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Heterozygous TERT (Telomerase reverse transcriptase) promoter mutations (TPMs) facilitate TERT expression and are the most frequent mutation in glioblastoma (GBM). A recent analysis revealed this mutation is one of the earliest events in gliomagenesis, however no appropriate human models have been engineered to study the role of this mutation in the initiation of these tumors. To address this, we established GBM models by introducing the heterozygous TPM in human induced pluripotent stem cells (hiPSCs) using a two-step targeting approach in the context of GBM genetic alterations, CDKN2A/B and PTEN deletion, and EGFRvIII overexpression. Orthotopic injection of neuronal precursor cells (NPCs) derived from hiPSCs with TPM into immunodeficient mice did not enhance tumorigenesis compared to TERT promoter wild type (TPW) NPCs at initial in vivo passage which we attribute to relatively long telomeres. We further show that the TPM mutation recruited GA-Binding Protein (GABPA) and engendered low-level TERT expression, resulting in enhanced tumorigenesis upon secondary passage and maintenance of short telomere length as has been reported in human GBM. RNA sequencing of harvested tumors grown as secondary spheres demonstrated upregulated proliferation and mitosis pathway signatures in TPM cells, consistent with their increased in vitro proliferation relative to TPW counterparts. Finally, when secondary TPM and TPW sphere cultures were reinjected into mice, only the TPM led to tumor formation. In summary, our novel GBM model illustrates a growth advantage imparted by heterozygous TPMs in the context of GBM driver mutations relative to isogenic controls, and thereby allows for the identification and validation of TERT promoter-specific vulnerabilities in a genetically accurate background.

Histone H3 K27M mutation is a hallmark mutation for H3 K27M-mutant diffuse midline glioma (DMG), but targeting this mutation has yet to achieve a significant survival benefit in clinical trials. Recent analyses revealed alterations in several genes, such as NF1 and PDGFRA, are observed in substantial subpopulations of H3 K27M-mutant DMG patients in addition to H3 mutation and recurrent TP53 mutations, indicating patient-to-patient tumor heterogeneity and the potential necessity of tailored target therapy for the treatment of this disease. Here, using our human induced pluripotent stem cells (iPSC)-derived glioma avatar platform, we designed DMG models by introducing TP53R248Q with or without heterozygous H3 K27M mutation in combination with further genetic modifications of either NF1 knockout or PDGFRAD842V overexpression to recapitulate DMG subpopulations. Mice with TP53R248Q; H3F3AK27M (QM) tumors survived significantly longer than those harboring QM;NF1-/- (QMN) tumors and QM; PDGFRAD842Voe (QMP) tumors. RNA-sequencing of those induced DMG (iDMG) neurospheres revealed altered patterns of upregulation of MAPK pathway genes both in QMN and QMP-iDMG neurospheres compared to their H3 wildtype counterparts with the same combinations of genetic alterations, suggesting that those additional mutations modifies the oncogenic signaling associated with H3 K27M mutation. Further, differential expression analysis comparing QMN and QMP-iDMG neurospheres revealed 405 differentially expressed genes. Gene set enrichment analysis showed upregulation of transcriptional programs related to mesenchymal signature in QMN-iDMG neurospheres and proneural signature in QMP-iDMG neurosphere as expected. These data show that NF1 deletion and PDGFRAD842V overexpression significantly alter gene expression in H3 K27M-mutant iDMG tumors, potentially opening up a new therapeutic avenue in these devastating tumors with patient-to-patient heterogeneity. Further work using these models will shed light on the development of tailored therapy based on detailed genetic information on each patient sample, such as combining targeted kinase inhibition with HDAC inhibitors that have shown promise in the clinic.

Glioblastomas (GBMs) are the most common malignant primary brain tumors, and the paucity of novel treatments warrants an investigation of its origins and development into aggressive, lethal tumors. Koga & Chaim et al. have recently shown that human pluripotent stem cells (hiPSCs) with different combinations of driver mutations can be differentiated into neural progenitor cells (NPCs) and engrafted into mice to form high grade gliomas (iHGGs). In this work, scRNA seq analysis was used to investigate the development of TP53−/−;PDGFRAΔ8–9 iHGGs, an avatar model that has been shown to recapitulate the proneural subtype of GBM. After re-engrafting the primary avatar cultures (secondary tumor stage), the TP53−/−;PDGFRAΔ8–9 iHGGs developed diverse transcriptional programming and acquired a subpopulation of cells with high expression of known GBM oncogenes, such as MYC, CDK4, and PDGFRA. Notably, when all datasets were aggregated, this oncogene amplifying transcriptional program became the largest source of variation between all stages and replicates of the TP53−/−;PDGFRAΔ8–9 iHGGs. Indicated by a larger total copy number variation (CNV), this oncogene-amplifying program was associated with a genomically unstable developmental state. Trajectory inference could track the development of this population from the initial primary culture of TP53−/−;PDGFRAΔ8–9 iHGG. Differential gene expression analysis identified distinct divergences in clonal evolution––e.g., high expression of the S100 protein family in one cluster––following the acquisition of this genomically unstable state. Lastly, genomic PCR was used to ascertain whether these changes in transcriptional programming were reflected in changes in DNA copy number and identified DNA amplifications of MYC and CDK4. Our scRNA seq analysis of the GBM avatar model platform provides novel insight into how oncogenic states in GBM develop from a small number of driver mutations.