Targeting the DNA damage response in combination with radiation enhances type I interferon (T1IFN)-driven innate immune signaling. It is not understood, however, whether DNA-dependent protein kinase (DNA-PK), the kinase critical for repairing the majority of radiation-induced DNA double-strand breaks in cancer cells, is immunomodulatory. We show that combining radiation with DNA-PK inhibition increases cytosolic double-stranded DNA and tumoral T1IFN signaling in a cyclic GMP-AMP synthase (cGAS)- and stimulator of interferon genes (STING)-independent, but an RNA polymerase III (POL III), retinoic acid-inducible gene I (RIG-I), and antiviral-signaling protein (MAVS)-dependent manner. Although DNA-PK inhibition and radiation also promote programmed death-ligand 1 (PD-L1) expression, the use of anti–PD-L1 in combination with radiation and DNA-PK inhibitor potentiates antitumor immunity in pancreatic cancer models. Our findings demonstrate a novel mechanism for the antitumoral immune effects of DNA-PK inhibitor and radiation that leads to increased sensitivity to anti–PD-L1 in poorly immunogenic pancreatic cancers.

Implications:

Our work nominates a novel therapeutic strategy as well as its cellular mechanisms pertinent for future clinical trials combining M3814, radiation, and anti-PD-L1 antibody in patients with pancreatic cancer.

This article is featured in Highlights of This Issue, p. 1011

Immune checkpoint inhibitors (ICI) have improved survival outcomes for numerous malignancies including melanoma (1), non–small cell lung cancer (2), and urothelial carcinoma (3). Only a minority of patients, however, achieve durable responses, and there is an unmet need to develop combinatorial treatment strategies to enhance ICI efficacy, especially in poorly immunogenic solid tumors such as pancreatic cancer (4, 5). Pancreatic cancers are immunologically “cold” tumors characterized by an immunosuppressive tumor microenvironment (6). One strategy to enhance antigenicity and adjuvanticity in the setting of poorly immunogenic solid tumors, such as pancreatic cancer, is with radiotherapy (7).

Radiation promotes systemic antitumoral immune responses and enhances ICI efficacy in both preclinical models (8–10) and patients (11, 12), including pancreatic cancer (13). Furthermore, radiation causes DNA damage which becomes aberrantly localized to the cytoplasm within micronuclei or as free cytosolic DNA where it is sensed by the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway resulting in a type 1 interferon (T1IFN)-driven inflammatory response (14, 15). Double-stranded DNA (dsDNA) fragments produced following radiation can also be transcribed by RNA polymerase III (POL III) into double-stranded RNA which triggers the retinoic acid-inducible gene I (RIG-I) and mitochondrial antiviral-signaling protein (MAVS) signaling pathway (16). Thus, the production of dsDNA following radiation is an important molecular target for enhancing antitumor immune responses.

The pharmacologic inhibition of DNA damage response (DDR) proteins such as ATM, ATR, CHK1/2, and PARP results in persistence of DNA damage as well as enhanced immunostimulatory effects of radiation (17–23). DNA-dependent protein kinase (DNA-PK) is required for nonhomologous end joining (NHEJ), and its catalytic subunit, DNA-PKcs, is overexpressed in human pancreatic cancers (24). Pharmacologic inhibition by the selective DNA-PK inhibitor M3814 (i.e., peposertib), currently in clinical trials, augments radiation efficacy (25–27). While prior studies have shown that DNA-PK inhibition reduces radiation-induced micronuclei generation to restrain the T1IFN response (28, 29), other studies have demonstrated DNA-PK inhibition enhances radiation-induced T1IFN expression to promote durable responses in immunogenic tumor models (30). It remains unclear whether DNA-PK is a therapeutic target in pancreatic cancer or how DNA-PK inhibition modulates T1IFN signaling.

In this study, we sought to identify the mechanisms of T1IFN generation in response to DNA-PK inhibitor and radiation in poorly immunogenic pancreatic cancers. Furthermore, we hypothesized that DNA-PK inhibitor would synergize with radiation to potentiate innate inflammatory signaling in pancreatic cancer cells. To test this hypothesis, we examined IFN signaling and found that combination therapy with the DNA-PK inhibitor M3814 and radiation induced T1IFN production and IFN stimulated genes such as CXCL9/10 and programmed death-ligand 1 (PD-L1). We investigated the cytosolic DNA ligand and the respective molecular sensors through which DNA-PK and radiation regulate innate immunity. Finally, based on the observed PD-L1 expression in response to DNA-PK inhibition and radiation, we tested whether the treatment efficacy could be potentiated in vivo by anti–PD-L1 treatment. The results of this study nominate a novel therapeutic strategy to maximize the efficacy of DNA-PK inhibitors, radiation, and immunotherapy in pancreatic cancer.

Reagents and cell lines

DNA-PK inhibitor, M3814 (peposertib) was acquired from Merck KGaA/EMD and Cancer Therapy Evaluation Program (CTEP). The murine PD-L1 blocking antibody (10F.9G2, catalog no. BE0101) and its respective IgG1 isotype control (LTF-2, catalog no. BE0090) were acquired from BioXCell. The CD4 (D7D2Z, catalog no. 25229), CD8a (D4W2Z, catalog no. 98941), and Granzyme B (D6E9W, catalog no. 46890) antibodies for IHC staining were purchased from Cell Signaling Technology. For in vitro experiments, M3814 was dissolved in dimethyl sulfoxide (Sigma-Aldrich) and stored in aliquots at -20°C. The POL III inhibitor, ML-60128, was purchased from Focus Biomolecules. The human pancreatic cancer cell lines, Panc1 and MiaPaCa2, and murine pancreatic cancer cell line, mT4, were grown in DMEM (Gibco/Invitrogen) + 10% FBS (Hyclone) + 100 units/mL penicillin +100 μg/mL streptomycin. The human pancreatic cancer cell line Capan1 was cultured in Improved Minimum Essential Medium (IMEM) supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. The human pancreatic cancer cell line, BxPC3, and murine pancreatic cancer cell line, KPC2, were grown in RPMI-1640 with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. All human pancreatic cancer cell lines were purchased from ATCC. KPC2 (also known as 65.671) and mT4 cells were obtained through collaboration with Drs. Pasca di Magliano (University of Michigan) and David Tuveson (Cold Spring Harbor Laboratory; ref. 31). All cell lines were tested for Mycoplasma every 3 months and authenticated by short tandem repeat profiling. mT4 cells were transfected with pCI-neo-mOVA plasmid by Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The cells were selected with G418, and ovalbumin expression was confirmed by flow cytometry and used for in vitro antigen presentation assay.

Irradiation

Irradiations were performed using a Philips RT250 (Kimtron Medical) at a dose rate of approximately 2 Gy/minute at the University of Michigan Rogel Comprehensive Cancer Center Experimental Irradiation Shared Resource (Ann Arbor, MI). The beam energy was 225 kilovolts. Dosimetry was performed using an ionization chamber connected to an electrometer system that is directly traceable to a National Institute of Standards and Technology calibration. For tumor irradiation, animals were anesthetized with isoflurane and positioned such that the apex of each flank tumor was at the center of a 2.4-cm circular aperture in a custom lead holder which shielded the rest of the mouse from radiation. Light fields were used for geometric verification of tumor positioning.

