PRMT7 ablation stimulates anti-tumor immunity and sensitizes melanoma to immune checkpoint blockade

Abstract

Despite the success of immune checkpoint inhibitor (ICI) therapy for cancer, resistance and relapse are frequent. Combination therapies are expected to enhance response rates and overcome this resistance. Herein, we report that combining PRMT7 inhibition with ICI therapy induces a strong anti-tumor T cell immunity and restrains tumor growth in vivo by increasing immune cell infiltration. PRMT7-deficient B16.F10 melanoma exhibits increased expression of genes in the interferon pathway, antigen presentation, and chemokine signaling. PRMT7 deficiency or inhibition with SGC3027 in B16.F10 melanoma results in reduced DNMT expression, loss of DNA methylation in the regulatory regions of endogenous retroviral elements (ERVs) causing their increased expression. PRMT7-deficient cells increase RIG-I and MDA5 expression with a reduction in the H4R3me2s repressive histone mark at their gene promoters. Our findings identify PRMT7 as a regulatory checkpoint for RIG-I, MDA5, and their ERV-double-stranded RNA (dsRNA) ligands, facilitating immune escape and anti-tumor T cell immunity to restrain tumor growth.

Keywords: CP: Cancer; CP: Immunology; DNMTs; PRMT7; RLR pathway; arginine methylation; cytotoxic T cells; dsRNA; immune checkpoint inhibitors; melanoma.

Figure 1:. Deletion of PRMT7 sensitizes B16.F10 melanomas to ICIs.

(A) Western blot showing the expression of PRMT7 in sgCTL and sgPRMT7-1 and 2 targeted B16.F10 melanoma cells. β-actin is the loading control. Molecular mass markers are indicated in the left in kDa. One representative image out of three is shown. (B) PRMT7 mRNA levels for clones in (A) measured by RT-qPCR. Data are mean ± SD. Data representative for four independent experiments. Statistical significance was calculated by unpaired student t test (****p <0.0001). (C) Left panel: Tumor volume averaged for each group at each time point for sgCTL, sgPRMT7-1 and sgPRMT7-2 B16 cells injected into C57BL/6J mice without ICI treatment. Right panel: Kaplan-Meier survival curves were assessed at indicated time points. All groups reached the endpoint on the same day. Data are mean ± SEM; n = 8-10 mice per group.; p values were determined using multiple t test (* p <0.05; **p <0.01; ns: non-significant). (D) Left panel: Tumor volume averaged for each group at each time point for sgCTL, sgPRMT7-1 and sgPRMT7-2 B16 cells injected into C57BL/6J mice treated intraperitoneally with anti-CTLA-4 and anti-PD-1 (ICI) at day 3, 6, 9 and 12 (black triangles). Right panel: Kaplan-Meier survival curve was assessed at indicated time points. Data are mean ± SEM; n = 8-10 mice per group. Representative of two to three independent experiments is shown; p values were determined using multiple t test (**p <0.01; ***p <0.001; ****p <0.0001). (E-F) Upper panels: Tumor volume averaged for each group at each time point for sgCTL, sgPRMT7-1 and sgPRMT7-2 B16 cells injected into C57BL/6J mice treated intraperitoneally with anti-CTLA-4 alone (E) or anti-PD-1 (F) as performed in (D). Lower panels: Kaplan-Meier survival curve was assessed at indicated time points. Data are mean ± SEM; n = 6-7 mice per group. p values were determined using multiple t test (*p <0.05; **p <0.01; ns: non-significant). (G) Upper panel: Tumor volume averaged for each group at each time point for sgCTL cells injected into C57BL/6J mice treated or not with anti-CTLA-4 and anti-PD-1 (ICI) followed by intratumoral injection of DMSO or PRMT7 inhibitor (SGC3027) at indicated time points (day 7, 8, 9 and 10) as presented in the schema. One representative experiment is shown. Data are mean ± SEM; n = 10 mice per group. p values were determined using multiple t test (**p <0.01; ****p <0.0001; ns: non-significant). Lower panels: Kaplan-Meier survival curve was assessed at indicated time points. Data are mean ± SEM; n = 10 mice per group. p values were determined using multiple t test (***p <0.001; ****p <0.0001; ns: non-significant).

Figure 2:. Deletion of PRMT7 in melanomas increases immune cell infiltration and increases melanocytic plasticity.

