ACT001 reduces the expression of PD-L1 by inhibiting the phosphorylation of STAT3 in glioblastoma

Abstract

ACT001, which is derived from an ancient anti-inflammatory drug, has been shown to cross the blood-brain barrier in preclinical studies and has demonstrated anti-glioblastoma (GBM) activity in clinical trials. However, its pharmacological potential for anti-GBM immune response modulation remains unclear. The chemical structure of ACT001 indicates that it may bind to STAT3 and thus modulate antitumor immune response. Methods: Bioinformatics and immunohistochemistry (IHC) were used to assess STAT3 and PD-L1 expression in gliomas. Western blotting, RT-PCR and immunofluorescence were used to detect PD-L1 and p-STAT3 expression in glioma cells exposed to ACT001. Chromatin immunoprecipitation, an ACT001-Biotin probe, and a dual-luciferase reporter assay were used to detect direct modulation. The in vivo efficacy of ACT001 was evaluated in GL261 murine glioma model. Survival analyses were conducted using the log-rank (Mantel-Cox) test. Results: Bioinformatic analysis of 1,837 samples from 4 public glioma datasets showed that STAT3 mRNA expression was correlated with the degree of malignancy and therapeutic resistance and that STAT3 mRNA expression was related to immunosuppression, leukocyte infiltration, and PD-L1 expression. IHC staining of 53 tissue samples confirmed that relatively high phosphorylated STAT3 and PD-L1 protein expression was associated with a relatively advanced World Health Organization (WHO) glioma grade. Next, we confirmed that ACT001 treatment reduced PD-L1 expression and STAT3 phosphorylation. An ACT001-biotin probe was used to verify that ACT001 bound to STAT3. We also demonstrated that STAT3 bound to the PD-L1 promoter. The inhibition of PD-L1 expression and STAT3 phosphorylation by ACT001 could be rescued by STAT3 overexpression. Additionally, ACT001 inhibited GBM growth and decreased PD-L1 expression in vivo. The expression of the M2 markers CD206 and CD163 was decreased, while that of the antitumor immune markers iNOS and IFNγ was increased by ACT001 in vivo. Conclusion: Our results demonstrate that STAT3 plays a key role in immunosuppression of glioma and is inhibited by ACT001. ACT001 inhibits PD-L1 transcription and modulates anti-tumor immune response in glioma bearing mice. These findings will help us to understand the mechanism of ACT001 in GBM therapy.

Keywords: ACT001; PD-L1; glioblastoma; immunosuppression; p-STAT3.

Figure 1

STAT3 mRNA is related to the malignancy of glioma and therapeutic resistance. STAT3 mRNA expression in nontumor tissue versus glioma tissue of varying WHO grades in the TCGAmic (A), TCGAseq (B), CGGAseq (C), and GSE16011 (D) datasets. The types of glioma evaluated included astrocytoma (A), oligodendroastrocytoma (OA), oligodendroglioma (O), anaplastic astrocytoma (AA), anaplastic oligodendroastrocytoma (AOA), anaplastic oligodendroglioma (AO) and primary GBM (pGBM). STAT3 mRNA in pGBM of different subtypes in the TCGAmic (E), TCGAseq (F) and CGGAseq (G) datasets. H. STAT3 mRNA in secondary GBM (sGBM) and pGBM. Survival curves for glioma (L), pGBM treated with chemoradiation (I), primary glioma (pGlioma) with radiation (J) and pGlioma with chemoradiation (K) based on STAT3 mRNA levels. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; NS, not significant.

Figure 2

p-STAT3 and PD-L1 protein levels are positively correlated with WHO glioma grades. Representative images of p-STAT3 (Tyr705) expressed on nontumor (A), astrocytoma (B, abbreviated as A), anaplastic oligodendroastrocytoma (C, AOA) and glioblastoma (D, GBM) tissue samples. Representative images of PD-L1 expressed on nontumor (E), oligodendroastrocytoma (F, OA), anaplastic oligodendroglioma (G, AO) and glioblastoma (H, GBM). I and J show images of entire tissue microarrays. K and L. Nontumor tissue exhibited the lowest protein expression of p-STAT3 and PD-L1. P-STAT3 and PD-L1 protein levels positively correlated with WHO glioma grades. *, p<0.05; ***, p<0.001; NS, not significant.

