ARIH1 activates STING-mediated T-cell activation and sensitizes tumors to immune checkpoint blockade

Abstract

Despite advances in cancer treatment, immune checkpoint blockade (ICB) only achieves complete response in some patients, illustrating the need to identify resistance mechanisms. Using an ICB-insensitive tumor model, here we discover cisplatin enhances the anti-tumor effect of PD-L1 blockade and upregulates the expression of Ariadne RBR E3 ubiquitin-protein ligase 1 (ARIH1) in tumors. Arih1 overexpression promotes cytotoxic T cell infiltration, inhibits tumor growth, and potentiates PD-L1 blockade. ARIH1 mediates ubiquitination and degradation of DNA-PKcs to trigger activation of the STING pathway, which is blocked by the phospho-mimetic mutant T68E/S213D of cGAS protein. Using a high-throughput drug screen, we further identify that ACY738, less cytotoxic than cisplatin, effectively upregulates ARIH1 and activates STING signaling, sensitizing tumors to PD-L1 blockade. Our findings delineate a mechanism that tumors mediate ICB resistance through the loss of ARIH1 and ARIH1-DNA-PKcs-STING signaling and indicate that activating ARIH1 is an effective strategy to improve the efficacy of cancer immunotherapy.

Fig. 1. ARIH1 accumulates after treatment of 4T1 tumor models with a combination of cisplatin and anti-PD-L1 antibody.

a, b Tumor growth curves and tumor weights upon subcutaneous injection of 5 × 10⁵ 4T1 cells into female BALB/c mice (6–8 week old) treated with vehicle, cisplatin alone, anti-PD-L1 alone and cisplatin+anti-PD-L1. n = 6 mice/group. Data represent means ± SEM. a ^**P < 0.01 (P = 0.0052), ^****P < 0.0001. b ^**P < 0.01 (P = 0.0014), ^***P < 0.001 (P = 0.0007), ^****P < 0.0001. c The survival curves of tumor bearing mice with indicated treatments. n = 7 mice/group. Log-rank test, ^*P < 0.05 (P = 0.0351), ^**P < 0.01 (P = 0.0023). d, e. Representative images of tumor CD8 and GzmB IHC staining of the mice as in (a). The percent of each expression pattern was quantified (e). Scale bar, 60 μm. n = 6 mice/group. Data represent means ± SEM, ^***P < 0.001 (P = 0.0002), ^****P < 0.0001. f Representative ARIH1 IHC staining for tumors of the mice as in (a). The percent of expression pattern was quantified. Scale bar, 60 μm. n = 6 mice/group. Data represent means ± SEM, ^*P < 0.05 (P = 0.0233). g Immunoblots analysis of the ARIH1 protein levels in the indicated tumor lysates of mice as in (a). h–j Tumor growth, final tumor image and tumor weights of Ctrl-KD and Arih1-KD 4T1 cells in female BALB/c mice (n = 7 per group, 6–8 week old) with indicated treatments. Data represent means ± SEM. h ^**P < 0.01, ^****P < 0.0001. j ^*P < 0.05, ^****P < 0.0001, ns, not significant. Data shown in a and h are representative of three independent experiments. For a, h data, Two-way ANOVA test. For b, e, j data, One-way ANOVA test. For f data, Two-tailed t-test. Source data are provided as a Source Data file.

Fig. 2. ARIH1 enhances anti-PD-L1 antibody-induced anti-tumor immunity in 4T1 tumor models.

a Tumor growth curves in female BALB/c mice (6–8 week old) with control (CTRL) and Arih1-overexpressing (Arih1^-WT-OE) tumors treated with PD-L1 or isotype mAbs intraperitoneally (i.p.) starting on day 7 and then every three days after subcutaneous inoculation of 5 × 10⁵ 4T1 cells. n = 6 mice/group. Data represent means ± SEM, ^****P < 0.0001. b, c Representative image of tumors and tumor weights of tumor bearing mice at Day25 with the indicated treatments. n = 6 mice/group. Data represent means ± SEM, ^*P < 0.05 (P = 0.0134), ^***P < 0.001 (P = 0.0002). d, e Representative figures and quantification of tumor infiltrating CD8⁺ T cells and CD8⁺GzmB⁺ T cells of the mice as in a. n = 6, 6, 6, 4 mice/group. Data represent means ± SEM. d ^*P < 0.05, ^***P < 0.001 (P = 0.0001), ^****P < 0.0001. e ^**P < 0.01. f, g Representative images of tumor CD8 and GzmB IHC staining of mice as in a. The percent of each expression pattern was quantified (g). Scale bar, 60 μm. n = 6, 6, 6, 4 mice/group. Data represent means ± SEM, ^****P < 0.0001. h–j Representative tumor growth, image of tumors, and tumor weights in female BALB/c mice (6–8 week old) bearing CTRL, Arih1^-WT-OE, and Arih1^-C355S-OE 4T1 tumors with the indicated treatments. n = 5 mice/group. Data represent means ± SEM. h ^****P < 0.0001, ns, not significant. j ^**P < 0.01 (P = 0.0059), ^****P < 0.0001, ns, not significant. k. The survival curves for mice (n = 7 per group) with the indicated treatments. Log-rank test, ^***P < 0.001, ns, not significant. l, m Quantification of FACS data for tumor infiltrating CD8⁺ T cells and CD8⁺GzmB⁺ T cells of mice as in h. n = 5, 5, 4, 5 mice/group. Data represent means ± SEM, ^**P < 0.01 (P = 0.0020), ^***P < 0.001 (P = 0.0006), ^****P < 0.0001, ns, not significant. Data shown in a and h are representative of three independent experiments. For a, h data, Two-way ANOVA test. For d, e, g, j, l, m data, One-way ANOVA test. For c data, Two-tailed t-test. Source data are provided as a Source Data file.

