Pharmacological Wnt ligand inhibition overcomes key tumor-mediated resistance pathways to anti-PD-1 immunotherapy

Abstract

While immune checkpoint blockade is associated with prolonged responses in multiple cancers, most patients still do not benefit from this therapeutic strategy. The Wnt-β-catenin pathway is associated with diminished T cell infiltration; however, activating mutations are rare, implicating a role for autocrine/paracrine Wnt ligand-driven signaling in immune evasion. In this study, we show that proximal mediators of the Wnt signaling pathway are associated with anti-PD-1 resistance, and pharmacologic inhibition of Wnt ligand signaling supports anti-PD-1 efficacy by reversing dendritic cell tolerization and the recruitment of granulocytic myeloid-derived suppressor cells in autochthonous tumor models. We further demonstrate that the inhibition of Wnt signaling promotes the development of a tumor microenvironment that is more conducive to favorable responses to checkpoint blockade in cancer patients. These findings support a rationale for Wnt ligand-focused treatment approaches in future immunotherapy clinical trials and suggest a strategy for selecting those tumors more responsive to Wnt inhibition.

Keywords: Wnt inhibition; Wnt-β-catenin pathway; anti-PD-1 resistance; dendritic cells; indoleamine 2,3-dioxygenase; melanoma; myeloid-deriver suppressor cells; non-small cell lung cancer; regulatory T cells; tumor immunotherapy.

Figure 1.. Transcriptomic analysis of Wnt ligands, receptors, and regulators in anti-PD-1 resistance

(A) Schematic of the canonical Wnt signaling pathway and the mechanism of action of OMP-18R5, which blocks Fzd receptors; OMP-54F28 scavenges Wnt ligands; and ETC-159 blocks Wnt ligand secretion by inhibiting PORCN. (B) Wnt ligand, receptor, and target-focused heatmap generated from RNA-seq analysis of the BP mouse model after aPD1 escape. M, mouse; aPD1, anti-PD-1 antibody. (C) Reanalysis of RNA-seq data from biopsies of responding (red) and nonresponding (blue) metastatic melanoma patients focused on Wnt ligands, receptors, and regulators (Hugo et al., 2016). AU, arbitrary units. A Student’s two-tailed t test was used to compare aPD1 responders versus non-responders. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (D) Volcano plot of NanoString Wnt pathway analysis of the top 30 differentially expressed genes in metastatic melanoma patients (n = 12 responders, 12 non-responders) prior to receiving aPD1 monotherapy. The Benjamini-Yekutieli method of correction was used for controlling the false discovery rate (FDR), and p values were adjusted based on the total number of comparisons made across the entire gene panel. See also Tables S1 and S2.

Figure 2.. Wnt ligand inhibition reverses Treg generation via IDO1 inhibition and enhances anti-PD-1 therapy in a syngeneic melanoma model

(A) Luciferase assay of the TCF-LEF-luc 293T cell line treated with OMP-18R5 or OMP-54F28. Each condition was performed in triplicate. Data are representative of two independent experiments. (B) IDO1 western blot analysis of DCs treated with Wnt5a with or without OMP-18R5 or OMP-54F28. Data are representative of two independent experiments. (C) DCs were treated with Wnt5a with or without OMP-18R5 followed by co-culture with naive CD4⁺ T cells derived from FoxP3-GFP reporter mice. CD4⁺GFP⁺ Tregs were quantified by flow cytometry. (D) DCs were treated with conditioned media derived from C59-treated versus control-treated BP melanoma cells followed by co-culture with FoxP3-GFP naive CD4⁺ T cells. CD4⁺GFP⁺ Tregs were quantified by flow cytometry. For (C) and (D), each condition was performed in triplicate. Data are representative of two independent experiments. (E) Primary tumor growth of syngeneic implanted BP tumors treated with either IgG isotype control, aPD1 alone, OMP-18R5 alone, OMP-54F28 alone, OMP-18R5/aPD1, or OMP-54F28/aPD1 (n = 6/group). All associated data are representative of two independent experiments. (F) Quantification of lung metastasis in mice related to (E) by H&E microscopy (n = 3/group). (G) Quantification of tumor-infiltrating CD8⁺ T cells in mice related to (E) by IHC (n = 3/group). (H) Flow cytometric analysis of intra-tumoral CD8⁺ T cells and CD4⁺FoxP3⁺ T cells in mice related to (E). Data are expressed as CD8⁺ T cell/CD4⁺FoxP3⁺ T cell ratios. UT, untreated. CM, conditioned media; Ctrl, control. All statistical analyses are based on one-way ANOVA followed by a Tukey post hoc test. All data show mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S1.

