A multi-scale analysis of the mutagenicity of cisplatin and its modulation of acquired chemoresistance in high-grade serous ovarian cancer

项目总结。 高级别浆液性卵巢癌(HGSOC)占卵巢癌死亡的80%，原因是 超过一半的患者复发与铂类药物耐药有关。瘤内异质性 (Ith)被认为是抵抗的主要贡献者。ITH允许不同的表型景观在 在抵抗或耐受化疗方面可能提供某些克隆优势的细胞。顺铂，常规用药 用于HGSOC的治疗，形成铂-DNA(铂-DNA)加合物作为其主要的细胞毒性机制。然而， PT-DNA加合物是诱变的，特别是由于DNA错误地结合了错误的核苷酸 与这些加合物相反的聚合酶β(POLβ)和η(POLη)。尽管有证据表明POLβ、POLη和绕过 PT-DNA加合物在顺铂耐药中发挥作用，这是控制这种突变的分子机制 旁路手术尚不清楚。此外，HGSOC的特点是巨大的基因组不稳定导致肿瘤内 基因多样性，增加了获得抗药性的可能性。作为世界上最具诱变性的 药物，顺铂进一步增加这种遗传多样性，并诱导不必要的突变，可能会导致 出现了新的抗病毒克隆。我假设Pollβ和Pollη执行 顺铂和顺铂的致突变性调节繁殖的抗性克隆的从头开始 HGSOC中的抗性。这项提议试图定义使诱变剂能够 绕过铂-DNA加合物，并结合确定顺铂的致突变性如何调节 顺铂治疗期间HGSOC耐药性的获得。通过DNA条形码进行最近的血统追踪 技术使亚克隆结构的令人兴奋的定量分析在整个治疗过程中成为可能 癌症人口。然而，随后的分析破坏了条形码细胞样本，限制了进一步的研究。 科尔伯特通过一种特定于谱系的荧光基因表达来实现谱系追踪和分离 记者。我将利用科尔伯特对新出现的抵抗力进行一项史无前例的纵向研究 顺铂处理HGSOC细胞株的整个过程。这项提案将进行基因表达和 在整个治疗过程中对个体新生耐药谱系进行全基因组测序分析，以确定 赋予抗药性的关键从头突变，并了解抗药性谱系是如何获得的 随着时间的推移产生抵抗力。2015年卵巢癌行动会议描述了研究的必要性，目的是 更好地了解克隆多样性和基因组不稳定对获得性HGSOC抵抗的贡献。这 这项工作将通过独特地联合分子和系统水平的方法来解决这个问题，以提供表征 顺铂的致突变性。这项研究将提供一个整体的理解，为什么顺铂不能单独作为 一种细胞毒性药物，并诱导不良的诱变事件，以及这些从头突变可能是如何起源的 抵抗。这项工作将为改进个性化治疗和白金药物设计提供见解。

PROJECT SUMMARY. High-grade serous ovarian cancer (HGSOC) accounts for up to 80% of ovarian cancer deaths, due to recurrence in over half of patients associated with platinum-based chemoresistance. Intratumor heterogeneity (ITH) is thought to be the main contributor in resistance. ITH permits diverse phenotypic landscapes among cells that may provide certain clones advantages in resisting or tolerating chemotherapy. Cisplatin, routinely used in HGSOC treatment, forms platinum-DNA (Pt-DNA) adducts as its main cytotoxic mechanism. However, Pt-DNA adducts are mutagenic, particularly due to the misincorporation of incorrect nucleotides by DNA polymerases β (polβ) and η (polη) opposite these adducts. Despite evidence that polβ, polη, and the bypass of Pt-DNA adducts play roles in cisplatin resistance, the molecular mechanisms that govern this mutagenic bypass are unclear. In addition, HGSOC is characterized by vast genome instability that causes intratumor genetic diversity and augments the possibilities for the acquisition of resistance. As one of the most mutagenic drugs, cisplatin further increases this genetic diversity and induces unwanted mutations that may elicit the emergence of de novo resistant clones. I hypothesize that polβ and polη execute the mutagenic events of cisplatin, and the mutagenicity of cisplatin modulates the de novo origination of resistant clones that propagate resistance in HGSOC. This proposal seeks to define the molecular mechanisms that enable the mutagenic bypass of Pt-DNA adducts, and in conjunction, determine how the mutagenicity of cisplatin modulates the acquisition of HGSOC resistance during cisplatin treatment. Recent lineage tracing through DNA barcoding technologies have enabled exciting quantitative analyses of subclonal architecture throughout treatment of cancer populations. However, subsequent analyses destroy barcoded cellular samples, limiting further studies. COLBERT enables both lineage tracing and isolation through lineage-specific gene expression of a fluorescent reporter. I will employ COLBERT in an unprecedented longitudinal study of de novo resistance that emerges throughout cisplatin treatment of HGSOC cell line populations. This proposal will perform gene expression and whole genome sequencing analyses of individual de novo resistant lineages throughout treatment to determine key de novo mutations that confer resistance, and understand how resistant lineages came to acquire resistance over time. The 2015 Ovarian Cancer Action meeting described the need for studies that aim to better understand clonal diversity and genome instability contributing to acquired HGSOC resistance. This work will address this by uniquely uniting molecular and systems level approaches to provide characterizations of cisplatin’s mutagenicity. This study will provide a holistic understanding of how cisplatin fails to act solely as a cytotoxic drug and induces undesirable mutagenic events, and how these de novo mutations may originate resistance. This work will provide insights into improving personalized therapies and platinum drug design.