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The tumor suppressor kinase DAPK3 drives tumor-intrinsic immunity through the STING–IFN-β pathway

Abstract

Evasion of host immunity is a hallmark of cancer; however, mechanisms linking oncogenic mutations and immune escape are incompletely understood. Through loss-of-function screening of 1,001 tumor suppressor genes, we identified death-associated protein kinase 3 (DAPK3) as a previously unrecognized driver of anti-tumor immunity through the stimulator of interferon genes (STING) pathway of cytosolic DNA sensing. Loss of DAPK3 expression or kinase activity impaired STING activation and interferon (IFN)-β-stimulated gene induction. DAPK3 deficiency in IFN-β-producing tumors drove rapid growth and reduced infiltration of CD103+CD8α+ dendritic cells and cytotoxic lymphocytes, attenuating the response to cancer chemo-immunotherapy. Mechanistically, DAPK3 coordinated post-translational modification of STING. In unstimulated cells, DAPK3 inhibited STING K48-linked poly-ubiquitination and proteasome-mediated degradation. After cGAMP stimulation, DAPK3 was required for STING K63-linked poly-ubiquitination and STING–TANK-binding kinase 1 interaction. Comprehensive phospho-proteomics uncovered a DAPK3-specific phospho-site on the E3 ligase LMO7, critical for LMO7–STING interaction and STING K63-linked poly-ubiquitination. Thus, DAPK3 is an essential kinase for STING activation that drives tumor-intrinsic innate immunity and tumor immune surveillance.

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Fig. 1: DAPK3 regulates DNA-stimulated STING signaling.
Fig. 2: Tumor-expressed DAPK3 shapes immune surveillance.
Fig. 3: DAPK3 regulates response to cancer chemo-immunotherapy.
Fig. 4: DAPK3 inhibits STING K48-linked poly-ubiquitination.
Fig. 5: DAPK3 promotes STING K63-linked poly-ubiquitination.
Fig. 6: Phospho-proteomic profiling uncovers DAPK3 targets.
Fig. 7: DAPK3 phosphorylation of LMO7 is necessary for STING K63-linked poly-ubiquitination.

Data availability

Screening results are presented in Supplementary Table 1 and phospho-proteomics results in Supplementary Table 2. Mass spectrometry proteome and phosphoproteome data were deposited in MassIVE (identifier PXD023639) and ProteomeXchange (identifier PXD023637). Uncropped immunoblot images are provided in the manuscript. Source data are provided with this paper. Additional data will be made available from the corresponding author upon reasonable request.

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Acknowledgements

We thank J. Huang (LJI) for support for the HUVEC siRNA screen; S. Shresta (LJI) for Ifnar1-null mice; C. Benedict (LJI) for MEFs, NHDFs and hCMV; N. P. Restifo (NCI) for MCA205; J. Schlom (NCI) for MC38; S. Schoenberger (LJI) for B16F10; C. C. Hedrick (LJI) for LLC-RFP; Y. C. Liu (LJI) for pEF-neo-HA-Ub; D. Zajonc (LJI) for pGEX-4T-2; J. Day and K. Tanguay (LJI) for shRNA and siRNA constructs, and siRNA screen support; D. Freeman, B. McDonald and R. El Morabiti (LJI) for hCMV propagation; M. Diep, K. Foos and M. Kaur (LJI) for technical assistance; Z. Mikulski (LJI Microscopy Core Facility) for confocal microscopy support; A. Sethi (LJI Bioinformatics Core Facility) for pathway analysis support; LJI Flow Cytometry Core Facility for cell sorting (FACSAria-II Cell Sorter; supported by the Shared Instrumentation Grant (SIG) program no. S10 RR027366); and D. Araujo (LJI) for proofreading the manuscript. This work was supported by National Institutes of Health (NIH) grant nos. R01CA199376, U01DE028227 and U54CA260591 (S.S.); and NIH grant no. S10OD020025 and grant no. R01ES027595 (M.J.). C.-W.J.L. was supported by a Cancer Research Institute (CRI) Irvington Postdoc Fellowship. A.C. was supported by the UCSD Microbial Sciences Initiative Graduate Research Fellowship and by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology, through an institutional training grant from the National Institute of General Medical Sciences, grant no. T32 GM007752.

