Combining protein arginine methyltransferase inhibitor and anti-programmed death-ligand-1 inhibits pancreatic cancer progression

Abstract

Background: Immunotherapy targeting programmed death-1 (PD-1) or programmed death-ligand-1 (PD-L1) has been shown to be effective in a variety of malignancies but has poor efficacy in pancreatic ductal adenocarcinoma (PDAC). Studies have shown that PD-L1 expression in tumors is an important indicator of the efficacy of immunotherapy. Tumor cells usually evade chemotherapy and host immune surveillance by epigenetic changes. Protein arginine methylation is a common posttranslational modification. Protein arginine methyltransferase (PRMT) 1 is deregulated in a wide variety of cancer types, whose biological role in tumor immunity is undefined.

Aim: To investigate the combined effects and underlying mechanisms of anti-PD-L1 and type I PRMT inhibitor in pancreatic cancer in vivo.

Methods: PT1001B is a novel type I PRMT inhibitor with strong activity and good selectivity. A mouse model of subcutaneous Panc02-derived tumors was used to evaluate drug efficacy, toxic and side effects, and tumor growth in vivo. By flow cytometry, we determined the expression of key immune checkpoint proteins, detected the apoptosis in tumor tissues, and analyzed the immune cells. Immunohistochemistry staining for cellular proliferation-associated nuclear protein Ki67, TUNEL assay, and PRMT1/PD-L1 immunofluorescence were used to elucidate the underlying molecular mechanism of the antitumor effect.

Results: Cultured Panc02 cells did not express PD-L1 in vitro, but tumor cells derived from Panc02 transplanted tumors expressed PD-L1. The therapeutic efficacy of anti-PD-L1 mAb was significantly enhanced by the addition of PT1001B as measured by tumor volume (1054.00 ± 61.37 mm³ vs 555.80 ± 74.42 mm³, P < 0.01) and tumor weight (0.83 ± 0.06 g vs 0.38 ± 0.02 g, P < 0.05). PT1001B improved antitumor immunity by inhibiting PD-L1 expression on tumor cells (32.74% ± 5.89% vs 17.95% ± 1.92%, P < 0.05). The combination therapy upregulated tumor-infiltrating CD8+ T lymphocytes (23.75% ± 3.20% vs 73.34% ± 4.35%, P < 0.01) and decreased PD-1+ leukocytes (35.77% ± 3.30% vs 6.48% ± 1.08%, P < 0.001) in tumor tissue compared to the control. In addition, PT1001B amplified the inhibitory effect of anti-PD-L1 on tumor cell proliferation and enhanced the induction of tumor cell apoptosis. PRMT1 downregulation was correlated with PD-L1 downregulation.

Conclusion: PT1001B enhances antitumor immunity and combining it with anti-PD-L1 checkpoint inhibitors provides a potential strategy to overcome anti-PD-L1 resistance in PDAC.

Keywords: Combination therapy; Pancreatic ductal adenocarcinoma; Programmed death-ligand-1 blockade; Protein arginine methyltransferase; Tumor microenvironment.

Figure 1

Programmed death-ligand-1 expression in pancreatic ductal adenocarcinoma in vitro and in vivo. A: Panc02 cells were subcutaneously transplanted into C57BL/6 mice to establish pancreatic tumors (orange arrow); B: Programmed death-ligand-1 expression levels in cultured pancreatic cancer cells (in vitro) and tumors (in vivo). The mean fluorescence intensity was used to quantify protein expression levels, and the values were statistically analyzed by a two-sided t-test; the results are presented at the bottom. Data represent the mean ± SE (n = 3). ^aP < 0.05 vs in vitro condition. PD-L1: Programmed death-ligand-1; MFI: Mean fluorescence intensity.

Figure 2

The combination of PT1001B and programmed death-ligand-1 blockade inhibits the development of pancreatic ductal adenocarcinoma in mice. Panc02 cells were surgically transplanted into C57BL/6 mice. One week later, tumors reached approximately 100 mm³, and tumor-bearing mice were randomly divided into four groups that were treated with PT1001B (30 mg/kg body weight, once daily) or anti- programmed death-ligand-1 (PD-L1) mAb (200 μg/mouse, every 2 d for 5 intervals) alone or in combination via intraperitoneal injection for 37 d. A: The combination of PT1001B and anti-PD-L1 mAb significantly inhibited tumor growth; B: Tumors in different treated groups; C: Mouse body weight curve. The values were analyzed by one-way ANOVA. Data represent the mean ± SE (n = 4). ^aP < 0.05, ^bP < 0.01 vs control group. PD-L1: Programmed death-ligand-1.

Figure 3

PT1001B enhances antitumor immunity. Cell suspensions were prepared from pancreatic tumor tissues and spleens from the four groups of tumor-bearing mice and stained with CD45-, CD4-, CD8-, PD-L1-, and PD-1-specific antibodies. Stained cells were analyzed by flow cytometry. The left panel presents representative images, and the right panel shows the statistical analysis. A: Analysis of CD45-/PD-L1+ cells in the tumors; B: Analysis of CD45+/PD-1+ cells in the tumors; C: Analysis of CD45+/PD-1+ cells in the spleens; D: Analysis of CD4+ T cells and CD8+ T cells in the tumors; E: Analysis of CD4+ T cells and CD8+ T cells in the spleens. Differences in the percentages of cells were analyzed by one-way ANOVA. Data represent the mean ± SE (n = 4). ^aP < 0.05, ^bP < 0.01 vs control group; ^cP < 0.05, ^dP < 0.01 vs anti-PD-L1 group. PD-L1: Programmed death-ligand-1; PD-1: Programmed death-1.

Figure 4

Effect of PT1001B and anti-programmed death-ligand-1 mAb on tumor cell proliferation and apoptosis. A: Cell suspensions prepared from pancreatic tumor tissues were stained with annexin V and propidium iodide to analyze the apoptosis rate by flow cytometry; B: The four groups of tumors were sectioned and stained for Ki67 (brown); C: The four groups of tumors were sectioned and subjected to TUNEL assay (brown). Representative images are shown. a: Control; b: PT1001B; c: Anti-programmed death-ligand-1 (PD-L1) mAb; d: Anti-PD-L1 mAb + PT1001B. Scale bars = 100 µm. The populations of apoptotic cells, Ki67-positive, or TUNEL-positive cells were analyzed by one-way ANOVA. Data represent the mean ± SE (n = 4). ^aP < 0.05, ^bP < 0.01 vs control group; ^cP < 0.05, ^dP < 0.01 vs anti-PD-L1 group. PI: Propidium iodide; FITC: Fluorescein Isothiocyanate; PD-L1: Programmed death-ligand-1; TUNEL: Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling.

Figure 5

Immunofluorescence analysis of protein arginine methyltransferase and programmed death-ligand-1 in tumor tissue. Protein arginine methyltransferase 1 is shown in red, programmed death-ligand-1 is shown in green, and DAPI staining indicates the nucleus. Scale bars = 100 µm. PRMT: Protein arginine methyltransferase; PD-L1: Programmed death-ligand-1; DAPI: 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride.