P01.32.b a Recurrent Single Nucleotide Mutation in Tbr1 Promotes Stemness in Group 4 Medulloblastoma

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 TBR1G275C 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 TBR1G275C’s DNA binding sites we perform ChIP-seq in a TBR1G275C patient tumour sample and in a TBR1WT group 4 MB sample. To observe phenotypic effects of TBR1G275C, we generate cortical organoids from human iPSCs edited with CRISPR-Cas9 to harbour a homozygous TBR1G275C 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 TBR1G275C tumours map to the most stem-like compartment of the cerebellar rhombic lip. ChIP-seq reveals changes in the preferred DNA binding sites of TBR1G275C, 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. TBR1G275C 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 TBR1G275C 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.