Clonogenic survival assays

Cells treated with M3814 and/or radiation were processed for clonogenic survival as previously described (32). M3814 was given for 25 hours, beginning 1 hour prior to radiation. Radiation survival curves were normalized for M3814 toxicity, and the radiation enhancement ratio (13) was calculated as the ratio of the mean inactivation dose under control conditions divided by the mean inactivation dose after drug exposure. A value significantly greater than 1 indicates radiosensitization. Cytotoxicity in the absence of radiation treatment was calculated by normalizing the plating efficiencies of M3814-treated cells to non–M3814-treated cells.

Genetically modified cell lines

Human cGAS-, STING-, or TANK-binding kinase (TBK1)-knockout cells and murine cGas-, Sting-, or Tbk1-knockout cells were generated with CRISPR/Cas9 technology. Panc1 or mT4 cells were transduced with lenti-CRISPR lentiviral constructs encoding Cas9 and the control, cGAS, STING, or TBK1 (cGas, Sting, or Tbk1) guide RNAs. Positive cells were selected by adding puromycin (2 μg/mL) at 48 hours after lentiviral infection. Single-cell clones were selected and expanded. POLR3A-, RIG-I–, or MAVS-silenced murine and human pancreatic cancer cell lines were generated by infecting the cells with lentivirus expressing shRNA targeting Ctrl, POLR3A, RIG-I or MAVS (pLenti-CMV-Puro-Luciferase). The cells were then selected with puromycin to generate stable cell lines.

Western blotting

Western blotting was performed as previously described (33). Whole-cell extract was prepared by sonicating cells in a SDS sample buffer containing protease inhibitor and phosphatase inhibitor. After boiling at 95°C for 10 minutes, samples were separated by SDS-PAGE and transferred to PVDF membranes (Millipore). The following primary antibodies were used in this study: anti–β-actin (sc-47778, Santa Cruz Biotechnology), anti-TBK1 (D1B4, Cell Signaling Technology), anti-STING (D2P2F, Cell Signaling Technology), anti-human cGAS (D1D3G, Cell Signaling Technology), anti-POLR3A (A10737, ABclonal), anti-RIG-I/DDX58 (A0550, ABclonal), anti-MAVS (A5764, ABclonal).

IFNß1–GFP reporter assay

T1IFN was measured using a reporter-promotor plasmid as previously described (20, 28). Briefly, the pIFNß1-GFP reporter containing the IFNß1 promoter was stably transfected into Panc1 cells, and positive cells were selected with 50 μg/mL hygromycin. Upon M3814 and/or radiation treatment, single-cell suspensions were generated by trypsin and resuspended in PBS; GFP levels were measured by flow cytometry (BD Biosciences). After subtracting background GFP expression (baseline), technical triplicates were averaged for each experiment and the mean change in median fluorescence intensity (MFI) for indicated treatments was determined.

qRT-PCR

Total RNA was isolated from cells by column purification (RNeasy Mini Kit, Qiagen) with DNase treatment. cDNA was synthesized using SuperScript III First-strand Synthesis System for RT-PCR (Invitrogen) with Oligo (27). qPCR was performed on cDNA using Fast SYBR Green Master Mix (Thermo Fisher Scientific) on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). Fold changes in mRNA expression were calculated by the ΔΔCt method using β-actin as an endogenous control. All fold changes are expressed normalized to the untreated control. PCR was performed with the following primers: Cxcl9 (Forward): 5′-TGTGGAGTTCGAGGAACCCT-3′, Cxcl9 (Reverse): 5′-TGCCTTGGCTGGTGCTG-3′; Cxcl10 (Forward): 5′-AGAACGGTGCGCTGCAC-3′, Cxcl10 (Reverse): 5′-CCTATGGCCCTGGGTCTCA-3′; Mx1 (Forward): 5′-GGGGAGGAAATAGAGAAAATGAT-3′, Mx1 (Reverse): 5′-GTTTACAAAGGGCTTGCTTGCT-3′; Isg15 (Forward): 5′-TGGAAAGGGTAAGACCGTCCT-3′, Isg15 (Reverse): 5′-GGTGTCCGTGACTAACTCCAT-3′; Polr3a (Forward): 5′-TGATGCTGGCCTCCTTTGAA-3′, Polr3a (Reverse): 5′-AGCTTGAAGAGCCCAGTTCC-3′; Rig-I (Forward): 5′-AAGAGCCAGAGTGTCAGAATCT-3′, Rig-I (Reverse): 5′-AGCTCCAGTTGGTAATTTCTTGG-3′); Mavs (Forward): 5′-GAATCCAGGTAGACGAAAGCC-3′, Mavs (Reverse): 5′-GCCTACTACGGTACAGCATCAC-3′); Human IFNβ (Forward): 5′-ATGACCAACAAGTGTCTCCTCC-3′, Human IFNβ (Reverse): 5′-GCTCATGGAAAGAGCTGTAGTG-3′; Human PD-L1 (Forward): 5′-TGGCATTTGCTGAACGCATTT-3′, Human PD-L1 (Reverse): 5′-TGCAGCCAGGTCTAATTGTTTT-3′; Mouse IFNβ (Forward): 5′-CCCTATGGAGATGACGGAGA-3′, Mouse IFNβ (Reverse): 5′-CTGTCTGCTGGTGGAGTTCA-3′; Mouse Pd-l1 (Forward): 5′-GCTCCAAAGGACTTGTACGTG-3′, Mouse Pd-l1 (Reverse): 5′-TGATCTGAAGGGCAGCATTTC-3′.

Immunofluorescence

For micronuclei staining, cells were seeded onto coverslips and treated with M3814 and/or radiation. Coverslips were mounted with a drop (about 20 μL) of ProLong Gold Antifade with DAPI (Invitrogen). Images were captured using an Olympus IX71 FluoView confocal microscope (Olympus America) with a 60× oil objective. Micronucleated cells were classified manually by distinct staining by DAPI of structures outside of the main nucleus. To enumerate micronuclei these structures were counted manually for each field and expressed as a percentage of total cells within the field. At least 200 cells from each treatment condition were evaluated. For cytosolic dsDNA immunostaining, the previously described protocol was used (34) with minor modifications. Cells were grown and treated on cover slips in 12-well dishes. Briefly, cells were rinsed with PBS, and fixed in 4% PFA in PBS for 20 minutes. Cells were then washed with PBS three times for 5 minutes each and permeabilized using 0.2% Triton X-100 in PBS for 10 minutes. After washing with 1X PBS + 0.1% Tween-20 (PBST) three times for 5 minutes, cells were incubated in 50% formamide diluted in PBS at room temperature for 10 minutes in a staining jar. While incubating the slides, a separate portion of 50% formamide was microwaved for approximately 10 seconds or until it reached 75°C. After incubation for 10 minutes at room temperature, slides were immersed in the heated formamide for 15 minutes; the jar containing the slides and heated formamide was placed in a circulating water bath (preheated to 75°C) to minimize temperature fluctuations. After heating, cells were immediately transferred into 1X TBS and washed with 1X TBS three times. Cells were further incubated in 60 μL of boiled RNase A (1 mg/mL) for 1 hour at 37°C and washed with 1X PBS three times for 5 minutes each. For immunostaining, cells were first put into blocking buffer containing 1% BSA + 2% goat serum in PBS at room temperature for 1 hour. Cells were then incubated with 60 μL of dsDNA antibody (Chemicon, MAB1293, 10 μg/mL) and anti-mouse Tom20 antibody (Cell Signaling Technology, D8T4N, 1:200) in 1% BSA + 2% goat serum + 0.1% saponin in a humidified chamber overnight at 4°C. After washing with PBST three times, cells were stained with secondary antibodies goat anti-mouse IgG antibody conjugated Alexa Fluor 594 and goat anti-rabbit IgG antibody conjugated Alexa Fluor 488 (Invitrogen) in 1% BSA + 2% goat serum + 0.1% saponin in a humidified chamber for 1 hour at room temperature in the dark. Cells were finally washed with PBST three times, and coverslips were mounted with a drop (about 20 μL) of ProLong Gold Antifade Mounting with DAPI (Invitrogen) for 10 minutes in the dark. Samples were imaged with an Olympus IX71 FluoView confocal microscope (Olympus America) with a 60× oil objective. Fields were chosen at random based on DAPI staining.

