The chemotherapeutic agent DMXAA potently and specifically activates the TBK1-IRF-3 signaling axis

Abstract

Vascular disrupting agents (VDAs) represent a novel approach to the treatment of cancer, resulting in the collapse of tumor vasculature and tumor death. 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a VDA currently in advanced phase II clinical trials, yet its precise mechanism of action is unknown despite extensive preclinical and clinical investigations. Our data demonstrate that DMXAA is a novel and specific activator of the TANK-binding kinase 1 (TBK1)-interferon (IFN) regulatory factor 3 (IRF-3) signaling pathway. DMXAA treatment of primary mouse macrophages resulted in robust IRF-3 activation and approximately 750-fold increase in IFN-beta mRNA, and in contrast to the potent Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS), signaling was independent of mitogen-activated protein kinase (MAPK) activation and elicited minimal nuclear factor kappaB-dependent gene expression. DMXAA-induced signaling was critically dependent on the IRF-3 kinase, TBK1, and IRF-3 but was myeloid differentiation factor 88-, Toll-interleukin 1 receptor domain-containing adaptor inducing IFN-beta-, IFN promoter-stimulator 1-, and inhibitor of kappaB kinase-independent, thus excluding all known TLRs and cytosolic helicase receptors. DMXAA pretreatment of mouse macrophages induced a state of tolerance to LPS and vice versa. In contrast to LPS stimulation, DMXAA-induced IRF-3 dimerization and IFN-beta expression were inhibited by salicylic acid. These findings detail a novel pathway for TBK1-mediated IRF-3 activation and provide new insights into the mechanism of this new class of chemotherapeutic drugs.

Figure 1.

DMXAA preferentially induces IRF-3–mediated gene expression. (A) Peritoneal macrophages from C57BL/6 mice were stimulated for 2 h with 100 ng/ml LPS or increasing concentrations of DMXAA, as shown. Total RNA was extracted and subjected to reverse transcription followed by quantitative real-time PCR, as described in Materials and methods. (B) Primary mouse macrophages were stimulated with medium alone, 100 ng/ml LPS, or 100 μg/ml DMXAA for the indicated times. Total protein was collected and subjected to SDS-PAGE, followed by Western blotting with anti-IκBα antibody. Protein molecular masses (in kD) appear at the right. (C) RAW 264.7 macrophages were stimulated with medium alone, 10 ng/ml LPS, or 1 mg/ml DMXAA for the indicated times. Nuclear extracts were prepared and subjected to EMSA with an NF-κB–specific labeled oligonucleotide. (D) Peritoneal macrophages from C57BL/6 mice were stimulated with medium alone, 100 ng/ml LPS, or 100 μg/ml DMXAA for the indicated times. Western blotting for activated MAPK was performed on whole-cell lysates. β-Actin was used as a loading control. Protein masses (in kD) appear at the right. (E) Peritoneal macrophages from TLR4^+/+ and TLR4^−/− were stimulated with medium alone, 100 ng/ml LPS, or 100 μg/ml DMXAA for 2 h. Total RNA was collected and analyzed by quantitative real-time PCR, as in A. Results represent the mean ± SE for at least three independent experiments. **, P < 0.01.

Figure 2.

