SU5416, a VEGF receptor inhibitor and ligand of the AHR, represents a new alternative for immunomodulation

Abstract

The experimental compound SU5416 went as far as Phase III clinical trials as an anticancer agent, putatively because of its activity as a VEGFR-2 inhibitor, but showed poor results. Here, we show that SU5416 is also an aryl hydrocarbon receptor (AHR) agonist with unique properties. Like TCDD, SU5416 favors induction of indoleamine 2,3 dioxygenase (IDO) in immunologically relevant populations such as dendritic cells in an AHR-dependent manner, leading to generation of regulatory T-cells in vitro. These characteristics lead us to suggest that SU5416 may be an ideal clinical agent for treatment of autoimmune diseases and prevention of transplant rejection, two areas where regulatory ligands of the AHR have shown promise. At the same time, AHR agonism might represent a poor characteristic for an anticancer drug, as regulatory T-cells can inhibit clearance of cancer cells, and activation of the AHR can lead to upregulation of xenobiotic metabolizing enzymes that might influence the half-lives of co-administered chemotherapeutic agents. Not only does SU5416 activate the human AHR with a potency approaching 2,3,7,8-tetrachlorodibenzo-p-dioxin, but it also activates polymorphic murine receptor isoforms (encoded by the Ahr(d) and Ahr(b1) alleles) with similar potency, a finding that has rarely been described and may have implications in identifying true endogenous ligands of this receptor.

Figure 1. Screen of small molecule library for AHR agonists.

A. A collection of 4160 compounds was screened for the induction of the DRE-driven luciferase in the human hepatoma 101L cell line. In 384-well plates, 100 µL media containing 70% confluent 101L cells was incubated with 10 µM of each test compound (1% v/v DMSO) for 24 hours. Dotted line indicates 3-fold induction. B. Screen for agonists of the AHR^d. The AHRd-15 cell line was treated with 1 µM of the 98 compounds identified from the primary screen, 2 nM TCDD or DMSO and EROD activity was determined. Dashed line indicates 5-fold induction.

Figure 2. Induction of DRE-mediated transcription by SU5416 is AHR dependent. A.

The AHR-mutant C35 cell line was transfected with the AHR^b, lacZ gene and a 3×DRE-Luc construct. Controls were transfected with the empty pSPORT vector plus the reporter constructs. After 24 h, the cells were treated with 3 µM SU5416 or 0.3% (v/v) DMSO, then incubated for 18 more h. Induction of AHR activity was determined by normalizing the luciferase activity to β-galactosidase activity. White bars: Empty vector. Grey bars: AHR. Error bars: SD; (n = 3). B. Induction of DRE-mediated transcription by SU5416 is ARNT dependent. The ARNT-deficient C4 cell line was transfected with the human ARNT or the pSPORT parent vector. These cells were also co-transfected, treated and assayed as in A. White bars: Empty vector. Grey Bars: ARNT. Error bars: SD; (n = 3). C. SU5416 is a ligand of the AHR. The hepatic cytosolic fraction from C57BL/6J mice was incubated with 1 nM of the radioligand ¹²⁵BR₂N₃DpD, in the presence of increasing concentrations of competitor, SU5416, TCDD, BNF or 1,2-Benzanthracene. Ordinate: Specifically bound radioligand in the presence of competitor divided by specifically bound radioligand in the absence of competitor. Abscissa: The concentration of competing ligand, represented as log of molar concentration. Each data point represents the average of two determinations. Competitive binding to the C57BL/6J cytosol produced the IC₅₀ values of SU5416 = 2.1 nM, TCDD = 1.5 nM, BNF = 2.8 nM, and 1,2-Benzanthracene = 13.7 nM.

Figure 3. In Vitro dose response curves. A.

Rat hepatoma cell lines bearing the murine AHR^d (AHR^d-15) or the rat AHR^b (5L) were incubated with increasing doses of TCDD in 96-well plates. After 40 hours, EROD activity was performed from whole cell lysate. (n = 3). B. Same assay as in A, with increasing doses of SU5416. (n = 3). C. Same assay as in A, with increasing doses of BNF. (n = 2). D. Male DBA/2J and C57BL/6J mice (6 weeks old) were orally administered 120 mg/kg BNF, the indicated doses of SU5416 or the vehicle corn oil. Following 48 hours, hepatic microsomal proteins were isolated and used for the assessment of EROD activity. Animals were treated in groups of 4. Error bars: S.D.

Figure 4. Splenocytes from wild-type and AHR^d mice analyzed by qPCR for CYP1A1.

Spleens from these mice were harvested and suspended in culture media, and exposed to titrating doses of A) TCDD B) SU5416. After 4 hours they were analyzed by qPCR for CYP1A1 analysis. The curves are normalized from 0 to 100% response. Each graph is representative of 3 independent experiments. C–D. Cells transfected with AHR containing a valine point-mutation show similar ED₅₀ to Cos-1 cells with AHR^b isoform. Cos-1 cells were transfected with an AHR containing the same point mutation (valine for alanine) thought to be responsible for the low affinity of the AHR^d isoform compared to AHR^b, and compared to the wild-type AHR response. These cells also harbor a luciferase gene next to the DRE. C. Cos-1 cells were exposed to TCDD. D. Cos-1 cells were exposed to SU5416. The graphs represent normalized data from 0 response to 100% response. They are representative of 2 independent experiments.

Figure 5. SU5416 upregulates CYP1A1.

A. Spleens were harvested from mice and processed in the standard fashion. Cells were incubated for 4 hours in culture with titrating doses of SU5416 as indicated, and afterwards mRNA was measured for CYP1A1. B. SU5416 upregulates CYP1B1 and IDO. Same assay as A, but mRNA for CYP1B1 and IDO were measured. C. Upregulation of CYP1A1 mRNA is dependent on the AHR. Splenocytes from C57BL/6J or AHR^−/− mice were exposed to media, IFN-γ 100 ng/ml, or SU5416 500 nM for 4 hours. mRNA was then harvested and assayed for CYP1A1. nd represents “not detected”. *** - p≤0.001. D. IDO upregulation by SU5416 is AHR-dependent. Same assay as in C, but IDO mRNA was assessed. * - p≤0.05. * - p≤0.01. E. SU5416 induces IDO in the pDC/T cell coculture to a greater extent than TCDD. pDC/T cell coculture was utilized as described previously . Culture was performed for 5 days with SU5416 500 nM, TCDD 10 nM, FICZ 100 nM, or control, at which point mRNA was harvested and measured for IDO. * - p≤0.05. *** - p≤0.001. F. SU5416 induces FoxP3 in the pDC/T cell coculture to a greater extent than TCDD. Same assay as in E, but mRNA was assayed for FoxP3. * - p≤0.05. ** - p≤0.01. G. SU5416 enhances FoxP3 expression and CD39 on naïve T cells in the presence of TGF-β. Naïve T cells were placed in culture with DMSO (1∶4×10⁴ dilution), TGF-β (2 ng/ml), or TGF-β (2 ng/ml) and SU5416 (250 nM), harvested on day 3, and analyzed by flow cytometry. All of the above figures are representative of 3 independent experiments.