PROJECT SUMMARY/ABSTRACT Transformation of normal cells to cancer cells involves complex changes influenced by oncogenes, inactivation of tumor suppressors, and disrupted signaling pathways, imposing a metabolic demand that is sustained through alterations in critical metabolic pathways and the availability of nutrients in the tumor microenvironment to support tumor expansion. Previously, we have shown that the electron transport chain (ETC) is necessary for in vivo tumor initiation and progression. However, the exact reasons behind the crucial role of the ETC in tumor growth remain somewhat unclear. We hypothesize the ETC supports tumor growth by maintaining oxidative tricarboxylic acid (TCA) cycle activity by replenishing NAD+ and converting succinate to fumarate for production of lipids, nucleotides, heme, and glutathione. However, a comprehensive understanding of the TCA cycle's importance in tumor growth has not been achieved thus far. Hence, we chose to focus our investigation on malate dehydrogenase 2 (MDH2), a key enzyme involved in the final step of the TCA cycle, to explore its significance in tumor growth. MDH2 has two distinct functions involving conversion of malate to oxaloacetate and cytosolic NAD+ generation through the malate-aspartate shuttle. Preliminary data, demonstrates that the absence of MDH2 impaired orthotopic tumor growth of murine lung adenocarcinoma (LUAD) cells in immunocompetent mice. This proposal outlines our plan to further investigate the necessity of MDH2 in tumor initiation and progression using a novel MDH2 floxed mouse line with the Kras+/LSL-G12D; Trp53fl/fl (KP) mouse model of LUAD. Furthermore, as tumors progress, they become hypoxic concomitant with a decline in ETC function and mitochondria NAD+ regeneration. Moreover, MDH2 plays a role in regenerating cytosolic NAD+. Thus, we will address whether cytosolic or mitochondrial NAD+ acts as a limiting factor for LUAD in the KP mouse model. To address this, we have also generated conditional mice expressing cytoplasmic or mitochondrial Lactobacillus brevis H2O-forming NADH oxidase (LbNOX) enzymes, which convert NADH back to NAD+ in the cytosol or mitochondria, respectively. To complement these experiments, we will perform CRISPR knockouts of MDH2 or express CytoLbNOX and MitoLbNOX in orthotopic lung cancer mouse models using mouse cell lines derived from the KP mouse model. A key set of experiments will assess whether MDH2 is required for normal alveolar epithelial cell development and function in adult mice. These investigations will enhance our understanding of the necessity of mitochondrial metabolism dependent TCA cycle and NAD+ regeneration in supporting lung tumor growth and normal lung epithelial cell function.