PROJECT SUMMARY/ABSTRACT The CIC-DUX4 fusion oncoprotein is the molecular hallmark of a highly lethal subset of undifferentiated sarcomas that affects children, adolescents, and young adults (AYA). We have recently revealed critical transcriptional programs downstream of CIC-DUX4 that drive malignant phenotypes, including tumor growth and metastasis. Additionally, recent studies demonstrate that Capicua (CIC) protein expression is directly modulated by the mitogen-activated protein kinase (MAPK)-signaling pathway. The long-term goal of our laboratory is to identify and dissect the critical molecular pathways that regulate fusion oncoprotein stability and expression in human sarcoma. Our studies will reveal fusion oncoprotein-specific vulnerabilities that can facilitate precision-based therapeutic design to improve outcomes for patients with universally lethal forms of sarcomas. The objective of this proposal is to fuse our expertise in Capicua (CIC) biology and MAPK signaling to develop the first precision-based therapeutic approach for CIC-DUX4 sarcomas. We hypothesize, that genetic or pharmacologic MAPK activation will lead to the degradation of the CIC-DUX4 oncoprotein. Our rationale is based on our prior studies demonstrating MAPK-mediated regulation of wild-type (non-fused) CIC protein expression in the context of human cancer. Our specific aims will test the following hypotheses: (Aim 1) human ERK physically interacts with nuclear CIC-DUX4; (Aim 2) MAPK-ERK pathway activation decreases CIC-fused oncoprotein stability, leading to degradation; and (Aim 3) pharmacologic activation of the MAPK pathway decreases tumor cell survival through rapid degradation of the CIC-DUX4 fusion oncoprotein. The significance of these findings are highly impactful, as there are no effective therapeutic strategies to target CIC- DUX4 sarcomas, which remain lethal in the metastatic setting. Our proposal is innovative because we are leveraging our expertise in MAPK-signaling, transcription factor fusions, and Capicua biology to develop a precision-based therapeutic approach to directly target and degrade a lethal fusion oncoprotein in sarcoma, an unmet scientific and clinical need.