53BP1 loss elicits cGAS-STING-dependent antitumor immunity in ovarian and pancreatic cancer

Abstract

53BP1 nucleates the anti-end resection machinery at DNA double-strand breaks, thereby countering BRCA1 activity. Loss of 53BP1 leads to DNA end processing and homologous recombination in BRCA1-deficient cells. Consequently, BRCA1-mutant tumors, typically sensitive to PARP inhibitors (PARPi), become resistant in the absence of 53BP1. Here, we demonstrate that the 'leaky' DNA end resection in the absence of 53BP1 results in increased micronuclei and cytoplasmic double-stranded DNA, leading to activation of the cGAS-STING pathway and pro-inflammatory signaling. This enhances CD8⁺ T cell infiltration, activates macrophages and natural killer cells, and impedes tumor growth. Loss of 53BP1 correlates with a response to immune checkpoint blockade (ICB) and improved overall survival. Immunohistochemical assessment of 53BP1 in two malignancies, high grade serous ovarian cancer and pancreatic ductal adenocarcinoma, which are refractory to ICBs, reveals that lower 53BP1 levels correlate with an increased adaptive and innate immune response. Finally, BRCA1-deficient tumors that develop resistance to PARPi due to the loss of 53BP1 are susceptible to ICB. Therefore, we conclude that 53BP1 is critical for tumor immunogenicity and underpins the response to ICB. Our results support including 53BP1 expression as an exploratory biomarker in ICB trials for malignancies typically refractory to immunotherapy.

Fig. 1. 53BP1 depletion promotes immune cell infiltration.

Pie chart demonstrating TP53BP1 mRNA expression in HGSOC (a) and PDAC (b) from the TCGA. Tumor mutational burden is inversely proportional with TP53BP1 expression in HGSOC (c) and PDAC (d) cancers. n = 16 for high expression, n = 68 for low expression in (c), n = 23 for high expression, n = 24 for low expression in (d). Boxplots represent the median and interquartile range, and the whiskers denote minimum and maximum values. Wilcoxon test. Lollipop chart demonstrating p-values for Spearman correlation between abundance of activated CD8⁺ T-cells and expression of the indicated gene in HGSOC (e) and PDAC (f), Spearman correlation analysis. g Kaplan−Meier curves depicting overall survival of patients with high or low expression of TP53BP1. n = 16 for high expression and n = 68 for low expression. h KEGG pathway analysis comparing RNA-sequencing profiles of KPC Trp53bp1 KO to KPC malignant cells from subcutaneous allograft tumors from immunocompetent mice. n = 5 mice per group. i GSEA comparing RNA-sequencing profiles of KPC Trp53bp1 KO to KPC malignant cells from subcutaneous allograft tumors from immunocompetent mice. The vertical axis represents the top 10 ranked pathways in the Hallmark database. Interferon and inflammatory pathways are highlighted in red. n = 5 mice per group. Source data are provided as a Source Data file.

Fig. 2. 53BP1 depletion inhibits tumor growth and promotes immune cell infiltration.

Immunocompetent wildtype mice were injected subcutaneously (a) or orthotopically (b) with the indicated derivatives of KPC cells. Analysis of serial changes in tumor volumes. n = 13 in control group and n = 12 in Trp53bp1 KO group (a), n = 5 mice per group (b). c Immunocompetent wildtype mice were injected subcutaneously with the indicated derivatives of ID8 cells. Analysis of serial changes in tumor volumes. n = 15 mice per group. d ID8 tumor cells were injected into age-matched immunocompetent wildtype mice or mice previously implanted with ID8 Trp53bp1 KO tumor cells and cured. Tumor volumes at day 20 and 25. n = 5 mice per group. e Representative immunohistochemistry (IHC) images for CD8⁺ T cells. Scale bar, 0.050 mm. f Quantification of IHC data from subcutaneously implanted derivative KPC cells. n = 7 mice per group. g Quantification of IHC data from orthotopically implanted derivative KPC cells. n = 5 mice per group. h Nude mice were injected subcutaneously with indicated derivative KPC cells. Analysis of serial changes in tumor volumes. n = 10 mice per group. i, Representative IHC images for F4/80⁺ cells. Scale bar, 0.100 mm. j Quantification of IHC data. n = 7 mice per group. k Quantification of IHC data from orthotopically implanted derivative KPC cells. n = 5 mice per group. l Representative IHC images for CD86⁺ cells. Scale bar, 0.100 mm. m Quantification of IHC data. n = 7 mice per group. n Representative IHC images for NKp46⁺ cells. Scale bar, 0.100 mm. o Quantification of IHC data. n = 7 mice per group. p NSG mice were inject subcutaneously with indicated derivative KPC cells. Analysis of serial changes in tumor volumes. n = 10 mice per group. q NSG mice were injected subcutaneously with indicated derivative ID8 cells. Analysis of serial changes in tumor volumes. n = 10 mice per group. Data represent mean ± SEM; two-tailed unpaired t-test for (a, c, d, h). Data represent median, two-tailed unpaired t-test for (b, f, g, j, k, m, o). Data represent mean ± SEM for (p, q.). Source data are provided as a Source Data file.

