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Defining the function of Complex I truncating mutations in cancer

定义复合物 I 截短突变在癌症中的功能

基本信息

批准号：
10563387
负责人：
Payam Gammage
金额：
$ 65.82万
依托单位：
SLOAN-KETTERING INST CAN RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-01-01 至 2027-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10563387
关键词：
Affect Alleles Binding Biochemical Cancer cell line Cell Line Cell physiology Cells Code Colon Carcinoma Colorectal Cancer Complex Computational algorithm Computer Analysis Consumption Cytidine Cytochrome c Reductase Cytosine DNA DNA Binding Domain DNA Sequence Alteration Data Data Set Dependence Development Engineering Enzymes Generations Genes Genetic Genetic Engineering Genetic Induction Genetic Transcription Genomics Germ-Line Mutation Glucose Impairment Induced Mutation Investigation Isotopes Malignant Neoplasms Malignant neoplasm of thyroid Metabolic Metabolic Pathway Metabolism Methods Mitochondria Mitochondrial DNA Modeling Molecular Profiling Mutation Nuclear Outcome Oxidative Phosphorylation Oxygen Consumption Pathogenicity Pathway interactions Pattern Phenotype Physiological Point Mutation Proliferating Proteins RNA Recurrence Renal carcinoma Residual state Sampling Technology Testing Therapeutic Time Tissues Variant analytical method base editing base editor cancer cell cancer genome cell type colon cancer cell line dimer dosage driver mutation experience experimental study genetic manipulation heteroplasmy innovation interdisciplinary approach metabolic phenotype metabolomics mitochondrial DNA mutation molecular phenotype mutant novel novel therapeutic intervention novel therapeutics pressure single cell sequencing single-cell RNA sequencing tool transcriptomics tumor tumor metabolism

项目摘要

PROJECT SUMMARY/ABSTRACT Highly disruptive truncating mutations to protein-coding genes in mitochondrial DNA (mtDNA) affect nearly 10% of all cancers and predominantly arise heteroplasmically, affecting a fraction of the total mtDNA pool. Although decades of investigation into pathogenic mtDNA variants in the germline have established that they profoundly disrupt normal mitochondrial oxidative phosphorylation, the effects of such mutations in cancer cells are largely unknown. The fundamental barrier to rigorous interrogation of mtDNA mutations in cancer cells has been a lack of tools for genetically engineering mtDNA. Recently, a new mtDNA-editing technology pairing TALE binding domains to a DddAtox cytosine base editor (DdCBE) has been successfully used to introduce point mutations into mtDNA, revolutionizing the ability to genetically manipulate mtDNA with high precision. In parallel, our team recently discovered that truncating mtDNA mutations are under strong positive selection in specific genetic contexts (subunits of NADH dehydrogenase/Complex I, “CI”) and cancer lineages (colorectal, kidney, and thyroid cancers), and that the heteroplasmic dosage and transcriptional phenotype of these mutations are readily detectable in single cell sequencing data. These convergent discoveries motivated our team to engineer DdCBEs to introduce truncating mutations to several CI and non-CI mtDNA genes in cell lines, enabling for the first time a functional interrogation of truncating mtDNA mutations in cancer cells. Using these tools, we propose integrative computational/experimental studies to test the overarching hypothesis that CI-truncating mutations produce physiologically significant and therapeutically actionable metabolic changes in tumors. In Aim 1, we will computationally investigate mtDNA mutation patterns across ~100,000 tumor samples, identifying recurrent mutant alleles and co-incident driver mutations in nuclear DNA. In parallel, we will express DdCBEs to model CI- and non-CI truncating mutations in colorectal cancer cell lines, and define the molecular phenotypes conferred by CI truncating mutations using transcriptomic, metabolomic, and isotope tracing experiments. Our Preliminary Data indicates that the phenotype of CI-truncating mutations depends on their heteroplasmic dosage. Thus, in Aim 2 we will use transient expression of DdCBEs to produce isogenic panels of colorectal cancer cell lines at characteristically distinct mutation dosages. Using a combination of single cell and bulk molecular profiling, we will test the hypothesis that CI-truncating mutations rewire tumor cell metabolism towards a pro-proliferative configuration in a dosage-sensitive manner. Finally, hypothesizing that CI-truncating mutations induce genetic dependencies absent in mtDNA-wild-type cells, Aim 3 will use DdCBE-engineered cell line models to identify, validate, and mechanistically study novel synthetic lethalities associated with CI-truncating mutations. The results of these studies will deliver a new, detailed understanding of the function, dosage sensitivity, and therapeutic vulnerability of one of the most common genetic insults in the cancer genome.

项目概要/摘要线粒体 DNA (mtDNA) 中蛋白质编码基因的高度破坏性截短突变影响近 10% 是所有癌症中的一种，并且主要是异质性产生的，影响了线粒体 DNA 总量的一小部分。虽然对种系中致病性 mtDNA 变异的数十年研究表明，它们深刻地影响了破坏正常的线粒体氧化磷酸化，这种突变对癌细胞的影响很大程度上是未知。严格研究癌细胞 mtDNA 突变的根本障碍是缺乏线粒体 DNA 基因工程工具。最近，一种新的线粒体DNA编辑技术与TALE结合配对 DddAtox 胞嘧啶碱基编辑器 (DdCBE) 的结构域已成功用于引入点突变转化为 mtDNA，彻底改变了高精度基因操纵 mtDNA 的能力。与此同时，我们的团队最近发现截短mtDNA突变在特定遗传中处于强正选择之下环境（NADH 脱氢酶/复合物 I，“CI”的亚基）和癌症谱系（结直肠癌、肾癌和甲状腺癌）癌症），并且这些突变的异质剂量和转录表型很容易可在单细胞测序数据中检测到。这些趋同的发现激励我们的团队设计 DdCBE 向细胞系中的多个 CI 和非 CI mtDNA 基因引入截短突变，首次实现对癌细胞中 mtDNA 突变截断的功能性研究。使用这些工具，我们建议综合计算/实验研究来检验 CI 截断突变的总体假设在肿瘤中产生具有生理意义和治疗上可行的代谢变化。在目标 1 中，我们将通过计算研究约 100,000 个肿瘤样本的 mtDNA 突变模式，识别复发性肿瘤突变等位基因和核 DNA 中同时发生的驱动突变。同时，我们将表达 DdCBE 来建模 CI- 和非 CI 截短结直肠癌细胞系突变，并定义赋予的分子表型通过 CI 截断突变，使用转录组学、代谢组学和同位素示踪实验。我们的初步数据表明 CI 截短突变的表型取决于其异质剂量。因此，在目标 2 我们将使用 DdCBE 的瞬时表达来产生结直肠癌细胞系的同基因组特征不同的突变剂量。通过结合单细胞和本体分子分析，我们将检验 CI 截短突变将肿瘤细胞代谢重新连接至促增殖的假设以剂量敏感的方式配置。最后，假设 CI 截短突变会诱导遗传 mtDNA 野生型细胞中不存在依赖关系，Aim 3 将使用 DdCBE 工程细胞系模型来识别，验证并从机制上研究与 CI 截断突变相关的新型合成致死率。这这些研究的结果将对功能、剂量敏感性和癌症基因组中最常见的遗传损伤之一的治疗脆弱性。