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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.

项目总结/文摘