Figure 3.. OMP-18R5 and ETC-159 enhance Anti-PD-1 therapy in an autochthonous melanoma model while inhibiting DC b-catenin signaling

(A) Autochthonous BP mice were treated with either IgG isotype ctrl, aPD1, OMP-18R5, or anti-PD-1 + OMP-18R5/aPD1 after the development of primary melanomas (n = 6–7/group). Primary melanoma volumes were recorded every 3 days. All associated data are representative of two independent experiments. (B) Quantification of lung metastasis in mice related to (A) by H&E microscopy (n = 5/group). (C) Flow cytometric analysis of intra-tumoral CD8⁺ T cells and CD4⁺FoxP3⁺ T cells in mice related to (A). Data are expressed as CD8⁺ T cell/CD4⁺FoxP3⁺ T cell ratios. (D) Quantification of TRP2-specific CD8⁺ T cells related to (A) based on IFN-γ ELISpot. (E) Primary melanoma volumes in autochthonous BP mice treated with anti-CD8 depleting mAb followed by treatment with either IgG isotype ctrl, aPD1, OMP-18R5, or OMP-18R5/aPD1 (n = 5/group). (F) Autochthonous BP mice were treated with either IgG isotype ctrl, aPD1, delayed ETC-159/IgG ctrl, or delayed ETC-159/aPD1. ETC-159 was initiated at aPD1 escape (black arrow, day 14) (n = 6/group). All associated data are representative of two independent experiments. (G) Flow cytometric analysis of intra-tumoral CD8⁺ T cells and Tregs related to (F). (Right) Representative flow dot plots. (H and I) qRT-PCR analysis of β-catenin target gene expression, Tcf7 and Ccnd1, by tumor-associated DCs (H) or by tumor tissue (I) harvested from mice described in (F) (n = 3/group). ns, non-significant. Student’s two-tailed t test comparing aPD1 monotherapy groups versus combination therapy. All data show mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S2 and S3.

Figure 4.. Immunotherapeutic properties of OMP-18R5 extend to an autochthonous model of NSCLC

(A) Cre-expressing adenoviral vector was administered intra-nasally to autochthonous KP mice, inducing primary lung tumor development. At 10 weeks, mice were initiated on IgG ctrl, aPD1, OMP-18R5, or OMP-18R5/aPD1 (n = 6/group). All associated data are representative of two independent experiments. (B) Micro-CT quantification of tumor volume at weeks 10, 14, and 17 in (A). (Left) Final tumor volumes. Each point represents an individual lung tumor. (Right) Representative images from each treatment group. Red arrows indicate lung tumors. Statistical analyses for (A) and (B) are based on one-way ANOVA followed by a Tukey post hoc test. (C) Lung weights related to (A). Weights are normalized to lungs harvested from age-matched non-tumor-bearing mice. (D) Representative H&E sections from each treatment group taken at week 17. Scale bars, 2,000 μm. (E) IHC quantification of intra-tumoral CD8⁺ T cells in the experiment described in (A) (n = 5/group). (F) Quantification of KP cell line-derived antigen-specific splenic CD8⁺ T cells harvested in (A) based on IFN-γ ELISpot (n = 3–5/group). (G) Kaplan-Meier survival analysis of mice related to (A). Statistical analysis based on log-rank test, p = 0.06. A Student’s two-tailed t test was performed when comparing aPD1 monotherapy groups versus combination therapy. All data show mean ± SEM. **p < 0.01, *p < 0.05. See also Figure S4.

