个 酵母基因组不稳定性抑制基因的全面网络和在硅胶中的证明 这些酵母地理信息系统基因的人类同源基因在许多癌症中都发生了遗传和/或表观遗传改变 类型。从酵母遗传研究中，我们还识别并排序了合成致死(SL)合作伙伴 非必要的地理信息系统基因。靶向SL基因交互作用用于合理的癌症治疗的可行性 PARP抑制剂在乳腺癌和卵巢癌维持治疗中的应用 有BRCA1或BRCA2缺陷。为了开发更多的SL相互作用用于癌症治疗，这项提议旨在 利用在酵母中开发的关于GIS基因及其SL合作伙伴的丰富知识来指导 针对导致癌症GCR的人类地理信息系统基因缺陷的治疗方法的发展。具体地说， 拟议的研究将集中在人瓣内切酶1(FEN1)，它是酵母RAD27的同源物，它 与具有多种功能的地理信息系统基因具有最高数量的SL-互作。FEN1处理冈崎 在滞后的链DNA合成过程中的片段，并在长补丁碱基切除修复中起作用，但本身不是 必不可少的。 针对FEN1-SL伙伴地理信息系统基因，我们建议开展以下几个方面的研究：目的1)拓展 目前正在对细胞系和小鼠进行CRISPR-Dropr筛选和验证研究，以(A)在 哪些缺陷会导致对我们专有的高度有效和特异的FEN1抑制剂的敏感性，以及(B) 定义FEN1 SL伙伴基因以及可被FEN1抑制剂靶向的癌症组学特征 诱导SL；目标2)将信息学和基于细胞的验证方法相结合，深入研究癌症 组学和基因重要性数据，以确定适合治疗的癌症的谱系和特征 以FEN1抑制剂为靶点；目的3)应用一系列基于遗传学的方法来研究 FEN1抑制剂获得性耐药机制及对FEN1与PARP耐药性的比较 抑制剂；以及，目的4)确定抑制FEN1对DNA复制、分叉稳定性和 BRCA1和BRCA2突变细胞的染色体完整性以验证FEN1抑制剂诱导 对HRR缺陷的癌细胞复制造成不可修复的损害，从而导致致命性。这些机械式的 研究将扩展到其他经过验证的FEN1-SL伙伴基因。这些项目将大大加快 开发针对癌症基因组不稳定的FEN1抑制剂并将建立一个实验平台 用于评估和开发拟议研究将确定的其他潜在治疗靶点。

PROJECT SUMMARY Gross chromosomal rearrangements (GCRs), a type of genome instability, are often seen in inherited and sporadic cancers and are an important driver of malignant progression. For example, hereditary breast and ovarian cancers resulting from BRCA1 and BRCA2 germline mutations suffer from homologous recombination repair (HRR) defects that increase GCRs in model systems. Our previous studies have established a comprehensive network of genome instability suppressor (GIS) genes in yeast and demonstrated in silico that human homologs of these yeast GIS genes are genetically and/or epigenetically altered across many cancer types. From yeast genetic studies, we have also identified and rank-ordered synthetic lethal (SL) partners of non-essential GIS genes. The feasibility of targeting SL genetic interactions for rational cancer therapy has been supported by the application of PARP inhibitors for maintenance therapy of breast and ovarian cancers with BRCA1 or BRCA2 defects. To exploit additional SL interactions for cancer therapy, this proposal aims to leverage the wealth of knowledge on GIS genes and their SL partners developed in yeast to guide the development of therapeutics that target human GIS gene defects that cause GCRs in cancer. Specifically, the proposed studies will focus on the human Flap Endonuclease 1 (FEN1), the homolog of yeast RAD27, which has the highest number of SL-interactions with GIS genes of a variety of functions. FEN1 processes Okazaki fragments during lagging strand DNA synthesis and acts in long-patch base excision repair but is itself non- essential. To develop this nuclease as a target for treating cancers with defects in BRCA1, BRCA2 and other FEN1-SL partner GIS genes, we propose to carry out the following lines of research: AIM 1) To expand ongoing CRISPR-dropout screens and validation studies in cell lines and mice to (a) identify human genes in which defects cause sensitivity to our proprietary FEN1-inhibitors of highly potency and specificity and (b) define FEN1 SL-partner genes as well as cancer omics signatures that can be targeted with FEN1 inhibitors to induce SL; AIM 2) To combine informatics and cell-based validation approaches for a deep-dive into cancer omics and gene essentiality data to identify the spectrum and signatures of cancers amenable to therapeutic targeting with FEN1 inhibitors; AIM 3) To apply an array of genetics-based approaches to investigate mechanisms for acquired resistance to FEN1 inhibitors and to compare resistance to FEN1 versus PARP inhibitors; and, AIM 4) To determine the effects of FEN1 inhibition on DNA replication, fork stability, and chromosome integrity in BRCA1 and BRCA2 mutant cells to test the hypothesis that FEN1 inhibitors induce irreparable damage to replication forks in HRR-deficient cancer cells to cause lethality. These mechanistic studies will be extended to other validated FEN1-SL partner genes. These projects will greatly accelerate the development of FEN1 inhibitors to target cancer genome instability and will establish an experimental platform for evaluation and development of other potential therapeutic targets to be identified by the proposed research.