Targeting the aryl hydrocarbon receptor (AhR) with BAY 2416964: a selective small molecule inhibitor for cancer immunotherapy

Abstract

Background: The metabolism of tryptophan to kynurenines (KYN) by indoleamine-2,3-dioxygenase or tryptophan-2,3-dioxygenase is a key pathway of constitutive and adaptive tumor immune resistance. The immunosuppressive effects of KYN in the tumor microenvironment are predominantly mediated by the aryl hydrocarbon receptor (AhR), a cytosolic transcription factor that broadly suppresses immune cell function. Inhibition of AhR thus offers an antitumor therapy opportunity via restoration of immune system functions.

Methods: The expression of AhR was evaluated in tissue microarrays of head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). A structure class of inhibitors that block AhR activation by exogenous and endogenous ligands was identified, and further optimized, using a cellular screening cascade. The antagonistic properties of the selected AhR inhibitor candidate BAY 2416964 were determined using transactivation assays. Nuclear translocation, target engagement and the effect of BAY 2416964 on agonist-induced AhR activation were assessed in human and mouse cancer cells. The immunostimulatory properties on gene and cytokine expression were examined in human immune cell subsets. The in vivo efficacy of BAY 2416964 was tested in the syngeneic ovalbumin-expressing B16F10 melanoma model in mice. Coculture of human H1299 NSCLC cells, primary peripheral blood mononuclear cells and fibroblasts mimicking the human stromal-tumor microenvironment was used to assess the effects of AhR inhibition on human immune cells. Furthermore, tumor spheroids cocultured with tumor antigen-specific MART-1 T cells were used to study the antigen-specific cytotoxic T cell responses. The data were analyzed statistically using linear models.

Results: AhR expression was observed in tumor cells and tumor-infiltrating immune cells in HNSCC, NSCLC and CRC. BAY 2416964 potently and selectively inhibited AhR activation induced by either exogenous or endogenous AhR ligands. In vitro, BAY 2416964 restored immune cell function in human and mouse cells, and furthermore enhanced antigen-specific cytotoxic T cell responses and killing of tumor spheroids. In vivo, oral application with BAY 2416964 was well tolerated, induced a proinflammatory tumor microenvironment, and demonstrated antitumor efficacy in a syngeneic cancer model in mice.

Conclusions: These findings identify AhR inhibition as a novel therapeutic approach to overcome immune resistance in various types of cancers.

Keywords: Drug Evaluation, Preclinical; Immune Checkpoint Inhibitors; Immunotherapy; Indoleamine-Pyrrole 2,3,-Dioxygenase; Metabolic Networks and Pathways.

Figure 1

AhR is expressed in tumor cells and tumor-infiltrating immune cells in various cancers. (A–C) Representative IHC images of AhR expression in tissue samples of (A) HNSCC (n=16), (B) NSCLC (n=29) and (C) CRC (n=55). Brown color indicates AhR staining. Scale bars are included in the figures. (D) AhR expression level and (E) localization in tumor samples shown in panels A-C. AhR, aryl hydrocarbon receptor; CRC, colorectal cancer; HNSCC, head and neck squamous cell carcinoma; IHC, immunohistochemistry; NSCLC, head and neck squamous cell carcinoma.

Figure 2

BAY 2416964 inhibits AhR ligand-induced CYP1A1 transcription and AhR translocation into the nucleus. (A) Molecular structure and characteristics of BAY 2416964 identified in the optimized cellular screening cascade described in online supplemental figure S1. (B–C) Effect of BAY 2416964 on luciferase expression in (B) human U87 cells and (C) mouse Hepa1c1c7 cells in the presence (antagonism) or absence (agonism) of 150 µM or 200 µM KA, respectively, after 20 hours (representative experiments of (B) n=33 and (C) n=34). (D) Unbound BAY 2416964 plasma level in relation to the determined mean cellular unbound IC₅₀ value (as determined by CYP1A1 inhibition in KA-stimulated mouse splenocytes, indicated with a blue dotted line) after repeated dosing with BAY 2416964 at 30 mg/kg (QD) in B16F10-OVA tumor-bearing mice (n=2–3 mice/time point). (E, F) Inhibition of AhR-induced CYP1A1 expression in BAY 2416964-treated (E) human U937 cells and (F) freshly isolated mouse splenocytes on stimulation with 200 µM KA after 4 hours (representative experiments of n=5). (G) AhR translocation from the cytosol to the nucleus induced with 100 nM TCDD after 4 hours in human Hep G2 cells treated with BAY 2416964 (n=4). N, nucleus; C, cytosol. (H) Direct target engagement of BAY 2416964 with AhR, as determined using CETSA. AhR, aryl hydrocarbon receptor; CETSA, cellular thermal shift assay; DMSO, dimethyl sulfoxide; KA, kynurenic acid; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Figure 3

