6093 Characterising KBTBD4 mutations in paediatric medulloblastoma

Objectives Medulloblastoma (MB) is the most prevalent paediatric malignant brain tumour.¹ ² Survival rates vary, and of the four molecular subgroups, MBgroup3 has the poorest prognosis, frequently metastatic at diagnosis.³ Recent studies have suggested the role of hotspot mutations in the Kelch domain of KBTBD4 contributing to the dismal outcomes and treatment failure in this group.⁴ This project aimed to interrogate the role of KBTBD4 mutations in MBgroup3 tumorigenesis and investigate their impact in chemotherapy resistance utilising a number of in vitro experiments. Methods Novel MB cell lines (D425 and D283) harbouring doxycycline inducible KBTBD4 mutations were utilised to investigate the proliferative, morphological and migratory changes associated with the mutations, using microscopy, scratch and CellTiter-Glo assays. RNA-seq data was analysed to identify biological pathways upregulated in KBTBD4 mutant versus wildtype models. Chemotherapy resistance associated with KBTBD4 mutant cell lines was investigated using standard of care agents (cisplatin, cyclophosphamide, lomustine), then dose response curves and IC50 values were compared. Data analysis was performed using GraphPad Prism (version 6.07). Results In D283 cells, KBTBD4 mutations induced changes to morphology and migratory capacity, compared to the corresponding wildtype lines. The scratch assay demonstrated a significant difference in the wound closure, and therefore migration of the KBTBD4 mutant versus wildtype cells. Figure 1 highlights these results as a grouped bar chart, demonstrating the% wound closure for wildtype and mutant KBTBD4 cells over a 48hr period. Gene set enrichment analysis of RNA-seq data demonstrated KBTBD4 mutations promoted upregulation of genesets involved in Epithelial Mesenchymal Transition (EMT), and KEGG pathway analysis revealed upregulation of downstream signalling pathways involved in EMT and angiogenesis. However, investigations did not suggest KBTBD4 mutations significantly increased proliferative capacity of either the D283 or D425 cell lines and did not reveal a convincing chemoresistant phenotype in the cells. Further investigations should be carried out to validate this. Conclusion The results from this project have established KBTBD4 as a novel driver of MBgroup3 tumorigenesis, driving a migratory change to the cells, promoting EMT and angiogenesis to enhance metastatic potential. Future research should be conducted to explore this mechanism further, to direct the development of targeted therapeutics and optimise treatment of group 3 disease. References Suk Y, Gwynne WD, Burns I, Venugopal C, Singh SK. Childhood medulloblastoma: an overview. Methods in Molecular Biology 2022. Northcott PA, Robinson GW, Kratz CP, Mabbott DJ, Pomeroy SL, Clifford SC. Medulloblastoma. Nature Reviews Disease Primers. 2019. Hovestadt V, Ayrault O, Swartling FJ, Robinson GW, Pfister SM, Northcott PA. Medulloblastomics revisited: biological and clinical insights from thousands of patients. Nature Reviews Cancer. 2020. Chen Z, Ioris RM, Richardson S, Van Ess AN, Vendrell I, Kessler BM. Disease-associated KBTBD4 mutations in medulloblastoma elicit neomorphic ubiquitylation activity to promote CoREST degradation, Cell Death & Differentiation, 2022.