Novel GABAAR antagonists target networked gene hubs at the leading edge in high-grade gliomas

Abstract

Background: Ion channel activity underlying biological processes that drive high-grade gliomas (HGG) is largely unknown. We aimed to determine the networking of ion channel genes and validate their expression within HGG patient tumors, to identify ion channel-targeting drugs that would inhibit tumor-promoting processes.

Methods: We used weighted gene co-expression network analysis (WGCNA) of RNAseq data to identify ion channel gene hubs in diffuse midline glioma (DMG) and glioblastoma. Using scRNA-seq, spatial transcriptomics, and immunohistochemistry, we characterized the expression of identified hubs within patient tumors, validating their role by testing the efficacy of ion channel inhibitors alone or in combination with radiation and temozolomide on the growth and invasion of patient-derived glioblastoma explant organoids (GBOs).

Results: Network analysis revealed a preserved HGG "neuronal regulation" module, containing the greatest number of ion channels, with its corresponding genes concentrated at the tumor's leading edge. Hubs within this module included γ-Aminobutyric-acid type A receptor (GABAAR) genes GABRA1 (α1) and GABRG2 (γ2), which immunohistochemically colocalized with GABAergic synaptic markers at the leading edge. GBOs failed to retain this synaptic architecture but expressed a glioblastoma hub GABRA5 (α5), a component of extrasynaptic GABAARs. S44819, an α5-GABAAR antagonist strongly inhibited GBO invasion, with GABA(A)-compound 1b, a partial antagonist of GABAARs, robustly inhibiting GBO proliferation and invasion. Moreover, combined with standard-of-care (SOC) regimens, the anti-invasive properties of both compounds were enhanced in GBOs.

Conclusions: Our co-expression network analysis identified key ion channels at the leading edge in HGGs, which can be targeted by GABAAR-acting drugs to disrupt tumor progression.

Keywords: GABAARs; WGCNA; high-grade glioma; leading edge; synapse.

Figure 1.

Network analysis of DMG and glioblastoma identifies neuronal regulation associated network module that is expressed at the tumor’s leading edge. A, B, Network analysis was carried out independently for DMG and glioblastoma using WGCNA. The modular architecture was visualized employing a topological overlap matrix (i.e., heatmap and dendrogram) in A, DMG, and B, glioblastoma coexpression networks. Color bands signify identified functional modules (rows and columns) of highly connected genes. Red intensity across the diagonal signifies a high correlation. Black boxes represent the identified modules in DMG (nine modules) and glioblastoma networks (eight modules). C, D, Heatmaps representing the relative expression (min-max normalized by cell type) of neuronal regulation genes (columns) across cell types in C, two DMG and D, four glioblastoma scRNA-seq datasets. E, F, The spatial expression pattern of four tumor regions defined by Puchalski et al., (2018) in a representative E, DMG and F, glioblastoma patient tumor alongside the neuronal regulation module gene signature in the same G, DMG and H, glioblastoma tumors. I, J, Heatmaps representing the relative expression (min-max normalized by region) of neuronal regulation module genes (columns) in tumor regions (rows) for I, five DMG and J, four glioblastoma tissues profiled by spatial transcriptomics.

Figure 2.

GABA _A R gene expression in HGG patient tumors. Log₂ gene expression in A, DMG (n = 31), B, glioblastoma (n = 151). The dotted line signifies a log₂ gene expression of >5 where the gene is considered expressed. Red lettering denotes neuronal regulation gene hubs. C, D, Heatmaps representing the normalized expression (min-max normalized by cell type) of GABA_AR genes (rows) across cell types (columns) C, two DMG and D, four glioblastoma single-cell RNA-seq datasets. Red lettering denotes gene hubs. E, GABA_Aα1 and GABA_Aγ2 expression within primary HGG tumor samples. Quadruple immunofluorescence for GABA_Aα1 (red) and GABA_Aγ2 (green), OLIG2 (gray; tumor marker), and DAPI (blue; nuclear stain) in DMG (E_1-3) and glioblastoma (E_4-6) tumors shows GABA_Aα1 and GABA_Aγ2 expression within patient tumor samples. Images are maximum intensity z-projections of single plane scans taken at 0.5 µm increments through 4-5 µm of tumor using a x40 objective. Top right inserts are taken from single plane images, note GABA_Aα1 and GABA_Aγ2 colocalization in OLIG2+/- cells. F, Quantification of the percentage of OLIG2+ cells co-expressing either GABA_Aα1 or GABA_Aγ2 in DMG and glioblastoma patient tissue. *** P<.001, Mann-Whitney.

