PROJECT SUMMARY/ABSTRACT Mutations in the KRAS GTPase are some of the most frequent gain-of-function alterations in cancer. KRAS mutations are found in 25% of patients with lung cancer and nearly half of these involve a G12C substitution. Lung cancer is the leading cause of cancer related mortality and, considering that more than 250,000 people are diagnosed each year with lung cancer in the US, developing therapeutic strategies that directly targeting KRAS oncoprotein is of paramount importance. The discovery of inhibitors that selectively target KRAS G12C has been one of the most exciting recent developments in precision oncology. We previously showed that the inhibitors trap the oncoprotein in an inactive (GDP-bound) state by blocking its reactivation by nucleotide exchange and that such inhibition requires the breakdown of GTP by mutant KRAS in cancer cells. Inactive state selective KRAS G12C inhibitors (e.g., sotorasib and adagrasib) have a 37-43% objective response rate (ORR) and a 6-8 month median progression free survival (PFS) in patients with lung cancer. Sotorasib was approved by the food and drug administration (FDA) as a second line treatment in patients with KRAS G12C- mutant lung cancer. Despite these early successes more work is needed if we are to dramatically affect the survival of patients with KRAS mutant cancer. The current proposal seeks to identify factors responsible for modulating drug adaptation and resistance in patients and to identify improved KRAS-directed therapeutic approaches. The first part of the proposal builds on our preclinical work showing that isogenic lung cancer cells bypass G12C inhibition in a non-uniform manner. Our preliminary data suggest this occurs because of the conformation-specific nature of available G12Ci and the synthesis of new KRAS G12C in some drug-treated cells. In Aim 1, we will determine the heterogeneity in response and adaptation to treatment with inhibitors targeting either the active or the inactive conformation of mutant KRAS. The studies will be performed in parallel in clinical specimens and in patient derived models to ensure robustness and reproducibility. Aim 2 builds on our recent effort to study acquired resistance in patients treated with sotorasib or adagrasib. Here we will focus on alterations that cause resistance to emerging active state selective drugs, relying initially on patient-derived models and then on clinical specimens, once these drugs enter clinical testing in late 2022. In addition, prospective studies with barcoded populations will investigate the clonal interactions that guide the selection of resistance-causing alterations. In Aim 3 we will study the mechanisms by which the alterations or phenotypes identified in the preliminary data or in Aims 1 and 2 lead to resistance. The early success of KRAS G12C inhibitors has inspired the development of additional KRAS inhibitors that are entering clinical trials and promise to improve the survival and quality of ...