PROJECT SUMMARY/ABSTRACT We are in desperate need of novel therapies for treating lung cancer, particularly ones that can overcome treatment resistance. Intra-tumoral bacteria are emerging as a cause of therapy resistance in cancer. We recently discovered that a specific bacteria, Chryseobacterium indologenes, identified within human lung cancers, leads to resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI). The third-generation EGFR-TKI osimertinib is the standard of care for first-line treatment of patients with advanced EGFR-mutated lung cancer. Mechanisms of resistance to osimertinib are poorly understood. In this proposal we seek to understand the mechanisms by which intra-tumoral Chryseobacterium (ITC) leads to resistance to osimertinib. The long-term objective of this proposal is to develop new therapies targeting the effects of ITC to prevent resistance to EGFR-TKIs. The objective of this proposal is to identify the mechanism responsible for the effect of ITC on sensitivity to osimertinib, and to determine if other species of Chryseobacterium also lead to EGFR-TKI resistance. Our central hypothesis is that ITC leads to resistance to osimertinib via secreted proteins in some patients with EGFR-mutated lung cancer. In Aim 1, we will validate the candidate proteins that we previously identified in Chryseobacterium preconditioned medium (C-PCM) as potentially responsible for osimertinib resistance using fast protein liquid chromatography and mass spectrometry, by testing the purified candidate proteins in EGFR-mutated lung cancer cell lines in the presence/absence of osimertinib. Next, we will determine the mechanism by which ITC affects resistance to EGFR-TKIs, by investigating the effects of C-PCM on (1) drug efflux, (2) re-activation of the EGFR pathway or (3) activation of molecular pathways other than EGFR. These experiments will help us understand how Chryseobacterium indologenes leads to osimertinib, allowing us to eventually develop new therapies to prevent or overcome osimertinib resistance. In Aim 2, we will determine if Chryseobacterium species other than indologenes lead to EGFR-TKI resistance in PC9 cells. We will obtain commercially available Chryseobacterium meningoseptica, angstadti, gleum, indoltheticum, scophthalmum, diehli, shigense, and balustinum species which will be cultured and genotyped using PCR. Preconditioned medium (PCM) from each species will then be serially diluted and added to PC9 cells with and without osimertinib, and effects on cell growth will be determined using cell proliferation assays. These experiments will help us understand which other Chryseobacterium species lead to osimertinib and may shed additional light on mechanisms of osimertinib resistance. The proposed research is potentially transformative as it may lead to the discovery of a new approach to overcoming resistance to EGFR-TKIs in patients with EGFR- mutated lung cancer through targeting specific tumor-mic...