MDB-14. MULTI-OMICS OF HUMAN MEDULLOBLASTOMAS AND GENETIC LINEAGE TRACING IN VIVO IDENTIFY REGULATORS OF STEM-CELL MAINTENANCE

Abstract

Abstract Recent studies implicate the glutamatergic lineage of the rhombic lip (RL) as the origin of group 3/4 medulloblastomas (MBs). MB stem cells resemble stalled RL progenitors and targeting common pluripotency factors induces differentiated phenotypes. However, the genetic and epigenetic regulators of stemness in MB remain incompletely understood. We profiled tissues resected from 38 patients who underwent surgery for MB at UCSF via single-nucleus RNA sequencing (snRNA-seq). Of these, 13 were also subjected to single-cell assay for transposase-accessible chromatin by sequencing (scATAC-seq). These assays generated 560,509 and 71,276 cells respectively, after quality control. Motif and latent-space analyses were used to infer cell-type specific transcription-factor expression and binding, and associated cis-regulatory enhancers. Further, we performed single-cell genetic lineage tracing on xenografts obtained from two distinct group-3/4 patient-derived lines, engineered to express heritable barcodes. We trained a machine-learning classifier on public snRNA-seq of human RL and classified Nestin-high quiescent stem-like cells, Nestin-moderate/ASPM-high proliferating progenitor cells, and glutamatergic lineage cells at various stages of differentiation in our MB clinical specimens and xenografts. Integrated analysis of these datasets maps the transition from quiescent to proliferating stem-like MB cells to more differentiated cell types at the level of transcription-factor activity and binding. We found that both the expression and binding of epigenetic regulators SUZ12, EZH2, DNMT1, and BRD7/8 were high in stem cells, but attenuated during lineage commitment. This was also the case for genes known to maintain neural stem cells in the developing brain, e.g. LIN28, FOXO3, HES6, LHX1/2/4, NUP98. Comparative analysis of developmental and MB gene expression, with motif analysis, implicates a conserved embryonal mechanism for indefinite replicative division in MB stem-like cells. These studies provide a single-cell atlas of transcriptomic and epigenetic cellular states in human MB, a map of MB cellular hierarchical organization and transcription-factor regulation of stem-cell maintenance in vivo.