Abstract High-grade gliomas of childhood occur in a striking spatiotemporal pattern, with tumors of the midline occurring chiefly in the first decade of life and tumors of the cerebral lobes occurring later in adolescence and young adulthood. This pattern of gliomagenesis bespeaks dysregulation of a developmental process, and maps well onto discrete waves of developmental myelination of the nervous system, a process that spans over 3 decades in humans. Increasing data from our group and from others implicate myelin-forming precursor cells as putative cells of origin for the most common and best-studied diffuse midline glioma, diffuse intrinsic pontine glioma (DIPG). DIPG is a fatal pediatric cancer peaking in incidence at age 6-7 years with a median survival of only 9 months and 5-year survival of less than 1%. DIPG affects 300-400 children each year in the US and represents the leading cause of brain tumor-related death in children. Therapeutic options are limited, and effective treatment is direly needed. During the early part of this decade, next-generation sequencing technologies enabled a comprehensive view of the DIPG genome. DIPG and other diffuse gliomas of the midline structures of the central nervous system, in particular the thalamus and spinal cord, exhibit a specific and highly recurrent mutation in histone-3 (H3K27M). The H3K27M mutation, occurring in genes encoding histone H3.3 and H3.1, affect ~80% of DIPG cases and the majority of thalamic and spinal cord gliomas as well. The H3K27M mutation causes perturbation of Polycomb repressive complex 2 (PRC2) function, resulting in global hypomethylation of K27 in all H3 variants. The broad epigenetic dysregulation and characteristic gene expression changes that ensue are believed to be central to DIPG oncogenesis. We have generated a panel of patient-derived DIPG cultures and correlate orthotopic xenograft models and have used these new resources for drug screening efforts. DIPG cells are vulnerable to HDAC inhibition, which we found restores H3 trimethyl mark and normalizes gene expression, an unexpected finding that may be explained by actetyl groups on nearby residues sterically interfering with the interaction between PRC2 and the aberrant methionine. We have also found that DIPG is vulnerable to transcriptional disruption using bromodomain inhibition or CDK7 blockade. Targeting oncogenic transcription through either of these methods synergizes with HDAC inhibition, and DIPG cells resistant to HDAC inhibitor therapy retain sensitivity to CDK7 blockade. A clinical trial of panobinostat for DIPG is ongoing, and follow-on trials of combination therapy hold promise. Other forms of diffuse midline gliomas, including H3K27M-mutant gliomas of the spinal cord and thalamus, may respond similarly to therapeutic strategies targeting the regulation of gene expression. Identification of super-enhancers in DIPG highlights oligodendroglial lineage genes, supporting the hypothesis that precursor cells in the oligodendroglial lineage are the cell of origin, and reveals unexpected mechanisms mediating tumor viability and invasion, including potassium channel function and EPH receptor signaling. Also highlighted by super-enhancer analysis are genes traditionally involved in neuronal communication. Normal oligodendroglial lineage precursor cell function is critically regulated by interactions with active neurons during development and as part of ongoing neural plasticity. We have recently shown that neuronal activity robustly promotes the proliferation of both normal and neoplastic glial cells. A key mechanism mediating neuronal influences on glioma growth is the activity-regulated secretion of the synaptic protein neuroligin-3 into the glioma microenvironment. Blocking neuroligin-3 secretion by inhibiting the enzyme responsible for its cleavage and release from normal synapses has proven robustly effective in slowing the growth of DIPG and other gliomas in preclinical models. Targeting neuron-glioma interactions represents a promising and largely unexplored avenue for glioma therapy. A therapeutic strategy is thus emerging that involves a multipronged approach, targeting the cell-intrinsic vulnerabilities of histone-3 mutant gliomas together with key microenvironmental targets central to glioma growth and progression. Increasingly effective therapy for these deadly brain cancers of childhood will be enabled by answers to outstanding basic questions, such as why H3K27M-mutant gliomas occur only in midline structures, how the tumors hijack mechanisms of normal neural development and plasticity, and what mechanisms the tumor utilizes to evolve resistance to therapy. Citation Format: Michelle Monje. Histone-3K27M mutant diffuse midline gliomas of childhood: Epigenetic pathogenesis and therapeutic opportunities [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr PL05-03.