(A-D) Tumors were digested into a single cell suspension and their immune cell composition analyzed. Quantification of Myeloid-Derived Suppressor Cell (MDSC) populations such as Granulocytic MDSC (G-MDSC, F4/80^neg) (A), Monocytic MDSC (M-MDSC, F4/80^pos) (B), total T cells (C) and non-lymphatic dendritic cells (NLT DC) (D) in sgCTL (black) and sgPRMT7-1 (red) B16.F10 tumors. (A-D) The data represents the mean ± SD and is from two to three independent experiments with a minimum of 3 mice per group. Each dot represents one mouse. Statistical significance was calculated using paired student t test. (*p <0.05; **p <0.01; ns: non-significant). (E) Representative flow cytometry plots using anti-CD45, anti-CD3 and anti-CD8 antibodies in sgCTL and sgPRMT7-1 B16.F10 tumors. (F) Quantification graphs from (E) showing frequencies of double positive CD3^pos, CD8^pos cells (gated on CD45^pos cells) in sgCTL (black) and sgPRMT7-1 (red) B16.F10 tumors. Cells were gated as indicated and the relative percentage of cells shown; n=13 animals per group; data from three independent experiments. Statistical significance was calculated using paired student t test (****p <0.0001). (G) Representative immunofluorescent images of sgCTL and sgPRMT7-1 tumor sections treated with anti-CTLA-4 and anti-PD-1 in vivo and stained with anti-CD8α antibody at 10x magnification. DAPI, 4’,6-diamidino-2-phenylindole, was used to visualize nuclei by Zeiss confocal microscopy. A minimum of 3 biological replicates were used for each experiment. (H) Immunofluorescence intensity of the CD8 staining done in (G) using ImageJ software. Bar graphs show fluorescence mean intensity ± SEM. Statistical significance was calculated using unpaired student t test (****p <0.0001). (I) Representative pictures of subcutaneous sgCTL and sgPRMT7-1 derived-melanomas in C57BL/6J mice treated with anti-CTLA-4 and anti-PD-1 (day 21, top) and corresponding representative images of H&E-stained tumor sections (day 21, bottom). Black arrowheads indicate the pigmented areas. (J) RT-qPCR analysis of Mitf, Gp100 and Melan-A mRNA transcripts in sgCTL, sgPRMT7-1 and sgPRMT7-2 B16.F10 cells. Data are mean ± SD. Data representative for 3 independent experiments. Statistical significance was calculated by unpaired student t test (**p <0.01; ***p <0.001; ****p <0.0001). (K) Western blot showing the expression of the indicated proteins (PRMT7, MITF, GP100 and Melan-A) in sgCTL, sgPRMT7-1 and sgPRMT7-2 B16.F10 melanoma cells. β-actin was used as the loading control. Molecular mass markers are indicated in the left in kDa. Data are representative of three independent experiments. Cell pellet representative images were shown (bottom; note the black pellet in sgPRMT7 clones).

Figure 3:. PRMT7 loss increases IFN pathway, antigen presentation and chemokine production

(A) siLuc and siPRMT7 B16.F10 cells (n=3 per group) were subjected to RNA-seq analysis. Heat map showing expression value (z-score based on cufflink count) of IFN genes, antigen processing and chemokine signaling genes with or without IFN-γ treatment (100 ng/ml) for 24h. (B) Gene set enrichment analysis of IFN-γ signaling pathway antigen processing and presentation and chemokine signaling pathway in siLuc and siPRMT7 cells. (C-G) RT-qPCR validation of genes identified from the RNA-seq dataset. Fold-change analysis using some selected genes: Cxcl1(C), Cxcl2 (D), Ccl2 (E), Ccl5 (F) and Ccl8 (G) before and after IFN-γ treatment in siLuc (black) and siPRMT7 (red) B16.F10 cells. The fold-change in gene expression levels, before and after treatment, were calculated using the comparative cycle threshold (DDCT) method and values were normalized to Gapdh mRNA levels as an internal control. Triplicates were used per biological sample. Bar graphs represent the mean fold-change ± SD. Statistical significance was calculated by unpaired student t test (**p <0.01; ****p <0.0001). (H) Heat map showing expression value (z-score based on cufflink count) of all genes categorized in GO term ‘MHC protein complex’ in siLuc and siPRMT7 B16.F10 cells. (I) RT-qPCR analysis of genes implicated in antigen presentation (Nlrc5, Psmb9, B2m and Tap1) in siLuc and siPRMT7 B16.F10 cells. Bar graphs represent the mean fold-change ± SD. Data are representative of three independent experiments. Statistical significance was calculated by unpaired student t test (*p <0.1; **p <0.01). (J) RT-qPCR analysis of same transcripts analyzed in (I) in B16.F10 cells treated with the indicated PRMT inhibitors for 48h (SGC3027: 10 μM; EPZ015666: 5 μM; MS023: 600 nM and TP064: 3 μM). Bar graphs represent the mean fold-change ± SD. Data are representative of three independent experiments. Statistical significance was calculated by unpaired student t test (*p <0.1; **p <0.01; ***p <0.001; ****p <0.0001; ns: non-significant).

