Project 2: Targeting DNA replication in BRCA-associated breast cancer

项目总结 BRCA1、BRCA2和PALB2的种系突变易患乳腺癌，也增加了风险 卵巢癌、胰腺癌、前列腺癌和其他癌症。这3个基因的产物构成BRCA1-PALB2- BRCA2轴在DNA损伤反应中发挥重要作用，特别是在DNA双链断裂中 同源重组(HR)修复。此外，BRCA1和BRCA2也与 DNA损伤后DNA复制的调节，因为它们的突变细胞在S体内是有缺陷的 Checkpoint，它在损伤后减缓DNA合成，可能是为了有时间进行DNA修复和 最大限度地减少突变的产生。然而，这两种蛋白质调节DNA的机制 合成仍然难以捉摸，这种不受限制的DNA合成对复制保真度和 DNA损伤后的基因组稳定性仍不清楚。在我们的初步研究中，我们发现BRCA2相互作用 具有重要的DNA复制因子MCM10，并且BRCA2的丢失或BRCA2的中断- MCM10交互导致不受限制的复制分叉速度、减少分叉失速和减少原点触发 在DNA损伤和复制应激之后。此外，我们还发现BRCA1基因的缺失导致了 DNA损伤前后的复制分叉速度。在本提案中，我们将确定BRCA2和 BRCA1调节DNA复制动力学，不受限制的DNA合成对复制保真度和 BRCA和PALB2的治疗反应和复制应激诱导剂作为靶向治疗的可能性 突变的癌症。在目标1中，我们将定义BRCA2和BRCA1在复制调控中的机制 拓扑异构酶抑制剂诱导的复制应激后叉子速度、叉子失速和起火。在目标2中， 我们将设计三重阴性乳腺癌(TNBC)细胞株来评估复制动力学的影响 复制保真度的失调、癌细胞对拓扑异构酶抑制剂的反应和术后复发的可能性 治疗。在目标3中，我们将使用条件基因敲除和同基因小鼠模型来确定 BRCA1、BRCA2和PALB2缺失乳腺肿瘤的复制应激诱导物

PROJECT SUMMARY Germline mutations in BRCA1 and BRCA2 and PALB2 predispose to breast cancer and also increase the risks of ovarian, pancreatic, prostate, and other cancers. The products of the 3 genes constitute a BRCA1-PALB2- BRCA2 axis that plays essential roles in the DNA damage response, particularly in DNA double strand break repair by homologous recombination (HR). In addition, BRCA1 and BRCA2 have also been implicated in the regulation of DNA replication after DNA damage, as their mutant cells are defective in the intra-S phase checkpoint, which slows down DNA synthesis after the damage presumably to allow time for DNA repair and minimize the generation of mutations. However, the mechanisms by which the two proteins regulate DNA synthesis have remained elusive, and the impact of such “unrestrained” DNA synthesis on replication fidelity and genome stability after DNA damage remains unknown. In our preliminary studies, we found that BRCA2 interacts with an essential DNA replication factor, MCM10, and that either loss of BRCA2 or disruption of the BRCA2- MCM10 interaction results in unrestrained replication fork speed, reduced fork stalling, and reduced origin firing after DNA damage and replication stress. Moreover, we also found that loss of BRCA1 leads to reduced replication fork speed both before and after DNA damage. In this proposal, we will determine how BRCA2 and BRCA1 regulate DNA replication kinetics, the impact of unrestrained DNA synthesis on replication fidelity and therapy response, and the potential of replication stress inducers as a targeted therapy for BRCA and PALB2 mutant cancers. In Aim 1, we will define the mechanisms of BRCA2 and BRCA1 in the regulation of replication fork speed, fork stalling and origin firing after replication stress induced by topoisomerase inhibitors. In Aim 2, we will engineer triple negative breast cancer (TNBC) cell lines to assess the impact of replication kinetics dysregulation on replication fidelity, cancer cell response to topoisomerase inhibitors and potential relapse after treatment. In Aim 3, we will use conditional knockout and syngeneic mouse models to determine the efficacy of replication stress inducers on Brca1, Brca2 and Palb2 null mammary tumors.