53BP1-Mediated Regulation of DNA Repair and Chemoresistance

描述(申请人提供)：缺乏功能性BRCA1的癌细胞在HDR中有一个严重的缺陷，导致基因组不稳定，并使它们对PARP抑制剂(PARPI)和链间交联物(ICL)非常敏感。人们对使用这些药物治疗具有DNA修复途径功能缺陷的更大范围的散发性癌症非常感兴趣。然而，即使是BRCA1突变的癌症也会迅速对PARPI和ICL产生耐药性，从而导致肿瘤进展。了解在DNA修复存在明确缺陷的癌症中发生化疗耐药的分子机制对于开发克服耐药的方法并改善这些癌症患者的预后至关重要。我们最近在BRCA1突变细胞中发现了一种意想不到的化疗耐药机制。DNA修复和检查点蛋白53BP1的丢失逆转了BRCA1突变细胞中存在的DNA修复缺陷，并诱导了对PARPI和顺铂的耐药性。遗传性BRCA1相关乳腺癌和散发性“BRCA1样”乳腺癌的亚群均显示53BP1表达缺失。此外，在这些癌症中，53BP1的丢失与不良的临床结果有关。我们推测，53BP1功能的异常将缓解BRCA1突变和BRCA1样癌中存在的同源介导的DNA修复缺陷，并导致化疗耐药。为了研究这一假设，我们将：1)确定维持BRCA1突变细胞中存在的HDR缺陷所需的53BP1的功能元件。2)确定53BP1的相互作用伙伴在调节BRCA1突变细胞DNA修复缺陷中的作用；3)确定散发性癌症中53BP1的获得性丢失是否是HDR潜在缺陷的代偿性突变。前两个目标将通过分析具有临床相关的BRCA1零突变的基因定义的小鼠乳腺癌细胞系来实现。其中一些细胞系通过53BP1的体细胞突变获得了化疗耐药性。53BP1上游和下游的DNA因子在调节BRCA1-/-细胞系DNA修复表型中的作用将通过使用特异的DNA修复分析来仔细定义和验证。目的3将集中于53BP1在更常见的散发性乳腺癌中调节DNA修复表型的作用，在这些散发性乳腺癌中，BRCA1基因完整但功能受损。在这里，我们将系统地分析被证实存在53BP1功能缺陷的人基底样细胞系，以确定53BP1功能障碍的分子机制，以及它对DNA修复和化疗耐药的影响。细胞系中的任何发现都将在带有临床注释的人类癌症样本集上得到验证。通过这种方法，我们将对53BP1功能如何影响遗传性BRCA1突变和散发性BRCA1样癌的DNA修复表型和对PARPI和ICL化疗药物的敏感性有一个新的理解。这最终将导致克服耐药性的新方法，并专门针对这些预后较差的癌症。

DESCRIPTION (provided by applicant): Cancer cells lacking functional BRCA1 have a profound defect in HDR that leads to genomic instability and renders them exquisitely sensitive to PARP inhibitors (PARPi) and interstrand crosslinkers (ICLs). There is great interest in using these agents to treat a larger spectrum of sporadic cancers with functional defects in DNA repair pathways. However, even BRCA1-mutant cancers can rapidly develop resistance to PARPi and ICLs leading to tumor progression. Understanding the molecular mechanism underlying the development of chemoresistance in cancers with defined defects in DNA repair will be critical for developing methods to overcome resistance and improve the outcome of patients with these cancers. We have recently identified an unexpected mechanism of chemo-resistance in BRCA1-mutant cells. Loss of the DNA- repair and checkpoint protein 53BP1 reverses the DNA repair defect present in BRCA1-mutant cells and induces resistance to both PARPi and cisplatin. Subsets of both hereditary BRCA1-associated breast cancers and sporadic "BRCA1-like" breast cancers show loss of 53BP1 expression. Moreover, loss of 53BP1 is associated with poor clinical outcome in these cancers. We hypothesize that abnormalities in 53BP1 function will alleviate the defect in homology-mediated DNA repair present in BRCA1-mutant and "BRCA1-like" cancers, and lead to chemo-resistance. To investigate this hypothesis we will: 1) Determine the functional elements of 53BP1 required to maintain the HDR defect present in BRCA1-mutant cells. 2) Determine the role of interacting partners of 53BP1 in regulating the DNA repair defect in BRCA1-mutant cells and 3) Determine whether or not acquired loss of 53BP1 in sporadic cancers is a compensating mutation for an underlying defect in HDR. The first two aims will be achieved through the analysis of genetically defined sets of murine breast cancer cell lines with clinically relevant null mutations in BRCA1. Several of these cell lines hav acquired chemo-resistance through somatic mutations in 53BP1. The role of DNA factors upstream and downstream of 53BP1 in regulating the DNA repair phenotype in Brca1-/- cell lines will be carefully defined and validated through use of specific DNA repair assays. Aim 3 will focus on the role of 53BP1 in regulating the DNA repair phenotype in the more common sporadic breast cancers in which BRCA1 is genetically intact but functionally impaired. Here human basal-like cell lines identified to have defects in 53BP1 function will be systematically analyzed to determine the molecular mechanism of 53BP1 dysfunction, and its effect on DNA repair and chemo-resistance. Any findings in cell lines will be validated in clinically annotated sets of human cancer specimens. By this approach we will gain a new understanding of how 53BP1 function impacts the DNA repair phenotype and sensitivity of both hereditary BRCA1-mutant and sporadic "BRCA1-like" cancers to PARPi and ICL chemotherapeutic agents. This will ultimately lead to new approaches to overcome resistance and specifically target these poor-prognosis cancers.