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

项目摘要。 高级别浆液性卵巢癌（HGSOC）占卵巢癌死亡人数的80%，原因是 超过一半的患者复发与铂类药物耐药相关。瘤内异质性 (ITH)被认为是抵抗的主要因素。ITH允许不同的表型景观， 这些细胞可以提供某些克隆在抵抗或耐受化疗方面的优势。顺铂，常规 用于HGSOC治疗，形成铂-DNA（Pt-DNA）加合物作为其主要细胞毒性机制。然而，在这方面， Pt-DNA加合物具有致突变性，特别是由于DNA错误掺入不正确的核苷酸 聚合酶β（polβ）和η（polη）与这些加合物相对。尽管有证据表明，polβ，polη，以及 Pt-DNA加合物在顺铂耐药性中发挥作用，这是控制这种诱变的分子机制。 旁路不清楚。此外，HGSOC的特征在于大量基因组的不稳定性， 遗传多样性，并增加获得抗性的可能性。作为最具诱变性的 顺铂进一步增加了这种遗传多样性，并诱导不需要的突变， 新抗性克隆的出现。我假设polβ和polη执行了 顺铂的致突变性调节耐药克隆的从头起源， HGSOC中的电阻。该提案旨在定义使诱变剂能够在细胞中产生的分子机制。 绕过Pt-DNA加合物，并结合，确定顺铂的致突变性如何调节 在顺铂治疗期间获得HGSOC抗性。最近通过DNA条形码追踪谱系 这些技术使得能够在整个治疗过程中对亚克隆结构进行令人兴奋的定量分析， 癌症人群。然而，随后的分析破坏了条形码化的细胞样品，限制了进一步的研究。 COLBERT通过荧光标记的谱系特异性基因表达实现谱系追踪和分离。 记者.我将利用科尔伯特进行一项前所未有的纵向研究， 在整个顺铂处理HGSOC细胞系群体中。该提案将进行基因表达， 在整个治疗过程中对个体从头耐药谱系进行全基因组测序分析，以确定 关键的从头突变，赋予耐药性，并了解如何耐药谱系来获得 时间的阻力。2015年卵巢癌行动会议描述了研究的必要性， 更好地了解克隆多样性和基因组不稳定性有助于获得HGSOC抗性。这 工作将通过独特地结合分子和系统水平的方法来提供表征， 顺铂的致突变性这项研究将提供一个全面的了解顺铂如何未能单独作为 一种细胞毒性药物，并诱导不良的诱变事件，以及这些新生突变如何起源 阻力这项工作将为改善个性化治疗和铂类药物设计提供见解。

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.