PROPOSAL SUMMARY Diffuse intrinsic pontine gliomas (DIPGs) are aggressive brainstem tumors in children with no curative therapies available. DIPGs are canonically driven by recurrent mutations in the histone 3 gene (H3K27M). This substitution imparts broad dysregulation of the histone post-translational modifications (PTMs) that regulate the recruitment and initiation of transcriptional machinery. Processive transcription, or the mechanics of RNA Pol II as it actively transcribes across chromatin, is both dependent on and actively propagates chromatin states such as dynamic accessibility and transcription-associated PTMs (tPTMs). Disorders of transcription dynamics have demonstrated pathogenic roles in cancer development, and inhibition of transcription machinery is an effective therapy in these models. We have recently shown that the H3K27M mutation activates regulators of transcriptional elongation, including CDK9. We have demonstrated that inhibition of CDK9-dependent transcriptional elongation is an effective therapy in DIPG, but the contribution of processive transcription to DIPG oncogenic transformation is unknown. The overall hypothesis of this proposal is that the H3K27M mutation promotes CDK9-dependent nascent transcription, which in turn contributes to both the establishment of an oncogenic chromatin state as well as the adaptive response to standard-of-care radiation therapy. Using a combination of CRISPR-edited model systems, patient derived cultures, and both patient-derived xenograft and syngeneic engineered mouse models, we will test this hypothesis by 1) defining the role of processive transcription in H3K27M-mediated oncogenesis, 2) determining the impact of CDK9 inhibition on processive transcription, and 3) characterizing the role of transcriptional induction in response to ionizing radiation. Successful completion of the proposal will allow us to comprehensively map the impact of the H3K27M mutation on the nascent transcriptional landscape. This data will enable us to define a novel transcriptional framework for understanding DIPG’s chromatin-mediated oncogenesis, and it will demonstrate how exploiting this transcriptional dependence may be leveraged to improve the patient benefit derived from radiation therapy. The proposed career development plan leverages these studies to provide advanced training in the conduct of rigorous hypothesis-driven research, the molecular study of transcriptional regulation, and representative pre- clinical cancer modeling. The mentorship team reflects nationally-recognized senior scientists who possess both focused expertise in these areas of study as well as a strong commitment to my career development. The training plan outlines how I will refine my expertise through a combination of didactic course work, focused workshops, national meetings, and mentorship guidance. Collectively, this training platform will facilitate my transition to independence as a basic-translational researcher with a lo...