TERT promoter C228T mutation in neural progenitors confers growth advantage following telomere shortening in vivo

Abstract

Background: 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.

Method: 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. The impact of the mutation was evaluated through the in vivo passage and in vitro experiment and analysis.

Results: Orthotopic injection of neuronal precursor cells (NPCs) derived from hiPSCs with the TPM into immunodeficient mice did not enhance tumorigenesis compared to TERT promoter wild type NPCs at initial in vivo passage presumably due to relatively long telomeres. However, the mutation recruited GA-Binding Protein and engendered low-level TERT expression resulting in enhanced tumorigenesis and maintenance of short telomeres upon secondary passage as observed in human GBM. These results provide the first insights regarding increased tumorigenesis upon introducing a TPM compared to isogenic controls without TPMs.

Conclusion: Our novel GBM models presented the growth advantage of heterozygous TPMs for the first time in the context of GBM driver mutations relative to isogenic controls, thereby allowing for the identification and validation of TERT promoter-specific vulnerabilities in a genetically accurate background.

Keywords: TERT promoter; genome editing; glioma; neural progenitor cell; telomerase.

Fig. 1

Engineering a heterozygous TERT promoter mutation in hiPSC. a) Schema of CRISPR editing strategy to introduce heterozygous C228T TERT promoter mutation. b) Genotyping PCR and Sanger sequencing confirmation of the TERT promoter mutation. c) RT-qPCR of TERT in engineered hiPSC clones with a TPM or TPW reconstituted promoter region. Data represents mean ± SD.

Fig. 2

Impact of the TERT promoter mutation on tumorigenesis. a) Workflow of the in vivo experiments. b) Survival analysis of the mice implanted with PTEN^−/−; CKDKN2A/2B^−/−; EGFRvIII^OE; TPM or TPW NPCs. 3 mice for each NPC clone were injected for the analysis. c) H&E staining of the parental, TPW, and TPM primary tumors illustrating pseudopalisading necrosis (arrow). Scale bars indicate 100 µm. d) RT-qPCR of Pax6, PDGFRA, and GFAP in 4 injected NPC lines and 4 primary tumor sphere lines (2 TPW and 2 TPM). Data represents mean ± SD (*P < .05, n.s = not significant).

Fig. 3

Functional analysis of the TPW and TPW primary tumor spheres. a) Schema explaining the cell lines used. b) TERT RT-qPCR of the 4 biological replicates of the TPM and TPW primary tumor spheres. c) Telomerase activity of the 4 biological replicates of the TPM and TPW primary tumor spheres. (P: positive control, N: negative control). d) GABPA ChIP-qPCR of the 2 TPM and 2 TPW primary sphere cell lines. The quantitative results of qPCR from IgG ChIP and GABPA ChIP DNA using TERT promoter lesion primers (left half) and negative control primers (right half) are shown. e) Sanger sequencing results of ChIP-PCR products. f) Average telomere length in the TPM and TPW primary tumor spheres. Data represent mean ± SD (***P < .0001, n.s = not significant).

Fig. 4

Impact of the TERT promoter mutation upon serial in vivo passage. a) Workflow schema of in vivo experiments. b) Graph represents the number of hNuMA-positive cells at each level of sectioned brains in the mice implanted with the 4 TPM and 4 TPW primary sphere cell lines. Data represents mean ± SEM (*P < .05). c) Representative images of hNuMA, vimentin, and Ki-67 staining of the TPM and TPW secondary tumors in the sections 2.4 mm posterior to the bregma.

Fig. 5

Function of the TERT promoter mutation in serial passaged tumors. a) TERT RT-qPCR of the primary/secondary TPM and TPW tumor spheres. b) Differential gene expression analysis of the TPM (n = 3) and TPW (n = 3) secondary tumor spheres. Volcano plot showing gene expression comparison between the TPM and TPW tumor spheres, with significant genes colored in teal. c) Gene ontology networks of the genes differentially expressed between the TPM and TPW secondary tumor spheres are shown. d) Average telomere length in the TPM and TPW secondary spheres. All data represents mean ± SD (*P < .05, n.s = not significant).

Fig. 6

TERT promoter mutant tumors maintain growth capacity and telomere length during serial passage. a) Survival curve for tumor associated death of TPM and TPW secondary sphere-injected mice. b) Representative histological staining of the TPM tertiary tumor (left H&E, right human nuclei staining. Bar 5 mm). c) Relative TERT expression in the TPM secondary spheres compared to the tertiary tumor spheres. d) Relative telomere lengths in the TPM secondary and tertiary spheres. e) Growth rate comparison between the TPM tertiary (red) and secondary (black) spheres. All Data represents mean ± SD (*P < .05, n.s = not significant).