Abstract Group 3 and Group 4 medulloblastomas (MB) are among the most common and highly fatal pediatric brain cancers, characterized by marked cellular heterogeneity. Limited understanding of the molecular mechanism underlying this heterogeneity presents a profound therapeutic challenge. To understand how the molecular heterogeneity affects tumor biology, we generated a comprehensive single-nucleus multi-omic atlas integrating transcriptomic and chromatin accessibility profiles from 38 Group 3/4 MB spanning all eightmolecular (I-VIII) subtypes. Our analysis shows that these tumors are not fixed entities but instead exist along a continuous landscape of transcriptionally plastic cell states. We identify four principal axes of tumor identity—photoreceptor-like, MYC-driven, precursor-like, and unipolar brush cell–like—governed by interacting transcription factor gene regulatory networks that aberrantly recapitulate cerebellar and retinal developmental programs. Functional interrogation of CRX, EOMES and MYC established their central roles in determining and maintaining tumor cell-states. We show that CRX/NRL-mediated photoreceptor-like identity in Group 3 MB arises from a potentially direct repression of EOMES/LMX1A-driven unipolar brush cell identity, a feature of Group 4 tumors; and vice versa. Notably, clinically interesting intermediate tumors belonging to subtype VII, with the most favorable prognosis among all subtypes, exhibit bi-lineage differentiation trajectories within individual tumors. We identify PAX6 as a key regulator of this bi-lineage trajectory. Its lineage-reprogramming activity suppresses MYC-driven proliferation, induces tumor differentiation, and significantly extends survival in preclinical models, consistent with the favorable clinical outcome in subtype VII patients. Together, our findings establish transcription factor-driven cellular plasticity as a central mechanism underlying Group 3/4 medulloblastoma heterogeneity and identify lineage reprogramming as a clinically relevant and therapeutically exploitable vulnerability.
Silva et al. (Tue,) studied this question.