Figure 4:. PRMT7 regulates Ddx58 and Ifih1 transcription levels by promoting H4R3me2s histone mark establishment at their promoters.

(A) Western blot analysis of RIG-I, MDA5, IRF-3 and p-IRF-3 expression in total cell lysates isolated from sgCTL and sgPRMT7-1 cells transfected or not with poly (I:C) at 2.5 μg/ml for 24 h. β-actin was used as the loading control. Data are representative of two independent experiments. The molecular mass markers are indicated in the left in kDa. (B-E) RT-qPCR analysis of p-IRF-3 responsive genes (Ifnβ, Il-6, Cxcl9 and Cxcl10) in sgCTL and sgPRMT7-1 cells transfected or not with poly (I:C) at 2.5 μg/ml for 24 h. Bar graphs represent the mean fold-change ± SD. Data are representative of two independent experiments. Statistical significance was calculated by unpaired student t test (*p <0.1; **p <0.01; ns: non-significant). (F) RT-qPCR analysis of Ddx58, Ifih1, some IFN genes (Ifn-α, Ifn-β and Il-28) and selected ISGs (Oasl, and Isg15) in sgCTL and sgPRMT7-1 cells transfected or not with siRIG-I or si-MDA5 for 72h. Bar graphs represent the mean fold-change ± SD. Data are representative of two independent experiments. Statistical significance was calculated by unpaired student t test (*p <0.1; **p <0.01). (G) Chromatin was prepared from B16.F10 melanoma cells and analyzed by ChIP with a PRMT7-specific antibody (black bars). The immunoprecipitated chromatin fragments were then analyzed by qPCR with primers spanning Ddx58 and Ifih1 promoters. Results are normalized to input. β-actin served as a negative control. (H-I) Analyses of distribution of H4R3me2s (H) and H3K4me3 (I) at the promoter regions of Ddx58 and Ifih1. sgCTL and sgPRMT7-1 B16.F10 melanoma cells are represented in black and red bars, respectively. Chromatin was immunoprecipitated using anti-H4R3me2s and anti-H3K4me3. Anti-H3 and anti-H4 were used as controls for histone marks and IgG isotype was used for mock precipitation to exclude non-specific enrichment (grey bars). Subsequent qPCR was performed using promoter primer sets for Ddx58 and Ifih1. Data were represented as percentage of input. Experiments were repeated two times. Asterisks denote significance in an unpaired t test (*p <0.05; **p <0.01; ***p <0.001; ns: non-significant), and error bars denote SD. (J) Western blot analysis of acid-extracted histones using anti-H3, anti-H3R2me2s, anti-H3R8me2s, anti-H4 and anti-H4R3me2s in sgCTL, sgPRMT7-1 and sgPRMT7-2 B16.F10 melanoma cells. The data is representative of two independent experiments. Molecular mass markers are indicated in the left in kDa.

Figure 5:. PRMT7 loss induces “viral mimicry” by regulating ERVs, dsRNA accumulation and stress granule (SG) formation.

(A) RT-qPCR analysis of selected retrotransposons, IFNs and ISGs transcripts in sgCTL, sgPRMT7-1 and sgPRMT7-2 B16 cells. Bar graphs represent the mean fold-change ± SD. Data are representative of three independent experiments. Statistical significance was calculated by unpaired student t test (*p <0.1; **p <0.01; ***p <0.001; ****p <0.0001). (B) The assessment of both sense and antisense transcripts of selected ERVs (MuERV-L and IAP) using strand-specific primers for RT-PCR (TASA-TD technique) in sgCTL, sgPRMT7-1 and sgPRMT7-2 B16 cells. β-actin was used as a negative control for antisense transcription. A representative experiment is shown of three independent experiments. (C) dsRNA enrichment of MuERV-L IAP, MusD and Line-1 retrotransposons in sgCTL, sgPRMT7-1 and sgPRMT7-2 B16 cells by RT-qPCR analysis. RNase A treatment was used to digest ssRNAs and maintain the presence of dsRNAs. (D) Total RNA extracted from sgCTL and sgPRMT7-1 B16 cells were treated with Mock, RNase III or RNase A (under high salt condition: 350 mM NaCl), dotted on Hybond N+ membrane and immunoblotted with the J2 antibody and visualized by methylene for loading control. Dots are denoted by numbers: 1, 3, 5 for sgCTL and 2, 4, 6 for sgPRMT7-1 cells nontreated (dots 1 and 2), treated with RNase III (dots 3 and 4) or RNase A (dots 5 and 6). (E) Quantification of the J2 immunoblot presented in (D) using image J software and presented as a bar plot. (F) sgCTL and sgPRMT7-1 B16 cells were incubated with 0.5 mM sodium arsenite for 1h or 45°C (heat shock) treatment for 30 min. Cells were then fixed with 4% PFA and immunostained using anti-G3BP1 antibodies. A representative IF image is shown 60x magnification. DAPI, 4’,6-diamidino-2-phenylindole, was shown in blue as indicated. (G) The average number of SGs per cell of the staining done in (F) was quantified using image J software and presented as a bar plot (n=60 to 70 cells per condition). Bar graphs show mean intensity ± SEM. Statistical significance was calculated by unpaired student t test (****p <0.0001). (H) Western blot analysis of PKR, eIF2α and p- eIF2α expression in total cell lysates isolated from sgCTL and sgPRMT7-1 cells transfected with siCTL or siPKR for 72 h. β-actin was used as the loading control. Data are representative of two independent experiments. The molecular mass markers are indicated in the left in kDa. (I) RT-qPCR analysis of p-eIF2α target genes (Atf4, Bip, Xbp1) in sgCTL, sgPRMT7-1 cells transfected or not with siPKR for 72 h. Bar graphs represent the mean fold-change ± SD. Data are representative of two independent experiments. Statistical significance was calculated by unpaired student t test (*p <0.1; **p <0.01; ns: non-significant).

