Targeting DNA Ligase I in Ovarian Cancer

项目摘要 基因组维持途径的缺失为开发治疗方法提供了机会， 选择性地靶向癌细胞。此外，由于癌细胞的增殖性质， 复制相关的DNA损伤，更依赖于处理这种类型的 损害在最近的一项合作研究中，我们发现DNA连接酶I（LigI）的水平， 冈崎片段在复制叉，与卵巢癌的结果和耐药性。 此外，我们发现，卵巢癌细胞，其中有缺陷的同源依赖性修复（HDR）， 对LigI抑制敏感。在初步研究中，我们比较了LIG 1缺失与化学修饰的影响。 抑制作用在LIG 1缺失细胞中，有升高水平的XRCC 1，LigIII β的伴侣蛋白，和PARP 2 与新合成的DNA相关，与PARP和LigIII β依赖性备份途径一致， 连接冈崎片段，使细胞能够在缺乏LigI的情况下复制。与此相反， LigI抑制剂未导致XRCC 1或PARP 2水平升高。相反， Mre 11 Rad 50 Nbs 1复合物，启动同源依赖性修复（HDR）的切除步骤。在目标1中，我们 将使用遗传学，细胞生物学和生物化学方法的组合来确定 由LigI抑制诱导的HDR途径。由于HDR缺陷发生在遗传性和散发性的形式， 我们已经鉴定了LIG 1作为卵巢癌预后和治疗耐药性的生物标志物， 我们将检测单独的LigI抑制剂和与DNA修复抑制剂和/或 基因毒性化疗药物在基因定义的卵巢癌细胞系中的作用。基于现有的LigI 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 合成推定的LigI抑制剂，其被预测具有增强的效力和选择性，以及 改善的药理学性质。将平行评价推定的LigI抑制剂的活性 用纯化的人DNA连接酶进行的生物化学测定和用遗传上确定的卵巢癌细胞进行的基于细胞的测定， 癌细胞系。在目标3中，我们将首先检查现有的和新的LigI抑制剂的活性， 单独和与DNA修复抑制剂组合的遗传表征的患者来源的类器官， 用于治疗卵巢癌的化疗药物。随后，具有最高活性的组合将 在卵巢的小鼠异种移植物模型（包括患者来源的异种移植物）中评价。在一起，拟议的 这些研究将为复制相关的PARP依赖性和HDR途径提供机制性见解， 在LigI缺陷细胞中至关重要，开发改进的LigI抑制剂，并鉴定特异性DNA改变， 对单独的LigI抑制剂或与DNA损伤剂和/或DNA修复抑制剂的组合的敏感性 在临床前卵巢癌模型中，为进一步开发LigI抑制剂作为 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.