MDB-06. ALTERNATIVE SPLICING MEDIATED BY OTX2 DRIVES THE DEVELOPMENT OF GROUP 3 MEDULLOBLASTOMA

Abstract

Abstract BACKGROUND OTX2 is a transcription factor and known molecular hallmark of Group 3 medulloblastoma (MB). While previous studies have focused on its association with active enhancer elements to modulate the chromatin landscape, here, we demonstrate an unexpected role for OTX2 in regulating Group 3 MB alternative splicing (AS). METHODS We used TurboID to broadly interrogate the Group 3 MB OTX2 protein interactome and identify associations with splicing regulators. RNA-sequencing was performed to identify OTX2-driven AS events, a subset of which were validated in Group 3 and Group 4 MB patient tumors and the developing human cerebellar rhombic lip (RL). Splice blocking morpholinos were used to mimic OTX2-regulated splicing events and to determine their role in MB cell fate decisions both in vitro and in vivo. RESULTS OTX2 associates with the Large Assembly of Splicing Regulators (LASR) complex through protein-protein interactions and modulates the localization of a LASR protein to help stabilize the complex and sustain a Group 3 MB splicing program. Motif enrichment analysis demonstrates that members of the LASR complex are also significantly enriched near OTX2-regulated alternatively spliced exons. OTX2 is found on nascent mRNA, and its role in AS is independent of DNA-regulatory functions. OTX2 controls a pro-tumorigenic splicing program that is mirrored in the human cerebellar RL origins. Among the OTX2-regulated spliced genes, PPHLN1 is expressed in the most primitive rhombic lip stem cells, and targeting PPHLN1 splicing reduces tumor growth, while enhancing differentiation and survival in vivo. CONCLUSIONS While splicing defects are commonly observed in adult cancers, the functional role of AS in the most aggressive MBs has never been explored. These findings identify OTX2-mediated AS as a major determinant of cell fate decisions and highlight AS events as novel therapeutic targets to drive Group 3 MB differentiation.