Fig. 3. 53BP1 depletion upregulates immune checkpoints and synergizes with anti-PD1 antibody treatment.

Quantitative flow cytometry mean fluorescence intensity (MFI) for MHC-I (a) and PD-L1 (b) positivity from ID8 or ID8 Trp53bp1 KO tumors. n = 3 mice per group. c mRNA expression of Cd274 from ID8 or ID8 Trp53bp1 KO tumors. mRNA expression was normalized to Gapdh. n = 3 mice per group. d Western blot analysis of PD-L1 in ID8 and ID8 Trp53bp1 KO cells. 3 biological replicates. e Representative IHC images for PD-L1⁺ cells from indicated derivative ID8 tumors. Scale bar, 0.100 mm. f Quantification of IHC data from 3e. n = 5 mice per group. Quantification of IHC data from subcutaneously (g) or orthotopically (h) implanted derivative KPC cells. n = 7 mice per group (g), n = 5 mice per group (h). Immunocompetent wildtype mice were injected subcutaneously with the indicated derivatives of ID8 (i) or KPC (j) cells and treated with vehicle or anti-PD-1 antibody. n = 10 in IgG group and n = 11 in other three groups (i), n = 11 in IgG group and n = 12 in other three groups (j). Tumors from 3j were analyzed by flow cytometry for CD3⁺ (k), Ifn-γ (l), and Granzyme B (m). n = 8−9 mice per group. n Representative IHC images for Granzyme B⁺ cells. Scale bar, 0.100 mm. o Quantification of IHC data. n = 6 mice per group. p Dotplot with Spearman correlation test of abundance of activated CD8⁺ T-cells in pre-treatment biopsies of patients enrolled in the IMvigor210 trial in correlation to TP53BP1 mRNA expression level. q Kaplan−Meier curves depicting overall survival of patients with high (n = 143) or low (n = 205) expression of TP53BP1 treated with anti-PD1 therapy in patients from the IMvigor210 trial. r Association between expression of other genes in the Shieldin complex with CTL and overall survival. Data represent mean ± SEM; two-tailed unpaired t-test for (a−c, i, j). Data represent median, two-tailed unpaired t-test for (f−h). Data represent mean ± SEM; one-way ANOVA for (k−m, o). Source data are provided as a Source Data file.

Fig. 4. 53BP1 depletion activates the cGAS-STING pathway.

a Quantification of IF data for the indicated cell lines. Representative IF images in Supplementary Fig. 4a. n = 21 cells counted per group. b Quantification of cytoplasmic dsDNA foci in indicated cell lines (n = 488 cells). c Representative IF images for the indicated cell lines demonstrating cGAS-bound micronuclei. 3 biological replicates. 2’3’-cGAMP levels by ELISA in the indicated ID8 derivative (d) and KPC derivative (e) cell lines n = 3 independent experiments. f Western blot analysis of PD-L1 expression and cGAS-STING pathway activation in the indicated ID8 derivatives (left panel) or the indicated KPC derivatives (right panel). g mRNA expression of Cd274, Ccl5, and Ifnb1, in the indicated ID8 derivatives (left panel) or the indicated KPC derivatives (right panel). mRNA expression was normalized to Actb. n = 3 independent experiments. h Western blot analysis of PD-L1 expression and cGAS-STING pathway activation in the indicated ID8 derivatives. i mRNA expression of Cd274, Ccl5, and Ifnb1, in the indicated ID8 derivatives (left panel) or the indicated KPC derivatives (right panel). mRNA expression was normalized to Actb. n = 3 independent experiments. j Western blot analysis of PD-L1 expression and cGAS-STING pathway activation in the indicated ID8 derivatives. 3 independent experiments for (f, h, j). k mRNA expression of Cd274, Ccl5, and Ifnb1, in the indicated ID8 derivatives (left panel) or the indicated KPC derivatives (right panel). mRNA expression was normalized to Actb. n = 3 independent experiments. l Representative IHC images for PD-L1⁺ cells from indicated derivative ID8 Trp53bp1 KO tumors. Scale bar, 0.100 mm. m Quantification of IHC data. n = 5 mice per group. n Representative IHC images for CD8⁺ T-cells from indicated derivative ID8 Trp53bp1 KO tumors. Scale bar, 0.100 mm. o Quantification of IHC data. n = 5 mice per group. p Immunocompetent wildtype mice were injected subcutaneously with indicated derivative ID8 cells. Analysis of serial changes in tumor volumes. n = 10 mice per group. Data represent mean ± SD; two-tailed unpaired t-test for (a, b, g, i, k). Data represent median, two-tailed unpaired t-test for (d, e, m, o). Data represent mean ± SEM; two-tailed unpaired t-test for (p). Source data are provided as a Source Data file.

