Defining the function of Complex I truncating mutations in cancer

项目摘要/摘要 线粒体DNA(MtDNA)中蛋白质编码基因的高破坏性截断突变影响近10% 在所有癌症中，主要发生在异质性，影响到总mtDNA池的一小部分。虽然 几十年来对生殖系致病线粒体DNA变异的研究已经证实，它们深刻地 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。严格询问癌细胞中mtdna突变的根本障碍是缺乏。 用于基因工程线粒体DNA的工具。最近，一种新的线粒体DNA编辑技术配对故事绑定 DddAtox胞嘧啶碱基编辑程序(DdCBE)的结构域已成功用于引入点突变 转化为线粒体DNA，彻底改变了对线粒体DNA进行高精度基因操作的能力。同时，我们的团队 最近发现，截短mtdna突变在特定基因中处于强正向选择之下。 背景(NADH脱氢酶/复合体I的亚单位，“CI”)和癌症谱系(结直肠癌、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到。这些一致的发现激励我们的团队设计DdCBE 在细胞系中对几个CI和非CI mtDNA基因引入截断突变，首次启用 截断癌细胞mtdna突变的功能讯问。使用这些工具，我们建议 综合计算/实验研究以检验CI截断突变这一主要假设 在肿瘤中产生生理意义上和治疗上可操作的代谢变化。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，确定复发 核DNA中的突变等位基因和共生驱动突变。同时，我们将DdCBE表示为模型CI- 和非CI截短突变，并定义所赋予的分子表型 CI使用转录、代谢和同位素示踪实验截断突变。我们的预赛 数据表明，CI截断突变的表型取决于它们的异质剂量。因此，在 目的2我们将利用DdCBE的瞬时表达来制作结直肠癌细胞系的等基因面板 独特的突变剂量。使用单细胞和整体分子图谱相结合的方法，我们 将检验这一假设，即CI截短突变将肿瘤细胞的新陈代谢重新连接到促增殖的 以剂量敏感的方式进行配置。最后，假设CI截断突变导致基因 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， 验证并机械地研究与CI截断突变相关的新的合成致死性。这个 这些研究的结果将提供一个新的，详细的功能，剂量敏感性，和 癌症基因组中最常见的遗传侮辱之一的治疗脆弱性。

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.