PARPi Triggers the STING-Dependent Immune Response and Enhances the Therapeutic Efficacy of Immune Checkpoint Blockade Independent of BRCAness

Abstract

PARP inhibitors (PARPi) have shown remarkable therapeutic efficacy against BRCA1/2-mutant cancers through a synthetic lethal interaction. PARPi exert their therapeutic effects mainly through the blockade of ssDNA damage repair, which leads to the accumulation of toxic DNA double-strand breaks specifically in cancer cells with DNA repair deficiency (BCRAness), including those harboring BRCA1/2 mutations. Here we show that PARPi-mediated modulation of the immune response contributes to their therapeutic effects independently of BRCA1/2 mutations. PARPi promoted accumulation of cytosolic DNA fragments because of unresolved DNA lesions, which in turn activated the DNA-sensing cGAS-STING pathway and stimulated production of type I IFNs to induce antitumor immunity independent of BRCAness. These effects of PARPi were further enhanced by immune checkpoint blockade. Overall, these results provide a mechanistic rationale for using PARPi as immunomodulatory agents to harness the therapeutic efficacy of immune checkpoint blockade. SIGNIFICANCE: This work uncovers the mechanism behind the clinical efficacy of PARPi in patients with both BRCA-wild-type and BRCA-mutant tumors and provides a rationale for combining PARPi with immunotherapy in patients with cancer.

Fig. 1.. PARPi induces accumulation of cytosolic DNA and activates the STING signaling pathway

A, Representative images and quantitative analysis of PicoGreen staining in cells treated with DMSO or BMN673 (2 μM) for 48 hrs. DAPI (blue) was used to visualize the nuclei. Data represent mean ± s.e.m. of three independent experiments. B, Western blots (Left) and immunofluorescent images (Right) of phosphorylated IRF3 (p-IRF3) and phosphorylated TBK1 (p-TBK1) in cells treated with BMN673 (2 μM). The numbers represent the folds change of p-IRF3 and p-TBK1 from three independent experiments. For immunofluorescence staining, HeLa cells were treated with DMSO or BMN673 (2 μM) for 48 hrs. Data represent mean ± s.e.m. of three independent experiments. C, qPCR and ELISA evaluation of CCL5 and CXCL10 expression in cells under DMSO or BMN673 treatment. For ELISA analysis, HOC1 cells were treated with DMSO or BMN673 treatment for 48 hrs. Data represent mean ± s.e.m. of three independent experiments. D, qPCR evaluation of CCL5 and CXCL10 levels in HeLa cells with depletion of STING (siSTING), TBK1 (siTBK1), IRF3 (siIRF3), cGAS (sicGAS), CtIP (siCtIP), BLM (siBLM), MRE11 (siMRE11), or EXO1 (siEXO1). Cells were treated with DMSO or BMN673 (2 μM) for 48 hrs. Data represent mean ± s.e.m. of three independent experiments. Scale bar, 10 μm, *, p<0.05; **, p<0.01.

Fig. 2.. PARPi activates STING signaling and immune checkpoint in vivo

A, Schematic of PARPi treatment in nude and syngeneic mice bearing intraperitoneal (i.p.) ID8 tumors. Mice were treated with BMN673 (0.33 mg/kg) daily by oral administration 7 days after ID8 inoculation until euthanization (n=5). B, Representative bioluminescence images of i.p. tumors in immune-deficient (Left) and immune-proficient (Right) mice on day 7 and day 21 of ID8 cell inoculation and survival curves. C, Representative images and quantitative analysis of phosphorylated Irf3 (p-Irf3), Sting, CD8 and PD-L1 in ID8 tumors 30 days after inoculation. DAPI (blue) was used to visualize the nuclei. D, ELISA evaluation of Ccl5 levels in ascites from C57BL/6 mice when euthanization was performed (Left) and qPCR evaluation of Ccl5 and Cxcl10 levels in CT26 tumors from BALB/C mice 22 days after tumor cell inoculation (Right). Dashed square, area for magnification. Scale bar, 50 μm, *, p<0.05; **, p<0.01; ***, p<0.001; n.s. not significant.

Fig. 3.. Immune checkpoint blockade targeting PD-1/PD-L1 pathway potentiates therapeutic efficacy of PARPi in syngenic mouse models

A, Combination treatment in C57BL6 mice bearing ID8 tumors. Schematic of treatment (Left). i.p. injections of isotype control IgG (200 μg/mouse) and anti-PD-L1 antibody (aPD-L1, 200 μg/mouse) started at day 7 and stopped at day 28 after ID8 cell inoculation. BMN673 (0.33 mg/kg) was orally administered daily. Representative bioluminescence images of ID8 tumors after 7 and 21 days of inoculation (Middle). Statistical analysis of bioluminescence changes over time (mean ± s.e.m) or at the endpoint (Day 21, each dot represents one mouse) (Right) (n=5). B, Survival curves of mice with temporary (Left, started on day 7 after ID8 inoculation and stopped on day 28) (n=5) or continuous treatment (Right, started on day 7 after inoculation and continued until the mice were euthanized) (n=10). C, Representative images and tumor volume measurements of CT26 tumors in BALB/c mice with continuous treatment (n=5). D, Percentage of Cd8+ cells in ID8 or CT26 tumors after treatments. *, p<0.05; **, p<0.01; ***, p<0.001; n.s. not significant.

Fig. 4.. An intact immune system is required for the therapeutic benefits of combining PARPi with anti-PD-L1

A, Representative images and statistical analysis of endpoint bioluminescence in athymic nude mice bearing ID8 i.p. tumors. Data represent mean ± s.e.m, (n=5). i.p. injection of isotype control IgG (200 μg/mouse) and anti-PD-L1 (aPD-L1, 200 μg/mouse) were stated at day 7 and continued until mice were euthanized. BMN673 (0.33 mg/kg) was orally administered daily. B, Survival curves of athymic nude mice with ID8 i.p. tumors. Treatment was started on day 7 and continued until mice were euthanized. C, Representative images and tumor volume measurements of CT26 tumors in athymic nude mice with the indicated treatments (n=5). n.s., not significant. D, Representative images and statistical analysis of bioluminescence in Wildtype (WT) or STING KO (KO, Tmem173 gt/J) mice bearing ID8 i.p. tumors. i.p. injection of Vehicle (isotype control IgG, 200 μg/mouse) and anti-PD-L1 (aPD-L1, 200 μg/mouse) were stated at day 7 and continued. BMN673 (0.33 mg/kg) was orally administered daily.