Project Summary/Abstract Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK), is an oncogenic driver that is frequently overexpressed or mutated in non-small cell lung cancer (NSCLC). Many EGFR inhibitors are used to treat NSCLC, but drug resistance is common. Necitumumab (Neci), an EGFR-directed monoclonal antibody, is approved for a subset of NSCLC harboring wild type (WT) EGFR but its efficacy is very limited. Patients whose tumors harbor mutated EGFR often derive significant benefit from EGFR tyrosine kinase inhibitors (TKIs), but resistance invariably develops. Mechanism of resistance to EGFR inhibitors is complex and not fully known. Our objective in this project is to evaluate a new agent for overcoming drug resistance in NSCLC using preclinical models. The project builds on our recent discovery that a recombinant human protein, PEPDG278D, induces the degradation of EGFR and HER2 by binding to their extracellular domain. HER2 is closely related to EGFR and a key contributor to drug resistance in NSCLC. PEPDG278D is an enzymatically inactive mutant of human peptidase D, also known as prolidase important for collagen metabolism. We also find that PEPDG278D induces the degradation of mutated EGFR and HER2 that occur in NSCLC and strongly inhibits NSCLC cells and tumors resistant to current EGFR inhibitors. We envision that targeted degradation of EGFR and HER2 is more effective than only inhibiting the activation or activity of their tyrosine kinase, as kinase-independent functions are also important for their oncogenic signaling. Our overall hypothesis is that NSCLC cells and tumors resistant to current EGFR inhibitors are vulnerable to targeted degradation of EGFR and HER2 by PEPDG278D and that PEPDG278D induces the degradation of not only WT EGFR and HER2 but also a broad spectrum of clinically relevant mutants. We will test the hypothesis in three specific aims: 1) to assess PEPDG278D for inhibition of oncogenic signaling and antitumor activity in NSCLC cells and tumors expressing WT EGFR, and to compare PEPDG278D to Neci; 2) to determine if PEPDG278D inhibits the oncogenic signaling of a broad spectrum of EGFR and HER2 mutants in NSCLC cells by inducing their degradation and to compare PEPDG278D to osimertinib (Osi) which is a third generation EGFR TKI and the preferred EGFR TKI for NSCLC patients; and 3) to determine if Osi-resistant NSCLC cells and tumors are sensitive to PEPDG278D and if combining the two agents enhances therapeutic outcome. PEPDG278D and Osi complement each other mechanistically in targeting EGFR mutants. The proposed research is significant, because it addresses drug resistance in NSCLC which remains a major clinical problem. We expect 1) to show that PEPDG278D is therapeutically superior to Neci and Osi; 2) to establish the novel concept that inducing the degradation of EGFR and HER2 is an effective strategy for overcoming drug resistance in NSCLC. These findings may generate strong enthusiasm...