Examining the Role of RNF168 Activity in BRCA1 Mutant Cancers

项目摘要/摘要 我的职业目标是开发一个独立的研究项目，专注于DNA损伤信号及其如何 可用于治疗癌症。我在这里提出的工作是探索DNA损伤信号 BRCA1突变癌症的机制及其对PARP抑制反应的影响。致病BRCA1 突变会增加癌症发展的风险，并损害同源重组(HR)DNA损伤 反应途径。这种HR缺陷使其容易受到DNA损伤剂的攻击，包括PARP抑制剂。 PARP抑制剂的疗效受到耐药性发展的限制，通常是由HR的恢复引起的。在……里面 BRCA1突变体的表达可促进肿瘤、PARP抑制剂耐药性和HR活性的提高 BRCA1蛋白。已经检测到几种类型的截短的低构象BRCA1蛋白，尽管 DNA损伤和HR活性的定位机制尚未建立。最近，我们确定了 野生型BRCA1对DNA损伤的募集可以通过RNF168依赖和非依赖进行 涉及BRCA1环和BRCT结构域的通路。在初步数据中，我们发现RNF168耗尽 对BRCA1突变癌症的部分但不是全部的肿瘤生长有损害作用。此外，我们发现一些癌细胞 HR完全依赖于RNF168途径，而其他的则依赖于相互作用的途径。这些发现 符合转基因小鼠的研究，不同的BRCA1突变等位基因产生不同的表型 在RNF168基因敲除后。在这里，我们探索BRCA1突变特异性差异产生的可能性 招募不同亚型BRCA1蛋白对DNA损伤的能力。我们将使用细胞系，病人- 衍生异种移植和转基因小鼠模型系统，以解决如何 RNF168通路在不同BRCA1的肿瘤进展、DNA修复和PARP抑制反应中的作用 变异的背景。具体来说，我们假设在表达BRCA1环的癌症中RNF168的缺失 含有结构域的亚型蛋白将阻碍肿瘤进展，消除HR活性，并恢复 对PARP抑制剂的敏感性。为了验证这一假设，我们开发了PARP抑制剂敏感和耐药细胞 表达低形态BRCA1蛋白的品系和患者来源的异种移植模型。此外，我们生成了 同基因表达系统以及带有Rnf168和各种BRCA1突变的转基因小鼠。使用这些 模型，我们将检测RNF168基因缺失对肿瘤发生、生长和定位的影响。 BRCA1亚型和其他DNA修复蛋白，以及对PARP抑制的反应。这些研究将提供 从机制上洞察癌症发生的修复过程并评估RNF168的潜在疗效 路径靶向疗法。此外，这项提案的资金将帮助我获得一个独立的 研究人员定位并为未来R01应用奠定基础，以构建可持续的研究计划 专注于DNA修复和癌症生物学。

PROJECT SUMMARY/ABSTRACT My career goal is to develop an independent research program focused on DNA damage signaling and how it can be exploited for the treatment of cancer. The work that I propose here explores DNA damage signaling mechanisms in BRCA1 mutant cancers and the implications for PARP inhibitor response. Pathogenic BRCA1 mutations increase the risk of cancer development and impair the homologous recombination (HR) DNA damage response pathway. This HR defect confers vulnerability to DNA damaging agents, including PARP inhibitors. PARP inhibitor efficacy is limited by the development of resistance, often caused by the restoration of HR. In BRCA1 mutant cancers, PARP inhibitor resistance and HR activity can be promoted by expression of mutant BRCA1 proteins. Several types of truncated hypomorphic BRCA1 proteins have been detected, though the mechanism of localization to DNA damage and HR activity has not been established. Recently, we determined that wild-type BRCA1 recruitment to DNA damage can proceed through RNF168-dependent and -independent pathways involving the BRCA1 RING and BRCT domains. In preliminary data, we found that RNF168 depletion impairs tumor growth for some, but not all, BRCA1 mutant cancers. Additionally, we show that some cancer cells are entirely reliant on the RNF168 pathway for HR, whereas others rely on the reciprocal pathway. These findings are in line with transgenic mouse studies where different Brca1 mutant alleles produced contrasting phenotypes upon RNF168 knockout. Here, we explore the possibility that BRCA1 mutation-specific differences arise from the capacity to recruit various hypomorphic BRCA1 proteins to DNA damage. We will use cell line, patient- derived xenograft, and transgenic mouse model systems to address the fundamental question of how the RNF168 pathway contributes to tumor progression, DNA repair, and PARP inhibitor response in different BRCA1 mutant backgrounds. Specifically, we hypothesize that depletion of RNF168 in cancers expressing BRCA1 RING domain-containing hypomorphic proteins will impair tumor progression, eliminate HR activity, and restore sensitivity to PARP inhibitors. To test this hypothesis, we developed PARP inhibitor sensitive and resistant cell line and patient-derived xenograft models that express hypomorphic BRCA1 proteins. Additionally, we generated isogenic expression systems as well as transgenic mice with Rnf168 and various Brca1 mutations. Using these models, we will examine the effects of genetic depletion of RNF168 on tumorigenesis and growth, localization of BRCA1 hypomorphs and other DNA repair proteins, and response to PARP inhibition. These studies will provide mechanistic insight into the repair processes occurring in cancers and assess the potential efficacy of RNF168 pathway-targeted therapeutics. Moreover, funding of this proposal will help me to secure an independent investigator position and lay the foundation for future R01 applications to build a sustainable research program focused on DNA repair and cancer biology.