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.