Compensatory mechanisms that promote homologous recombination in BRCA1 mutant cancers

项目总结 BRCA1蛋白在同源重组(HR)中发挥作用，这是一种DNA修复过程，使用 未受损的姐妹染色单体对DNA双链断裂(DSB)进行高保真修复。遗传 在小鼠中敲除HR基因表明BRCA1和HR过程对生存至关重要。相比之下， BRCA1野生型等位基因的丢失提供了生长优势，细胞在癌症期间得到了积极的选择 生殖系BRCA1突变患者的启动。在我们的初步分析中，我们表明BRCA1 突变等位基因总是保留在来自生殖系突变患者的癌症中。有趣的是，BRCA1 突变等位基因已被证明在BRCA1缺失背景下延长胚胎存活率，提示部分 拯救全长BRCA1活性。在我们最近发表的研究中，我们表明BRCA1突变的等位基因是 能够在BRCA1突变的癌症中产生截断的蛋白质并促进残留的HR。在这份提案中， 我们将研究BRCA1突变等位基因和HR对BRCA1突变的细胞和肿瘤存活率的重要性 癌症。此外，HR DNA修复缺陷的细胞，如缺乏功能性BRCA1或 BRCA2对铂和PARP抑制(PARPI)高度敏感。然而，新出现的数据表明， PARPI治疗可能只使BRCA1突变携带者的一部分受益。在这项建议的第二部分，我们 将揭示使心率升高到铂和PARPI耐药所需水平的机制。我们证明了 BRCA1185delAG和BRCA1C61G等位基因产生的蛋白质不能与BARD1相互作用，缺乏泛素连接酶 活性，并且只能提供中等的铂和PARPI抗性。我们假设 BRCA1-BARD1泛素连接酶活性对于治疗所需的更强健的HR水平很重要 抵抗。在初步数据中，我们确定RNF7和RNF207是新的泛素调节因子，证明了 PARPI抗性BRCA1185delAG和BRCA1C61G克隆的表达增加并补偿泛素 连接酶缺陷-BRCA1蛋白。我们将操纵泛素连接酶蛋白的表达并测量 DNA修复、PARPI和顺铂的体内外敏感性。导致抗药性的蛋白质将是 评估其在PDX模型和原发肿瘤中的表达。使用这些方法，我们将解决 具体目的如下：1)研究BRCA1突变等位基因在维持HR和肿瘤存活率中的作用； 2)探讨HR促进治疗抵抗的机制。我们的工作最终将 有助于开发预测治疗反应和揭示化疗新靶点的生物标记物 敏化。

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.