Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses

Abstract

We show that DNA methyltransferase inhibitors (DNMTis) upregulate immune signaling in cancer through the viral defense pathway. In ovarian cancer (OC), DNMTis trigger cytosolic sensing of double-stranded RNA (dsRNA) causing a type I interferon response and apoptosis. Knocking down dsRNA sensors TLR3 and MAVS reduces this response 2-fold and blocking interferon beta or its receptor abrogates it. Upregulation of hypermethylated endogenous retrovirus (ERV) genes accompanies the response and ERV overexpression activates the response. Basal levels of ERV and viral defense gene expression significantly correlate in primary OC and the latter signature separates primary samples for multiple tumor types from The Cancer Genome Atlas into low versus high expression groups. In melanoma patients treated with an immune checkpoint therapy, high viral defense signature expression in tumors significantly associates with durable clinical response and DNMTi treatment sensitizes to anti-CTLA4 therapy in a pre-clinical melanoma model.

Figure 1. DNMT inhibitors upregulate immune genes in ovarian cancer cell lines

A) Levels of immune genes in four EOC cell lines and DKO colon cancer cell line (DNMT1−/−, DNMT3B−/−) relative to its parental HCT116 line. Y-axis = log₂ Aza/Mock fold change from microarray data. Dotted line denotes twofold change. B) qRT-PCR validation of immune genes in EOC cells treated for 72 hours: Mock, 500 nM Aza, or 500 nM− 3 μM carboplatin and rested for 7 days before assaying (Day 10). IC50s: A2780 (Aza = 848 nM, Carb = 457 nM), Hey (Aza = 4.1 μM, Carb = 12.2 μM), TykNu (Aza = 491 nM, Carb = 986.2 nM). C-D. qRT-PCR validation of interferon response genes in the A2780 (C) and TykNu (D) EOC lines treated with no drug (Mock), 500 nM Aza (Aza), or 100 nM Decitabine (Dac) for 3 days, and rested for 4 (Day 7) or 7 (Day 10) days before assaying. Y-axis = fold change over mock. Data in B-D are represented as mean +/− S.E.M of 3 biological replicates. * = p≤0.05. See also Figure S1.

Figure 2. DNMTis upregulate immune signaling through secreted interferon

A) Schematic of interferon pathway. Protein symbols outlined in text. B) Treatment of recipient A2780 or TykNu cells with media from cells treated with Mock or Aza, +/− addition of anti-IFNβ. Y-axis = qRT-PCR fold Aza/Mock of ISGs. * = p≤0.05 and ^ = p≤0.05, respectively, for Mock or Aza media with versus without anti-IFNβ versus Mock or Aza media plus anti-IFN β; C) ELISA of IFNβ in media from TykNu cells at Day 10 from studies in B. D) Treatment of EOC cells with Aza as in Figure 1c,d, but in the presence of 2 μM Ruxolitinib (Rux). qRT-PCR fold changes for ISGs. * = p≤0.05 for fold Aza over Mock and ^ = p≤0.05, Mock or Aza versus Mock + Rux or Aza + Rux. Data = mean +/− S.E.M. of three (B, D) or four (C) biological replicates. See also Figure S2.

Figure 3. Aza induces immune signaling through dsRNA activation of secreted interferon

A) Blocking IFNAR2 (αIFNAR2) or B) IFNβ in TykNu cells treated vs. non-treated with Aza as in Figures 1c,d; parentheses = U/mL of antibody. Y-axis = qRT-PCR for ISGs. * = p≤0.05 for A), Mock or Aza - anti-IFNAR2 versus + anti-IFNAR2, B) Mock or Aza with no anti-IFNβ versus with anti-IFNβ. C) Immunoblotting for cleaved PARP with β-actin loading control. Fold change shown for cleaved/total PARP ratio, normalized to β-actin for each dose of anti-IFNAR2 (triangles = 0-1.25 U/mL). Aza compared to Mock = 1. D,E) Indicated nucleic acids from the cytoplasm of A2780 or TykNu treated cells with no drug (Mock) or 500 nM Aza (Aza) for 3 days and rested without drug for 4 days before transfection into recipient HT29 cells. Y-axis = fold change, Aza/Mock, for IFNβ1 transcript (D) or ISG transcripts (E) induced in HT29s. No Tx = no transfection, Cyto DNA = Cytoplasmic DNA, Cyto RNA = Cytoplasmic RNA excluding ribosomal RNA. * = p≤0.05 for D) fold change Aza vs Mock, E) Aza / Mock. Data in A,B,D,E is mean +/− S.E.M. of three biological replicates. See also Figure S3.

