Diffuse midline gliomas (DMG) are highly aggressive malignant brain tumours that arise in children. Despite decades of effort and clinical trials, the prognosis of DMG remains invariably fatal. Further, they are one of many childhood cancers recognised as diseases of dysregulated development. The current standard of care is for patients to receive radiotherapy. Other therapies are offered in an adjuvant setting should actionable targets be identified through molecular genetic profiling. DMGs are driven by K27M mutations in histone 3 variant genes, HIST1H3B or H3F3A encoding Histone 3.1 and 3.3, respectively. The K27M mutation prevents tri-methylation at residue K27 and results in widespread epigenetic dysregulation and a stalled developmental phenotype. Published work has not yet identified a means to overcome stalled differentiation in these malignant brain tumours. H3K27M mutations halt the differentiation process of oligodendroglial progenitor cells (OPC) to oligodendrocytes. OPCs have been identified as the putative cell-of-origin of DMG as determined via single cell RNA-sequencing. The aim of our research is to target the cells-of-origin of DMG and reprogram their cell fate trajectories towards mature glial cells. To investigate potential druggable targets, we will use a small epigenetic library of 775 compounds and assess differentiation and proliferation in patient-derived DMG cell lines. Altered differentiation will be quantified by a global loss-in-signal of neural progenitor stem cell markers Nestin and SOX2 using image-based analysis. Proliferation will be quantified by changes in nuclear staining with DAPI. We have established proof-of-concept for assessment of patient-derived DMG cell lines in 384-well plates with fluorescence and bright-field image analysis. Pipeline design, optimisation, and analysis of proliferation and differentiation will be presented. Future experiments will validate drugs identified from the drug screen and explore effects in DMG cells in vitro and in tumorigenic models in vivo, as well as assessing changes in chromatin accessibility and gene expression. The areas of immunotherapy, targeted therapy and chemotherapy have shown promise in the last three years to prolong the life of DMG patients or lessen their symptoms, but it is still an early area of research that requires more mechanistic and functional understanding to find a reliable treatment.