Quantification of cytosolic dsDNA

Cytosolic extracts from cultured cells were isolated as previously described with minor modifications (8). Briefly, cells were lysed in 10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 1.5 mmol/L MgCl2, 0.34 M sucrose, 10% volume for volume (v/v) glycerol, plus protease inhibitors (Roche) for 5 minutes on ice with 0.1% (v/v) Triton X-100, and nuclei were removed by low-speed centrifugation (1,500 × g, 10 minutes). Cytosolic extracts were treated with 1 mg/mL proteinase K at 55°C for 1 hour. After phenol/chloroform (phenol:chloroform:isoamyl alcohol 25:24:1, Invitrogen) extraction, the aqueous supernatants were incubated with 500 μg/mL DNase-free RNase A (Roche) for 30 minutes at 37°C, and then followed by a second phenol/chloroform extraction. The DNA in the aqueous phase was precipitated by chilled 100% ethanol and then washed with 70% ethanol at max speed. After air-drying, DNA was resuspended in Tris-EDTA (TE) buffer and referred to as cytosolic dsDNA. Cytosolic dsDNA was quantified using the SpectraMax QuantTM AccuClear Nano dsDNA Assay kit (Molecular Devices, Part#: R8356) and plate reader (Biotek Cytation 3 imaging reader). The amounts of cytosolic dsDNA were normalized to the cell number of each group and the relative levels of cytosolic dsDNA were normalized to the untreated group.

Flow cytometry analysis

To analyze cell surface PD-L1 expression, human or murine cells were trypsinized and resuspended in 100 μL of cell staining buffer (#420201, BioLegend) and incubated with PE-conjugated anti-human PD-L1 antibody (#329708, BioLegend), or anti-mouse PE-labeled PD-L1 antibody (#124308, BioLegend) for 1 hour at room temperature. Stained cells were washed in the staining buffer and analyzed by flow cytometry (BD Biosciences). The PD-L1 expression levels on the cell surface were analyzed in FlowJo 7.6. Following gating, background-corrected PD-L1 median fluorescence intensity (i.e., PD-L1 MFI minus isotype control MFI for each treatment condition) was calculated. mT4-ova cells were analyzed by flow cytometry for murine OVA257–264 (SIINFEKL) peptide bound to H-2Kb (Clone 25-D1.16) mAb (Invitrogen). For cell cycle analysis, single cell suspensions were generated by trypsinization and fixed with ice-cold 70% ethanol. Cells were then permeabilized with 0.25% Triton X-100/PBS solution and stained with pH3 (Serine 10) antibody conjugated to Alexa Fluor 488 (Invitrogen) in 1% BSA/PBS solution. After washing in PBS, cells were incubated in PBS containing 50 μg/mL propidium iodide (Santa Cruz Biotechnology) and 100 μg/mL RNase A (Roche). Flow cytometry was performed and analyzed using FlowJo 7.6 software. Single cells and G1/S/G2 peaks were manually gated. The gates for pH3-positive cells were chosen by comparison with a population in which the antibody was omitted during processing.

In vivo mouse models

Eight- to 10-week-old athymic (NU/J), wild-type C57BL/6 mice were obtained from the The Jackson Laboratory. mT4 pancreatic cancer cells (106) were subcutaneously injected to the left and right flanks of female C57BL/6 or athymic mice. M3814 was dissolved in 0.5% Methocel, 0.25% Tween-20, 300 mmol/L Na-Citrate pH2.5, and orally dosed. Anti–PD-L1 and IgG1 isotype control were given intraperitoneally 100 μg/mouse every 3 days after tumors reached approximately 75 mm3. A single fraction of 5 Gy or 8 Gy was given when tumors reached approximately 100 to 150 mm3. Tumor diameters were measured using calipers two times per week. Tumor volume was calculated according to the equation: TV = π/6 (ab2), where a and b are the longer and shorter dimensions of the tumor, respectively and TV is the tumor volume. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) Committee of the University of Michigan.

Statistical analyses

Unless otherwise stated, all data are presented as mean ± SD. When assessing statistical significance between two treatment groups, continuous variables were analyzed using the unpaired Student t test and Mann–Whitney test for normally and nonnormally distributed data, respectively. In cases of more than two groups, ANOVA with the Tukey post-comparison test or Kruskal–Wallis analysis was used. Differences in the time taken to reach two times the tumor volume at the start of treatment (i.e., tumor volume doubling time) were examined using the log-rank test. P < 0.05 were considered statistically significant and are denoted in the figures as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. NS represents not significant (i.e., P > 0.05). All tests were two-sided. All statistical analyses were performed using GraphPad Prism 8 (GraphPad Inc.) statistical software.

DNA-PK inhibition enhances radiation efficacy in a syngeneic model of pancreatic cancer

DNA-PK inhibitors are effective radiosensitizers in several cancer cell types (27, 30, 35, 36). To investigate the ability of M3814 to radiosensitize pancreatic cancer cells, clonogenic survival assays were performed following M3814 and radiation treatment in both human (MiaPaCa2 and Panc1) and mouse (mT4 and KPC2) pancreatic cancer cells. While M3814 treatment alone (no radiation) did not affect the surviving fraction of these four cell lines, we observed significant and concentration-dependent radiosensitization by M3814 in all cell lines (Supplementary Fig. S1A–S1D), indicating that M3814 is a potent radiosensitizer in pancreatic cancer models.

Emerging evidence suggests that radiation exerts antitumor activity not only through inducing DNA damage, but also by activating antitumor immune responses (37, 38). This effect is further augmented by DDR inhibitors, including inhibitors of ATM, ATR, and PARP (39). DNA-PK inhibition enhances antitumor immunity when combined with radiation in immunogenic tumor models (30). To examine this in poorly immunogenic pancreatic tumors, mT4 murine pancreatic cancer tumors were established in syngeneic fully immunocompetent (C57BL/6) mice and treated with the DNA-PK inhibitor M3814 and a single fraction of radiation (Fig. 1A). M3814 monotherapy was associated with slightly slower tumor progression compared with untreated controls (Fig. 1B). Similarly, radiation alone also inhibited tumor growth as compared with untreated controls. However, the combination of radiation with M3814 significantly inhibited tumor growth compared with radiation alone, and extended the time to tumor doubling (Fig. 1C; Supplementary Fig. S1E).

Figure 1.

DNA-PK inhibition enhances radiation efficacy in syngeneic models of pancreatic cancer. A, Schematic showing schedules of the DNA-PK inhibitor, M3814, and radiation. M3814 (25 mg/kg) was administered approximately 1 hour before RT on day 0 (8 Gy × 1) and alone on days 1 to 4. mT4 tumor size (B) and tumor volume doubling time (C) implanted in C57BL/6 mice treated with M3814 1 hour preradiotherapy on day 0, followed by M3814 treatment from days 1 to 4, n = 12 to 18 tumors per treatment group. mT4 tumor size (D) and tumor volume doubling time (E) implanted in athymic nude mice (n = 5 per group). Statistical significance was determined using Student two-tailed t test or log-rank test. **, P < 0.01. RT, radiotherapy; Ctrl, control.

