PROJECT SUMMARY/ABSTRACT Small cell lung cancer (SCLC) is a high-grade neuroendocrine tumor currently without any approved targeted therapies. Inhibitors of the histone demethylase LSD1 are currently in clinical trials in SCLC inspired by strong preclinical data demonstrating that some SCLCs are highly and selectively sensitive to LSD1 inhibition. However, only a minority of SCLCs are highly sensitive to LSD1 inhibition, while most SCLCs are inherently resistant. The molecular basis for why some SCLCs are highly dependent on LSD1 is not understood. Understanding the causative mechanisms for why some SCLCs are highly sensitive to LSD1 inhibitors could identify predictive biomarkers to select patients more likely to respond to LSD1 inhibitors or identify rational combination strategies to make LSD1 inhibitors more effective. Using unbiased positive selection CRISPR/Cas9 loss of function screening with LSD1 inhibitors, we have uncovered novel causative mechanisms by which LSD1 inhibitors regulate SCLC, which include mechanisms by which LSD1 regulates neuroendocrine differentiation in SCLC, target genes that are bound and repressed by LSD1 and required for LSD1 inhibitor sensitivity, and also identified a rational combination strategy to overcome resistance to LSD1 inhibitors. The broad long-term objective of our proposal is to elucidate the molecular mechanisms by which LSD1 promotes SCLC tumorigenesis and neuroendocrine differentiation (aims 1 and 3), and test a rational combination strategy to make LSD1 inhibition more effective in SCLC (aim 2). To accomplish this, we will combine in vitro mechanistic approaches to rigorously dissect the mechanisms by which LSD1 drives SCLC proliferation and neuroendocrine differentiation with an in vivo autochthonous immunocompetent SCLC genetically-engineered mouse model (GEMM) that we developed using CRISPR/Cas9 that can be used to delete LSD1 at tumor initiation and study its function during SCLC tumorigenesis. Using these LSD1 isogenic SCLC GEMMs, we will interrogate how loss of LSD1 blocks neuroendocrine differentiation, promotes lineage plasticity, and increases tumor immunogenicity. Together, these studies will provide important biological insights into how SCLCs utilize LSD1 to drive tumorigenesis and neuroendocrine differentiation, which ultimately could lead to the identification of predictive biomarkers to select patients more likely to respond to LSD1 inhibitors. Lastly, this research could provide the preclinical foundation for a rational combination therapeutic strategy to make LSD1 inhibitors more effective for SCLC patients.