Targeting Wnt signaling for improved glioma immunotherapy

Abstract

Introduction: Despite aggressive standard-of-care therapy, including surgery, radiation, and chemotherapy, glioblastoma recurrence is almost inevitable and uniformly lethal. Activation of glioma-intrinsic Wnt/β-catenin signaling is associated with a poor prognosis and the proliferation of glioma stem-like cells, leading to malignant transformation and tumor progression. Impressive results in a subset of cancers have been obtained using immunotherapies including anti-CTLA4, anti-PD-1, and anti-PD-L1 or chimeric antigen receptor (CAR) T cell therapies. However, the heterogeneity of tumors, low mutational burden, single antigen targeting, and associated antigen escape contribute to non-responsiveness and potential tumor recurrence despite these therapeutic efforts. In the current study, we determined the effects of the small molecule, highly specific Wnt/CBP (CREB Binding Protein)/β-catenin antagonist ICG-001, on glioma tumor cells and the tumor microenvironment (TME)-including its effect on immune cell infiltration, blood vessel decompression, and metabolic changes.

Methods: Using multiple glioma patient-derived xenografts cell lines and murine tumors (GL261, K-Luc), we demonstrated in vitro cytostatic effects and a switch from proliferation to differentiation after treatment with ICG-001.

Results: In these glioma cell lines, we further demonstrated that ICG-001 downregulated the CBP/β-catenin target gene Survivin/BIRC5-a hallmark of Wnt/CBP/β-catenin inhibition. We found that in a syngeneic mouse model of glioma (K-luc), ICG-001 treatment enhanced tumor infiltration by CD3⁺ and CD8⁺ cells with increased expression of the vascular endothelial marker CD31 (PECAM-1). We also observed differential gene expression and induced immune cell infiltration in tumors pretreated with ICG-001 and then treated with CAR T cells as compared with single treatment groups or when ICG-001 treatment was administered after CAR T cell therapy.

Discussion: We conclude that specific Wnt/CBP/β-catenin antagonism results in pleotropic changes in the glioma TME, including glioma stem cell differentiation, modulation of the stroma, and immune cell activation and recruitment, thereby suggesting a possible role for enhancing immunotherapy in glioma patients.

Keywords: ICG-001; NanoString gene expression; Wnt signaling, pathway; differentiation; glioma; immunotherapy; proteomics.

Figure 1

Cytostatic effect of ICG-001 in human and mouse glioma cell lines in vitro. (A) Growth kinetics of human PBT glioma lines (PBT135, PBT144, and PBT147) treated with ICG-001 (0, 5, 10 µM) for 7 days (n=3); and (B) mouse glioma cells (K-luc, GL261-IL13, and GL261) treated with ICG-001 (0-10 µM) for 4 days (n=3). Scale bars represent SD of triplicate samples from 2 independent experiments. (C) Expression of target gene KIF20 post-ICG-001 treatment (5, 10 µM) by RT-PCR using KIF20A-specific primers (n=3). (D–F) Live images taken by IncuCyte of PBT017 glioma expressing red fluorescent protein untreated or treated with ICG-001 (0, 4, 8 µM). (G) Analysis and quantification of IncuCyte images (10x) for PBT017.RFP (PBT017 expressing red fluorescent protein-RFP) glioma cells and PBT030 quantified using phase images. P values denoted by *<0.05, **<0.01, ***<0.001, ****<0.0001, *****<0.00001.

Figure 2

Expression of the target genes BIRC5/Survivin and EphB2 post-ICG-001 treatment (0, 5, 10 mM) as detected by RT-PCR and Luciferase Reporter Assay. (A) Survivin and EphB2 mRNA levels in PBT017 and PBT030 human glioma lines and mouse GL-261 and K-Luc lines treated with ICG-001 for 3 days (0, 5, 10 μM). (B) Luciferase reporter assay indicating downregulation of luciferase reported gene in PBT017 and PBT030 cell lines upon treatment with ICG-001 at 0, 5, and 10 μM in triplicates. (C) Survivin/BIRC5 relative expression upon treatment with ICG001. P values denoted by *<0.05, **<0.01, ***<0.001, ****<0.0001, *****<0.00001.

Figure 3

NanoString analysis of human metabolic pathways after treatment with ICG-001. (A) Multidimensional scaling (MDS) plot of visual representation of the patterns of proximities among PBT030 and PBT147 human cell lines treated with ICG-001 at 2 time points (24 hrs & 72 hrs) and 3 doses (0µM, 5µM, 10 µM) [n=2] separately on the Y-axis. X-axis indicated clustering of PBT030 and PBT147 cell lines each separately. (B) Volcano plot showing difference in expression for PBT030 between ICG-001 treated vs untreated, with the x-axis showing log2 fold change and the y-axis showing the negative log10 of p-adj value and heatmap; mRNA represented by red circles for significantly upregulated genes and blue circles for significantly down-regulated human genes. Full list of differentially expressed genes is shown in Supplementary Table 2 (C) Volcano plot showing difference in expression for PBT147 between ICG-001 treated vs untreated, with the x-axis showing log2 fold change and the y-axis showing the negative log10 of p-adj value and heatmap; mRNA represented by red circles for significantly upregulated genes and blue circles for significantly down-regulated genes. Full list of differentially expressed genes are shown in Supplementary Table 2 . (D) Heat map of differentially expressed genes when comparing PBT030 and PBT147 cell lines (selected glioma gene list). (E, F) Bar chart of functional analysis showing the top 10 significance score predicted using ROSALIND Gene Set Analysis with NanoString annotations for PBT030 and PBT147 human cell lines treated with ICG-001.