Figure 1.

DNA-PK inhibition enhances radiation efficacy in syngeneic models of pancreatic cancer. A, Schematic showing schedules of the DNA-PK inhibitor, M3814, and radiation. M3814 (25 mg/kg) was administered approximately 1 hour before RT on day 0 (8 Gy × 1) and alone on days 1 to 4. mT4 tumor size (B) and tumor volume doubling time (C) implanted in C57BL/6 mice treated with M3814 1 hour preradiotherapy on day 0, followed by M3814 treatment from days 1 to 4, n = 12 to 18 tumors per treatment group. mT4 tumor size (D) and tumor volume doubling time (E) implanted in athymic nude mice (n = 5 per group). Statistical significance was determined using Student two-tailed t test or log-rank test. **, P < 0.01. RT, radiotherapy; Ctrl, control.

Close modal

Radiation efficacy in vivo relies on adaptive immune responses (8, 9). We next evaluated whether the combination of DNA-PK inhibition and radiotherapy also required an adaptive immune response. To examine this, we established mT4 murine pancreatic tumors in immunodeficient athymic nude mice and treated them as indicated (Fig. 1A). M3814 monotherapy failed to slow tumor progression, and radiation showed limited efficacy (Fig. 1D). The combination of M3814 plus radiation failed to significantly delay tumor growth compared with radiation alone (Fig. 1D). Neither radiotherapy, M3814, nor the combination extended the time to tumor doubling in immunodeficient mice (Fig. 1E; Supplementary Fig. S1E). Collectively, these findings suggest that an intact immune system is required for the therapeutic efficacy of DNA-PK inhibition with radiation in vivo.

DNA-PK inhibition and radiation synergistically promote T1IFN expression and signaling

Radiation alone, or in combination with DDR inhibitors induces interferon signaling to promote antitumor immune responses (28, 30, 40). Given that the combination of M3814 with radiation requires an intact immune system for efficacy (Fig. 1), we hypothesized that M3814 would synergize with radiation to induce T1IFN production. To test this hypothesis, Panc1 cells stably expressing a GFP reporter driven by the human IFNβ1 promoter (20, 28) were treated with M3814 and/or radiation. Radiation, but not M3814, modestly induced IFNβ1 promoter driven GFP expression (Fig. 2A). Furthermore, the combination treatment increased T1IFN reporter activity in a concentration- and time-dependent manner compared with radiation alone (Fig. 2A). IFNβ1 mRNA levels in Panc1 and KPC2 cells peaked 12 hours after the initiation of M3814 and radiation treatment (Supplementary Fig. S2A). We next investigated whether M3814 and radiation modulated the expression of the IFN stimulated genes CXCL9 and CXCL10 in Panc1 cells. Consistent with prior studies (20, 38), we found increased expression of these IFN stimulated genes (Fig. 2B) in response to radiation. The combination of M3814 and radiation further increased the expression of IFN stimulated genes as compared with radiation alone (Fig. 2B). M3814 also enhanced radiation-induced expression of IFN stimulated genes in KPC2 cells (Fig. 2C). T1IFN facilitates antigen presentation by increasing the expression of MHCI on tumor cells. To define the functional consequences of DNA-PK inhibition and/or radiation on antigen presentation, we transduced the murine mT4 pancreatic cancer cell line with ovalbumin and examined ovalbumin peptide presentation within MHCI (20). As expected, radiation augmented antigen presentation as compared with control, an effect that was further enhanced by M3814 (Supplementary Fig. S2B). Collectively, these findings indicate that the combination of DNA-PK inhibition and radiation increases T1IFN expression and its downstream responses.

Figure 2.

DNA-PK inhibition and radiation synergistically promote T1IFN expression and signaling. A, GFP MFI of IFNβ1 promoter-reporter Panc1 cells at days 1, 2, and 3 after treatment with M3814 (100 nmol/L and 1 μmol/L) and/or radiation (8 Gy). Data represent three independent experiments with each performed in technical triplicate. B, qPCR for CXCL9 and CXCL10 in Panc1 cells at 3 days after treatment with radiation (8 Gy) and/or treatment M3814 (1 μmol/L). C, qPCR for Cxcl9, Cxcl10, Isg15, and Mx1 in KPC2 cells at 3 days after treatment with radiation (8 Gy) and/or treatment M3814 (1 μmol/L). Cell surface PD-L1 expression in Panc1 (D) and KPC2 (E) cells receiving the specified treatments. Three independent experiments; Statistical significance was determined using two-tailed, unpaired t tests. *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001. Ctrl, control; RT, radiotherapy.

Figure 2.

DNA-PK inhibition and radiation synergistically promote T1IFN expression and signaling. A, GFP MFI of IFNβ1 promoter-reporter Panc1 cells at days 1, 2, and 3 after treatment with M3814 (100 nmol/L and 1 μmol/L) and/or radiation (8 Gy). Data represent three independent experiments with each performed in technical triplicate. B, qPCR for CXCL9 and CXCL10 in Panc1 cells at 3 days after treatment with radiation (8 Gy) and/or treatment M3814 (1 μmol/L). C, qPCR for Cxcl9, Cxcl10, Isg15, and Mx1 in KPC2 cells at 3 days after treatment with radiation (8 Gy) and/or treatment M3814 (1 μmol/L). Cell surface PD-L1 expression in Panc1 (D) and KPC2 (E) cells receiving the specified treatments. Three independent experiments; Statistical significance was determined using two-tailed, unpaired t tests. *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001. Ctrl, control; RT, radiotherapy.

Close modal

Although T1IFN promotes innate and adaptive antitumor immunity, homeostatic negative feedback mechanisms act to temporally and spatially constrain this inflammatory response (7). PD-L1 upregulation following T1IFN signaling is an established inhibitory feedback mechanism. Given that PD-L1 is an IFN-response gene, and M3814 potentiates radiation-induced T1IFN production, we investigated whether the combination of M3814 with radiation would promote PD-L1 expression. We found that treatment with M3814 alone did not affect PD-L1 expression, while radiation increased cell surface PD-L1 levels in a time-dependent manner. The combination of M3814 and radiation significantly increased PD-L1 expression compared with radiation alone, demonstrating the synergistic effect in multiple human and mouse pancreatic cancer cell lines (Fig. 2D and E; Supplementary Fig. S2C). PD-L1 expression in response to radiation and M3814 treatment was preceded by T1IFN expression (Supplementary Fig. S2A). These results demonstrate that T1IFN produced in response to the combination of M3814 and radiation initially stimulates an immune response that is subsequently constrained by an immune inhibitory negative feedback response.

DNA-PK inhibition and radiation increase cytosolic dsDNA

We next investigated the mechanism by which M3814 enhances radiation-induced T1IFN signaling in pancreatic cancer cells. Formation of micronuclei in response to radiation, which is attributed to acentric chromosome fragments lost during anaphase/mitosis, is thought to promote T1IFN signaling (28, 41). Treatment with radiation alone, as expected, significantly increased micronucleus formation; however, combined treatment with M3814 and radiation resulted in a decreased frequency and number of micronuclei in both human and mouse pancreatic cancer cells (Fig. 3A–C; Supplementary Fig. S3A and S3B). These findings are consistent with a prior report demonstrating reduced micronuclei formation due to impaired mitotic progression (28). Indeed, treatment with M3814 enhanced radiation-induced G2 arrest, a finding consistent with the observed reduction in micronuclei given that mitotic progression is required for micronucleus generation (Supplementary Fig. S3C).

