DNA Sensing in Mismatch Repair-Deficient Tumor Cells Is Essential for Anti-tumor Immunity

Abstract

Increased neoantigens in hypermutated cancers with DNA mismatch repair deficiency (dMMR) are proposed as the major contributor to the high objective response rate in anti-PD-1 therapy. However, the mechanism of drug resistance is not fully understood. Using tumor models defective in the MMR gene Mlh1 (dMLH1), we show that dMLH1 tumor cells accumulate cytosolic DNA and produce IFN-β in a cGAS-STING-dependent manner, which renders dMLH1 tumors slowly progressive and highly sensitive to checkpoint blockade. In neoantigen-fixed models, dMLH1 tumors potently induce T cell priming and lose resistance to checkpoint therapy independent of tumor mutational burden. Accordingly, loss of STING or cGAS in tumor cells decreases tumor infiltration of T cells and endows resistance to checkpoint blockade. Clinically, downregulation of cGAS/STING in human dMMR cancers correlates with poor prognosis. We conclude that DNA sensing within tumor cells is essential for dMMR-triggered anti-tumor immunity. This study provides new mechanisms and biomarkers for anti-dMMR-cancer immunotherapy.

Keywords: DNA sensing; MLH1; MSI; STING; T cell infiltration; cGAS; cancer; checkpoint blockade; cytosolic DNA; mismatch repair.

Figure 1.. dMLH1 tumor cell-intrinsic STING pathway regulates tumor progression

(A) Different generations of 4T1 cells with dMLH1 were inoculated into WT BALB/C mice (n=5). *P = F.18 between 4T1-Mlh1^−/− D104 vs. 4T1; **P=0.0034 between 4T1-Mlh1^−/− D172 vs. 4T1. (B) Different generations of B16-OVA cells with dMLH1 were inoculated into WT C57BL/6 mice (n=4). *P = 0.0116 at end point between B16-OVA- Mlh1^−/− D169 vs. B16-OVA. (C) B16-OVA cells with dMLH1 (D170) were inoculated into WT and Sting^−/− mice (n=5). n.s, P = 0.5024 at end point between the two groups. (D) B16-OVA cells with dMLH1 (D167), and double deficiency cells of Mlh1 plus Sting (D164 and D197) were inoculated into WT C57BL/6 mice (n=4). *P=0.0253 at end point between B16-OVA-Mlh1^−/− D167 vs. B16-OVA-Mlh1^−/−-Sting^−/− D197. (E) 4T1 cells with dMLH1 (D166), and double deficiency cells of Mlh1 plus Sting (D169) were inoculated into WT BALB/C mice (n=5). ***P=0.0004 at end point between the two groups. (F) B16-OVA cells with dMLH1 (D170) were inoculated into WT and IFNAR1^−/− mice (n=5). ***P<0.0001 at indicated point between the two groups. Data are represented as mean ± SEM. Tumor size was measured twice weekly. Experiments were repeated at least 2 times. Unpaired t test was used to determine significance.

Figure 2.. Type I IFN signal pathway is activated in dMLH1 tumor cells

(A) ISGs expression at the mRNA level in cultured cells (n=3) was determined by qPCR. Relative expression fold change of representative ISG (Isg15) was shown. (also see Figure S2A). (B) Phosphorylation of STAT1 at Y701 was shown by WB. (C) IFN-β was quantified by ELISA in the supernatant of indicated cell lines (n=3). (D and E) Isg15 and IFN-β were quantified by qPCR and ELISA, respectively, in Sting or cGAS-deficient cells lines (n=3). (F) Isg15 and phosphorylation of STAT1 at Y701 were determined in H460 cells (n=3). (G) The plot shows the ranked p values (-log10 scaled) estimated from Wilcoxon rank test comparing gene expression levels in MSI-H versus MSS colorectal tumors from the TCGA database. The test was one-sided, to test if genes in MSI-H are expressed at higher levels than in MSS tumors. Red vertical lines marked the type I IFN induced ISGs (see Table S1 for ISGs list and expression). Data are represented as mean ± SEM. Representative data of over 2 independent experiments are shown. Unpaired t test was used to determine significance in A-F. Wilcoxon rank sum test was used to determine significance in G.