Figure 4

Proteomics analysis of PBT147 and PBT030 cell lines treated with ICG-001 (0-10 µM) for 72h. (A) Up-regulated protein density was calculated by the difference in log normalized levels for each protein between the four treated samples (ICG001 5, 10 mM at 24 and 72h) and the appropriate untreated sample control. (B) Calculating the difference in log levels corresponds to the log of the fold changes in each pathway. For both cell lines, the log fold-change is plotted for each protein at 5 µM against the log fold-change at 10 µM. These plots are shown in (A), with the points color-coded by ICG001 concentrations. This highlights a positive linear relationship between the log fold changes at 5µM and 10µM in both cell lines. The dose effect is significantly greater in PBT030 than PBT147, with the same increase in dose, usually resulting in a greater fold-change.

Figure 5

IHC analysis of immune cells in syngeneic models of subcutaneous glioma (K-Luc). (A–C, E–G, I–K) Mice bearing subcutaneous K-luc glioma tumors were treated with ICG-001 (50 mg/kg/day) for 7, 14, or 21 days using Alzet minipumps. At the end of treatment, tumor tissue was excised and IHC stained for CD3 cells to evaluate recruitment and patterns of CD3, CD8, and CD31 distribution. (D, H, L) Quantification of tumor coverage was performed using QuPath for mouse CD3, CD8, and CD31 positive cells on days 7, 14, 21 post minipump implantation. Pairwise comparison was performed to access the CD31 expression on days 7, 14 and 21 (P-value 0.314, 0.624, 0.0016 respectively). Control animals were treated with no pumps. Scale bars are SD of duplicate sections (n=4). (M) NanoString analysis of mouse immune panel genes in subcutaneous K-Luc tumors at days 7 and 21, as compared with untreated controls (untreated tumors were also collected on days 7 and 21).

Figure 6

NanoString analysis of nCounter mouse Pan Cancer immune profile gene expression within ICG-001, CAR T, CAR T + ICG-001 and ICG-001 + CAR T treatment groups. (A) Multidimensional scaling (MDS) plot for the samples treatments with ICG001[MI] (ICG001 only), 3 treatments with CAR Ts [MC] (CART only), 2 treatments with ICG001 and then CAR Ts [MIC] (ICG001 first then CART), and 2 treatments with CAR Ts and ICG001 [MCI] (CART first then ICG001). (B) Volcano plot showing difference in expression of |Fold Change|>1.5 & p-value<0.05 between ICG001-CAR T and CAR T-ICG001 treated mice, with the x-axis showing log2 fold change using cutoff and the y-axis showing the negative log10 of p-value for the 11 selected mouse brain tumor genes of interest. (C) Heatmap of differential expression gene comparison between ICG001 + CAR T and CAR T + ICG001 using filtering |FC |≥ 1.5 & p-value ≤ 0.05 label highlighted. (D) The top statistic significant ROSALIND oncology collection biologic process based on the differential expression gene expression comparison between ICG001 + CAR T and CAR T + ICG001. (E) The heatmap of differential expression gene comparison between ICGCART & CARTICG for the 11 selected brain tumor genes of interest which are also labelled on Volcano plot. (F) The description and log2(fold change) & p-value for the 11 selected brain tumor genes of interest.

Figure 7

NanoString nCounter mouse cell line PanCancer immune profiling: Genes previously shown to be characteristic of various cell populations by measuring cells population abundance based on the NanoString Cell Type Profiling Module. (A–C) Bar graph charts showing abundance scores of CD45, NK-CD56, and Macrophage cell type populations across all samples for genes that are characteristic and grouped by cell population abundance. (D) Heat map of gene clustering and cell profile in groups treated with ICG001 (MI), CAR T + ICG001 (MCI1, MCI2), ICG001+ CAR Ts (MIC1, MIC2), and CAR Ts only (MC1, MC2, MC3). Genes previously shown to be characteristic of CD45, NK-CD56, and Macrophage cell population abundance across all samples for ICG001+ CAR Ts (MIC1, MIC2) shows positive Z-score differently comparing from samples of ICG001 (MI), CAR T + ICG001 (MCI1, MCI2), and CAR Ts only (MC1, MC2, MC3) with negative Z-score.