Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

Abstract

Cell-intrinsic mechanisms of immunogenicity in ovarian cancer (OC) are not well understood. Damaging mutations in the SWI/SNF chromatin remodeling complex, such as SMARCA4 (BRG1), are associated with improved response to immune checkpoint blockade; however, the mechanism by which this occurs is unclear. We found that SMARCA4 loss in OC models resulted in increased cancer cell-intrinsic immunogenicity, characterized by up-regulation of long-terminal RNA repeats, increased expression of interferon-stimulated genes, and up-regulation of antigen presentation machinery. Notably, this response was dependent on STING, MAVS, and IRF3 signaling but was independent of the type I interferon receptor. Mouse ovarian and melanoma tumors with SMARCA4 loss demonstrated increased infiltration and activation of cytotoxic T cells, NK cells, and myeloid cells in the tumor microenvironment. These results were recapitulated in BRG1 inhibitor-treated SMARCA4-proficient tumor models, suggesting that modulation of chromatin remodeling through targeting SMARCA4 may serve as a strategy to overcome cancer immune evasion.

Fig. 1.. Pan-cancer association of SMARCA4 expression with immune expression signatures.

Transcriptomic analysis of The Cancer Genome Atlas (TCGA) pan-cancer RNA-sequencing data. (A) Immune signatures [from Thorsson et al. (17) dataset] by SMARCA4 expression for all tumor types combined. (B) Signature scores for pro-inflammatory immune cell populations by SMARCA4 expression for all tumor types combined. (C) Signature scores and cell populations for immunomodulatory cell populations by SMARCA4 expression for all tumor types combined. (D) Heatmap of immune signatures in SMARCA4-low and SMARCA4-high tumors for each tumor type. Gene expression fold change of SMARCA4-low versus SMARCA4-high tumors is color coded according to the legend. (E) Immune signatures and cell populations by SMARCA4 expression derived from RNA-sequencing data for high-grade serous OCs obtained from TCGA. Statistical analysis was performed using Mann-Whitney U test, Fisher exact test, and Benjamini-Hochberg adjustment. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ACC, adrenocortical carcinoma; BLCA, bladder urothelial cancer; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LGG, brain low-grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; OC/OV, ovarian serous cystadenocarcinoma; PRAD, prostate adenocarcinoma; SKCM, skin cutaneous melanoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma.

Fig. 2.. Loss of function of SMARCA4 in OC cells leads to increased interferon response and antigen presentation gene activation.

(A) Up-regulated Hallmark and Gene Ontology (GO) pathways in ID8 cell single-guide SMARCA4 (sgSMARCA4) compared to those in sgNTC. Ribodeplete RNA sequencing was performed. Statistical analysis was based on hypergeometric test and performed using ClusterProfiler. IL-6, interleukin-6; JAK, Janus kinase; STAT3, signal transducer and activator of transcription 3; TNFA, tumor necrosis factor–α; FDR, false discovery rate. (B) Gene expression heatmap of type I IFN pathway–related genes in ID8 cells. Reads per kilobase of transcript per million mapped reads values were scaled to z-score for visualization. Gene expression fold change of sgSMARCA4 versus sgNTC cells is color coded according to the legend. (C) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) validation results for IFN genes in ID8 cells (sgNTC and four sgSMARCA4 clones). Expression levels were normalized to β-actin expression, and comparisons of mRNA expression levels were performed relative to control (sgNTC). n = 3 independent experiments. (D) MHC1 expression in ID8 cells with or without IFN-ɣ by flow cytometry. (E) PD-L1 expression in ID8 cells with or without IFN-ɣ by flow cytometry. MFI, median fluorescence intensity. Statistical analysis was performed using two-tailored unpaired t test [(C) to (E)]. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; n.s., not significant. Error bars represent ± SEM. Samples in duplicates [(A) and (B)] and triplicates [(D) and (E)]. KO, knockout; KO_1, target exon 14 clone 1; KO_2, target exon 14 clone 2; KO_3, target exon 23 clone 3; KO_4, target exon 23 clone 4; NTC, non-target control; NTC_1, NTC clone 1.

Fig. 3.. SMARCA4 loss-of-function mutation results in perturbation of chromatin accessibility at key immune genes.