Figure 3.

DNA-PK inhibition and radiation increase cytosolic dsDNA. A, Representative DAPI IF of Panc1 cells at 3 days following treatment with M3814 (1 μmol/L) and/or radiation (8 Gy). Arrowheads highlight micronuclei. Frequency (B) and distribution (C) of micronucleated Panc1 cells (i.e., 0, 1–2, ≥3) treated as in (A); error bars represent the SD of two independent experiments. D, Representative dsDNA IF in BxPC3 cells at 24 hours following treatment with M3814 (1 μmol/L) and/or radiation (8 Gy). Representative micrographs show DAPI-stained nuclei, cytosolic DNA, mitochondria marker Tom20, and the three channels combined. Magnification, ×400. Double-stranded cytosolic DNA quantification in Panc1 (E) and KPC2 (F) cells at 24 hours following treatment with M3814 (1 μmol/L) and/or radiation (8 Gy). Data represent three or four independent experiments. Mean ± SD bars shown. Statistical significance was determined using two-tailed, unpaired t tests. *, P < 0.05, **, P < 0.01, ***, P < 0.001. Ctrl, control; RT, radiotherapy; cyto-dsDNA, cytosolic dsDNA.

Figure 3.

DNA-PK inhibition and radiation increase cytosolic dsDNA. A, Representative DAPI IF of Panc1 cells at 3 days following treatment with M3814 (1 μmol/L) and/or radiation (8 Gy). Arrowheads highlight micronuclei. Frequency (B) and distribution (C) of micronucleated Panc1 cells (i.e., 0, 1–2, ≥3) treated as in (A); error bars represent the SD of two independent experiments. D, Representative dsDNA IF in BxPC3 cells at 24 hours following treatment with M3814 (1 μmol/L) and/or radiation (8 Gy). Representative micrographs show DAPI-stained nuclei, cytosolic DNA, mitochondria marker Tom20, and the three channels combined. Magnification, ×400. Double-stranded cytosolic DNA quantification in Panc1 (E) and KPC2 (F) cells at 24 hours following treatment with M3814 (1 μmol/L) and/or radiation (8 Gy). Data represent three or four independent experiments. Mean ± SD bars shown. Statistical significance was determined using two-tailed, unpaired t tests. *, P < 0.05, **, P < 0.01, ***, P < 0.001. Ctrl, control; RT, radiotherapy; cyto-dsDNA, cytosolic dsDNA.

Close modal

We therefore sought to identify alternative ligands that trigger the T1IFN response induced by the combination of M3814 and radiation. Cytosolic dsDNA released in irradiated cells is also able to activate T1IFN signaling pathways (8, 42). Thus, we evaluated cytosolic dsDNA levels in pancreatic cancer cells following treatment with M3814 and/or radiation. Immunofluorescence staining revealed a significantly higher accumulation of cytosolic dsDNA at 24 hours following radiation and M3814 treatment relative to control or radiation alone (Fig. 3D). In line with this observation, we quantified cytosolic dsDNA in Panc1 and KPC2 cells, and found that combined M3814 and radiation significantly increased cytosolic dsDNA levels compared with radiation alone (Fig. 3E and F). Taken together, these findings demonstrate that the T1IFN response to combination treatment with radiation and M3814 is associated with an increase in cytosolic dsDNA but not micronuclei.

DNA-PK inhibition and radiation enhance IFN signaling in a TBK1-dependent, but cGAS- and STING-independent manner

Considering that M3814 and radiation treatment enhanced cytosolic dsDNA (Fig. 3), we investigated the molecular pathways regulating T1IFN expression. TBK1 is a key signaling hub downstream of multiple DNA sensors, which activates IRF3/7 and leads to T1IFN production and subsequent induction of IFN stimulated genes (43). We therefore generated TBK1-deficient Panc1 IFNβ1-GFP reporter cells using CRISPR/Cas9 (Supplementary Fig. S4A) and treated the cells with radiation, M3814, or the combination. TBK1 deletion partially inhibited IFNβ1 promoter activity following treatment with radiation. Furthermore, T1IFN promoter activity was significantly reduced following combination treatment of M3814 and radiation in TBK1-null cells (Fig. 4A). Consistent with this finding, TBK1 deletion limited the effects of combined treatment with M3814 and radiation on CXCL9 and CXCL10 expression (Fig. 4B and C), as well as the cell surface expression of PD-L1 (Fig. 4D). Similarly, we used TBK1-deficient mT4 cells (Supplementary Fig. S4B) and found that TBK1 deletion abrogated the cell surface expression of PD-L1 following combined treatment with M3814 and radiation as compared with radiation alone (Fig. 4E). Taken together, these findings demonstrate that TBK1 is required for T1IFN signaling following treatment with DNA-PK inhibitor and radiation.

Figure 4.

DNA-PK inhibition and radiation enhance interferon signaling in a TBK1-dependent, but cGAS- and STING-independent manner. A, GFP MFI of control (sgCtrl) or TBK1-deleted (sgTBK1) Panc1-IFNβ1 cells at 3 days after radiation (8 Gy) and/or treatment with M3814 (1 μmol/L). qPCR of CXCL9 (B), CXCL10 (C) in control or TBK1-deleted Panc1 cells 3 days after treatment with radiation (8 Gy) and/or M3814 (1 μmol/L). Cell surface PD-L1 of control or TBK1-deleted Panc1 cells (D) and mT4 cells (E) at 3 days following treatment with M3814 (1 μmol/L) and/or radiation (8 Gy). F, GFP MFI in indicated (sgCtrl, sgSTING, or sgcGAS) Panc1-IFNβ1 promotor reporter cells at 3 days following radiation (8 Gy) and/or M3814 (1 μmol/L). qPCR for IFNα2 (G), CXCL9 (H), CXCL10 (I) in indicated (sgCtrl, sgSTING, or sgcGAS) Panc1 cells 3 days after treatment with radiation (8 Gy) and/or M3814 (1 μmol/L). J, Cell surface PD-L1 in Panc1 cells (sgCtrl, sgSTING, or sgcGAS) at 3 days following treatment with M3814 (1 μmol/L) and/or radiation (8 Gy). Data represent three independent experiments. Mean ± SD bars shown. Statistical significance was determined using two-tailed, unpaired t tests. *, P < 0.05, **, P < 0.01, ***, P < 0.001. Ctrl, control; RT, radiotherapy; NS, not significant.

Figure 4.

