53BP1-Mediated Regulation of DNA Repair and Chemoresistance

描述（由申请人提供）：缺乏功能性BRCA 1的癌细胞在HDR方面存在严重缺陷，导致基因组不稳定，并使其对PARP抑制剂（PARPi）和链间交联剂（ICL）非常敏感。人们对使用这些药物治疗更广泛的具有DNA修复途径功能缺陷的散发性癌症非常感兴趣。然而，即使是BRCA 1突变型癌症也会迅速对PARPi和ICL产生耐药性，导致肿瘤进展。了解具有DNA修复缺陷的癌症中化疗耐药性发展的分子机制对于开发克服耐药性和改善这些癌症患者结局的方法至关重要。我们最近发现了BRCA 1突变细胞中一种意想不到的化学抗性机制。DNA修复和检查点蛋白53 BP 1的丢失逆转了BRCA 1突变细胞中存在的DNA修复缺陷，并诱导对PARPi和顺铂的抗性。遗传性BRCA 1相关乳腺癌和散发性“BRCA 1样”乳腺癌的亚组均显示53 BP 1表达缺失。此外，53 BP 1的缺失与这些癌症的不良临床结果相关。我们假设53 BP 1功能的异常将减轻BRCA 1突变型和“BRCA 1样”癌症中存在的同源介导的DNA修复缺陷，并导致化疗耐药性。为了研究这一假设，我们将：1）确定维持BRCA 1突变细胞中存在的HDR缺陷所需的53 BP 1的功能元件。2)确定53 BP 1的相互作用配偶体在调节BRCA 1突变细胞中的DNA修复缺陷中的作用，以及3）确定散发性癌症中53 BP 1的获得性缺失是否是HDR中潜在缺陷的补偿突变。前两个目标将通过分析BRCA 1中具有临床相关无效突变的遗传定义的小鼠乳腺癌细胞系来实现。这些细胞系中的几种通过53 BP 1中的体细胞突变获得了化学抗性。53 BP 1的上游和下游的DNA因子在调节Brca 1-/-细胞系中的DNA修复表型中的作用将通过使用特异性DNA修复测定来仔细定义和验证。目标3将集中在53 BP 1在调节BRCA 1基因完整但功能受损的更常见的散发性乳腺癌中的DNA修复表型中的作用。在这里，将系统地分析被鉴定为具有53 BP 1功能缺陷的人类基底样细胞系，以确定53 BP 1功能障碍的分子机制，以及其对DNA修复和化学抗性的影响。将在临床注释的人类癌症标本集中验证细胞系中的任何发现。通过这种方法，我们将对53 BP 1功能如何影响DNA修复表型以及遗传性BRCA 1突变体和散发性“BRCA 1样”癌症对PARPi和ICL化疗药物的敏感性有新的认识。这将最终导致克服耐药性的新方法，并专门针对这些预后不良的癌症。

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.