MULTI-OMIC INTEGRATION REVEALS INTRA-TUMOUR HETEROGENEITY AND IDENTIfiES DISULfiRAM AND COPPER AS A SYNERGISTIC THERAPY IN PAEDIATRIC EPENDYMOMA

Abstract AIMS Ependymoma (EPN) is the second most malignant paediatric brain tumor. The PF-A subgroup, associated with a hypoxic microenvironment and overexpression of the epigenetic regulator EZHIP, has a dismal survival rate of 50%, with clinical trials failing to demonstrate a significant survival advantage from chemotherapy to date. We present a multi-omic integration investigating whether spatially distinct tumour microenvironments represent targetable metabolic niches in PF-A EPN and evaluate the broader efficacy of our lead drug combination. METHODS Surgical sampling of spatially-distinct regions from eight PF-A patients was performed, with metabolites and RNA simultaneously extracted and analysed using LC-MS and RNA-seq. Integration of metabolites and RNA was performed using Metscape3 with functional assays evaluating proliferation and invasion in 2D and 3D patient-derived in-vitro cell models. qPCR and LC-MS were used to analyse post-treatment changes of our lead drug combination under hypoxia and normoxia. RESULTS Multi-omic integration identified 124 dysregulated metabolic pathways, demonstrating heterogeneity within and across PF-A tumours. Based on the identified metabolically relevant genes, Disulfiram (Dsf) and Cu2+ were highlighted as potential therapeutic agents showing impaired metabolic viability and invasion in 2D and 3D models of PF-A EPN, with limited chemosensitivity observed in human cerebellar astrocytes. Under normoxic conditions, LC-MS showed that Dsf/Cu2+ disrupts mitochondrial fatty acid oxidation and membrane lipid in- tegrity, whereas qPCR analysis revealed changes in EPOP, EZH2 and PSMB7, implicating EZHIP-mediated regu- lation and proteasomal alterations. Furthermore, this combination also demonstrated efficacy in AT/RT, medul- loblastoma and paediatric glioma in-vitro models, suggesting pan-cancer potential. CONCLUSION This is the first instance where multi-omic data integration and intra-tumor heterogeneity have been inves- tigated for paediatric EPN, revealing novel therapeutic targets in the context of gene-metabolite correlations. Ongoing in-vivo studies using the MAF-928 model are assessing drug tolerability, biodistribution and pathway dependencies. Additionally, radiosensitization potential is being evaluated to reduce radiation-associated toxi- cities in children and enhance clinical translation.