Compensatory mechanisms that promote homologous recombination in BRCA1 mutant cancers

项目摘要 BRCA 1蛋白在同源重组（HR）中发挥作用，这是一种DNA修复过程， 未受损的姐妹染色单体进行DNA双链断裂（DSB）的高保真修复。遗传 在小鼠中敲除HR基因证明Brca 1和HR过程对于存活力是关键的。与此相反， BRCA 1野生型等位基因的缺失提供了生长优势，并且细胞在癌症期间被积极选择。 在生殖系BRCA 1突变的患者中启动。在我们的初步分析中，我们发现BRCA 1 突变等位基因总是保留在来自具有生殖系突变的患者的癌症中。有趣的是，Brca 1 突变等位基因已被证明在Brca 1无效背景下延长胚胎活力，这表明部分 拯救全长Brca 1活性。在我们最近发表的研究中，我们表明BRCA 1突变等位基因是 能够在BRCA 1突变型癌症中产生截短蛋白并促进残留HR。在这一提议中， 我们将研究BRCA 1突变体等位基因和HR对BRCA 1突变体中细胞和肿瘤活力的重要性， 癌的此外，缺乏HR DNA修复的细胞，如缺乏功能性BRCA 1或BRCA 2的细胞， BRCA 2对铂和PARP抑制（PARPi）高度敏感。然而，新出现的数据表明， PARPi疗法可能仅对BRCA 1突变携带者的一部分有益。在本建议的第二部分，我们 将揭示将HR提高到铂和PARPi抗性所需水平的机制。我们证明了 由BRCA 1185 delAG和BRCA 1C 61 G等位基因产生的蛋白不能与BARD 1相互作用，缺乏泛素连接酶 活性，并且仅能够提供中等的铂和PARPi抗性。我们假设 BRCA 1-BARD 1泛素连接酶活性对于治疗所需的更稳健的HR水平很重要 阻力在初步数据中，我们鉴定RNF 7和RNF 207为新的泛素调节剂， PARPi抗性BRCA 1185 delAG和BRCA 1C 61 G克隆中的表达增加，并补偿泛素 连接酶缺陷-BRCA 1蛋白。我们将操纵泛素连接酶蛋白的表达， 体外和体内DNA修复、PARPi和顺铂敏感性。有助于抵抗的蛋白质将被 评估PDX模型以及原发性肿瘤中的表达。使用这些方法，我们将解决 以下具体目的：1）研究BRCA 1突变等位基因在维持HR和癌症存活中的作用; （2）研究促进HR的机制对治疗抵抗的作用。我们的工作最终能够 有助于开发生物标志物，预测治疗反应并揭示化疗的新靶点， 致敏

PROJECT SUMMARY The BRCA1 protein functions in homologous recombination (HR), a DNA repair process that uses the undamaged sister chromatid to carry out high fidelity repair of DNA double strand breaks (DSBs). Genetic knockout of HR genes in mice demonstrated that Brca1 and the process of HR are critical for viability. In contrast, loss of the BRCA1 wild-type allele provides a growth advantage and cells are positively selected during cancer initiation in patients with germline BRCA1 mutations. In our preliminary analyses, we show that the BRCA1 mutant allele is invariably retained in cancers derived from patients with germline mutations. Interestingly, Brca1 mutant alleles have been shown to prolong embryonic viability in Brca1 null backgrounds, suggesting partial rescue of full-length Brca1 activity. In our recently published studies, we show that BRCA1 mutant alleles are capable of generating truncated proteins and promote residual HR in BRCA1 mutant cancers. In this proposal, we will investigate the importance of BRCA1 mutant alleles and HR for cell and tumor viability in BRCA1 mutant cancers. Furthermore, cells that are deficient in HR DNA repair, such as those lacking functional BRCA1 or BRCA2, are highly sensitive to platinum and PARP inhibition (PARPi). However, emerging data indicate that PARPi therapy may benefit only a subset of BRCA1 mutation carriers. In the second part of this proposal, we will uncover mechanisms that elevate HR to a level required for platinum and PARPi resistance. We show that proteins generated from BRCA1185delAG and BRCA1C61G alleles fail to interact with BARD1, lack ubiquitin ligase activity, and are only capable of providing moderate platinum and PARPi resistance. We hypothesize that BRCA1-BARD1 ubiquitin ligase activity is important for more robust levels of HR that is necessary for therapy resistance. In preliminary data, we identified RNF7 and RNF207 as novel ubiquitin regulators that demonstrated increased expression in PARPi resistant BRCA1185delAG and BRCA1C61G clones, and compensate for ubiquitin ligase deficient-BRCA1 proteins. We will manipulate the expression of ubiquitin ligase proteins and measure DNA repair, PARPi and cisplatin sensitivity in vitro and in vivo. Proteins that contribute to resistance will be assessed for expression in PDX models as well as primary tumors. Using these approaches, we will address the following Specific Aims: 1) investigate the role of BRCA1 mutant alleles in maintaining HR and cancer viability; and 2) investigate HR-promoting mechanisms contributing to therapy resistance. Our work will ultimately contribute to the development of biomarkers that predict therapy response and reveal novel targets for chemo- sensitization.