Figure 3.

GABRA1 and GABRG2 are expressed at the leading edge with GABAergic synaptic markers in DMG tumors A, The spatial expression pattern of gene signatures for the leading edge region defined by Puchalski et al., (2018) within a representative DMG tumor with spatial plots of GABRA1 and GABRG2 expression. B, Manders’ Overlap coefficient across the four regions for each GABA_AR subunit transcript in five DMG tumors. C, Spatial plots of tumor (OLIG2), neuronal (NFH) and GABAergic synapse marker genes (GAD1/GAD67, GAD2/GAD65, VGAT and GPHN/Gephyrin) in DMG. D, Manders’ Overlap coefficient across the four regions for each marker gene in five DMG tumors. E_1-3, Quadruple immunofluorescence for GABA_Aα1 (red), the presynaptic marker GAD65/67 (green), the neuronal marker NFH (blue) and OLIG2 (gray) in three DMG tumors. Note close apposition of GABA_Aα1 and GAD65/67 (arrows). F_1-3, Quadruple immunofluorescence for GABA_Aγ2 (red), the presynaptic marker GAD67 (green), NFH (blue) and OLIG2 (gray) in three DMG tumors. Note close apposition of GABA_Aγ2 and GAD67 (arrows). G_1-5, Immunofluorescence images of DMG patient tissue showing the postsynaptic marker, gephyrin (red) in close apposition to presynaptic markers GAD65/67 (green) and VGAT (blue) adjacent to OLIG2 + cells (arrows). H_1-5,  Immunofluorescence images of DMG patient tissue showing GABA_Aα1 (red) and GABA_Aγ2 (green) apposing VGAT (blue; arrows). All confocal images are single plane taken with a x40 objective.

Figure 4.

GABRA1 and GABRG2 are expressed at the leading edge with GABAergic synaptic markers in glioblastoma tumors A, The spatial expression pattern of gene signatures for the leading edge region defined by Puchalski et al., (2018) within a representative glioblastoma tumor with spatial plots of GABRA1 and GABRG2 expression. B, Manders’ Overlap coefficient across the four regions for each GABA_AR subunit transcript in four glioblastoma tumors. C, Spatial plots of tumor (OLIG2), neuronal (NFH) and GABAergic synapse marker genes (GAD1/GAD67, GAD2/GAD65, VGAT and GPHN/Gephyrin) in glioblastoma. D, Manders’ Overlap coefficient across the four regions for each marker gene in four glioblastoma tumors. E_1-3, Quadruple immunofluorescence for GABA_Aα1 (red), the presynaptic marker GAD65/67 (green), the neuronal marker NFH (blue) and OLIG2 (gray) in three glioblastoma tumors. Note close apposition of GABA_Aα1 and GAD65/67 (arrows). F_1-3, Quadruple immunofluorescence for GABA_Aγ2 (red), the presynaptic marker GAD67 (green), NFH (blue) and OLIG2 (gray) in three glioblastoma tumors. Note close apposition of GABA_Aγ2 and GAD67 (arrows). G_1-5, Immunofluorescence images of glioblastoma patient tissue showing the postsynaptic marker, gephyrin (red) in close apposition to presynaptic markers GAD65/67 (green) and VGAT (blue) adjacent to OLIG2+ cells (arrows). H_1-5, Immunofluorescence images of glioblastoma patient tissue showing GABA_Aα1 (red) and GABA_Aγ2 (green) apposing VGAT (blue; arrows). All confocal images are single plane taken with a x40 objective.

