Project Summary The overarching goal of this proposal is to define the interplay of epigenetics and developmental context in driving pediatric high-grade gliomas (pHGG), universally lethal tumors defined by histone mutations, so as to identify better targeted treatments. This work utilizes an innovative cerebral organoid-based model that allows side-by-side comparison of oncohistone mutants and dynamic modulation of oncogene expression in a three- dimensional human context. Leveraging this innovative model, Dr. Graham will use cutting-edge single cell profiling techniques and preclinical glioma modeling to address unanswered questions that currently hamper therapeutic development for these patients. In Aim 1, chromatin profiling and single cell RNA sequencing will be used to investigate the distinct effects of two different oncohistones (H3.3K27M and H3.3G34V) during neural development. In Aim 2, the timing and order of mutations will be manipulated to evaluate the impact of cellular and mutational context on tumor phenotype. Finally, in Aim 3, orthogonal genetic “rescue” and pharmacologic inhibition approaches will be used to interrogate the role of these mutations in tumor maintenance. This work, as well as Dr. Graham's career goal, is well-aligned with the NINDS mission to seek basic knowledge about the brain and to translate that knowledge into clinical impact. Through the proposed studies and the accompanying career development plan, Dr. Graham will gain essential training in epigenetics, bioinformatics and preclinical glioma studies, critical gaps in her current skillset. Dr. Graham is an Instructor of Neuro-Oncology under the mentorship of Dr. Ingo Mellinghoff at Memorial Sloan Kettering Cancer Center (MSK). Dr. Mellinghoff is a leader in translational glioma research with a strong track record of mentoring trainees to independence. He and Dr. Graham have assembled an exceptional Advisory Committee with expertise in chromatin biology, translational oncology, and bioinformatic analyses: Dr. Kristian Helin, Dr. Ross Levine and Dr. Nicholas Socci. MSK provides an outstanding environment for cultivating budding careers in biomedical research, with unparalleled resources, support, and opportunities for collaboration. Upon completion of the proposed work, Dr. Graham will be ideally positioned and uniquely qualified for a career as an independent investigator elucidating fundamental aspects of epigenetic regulation in neural lineage commitment and applying her findings to address the unmet needs in the treatment of malignant glioma.