PROJECT SUMMARY Nearly 2 million people are diagnosed with lung cancer each year and it is the leading cause in cancer related death worldwide. Standard of care for non-small cell lung cancer (NSCLC) patients has changed little over the past several years however targeted therapies and immune checkpoint inhibitors have recently become a promising option. The most common oncogenic drivers in NSCLC are mutations in EGFR and KRAS which upregulate a myriad of downstream signaling pathways that promote tumor cell growth. Until recently, efforts to develop therapeutics that directly target EGFR or KRAS have largely been met by failure. Specifically, development of farnesyl transferase inhibitors and downstream pathway inhibitors like MEK and RAF, have not shown improvement in survival in the clinic and resistance mechanisms are described. The discovery of KRASG12C mutant inhibitors like AMG 510 and MRTX849 is encouraging as they have both shown promising signs of clinical activity and promise to transform treatment of KRAS mutant cancer. These inhibitors work by covalently binding to the reactive Cys12 locking KRAS in its inactive GDP-bound state. However, as with previously targeted therapies, mechanisms of resistance are beginning to be described. Utilizing precision modeling in mice, I will test the hypothesis that KRAS allelic imbalance and genetic determinants in NSCLC drive tumor progression and confer unique responses to targeted therapies. Since our lab has developed LSL-Kras allelic series that allows for selective targeting of the WT Kras allele, in Aim 1, I will use CRISPR-based genome editing technology to knockout WT Kras in vivo and measure effects in tumor burden and G12C inhibitor response. Further, I aim to understand how WT KRAS signaling contributes to the tumor immune microenvironment and how it affects targeted treatment response. In Aim 2, I will use a patient data guided approach to elucidate how cooperative mutations in tumor suppressors effect tumor progression and G12C inhibitor response. This powerful genetic approach will allow me to directly interrogate ways in which KRASG12C targeted therapy can be affected. Identifying an effective approach to disrupt KRASG12C mutant NSCLC will have a profound impact on the clinical management of these patients. Thus, we believe our work will contribute significant pre-clinical data to developing safe and effective targeted therapies for NSCLC and other cancer types.