Decoding AMPK-dependent regulation of DNA methylation in lung cancer

项目摘要 肺癌是全球癌症相关死亡的主要原因。突变的KRAS基因引发的肺肿瘤 是最具侵略性和最难治疗的疾病之一，部分原因是KRAS驱动的新陈代谢 重新编程。更好地了解致癌KRAS代谢适应调节途径的努力- 驱动肿瘤将提供对肺癌进展的洞察，并确定可能是 在治疗上有针对性地提高患者的存活率。邵氏实验室最近显示了AMP的要求- 活化蛋白激酶促进KRAS致癌非小细胞肺癌的生长 (NSCLC)。AMPK是细胞和生物体新陈代谢的主要调节器，是细胞的传感器。 当能量水平较低时，通过改变新陈代谢来补充能量。而Shaw实验室和其他人已经证明 AMPK信号为癌细胞提供了适应新陈代谢压力的灵活性，即表观遗传学 AMPK促进代谢改变和肺癌生长的机制仍然知之甚少。 初步研究确定从头DNA甲基转移酶是AMPK的一种新底物。DNA甲基化 参与了许多正常的细胞过程，并在癌细胞中异常分布，导致一些 它们的攻击性特征。该提案解决了依赖AMPK的监管的后果 从头DNA甲基转移酶对DNA甲基化、代谢程序和肺癌生长的影响。第一, 这项工作旨在定义由依赖AMPK的磷酸化形式控制的甲基化特征 应用全基因组亚硫酸氢盐测序、切割标签和RNA测序的从头DNA甲基转移酶 化验。致瘤性和海马氏实时细胞代谢分析将决定这一调控 破坏DNA甲基化模式，产生促进肿瘤的表观遗传损伤和代谢 改装。此外，原生KRAS驱动的NSCLC小鼠株系的产生 丝氨酸到丙氨酸突变的从头DNA甲基转移酶的结构性敲入 磷酸受体丝氨酸将使测试从头DNA甲基转移酶的调节是否影响 肿瘤的发生、生长和转移。这项工作解决了两个标志之间的基本关系 如果成功，将导致代谢压力与肿瘤细胞之间的机械联系 以及它们可能如何引发持续的DNA甲基化变化。

Project Summary Lung cancer is the leading cause of cancer-related mortality worldwide. Lung tumors driven by mutant KRAS are among the most aggressive and refractory to treatment, due in part by KRAS-driven metabolic reprogramming. Efforts to better understand the pathways regulating metabolic adaptations in oncogenic KRAS- driven tumors will provide insight into lung cancer progression and identify vulnerabilities that could be therapeutically targeted to improve patient survival. The Shaw lab recently showed the requirement of AMP- activated protein kinase (AMPK) to promote the growth of oncogenic KRAS-driven non-small-cell lung cancer (NSCLC). AMPK is a master regulator of cellular and organismal metabolism that acts as a sensor of cellular energy by altering metabolism when energy levels are low. While the Shaw lab and others have demonstrated that AMPK signaling provides cancer cells with flexibility to adapt to metabolic stresses, the epigenetic mechanisms by which AMPK promotes metabolic alterations and lung tumor growth remain poorly understood. Preliminary studies identified a de novo DNA methyltransferase as a novel substrate of AMPK. DNA methylation is involved in many normal cellular processes and is abnormally distributed in cancer cells, contributing to some of their aggressive characteristics. This proposal addresses the consequences of AMPK-dependent regulation of the de novo DNA methyltransferase on DNA methylation, metabolic programs, and lung tumor growth. First, this work aims to define the methylation profile controlled by the AMPK-dependent phosphorylated form of the de novo DNA methyltransferase using whole-genome bisulfite sequencing, CUT&TAG, and RNA sequencing assays. Tumorigenicity and Seahorse real-time cell metabolic analyses will determine whether this regulation disrupts DNA methylation patterns in a manner that generates tumor-promoting epigenetic lesions and metabolic alterations. Additionally, the generation of autochthonous KRAS-driven NSCLC mouse lines expressing constitutive knock-in of the de novo DNA methyltransferase with a serine-to-alanine mutation at the putative phosphor-acceptor-serine will enable testing whether regulation of the de novo DNA methyltransferase impacts tumor initiation, growth, and metastasis. This work addresses a fundamental relationship between two hallmarks of cancer and if successful would lead to the mechanistic connection between metabolic stresses tumor cells face and how they may trigger sustained DNA methylation changes.