Figure 3.. MLH1 regulates accumulation of cytosolic DNA in tumor cells and tumor progression in vivo

(A) Isg15 expression and phosphorylation of STAT1 at Y701 were determined in MLH1-rescued cells. (B) IFN-β was quantified by ELISA in the supernatant of MLH1-rescued cells. (C) dsDNA was determined by PicoGreen dsDNA quantitation assay, and extra-nuclear dsDNA was counted. Statistical data are shown (also see Figure S3B). (D) Cytosolic DNA was isolated by a commercial kit and quantified by qPCR with genomic DNA specific primers. (E-G) Cytosolic DNA was isolated and quantified in human cancer cell lines H460, SW620 and HCT116. (H) 2×10⁶ 4T1-Mlh1^−/− cells with either rescued MLH1 or control vector were inoculated into WT BALB/C mice (n=5). Tumor size was measured twice weekly. Data are represented as mean ± SEM. Unpaired t test was used to determine significance.

Figure 4.. dMLH-mediated DNA sensing promotes T-cell priming independent of TMB

(A and B) BMDCs pre-educated with MC38-OVA and B16-OVA cells were co-cultured with OT-I T cells, then T-cell proliferation was determined. IFN-γ and TNF-α were quantified. Representative FACS histograms and statistic data are shown (also see Figures S4A and S4B). (C) Supernatants were added into co-culture system of BMDCs and OT-I T cells, then T-cell proliferation was determined. Representative FACS histograms and statistical data are shown. (D) B16-OVA cells were inoculated into WT C57BL/6 mice (n=4). One week later, cells from the spleen were isolated and re-stimulated by OT-I peptide or control SIY peptide in vitro. T-cell responses were determined by IFN-γ ELISPOT assay. (E and F) Fragmented tumor tissues derived from 4T1 cells with d Mlh1 (D129), double deficiency cells of Mlh1 plus Sting (D136), and double deficiency cells of Mlh1 plus cGAS (D144) were implanted into WT BALB/C mice (n=6–7). Eleven days later, tumor-infiltrating T cells were detected by FACS. Data are represented as mean ± SEM. Unpaired t test was used to determine significance.

Figure 5.. dMLH1-mediated DNA sensing enhances ICB therapy

(A) Fragmented tumor tissues derived from 4T1-Mlh1^−/− and 4T1-Mlh1^−/−+Mlh1 cells in Rag1 mice were implanted into WT mice (n=4–5), and treated with ICB drugs at day 11, 14 and 17. Tumor-bearing mice percentages are shown. (also see Figures S5A) (B) Fragmented tumor tissues derived from 4T1-Mlh1^−/− (D173) and 4T1-Mlh1^−/−Sting^−/− (D170) cells in Rag1 mice were implanted into WT mice (n=6–8), and treated with ICB drugs at day 7, 10, 13 and 16. *P=0.0156 between the two groups in 4T1-Mlh1^−/− model; ns, P=0.2467 between the two groups in 4T1-Mlh1^−/−Sting^−/− model. (C) MC38-OVA and MC38-OVA-dMLH1 cells were inoculated into Rag1/2C mice (n=9–10), followed by OT-I T-cell transfer at day 10, then ICB treatment was administrated at day 11, 14 and 17. (D) 4T1-HA and 4T1-HA-dMLH1 cells were inoculated into F1 mice (n=5) of Rag2× Rag2/OT-I mice, followed by CL4 T-cell transfer at day 10 and ICB treatment at day 11, 14, 17 and 20. (also see Figure S5D). Data are represented as mean ± SEM. The log rank test (Mantel–Cox) was used to assess the significance of differences in A. Unpaired t test was used to determine significance in B-D.

Figure 6.. impaired cGAS expression renders resistant to ICB therapy

(A) cGAS expression between MSS (n=321) and MSI-H (n=170) UCEC samples, ****p<0.0001 (see Table S2 for patient information). (B) CD8⁺ T cells in HCT116 tumor are detected by FACS (n=4–5). (C) The growth curves of HCT116 and HCT116+cGAS cells-derived tumors (n=5–7) are shown. *P=0.011 between the two groups in HCT116+cGAS model; ns, P=0.3140 between the two groups in HCT116 model. (D) Curves for disease-free survival are shown between the high and low expression of cGAS in UCEC samples with MSS (n=44) and MSI-H (n=24) (see Table S2 for patient information). (E) Curves for overall survival are shown between the high and low expression of cGAS and STING in pan-dMLH1 tumors (n=3–4). (F) cGAS and STING expression levels are shown between responders and non-responders treated with pembrolizumab. Data are represented as mean ± SEM. Unpaired t test was used to determine significant differences in A-C. The log rank test (Mantel–Cox) for D. The gehan wilcoxon test for E.