STEM-09. A GROUP 3 MEDULLOBLASTOMA STEM CELL PROGRAM IS MAINTAINED BY OTX2-MEDIATED ALTERNATIVE SPLICING

Abstract Group 3 medulloblastomas (MBs) are highly metastatic pediatric brain tumors with limited therapeutic options. The transcription factor orthodenticle homeobox 2 (OTX2) is a known driver and molecular hallmark of Group 3 MB. We previously demonstrated that OTX2 regulates Group 3 MB cell fate decisions that favor self-renewal or stemness and the retention of a primitive cerebellar rhombic lip signature. Here, we show that OTX2-regulated alternative splicing (AS) is a major driver of Group 3 MB progression. Surprisingly, the functional role of post-transcriptional events, such as AS, in Group 3 MB development has never been explored. This gene regulatory layer represents an untapped source of therapeutic targets. Using a combination of TurboID, RNA-sequencing and single cell RNA-seq, we characterized the Group 3 primary MB AS landscape with a focus on genes that control MB stem cells. OTX2 associates with the multimeric Large Assembly of Splicing Regulators (LASR) complex through protein-protein interactions and can directly or indirectly bind RNA. Together, OTX2 and LASR complex proteins oversee a pro-tumorigenic splicing program that is mirrored in the human cerebellar rhombic lip origins and is associated with Group 3γ MBs that exhibit the worst prognosis. Among our core set of OTX2-regulated spliced genes, periphilin 1 (PPHLN1) is expressed in the most primitive cerebellar rhombic lip stem cells, and mimicking PPHLN1 AS by pre-treating cells with a splice blocking antisense morpholino reduces tumor growth while increasing survival in vivo. We propose that OTX2-mediated AS is a key determinant of cell fate decisions favoring the maintenance of the stem cell state. Targeting transcription factors like OTX2 is notoriously difficult; thus, our work offers fresh opportunities to exploit MB-specific AS events therapeutically to abrogate tumor progression.