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.

项目摘要/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％在所有癌症中，主要出现异常出现，影响了总mtDNA池的一部分。虽然对种系中致病性mtDNA变异的数十年来，它们已经深刻地研究了它们破坏正常的线粒体氧化磷酸化，这种突变在癌细胞中的作用在很大程度上是未知。严格询问癌细胞中mtDNA突变的基本障碍一直缺乏基因工程mtDNA的工具。最近，一种新的MTDNA编辑技术配对故事绑定 DDDATOX胞嘧啶碱基编辑器（DDCBE）的域已成功用于引入点突变进入mtDNA，彻底改变了以高精度来操纵mtDNA的能力。同时，我们的团队最近发现，截断的mtDNA突变在特定的通用性中处于强阳性选择下上下文（NADH脱氢酶/复合物I，“ CI”的亚基）和癌症（结直肠，肾脏和甲状腺癌症），并且这些突变的异质剂量和转录表型很容易在单细胞测序数据中可检测。这些融合的发现将我们的团队与工程师DDCBES融合在一起将截短突变引入细胞系中的几个CI和非CI mtDNA基因，这是首次使对癌细胞中截短的mtDNA突变的功能询问。使用这些工具，我们建议综合计算/实验研究，以检验总体假设，即CI-TRUSS truncations突变在肿瘤中产生具有物理意义和热可起作用的代谢变化。在AIM 1中，我们将计算研究跨约100,000个肿瘤样品的mtDNA突变模式，鉴定了经常性核DNA中的突变等位基因和同伴驱动突变。同时，我们将表达DDCBES以建模CI- 结直肠癌细胞系中的非CI截断突变，并定义了分子表型。通过使用转录组，代谢组和同位素追踪实验截断突变。我们的初步数据表明，CI-trungust突变的表型取决于其异质剂量。那，在 AIM 2我们将使用DDCBE的瞬态表达来生成结直肠癌细胞系的等源图。特征上不同的突变剂量。结合单细胞和散装分子分析，我们将检验以下假设，即CI-C-trust突变将肿瘤细胞代谢重新促成促肿瘤的假设以剂量敏感的方式进行配置。最后，假设C-trunccation突变会诱导通用 MTDNA野生型细胞中不存在依赖性，AIM 3将使用DDCBE工程细胞系模型来识别，验证并机械研究与CI截断突变有关的新型合成致死性。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。