Fig. 5. 53BP1 loss mediated PARP inhibitor resistance is overcome with anti-PD-1 antibody treatment.

a Western blot analysis demonstrating generation of KPC Brca1 KO and KPC Brca1 Trp53bp1 double knockout (DKO) cells. b Quantification of clonogenic survival assay with KPC Brca1 KO and KPC Brca1 Trp53bp1 DKO cells, treated with PARP inhibitor (Olaparib). Data are represented as mean ± SD; two-tailed unpaired t-test of highest dose. 3 independent experiments for (a, b). c mRNA expression of Cd274, Ccl5, and Ifnb1, in the indicated cell lines. mRNA expression was normalized to Actb. n = 3 independent experiments. Data represent mean ± SD; two-tailed unpaired t-test. d Left: representative IHC images for CD8⁺ T cells. Scale bar, 0.100 mm. Right: quantification of IHC data. n = 8 mice per group. e Left: representative IHC images for F4/80⁺ cells. Scale bar, 0.100 mm. Right: quantification of IHC data. n = 8 mice per group. f Left: representative IHC images for CD86⁺ cells. Scale bar, 0.100 mm. Right: quantification of IHC data. N = 8 mice per group. g Left: representative IHC images for NKp46⁺ cells. Scale bar, 0.100 mm. Right: quantification of IHC data. n = 8 mice per group. h Left: representative IHC images for PD-L1⁺ cells. Scale bar, 0.100 mm. Right: quantification of IHC data. n = 8 mice per group. i mRNA expression of Cd274, Ccl5, and Ifnb1 from KPC Brca1 KO and KPC Brca1 Trp53bp1 DKO tumors. mRNA expression was normalized to Actb. n = 8 mice per group. Data represent median; two-tailed unpaired t-test for (d−i). j Immunocompetent wildtype mice were injected subcutaneously with KPC Brca1 KO or KPC Brca1 Trp53bp1 DKO cells and treated with vehicle (IgG) PARP inhibition (PARPi, Olaparib), anti-PD-1 antibody, or the combination of PARPi and anti-PD-1. Analysis of tumor volumes at Day 0, Day 14, and Day 21 of treatment. n = 8 in VEH and anti-PD-1 groups and n = 10 in the other groups were initially inoculated. Data represent mean ± SEM; one-way ANOVA analysis with Sidak post hoc test. Source data are provided as a Source Data file.

Fig. 6. Low 53BP1 expression correlates with increased immune cell infiltration in HGSOC and PDAC.

a Representative IF images processed for DAPI, 53BP1, CD8, NKp46, and PD-L1 from an independent cohort of 45 HGSOC tissue samples. 45 samples analyzed. Dotplot with Spearman correlation test of abundance of CD8⁺ T-cells (b), NKp46 NK cells (c) and PD-L1 expression (d) CD8⁺ T-cells in in 45 HGSOC tissue samples in correlation to TP53BP1 protein expression level. Dotplot with Spearman correlation test of abundance of CD8⁺ T-cells (e), NKp46 NK cells (f) and PD-L1 expression (g) in 38 PDAC tissue samples in correlation to TP53BP1 protein expression level. Spearman correlation analysis was performed for (b−g). Source data are provided as a Source Data file.