PROJECT SUMMARY. The aim of this project is to decode new components of the LKB1 tumor suppressor pathway to therapeutically target the 15-20% of non-small cell lung cancers (NSCLC) bearing mutations in this gene. Research over the last 20 years has revealed multiple direct linkages between genes mutated in human cancer and genes that control cell metabolism. The LKB1 tumor suppressor is a serine/threonine kinase mutationally inactivated in the familial cancer disease Peutz-Jeghers Syndrome, as well as being the third most frequently mutated gene in NSCLC, the most common cause of cancer deaths per year. To date, NSCLC patients with an LKB1 (STK11) mutation fare worse than any other specific genetic subset, exhibiting minimal response to chemotherapy and other targeted therapies. In recent years, multiple labs have found that STK11-mutant patients are highly resistant to checkpoint immunotherapies and have minimal therapeutic potential regardless of mutational burden. Twenty years ago, Dr. Shaw and others discovered that LKB1 directly phosphorylates the activation loop of the AMP-activated protein kinase (AMPK) and 12 related kinases, turning on their kinase activity. AMPK is a serine/threonine kinase activated by LKB1 under conditions of low intracellular ATP levels, after mitochondrial damage, or following loss of glucose or oxygen. AMPK plays a highly conserved role as an energy sensor and serves to restore metabolic homeostasis by downregulating mammalian target of rapamycin complex 1 (mTORC1) and parallel anabolic ATP-consuming processes (e.g., lipid biosynthesis), while upregulating catabolic ATP-restoring processes (like autophagy and fatty acid oxidation). Studies by the Shaw lab over the past 18 years have sought to understand the mechanistic basis for how AMPK and its related kinases function by decoding their direct substrates that mediate downstream effects on growth and metabolism. This may provide actionable targets for therapeutics aimed at this specific genetic LKB1-mutant subset of NSCLC. The Shaw lab has also used multiple genetically engineered mouse models of NSCLC to perform preclinical studies with novel cancer metabolism drugs they helped to develop. Building off prior Shaw lab findings and new clinical insights of LKB1-mutation intractability, two lines of research are proposed. First, a new in vivo autochthonous CRISPR screening technology will be utilized to directly query the tumorigenic potential of hundreds of candidate substrates of AMPK and its related kinases to identify those targets that are essential for tumor growth in Kras:LKB1 (“KL”) but not Kras (“K”) or Kras:p53 (“KP”) mutant tumors. This work builds off successful phospho-proteomics screens that identified novel substrates of AMPK and the AMPK-related SIK kinases critical for lung tumor cell survival. Secondly, to address the intrinsic resistance of KL-tumors to checkpoint immunotherapies, screening will be deployed in combination with biochemical dissecti...