Figure 6:. PRMT7-deficient B16 cells have decreased DNMT1, 3a, 3b expression and increased hypomethylation at ERV loci.

(A) RT-qPCR analysis of DNMT mRNAs (Dnmt1, Dnmt3a and Dnmt3b) in sgCTL, sgPRMT7 B16 melanoma cells. Bar graphs represent the mean fold-change ± SD. Data are representative of three independent experiments. Statistical significance was calculated by unpaired student t test (****p <0.0001). (B) Immunoblot of DNMT proteins (DNMT1, DNMT3a and DNMT3b) in sgCTL, sgPRMT7-1 and sgPRMT7-2 B16 cells. β-actin was used as the loading control. A representative experiment is shown out of three independent experiments. The molecular mass markers are indicated in the left in kDa. The DNMT bands are shown with arrowheads. The value below each panel corresponds to the quantification by image J of DNMTs expression compared to sgCTL normalized to one. (C) RT-qPCR analysis of MuERV-L IAP, MusD and Line-1 retrotransposons in sgCTL, sgPRMT7-1 B16 cells treated or not with 5-Aza for 72 h. Bar graphs represent the mean fold-change ± SD. Data are representative of three independent experiments. Statistical significance was calculated by unpaired student t test (**p <0.01; ns: non-significant). (D) Bisulfite sequencing analysis of regions in the 5’ LTRs of MuERV-L, IAP and MusD and the 5’ UTR of Line-1, using genomic DNA isolated from sgCTL and sgPRMT7-1 B16.F10 cells treated or not with 5-Aza for 72 h. Each horizontal line represents one analyzed clone and at least 7 clones are presented for each sample. The black filled circles represent methylated CpG sites, while the white open circles represent unmethylated CpG sites. The percentages of methylated CpGs are shown at the top of each group of clones. Note that the numbers of CpG sites in IAP clones vary from 9 to 12 due to sequence variations.

Figure 7:. PRMT7 expression is inversely correlated with the response to ICI in human melanoma patients.

(A) Plot of FPKM gene expression values showing the correlation between PRMT7 mRNA expression in patients treated with ICI therapy. n=28 cases grouped according to whether they receive complete (pink; n=5), partial (green; n=10) or progressive recovery (blue; n=13). The FPKM values were obtained from the GEO accession GSE78220. (B) Box plots showing the FPKM values for each case reported in (a). p values by Wilcoxon test are shown. (C) Plot of the FPKM gene expression values for PRMT7 showing the correlation between PRMT7 in patients before (upper panel) and during (lower panel) Nivolumab treatment. n=58 cases grouped according to whether they showed a complete response (CR, orange; n=3), partial response (PR, olive; n=8) or stable disease (SD, green; n=19), progressive disease (PD, blue; n=26). 2 patients were non evaluable (NE, pink; n=2). The FPKM values were obtained from the GEO accession GSE91061. (D) Box plots showing the FPKM values for each case reported in (C). p values by Wilcoxon test are shown.

Figure 8:. Proposed model for PRMT7 function in sensitizing melanoma to immunotherapy.

PRMT7 deletion or inhibition in melanoma enhances tumor immunogenicity and sensitivity to cancer immunotherapy by derepressing ERVs, activating RLR pathway, IFN response, antigen presentation and pro-inflammatory cytokines expression. This occurs through the decrease presence of H4R3me2s on Ddx58 and Ifih1 promoters and influencing DNA methylation on selected ERVs.