Abstract
BACKGROUND: Medulloblastoma (MB) is the most common pediatric brain cancer. It arises from the rhombic lip, the germinal zone for the glutamatergic cells of the cerebellum. A unique single-nucleotide mutation (pG275C) in T-box Brain 1 (TBR1), a key transcription factor in neuronal development, is recurrent in the most common subgroup of MB, group 4. The function of this mutation is unknown, but the specificity of the mutation, and increased expression of TBR1 in TBR1(G275C) tumours, suggest a gain of function. MATERIAL AND METHODS: Using bulk transcriptomic data on over 300 group 4 MB tumours, we identify differentially expressed genes (DEG), and leverage published single cell transcriptomic datasets of the developing human cerebellum to situate expression of these DEG in development. To identify changes in TBR1(G275C)’s DNA binding sites we perform ChIP-seq in a TBR1(G275C) patient tumour sample and in a TBR1(WT) group 4 MB sample. To observe phenotypic effects of TBR1(G275C), we generate cortical organoids from human iPSCs edited with CRISPR-Cas9 to harbour a homozygous TBR1(G275C) mutation. Organoids are fixed and stained for markers of neural stemness and differentiation. Performing Co-IP experiments on differentially tagged TBR1 proteins, we investigate the mechanisms behind observed effects of the mutation. RESULTS: Analysis of bulk transcriptomic data shows that upregulated genes in TBR1(G275C) tumours map to the most stem-like compartment of the cerebellar rhombic lip. ChIP-seq reveals changes in the preferred DNA binding sites of TBR1(G275C), with the mutant protein binding the promoters of genes which are developmentally regulated by HES family transcription factors, downstream effectors of Notch signalling that maintain stemness in neural development. TBR1(G275C) cortical organoids exhibit an increase in SOX2+ stem cell rosettes. Co-IP reveals an increase in TBR1 homodimerization caused by the G275C mutation. CONCLUSION: These data support a model in which the TBR1(G275C) mutation increases the stem cell population of group 4 MBs by changing the transcriptional targets of TBR1, potentially through alterations in its dimerization behaviour. Properties of stemness such as self renewal capacity are important to a cancer’s ability to replicate indefinitely, metastasize, and evade traditional therapies, and this project will lead to an increased understanding of the ways in which group 4 MB can achieve these properties.