ARID1A suppresses R-loop-mediated STING-type I interferon pathway activation of anti-tumor immunity

Abstract

Clinical trials have identified ARID1A mutations as enriched among patients who respond favorably to immune checkpoint blockade (ICB) in several solid tumor types independent of microsatellite instability. We show that ARID1A loss in murine models is sufficient to induce anti-tumor immune phenotypes observed in ARID1A mutant human cancers, including increased CD8+ T cell infiltration and cytolytic activity. ARID1A-deficient cancers upregulated an interferon (IFN) gene expression signature, the ARID1A-IFN signature, associated with increased R-loops and cytosolic single-stranded DNA (ssDNA). Overexpression of the R-loop resolving enzyme, RNASEH2B, or cytosolic DNase, TREX1, in ARID1A-deficient cells prevented cytosolic ssDNA accumulation and ARID1A-IFN gene upregulation. Further, the ARID1A-IFN signature and anti-tumor immunity were driven by STING-dependent type I IFN signaling, which was required for improved responsiveness of ARID1A mutant tumors to ICB treatment. These findings define a molecular mechanism underlying anti-tumor immunity in ARID1A mutant cancers.

Keywords: ARID1A; R-loops; STING; SWI/SNF complex; anti-tumor immunity; cancer immunotherapy; cytosolic DNA; type I IFN.

Figure 1.. ARID1A loss is sufficient to induce anti-tumor immunity in murine tumor models and recapitulates features of human ARID1A mutant anti-tumor immunity.

(A-B) Tumor growth curves of sgScramble and sgArid1a B16F10 and MC38 tumors. Data are represented as mean ± SEM. (C-D) Tumor weights on Day 15 or Day 17 post injection for B16F10 tumors or MC38 tumors. (E-I) Tumor infiltrating immune cell profile in sgScramble and sgArid1a B16F10 tumors. (J) Human endometrial cancer IHC samples with representative images of ARID1A IHC and multiplexed fluorescence IHC for CD8α (yellow) and DAPI (blue) in patient samples with ARID1A protein intact or ARID1A protein loss. MMR status assessed via IHC (scale bar = 100μM). (K-L) Quantification and flow cytometry contour plots of CD8+ T cell TNFα and IFNγ or Granzyme B and IFNγ staining following ex vivo PMA/ionomycin stimulation from sgScramble and sgArid1a B16F10 tumors. (M) TCGA cohort cytolytic scores in ARID1A mutant and non-mutant cancers. (N) Ki67 staining in CD8+ T cells from spleen, sgScramble, or sgArid1a B16F10 tumors. (O) PD1 staining in CD8+ T cells from sgScramble and sgArid1a B16F10 tumors. (P) CD8+ T cell exhaustion subsets quantified by cells per gram of B16F10 tumor. (Q) CD8+ T cell exhaustion subsets in sgScramble and sgArid1a B16F10 tumors. (R) Tumor infiltrating CD4+ T cell IFNγ and TNFα staining following ex vivo PMA/ionomycin stimulation. (S) Tumor infiltrating NK cell IFNγ and Granzyme B staining following ex vivo PMA/ionomycin stimulation. (T-U) Comparisons of tumor growth curves of sgScramble and sgArid1a B16F10 and MC38 tumors in Rag1 −/− and C57BL/6J mice. Data are represented as mean ± SEM. All data are represented as mean ± SD unless otherwise noted. Statistical analysis in Fig 1L was performed using Welch’s Two sample t-test.

Figure 2.. ARID1A loss induces a tumor transcriptome dominated by IFN response and is associated with altered CD8+ T cell states and T cell receptor clonality types.

(A) Heatmap of differentially expressed genes in sgArid1a and sgScramble B16F10 tumors. (B) GSEA Hallmarks enriched in sgArid1a B16F10 tumors. (C) GSEA curves of IFN Alpha and Gamma Response gene sets comparing ARID1A mutant versus non-mutant cancers from TCGA. (D) UMAP of CD8+ T cells sorted from sgScramble and sgArid1a tumors and subjected to scRNA-sequencing. (E-F) Cluster density and quantification as proportion of UMAP. (G) Anti-PD1 Response Signature gene set expression projected on UMAP. (H) GSEA Hallmarks enriched in C2 effector-like CD8+ T cells from sgArid1a tumors compared to sgScramble tumors. (I) CD8+ T cell metagene ISG score composed of the composite expression of genes from IFN Alpha and Gamma Response GSEA leading edge in C1–C5 projected on UMAP. (J) T cell receptor clonality classes projected onto UMAP with stacked bar plot quantification of classes. (K) Quantitation of the proportion of individual TCR clonotypes in sgArid1a and sgScramble tumors.

Figure 3.. ARID1A loss or cBAF inhibition induces a cancer-cell intrinsic IFN gene expression signature associated with anti-tumor immunity and immunotherapy response.