DNA-PK inhibition and radiation enhance interferon signaling in a TBK1-dependent, but cGAS- and STING-independent manner. A, GFP MFI of control (sgCtrl) or TBK1-deleted (sgTBK1) Panc1-IFNβ1 cells at 3 days after radiation (8 Gy) and/or treatment with M3814 (1 μmol/L). qPCR of CXCL9 (B), CXCL10 (C) in control or TBK1-deleted Panc1 cells 3 days after treatment with radiation (8 Gy) and/or M3814 (1 μmol/L). Cell surface PD-L1 of control or TBK1-deleted Panc1 cells (D) and mT4 cells (E) at 3 days following treatment with M3814 (1 μmol/L) and/or radiation (8 Gy). F, GFP MFI in indicated (sgCtrl, sgSTING, or sgcGAS) Panc1-IFNβ1 promotor reporter cells at 3 days following radiation (8 Gy) and/or M3814 (1 μmol/L). qPCR for IFNα2 (G), CXCL9 (H), CXCL10 (I) in indicated (sgCtrl, sgSTING, or sgcGAS) Panc1 cells 3 days after treatment with radiation (8 Gy) and/or M3814 (1 μmol/L). J, Cell surface PD-L1 in Panc1 cells (sgCtrl, sgSTING, or sgcGAS) at 3 days following treatment with M3814 (1 μmol/L) and/or radiation (8 Gy). Data represent three independent experiments. Mean ± SD bars shown. Statistical significance was determined using two-tailed, unpaired t tests. *, P < 0.05, **, P < 0.01, ***, P < 0.001. Ctrl, control; RT, radiotherapy; NS, not significant.

Close modal

The cytosolic dsDNA sensor cGAS and its adaptor STING have been implicated in the activation of TBK1 in response to both radiation and DDR inhibition (44–46). Given that both cGAS and STING were strongly expressed in human and murine pancreatic cancer cells (20), we assessed their requirement in M3814 and radiation mediated T1IFN signaling. Surprisingly, neither cGAS nor STING deletion significantly affected IFNβ1 reporter activity in response to radiation alone or radiation plus M3814 (Supplementary Fig. S4C; Fig. 4F). Furthermore, genetic deletion of cGAS and STING in Panc1 cells failed to attenuate the increased IFNα2 mRNA levels following radiation or combined M3814 and radiation (Fig. 4G). In line with this, neither cGAS nor STING deletion affected enhanced transcription of CXCL9 or CXCL10 following radiation and M3814 treatment (Fig. 4H and I). cGAS and STING were also dispensable for cell surface expression of PD-L1 following the combination of M3814 and radiation (Fig. 4J). Similar findings were reproduced in mT4 cells depleted of cGAS or STING (Supplementary Fig. S4D–S4G). These data demonstrate that M3814 and radiation activate TBK1 and T1IFN signaling in a cGAS/STING-independent manner.

DNA-PK inhibition and radiation activate T1IFN signaling in a POL III/RIG-I/MAVS–dependent manner

Alternative innate immune DNA sensors upstream of TBK1 include POL III which converts cytosolic dsDNA into RNA (16). We therefore treated IFNβ1 promoter reporter Panc1 cells with M3814 and/or radiation in the presence or absence of ML-60218, a pharmacologic inhibitor of POL III. As expected, M3814 and radiation significantly enhanced T1IFN reporter activity compared with radiation alone (Fig. 5A). However, pharmacologic inhibition of POL III prevented the synergy between radiation and M3814 (Fig. 5A). Consistent with this finding, POL III inhibition blocked the synergistic increase in cell surface PD-L1 expression following treatment with M3814 and radiation in both human and murine pancreatic cancer cells (Fig. 5B and C). To solidify this finding, we used short hairpin RNA (shRNA) to silence POLR3A (the largest subunit of POL III) in Panc1 cells (Supplementary Fig. S5A). Consistent with POL III inhibition, we found that silencing of POLR3A abrogated the increase in cell surface PD-L1 by M3814 and radiation (Fig. 5D). We next examined whether POLR3A loss impacted other IFN stimulated genes by quantifying transcription of CXCL9 and CXCL10 in Panc1 cells and found that silencing of POLR3A diminished the synergistic induction of CXCL9 and CXCL10 by M3814 and radiation (Fig. 5E and F). These observations were also confirmed in Polr3a-depleted KPC2 cells (Supplementary Fig. S5B–S5D). Interestingly, knockdown of Polr3a augmented radiation induced Cxcl10 expression in KPC2 but not Panc1 cells, suggesting Polr3 may modulate responses to radiation in a cell-type–specific manner. Collectively, these data suggest that the combination of DNA-PK inhibition and radiation regulates T1IFN signaling in a POL III–dependent manner.

Figure 5.

DNA-PK inhibition and radiation activate T1IFN signaling in an RNA POL III–dependent manner. A, GFP MFI of IFNβ1 promoter reporter Panc1 cells at 3 days after radiation (8 Gy) and/or M3814 (1 μmol/L) treatment in the absence or presence of the POL lII inhibitor ML-60218 (20 μmol/L per day for 3 days). PD-L1 MFI at 3 days in Panc1 cells (B) and KPC2 cells (C) after radiation (8 Gy) and/or treatment with M3814 (1 μmol/L) with or without POL III inhibitor ML-60218 (20μmol/L per day for 3 days). D, PD-L1 MFI in control or POLR3A shRNA silenced Panc1 cells treated with radiation (8 Gy) and/or M3814 (1 μmol/L). E and F, qPCR for CXCL9 and CXCL10 mRNA expression in control or POLR3A-silenced Panc1 cells 3 days after radiation (8 Gy) and/or treatment with M3814 (1 μmol/L; n = 3). G and H, qPCR for CXCL9 and CXCL10 in the indicated Panc1 cells (control, shRIG-I, or shMAVS) 3 days after radiation (8 Gy) and/or treatment with M3814 (1 μmol/L; n = 3). I, PD-L1 MFI in indicated Panc1 cells (control, shRIG-I, or shMAVS) 3 days after treatment with radiation (8 Gy) and/or M3814 (1 μmol/L). Mean ± SD bars shown. Statistical significance was determined using two-tailed, unpaired t tests. *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001. Ctrl, control, RT, radiotherapy; NS, not significant; d, days.

Figure 5.

DNA-PK inhibition and radiation activate T1IFN signaling in an RNA POL III–dependent manner. A, GFP MFI of IFNβ1 promoter reporter Panc1 cells at 3 days after radiation (8 Gy) and/or M3814 (1 μmol/L) treatment in the absence or presence of the POL lII inhibitor ML-60218 (20 μmol/L per day for 3 days). PD-L1 MFI at 3 days in Panc1 cells (B) and KPC2 cells (C) after radiation (8 Gy) and/or treatment with M3814 (1 μmol/L) with or without POL III inhibitor ML-60218 (20μmol/L per day for 3 days). D, PD-L1 MFI in control or POLR3A shRNA silenced Panc1 cells treated with radiation (8 Gy) and/or M3814 (1 μmol/L). E and F, qPCR for CXCL9 and CXCL10 mRNA expression in control or POLR3A-silenced Panc1 cells 3 days after radiation (8 Gy) and/or treatment with M3814 (1 μmol/L; n = 3). G and H, qPCR for CXCL9 and CXCL10 in the indicated Panc1 cells (control, shRIG-I, or shMAVS) 3 days after radiation (8 Gy) and/or treatment with M3814 (1 μmol/L; n = 3). I, PD-L1 MFI in indicated Panc1 cells (control, shRIG-I, or shMAVS) 3 days after treatment with radiation (8 Gy) and/or M3814 (1 μmol/L). Mean ± SD bars shown. Statistical significance was determined using two-tailed, unpaired t tests. *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001. Ctrl, control, RT, radiotherapy; NS, not significant; d, days.

