ABSTRACT Inactivating mutations in the LKB1/STK11 tumor suppressor are found across multiple epithelial tumor types including ~20% of non-small cell lung cancers (NSCLC) and ~5% of pancreatic ductal adenocarcinomas (PDA). In NSCLC, these mutations correlate with poor response to conventional, targeted, and immune-modulating treatment. In pancreatic tumorigenesis, LKB1 mutations are found in both in premalignant intraductal papillary mucinous neoplasms (IPMN) and in PDA and also portend a poor prognosis (Furukawa et al., 2007; Yang et al., 2015). Work from our group and others has shown that LKB1 is a central regulator of cell metabolism (Gurumurthy et al., 2009, Kottakis, et al., 2016). LKB1 loss results potentiates biosynthetic processes while reducing mitochondrial oxidative phosphorylation. The basis for metabolic reprogramming in LKB1 mutant cells is incompletely understood. This proposal seeks to explore the novel roles of LKB1 in the regulation of the mitochondria. Our preliminary data indicate that mitochondrial dysfunction is central to the pathogenesis of LKB1 mutant tumors and presents significant new therapeutic vulnerabilities. In preliminary studies, we find that LKB1 mutant (LKB1m) cancer cells exhibit a striking change in mitochondrial architecture and that LKB1 directly controls two fundamental processes of mitochondrial homeostasis—maintaining the balance between mitochondrial fusion and fission and activating the primary mechanism for repair or clearance of damaged mitochondria. These findings have important functional implications. Our preliminary studies indicate that restoration of mitochondrial fusion is lethal in LKB1m cancer cells; that these cells are hypersensitive to mitochondrial insults; and that defective mitochondrial quality control can be further exploited to restore anti-tumor immunity. The overarching concept emerging from these studies is these specific mitochondrial alterations are a hallmark of LKB1 mutant NSCLC and PDA, and that deciphering the mechanisms of mitochondrial regulation will open new therapeutic directions. The present proposal focuses one aspect of these mitochondrial phenotypes, the increase in mitochondrial fission resulting from LKB1 loss. Mitochondrial dynamics, the conversion between fusion and fission, configure mitochondria between tubular and fragmentated states, which have distinct metabolic outputs. Fusion leads to tightly packed cristae, efficient OXPHOS and fatty acid oxidation, whereas fission expands the cristae, reduces OXPHOS efficiency and promotes aerobic glycolysis. Our proposed studies will test the hypothesis that mitochondrial hyper-fragmentation resulting from LKB1 loss drives metabolic reprogramming essential for tumorigenesis. To this end, we will modulate mitochondrial dynamics using genetic and pharmacological approaches in novel in vitro and in vivo model systems, and we will test the resulting impact on metabolic homeostasis and tumor cell growth and survival. Resul...