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

mtDNA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。我实验室最近的突破性工作已经确定mtDNA突变是所有癌症的主要表型修饰剂，结直肠癌中的mtDNA突变与寿命的主要延长有关，这是mtDNA突变对癌症临床影响的第一个有力证据。然而，这种效应的机制细节，以及mtDNA突变在癌症发生和发展中的作用的基本问题仍然没有得到解决，缺乏实验mtDNA模型系统可用于进一步研究。迄今为止，该领域一直依赖于小鼠模型，这些模型经历mtDNA的随机和连续诱变，而不会重演疾病状态。运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mtDNA基因组工程方面的高度专业化和成熟的记录，我建议创建模型和方法来促进对癌症中线粒体遗传学的理解，解决目前发现限制的技术不足。以结直肠癌为初始范例，我将：i）使用我自己发明的先进mtDNA操作工具来创建具有可操作水平的癌症相关mtDNA突变（WP 1）的新的最先进的结直肠癌小鼠模型; ii）应用和修改最近开发的mtDNA诱变方法，以创造新的状态-携带与我在人类疾病中鉴定的mtDNA突变类似的mtDNA突变的结直肠癌模型（WP 2）; iii）开发方法来彻底改变mtDNA的遗传操作，允许创建任何mtDNA变体-远远超出目前的最先进水平（WP 3）。通过这个提议，我将回答结直肠癌如何被mtDNA突变状态修饰的基本问题（目标1），同时创建广泛适用的mtDNA工具和模型，以推动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).