Author information

Affiliations

Authors

Contributions

M.T. designed, optimized and performed in vitro and in vivo experiments and the siRNA screen in THP1-Blue ISG cells. C.-W.J.L. optimized and performed the siRNA screen in HUVECs and generated L929 reporter cells. A.C. optimized and performed phospho-proteomics in THP1-Blue ISG cells under the supervision of D.J.G. M.S. optimized and performed mass spectrometry analysis in HEK293T and L929 lysates and supported phospho-proteomics under the supervision of M.M. F.A. provided support for bioinformatics analyses. M.J. provided support for mass spectrometry studies. S.S. provided overall direction and supervision. M.T. and S.S. wrote the manuscript with input from co-authors.

Corresponding author

Correspondence to Sonia Sharma.

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The authors declare no competing interests.

Additional information

Peer review information Nature Immunology thanks Katherine A. Fitzgerald, Hong-Bing Shu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 DAPK3 is a positive regulator of STING signaling and some TLR pathways.

a, Schematic representation of the RNAi screen for IRF3 nuclear translocation. b, Immunostaining of IRF3 in HUVEC stimulated with poly (dA:dT) (1 μg/ml) (right panel) for 3 h. Scale bar, 100 μm. c, d, qRT-PCR of DAPK1/Dapk1, DAPK2/Dapk2, and DAPK3/Dapk3 in (c) human and (d) mouse cell lines. #, Not detected. e, Images of IRF3 localization in L929-mRuby-hIRF3 stimulated with poly (dA;dT) (1 μg/ml) for 3 h. Scale bar, 100 μm. f, g, (f) qRT-PCR of IFNB1 and (g) immunoblot of HUVEC transfected with indicated siRNA. h, i, (h) qRT-PCR of Ifnb1 and (i) immunoblot of BMDM transfected with indicated siRNA. (f, h) Cells were stimulated with poly (dA:dT) (0.2 μg/ml) for 4 h. j, k, (j) qRT-PCR of Il6 in L929-mRuby-hIRF3 and (k) IL6 inTHP1-Blue ISG transduced with indicated shRNA and stimulated as indicated in Fig. 1g-l. l, m, qRT-PCR of (l) Ifnb1 in L929-mRuby-hIRF3 and (m) IFNB1 inTHP1-Blue ISG transduced with indicated shRNA and transfected with poly (I:C)(LMW) and poly (I:C)(HMW)(0.1 μg/ml for L929-mRuby-hIRF3, 0.5 μg/ml for THP1-Blue ISG). n, o, qRT-PCR of (n) IFNB1 and (o) IL6 in THP1-Blue ISG transduced with indicated shRNA and stimulated with FSL-1(100 ng/ml), naked poly (I:C)(LMW)(10 μg/ml), or LPS(100 ng/ml) for 4 h. p, q, Immunoblot of (p) THP1-Blue ISG and (q) L929-mRuby-hIRF3 transduced with indicated shRNA. Data in (b-e, g, i, p, q) are representative or (f, h, j-o) the mean of three independent experiments. Values represent mean ± s.d. *P < 0.05, **P < 0.01, and ***P < 0.001. Statistical comparisons were conducted using two-tailed t-test (f, h, j-o).

Source data

Extended Data Fig. 2 Association of DAPK3 with outcomes in human cancer.

Kaplan-Meier survival analysis of pancreatic adenocarcinoma, uterine corpus endometrial carcinoma, and esophageal carcinoma comparing the top (high) and bottom (low) tertiles of patients with respect to DAPK3 expression levels as reported by TCGA data portal. Statistical comparisons were conducted using two-sided log-rank test58.

Extended Data Fig. 3 Teniposide and paclitaxel induce micronuclei formation and anti-tumor immunity to B16F10 tumors in a type I IFN signaling-dependent manner.

