Fig 2: Dynamic changes in immune signatures during BRAFi treatment in the mouse melanoma models. (A) Schematic representation of the experimental design used for the mouse melanoma model with or without BRAFi treatment. On day -7, 1 × 10^5 SMM102 tumor cells were inoculated on the backs of C57BL/6 mice, and the tumor-bearing mice were treated with saline (control), or vemurafenib (PLX4032) for the corresponding times and analyzed at the indicated time points (day 3, 6, 18, and 27). (B) The resistance curve model of SMM102 tumor to vemurafenib. (C) Dynamic changes in tumor-infiltrating lymphocyte and stromal cell abundance at different time points of BRAFi treatment in the mouse melanoma models (GSE161430). (D,E) Heatmap showing changes in the expression levels of T cell marker genes, cytokines and effector molecule genes, and immunotherapy target genes at different times of BRAFi treatment in the mouse melanoma model. (F) Anti-CD8 immunofluorescence and anti-GZMB immunohistochemistry staining of BRAFi-treated mouse melanoma tissues. (G,H) Quantification of CD8+ cells and GZMB+ cells. The results are presented as the mean ± SD (n = 5), and statistical tests were performed using one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Fig 3: 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 4: 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 5: Dynamic changes in the immune microenvironment during MAPK-targeted therapy in melanoma. (A) Heatmap of log2FC expression in tumor-infiltrating immune cells and stromal cells between MAPKi-treated and patient-matched baseline biopsies (GSE75313, GSE65185, and EGAS00001000992). The rows have been sorted according to the log2FC of the T cell. The right histogram shows the proportion of samples with downregulated infiltrating cell abundance in the resistant biopsies compared to the baseline biopsies (B) Boxplot showing differences in log2FC expression of T cell and cytotoxicity scores between on-treatment and resistance biopsies. The log2FC was obtained by comparison with patient-matched baseline biopsy samples. Statistical analysis was performed using the Wilcoxon test. (C) The heatmap shows the changes in the expression of T cell marker genes, cytokines and effector molecule genes, and immunotherapy-related genes in the treated biopsies relative to baseline biopsies during MAPKi treatment. The order of the rows is consistent with that shown in (A). (D) Boxplot showing differences in log2FC expression of CD8A, CD8B, GZMB, and NKG7 between on-treatment and resistance biopsies. The log2FC was obtained by comparison with patient-matched baseline biopsy samples. Statistical analysis was performed using the Wilcoxon test. (E) Kaplan-Meier plots showing progression-free survival (PFS) of anti-PD1 treated melanoma patients, stratified by using the optimal cutoffs for T cell, CD8+ T cell, and cytotoxic lymphocyte infiltrating abundance (PRJEB23709). The optimal cutoff is defined as the point with the smallest p-value (log-rank test) split. (F) Kaplan-Meier plots showing overall survival (OS) of anti-PD1 treated melanoma patients, stratified by using the optimal cutoffs for T cell, CD8+ T cell, and cytotoxic lymphocyte infiltrating abundance (PRJEB23709). The optimal cutoff is defined as the point with the smallest p-value (log-rank test) split. ∗p < 0.05; ∗∗p < 0.01; ∗∗p < 0.001; *∗∗∗p < 0.0001. On-Tx: biopsies on-treatment of MAPKi; DP: biopsies resistant with MAPKi.