Abstract Medulloblastoma (MB) is a heterogenous cerebellar tumor that comprises four molecular and clinically significant subgroups. MB is the most common brain malignancy in children with a five-year survival of 70%, however, Group 3 MB patients have the poorest survival due to the amplification of MYC and overexpression of key oncogenes that underly the Group 3 phenotype. Recently, epigenetic modifications bearing super-enhancer, H3K27ac, marks have been suggested to contribute to transcriptional dependencies that define Group 3 MB and represents an emerging target for therapeutic intervention. Currently, the mechanism that establishes and maintains these modifications to the DNA architecture remains unclear. In our study, we hypothesize that an epigenetic modifying protein, JMJD6, regulates the promoter-pause release associated with super-enhancer mediated oncogene addiction in Group 3 MB. This hypothesis is based on evidence in the literature showing that JMJD6 regulates the transcriptional pause release at enhancer regions. Moreover, the promoter-pause release has been shown to be critical for super-enhancer mediated transcription. In our preliminary studies, we demonstrate elevated levels of JMJD6 in Group 3 MB and survival analysis indicating JMJD6 as a predictor for poor prognosis in MB patients. Immunoprecipitation revealed that JMJD6 forms complexes with proteins involved in maintaining and regulating the promoter-pause at super-enhancers. Inducible knockdown of JMJD6 decreased MYC expression, cellular proliferation and stemness in vitro while inhibiting tumor formation and promoting survival in mice. Together, these studies identify JMJD6 as a promising therapeutic target for treating aggressive epigenetically driven Groups 3 MB tumors. Citation Format: Matthew Kling, Sutapa Ray, Don W. Coulter, Nagendra Chaturvedi. JMJD6 regulates super-enhancer mediated transcriptional dependencies in group 3 medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6050.