Atrt-08. the single-cell landscape of pineoblastoma identifies developmental origins and exposes tumorigenic dependencies

Abstract Pineoblastoma (PB), a rare and aggressive brain tumor affecting children, presents with a highly variable age distribution and treatment outcome. Our recent bulk tumor analyses of DNA methylation and mutational landscapes uncovered four discrete PB molecular subgroups (PB-miRNA1, PB-miRNA2, PB-MYC/FOXR2, and PB-RB), providing a major advance in our understanding of biological and clinical heterogeneity. However, developmental origins of PB subgroup heterogeneity and mechanisms governing how specific genetic alterations promote malignancy remain unknown. We conducted single-nucleus RNA sequencing of 38 primary tumors, including cases from all subgroups, to uncover intra-tumoral heterogeneity, developmental origins and expose selective dependencies. Transcriptional programs driving intra-tumoral heterogeneity showed subgroup-specific patterns such as mRNA splicing associated with PB-miRNA1/2 and phototransduction in PB-RB and PB-MYC/FOXR2. Next, we created a single-cell transcriptional atlas of the murine pineal gland across 11 developmental stages and found significant associations between PB subgroups and specific differentiation states of the pinealocyte lineage, suggesting subgroup-specific developmental origins. Characterization of pineal development informed generation of biologically faithful disease models, including a novel genetically engineered mouse model of the PB-RB subgroup. PB-Rb1 mouse tumors were histologically and molecularly validated for their fidelity to human tumor counterparts, exhibiting up-regulation of key pinealocyte lineage markers that are diagnostic in patients. Using cell lines derived from our PB-Rb1 mouse model we identified several transcription factor dependencies we believe are high-jacked during normal pinealocyte development. Our findings inform the developmental origins of pineoblastoma as well as provides candidates for potential therapeutic targets.