ATR inhibitor AZD6738 enhances the antitumor activity of radiotherapy and immune checkpoint inhibitors by potentiating the tumor immune microenvironment in hepatocellular carcinoma

Abstract

Background: Radioimmunotherapy has a promising antitumor effect in hepatocellular carcinoma (HCC), depending on the regulatory effect of radiotherapy on tumor immune microenvironment. Ionizing radiation (IR)-induced DNA damage repair (DDR) pathway activation leads to the inhibition of immune microenvironment, thus impairing the antitumor effect of radioimmunotherapy. However, it is unclear whether inhibition of the DDR pathway can enhance the effect of radioimmunotherapy. In this study, we aim to explore the role of DDR inhibitor AZD6738 on the combination of radiotherapy and immune checkpoint inhibitors (ICIs) in HCC.

Methods: C57BL/6 mouse subcutaneous tumor model was used to evaluate the ability of different treatment regimens in tumor growth control and tumor recurrence inhibition. Effects of each treatment regimen on the alterations of immunophenotypes including the quantification, activation, proliferating ability, exhaustion marker expression, and memory status were assessed by flow cytometry.

Results: AZD6738 further increased radiotherapy-stimulated CD8⁺ T cell infiltration and activation and reverted the immunosuppressive effect of radiation on the number of Tregs in mice xenografts. Moreover, compared with radioimmunotherapy (radiotherapy plus anti-PD-L1 (Programmed death ligand 1)), the addition of AZD6738 boosted the infiltration, increased cell proliferation, enhanced interferon (IFN)-γ production ability of TIL (tumor-infiltrating lymphocyte) CD8⁺ T cells, and caused a decreasing trend in the number of TIL Tregs and exhausted T cells in mice xenografts. Thus, the tumor immune microenvironment was significantly improved. Meanwhile, triple therapy (AZD6738 plus radiotherapy plus anti-PD-L1) also induced a better immunophenotype than radioimmunotherapy in mice spleens. As a consequence, triple therapy displayed greater benefit in antitumor efficacy and mice survival than radioimmunotherapy. Mechanism study revealed that the synergistic antitumor effect of AZD6738 with radioimmunotherapy relied on the activation of cyclic GMP-AMP synthase /stimulator of interferon genes (cGAS/STING) signaling pathway. Furthermore, triple therapy led to stronger immunologic memory and lasting antitumor immunity than radioimmunotherapy, thus preventing tumor recurrence in mouse models.

Conclusions: Our findings indicate that AZD6738 might be a potential synergistic treatment for radioimmunotherapy to control the proliferation of HCC cells, prolong survival, and prevent tumor recurrence in patients with HCC by improving the immune microenvironment.

Keywords: immunology; oncology; radiotherapy; tumors.

Figure 1

AZD6738 improves immune microenvironment in mice hepatocellular carcinoma (HCC) tissues after radiotherapy. (A) Representative contour plots depicting the percentages of tumor-infiltrating (TIL) Tregs (CD25⁺Foxp3⁺), and the quantitation of the number of TIL Tregs per 10⁴ cells stained at days 8 and 14 was shown. (B) Representative contour plots and quantification of CD8⁺/CD3⁺ ratios at days 8 and 14. (C) Quantitation of PD-1 and Tim3 median fluorescence intensity in TIL CD8⁺ T cells at days 8 and 14. (D) Representative contour plots and quantification of the percentage of TIL PD-1⁺ CD8⁺ T cells at days 8 and 14. (E) Representative contour plots and quantification of TIL CD8⁺ T cells that elicit IFN-γ following stimulation with PMA/ionomycin at days 8 and 14. Data represent the mean±SD for (A)-(E), statistical analysis was performed by using one-way analysis of variance with Tukey’s multiple comparisons test. *p<0.05; **p<0.005;+ ****p<0.0001. IFN-γ, interferon-γ; IR, ionizing radiation; PMA, phorbol 12-myristate 13-acetate.

Figure 2

Addition of AZD6738 potentiates radiation combined with anti-PD-L1 therapy in mice tumor growth control and survival improvement. (A) Schematic showing schedules of ionizing radiation (IR), AZD6738, and anti-PD-L1 treatment. (B–C) Response of the subcutaneous tumors (B) or individual tumor volumes (C) to the indicated treatment regimens. n=12 in each group. Data represent the mean±SEM. (D) Survival plots for each treatment regimens were shown. (E) Response of the orthotopic hepatocellular carcinoma (HCC) tumors to treatment with the indicated treatment regimens. n=5 in each group. Data represent the mean±SEM. (F) Representative images of mice liver MRI before treatment and the corresponded isolated liver samples after treatment. For (B), statistical analyses were performed using a mixed-effects model, followed by Tukey’s multiple comparison test. For (D), survival data were compared using the log-rank Mantel-Cox test. For (E), statistical analyses were performed using one-way analysis of variance with Tukey’s multiple comparisons test. *p<0.05; ***p<0.0005.

