PROJECT SUMMARY/ABSTRACT BRAF activating mutations occur in approximately 10% of metastatic colorectal cancers (CRCs) and are associated with worse prognosis due to an inferior response to standard chemotherapies. Current standard of care for patients with metastatic BRAFV600E CRC who have received prior therapy is BRAF inhibitor (BRAFi) plus EGFRi treatment. Unfortunately, responses are not durable and the median overall survival of patients receiving combination therapy remains dismal at roughly 9 months. Therefore, improving response to BRAFi plus EGFRi therapy in metastatic BRAF mutant CRC represents a critical unmet clinical need. With the use of more potent targeted therapies, shifts in cell identity due to epithelial cell transdifferentiation is an emerging cause of therapy resistance. In a subset of lung and prostate cancers, lineage transition from adenocarcinoma to a neuroendocrine phenotype is a mechanism of resistance to EGFRi and anti-androgen targeted therapies, respectively. However, the phenomenon of “therapy-induced transdifferentiation” has not been examined in CRC. It was recently determined that BRAF mutant CRC uniquely contains enteroendocrine cell (EEC) progenitors that promote cancer cell survival via secreted factors and that the differentiation state of these tumors is regulated by the lysine demethylase LSD1. In new preliminary studies, BRAFi alone or in combination with EGFRi is shown to further increase the number of EEC progenitors in BRAF mutant CRC. BRAFi treatment is also found to induce signal transducer and activator of transcription 3 (STAT3) to interact with LSD1 resulting in EEC progenitor enrichment. Consequently, inhibition or knockdown of either STAT3 or LSD1 blocks BRAFi- induced enrichment of EEC progenitors. EECs are intestinal neuroendocrine cells and the finding that this population expands after BRAFi provides strong scientific premise for the overall hypothesis that this lineage promotes therapy resistance in BRAF mutant CRC. It is hypothesized that BRAFi promotes therapy resistance by transdifferentiation of BRAF mutant CRCs to an EEC progenitor state through STAT3 and LSD1 activation. It is further hypothesized that inhibiting LSD1 is a promising strategy to block BRAFi-induced transdifferentiation and to improve the durability of tumor control with BRAFi plus EGFRi treatment. In Aim 1, the mechanism by which BRAFi promotes transdifferentiation in BRAF mutant CRC will be determined by focusing on how LSD1 and STAT3 interact to regulate downstream gene expression pathways. In Aim 2, a clinically relevant LSD1 inhibitor will be used in a syngeneic mouse model of metastatic BRAF mutant CRC and human PDX models to determine the effect of LSD1 inhibition on BRAFi plus EGFRi-induced transdifferentiation, the tumor immune microenvironment, and efficacy of targeted therapy in vivo. Overall, the proposed work has the potential to develop an epigenetic inhibitor-based approach to block therapy-induced transd...