Premalignant Nf1,Trp53-null Oligodendrocyte Precursor Cells BecomeStalled in a Heterogeneous State of Replication Stress BeforeGliomagenesis

Cancer evolves from premalignant clones that accumulate mutations and adopt unusual cell states to achieve transformation. Previously, we pinpointed the oligodendrocyte precursor cell (OPC) as a cell-of-origin for glioma, but the early changes of mutant OPCs during premalignancy remained unknown. Using mice engineered for inducible Nf1-Trp53 loss in OPCs, we acutely isolated labeled mutant OPCs by laser-capture microdissection and determined gene-expression changes by bulk RNA sequencing and a fluctuation analysis, called stochastic profiling, which uses RNA-sequencing measurements from random pools of 10 mutant cells. At 12 days after Nf1-Trp53 deletion, while bulk differences were mostly limited to mitotic hallmarks and genes for ribosome biosynthesis, stochastic profiling revealed a spectrum of stem-progenitor (Axl, Aldh1a1), proneural, and mesenchymal states as potential starting points for gliomagenesis. At 90 days, bulk sequencing detected very few differentially expressed transcripts, whereas stochastic profiling revealed cell states for neurons and mural cells that do not give rise to glial tumors, suggesting cellular dead-ends for gliomagenesis. Importantly, we identified mutant OPCs that strongly expressed key effectors of nonsense-mediated decay (Upf3b) and homology-dependent DNA repair (Rad51c, Slx1b, Ercc4) along with DNA-damage markers suggesting transcription-associated replication stress. Analysis of 10-cell transcriptomes at 90 days identified a locus of elevated gene expression containing an additional repair endonuclease (Mus81) and Rin1, a Ras-Raf antagonist and possible counterbalance to Nf1 loss. At 150 days, Rin1 was microdeleted in some gliomas and downregulated in all others. Replication stress may pose a considerable bottleneck that must be resolved for gliomas to initiate. Statement of significanceIn situ stochastic profiling of heterogeneous cell states in a mouse model of glioma uncovers regulatory confusion in a glioma cell-of-origin and defines a state of replication stress that precedes tumor initiation.