Figure 3

Triple therapy impacts T cell infiltration in mice tumors and spleen. (A) Schematic shows schedules of triple therapy including ionizing radiation (IR), AZD6738, and anti-PD-L1. The red frames indicated the time points for lymphocyte analyses in mice tumors or spleens. (B) Quantitation of the number of tumor-infiltrating lymphocyte (TIL) CD8⁺ T cells per 10⁴ cells at days 8 and 14. (C) Quantitation of CD8⁺/CD3⁺ ratios in mice tumors. (D) Quantitation of the number of TIL Tregs per 10⁴ cells. (E) Quantitation of CD8⁺/Treg ratios in mice tumors. (F) Quantitation of CD8⁺/Treg ratios in mice spleens. Data represent the mean±SD. For (B)–(F), statistical analysis was performed by using one-way analysis of variance with Tukey’s multiple comparisons test. *p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001.

Figure 4

Triple therapy promotes the proliferation of splenic and tumor-infiltrating T cells. (A–B) Representative contour plots of Ki67⁺ expression on tumor-infiltrating lymphocyte (TIL) and splenic CD8⁺ T cells at day 8. (C–D) Quantitation of the percentages of proliferating (Ki67⁺) TIL CD8⁺ T cells (C) and TIL CD4⁺ T cells (D) at days 8 and 14. (E–F) Quantitation of the percentages of proliferating (Ki67⁺) splenic CD8⁺ T cells (E) and splenic CD4⁺ T cells (F) at days 8 and 14. Data represent the mean±SD. For (C)–(F), statistical analysis was performed by using one-way analysis of variance with Tukey’s multiple comparisons test. *p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001. IR, ionizing radiation.

Figure 5

Triple therapy attenuates coexpression of CD8⁺ T cell exhaustion markers and promotes CD8⁺ T cell effector function in mice tumors. (A–B) Representative contour plots depicting PD-1 and LAG-3 coexpression and PD-1 and Tim-3 coexpression on tumor-infiltrating lymphocyte (TIL) CD8⁺ T cells at day 8. (C–D) Quantitation of the percentage of TIL CD8⁺ T cells coexpressing PD-1 and LAG-3 (C), and PD-1 and Tim-3 (D) at days 8 and 14. (E–F) Representative contour plots depicting IFN-γ expressing CD8⁺ T cells in mice spleens and tumors following stimulation with PMA/ionomycin at day 8. (G–H) Quantitation of the percentage of TIL (G) and splenic (H) CD8⁺ T cells that expressed IFN-γ at days 8 and 14. Data represent the mean±SD. For (C), (D), (G) and (H), statistical analysis was performed by using one-way analysis of variance with Tukey’s multiple comparisons test. *p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001. IFN-γ, interferon-γ; IR, ionizing radiation.

Figure 6

AZD6738 promotes radioimmunotherapy antitumor effect via activating cGAS/STING signaling pathway. (A) Several key proteins of cGAS/STING pathway were detected in Hepa 1–6 subcutaneous tumors from mice treated with radioimmunotherapy or triple therapy group. (B) Response of the Hepa 1–6 subcutaneous tumors to the indicated treatments. n=12 in each group. Data represent the mean±SEM. Statistical analysis was performed by using mixed-effects model, followed by Tukey’s multiple comparison test. ***p<0.0005. IR, ionizing radiation.

Figure 7

Triple therapy promotes immune memory activation in mice tumors. (A) Representative contour plots depicting CD62L and CD44 expression in tumor-infiltrating lymphocyte (TIL) CD8⁺ T cells at day 8. (B–C) Quantitation of the percentage of TIL CD8⁺ T cell central memory (T_CM, CD62L⁺CD44⁺) cells or effector memory (T_EM, CD62L^-CD44⁺) cells at days 8 and 14. (D) Mice tumors were re-challenged with Hepa 1–6 cells in the contralateral flank on the 20th day. Data represent the curve for the mean tumor volumes. Data represent the mean±SD. For (B) and (C), statistical analysis was performed by using one-way analysis of variance with Tukey’s multiple comparisons test. For (D), statistical analysis was performed using mixed-effects model, followed by Tukey’s multiple comparison test. *p<0.05; **p<0.005; ***p<0.0005. IR, ionizing radiation.