INSM1 governs a neuronal progenitor state that drives glioblastoma in a human stem cell model

Glioblastoma (GBM) is an aggressivea lethal brain tumorcancer marked by functional plasticity driven by the interplay between tumor cell-intrinsic mutations and -extrinsic heterogeneity, enabling rapid evolutiontheir interplay with developmental programs. To investigate how canonical GBM glioblastoma mutations promote functional plasticity, we have developeded an isogenic human neural stem cell (NSC) model of glioblastoma GBM by sequential addition of TERT promoter, TP53, and PDGFRA point mutations. TP53 loss-of-function increasesd TERT TERT expression during serial mutagenesis, but only triple mutant NSCs reliably formed lethal brain tumors in vivo that recapitulate glioblastomaGBM. Tumor cell evolution triggersed stress-related metabolic changes and transitionsed toward a neuronal progenitor network driven by transcription factor INSM1. INSM1 is highly expressed in human glioblastoma GBM tumors and, during cortical development, in intermediate progenitor cells (IPC), which give rise to neurons. Remarkably, INSM1 knockdown in triple mutant NSCs and primary glioblastoma GBM cells disrupted disrupts oncogenic gene expression and function and inhibited inhibits the in vivo tumorigenicity of triple mutant NSCs, highlighting the functional importance of an intermediate progenitor cellIPC-like cell state in glioblastoma 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.