Studies of DNA interstrand crosslink repair to improve crosslinking drug therapie

描述(申请人提供)：产生DNA交联物的药物是最有效的癌症化疗药物之一。这些药物属于几类双功能分子，可以产生DNA单加合物、链内和链间交联物，是许多恶性肿瘤的标准护理。链间交联(ICL)阻止了许多DNA交易，被认为是大多数交联药物疗效的细胞毒性损伤。然而，它们的疗效因细胞类型或患者而异。与这些化合物相关的主要挑战有两个：1)剂量限制毒性，主要是在血液中；2)获得性耐药性。这项建议的目的是描述ICL修复的机制，以更好地理解并最终改进以交联剂为基础的化疗的作用模式。ICL在S期内和期外进行修复。复制非依赖性ICL修复(RIR)对于处理过的哺乳动物细胞的存活是非常重要的。拟议的研究专门讨论了在没有其他DNA损伤的情况下ICL修复的机制，因此专注于由交联剂引发的最具临床相关性的修复反应。具体地说，我们建议鉴定RIR中涉及的核酸酶(Aim 1)和DNA聚合酶(Aim 2)。最后，我们建议评估复制依赖和非依赖ICL修复对交联剂疗效的影响。我们假设，靶向这些因子可以增加肿瘤细胞对交联剂的敏感性，减少耐药性的发生。我们的方法将结合无细胞提取物中的生物化学与正常细胞和肿瘤细胞中创新的ICL修复分析。我们预计，更好地了解ICL修复的分子机制将有助于阐明ICL损伤对交联剂毒性的贡献，以及DNA修复对交联剂治疗耐药性的影响。

DESCRIPTION (provided by applicant): Drugs that generate DNA crosslinks are among the most effective cancer chemotherapeutic agents. These drugs fall in several classes of bi-functional molecules that generate DNA mono-adducts, intrastrand and interstrand crosslinks and are the standard of care for many malignancies. Interstrand crosslinks (ICLs) block many DNA transactions and are thought to be the cytotoxic lesions responsible for most crosslinking drug efficacy. Their efficacy however, varies among cell types or patients. There are two major challenges associated with these compounds: 1) dose-limiting toxicity, primarily in the blood, and 2) acquired resistance. The goal of this proposal is to characterize the mechanisms of ICL repair to better understand and ultimately improve the mode of action of crosslinking agent-based chemotherapy. ICLs are repaired during and outside of S-phase. Replication-independent ICL repair (RIR) is robust and critical for survival in treated mammalian cells. The proposed studies specifically address the mechanism of ICL repair in the absence of other DNA lesions and therefore focus on the most clinically relevant repair reaction triggered by crosslinking drugs. Specifically, we propose to identify the nucleases (Aim 1) and DNA polymerases (Aim 2) involved in RIR. Finally, we propose to evaluate the impact of replication-dependent and -independent ICL repair on crosslinking drugs efficacy. We hypothesize that targeting these factors could increase the sensitivity of tumor cells to crosslinking agents and reduce the incidence of resistance. Our approach will combine biochemistry in cell-free extracts with innovative ICL repair assays in normal and tumor cells. We anticipate that a better understanding of the molecular mechanisms of ICL repair will shed light on the contribution of ICL lesions to crosslinking drug toxicity and on the impact of DNA repair on resistance to crosslinking therapy.