Identifying recurrent driver mutations in skin cancers by targeted UV damage sequencing

通过靶向紫外线损伤测序识别皮肤癌中的复发性驱动突变

• 批准号: 10645759
• 负责人: John J Wyrick
• 金额: $ 25.66万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-10 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10645759
• 关键词: Adenine; Affect; BRAF gene; Binding; Binding Sites; Bioinformatics; Cancer Etiology; Cancer cell line; Cell Line; Cell Proliferation; Cell Survival; Cells; Codon Nucleotides; DNA; DNA Binding; DNA Damage; DNA mapping; Data; Data Set; Early Promoters; Elements; Excision Repair; Exons; Exposure to; Family; Frequencies; Gene Proteins; Genes; Genome; Growth; Human; Human Genome; In Vitro; Individual; Lesion; Link; Malignant Neoplasms; Malignant neoplasm of urinary bladder; Maps; Measures; Methods; Molecular; Mutate; Mutation; Nucleotide Excision Repair; Nucleotides; Oncogenic; Pathway interactions; Patients; Phosphotransferases; Proteins; Protocols documentation; Publishing; Pyrimidine; Pyrimidine Dimers; Recurrence; Resolution; Role; Sampling; Site; Skin; Skin Cancer; Testing; Thymine; Triage; Tumor-Derived; UV Radiation Exposure; UV induced; Ultraviolet Rays; Untranslated RNA; Yeasts; base; bioinformatics pipeline; cancer cell; cancer genome; cancer type; candidate identification; carcinogenesis; carcinogenicity; driver mutation; endonuclease; genome sequencing; genomic locus; keratinocyte; melanocyte; melanoma; melanomagenesis; repaired; response; transcription factor; tumor; ultraviolet; ultraviolet damage; ultraviolet irradiation

Abstract: Skin cancers, such as melanoma, are among the most mutated human cancers due to the mutagenic action of ultraviolet (UV) light. The high mutation load in skin cancers makes it challenging to identify driver mutations in these tumors, particularly in non-coding DNA. For example, recent genome sequencing efforts have identified dozens of recurrent exon mutations and hundreds of recurrent non-coding mutations in melanomas. However, current evidence suggests that only a subset of these are under carcinogenic selection and contribute to melanomagenesis. Many recurrent mutation sites are found in the DNA-binding sites of E26 transformation- specific (ETS) family of transcription factors (TFs). We and others have recently shown that ETS and other TFs induce high levels of UV damage, both in UV-exposed cells and in vitro, which can potentially explain the presence of recurrent mutations at their binding sites. To test this hypothesis, we have developed new capture- sequencing methods for mapping UV damage at targeted sites in the human genome. We propose to use these methods to map DNA damage in UV-irradiated skin cells at sites of recurrent mutations in skin cancers. The overall objective of this proposal is to use targeted UV damage sequencing to distinguish between recurrent mutations that are simply caused by elevated levels of UV damage (often at TF binding sites) from those that cannot be explained by UV damage, and therefore are more likely to be oncogenic mutations. In Aim I, we will use the CPD-capture-seq method to map the formation and repair of UV-induced cyclobutane pyrimidine dimers (CPDs) in UV-irradiated primary skin cells and cancer cell lines. In parallel, will develop a new method, known as UVDE-capture-seq, to map less common 6-4 photoproducts (6-4PPs) and atypical photoproducts at sites of recurrent mutations in UV irradiated cells. In Aim II, we will develop a bioinformatics pipeline to use these capture sequencing data sets to identify candidate non-coding driver mutations by triaging recurrent mutations associated with elevated UV damage. While it is possible that a few of the recurrent mutations associated with elevated UV damage could contribute to carcinogenesis, multiple lines of evidence suggest that the vast majority of these are passenger mutations. In parallel, we will functionally characterize candidate non-coding driver mutations identified from our preliminary CPD-capture-seq data. We will also determine whether UV damage induction due to transient ETS TF binding causes a subset of recurrent exon mutations in melanoma and other skin cancers. Successful completion of these aims will provide a new method for identifying driver mutations in skin cancers, particularly in non-coding DNA. Importantly, the methods developed in this proposal could be adapted to analyze different classes of DNA damage associated with recurrent mutation sites in other cancer types.

摘要： 皮肤癌，例如黑素瘤，是由于以下物质的诱变作用而突变最多的人类癌症之一： 紫外（UV）光。皮肤癌中的高突变负荷使得识别皮肤癌中的驱动突变具有挑战性。 这些肿瘤，特别是非编码DNA。例如，最近的基因组测序工作已经确定， 黑色素瘤中有几十种复发性外显子突变和几百种复发性非编码突变。然而，在这方面， 目前的证据表明，只有一个子集，这些是在致癌选择，并有助于 黑色素瘤形成在E26转化的DNA结合位点发现了许多重复突变位点- 转录因子（TF）的特异性（ETS）家族。我们和其他人最近表明，ETS和其他TF 诱导高水平的紫外线损伤，无论是在紫外线暴露的细胞和体外，这可以潜在地解释 在其结合位点存在复发性突变。为了验证这一假设，我们开发了新的捕获- 用于在人类基因组中的靶位点绘制UV损伤的测序方法。我们建议使用 这些方法用于绘制皮肤癌复发突变位点处紫外线照射皮肤细胞中的DNA损伤。 该提案的总体目标是使用有针对性的紫外线损伤测序来区分 复发性突变仅仅是由紫外线损伤水平升高引起的（通常在TF结合位点）， 那些不能用紫外线损伤解释的，因此更可能是致癌突变。在 目的一、利用CPD捕获序列方法研究紫外光诱导的环丁烷的形成和修复 嘧啶二聚体（CPD）在UV照射的原代皮肤细胞和癌细胞系中的作用。与此同时，将开发一个 一种新的方法，称为UVDE-capture-seq，用于绘制不太常见的6-4光产物（6- 4PP）和非典型的 在紫外线照射的细胞中的复发突变位点的光产物。在目标II中，我们将开发一个生物信息学 使用这些捕获测序数据集来鉴定候选非编码驱动突变， 筛选与紫外线损伤升高相关的复发性突变。虽然有可能是一些 与紫外线损伤升高相关的复发性突变可能有助于致癌作用， 有证据表明，绝大多数是乘客突变。与此同时，我们将在功能上 表征从我们的初步CPD-捕获-seq数据中鉴定的候选非编码驱动突变。我们 还将确定由于瞬时ETS TF结合引起的UV损伤诱导是否会导致 黑色素瘤和其他皮肤癌中的复发性外显子突变。成功实现这些目标将 提供了一种新的方法，用于识别皮肤癌中的驱动突变，特别是在非编码DNA中。 重要的是，该提案中开发的方法可以适用于分析不同类型的DNA 与其他癌症类型中的复发突变位点相关的损伤。