Studies of DNA interstrand crosslink repair to improve crosslinking drug therapie

描述（由申请人提供）：产生DNA交联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，它们产生DNA单加合物、链内和链间交联，是许多恶性肿瘤的标准治疗药物。链间交联（ICLs）阻断了许多DNA交易，被认为是导致大多数交联药物疗效的细胞毒性病变。然而，它们的功效因细胞类型或患者而异。与这些化合物相关的两个主要挑战是：1)剂量限制性毒性，主要是血液毒性；2)获得性耐药性。本提案的目的是表征ICL修复的机制，以更好地理解并最终改进基于交联剂的化疗的作用模式。在s期和s期外，ICLs都得到了修复。复制非依赖性ICL修复（RIR）在处理过的哺乳动物细胞中是稳健且对存活至关重要的。拟议的研究专门研究了在没有其他DNA损伤的情况下ICL修复的机制，因此重点关注交联药物引发的最具临床相关性的修复反应。具体来说，我们建议鉴定参与RIR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性和非依赖性ICL修复对交联药物疗效的影响。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。我们的方法将结合生物化学的无细胞提取物和创新的ICL修复测定在正常和肿瘤细胞。我们预计，对ICL修复的分子机制的更好理解将有助于阐明ICL病变对交联药物毒性的贡献，以及DNA修复对交联治疗耐药性的影响。

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. PUBLIC HEALTH RELEVANCE: The proposed research is highly relevant to public health. A better understanding of the enzymes responsible for DNA interstrand crosslink (ICL) repair is warranted to unravel the mechanism of action of drugs that generate ICLs work. It will also provide insight into why these drugs, which are routinely used in chemotherapeutic protocols, have variable efficacy among cell types or patients. It can help explain how resistance to these drugs arises. Finally, the proposed research will identify potential targets to sensitize cells and to prevent resistance to crosslinking drugs.