ABSTRACT Despite notable success in EGFR-driven lung cancer, precision therapeutics have failed in EGFR-driven gliomas, the most common and deadly primary brain tumors. Reasons for failure of EGFR therapies in this clinical context include the lack of preclinical models that faithfully recapitulate the biology of EGFR-driven gliomas, including intra-tumor heterogeneity, drugs specifically designed to target invasive brain tumor cells located behind and intact blood-brain barrier (BBB), and adaptive drug resistance. Here we will develop and molecularly credential novel, EGFR-driven human glioma models for use in preclinical development of EGFR tyrosine kinase inhibitor (TKI)-based therapies. The foundation of the proposal comes from the Furnari Lab, who developed a novel platform (iGBM) for engineering glioma models using CRISPR genome editing and has established intra-tumor genetic heterogeneity as a symbiotic driver of tumorigenesis. The Miller Lab has extensive experience in small molecule experimental therapeutics using genetically engineered gliomas model and next-generation sequencing. He also used a novel chemical proteomics method, multiplex inhibitor beads coupled with mass spectrometry, to assess the glioma kinome en masse and showed that dynamic kinome reprogramming contributes to targeted drug resistance in glioma models. He is now at the University of Alabama at Birmingham, where local collaborators have extensive experience with biologically faithful human patient-derived xenograft (PDX) models. The O’Rourke Lab is a pioneer in development of sophisticated glioblastoma organoid (GBO) models that faithfully recapitulate the biology of molecularly and cellularly heterogeneous human tumors. In this Multi-PI project, we will combine our expertise to address the following Aims: (1) To develop novel genetically engineered human models driven by the most common EGFR extracellular domain mutations. We will then biologically and molecularly credential these models against genetically-matched PDX and GBO using genomics, epigenomics, transcriptomics, and kinome proteomics, and therapeutically challenge them using a panel of EGFR TKI, including one designed to specifically target invasive glioma cells behind the intact BBB. (2) To credential heterogeneous EGFR mutant iGBM models via biological, molecular, and EGFR TKI therapeutic profiling. We will thus develop human models with defined driver mutations that will be useful adjuncts to PDX/GBO for preclinical drug development. Models will be used to develop future rational combination therapies that combat drug resistance and enhance EGFR TKI efficacy. This work will therefore help realize the unmet need of precision therapeutics in neuro-oncology.