Evaluating the mechanism by which the DYNLL1-MRE11 complex regulates DNA end resection and genome stability.

项目总结 BRCA缺乏症与PARPI的合成致死相互作用正在不同的治疗中被利用 临床情况，包括卵巢癌、胰腺癌和前列腺癌)。然而，PARPI已经出现了抵抗 作为BRCA1/2基因缺陷的卵巢癌治疗的一个棘手的临床问题。分析了 独立研究表明BRCA1突变肿瘤中PARPI耐药相关基因丢失 无论是促进NHEJ还是抑制HR，在BRCA1活性缺失的情况下，HR都能部分恢复。 53BP1作为主调控因子出现，为DSB带来多个复合体。失去这些因素中的任何一个 在BRCA1突变细胞中引起PARPI耐药。因此，进一步了解影响经济增长的因素 DNA末端的处理对途径选择至关重要，在癌症生物学中具有巨大的相关性。 我们的实验室此前已经发现，在卵巢癌细胞中，DYNLL1的丢失是一个结构性因素 与53BP1结合，也会导致HR和PARPI抗性增强。我们发现DYNLL1pS88直接结合 结合并抑制Mre11，从而阻断DNA末端切除的启动。然而，如何在 带有Mre11的DYNLL1会削弱其核酸酶活性或其对病灶的募集仍未被发现。此外， 这种相互作用是如何在DNA损伤反应的背景下被调节的，例如， 激酶/磷酸酶(S)调节DYNLL1 S88残基的磷酸化以调节其与DYNLL1的相互作用 需要对Mre11进行调查。 我们在这项应用中的总体目标是确定S调节修复的分子机制 通过进一步了解末端切除蛋白是如何调控的来选择途径。我们的中心假设是 DYNLL1的细胞周期特异性调控促进BRCA1/BARD1介导的MRE11泛素化， 从而便于S期DSB的末端切除。 我们的目的是了解DYNLL1介导末端切除和HR的机制。我们将首先 研究BRCA1和DNA-PKcs之间的动态变化及其对DYNLL1和Mre11活性的调节 在细胞周期的背景下。这些末端切除因子的动态变化及其调控研究 DSB修复在对抗BRCA1突变肿瘤中的PARPI耐药方面具有重要的临床意义。 因此，我们建议确定是否涉及弱化DNA末端的机制的改变 切除可促进高级别浆液性卵巢癌(HGSOC)对PARPI耐药的发展 患者来源的异种移植(PDX)和原发肿瘤。我们期望这些研究将进一步推动我们的 了解末端切除机械是如何管理的，以确保适当的修复途径是 激活了。进一步了解这些末端切除因子以及它们在癌症中是如何失调的可能 引导我们找到对抗PARPI耐药癌症的相关治疗目标。

PROJECT SUMMARY The synthetic lethal interaction of BRCA deficiency with PARPi is being exploited therapeutically in diverse clinical contexts, including ovarian, pancreatic, and prostate cancers). However, PARPi resistance has emerged as a vexing clinical problem for the treatment of BRCA1/2-deficient ovarian cancer carcinomas. Analyses of PARPi resistance in BRCA1-mutant tumors across independent studies suggest that loss of genes involved in either promoting NHEJ or suppressing HR leads to the partial restoration of HR in the absence of BRCA1 activity. 53BP1 emerged as the master regulator that brings multiple complexes to the DSB. Loss of any of these factors causes PARPi resistance in BRCA1 mutant cells. Therefore, further understanding of factors that regulate the processing of DNA ends that are crucial for pathway choice have tremendous relevance in cancer biology. Our lab has previously identified that in ovarian cancer cells, loss of DYNLL1, a factor that is constitutively bound to 53BP1, also results in enhanced HR and PARPi resistance. We found that DYNLL1pS88 directly binds to and inhibits MRE11, thereby blocking the initiation of DNA end resection. However, how the interaction of DYNLL1 with MRE11 impairs its nuclease activity or its recruitment to foci remains unexplored. Furthermore, how this interaction is regulated in the context of the DNA damage response, for example, which kinase/phosphatase(s) regulates the phosphorylation of S88 residue of DYNLL1 to modulate its interaction with MRE11 needs to be investigated. Our overall objectives in this application are to identify the molecular mechanism(s) regulating repair pathway choice by further understanding how end resection proteins are regulated. Our central hypothesis is that cell cycle-specific regulation of DYNLL1 promotes BRCA1/BARD1 mediated ubiquitination of MRE11, thereby facilitating end resection at DSBs in S phase. We aim to understand the mechanism by which DYNLL1 mediates end resection and HR. We will first investigate the dynamics between BRCA1 and DNA-PKcs and how it regulates the DYNLL1 and MRE11 activity in the context of the cell cycle. Investigating the dynamics of these end resecting factors and their regulation in DSB repair bear significant clinical relevance in combating PARPi resistance in BRCA1-mutant tumors. Therefore, we propose to determine whether alterations in mechanisms involved in attenuated DNA end resection may drive development of PARPi resistance in the high grade serous ovarian cancer (HGSOC) using patient derived xenografts (PDXs) and primary tumors. We expect that these studies will further our understanding of how end resection machinery is regulated to ensure that the appropriate repair pathway is activated. Further understanding of these end resection factors and how they are dysregulated in cancer may lead us to a relevant therapeutic target to combat PARPi resistant cancers.