DMXAA is a potent and specific activator of TBK1. (A) Peritoneal macrophages from C57BL/6 mice were stimulated with medium alone, 100 ng/ml LPS, or 100 μg/ml DMXAA for the indicated times. Total protein was collected and subjected to native PAGE, followed by Western blotting with an anti–IRF-3 antibody. (B) Bone marrow–derived macrophages were stimulated for 90 min with medium alone, 200 ng/ml LPS, or 100 μg/ml DMXAA. Whole-cell lysates were subjected to immunoprecipitation with anti-TBK1 pAb, and immunoprecipitates were subjected to an in vitro kinase assay using the GST–C-terminal IRF-3 wild-type or A7 mutant as the substrate, as described in Materials and methods. Levels of TBK1 in immunoprecipitates were examined by Western blotting using anti-TBK1 mAb. Recombinant TBK1 and IKKβ were also included as controls with GST–IRF-3 or IκBα substrates. Protein molecular mass appears at the right. Results shown are representative of four independent experiments. (C) Peritoneal macrophages from IRF-3^+/+ and IRF-3^−/− were exposed to medium alone or 100 μg/ml DMXAA for 24 h. Supernatants were collected, and cytokine concentrations were determined by Luminex bead assay. Results are representative of three independent experiments. (D) TBK1^+/+ and TBK1^−/− MEFs were stimulated with medium alone, 100 ng/ml LPS, or 100 μg/ml DMXAA for 2 h. Total RNA was harvested and subjected to reverse transcription. Relative gene expression was measured by quantitative real-time PCR. Results represent the mean ± SE of at least three separate experiments. (E) TBK1^+/+ and TBK1^−/− MEFs were stimulated with 10 μg/ml LPS or 100 μg/ml DMXAA for 10 or 60 min. Whole-cell lysates were subjected to SDS-PAGE and probed with the indicated antibodies. Protein molecular masses appear at the right. Results are representative of three independent experiments. (F) IKKβ^+/+ and IKKβ^−/− MEFs were stimulated for 3 h with medium alone, 100 μg/ml DMXAA, 10 μg/ml or poly I:C in the presence of Fugene 6. IFN-β expression was measured by real-time PCR. Results are representative of three independent experiments. (G) Peritoneal macrophages isolated from IKKɛ^+/+ or IKKɛ^−/− mice were stimulated with 100 μg/ml DMXAA for 24 h. Supernatants were collected, and RANTES expression was determined by ELISA. Results shown are mean ± SD for three independent experiments.

Figure 3.

DMXAA signaling is TLR- and IPS-1–independent. (A and B) Peritoneal macrophages from MyD88^+/+ or MyD88^−/− mice were stimulated with 100 ng/ml LPS or 100 μg/ml DMXAA. After a 2-h incubation (left), total RNA was collected, and gene expression was assessed by quantitative real-time PCR. ELISA results (right) were generated from supernatants collected 24 h after exposure to medium, 100 ng/ml LPS, or 100 μg/ml DMXAA. (C) TRIF^+/+ and TRIF^−/− MEFs were stimulated with 100 μg/ml DMXAA or 100 μg/ml poly I:C for 24 h. Supernatants were collected, and RANTES levels were measured by ELISA. (D) Wild-type or MyD88^−/−/TRIF^−/− peritoneal macrophages were stimulated with 100 mg/ml DMXAA, 100 ng/ml LPS, or 200 hemagglutination U/ml Sendai virus for 24 h. RANTES expression was measured by ELISA. RIG-I^+/+ and RIG-I^−/− (E) or IPS-1^+/+ and IPS-1^−/− (F) MEFs were treated for 24 h with medium alone, 1 μg/ml LPS, 100 μg/ml DMXAA, or 10 μg/ml poly I:C in the presence of Fugene 6. Supernatants were collected, and RANTES levels were assessed by ELISA. Results shown are the mean ± SE of at least three independent experiments. **, P < 0.01; ***, P < 0.001.

Figure 4.

DMXAA pretreatment of macrophages induces a state of refractoriness upon reexposure to either LPS or DMXAA. Total RNA (A) or protein (B) from C57BL/6 peritoneal macrophages pretreated with medium alone, 100 ng/ml LPS, or 100 μg/ml DMXAA, washed, and restimulated with medium, LPS, or DMXAA was collected and subjected to quantitative real-time PCR (A) or native PAGE, followed by Western blotting for IRF-3 (B). Results shown are the mean ± SE of one representative experiment (n = 3).

Figure 5.

Sensitivity of DMXAA-induced gene expression and IRF-3 activation to SA. (A) Peritoneal macrophages from C57BL/6 mice were pretreated with medium alone or increasing concentrations of SA for 1 h and stimulated with LPS or DMXAA for an additional 2 h. Total RNA was harvested, and gene expression was assessed by quantitative PCR. Results represent the mean ± SE of at least three separate experiments. **, P < 0.01; ***, P < 0.001. (B) Macrophages were pretreated with or without 16 mM SA for 1 h and stimulated with 10 or 100 ng/ml LPS, or 10 or 100 μg/ml DMXAA, for an additional 1 h. Total protein was harvested and subjected to native PAGE (top) or SDS-PAGE (bottom), followed by Western blot analysis for IRF-3 and anti–phospho–IRF-3, respectively. Protein molecular mass appears at the right. Results shown are representative of three independent experiments.