Manipulating the mitochondrial genome to investigate the impact of mtDNA mutations in cancer

线粒体DNA突变存在于约60%的肿瘤中，长期以来在癌症生物学中被忽视。我实验室最近的开创性工作发现，mtDNA突变是所有癌症的主要表型修饰物，结直肠癌中的mtDNA突变与寿命的重大延长有关，这是mtDNA突变对癌症的临床影响的第一个强有力的证据。然而，这种效应的机制细节，以及线粒体DNA突变在癌症发生和发展中所起作用的基本问题仍然没有得到解决，缺乏实验的线粒体DNA模型系统来进一步研究这一问题。到目前为止，该领域一直依赖于经历随机和连续突变的mtDNA的小鼠模型，这些模型不能概括疾病状态。应用我在线粒体和癌症生物学交叉领域的专业知识，以及我在线粒体DNA基因组工程方面高度专业的、经过验证的跟踪记录，我建议创建模型和方法，以促进对癌症线粒体遗传学的理解，解决目前限制发现的技术缺陷。以结直肠癌为初始范式，我将：i)使用我自己发明的先进的mtDNA操纵工具来创建新的先进的结直肠癌小鼠模型，其中具有可操作的与癌症相关的mtDNA突变(WP1)；ii)应用和修改最近开发的mtDNA突变方法，以创建新的具有类似于我在人类疾病中发现的mtDNA突变的最先进的结直肠癌模型(WP2)；iii)开发方法来革命性地进行mtDNA的遗传操作，从而能够创建任何mtDNA变体-远远超出目前最先进的(WP3)。通过这项提议，我将回答关于线粒体DNA突变状态如何改变结直肠癌的基本问题(目标1)，同时创建广泛适用的线粒体DNA工具和模型，以推动对癌症的进一步研究，远远超出目前的技术限制(目标2)。

Mutations of mtDNA are present in ~60% of all tumours and have long been overlooked in cancer biology. Recent groundbreaking work from my lab has identified mtDNA mutations as a major phenotype-modifier across all cancers, and mtDNA mutations in colorectal cancer were linked to major extensions in lifespan, the first robust evidence for the clinical impact of mtDNA mutations in cancer. However, the mechanistic details of this effect, alongside fundamental questions of the role mtDNA mutations play in cancer initiation and progression remain unaddressed, with a lack of experimental mtDNA model systems available to investigate this further. To date, the field has relied on mouse models that undergo random and continuous mutagenesis of mtDNA, which do not recapitulate the disease state. Applying my expertise at the intersection of mitochondrial and cancer biology with my highly specialist, proven track record in mtDNA genome engineering, I propose creation of models and methods to advance understanding of mitochondrial genetics in cancer, addressing the present discovery-limiting technical shortfall. Taking colorectal cancer as an initial paradigm, I will: i) use advanced mtDNA manipulation tools of my own invention to create new state-of-the-art mouse models of colorectal cancer with manipulable levels of a cancer-relevant mtDNA mutation (WP1); ii) apply and modify recently developed mtDNA mutagenesis methods to create new state-of-the-art models of colorectal cancer bearing mtDNA mutations analogous to those I identified in human disease (WP2); iii) develop methods to revolutionise genetic manipulation of mtDNA, permitting creation of any mtDNA variant - well beyond the present state-of-the-art (WP3). Through this proposal, I will answer fundamental questions of how colorectal cancer is modified by mtDNA mutational state (Aim 1) while creating broadly applicable mtDNA tools and models to power further research into cancer far beyond present technical limits (Aim 2).