Close modal

RIG-I and MAVS have previously been implicated in POL III–mediated induction of T1IFN signaling (16, 47). To examine the hypothesis that DNA-PK inhibition and radiation activate IFN signaling through RIG-I and MAVS, we silenced these genes in Panc1 cells (Supplementary Fig. S5E). Knockdown of either RIG-I or MAVS prevented induction of CXCL9 and CXCL10 following treatment with M3814 and radiation (Fig. 5G and H). Furthermore, silencing of RIG-I or MAVS diminished cell surface expression of PD-L1 following treatment with M3814 and radiation (Fig. 5I), which is reminiscent of the effects of POL III inhibitor or POLR3A depletion. To confirm this finding, we conducted similar experiments in Rig-I and Mavs-depleted KPC2 cells (Supplementary Fig. S5F). Again, we found diminished Cxcl10 and Mx1 expression, as well as cell surface PD-L1 protein levels in response to radiation and M3814 treatment (Supplementary Fig. S5G–S5I). Taken together, these data suggest that DNA-PK inhibition and radiation regulate IFN signaling in a POL III/RIG-I/MAVS axis–dependent manner.

Recent findings suggest that the contribution of the cGAS/STING and RIG-I/MAVS pathway in radiation-induced T1IFN signaling is dependent on cell type and/or genetic background (22, 48). Human and mouse breast cancer cells MDA-MB-468 and 4T1 require the RIG-I/MAVS and cGAS/STING pathways to mediate radiation-induced T1IFN signaling, respectively (22). Consistent with this finding, induction of the T1IFN signaling pathway by radiation or radiation plus M3814 required STING signaling in 4T1 cells but not in MDA-MB-468 cells (Supplementary Fig. S5J). Furthermore, knockout of STING in mouse squamous cell carcinoma cells (NOOC1), but not human breast cancer cells (MDA-MB-231), significantly inhibited PD-L1 expression (Supplementary Fig. S5J). Taken together, our data support that the relative contributions of these two pathways in T1IFN signaling are cell-type–specific, and that RIG-I/MAVS signaling is the predominant mediator of radiation and M3814-induced T1IFN signaling in pancreatic cancer cells.

Immune checkpoint inhibition maximizes the efficacy of DNA-PK inhibition and radiation in pancreatic cancer tumors

Although combined treatment with M3814 and radiation promotes robust immune stimulation through T1IFN signaling, it also potentiates the activation of homeostatic negative feedback mechanisms such as adaptive upregulation of PD-L1 in pancreatic tumor cells (Fig. 2). We also observed radiation plus M3814 synergistically elevated cell surface PD-L1 levels in the parental MDA-MB-231, MDA-MB-468, 4T1, and NOOC1 cells (Supplementary Fig. S6A). Therefore, we hypothesized that combining anti–PD-L1 with DNA-PK inhibition and radiation would maximize tumor control in vivo. To test this, we established mT4 tumors in immunocompetent syngeneic mice and treated them with the combination of M3814, radiation, and anti–PD-L1 (Fig. 6A). A moderate dose of radiotherapy was used to permit observation of interactions between treatments. Single-agent treatment with M3814, radiation, or anti–PD-L1 had no significant antitumor activity (Fig. 6B and C). The combination of M3814 or anti-PD-L1 with radiation had minimal effects on tumor growth. Importantly, the combination of M3814, radiation, and anti–PD-L1 significantly inhibited tumor growth as compared with the other treatment groups (Fig. 6B and C). Consistent with these findings, the time until tumor volume doubling of the M3814, radiation, and anti–PD-L1–treated group was significantly prolonged compared with all other treatment groups (Fig. 6D). Notably, M3814 alone, or in combination with radiation and anti–PD-L1 therapy, did not adversely affect mouse weight (Supplementary Fig. S6B). In addition, we investigated the effects of combined therapy on the frequency of tumor-infiltrating CD4+, CD8+, and Granzyme B+ cells. The combination of M3814, radiation and anti–PD-L1 caused a significant increase in the number of CD4+, CD8+, and Granzyme B+ cells compared with radiation alone or radiation with M3814 (Supplementary Fig. S6C). Taken together, these results demonstrate that anti–PD-L1 therapy improves the antitumoral immune response induced by M3814 and radiation in otherwise poorly immunogenic pancreatic cancer.

Figure 6.

Immune checkpoint inhibition maximizes the efficacy of DNA-PK inhibition and radiation in pancreatic tumors. A, Schematic showing schedules of the DNA-PK inhibitor M3814, radiation, and αPD-L1 antibody treatment. M3814 (100 mg/kg) was orally administered approximately 1 hour before radiation (5 Gy) on day 0 as well as on days 1 to 4 and 7 to 11. Mouse αPD-L1 antibody (100 μg/mL) was intraperitoneally injected every 3 days. mT4 tumor growth following treatment with M3814, radiation, and/or αPD-L1. Data represent mean tumor volumes ± SD (B), individual tumor volumes with insets (n/n) providing the number of tumors with durable control/the number of total tumors (C), or tumor volume doubling time (D). n per arm (mice) = 12 (control), 16 (M3814), 14 (radiotherapy), 18 (M3814 + radiotherapy), 14 (αPD-L1), 16 (αPD-L1 + M3814), 14 (αPD-L1 + radiotherapy), and 20 (αPD-L1 + M3814 + radiotherapy). ****, P < 0.0001 (αPD-L1 + M3814 + radiotherapy vs. M3814 + radiotherapy), unpaired, two-way ANOVA. E, Model for POL III/RIG-I/MAVS/TBK1 pathway activation in response to radiation plus DNA-PK inhibition in pancreatic cancer cells. Inhibition of DNA-PK with the small-molecule inhibitor M3814 leads to increased cytosolic dsDNA in pancreatic cells which is recognized by POL III rather than cGAS, leading to activation of the RIG-I/MAVS/TBK1 pathway and subsequent expression of IFNβ and interferon stimulated genes such as CXCL9 and CXCL10 chemokines. IFNβ expression also promotes adaptive upregulation of PD-L1 which is therapeutically targeted with αPD-L1 antibody leading to enhanced antitumor immunity in pancreatic cancer models. Q3D, every 3 days.

Figure 6.

Immune checkpoint inhibition maximizes the efficacy of DNA-PK inhibition and radiation in pancreatic tumors. A, Schematic showing schedules of the DNA-PK inhibitor M3814, radiation, and αPD-L1 antibody treatment. M3814 (100 mg/kg) was orally administered approximately 1 hour before radiation (5 Gy) on day 0 as well as on days 1 to 4 and 7 to 11. Mouse αPD-L1 antibody (100 μg/mL) was intraperitoneally injected every 3 days. mT4 tumor growth following treatment with M3814, radiation, and/or αPD-L1. Data represent mean tumor volumes ± SD (B), individual tumor volumes with insets (n/n) providing the number of tumors with durable control/the number of total tumors (C), or tumor volume doubling time (D). n per arm (mice) = 12 (control), 16 (M3814), 14 (radiotherapy), 18 (M3814 + radiotherapy), 14 (αPD-L1), 16 (αPD-L1 + M3814), 14 (αPD-L1 + radiotherapy), and 20 (αPD-L1 + M3814 + radiotherapy). ****, P < 0.0001 (αPD-L1 + M3814 + radiotherapy vs. M3814 + radiotherapy), unpaired, two-way ANOVA. E, Model for POL III/RIG-I/MAVS/TBK1 pathway activation in response to radiation plus DNA-PK inhibition in pancreatic cancer cells. Inhibition of DNA-PK with the small-molecule inhibitor M3814 leads to increased cytosolic dsDNA in pancreatic cells which is recognized by POL III rather than cGAS, leading to activation of the RIG-I/MAVS/TBK1 pathway and subsequent expression of IFNβ and interferon stimulated genes such as CXCL9 and CXCL10 chemokines. IFNβ expression also promotes adaptive upregulation of PD-L1 which is therapeutically targeted with αPD-L1 antibody leading to enhanced antitumor immunity in pancreatic cancer models. Q3D, every 3 days.

