Project Summary/Abstract Transcription factor (TF) fusion genes create oncoproteins that drive tumorigenesis. While TF fusions represent cancer specific alterations, direct therapeutic targeting remains a clinical challenge. One example is the CIC-DUX4 TF fusion which defines an aggressive subset of round cell sarcoma in children and young adults. The clinical outcomes for CIC-DUX4 patients remain dismal due to high metastatic propensity and ineffective therapies. Currently, no therapies exist that direclty target the CIC- DUX4 fusion. To meet this need, we have recently identified a mechanistic link between the terminal MAPK signaling substrate, ERK, and CIC-DUX4. Specifically, ERK physically binds and phosphorylates CIC-DUX4 leading to rapid nuclear export, degradation of the fusion, and tumor cell death. Since MAPK signaling is a ubiquitous pathway expressed in both normal and malignant processes, we wondered how the CIC-DUX4 fusion could maintain its own expression. Through biochemical and molecular studies we have identified a key role for negative MAPK-ERK regulation in CIC-DUX4 sarcoma cells. Whereby, CIC-DUX4 transcriptionally upregulates the ERK specific phosphatase, DUSP6, to limit ERK activity and thus enable CIC-DUX4 expression. More recently, we made an unexpected finding that CIC-DUX4 expression could also downregulate RAS activity. Since RAS is a proximal MAPK substrate not targeted by DUSP6, we hypothesized that CIC-DUX4 was limiting MAPK-RAS-ERK signaling flux at multiple levels within this canonical signaling cascade. This proposal will define how CIC-DUX4 is regulating RAS activity, thus further sustaining CIC-DUX4 expression.