Poster Presentation 46th Lorne Genome Conference 2025

Molecular and Cellular Characterisation of the Novel EZHIP-expressing Subtype in Diffuse Midline Glioma  (#121)

Afraah Cassim 1 2 , Sarah Fox 1 , Yolanda Colino Sanguino 1 , Evangeline Jackson 3 4 , Ryan Duchatel 3 4 , Javad Nazarian 5 6 , Matthew Dun 3 4 , David Gallego Ortega 2 , Fatima Valdes Mora 1 2 7
  1. Childrens Cancer Institute, Randwick, NSW, Australia
  2. School of Biomedical Engineering, University of Technology , Sydney , NSW, Australia
  3. Cancer Signalling Research Group, School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle,, Newcastle, NSW, Australia
  4. Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, Australia
  5. DIPG/DMG Research Center Zurich, Children’s Research Center, , University Children’s Hospital , Zurich , Zurich, Switzerland
  6. Center for Genetic Medicine Research,, Children’s National Hospital, Washington , DC, USA
  7. School of Clinical Medicine, Faculty of Medicine & Health, University of New South Wales, Sydney , NSW, Australia

Background: Diffuse midline glioma (DMG) is a terminal paediatric brain cancer, with no current cure. DMG is primarily caused by the epigenetic histone mutation, H3K27M. In 2020, a novel DMG subtype was discovered for cases lacking H3K27M, where instead, these patients abnormally expressed the Enhancer of zeste inhibitory protein (EZHIP). Interestingly, EZHIP is a ‘mimic’ of H3K27M due to sequence similarities.  

We hypothesize that EZHIP is an oncogenic driver and that epigenetic similarities may exist between EZHIP-DMG and H3K27M-DMGs, which may concur with a common epigenetic therapy for DMG subtypes. Else, EZHIP-specific targeting approaches may be required.

We aim to establish the epigenetic profile of the novel EZHIP-DMG subtype in comparison to other DMGs and explore the EZHIP-specific effects to provide intuition on the best therapeutic strategy.

Methodology: To compare epigenetic profiles of DMG subtypes, patient-derived DMG cell lines expressing EZHIP or H3K27M, H3-wildtype (without EZHIP) and normal controls were used. Epigenetic profiling was performed using ATAC-sequencing, and ChIP-sequencing for 6 histone post translational modifications. Next, we ectopically expressed EZHIP in 2 DMG cell lines lacking EZHIP/H3K27M, to evaluate the direct pro-oncogenic role of EZHIP and compared the effects of cellular metabolism and colony formation.

Results: Epigenetically, EZHIP-DMG is similar to H3K27M-DMG subtypes, but with distinct characteristics in glial cell differentiation for EZHIP-DMG. In EZHIP-induced cell lines, a global reduction of the epigenetic H3K27me3 mark was detected compared to non-induced controls, similar to the H3K27M oncohistone effect. Additionally, EZHIP-induced cell lines displayed higher cell metabolism and colony formation compared to non-induced controls, confirming that EZHIP is pro-oncogenic.

Conclusion: This study suggests that EZHIP epigenetically mimics H3K27M and confirms that EZHIP promotes oncogenesis in DMG, with direct overall effects on the epigenome. Taken together, epigenetic similarities between EZHIP and H3K27M may be exploited as a therapeutic approach for EZHIP-DMGs.