Fig. 3. ARIH1 promotes the STING pathway activation.

a Heatmap for the qRT-PCR expression data of genes for 10 cytokines and 11 chemokines in CTRL and Arih1^-WT-OE 4T1 tumors (n = 3 per group). b qRT-PCR analysis for the gene expression of Ifnb1, Ifna2, Il-6, and Ccl5 as in a. n = 3 per group. Data represent means ± SEM, ^**P < 0.01 (P = 0.0013), ^***P < 0.001, ^****P < 0.0001. c Immunofluorescence analysis of dsDNAs and cGAS in CTRL and Arih1^-WT-OE 4T1 cells. The nuclei were stained with DAPI. Representative confocal images and quantitative data are shown. CTRL group (n = 31 cells), Arih1^-WT-OE group (n = 43 cells). Scale bar, 10 μm; insets: Scale bar, 2 μm. Data represent means ± SEM, ^**P < 0.01 (P = 0.0042), ^****P < 0.0001. d Immunoblots of markers in the STING pathway including total and phospho STING (S366 sites for human; S365 sites for mouse), total and phospho TBK1(S172) in lysates collected from breast cell lines (MCF-7 and MDA-MB-231) and 4T1 tumors (from Fig. 2a) with indicated treatments. e Immunofluorescent staining of IRF3 in CTRL and Arih1^-WT-OE 4T1 cells and their quantifications. The nuclei were stained with DAPI. Scale bar, 10 μm; insets: Scale bar, 5 μm. Data represent means ± SEM, ^****P < 0.0001. Each dot in the graph represents the percentage of counted nucleus IRF3 cells in each sample, and the total number of counted cells in each group is as follows: CTRL group (n = 135 cells), Arih1^-WT-OE group (n = 147 cells). For b, c, e data, Two-tailed t-test. Data shown in c–e are representative of three independent experiments. Source data are provided as a Source Data file.

Fig. 4. Knockdown of STING reverses the anti-tumor effect of ARIH1- enhanced PD-L1 blockade therapy.

a, b Representative tumor growth curves and final tumor weights of tumors (including CTRL, Arih1^-WT-OE, and Arih1^-WT-OE&Sting-KD) in female BALB/c mice (6–8 week old) with the indicated treatments after subcutaneous injection of 5 × 10⁵ 4T1 cells. n = 6 mice/group. Data represent means ± SEM. a ^****P < 0.0001. b ^*P < 0.05 (P = 0.0492), ^**P < 0.01 (P = 0.0027), ^***P < 0.001 (P = 0.0002), ns, not significant. c, d Representative figures and summary of frequency of tumor infiltrating CD8⁺ T cells and GzmB⁺CD8⁺ T cells of the mice as in a. n = 6, 6, 4, 6 mice/group. Data represent means ± SEM. c ^**P < 0.01 (P = 0.0083), ^***P < 0.001, ns, not significant. d ^*P < 0.05 (P = 0.0341), ^**P < 0.01 (P = 0.0022), ^***P < 0.001 (P = 0.0010), ns, not significant. e, f CD8 and GzmB IHC staining were performed in tumors of the mice as in a. The percent of each expression pattern was quantified f. Scale bar, 60 μm. n = 6, 6, 4, 6 mice/group. Data represent means ± SEM, ^****P < 0.0001, ns, not significant. g Immunoblot analysis of pSTING-S365, STING, and ARIH1 in indicated tumor lysates for the experiment described in a. h qRT-PCR measurement of ISGs for the tumors of the mice as in a. n = 6, 6, 4, 6 mice/group. Data represent means ± SEM, ^****P < 0.0001, ns, not significant. For a data, Two-way ANOVA test. For b–d, f, and h data, One-way ANOVA test. Source data are provided as a Source Data file.