Figure 3

STAT3 is related to immunosuppression and leukocyte infiltration in glioma. A. Heat maps correlating the expression of STAT3 mRNA and T cell coinhibitory molecules (PD-L1, CD86, HAVCR2, and LGALS9), the immune risk gene FCGR2B, and the immunosuppressive cytokine TGFβ in the TCGAmic, TCGAseq, CGGAseq, and GSE16011 datasets. STAT3 mRNA expression inversely correlated with that of the endogenous costimulatory molecule TNFSF9 (also named 4-1BBL). B. Heat maps correlating STAT3 mRNA expression with the immune score, tumor purity, and leukocytes infiltration in the TCGAmic and TCGAseq datasets.

Figure 4

Both p-STAT3 and PD-L1 levels are decreased by ACT001 treatment. A-C. PD-L1 mRNA expression in glioma cells treated with ACT001 as detected by RT-PCR. D. PD-L1, STAT3 and p-STAT3 (Tyr705) protein levels in glioma cells treated with ACT001 as detected by western blotting. E-G. Relative p-STAT3 (Tyr705) protein level compared to the total STAT3 protein level. H-J. Relative PD-L1 protein level compared to that of β-Actin. K-L. IF of PD-L1 and p-STAT3 in glioma cell lines. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; NS, not significant.

Figure 5

ACT001 binds to the STAT3 phosphorylation site. A-C. Glioma cell proteins in three different cell lines were detected via silver staining. Input refers to the whole protein lysate from the glioma cells. Negative refers to the ACT001-S-biotin probe solution. Positive refers to the proteins pulled down by the ACT001-biotin probe. D-F. Proteins of glioma cells were detected by western blotting using an anti-STAT3 primary antibody. G. PD-L1 and p-STAT3 (Tyr705) protein expression in glioma cells was detected by western blotting in different treatment arms. STAT3 pcDNA is a plasmid used to overexpress STAT3.

Figure 6

p-STAT3 binds to the PD-L1 promoter. A-C. ChIP was performed to verify that p-STAT3 binds to the PD-L1 promoter. The left lanes show the size of the ultrasonicated product. The right lanes show the PD-L1 promoter detected by agarose gel electrophoresis. Input refers to the whole lysate from glioma cells. The IgG group refers to the DNA pulled down by IgG. The p-STAT3 group refers to the DNA pulled down by p-STAT3. D-F. The PD-L1 promoter was PCR amplified. H, I. A dual-luciferase reporter assay was used to verify that PD-L1 transcription is promoted by STAT3. Vectors or overexpressing plasmids were transfected to glioma cells and the ratio of Firefly luciferase signal to Renilla luciferase signal was calculated.

Figure 7

Pharmacological mechanism of ACT001. ACT001 directly binds to STAT3 and inhibits the phosphorylation of STAT3. PD-L1 transcription, which is promoted by p-STAT3 binding to the promoter of PD-L1, is decreased by ACT001 suppressing STAT3 phosphorylation.

Figure 8

ACT001 decreases p-STAT3 and PD-L1 expression and inhibits the progression of glioma in vivo. A. Schematic diagram of implantation, luciferase imaging, and ACT001 administration. B. Survival curves of glioma bearing mice. The mice that remained alive at day 42 were sacrificed. C. Progression of tumor signals. Elimination of a tumor means that the signal for the tumor could not be detected compared to the background signal. Stable tumor means that the tumor signal was less than 5 times greater than the signals observed on day 7 and day 14. Progress is defined as a tumor signal that grows more than five-fold from the signals on days 7 and 14. PD-L1 (D and E) and p-STAT3 (F and G) were detected in tumor tissue from mice by IHC. *, p<0.05; **, p<0.01; NS, not significant.

Figure 9

ACT001 decreases M2 macrophage numbers and increases antitumor immune response in vivo. Red arrows, positive stainings. M2 macrophages were stained for the markers CD163 (A and B) and CD206 (C and D). T cell-mediated cytotoxicity was assessed by IFNγ (E and F) staining. M1 macrophage mediated antitumor response was assessed by iNOS (G and H) staining.