| Description |
Glioblastoma (GBM) is an aggressive, incurable brain tumor driven by heterogeneity between tumor cell-intrinsic and -extrinsic states for rapid evolution. To investigate how canonical GBM mutations promote functional plasticity, we developed an isogenic human neural stem cell (NSC) model of GBM by sequential addition of TERT promoter, TP53, and PDGFRA point mutations. TP53 loss-of-function increased TERT expression during serial mutagenesis, but only triple mutant NSCs reliably formed lethal in vivo tumors that recapitulate GBM. Tumor cell evolution began with stress-related metabolic changes and transitioned toward neuronal progenitor networks driven by transcription factor INSM1, which is highly expressed in human GBM tumors and intermediate progenitor cells during development that give rise to neuronal progenitors. Remarkably, inhibiting INSM1 in triple mutant NSCs caused reversal of oncogenic gene expression and functions to those of non-tumorigenic wildtype NSCs. These findings highlight the functional importance of an aberrant intermediate neuronal progenitor state in GBM pathogenesis.
Overall design: Proneural in vitro serial mutagenesis scRNA-seq: H1 human embryonic stem cells (hESCs) were CRISPR edited to create single mutant TERTp C228T and double mutant TERTp+TP53 G743A hESCs. Cells exposed to CRISPR reagents but non-mutagenized were wildtype (WT) controls. WT, single, and double mutant hESCs were differentiated to neural stem cells and transduced with empty vector or mutPDGFRA D842V expression construct to create 4 Proneural genotypes: WT, single mutant TERTp, double mutant TERTp+TP53, and triple mutant TERTp+TP53+mutPDGFRA (PRO) engineered neural stem cells (eNSCs). Proneural genotypes were uniquely labeled with hashtag oligos (HTOs) and pooled for multiplexed scRNA-seq. Proneural in vitro multiomic scRNA+scATAC-seq: 2 biological replicates (independent differentiation, same genetic clone) of WT and PRO eNSCs were profiled for joint gene expression and chromatin accessibility at single cell resolution. Each condition was submitted independently for sequencing. Proneural in vivo scRNA-seq: PRO eNSCs were intracranially xenografting in the striatum of athymic nude mice, which formed tumors and caused animal death within 109 days. Tumors harvested from 3 mice (mouse-35, mouse-38, mouse-42) 100 days post-injection were dissociated into single cells and tumor cells were isolated by FACS for double human nuclear antigen+/CD45- populations. Tumors cells from each mouse were submitted independently for scRNA-seq. PRO eNSC in vitro RNAi scRNA-seq: PRO eNSCs were transduced with two independent RNA inhibition (RNAi) lentivirus constructs targeting transcription factors INSM1, TCF4, or SOX4, versus non-target control plasmid. For each gene, RNAi conditions (shCtrl vs. 2x RNAi) were uniquely labeled with HTOs and pooled for multiplexed scRNA-seq, resulting in 3 datasets with 3 samples each. |
