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

项目摘要 BRCA1、BRCA2和PALB2的生殖系突变易患乳腺癌，也会增加风险。 卵巢癌、胰腺癌、前列腺癌和其他癌症。3个基因的产物构成BRCA1-PALB2- BRCA2轴在DNA损伤反应中起重要作用，特别是在DNA双链断裂中 同源重组（homologous recombination，HR）此外，BRCA1和BRCA2也参与了 DNA损伤后DNA复制的调节，因为它们的突变细胞在S期内有缺陷 检查点，在损伤后减缓DNA合成，可能是为了给DNA修复留出时间， 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 合成仍然是难以捉摸的，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然未知。在我们的初步研究中，我们发现BRCA2与 与一个重要的DNA复制因子，MCM10，和BRCA2的损失或破坏BRCA2- MCM10相互作用导致不受限制的复制叉速度、减少的叉停滞和减少的起点激发 在DNA损伤和复制应激之后。此外，我们还发现，BRCA1的缺失导致 复制叉的速度在DNA损伤之前和之后。在本提案中，我们将确定BRCA2和 BRCA 1调节DNA复制动力学，不受限制的DNA合成对复制保真度的影响， 治疗反应，以及复制应激诱导剂作为BRCA和PALB 2靶向治疗的潜力 突变型癌症在目标1中，我们将定义BRCA 2和BRCA 1在复制调节中的机制 拓扑异构酶抑制剂诱导的复制应激后的叉速度、叉停滞和起点激发。在目标2中， 我们将设计三阴性乳腺癌细胞系，以评估复制动力学的影响， 对复制保真度、癌细胞对拓扑异构酶抑制剂的反应和化疗后潜在复发的影响 治疗在目标3中，我们将使用条件性敲除和同基因小鼠模型来确定 复制应激诱导剂对Brca1、Brca2和Palb2无效乳腺肿瘤的作用。

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.