GSK3 acts as a switch for transcriptional programs in a model of low-grade gliomagenesis

Abstract

Mutations in isocitrate dehydrogenase (IDH)1/2 are defining drivers of low-grade gliomagenesis. However, mutant IDH alone is not sufficient for malignant transformation, and additional events are required for the development of low-grade glioma. While specific genetic lesions have been identified to contribute to low-grade gliomagenesis, less is known about the signaling pathways involved in the acquisition of malignancy. To identify prerequisites of IDH mutant tumorigenesis, we modulated pathways previously implicated in glioma initiation using a tractable in vitro model system for early IDH1^R132H-dependent gliomagenesis. Through the use of chemical compounds, we targeted WNT/GSK3, TGF-β and NOTCH-signaling, assessing their functional, transcriptional, and translational impacts. Expression of LGG-related marker L1CAM was affected by perturbation of all pathways, though only modulation of WNT/GSK3-signaling resulted in profound molecular transformation, including glioma-associated genes and programs regulating cellular architecture and cell replication. This was accompanied by altered cell morphology, migration capacity, and enhanced proliferation. Transcription factor RUNX2 was identified as a potential downstream effector, whose inhibition abrogated cell proliferation. Disrupted WNT/GSK3 signaling in a model system of early low-grade gliomagenesis fundamentally impacted cell fate, as demonstrated by a reshaped transcriptional landscape, aberrant transcription factor activity, extracellular matrix restructuring, and altered proliferation capacity. Our data suggests that GSK3 may play a central role during low-grade gliomagenesis, warranting further investigation.

Keywords: GSK3; Glioma; IDH mutation; IDHmut-gliomagenesis; WNT.

Fig. 1

GSK3 inhibition disturbs IDH1^R132H induced transcriptional programs. A Experimental Design. Chemical inhibition of WNT/GSK3 (CHIR99021), TGF-β (Repsox) and γ-secretase (YO-01027) in immortalized conditional IDH1^R132H astrocytes. B Flow cytometry after treatment with CHIR99021, Repsox and YO-01027 showed significantly changed expression of LGG-associated marker L1CAM (n = 2 biological and 3 technical replicates each). C Multidimensional scaling analysis of RNA-seq data showing segregation of CHIR99021- and Repsox-treated samples from control samples. D Heatmaps of all differentially expressed genes upon treatment with CHIR99021, Repsox, or YO-01027, showing most profound transcriptional reshaping after GSK3 inhibition. E Distribution of up- and downregulated DEGs after GSK3 inhibition, showing a higher fraction of downregulated genes, including those relevantin IDH^mut gliomas. F Gene expression of L1CAM and GSK3B is positively correlated in IDH^mut glioma patient cohorts (R = 0.29, p = 0.0017) (source: CGGA mRNA dataset, only primary IDH^mut glioma from cases included). Correlation was determined with Pearson’s correlation test. G GSK3B is significantly overexpressed in IDH^mut gliomas compared to IDH^wt gliomas (Wilcoxon test, p = 5.9 × 10^− 7; source: CGGA)

Fig. 2

Reduced migration and altered morphology upon GSK3 inhibition. A Gene ontology analysis of intersected RNA-seq and mass spectrometry data demonstrates significant enrichment for wound healing, cell adhesion and ECM related terms among downregulated genes and proteins after GSK3-inhibition with CHIR99021. B Heatmap of selected candidates within the GO wound healing family. PDGFRA is among the significantly downregulated genes affecting migratory capacity. C After inflicting a scratch wound, CHIR99021-treated IDH1^R132H astrocytes display decreased migration capacity over time, as shown in representative images. Wound width at different timepoints highlighted in grey (control) and light green (CHIR99021. Scale bar = 400 μm. Statistical analysis confirms that wound healing (migration) was significantly reduced after CHIR99021 treatment (non-linear regression curve analysis, p < 0.0001–0.0356) (n = 3 biological and 4 technical replicates each). D Immunofluorescence with DAPI (blue) and Phalloidin (red) staining demonstrates altered cell morphology upon GSK3 inhibition with reduced cell size and cell spreading. Scale bar = 20 μm. (n = 3 technical replicates). E Comparison of maximum cell diameters shows a significantly reduced cell diameter after treatment with CHIR99021 (p < 0.0001) (n = 3 biological replicates). F, G Simplified schematic of FAK/RhoA/ROCK interaction and corresponding heatmap of the respective genes upon modulation with CHIR99021

Fig. 3

Increased proliferative capacity and clonogenicity after GSK3 inhibition is associated with RUNX2 overexpression. A Gene ontology analysis of intersected RNA-seq and mass spectrometry data demonstrates significant enrichment for terms associated with cell proliferation among upregulated genes and proteins upon CHIR99021 treatment. B Cell cycle analysis using EdU flow cytometry showing a significant increase in S-phase (p = 0.0008–0.00001) along with a significant reduction of cells in G0/G1 (p = 0.00153–0.002919) and inconsistent reduction of G2/M (p = ns – 0.000025), indicating increased cell cycling (n = 3 biological and 3 technical replicates each). C Treatment of IDH1^R132H astrocytes with CHIR99021 significantly enhances clonogenic proliferation (p < 0.0001 – p = 0.0024) (n = 3 biological and 3 technical replicates each). D EnrichR transcription factor analysis reveals RUNX2 among the top 5 transcription factors regulating the expression of both up- and downregulated DEGs. E CPM expression profiles of the top transcription factors identified with EnrichR emphasizes RUNX2 as the only transcription factor significantly upregulated upon GSK3 inhibition

Fig. 4

RUNX2 knockdown in CHIR99021 treated cells decreases proliferation and clonal expansion potential. A Negative correlation of RUNX2 and L1CAM expression in IDH^mut glioma patients (R = –0.313, p = 0.000648) (source: CGGA, mRNA dataset; only primary IDH^mut gliomas included). Correlation was determined using Pearson’s correlation test. B RUNX2 is significantly overexpressed in IDH^wt compared to IDH^mut gliomas (p = 8.5 × 10^− 11) (source: CGGA). C High RUNX2 expression is associated with significantly impaired survival in patients with IDH^mut gliomas (source: Gliovis/CGGA). D qPCR analysis of RUNX2 expression after siRNA-mediated knockdown in CHIR99021-treated IDH^mut astrocytes shows reduced RUNX2 expression compared to control (n = 2 biological and n = 3 technical replicates per sample). E Cell cycle analysis with EdU flow cytometry after siRNA-mediated knockdown of RUNX2 in CHIR99021-treated cells demonstrates a significant decrease of cells in S phase (p = 0.0002–0.0047), a significant increased proportion of cells in G0/G1 (p = 0.0013–0.0161), and inconsistently enhanced proportions of cells in G2/M (p = 0.0331 – ns), (n = 3 biological and 3 technical replicates each). F RUNX2 knockdown in CHIR99021-treated astrocytes leads to significantly reduced clonal expansion potential (p = 0.0062–0.0182) (n = 3 biological and 3 technical replicates each). G Distribution of up- and downregulated DEGs after RUNX2 knockdown in CHIR99021-treated IDH^mut astrocytes, showing a higher fraction of downregulated genes, including several relevant in IDH^mut gliomas. H Ingenuity Pathway analysis of RUNX2-dependent DEGs highlightssignificant enrichment for proliferation-relevant functions, including cellular development, cell death and survival, and cellular growth and proliferation. I Heatmap of selected genes associated with cell death and survival from the siRUNX2 RNA-seq dataset