Examining the Role of RNF168 Activity in BRCA1 Mutant Cancers

项目总结/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，以及它是如何 可以用来治疗癌症我在这里提出的工作是探索DNA损伤信号 BRCA 1突变型癌症的机制和PARP抑制剂反应的影响。致病性BRCA 1 突变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缺陷赋予DNA损伤剂，包括PARP抑制剂的脆弱性。 PARP抑制剂的功效受到耐药性发展的限制，耐药性的发展通常由HR的恢复引起。 BRCA 1突变型癌症、PARP抑制剂抗性和HR活性可以通过突变型BRCA 1基因的表达来促进。 BRCA 1蛋白。已经检测到几种类型的截短的亚型BRCA 1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 野生型BRCA 1对DNA损伤的募集可以通过RNF 168依赖性和非依赖性 涉及BRCA 1 RING和BRCT结构域的通路。在初步数据中，我们发现RNF 168的消耗 对某些但不是全部BRCA 1突变型癌症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RNF 168通路，而其他依赖于相互通路。这些发现 与转基因小鼠研究一致，其中不同的Brca 1突变等位基因产生了相反的表型 在RNF 168敲除后。在这里，我们探讨BRCA 1突变特异性差异产生的可能性， 招募各种亚型BRCA 1蛋白以损伤DNA的能力。我们会用细胞系，病人- 衍生的异种移植物和转基因小鼠模型系统，以解决 RNF 168通路有助于不同BRCA中的肿瘤进展、DNA修复和PARP抑制剂反应1 变种人背景具体地说，我们假设在表达BRCA 1 RING的癌症中， 含结构域的亚型蛋白将损害肿瘤进展，消除HR活性， 对PARP抑制剂敏感。为了验证这一假设，我们开发了PARP抑制剂敏感和耐药细胞， 细胞系和患者来源的异种移植模型，表达亚型BRCA 1蛋白。此外，我们还生成了 等基因表达系统以及具有Rnf 168和各种Brca 1突变的转基因小鼠。使用这些 模型，我们将研究RNF 168基因缺失对肿瘤发生和生长的影响， BRCA 1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 深入了解癌症中发生的修复过程，并评估RNF 168的潜在疗效 通路靶向治疗。此外，这项建议的资金将有助于我确保一个独立的 研究人员定位并为未来的R 01应用奠定基础，以建立一个可持续的研究计划 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.