bioRxiv2025
Simple SummaryDiffuse midline gliomas (DMGs) are aggressive childhood brain tumours with no effective treatments. Over 80% of cases carry the histone H3K27M mutation, which alters chromatin structure and gene regulation and induce tumours growth. Immunotherapy, which uses the bodys immune system to fight cancer, relies on tumour- specific antigen molecules that immune cells can recognise. However, because DMGs have few mutations, finding suitable antigens for therapeutic purposes has been challenging. In this study, we investigated how the H3K27M mutation affects tumour antigen presentation in DMG. Using patient-derived DMG models, we found that H3K27M alters the landscape of antigens displayed on tumour cell surface, creating unique immune targets. We identified six immunogenic peptides, that triggered strong T cell responses. These antigens were absent when H3K27M was removed, confirming their link to the mutation. Our findings provide a blueprint for developing T cell-based immunotherapies for DMG, offering new hope for targeted treatments against this devastating disease. AbstractO_ST_ABSBackgroundC_ST_ABSDiffuse midline gliomas (DMGs) are among the most aggressive paediatric brain tumours, with the pathognomonic H3K27M mutation present in over 80% of cases. This mutation drives epigenetic dysregulation and transcriptional reprogramming, yet its impact on the tumour antigenic landscape remains poorly understood. Given the low mutational burden of DMG, an expanded search beyond neoantigens to include epigenetically dysregulated tumour-associated antigens (TAAs) is critical for advancing antigen-specific immunotherapies. MethodsTo assess how H3K27M influences antigenic landscape of DMG, we performed a comprehensive immunopeptidomic analysis using patient-derived DMG cell line models (SU-DIPG13 and BT245) that harbour the H3K27M mutation and their CRISPR-edited H3K27M-knockout (KO) counterparts. High-resolution mass spectrometry and bioinformatics were employed to define H3K27M-driven changes in the immunopeptidome. Functional T cell assays using HLA-matched healthy donor PBMCs were conducted to evaluate the immunogenicity of H3K27M-associated peptides. ResultsOur findings reveal that the H3K27M mutation reshapes the tumour antigenic landscape in a model-specific manner. While H3K27M knockout increased HLA-I expression in SU-DIPG13 but not BT245, immunopeptidomic profiling uncovered distinct shifts in the presentation of tumour-associated peptides, independent of direct effects on antigen processing machinery. Among these, we identified six immunogenic peptides, derived from SLITRK2, PRAME, XKR5, and CBX2, that elicited CD8 T cell responses in in vitro functional assays. Notably, PRAME, a well-characterised cancer-testis antigen was confirmed as an H3K27M-associated immunogenic target, reinforcing its therapeutic relevance. Peptides identified exclusively in H3K27M+ cells were absent in KO models, demonstrating a direct link between H3K27M-driven transcriptional dysregulation and tumour antigenicity. ConclusionsThis study provides the first systematic assessment of how H3K27M reshapes the antigenic landscape in DMG, uncovering novel, immunogenic tumour- associated peptides that could serve as targets for precision immunotherapy. By demonstrating that H3K27M mutation drives context-dependent antigen presentation, our findings establish a foundation for T cell-based therapies targeting H3K27M-associated antigens. These insights pave the way for next-generation personalised immunotherapies for this otherwise treatment-refractory disease. Key PointsH3K27M mutation induces expression of tumour-associated antigens in DMG H3K27M alters the DMG immunopeptidome without uniformly changing HLA-I levels PRAME- and CBX2-derived peptides are immunogenic and targetable by CD8 T cells Importance of StudyDiffuse midline gliomas (DMGs) are universally fatal paediatric brain tumours with limited treatment options and poor immune visibility. While the H3K27M mutation is a defining hallmark, its impact on tumour immunogenicity remains unclear. This study presents the first comprehensive to explore the effect of H3K27M-mution on the DMG immunopeptidome, revealing six immunogenic peptides derived from epigenetically dysregulated tumour- associated antigens, including SLITRK2, PRAME, XKR5, and CBX2. These antigens elicited CD8 T cell responses, establishing a direct link between H3K27M-driven transcriptional dysregulation and tumour antigenicity. By leveraging these altered antigens, we highlight actionable vulnerabilities for T cell-based immunotherapy.