Figure 5.

GABA _A R antagonism inhibits GBO proliferation and invasion A, Boxplots of NKCC1 and KCC2 expression in HGG tumors. Statistical analysis was performed using t-test: ****P<.0001. B, The spatial expression pattern of NKCC1 and KCC2 in glioblastoma. C, Manders’ Overlap coefficient of NKCC1 and KCC2 with studied GABA_AR gene hubs D, E, GABA_Aα1 (D₂) and GABA_Aα5 (E₂) are expressed with GABA_Aγ2 (D₃ and E₃) in GBOs and are concentrated at the periphery with VGAT (D₄, E₄). F, G, GBOs strongly express genes related to GABA_AR signaling (gephyrin F₂, G_2; VGAT F₄, G₄) and metabolism (GAD65/67, F₃, G₃). H, I, Representative images of GBOs derived from two patients H, in proliferation at days 0 and 20 (scale bar: 250 µm) and I, invading Matrigel at day 14 (scale bar: 1000 µm) following incubation with either 0.1% DMSO (vehicle control), 50 µM GABA(A) Compound 1b, a partial GABA_AR antagonist, or 10 µM S44819, a GABA_A α5βγ2 antagonist. J, Assay protocols to test the effects of GABA_AR antagonism on two patient-derived GBOs’ proliferation (J₁) and invasive potential (J₂). K, L, Quantification of GBO proliferation calculated as the fold-change (FC) of spheroid area (µm²) normalized to day 0 measurements derived from two patients during a 20-day drug incubation period with GABA_AR-drugs and vehicle control. All data represented as means±SEM. Statistical analysis was performed using Two-way ANOVA with Tukey’s multiple comparisons: *P<.05; **P<.01; ***P<.001; ****P<.0001. M, N, Quantification of GBO spheroid Matrigel invasion calculated as the FC in peak radial displacement (µm) normalized to day 0 in Matrigel measurements (see J₂). Between days 0-6 GBOs were treated with vehicle (0.1% DMSO), 50 µM Compound GABA(A)1b or 10 µM S44819. At day 6, organoids were embedded in Matrigel and were treated for an additional 14 days. All data represented as means±SEM. Statistical analysis was performed using Two-way ANOVA with Tukey’s multiple comparisons: *P<.05; ***P<.001; ****P<.0001.

Figure 6.

The effects of GABA _A R antagonism on GBO proliferation and invasion under clinically relevant treatment regimens A, Assay protocol to test the effects of GABA_AR antagonism with the Stupp protocol (as indicated in red) on two patient-derived GBOs’ proliferation and invasion. B, C, Quantification of B, methylated and C, unmethylated MGMT patient GBO proliferation calculated as the relative fold-change (FC) in spheroid area (µm²) normalized to day 0 measurements in vehicle (0.1% DMSO), 50 µM Compound GABA(A)1b and 10 µM S44819 conditions with or without the Stupp regimen (see A for protocol). All data represented as means±SEM. Statistical analysis was performed using Two-way ANOVA with Tukey’s multiple comparisons: ns=not significant; ****P<.0001. D, E, Representative images of D, methylated and E, unmethylated MGMT populations of GBOs in proliferation at day 0 and day 10 during radiation and drug administration protocols depicted in A. Scale bar: 250 µm. F, G, Quantification of GBO Matrigel invasion of F, methylated MGMT and G, unmethylated patient GBOs calculated as the peak radial displacement (µm) normalized to day 10 measurements (see A for protocol). All data represented as means±SEM. Statistical analysis was performed using Two-way ANOVA with Tukey’s multiple comparisons: *P<.05; ****P<.0001. H, Representative images of methylated (top panel) and unmethylated (bottom panel) MGMT populations of patient GBOs invading Matrigel at day 16 following vehicle (0.1% DMSO), 50 µM Compound GABA(A)1b and 10 µM S44819 administration with or without TMZ and radiation pretreatment (see A; scale bar: 1000 µm).