Investigating 53BP1 'dephosphorylation' as a critical determinant of PARP

项目摘要 53BP1对于DNA损伤的非同源末端连接(NHEJ)是必不可少的，并受到 可逆的磷酸化。我们最近报道了53BP1在T1609和T1609残基上的去磷酸化 在PP4C-PP4R3磷酸酶复合体的催化下，S1618在其焦点形成区(FFR)是必需的 它的招募在G1期间发生双链断裂(DSB)。独立的证据表明 在癌症的背景下，53BP1对DSB的去磷酸化依赖的招募可以被利用 心理治疗。BRCA1基因突变常见于乳腺癌和卵巢癌，它会损害HR和 使这些肿瘤对PARP抑制剂非常敏感。对PARP抑制剂的反应强烈 依赖于53BP1的功能。事实上，BRCA1突变细胞中53BP1的耗尽可以恢复同源 重组(HR)熟练，并使这些细胞对PARP抑制剂产生抗药性。其次，基因突变 在一例乳腺癌患者中发现了T1609/S1618序列中的53BP1，我们发现 这种突变破坏了53BP1在DNA损伤部位的募集，并诱导了对PARP抑制剂的耐药性。 重要的是，53BP1的功能缺陷(缺失或突变)增强了放射敏感性。因此 由于53BP1功能丧失而对PARP抑制剂产生耐药性的BRCA1突变肿瘤可能会 对放射治疗有反应。根据这些结果，我们假设PP4C-53BP1轴具有显著的 BRCA1突变肿瘤的治疗意义。我们观察到，磷酸化的 PP4R3C末端片段中的Ser840残基是形成PP4R3/53BP1所必需的 很复杂。在目标1中，我们将利用磷酸蛋白质组学方法来检测PP4C/PP4R3的特异性 介导的53BP1去磷酸化。使用体外结合试验和基于细胞的试验来确定 PP4R3与53BP1的相互作用是直接的，或受其他因素的调节。最后使用延时成像技术 研究PP4R3和53BP1在有丝分裂晚期和G1早期相互作用的动力学。我们的预赛 结果表明，CDK5介导的PP4R3上Ser840残基的磷酸化对 PP4R3与53BP1的相互作用。AIM 2将利用几种创新的化学遗传工具来系统地 研究CDK5与PP4/53BP1轴的关系。使用一种非常特定和强大的CDK5 我们将确定CDK5在有丝分裂过程中对PP4R3磷酸化的准确影响。53BP1焦点 在G1中形成，并广泛评估其对DSB修复的影响。此外，蛋白质组学与无偏见的 化学遗传学方法将使我们能够识别参与细胞DNA修复的其他CDK5底物。在……里面 目的鉴定和研究PP4R3-S840F的53BP1基因的零星突变对FFR的影响 CDK5基因突变及其对BRCA1突变型卵巢癌和乳腺癌细胞体外奥拉帕利敏感性的抑制作用 以及在原位植入的小鼠模型中选择的基因。我们将测试是否对奥拉帕利耐药 使用一种复杂的小动物辐照器进行放射治疗。

Project Summary 53BP1 is essential for non-homologous end-joining (NHEJ) of DNA lesions and is tightly regulated by reversible phosphorylation. We recently reported that dephosphorylation of 53BP1 at residues T1609 and S1618 in its foci-forming region (FFR), catalyzed by the PP4C-PP4R3 phosphatase complex, is necessary for its recruitment to double-strand breaks (DSB) during G1. Independent lines of evidence suggest that dephosphorylation-dependent recruitment of 53BP1 to DSB could be leveraged in the context of cancer therapy. Mutations in BRCA1, which are frequently found in breast and ovarian cancers, compromise HR and render these tumors exquisitely sensitive to PARP inhibitors. The response to PARP inhibitors is strongly dependent on the function of 53BP1. In fact, depletion of 53BP1 in BRCA1-mutant cells restores homologous recombination (HR) proficiency and renders these cells resistant to PARP inhibitors. Secondly, a mutation in 53BP1 within the sequence spanningT1609/S1618 was identified in a breast cancer patient and we found that this mutation disrupts 53BP1 recruitment to DNA damage foci, and induces resistance to PARP inhibitors. Importantly, the functional deficiency in 53BP1 (deletion or mutation) enhances radiosensitivity. Therefore BRCA1-mutant tumors that develop resistance to PARP inhibitors due to loss in 53BP1 function are likely to respond to radiotherapy. Based on these results we hypothesize that the PP4C-53BP1 axis has significant therapeutic implications specifically in BRCA1-mutant tumors. We have observed that phosphorylation of the Ser840 residue in a fragment in the C-terminus of PP4R3 is necessary for the formation of PP4R3/53BP1 complex. In Aim 1 we will utilize phosphoproteomic methods to examine the specificity of PP4C/PP4R3 mediated 53BP1 dephosphorylation. Use in vitro binding assays and cell based assays to determine whether the interaction of PP4R3 and 53BP1 is direct, or mediated by other factors. Finally use time-lapse imaging to examine the kinetics of interaction of PP4R3 and 53BP1 during late mitosis and early G1. Our preliminary results suggest that Cdk5 mediated phosphorylation of the Ser840 residue on PP4R3 is critical for the interaction of PP4R3 and 53BP1. Aim 2 will utilize several innovative chemical genetic tools to systematically investigate the connection of Cdk5 with the PP4/53BP1 axis. Using an extremely specific and potent Cdk5 inhibitor, we will determine the precise impact of Cdk5 on PP4R3 phosphorylation during mitosis. 53BP1 foci formation in G1, and broadly assess its impact on DSB repair. Furthermore proteomics coupled to an unbiased chemical genetic approach will allow us to identify other Cdk5 substrates involved in DNA repair in cells. In Aim 3 we will identify and investigate the impact of sporadic mutations in the FFR of 53BP1, a PP4R3-S840F mutation and inhibition of Cdk5 on olaparib sensitivity of BRCA1-mutant ovarian and breast tumor lines in vitro and for selected ones in orthotopically implanted mouse models. We will test whether olaparib-resistance can be overcome by radiation therapy using a sophisticated a small animal irradiator.