The rearranged during transfection (RET) gene encodes a protein tyrosine kinase (PTK). RET aberrations are found in various types of human cancer. Highly potent and selective RET protein tyrosine kinase inhibitors (TKIs) are in the later stage of clinical development for precision cancer treatment. A lesson learned from the previous experience is that the PTK-targeted cancer therapy often fails because of adaptive resistance evolved from the drug-tolerant, residual tumors. Results of our preliminary studies indicate that the RET-targeted cancer treatment is no exception to this rule. To prolong the suppression of tumors and the lives of patients, we must eliminate the residual tumor cells, identify target mutations that cause drug resistance, find or develop drugs effective against different drug-resistant mutants, and discover alternative signaling mechanisms that promote tumor progression. The overall goal of this project is to improve the targeted therapy for RET aberration- associated cancer. The objectives of this project are to map the landscape of drug-resistant RET kinase mutations, identify drugs effective against mutant RET, determine the structures of RET kinase variants, characterize the structural bases of interaction between RET and its inhibitors, and evaluate a method to minimize the residual lung tumors. Specific Aim 1 is to identify and characterize RET mutations resistant to selective RET TKIs in preclinical models and cancer patients, and find potential secondary drugs. Specific Aim 2 is to determine the crystal structures of drug-resistant RET mutant proteins and RET-inhibitor complexes. Specific Aim 3 is to test RET mutant-sensitive TKIs in vivo and evaluate a method for reducing the residual fibrotic stroma and the associated residual tumor cells. Taken together, this study will provide a RET mutation- TKI toolkit for precision management of RET-aberrant cancers to maximize the benefit of RET-targeted cancer therapy, lay the foundation for developing a continuous pipeline of mutant-effective RET TKIs, and find a method to reduce the residual drug-tolerant tumors to delay tumor recurrence. The outcomes of this project will have positive translational impact on prolonging the survival of patients with cancer.