Fig. 5. ARIH1-mediated degradation of DNA-PKcs promotes STING pathway activation.

a HEK293T cells were transfected with ARIH1^-WT-HA or ARIH1^-C357S-HA for 36 h. Proteins that co-immunoprecipitated (Co-IP) with ARIH1 were analyzed by mass spectrometry. DNA repair-related proteins are shown in heatmap. b Co-IP of ARIH1 with DNA-PKcs in HEK293T cells. Endogenous DNA-PKcs was immunoprecipitated using anti-DNA-PKcs, and the immunoprecipitates were analyzed with anti-ARIH1. lgG, immunoglobulin G. c Immunoblot (IB) analysis for ubiquitination of DNA-PKcs from HEK293T cells co-transfected with the indicated constructs. d In vitro ubiquitination assay of purified DNA-PKcs. The reactions were performed with purified His-ubiquitin, GST-UBA1 (E1), His-UBCH7 (E2), and GST-ARIH1^-WT or its ligase-dead mutant (GST-ARIH1^-C357S) or in the absence of UBA1, UBCH7, ubiquitin, ARIH1 or DNA-PKcs. e, f. IB analysis of DNA-PKcs levels in U2OS cells. The cells were incubated with small interfering RNAs (siRNAs) against ARIH1 (f), or transfected with ARIH1-HA (e). g IB analysis of DNA-PKcs in U2OS cells transfected with the indicated constructs. Cells were treated by MG132, Bafilomycin or NH₄Cl+Leup for 6 h. h Immunofluorescent staining of DNA-PKcs and Lamp2 in CTRL and Arih1^-WT-OE 4T1 cells and their quantifications. The nuclei were stained with DAPI. Scale bar, 10 μm; insets: Scale bar, 2 μm. Data represent means ± SEM, ^****P < 0.0001. Each dot in the graph represents the percentage of counted cells with co-localization in each sample, and the total number of counted cells in each group is as follows: CTRL group (n = 381 cells), Arih1^-WT-OE group (n = 321 cells). i IB analysis of pSTING-S366, pTBK1-S172 and DNA-PKcs in HEK293T-STING cells co-transfected the indicated constructs. j cGAS-KD MCF-7 cells were transfected with cGAS WT or the phosphorylation-mimic mutants and ARIH1-HA. WCLs were analyzed by immunoblotting. k qRT-PCR measurement of ISGs expression in cGAS-KD HeLa cells transfecting with cGAS WT or the phosphorylation-mimic mutants and ARIH1-HA. IFNB1 (n = 3/group), IFNA2 (n = 3/group), IL6 (n = 4/group). Data represent means ± SEM, ^***P < 0.001 (P = 0.0002), ^****P < 0.0001. In e–g, the numbers under the blots represent the gray scale quantification (DNA-PKcs/Actin). For k data, One-way ANOVA test. For h data, Two-tailed t-test. Data shown in b, c, e–h, and j, k are representative of two independent experiments. Data shown in a, d and i are representative of three independent experiments. Source data are provided as a Source Data file.

Fig. 6. The small molecule ACY738 increases ARIH1 protein levels and enhances PD-L1 blockade therapy in 4T1 tumor models.

a High-throughput screening of 8207 drug or drug candidates was performed to screen for ARIH1 enhancers that increase ARIH1 levels. After treatment with the drugs at 5 μM for 24 h, luciferase as a reporter to detect ARIH1 protein levels following exposing to the substrate (10 μM) in 384-well plates. Hit compounds are shown as red dots. b Immunoblots (IB) analysis for the ARIH1 levels in MDA-MB-231 and 4T1 cells treated with 1 µM ACY738 at the indicated time points. The numbers under the blots represent the gray scale quantification (ARIH1/Actin and ARIH1/Tubulin). c Tumor growth curves from subcutaneous injection of 5 × 10⁵ 4T1 cells in the female BALB/c mice (6–8 week old) treated with vehicle, anti-PD-L1 alone, ACY738 alone, and ACY738+anti-PD-L1. n = 6 mice/group. Data represent means ± SEM, ^**P < 0.01 (P = 0.0012), ^***P < 0.001 (P = 0.0005), ^****P < 0.0001. d–f. Representative image, final tumor weights, and survival curves of tumor bearing mice with the indicated treatments. n = 6 mice/group. Data represent means ± SEM, e ^*P < 0.05 (P = 0.0151), ^***P < 0.001 (P = 0.0010). Log-rank test (f), ^*P < 0.05 (P = 0.0200), ^**P < 0.01. g, h Representative images and quantification of spontaneous lung metastases and lung weights of BALB/c mice (6–8 week old) at day 16 with the indicated treatments after intravenous injection of 1 × 10⁵ 4T1 cells. Scale bar, 400 μm. n = 5 mice/group. Data represent means ± SEM, ^**P < 0.01, ^***P < 0.001 (P = 0.0008), ^****P < 0.0001. i Quantification of tumor-infiltrating CD8⁺ T cells of the mice as in c. n = 6 mice/group. Data represent means ± SEM, ^**P < 0.01 (P = 0.0018), ^***P < 0.001. j, k The CD8 and GzmB IHC staining (j) was performed in tumors of the mice as in c. The percent of each expression pattern was quantified (k). Scale bar, 60 μm. n = 6 mice/group. Data represent means ± SEM, ^***P < 0.001, ^****P < 0.0001. l IB analysis of total and phospho STING (S365), total and phospho TBK1(S172), and ARIH1 in tumor lysates of the mice as in c. For c data, Two-way ANOVA test. For e, h-i, and k data, One-way ANOVA test. Data shown in a, b and i are representative of three independent experiments. Source data are provided as a Source Data file.