De Micheli, A. J.; de Biagi-Junior, C. O. A.; Lo Cascio, C.; Machaalani, C.; Postlmayr, A.; Katiyar, S.; Maas, R.; Kancherla, V.; Reimann, R.; Zapotocky, M.; Nobre, L. F.; Eder, S. K.; Rozowsky, J. S.; Ribierre, T.; Zenk, F.; Resnick, A.; Clarke, M.; Gojo, J.; Tabori, U.; Hawkins, C.; Jones, C.; Cavalli, F. M. G.; Filbin, M. G.; Guerreiro Stucklin, A. S.

Pediatric cancers are thought to arise from dysregulation of developmental programs, otherwise tightly regulated in time and space. Infant-type hemispheric gliomas (IHGs) arise in early childhood, driven by characteristic ALK/ROS1/MET/NTRK receptor tyrosine kinase (RTK) gene fusions. We dissected the cellular hierarchies of 24 fusion-positive gliomas, spanning infants through adolescents, using single-cell and single-nucleus RNA/ATAC-seq, and spatial transcriptomics. We identified five cancer cell states, with radial glia-like cells at the apex of a neoplastic hierarchy resembling neuronal- and glial-like trajectories. Neuronal-like cells were enriched in most IHGs but diminished in ROS1-fused IHGs and older patients. Integration of chromatin profiling revealed FOS/JUN-driven oncogenic programs and high inferred plasticity across all cancer cell populations. Myeloid cells, the most abundant non-neoplastic population, comprised distinct subgroups, suggesting context-dependent functions. Despite lacking high-order structure, spatial transcriptomics revealed discrete cellular niches within IHGs. Collectively, our findings elucidate the cellular states and developmental programs underlying IHGs and RTK-fused gliomas in older patients, opening new avenues for research and therapy innovation.