(A-C) GSEA of Hallmark gene sets enriched in sgArid1a B16F10, sgArid1a MC38, and BD98 treated B16F10s, respectively. (D) Venn diagram of significantly upregulated genes in sgArid1a B16F10, sgArid1a MC38, and Arid1a^−/− MEFs with 57 commonly upregulated ISGs used to construct the ARID1A-IFN signature. (E) GSEA curves of ARID1A-IFN signature enrichment comparing ARID1A mutant vs non-mutant cancers from TCGA. (F) Pie chart of ARID1A mutation types in TCGA cohorts analyzed in this manuscript. (G) Tumor RPPA ARID1A levels between non-mutant, ARID1A nonsense/FS deletion, and ARID1A missense mutations in Colon, Stomach, and Uterine TCGA cohorts. (H-I) ARID1A-IFN signature and immune cytolytic scores between non-mutant, ARID1A nonsense/FS deletion, and ARID1A missense mutations in Colon, Stomach, and Uterine TCGA cohorts. (J) Survival pot of colon cancer patients stratified by top and bottom 20% of patients for ARID1A-IFN signature score. (K) ARID1A-IFN signature scores for non-mutant and ARID1A mutant colon cancer patients stratified by survival status at 100 months. (L) ARID1A-IFN signature scores association with Anti-PD1 response in stomach cancer patients across RECIST clinical response groups. Statistical analysis in Fig 3G–I were performed using Wilcoxon Rank Sum Test, Fig 3J log-rank test survival test, Fig 3K via two-way ANOVA, and Fig 3L via one-way ANOVA.

Figure 4.. ARID1A loss activates Type I IFN pathway and enhanced response to IFNγ.

(A) Heatmaps of ISGs commonly upregulated in sgArid1a or BD98 treated B16F10 cells and their expression following anti-IFNAR treatment in each condition. (B-C) Western blots of ISGs RIG-I, STAT1, and ISG15 in sgArid1a (B) and BD98 treated (C) B16F10 cells with or without anti-IFNAR treatment. (D-E) qPCR for upregulated ISGs in sgArid1a B16F10s (D) or sgArid1a MC38s (E) with or without treatment with listed IFN blocking antibodies normalized to sgScramble mRNA levels. All sgArid1a to sgScramble comparisons are statistically significant at p >0.01 confidence and all sgArid1a vs sgArid1a + IFN blocking antibody comparisons significance results are shown above the IFN blocking antibody condition bar. (F) Median fluorescence intensity (MFI) quantification and representative histograms for ISRE-GFP signal in vehicle, BD98 treated, or BD98 treated + IFN blocking antibodies in ISRE-GFP reporter MEFs. (G) IFNβ ELISA results from sgScramble and sgArid1a B16F10 and MC38 cell supernatants. (H) IFNβ ELISA results from vehicle or BD98 treated MEFs supernatants. (I) Clustered heatmap of gene expression for IFNγ responsive genes in untreated or IFNγ treated sgScramble or sgArid1a B16F10 cells. (J) Heatmap of selected genes whose expression is higher in sgArid1a B16F10 cells following IFNγ treatment. (K-L) Flow cytometry histograms and median fluorescence intensity quantifications of MHC Class I and II in sgScramble or sgArid1a B16F10 cells with or without IFNγ treatment. (M) Quantification of percentage of dead B16F10s following co-culture assay between P14 CD8+ T cells and GP33 pulsed B16F10s with or without IFNγ pre-treatment of B16F10s. All data are represented as mean ± SD. Statistical analysis in Fig 4D–E was performed using two-way ANOVA and Fig 4F statistical analysis using one-way ANOVA.

Figure 5.. ARID1A loss or cBAF inhibition induces R-loop derived cytosolic DNA that drives the ARID1A IFN Signature.

(A) Immunofluorescence of dRNH1 and DAPI and quantification of dRNH1-GFP (RNA:DNA hybrids) in B16F10 in sgScramble and sgArid1a B16F10s or B16F10 cells treated with BD98 or BRM014 for 72hrs (scale bar = 5μM). (B) Immunofluorescence of ssDNA and DAPI and quantification of cytosolic ssDNA in sgScramble and sgArid1a B16F10s or B16F10 cells treated with BD98 or BRM014 for 72hrs (scale bar = 20μM). (C) Immunofluorescence of dRNH1 and DAPI and quantification of dRNH1 in sgScramble and sgArid1a B16F10 cells with or with overexpression of TREX1 or RNASEH2B (scale bar = 5μM). (D) Immunofluorescence of ssDNA and DAPI and quantification of cytosolic ssDNA in sgScramble and sgArid1a B16F10 cells with or with overexpression of TREX1 or RNASEH2B. (E) Western blots of ISGs STAT1 and ISG15 induced in sgArid1a B16F10s with or without overexpression of TREX1 or RNASEH2B (scale bar = 10μM). (F) Immunofluorescence of ssDNA and DAPI and quantification cytosolic ssDNA in MEFs treated with vehicle or BD98 with or without overexpression of TREX1 or RNASEH2B (scale bar = 30μM). (G) Western blots of ISGs RIG-1, STAT1, and ISG15 induced with BD98 treatment in MEFs with or without overexpression of TREX1 or RNASEH2B. (H) IFNβ ELISA using supernatants from MEFs treated with vehicle or BD98 with or without overexpression of TREX1 or RNASEH2B. Data are represented as mean ± SD. Vehicle and BD98 data is identical to Figure 4H. (I) Western blots of ISGs RIG-1, STAT1, and ISG15 induced with in sgArid1a MC38s with or without overexpression of TREX1 or RNASEH2B. (J) IFNβ ELISA using supernatants from sgScramble or sgArid1a MC38s treated with or without overexpression of TREX1 or RNASEH2B. Data are represented as mean ± SD. sgScramble and sgArid1a data is identical to Figure 4G. Statistical analyses for Figures 5A–D, 5F, 5H, and 5J were performed via one-way ANOVA.

