Development of treatment resistance is a major impediment to effective cancer therapy. KRASG12C inhibitors (KRASi) have been recently approved by FDA and represents a promising new targeted therapy for cancer types with KRAS mutation, one of the most frequent genetic mutations observed in human cancers. However, as is the case with standard therapy, majority of tumors that initially respond to KRASi quickly develop resistant disease. Unfortunately, the underlying mechanisms of KRASi resistance are poorly understood. To tackle this challenging problem, this application will capitalize on the PI's track record and expertise in the area of KRAS signaling and biology and propose a comprehensive research program focusing on understanding the mechanisms of acquired resistance to KRAS targeted therapy. This application will identify new actionable therapeutic targets and approaches to overcome resistance, thus greatly improving the clinical outcome of patients with KRAS mutated cancers. To achieve this, the Yao laboratory has developed in vitro and in vivo model systems that will be employed to investigate hypotheses regarding mechanisms driving the development of KRASi resistance. The in vitro model system consists of KRAS dependent and independent cell cultures derived from genetically engineered mouse models of pancreatic cancer as well as human cancer cell lines of pancreatic and colon origin. The in vivo model system consists of patient-derived colon cancer models that developed resistance to KRASi, as well as genetically engineered mouse models of pancreatic cancer driven by KRASG12C. The major knowledge gaps to be addressed are that: i) what molecular events are activated to enable bypass of KRAS dependency in tumors treated with KRASi; ii) how these molecular events function to drive tumor survival and maintenance in response to KRASi; and iii) whether pharmaceutically targeting the resistant mechanism can achieve durable therapeutic responses in combination with KRASi. These studies will unveil a previously unknown mechanism by which tumor cells become resistant to KRAS targeted therapy and may define an innovative therapeutic option for KRASi-resistant patients. The proposed work comprises an essential step toward our long-term goal of developing effective therapy for patients with KRAS mutated cancer, in align with the mission of the National Cancer Institute (NCI) RAS initiative (“Kill RAS”).