Close modal

In this study, we found that the therapeutic efficacy of DNA-PK inhibition with radiation is dependent on an intact immune system. The immune-dependent therapeutic effects of this combination involve potentiation of radiation-induced T1IFN responses by DNA-PK inhibition. Furthermore, we demonstrate that the induction of T1IFN signaling by DNA-PK inhibitor and radiation occurs through a cGAS-/STING-independent but POL III–, RIG-I–, MAVS-, and TBK1-dependent mechanism (Fig. 6E). This results in both upregulation of inflammatory T1IFN signaling as well as immune checkpoints including PD-L1. We demonstrate that these mechanisms can be therapeutically leveraged by simultaneously inducing inflammatory T1IFN signaling with M3814 and radiation and inhibiting immunosuppressive signaling with a PD-L1 blocking antibody. This work nominates a novel combinatorial approach to increase the immunogenicity of pancreatic cancer.

Previous studies have evaluated targeting the DDR to enhance the immunostimulatory effects of radiation. These studies have found that multiple DDR inhibitors synergize with radiation by increasing micronuclei and cytosolic DNA which can agonize cytosolic innate immune sensing pathways (8, 28). It is unclear which specific DDR targets will be most effective in stimulating T1IFN driven antitumor immune responses either alone or in conjunction with radiation. Genetic and pharmacologic inhibition of homologous recombination stimulates antitumor immune responses alone and in conjunction with radiotherapy (49). There are conflicting reports regarding the immunogenicity of NHEJ inhibition. Recent reports suggested that NHEJ inhibition through XRCC4 knockout suppresses interferon signaling (29), but other studies have suggested that XRCC4 is required for IFN signaling (50). Pharmacologic inhibition of DNA-PK, a critical mediator of NHEJ, can in conjunction with radiation inducing T1IFN responses in colorectal cancer and melanoma models (30). Our work confirms and mechanistically extends studies suggesting that DNA-PK inhibition and radiation coordinate to promote T1IFN responses.

Mechanistically, we identified that the combination of DNA-PK inhibitor and radiation did not utilize the DNA sensing cGAS/STING pathway to induce T1IFN in pancreatic cancer. This extends recent reports that p53 mutations in pancreatic cancer restrain cGAS/STING signaling (51). DNA viruses can activate the RNA sensor RIG-I through POL III–mediated conversion of dsDNA to RNA (29, 52). Radiotherapy activates multiple innate immune pattern recognition receptors including cGAS and RIG-I (8, 28, 53, 54). Our work is consistent with the recent finding that RIG-I/MAVS mediates T1IFN signaling following radiation in pancreatic tumor cell models (22). Similarly, we also identified that POL III, RIG-I, and MAVS were required for T1IFN induction following treatment with DNA-PK inhibitor and radiation in multiple human and mouse pancreatic cancer cells. POL III has been shown to convert AT-rich DNAs to RNA which initiates T1IFN signaling in a MAVS-dependent manner (47). Future studies are needed to determine if M3814 augments radiation-induced production of AT-rich DNA fragments which activate T1IFN signaling through this pathway. Our finding is of significance in that it extends the mechanism of recent studies showing that DNA-PK inhibition and radiation induce T1IFN. Specifically, we show that RNA POL III, RIG-I, and MAVS are required for T1IFN responses to combined therapy with DNA-PK inhibition with radiation. In contrast, radiation-induced T1IFN signaling required RIG-I and MAVS but not RNA POL III. Radiation causes cytoplasmic accumulation of mitochondrial RNA, which triggers an RNA POL III–independent, but RIG-I–MAVS–dependent immune response (54). Future studies are also needed to understand whether the combination of radiation and M3814 relies on mitochondrial or nuclear damage to modulate T1IFN signaling.

We found that an intact immune system was required for the therapeutic efficacy of combined treatment with DNA-PK inhibition and radiation. This confirms recent reports that CD8+ T cells are required for the therapeutic efficacy of combined treatment with DNA-PK inhibitor and radiation (30). While combination therapy was more efficacious in immune competent animals, its antitumoral effects were restrained by T1IFN-driven adaptive PD-L1 expression and thus maximized by treatment with anti–PD-L1 therapy. This underscores the need for combinatorial therapeutic strategies for poorly immunogenic tumors, such as pancreatic cancer, in contrast to highly immunogenic tumors which are responsive to treatment with DNA-PK inhibitor and radiation (30). These studies have important implications for the design of future clinical trials as well as the interpretation of several ongoing early-stage clinical trials evaluating radiation in combination with M3814 in solid tumors (e.g., NCT03770689, NCT04068194) including pancreatic cancer (NCT04172532), as well as in combination with immunotherapy (NCT03724890). The results of our study imply that poorly immunogenic cancers such as pancreatic adenocarcinoma will require a combined treatment strategy with DNA-PK inhibition, radiation, and immune checkpoint inhibition to achieve effective antitumoral immune responses.

In summary, this study demonstrates a novel mechanism whereby combining DNA-PK inhibition with radiation potentiates IFN signaling in pancreatic cancer cells through a cGAS- and STING-independent, but POL III–, RIG-I–, MAVS-, and TBK1-dependent mechanism. These findings support the development of a combined therapeutic approach consisting of DNA-PK inhibition, radiation, and immune checkpoint inhibition to achieve maximal antitumoral immune responses in otherwise poorly immunogenic pancreatic cancer.

M.T. McMillan reports grants from Alpha Omega Alpha Carolyn L Kuckein Student Research Fellowship during the conduct of the study. L.A. Parsels reports grants from NIH during the conduct of the study; and grants from NIH outside the submitted work. M.A. Morgan reports grants from NIH during the conduct of the study; and grants and personal fees from AstraZeneca outside the submitted work. M.D. Green reports grants from NCI during the conduct of the study. No disclosures were reported by the other authors.

W. Wang: Formal analysis, validation, methodology, writing–original draft. M.T. McMillan: Formal analysis, validation, investigation, methodology, writing–original draft, writing–review and editing. X. Zhao: Formal analysis, validation, investigation. Z. Wang: Formal analysis, validation. L. Jiang: Investigation, methodology. D. Karnak: Resources. F. Lima: Formal analysis, investigation, methodology. J.D. Parsels: Formal analysis, validation, investigation. L.A. Parsels: Validation, methodology. T.S. Lawrence: Conceptualization, supervision, funding acquisition, writing–review and editing. T.L. Frankel: Supervision, investigation. M.A. Morgan: Conceptualization, resources, supervision, funding acquisition, writing–original draft, writing–review and editing. M.D. Green: Conceptualization, formal analysis, funding acquisition, writing–original draft, writing–review and editing. Q. Zhang: Conceptualization, resources, formal analysis, supervision, validation, investigation, methodology, writing–original draft, writing–review and editing.

This work was supported by R01CA240515 (to M.A. Morgan); U01CA216449 (to T.S. Lawrence); P30CA046592, an Alpha Omega Alpha Carolyn L. Kuckein Student Research Fellowship (to M.T. McMillan); I01 BX005267 (to M.D. Green); R21CA252010 (to M.D. Green) and R50CA251960 (to L.A. Parsels).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data