Targeting DNA Ligase I in Ovarian Cancer

项目摘要 基因组维持途径的异常为开发治疗方法提供了机会 选择性地靶向癌细胞。此外，由于癌细胞的增殖特性，它们很容易受到影响。 复制相关的DNA损伤，更多地依赖于处理这种类型的 损坏。在最近的一项合作研究中，我们发现DNA连接酶I(LIGI)的水平，连接的主要酶 复制分叉处的Okazaki片段与卵巢癌的预后和治疗耐药相关。 此外，我们发现卵巢癌细胞在同源依赖修复(HDR)中存在缺陷， 对Ligi抑制敏感。在初步研究中，我们比较了LIG1缺失和化学药物的影响 抑制力。在LIG1缺失细胞中，LigIII的配对蛋白XRCC1和PARP2水平升高 与新合成的相关，与依赖PARP和LigIIIDNA的备份途径一致 连接冈崎片段，使细胞能够在没有LIGI的情况下复制。相比之下，与 LIGI抑制剂并未导致XRCC1或PARP2水平升高。取而代之的是，体内的 Mre11Rad50Nbs1复合体，启动同源依赖修复(HDR)的切除步骤。在目标1中，我们 将使用遗传学、细胞生物学和生化方法的组合来确定 LIGI抑制诱导的HDR通路。由于HDR缺乏症既有遗传性的，也有零星的 卵巢癌，我们已经确定LIG1是卵巢癌预后和治疗耐药的生物标志物， 我们将单独检测LIGI抑制剂的活性，以及与DNA修复抑制剂和/或 基因定义的卵巢癌细胞系中的基因毒性化疗药物。在我们现有Ligi的基础上构建 在目标2中，我们将使用分子建模和药物化学的组合来设计和 合成可能的LIGI抑制剂，预计具有增强的效力和选择性以及 改善了药理作用。我们将同时评估可能的LIGI抑制剂的活性。 用纯化的人DNA连接酶进行生化分析和用基因定义的卵巢进行细胞分析 癌细胞系。在目标3中，我们将首先检查现有的和新的LIGI抑制剂在 基因特征的患者衍生的有机类化合物单独和与DNA修复抑制剂和 用于治疗卵巢癌的化疗药物。随后，具有最高活性的组合将 在小鼠卵巢异种移植模型中进行评估，包括患者来源的异种移植。总而言之，建议的 研究将提供对复制相关的PARP依赖和HDR途径的机械性见解 在LIGI缺陷细胞中是关键的，开发改进的LIGI抑制物并识别特定的DNA改变 对LIGI抑制剂单独或与DNA损伤剂和/或DNA修复抑制剂联合使用的敏感性 在临床前卵巢癌模型中，为进一步开发LIGI抑制剂作为一种 卵巢癌的新疗法。

PROJECT ABSTRACT Abnormalities in genome maintenance pathways offer an opportunity to develop therapeutic approaches that selectively target cancer cells. Furthermore, because of their proliferative nature, cancer cells are susceptible to replication-associated DNA damage and more dependent upon the pathways that deal with this type of damage. In a recent collaborative study, we found that the levels of DNA ligase I (LigI), the major enzyme joining Okazaki fragments at the replication fork, correlates with outcome and therapy resistance in ovarian cancer. Further, we found that ovarian cancer cells, which have a defect in homology-dependent repair (HDR), are sensitive to LigI inhibition. In preliminary studies we compared the effects of LIG1 deletion with chemical inhibition. In LIG1 null cells, there were elevated levels of XRCC1, the partner protein of LigIII, and PARP2 associated with newly synthesized DNA, consistent with the PARP- and LigIII-dependent back-up pathway that joins Okazaki fragments and enables cells to replicated in the absence of LigI. In contrast, incubation with the LigI inhibitor did not result in increased levels of XRCC1 or PARP2. Instead, there were elevated levels of the Mre11Rad50Nbs1 complex, that initiates the resection step of homology-dependent repair (HDR). In aim 1, we will use a combination of genetic, cell biological and biochemical approaches to determine the mechanism of the HDR pathway induced by LigI inhibition. Since HDR deficiency occurs in both inherited and sporadic forms of ovarian cancer and we have identified LIG1 as a biomarker of outcome and therapy resistance in ovarian cancer, we will examine the activity of the LigI inhibitor alone and in combination with DNA repair inhibitors and/or genotoxic chemotherapeutics in genetically-defined ovarian cancer cell lines. Building upon our existing LigI inhibitors in aim 2, we will use a combination of molecular modeling and medicinal chemistry to design and synthesize putative LigI inhibitors that are predicted to have enhanced potency and selectivity as well as improved pharmacological properties. The activity of the putative LigI inhibitors will be evaluated in parallel biochemical assays with purified human DNA ligases and cell-based assays with genetically-defined ovarian cancer cell lines. In aim 3, we will first examine the activity of existing and new LigI inhibitors for activity in genetically-characterized patient-derived organoids alone and in combination with DNA repair inhibitors and chemotherapeutics used to treat ovarian cancer. Subsequently, combinations with the highest activity will evaluated in mouse xenograft models of ovarian, including patient-derived xenografts. Together, the proposed studies will provide mechanistic insights into replication-associated PARP-dependent and HDR pathways that are critical in LigI deficient cells, develop improved LigI inhibitors and identify specific DNA alterations that confer sensitivity to the LigI inhibitor alone or in combination with DNA damaging agents and/or DNA repair inhibitors in preclinical ovarian cancer models, providing the rationale for the further development of LigI inhibitors as a novel therapeutic for ovarian cancer.