(A) Changes in genomic site accessibility in sgSMARCA4 (KO1) versus sgNTC cells. Log₂ fold change and FDR-adjusted P value (Wald test P values from DESeq2 with Benjamini-Hochberg correction). (B) Gene set enrichment analysis (GSEA) of immune pathways with increased accessibility in sgSMARCA4 versus sgNTC cells. Kolmogorov-Smirnov statistic with Benjamini-Hochberg correction. Exact q values indicated in each panel. (C) Changes in chromatin accessibility at transcription start sites (TSSs) of ISGs (CXCL10 and CCL2) in sgSMARCA4 versus sgNTC cells. (D) Motifs enriched in open chromosomal regions affected by SMARCA4 deficiency. P value and binomial test were performed using Homer2. Experiments performed in duplicates. GO, Gene Ontology; KO, knockout; KO_1, target exon 14 clone 1; NTC, non-target control; TF, transcription factor.

Fig. 4.. Increase in ISGs is not dependent on signaling through the type I IFN receptor in sgSMARCA4 OC cells.

(A) IFNAR-1 neutralizing assay in ID8 single-guide NTC (sgNTC) compared to that in sgSMARCA4 cells (four sgSMARCA4 clones). Cells were treated with Mock or IFNAR-1 antibody for 48 hours at 10 μg/ml. (B) IRF3 expression levels in sgSMARCA4 versus sgNTC cells by qRT-PCR. (C) IRF3 expression in doxycycline-inducible short hairpin NTC (shNTC) and shIRF3-transfected sgSMARCA4 cells by qRT-PCR. (D) qRT-PCR quantification of ISGs in ID8 sgNTC and sgSMARCA4 cells. The latter were transfected with shIRF3 or shNTC (with and without POLYI:C). (E) Enriched IRF motif at TSSs of ISG locus from ATAC-seq analysis of ID8 sgNTC versus sgSMARCA4 cells. Experiments performed in duplicates. (F) Analysis of publicly available ChIP-Atlas data of IRF3 DNA binding sites on ISGs in murine immune cell lines. Statistical analysis was performed using two-tailored unpaired t test [(A) to (D)]. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Error bars represent ± SEM. n = 3 independent experiments in (A) to (D). Expression levels were normalized to β-actin expression, and comparisons of mRNA expression levels were performed relative to control [sgNTC_1 in (A), (B), and (D) and sgSMARCA4 transfected with shNTC in (C), as indicated]. KO, knockout; KO_1, target exon 14 clone 1; KO_2, target exon 14 clone 2; KO_3, target exon 23 clone 3; KO_4, target exon 23 clone 4; LPS, lipopolysaccharide stimulated, NTC, non-target control; NTC_1 = NTC clone 1; POLYI:C, polyinosinic-polycytidylic acid stimulated.

Fig. 5.. Increase in ISGs is associated with production of TEs and up-regulation of dsRNA.

(A) Volcano plots for differential expression of TEs in ID8 cells. (B) Heatmap of long terminal repeats (LTR) with adjusted P < 0.05. (C) Representative pictures (top) of double-stranded RNA (dsRNA) identification by immunofluorescence (IF) in sgSMARCA4 and sgNTC ID8 cells with quantification (bottom). RNAse, ribonuclease. (D) qRT-PCR quantification of ISGs in ID8 sgNTC cells and sgSMARCA4 cells transfected with shMAVS or shNTC. (E) MAVS expression in doxycycline-inducible shNTC and shMAVS-transfected sgSMARCA4 cells by qRT-PCR. Expression levels were normalized to β-actin expression, and comparisons of mRNA expression levels were performed relative to control [sgSMARCA4 transfected with shNTC in (C) and sgNTC_1 in (D), as indicated]. Statistical analysis was performed using two-tailored unpaired t test [(C) to (E)]. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Error bars represent ± SEM. Samples in duplicates [for (A) and (B)]. n = 3 independent experiments [in (C) and (D)]. Red line in (A) represents a cutoff of an adjusted P value of <0.05. KO, knockout; KO_1, target exon 14 clone 1; KO_2, target exon 14 clone 2; KO_4, target exon 23 clone 4; NTC, non-target control; NTC_1, NTC clone 1.

Fig. 6.. sgSMARCA4 leads to increased ovarian tumor immunogenicity in vivo.