Figure 5.. OMP-18R5 suppresses the enzymatic production of kynurenine (Kyn) in the tumor microenvironment and outperforms IDO inhibition in vivo

(A) Schematic representation of Wnt ligand-driven DC fatty-acid oxidation (FAO)/heme biosynthesis, resulting in protoporphyrin IX (PpIX) generation and tryptophan (Trp)-degrading enzymatic activity. (B) DCs treated with Wnt5a with or without OMP-18R5 prior to incubation with aminolevulinic acid (ALA) and flow cytometric quantification of PpIX levels. Performed in triplicate. (Right) Representative PpIX flow histogram. Data are representative of three independent experiments. (C) Autochthonous BP mice were treated with either IgG isotype ctrl, aPD1, OMP-18R5, epacadostat, OMP-18R5/aPD1, or epacadostat/aPD1 after the development of primary melanomas (n = 9–11/group). (D) Quantification of TRP2-specific CD8⁺ T cells derived from (C) based on IFN-γ ELISpot. (E) LC-MS/MS analysis of Kyn and Trp from tumor, TDLNs, and non-tumor draining LN (NDLN) tissues following treatment with either OMP-18R5 or epacadostat (n = 4/group). Data are representative of two independent experiments. (F) Flow cytometric analysis of intra-tumoral Tregs derived from tumor tissues harvested from (C) (n = 4/group). Data are representative of two independent experiments. (G) Autochthonous BP mice were treated with either IgG isotype ctrl, aPD1, delayed ETC-159/aPD1, or delayed epacadostat/aPD1 where ETC-159 and epacadostat were initiated at aPD1 escape (black arrow, day 0) (n = 6/group). Tumor volumes were normalized to IgG isotype control group. All statistical analyses were based on one-way ANOVA followed by a Tukey post hoc test. All data show mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S5.

Figure 6.. Wnt ligand inhibition suppresses PMN-MDSC influx into tumors

(A and B) CXCL5 (A) and (B) YAP1 western blot analysis of the BP melanoma cell line treated with Wnt5a with or without OMP-18R5. Data are representative of three independent experiments. (C) CXCL5 IHC of resected BP melanoma tissues following treatment with IgG control, aPD1, or OMP-18R5/aPD1. Data are representative of two independent experiments. Scale bar, 20 μm. (D) qRT-PCR analysis of Cxcl5 expression levels in autochthonous BP melanomas before aPD1 escape, following aPD1 escape, and after delayed treatment with ETC-159 (dETC-159) (n = 3/group). Data are representative of two independent experiments. (E) Flow cytometry analysis of intra-tumoral CD11b⁺Ly6G⁺Ly6C^loF4/80⁻ PMN-MDSCs normalized by tumor size. Data are representative of two independent experiments. (F) Gr-1 IHC analysis of autochthonous BP melanoma tumors treated with IgG control, aPD1, or OMP-18R5/aPD1. Data are representative of three independent experiments. Scale bar, 50 μm. (G) Flow cytometry analysis of intra-tumoral CD11b⁺Ly6G⁺Ly6C^loF4/80⁻ PMN-MDSCs/g of tumor in the LLC model following treatment with IgG control, aPD1, or OMP-18R5/aPD1 (n = 3/group). (Below) Representative flow dot plots. Data are representative of two independent experiments. A Student’s two-tailed t test was used to compare aPD1 monotherapy groups versus combination therapy. All data show mean ± SEM. *p < 0.05, ***p < 0.001. See also Figures S6A and S6B.

Figure 7.. Wnt ligand inhibition with ETC-159 promotes the development of a more favorable tumor immune microenvironment in patients

(A) Schematic of patient tumor biopsies prior to and following exposure to ETC-159. (B) NanoString analysis of immune-related gene expression. Heatmaps illustrate log fold change in gene expression between pre- and post-treatment tumor specimens derived from two cancer patients. See also Figure S6C and Tables S3 and S4.