Figure 4. Aza activates dsRNA sensors to induce interferon signaling

A) Effects on IFNβ1 transcripts, at 24 hours, in HT29 recipient cells transfected with nucleic acid fractions, treated with RNaseIII, from A2780 as in Figure 3d,e. IFNβ1 transcripts were measured at 24 hours. * = p≤0.05 for fold change over untreated; ^ = p≤0.05 for Mock or Aza + versus - RNAseIII. B) Western blots for MDA5, RIG-I, and TLR3 in A2780 cells at four (Day 7) and seven (Day 10) days after Mock vs 500 nM Aza (Aza) for 3 days. C) Knockdown upon lentiviral infection, with puromycin selection, of A2780 and TykNu with shGFP, shTLR3, and shSTING hairpins. Immunoblotting with anti-TLR3, anti-STING and anti-β-Actin. Densitometry fold change, normalized to mock or shGFP, shown at the bottom of the gels. D) q-RT-PCR for ISGs from B and C. * = p≤0.05 Aza over Mock; ^ = p≤0.05 shGFP versus each shRNA sensor with mean fold change +/− S.E.M. of three biological replicates. See also Figure S4.

Figure 5. Aza upregulates sense and antisense ERV transcripts

RNA was isolated from cells at last (Day 3), one (Day 4), three (Day 7) and seven (Day 10) days after Mock or 500 nM Aza (Aza) for 3 days. A) Total number of molecules for all ERV genes; error bars = S.E.M for 4 independent experiments. Numbers above bars = significant data for indicated days. Gray = Mock, Black = Aza. B) qRT-PCR of ERV genes in A2780 cells for 4 independent experiments. Y-axis= fold increases for Aza/Mock +/− S.E.M and normalized to Mock = 1. White bars = non-significant and colored bars = significant ERV gene induction (p<0.05). C) TASA-TD PCR amplified sense and antisense transcripts of the env-Fc2 (731 bp) and Syncytin-1 (202 bp) genes from first strand cDNA. Aza treated A2780, Hey, and TykNu, and HCT116 and DKO cells are indicated. Ratios of sense (s) and antisense (as) determined by ImageJ. PCR primers = gene specific (GS); TAG. β-actin sense 399 bp amplification product = negative control for as transcripts (Chen et al., 2004). See also Figure S5.

Figure 6. Aza upregulates ERV transcripts, but not proteins, through DNA demethylation

A) env-Fc2 and erv-9-1 ERV gene total number of molecules, assayed by qRT-PCR, for DKO (DNMT1−/−, DNMT3B−/−) and parental HCT116 cells. Y-axis = mean +/− SEM for n = 6 biological replicates. * = p≤0.05 for DKO versus HCT116. B) DNA methylation changes in ERVs in A2780 cells treated with Mock or 500 nM Aza for 3 days at post-treatment day 4 (Day 7), or 7 (Day 10). Bisulfite treated DNA was amplified and digested with the AciI enzyme producing 155 and 44 bp fragments of methylated DNA while unmethylated DNA does not digest (189 bp fragment). “U” = unmethylated band, “M” = methylated band. C) DNA from B) was subjected to Methylation-specific PCR for Fc2 family members on chromosomes 7 and 11. U” = unmethylated, “M” = methylated. IVD = in vitro methylated DNA. D) Syncytin-1 and ERV-3 protein levels in EOC cells treated as in B). Fold change for densitometry by ImageJ for Aza vs Mock cells normalized to β-actin protein levels E) Transfection of full-length env genes from EnvW2, ERV-3, or Syncytin-1 or EGFP and ER controls in TykNu cells. qRT-PCR was performed for ISGs 7 days after transfection. Dotted black line indicates 1. Y-axis = mean +/− S.E.M fold change of three biological replicates for overexpression/ Mock. * = p≤0.05. See also Figure S6.

Figure 7. Aza-upregulated viral defense genes are significantly correlated with ERVs in primary tumors and correlate with sensitivity to immune therapy

A) Heatmap comparing basal levels of viral defense genes and ERVs in primary EOC. The cut-off for lower or higher ERVs was the mean control tissue value of 237.57 +/− 83.05 molecules/ng RNA. Mean ISGs of the high ERV ovarian tumor (T) cohort (n=10) is 12.65-fold higher than the mean of ISGs of the low ERV cohort (n=9). The (*) denotes that 8 of 10 high ERV tumors had significantly higher ISG expression compared to the low ERV tumors. ISG expression is organized according to low and high ERV expression cohorts in arbitrary units; color code from blue to red shows increasing ISG expression. For clusters (k=6), differences are significant between the high ERV expression (2.5 +/− 0.37) and the low ERV expression cohort (5.33 +/− 0.28). B) Interferon stimulated, viral defense genes upregulated at least twofold by Aza in EOC cell lines (right y-axis) were used to cluster EOC tumors for RNA-Seq data (blue = low; red=high) from The Cancer Genome Atlas (TCGA). EOC TCGA subtypes are shown: DIF (differentiated), IMR (immune reactive), MES (mesenchymal), and PRO (proliferative) C), D) Viral defense gene signature is upregulated in tumors from anti-CTLA-4 treated metastatic melanoma patients who derived durable clinical benefit (complete response, partial response, or progression free-survival > 6 months as previously described (Snyder et al., 2014)) compared to those without benefit. Tumors collected pre-CTLA-4 treatment and shortly post-treatment are shown. E), F) Tumor responses of mice injected with B16-F10 cells and treated with either PBS, anti-CTLA-4, Aza, or both anti-CTLA-4 and Aza. Data represent results from one of two independent experiments with identical results, each with n = 10 per arm.