Figure 6.. ARID1A loss induced ISG induction and anti-tumor immunity is dependent on cGAS-STING cytosolic DNA sensing Pathway.

(A) Western blot of ARID1A, cGAS, STING, and ISG15 in sgScramble sgArid1a, sgArid1a/sgCgas (cGAS dKO) and sgArid1a/sgSting (Sting dKO) B16F10s. (B) Heatmap of genes induced in sgArid1a that are dependent on cGAS-STING. (C) Over-representation analysis of sgArid1a induced cGAS-STING dependent genes against Hallmark gene sets. (D) B16F10 tumor growth curves comparing sgScramble, sgArid1a, and Sting dKO genotypes. Data are represented as mean ± SEM. (E-F) Tumor infiltrating immune populations whose significant increase in sgArid1a tumors are rescued in Sting dKO tumors. (G-H) Quantification and flow cytometry contour plots of CD8+ T cell TNFα and IFNγ or Granzyme B and IFNγ staining following ex vivo PMA/ionomycin stimulation in listed tumor genotypes. (I) Quantification and flow cytometry contour plots of CD4+ T cell IFNγ and TNFα staining following ex vivo PMA/ionomycin stimulation in listed tumor genotypes. All data are represented as mean ± SD unless otherwise noted. Statistical analysis in Fig 6E and Fig6E–I were performed via one-way ANOVA.

Figure 7.. ARID1A deficient anti-tumor immunity and immune checkpoint blockade response is Type I IFN dependent.

(A) Tumor growth curves in sgArid1a and sgScramble B16F10 with or without anti-IFNAR treatment. Data are represented as mean ± SEM. (B-C) Quantification of tumor infiltrating immune populations whose significant increase in sgArid1a tumors are rescued in sgArid1a tumors treated with anti-IFNAR. (D) Quantification of GZMB/IFNγ+ NK cells in sgArid1a or sgScramble tumors with and without anti-IFNAR treatment. (E) Quantification of TNFα/IFNγ positive CD4+ T cells in sgArid1a or sgScramble tumors with and without anti-IFNAR treatment. (F) Quantification and flow cytometry contour plots of TNFα/IFNγ positive CD8+ T cells in sgArid1a or sgScramble tumors with and without anti-IFNAR treatment. (G) Individual B16F10 tumor growth curves of sgScramble and sgArid1a tumors treated with anti-PDL1 and anti-CTLA4 or sgArid1a tumors treated with anti-IFNAR, anti-PDL1, and anti-CTLA4. (H) Kaplan-Meier survival curve of mice flank injected with sgScramble or sgArid1a B16F10 tumors treated with anti-PDL1 and anti-CTLA4 or sgArid1a tumors treated with anti-IFNAR, anti-PDL1, and anti-CTLA4. (I) Individual CT26 tumor growth curves of sgScramble and sgArid1a tumors treated with anti-PDL1 or sgArid1a tumors treated with anti-IFNAR and anti-PDL1. (J) Kaplan-Meier survival curve of mice flank injected with sgScramble or sgArid1a CT26 tumors treated with anti-PDL1 or sgArid1a tumors treated with anti-IFNAR and anti-PDL1. (K) Individual MC38 tumor growth curves of sgScramble and sgArid1a tumors treated with anti-PDL1 or sgArid1a tumors treated with anti-IFNAR and anti-PDL1. (L) Kaplan-Meier survival curve of mice flank injected with sgScramble or sgArid1a MC38 tumors treated with anti-PDL1 or sgArid1a tumors treated with anti-IFNAR and anti-PDL1. Statistical analysis in Fig 7B–F was performed via two-way ANOVA. All data are represented as mean ± SD unless otherwise noted. Statistical analysis in Fig 7H, 7J, and 7L were performed using the log-rank survival test.