BAY 2416964 induces proinflammatory effects in various human immune cell subsets in vitro. (A) CYP1A1 expression in 200 µM KA-treated human immune cell subsets from 3 to 5 donors. (B) Effect of BAY 2416964 (300nM) on the expression of immunoregulatory genes in KA-treated, LPS-stimulated human primary monocytes, as determined by RNAseq analysis. (C) Expression of AhR target genes and inflammatory cytokines in human primary monocytes (isolated from six different donors) treated with LPS, LPS and KA, or LPS+KA+BAY 2416964 (300 nM), as determined by RNAseq analysis. (D, E) Effect of BAY 2416964 (1 nM–10 µM) on (D) AHRR and (E) CYP1A1 expression (fold change vs vehicle) in 200 µM KA-treated, 10 ng/mL LPS-stimulated monocytes after treatment with BAY 2416964 (1 nM–10 µM), as determined by qRT-PCR. (F) TNF-α production after 24 hours in 200 µM KA-treated, 10 ng/mL LPS-stimulated monocytes on treatment with BAY 2416964 (1 nM–10 µM) (one representative of 6 donors). Statistical analyses were performed using one-way ANOVA and Dunnett’s multiple comparisons test. **p<0.01, ***p<0.001 compared with LPS+KA. (G) IL-2 production by human T cells cocultured with LPS and IFN-γ-matured moDCs treated with 1 nM–1 µM BAY 2416964 or epacadostat for 3 days (representative of n≥3). *p<0.05, **p<0.01, ***p<0.001 compared with control. (H) IL-2 production by T cells cocultured with LPS and IFN-γ-matured moDCs treated with 3 nM–1 µM BAY 2416964 alone or in combination with 3 µg/mL anti-PD-1 (n=4). *p<0.05, **p<0.01, ***p<0.001 compared with the respective concentration of isotype control; ^##p<0.01, ^###p<0.001 compared with anti-PD-1 alone. (I) Effect of BAY 2416964 (3 nM–1 µM) on IFN-γ production in naïve CD4⁺ T cells stimulated with CD3, CD28, and IL-2 in the absence or presence of 5 ng/mL TGF-β (n=3). *p<0.05, **p<0.01, ***p<0.001 compared with control. Statistical analyses were performed using the estimated linear model and corrected for family-wise error rate using Sidak’s method. DCs, dendritic cells; KA, kynurenic acid; LPS, lipopolysaccharide; moDCs, monocyte-derived DCs; PBMCs, peripheral blood mononuclear cells.

Figure 4

BAY 2416964 decreases tumor growth in an immune cell-dependent manner. (A) IFN-γ production on treatment with different doses of BAY 2416964 in OVA peptide SIINFEKL (H2–Kb)-pulsed mouse bone marrow-derived dendritic cells cultured with transgenic CD8⁺ OT-I T cells in vitro (n=3). (B) Tumor growth in the syngeneic B16F10-OVA melanoma mouse model in one of 5 experiments (n=8 mice/group) treated with vehicle or BAY 2416964 at 30 mg/kg (QD, p.o.). Statistical analyses were performed using two-way ANOVA and Tukey multiple comparison test. ***p<0.001 vs vehicle control. (C) Immune composition of the B16F10-OVA tumor microenvironment on treatment with BAY 2416964 (30 mg/kg, QD). Statistical analyses were performed using the Mann Whitney test. *p<0.05; **p<0.01 vs control. (D) Tumor growth in B16F10-OVA tumor-bearing immunodeficient NSG mice treated with vehicle or BAY 2416964 at 30 mg/kg (QD, p.o.). (E) Body weight of the B16F10-OVA tumor-bearing mice shown in A. ANOVA, analysis of variance; KA, kynurenic acid.

Figure 5

Proinflammatory activity of BAY 2416964 enhances antigen-specific T cell killing of human tumor spheroid in vitro. (A) Expression of IFN-γ, IL-17, IL-2, IL-6, and TNF-α in a coculture of H1299 NSCLC cells, primary human PBMCs, and primary human fibroblasts treated with 0.37, 1.1, or 3.3 µM BAY 2416964 or epacadostat determined using BioMAP Oncology Panel. Statistical analyses were performed using the estimated linear model and corrected for family-wise error rate using Sidak’s method. *p<0.05, **p<0.01 vs vehicle control. (B, C) Effect of BAY 2416964 (0.1 nM–10 µM) on (B) IL-2 and (C) granzyme B production by MART-1 T cells cocultured with COLO-800 tumor spheroids for 4 days. Granzyme B concentrations were normalized to vehicle-treated control samples. Statistical analyses were performed using one-way ANOVA and Dunnett’s multiple comparisons test. *p<0.05, ***p<0.001 vs vehicle control. (D) CYP1A1 and (E) TIPARP expression (fold change vs vehicle) in COLO-800 spheroid—MART-1 T cell cocultures after 4 days in culture as analyzed by RT-PCR. Statistical analyses were performed using one sample t-test with a hypothetical value of 1 and assumed Gaussian distribution. *p<0.05; ***p<0.001 vs vehicle-treated control. (F) Effect of BAY 2416964 (0.3 nM–10 µM) on the cytotoxic capacity of MART-1 T cells to kill human COLO-800 tumor spheroids measured as absolute tumor cell counts of COLO-800 spheroids after 4 days of coculture with MART-1 T cells determined by flow cytometry. One representative experiment with six technical replicates per condition is shown (n=4). Statistical analyses were performed using one-way ANOVA and Dunnett multiple comparisons test. *p<0.05, ***p<0.001 vs control (COLO-800 spheroid only). (G) Effect of BAY 2416964 (5–500 nM) on the absolute MART-1 T cell count after 4 days of coculture with COLO-800 spheroids determined by flow cytometry. Statistical analyses were performed using one-way ANOVA and Dunnett’s multiple comparisons test. **p<0.01 vs vehicle-treated control. ANOVA, analysis of variance; NSCLC, non-small cell lung cancer; PBMCs, peripheral blood mononuclear cells.