(A) Workflow for the OC tumor model. C57BL/6 (Cg)–Tyr^c-2J/J mice were inoculated intraperitoneally with 10 million ID8 sgNTC or sgSMARCA4 (KO_4) tumor cells, and spectral flow cytometry was performed 21 days later on harvested ascites samples. ip, intraperitoneal. (B) Frequency of tumor PD1⁺CD4⁺ T cells and PD1⁺CD8⁺ T cells. (C) Frequency of NK1.1⁺ cells expressing Granzyme B⁺. (D) Frequency of tumor dendritic cells (of CD45⁺ cells) and MHCII-expressing dendritic cells. (E) Frequency of tumor macrophages (of CD45⁺ cells) and PD-L1–expressing macrophages. (F) In vivo bioluminescence imaging of tumor burden in sgSMARCA4 versus sgNTC tumors (unpaired t test of area under the curve). Statistical analysis was performed using two-tailored unpaired t test [(B) to (F)]. *P < 0.05, **P < 0.01 and ****P < 0.0001. Error bars represent ± SEM. n = 10 mice per group. KO, knockout; KO_4, target exon 23 clone 4; NTC, non-target control; NTC_1, NTC clone 1.

Fig. 7.. Loss of SMARCA4 up-regulates innate and adaptive immune response in melanoma.

(A) Workflow for the B16-F10 tumor model. C57BL/6 (Cg)–Tyr^c-2J/J mice were inoculated subcutaneously with 150,000 shNTC, sgEV, shSMARCA4_1, shSMARCA4_2, or sgSMARCA4 tumor cells, and spectral flow cytometry was performed 21 days later on harvested tumor samples. (B) Frequency of tumor CD8⁺ T cells and MFI of ICOS⁺-expressing CD8⁺ T cells in B16-F10 model. (C) Frequency of NK1.1⁺ cells in B16-F10 knockout and knockdown models (of CD45⁺ cells). (D) MFI of MHCII in macrophages in B16-F10 SMARCA4 knockout and knockdown models. (E) Tumor volume of shSMARCA4 versus shNTC B16 tumors (unpaired t test of final time points). n = 5 mice per group, three biological replicates. (F) Frequency of NK1.1⁺ cells in shSMARCA4 versus shNTC tumors (of live cells) in RAG2^−/− mice. (G) MFI of MHCII-expressing macrophages in B16-F10 shSMARCA4 versus shNTC tumors in RAG2^−/− mice. (H) Tumor volumes of shSMARCA4 versus shNTC B16 tumors in RAG2^−/− mice (unpaired t test of final time points). n = 3 to 5 mice per group, three biological replicates. Statistical analysis was performed using two-tailored unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent ± SEM. Dox, doxycycline; EV, empty vector; NTC, non-target control; SC, subcutaneous.

Fig. 8.. Therapeutic targeting of BRG1 recapitulates the immunogenic effects of sgSMARCA4.

(A) qRT-PCR quantification of IFN gene expression using ID8 parental cell treated with 1 μM of BRG1/BRM inhibitor or vehicle [dimethyl sulfoxide (DMSO)]. Expression levels were normalized to β-actin expression, and comparisons of mRNA expression levels were performed relative to control (vehicle). (B) Workflow for the OC tumor model and treatment. C57BL/6 mice were inoculated intraperitoneally with 2 million ID8 parental cells and treated with oral BRG1/BRM inhibitor (20 mg/kg) or vehicle DMSO and Solutol HS in 10 mM citrate buffer (pH 6 in a ratio of 5:20:75, respectively) starting on day 5 for a total of eight doses (4 days on, 3 days off × 2 weeks). Flow cytometry was performed 3 weeks after inoculation on harvested ascites samples. (C) Frequency of tumor CD3⁺ cells and CD8⁺ T cells (of CD45⁺ cells). (D) Frequency of tumor ICOS⁺CD4⁺ T cells and PD1⁺ CD4⁺ T cells. (E) Frequency of tumor ICOS⁺CD8⁺ T cells, PD1⁺CD8⁺ T cells and Granzyme B–expressing CD8⁺ T cells. (F) MFI of activated (CD11b⁺) NK1.1⁺ cells. (G) MFI of MHCII-expressing tumor macrophages. (H) Frequency of tumor dendritic cells (of CD45⁺ cells) and MHCII-expressing dendritic cells. (I) In vivo bioluminescence imaging of tumor burden in vehicle versus BRG1/BRMi-treated tumors (unpaired t test of area under the curve). (J) Mouse weights in ID8 tumors treated with BRG1/BRM inhibitor or vehicle. Weights normalized to baseline weight (day 5). Statistical analysis was performed using two-tailored unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Error bars represent ± SEM. n = 10 mice per group in (C) to (H) and (J) and n = pooled 20 mice per group from two independent experiments in (I). BRG1/BRMi, BRG1/BRM inhibitor.