CRISPR screening in human iPSC-derived glioblastoma models reveals RNA-binding protein dependencies associated with TERT promoter mutation

IDH-wildtype glioblastoma (GBM) is a highly aggressive adult brain tumor with an extremely poor prognosis, characterized by multilayered intratumor heterogeneity. Telomerase reverse transcriptase (TERT) promoter mutation, the most frequent driver mutation in GBM, is homogeneously present within tumors, offering the potential for comprehensive and selective eradication of heterogeneous GBM populations. However, targeting cells expressing TERT in a mutation-dependent manner has not been feasible, largely due to the adverse effects of existing telomerase inhibitors and the inability to block GABPA, a transcription factor that selectively activates the mutant promoter. These obstacles highlight the need to identify novel actionable vulnerabilities. Previous GBM models, such as patient-derived xenografts and genetically engineered mouse models, have failed to provide such insights due to lack of experimental standardization and human-to-mouse variations in telomere biology and TERT promoter. To bypass these challenges, we leveraged human iPSCs to engineer GBM models harboring either wildtype or mutant TERT promoter, enabling discovery of synthetic lethal therapeutic targets. Performing a pooled CRISPR knockout screen targeting 1078 RNA-binding proteins (RBPs), we identified 40 RBPs whose depletion selectively impaired survival of TERT promoter-mutant (TPM) cells without affecting wildtype (TPW) cells. Intriguingly, these included 3 RBPs that form a methyltransferase complex governing N 6 -methyladenosine (m 6 A) modification; METTL14, METTL3, and RBM15. Functional analyses showed that METTL14 depletion downregulates GABPA mRNA level independent of TERT promoter status but reduces TERT expression specifically in TPM cells. m 6 A-targeted eCLIP-seq revealed m 6 A-independent regulation of GABPA and TERT, indicative of regulatory factor(s) mediating this gene expression cascade. Integrated analysis of m 6 A-eCLIP-seq and RNA-seq uncovered enrichment of transcription factors (TFs) among METTL14 target genes for m 6 A methylation, identifying potential mediators linking METTL14 with GABPA and TERT. These TFs are downregulated upon METTL14 depletion and m 6 A-methylated, with public databases showing ChIP-seq peaks at the GABPA promoter and highly correlated expression with METTL14 and GABPA. We hypothesize that m 6 A methyltransferase machinery stabilizes transcripts of these TFs via m 6 A methylation, driving GABPA transcription and sustaining TERT expression in TPM cells. This study uncovers a novel epitranscriptomic axis regulating TERT and lays the groundwork for developing new therapeutic strategies by leveraging stem cell-derived disease models.