Defining the function of Complex I truncating mutations in cancer

项目总结/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% 所有的癌症和主要出现异质性，影响总mtDNA池的一部分。虽然 几十年来对生殖系中致病性mtDNA变异的研究已经证实， 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主要是 未知对癌细胞中mtDNA突变进行严格研究的根本障碍是缺乏 基因工程mtDNA的工具。最近，一种新的mtDNA编辑技术配对TALE结合， DddAtox胞嘧啶碱基编辑器（DdCBE）的结构域已成功用于引入点突变 这是一个革命性的技术，可以高精度地对线粒体DNA进行基因操作。同时，我们的团队 最近发现，mtDNA截短突变在特定的遗传背景下受到强烈的正选择， 背景（NADH脱氢酶/复合物I的亚基，“Cl”）和癌症谱系（结肠直肠、肾脏和甲状腺）的差异。 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到。这些趋同的发现促使我们的团队设计DdCBE 将截短突变引入细胞系中的几个CI和非CI mtDNA基因，首次使 在癌细胞中截断mtDNA突变的功能性询问。利用这些工具，我们建议 综合计算/实验研究，以测试总体假设，CI截短突变 在肿瘤中产生生理学上显著的和治疗上可行的代谢变化。在目标1中，我们 通过计算研究约100，000个肿瘤样本的mtDNA突变模式， 突变等位基因和核DNA中的一致驱动突变。并行地，我们将DdCBE表示为模型CI-1。 和非C1截短突变，并定义赋予的分子表型 通过使用转录组学、代谢组学和同位素示踪实验的CI截短突变。我们的初步 数据表明，CI截短突变的表型取决于它们的异质剂量。因此在 目的2：我们将使用DdCBE的瞬时表达来产生大肠癌细胞系的等基因组， 独特的突变剂量使用单细胞和批量分子分析的组合，我们 将检验CI截短突变将肿瘤细胞代谢重新导向促增殖的假设。 以剂量敏感的方式配置。最后，假设CI截短突变诱导遗传性 依赖性在mtDNA-野生型细胞中不存在，Aim 3将使用DdCBE工程化的细胞系模型来鉴定， 验证和机械地研究与CI截短突变相关的新的合成致死性。的 这些研究的结果将提供一个新的，详细的了解功能，剂量敏感性， 癌症基因组中最常见的遗传损伤之一的治疗脆弱性。

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.