a, b, (Left) Immunoblot and (right) in vitro cell growth of (a) MCA205 and (b) B16F10 transduced with indicated shRNA. c, Flow cytometry of tumor-infiltrating CD8+T cells and CD103+CD8α+DCs in MCA205 tumor suspensions isolated from WT or Ifnar1-KO mice on Day 6 (n = 6 per group). d, Confocal fluorescence microscopy of MCA205 stably expressing cGAS-Clover. Scale bar, 10 μm. e, qRT-PCR of Ifnb1 in unstimulated MCA205 and B16F10 transduced with indicated shRNA. f, (Left) Immunoblot and (right) qRT-PCR of Ifnb1 in shDapk3#1-transduced MCA205 ectopically expressing V5-tagged DAPK3(WT) or DAPK3(D161A). Cells were stimulated with 2′,3′-cGAMP, 3′,3′-cGAMP, c-di-GMP (20 μg/ml for all three agonists) or DMXAA (50 μg/ml) for 4 h. g, Confocal fluorescence microscopy of B16F10 stably expressing cGAS-Clover. Cells were treated with teniposide (10 μM) for 24 h or paclitaxel (100 nM) for 72 h. Scale bar, 10 μm. h, (Left) Apoptosis measured in shRNA-transduced B16F10 treated with teniposide (10 μM) for 24 h or (right) paclitaxel (100 nM) for 48 h. i, Tumor volume of B16F10 subcutaneously transplanted into WT and Ifnar1-KO mice and treated with teniposide or paclitaxel (n = 6 for vehicle, n = 7 for teniposide and paclitaxel). Tumor size on Day 15 is represented (right panel). Data are representative (a-d, g-i) or the mean (a, b, e, f) of three independent experiments. Values represented mean ± s.d. *P < 0.05, **P < 0.01, and ***P < 0.001. Statistical comparisons were conducted using two-tailed t-test (a-c, e, f, h, i).

Source data

Extended Data Fig. 4 Flow cytometry gating strategy for tumor-infiltrating leukocytes.

Tumor single cell suspensions were stained with different fluorophore-conjugated antibodies and analyzed by flow cytometry.

Extended Data Fig. 5 DAPK3 does not directly phosphorylate STING or TBK1.

a-d, (a) qRT-PCR of Sting1 and Dapk3, (b) immunoblot, (c) IRF3 nuclear translocation, and (d) p65 nuclear translocation in L929-mRuby-hIRF3 transduced with indicated shRNA stimulated with poly (dA:dT) (0.5 μg/ml) or VACV70 (2 μg/ml) for 3 h. e, Immunoblot of L929-mRuby-hIRF3 transfected with indicated siRNA. f, Immunoblot of L929-mRuby-hIRF3 transduced with indicated shRNA stimulated with VACV70 (2 μg/ml) for 2 h and 4 h. g, Immunoblot of HUVEC stably expressing V5-tagged DAPK3(D161A), DAPK3(T180A), or luciferase. Cells were infected at MOI = 5, 2, or 1. h, Immunoblot of THP1-Blue ISG stably expressing V5-tagged DAPK3(WT) or DAPK3(D161A). i, qRT-PCR of Ifnb1 in L929-mRuby-hIRF3 pre-treated with DAPK inhibitors for 3 h prior to 2′,3′-cGAMP stimulation (10 μg/ml) for 4 h. j, qRT-PCR of IFNB1 in THP1-Blue ISG pre-treated with DAPK inhibitors (50 μM) for 6 h prior to 2′,3′-cGAMP or c-di-GMP stimulation (10 μg/ml for both) for 4 h. k, l, In vitro kinase assay of (k) GST-tagged human STING C-terminus (aa 149-379) and (l) GST-tagged human TBK1(K38M). Peptides were incubated with GST-tagged DAPK3 or TBK1 in the presence of [γ-32P] ATP. Data in (b, e-h, k, l) are representative or (a, c, d, i, j) mean of three independent experiments. Values represent mean ± s.d. *P < 0.05, **P < 0.01, and ***P < 0.001. Statistical comparisons were conducted using two-tailed t-test (a, c, d, i, j).

Source data

Extended Data Fig. 6 DAPK3 is not involved in STING trafficking from ER to Golgi.

a, Confocal fluorescence microscopy of THP1-Blue ISG transduced with indicated shRNA and unstimulated or stimulated with 2′,3′-cGAMP (25 μg/ml) for 3 h. Scale bar, 15 μm. b, (Upper) Co-localization of STING/Calreticulin and (lower) STING/GM130 analyzed using Image J software. Data are pooled from three independent experiments (n > 1,500 cells for unstimulated 32 images and cGAMP-stimulated 73 images). c, (Upper) Confocal fluorescence microscopy of THP1-Blue ISG stably expressing GFP-tagged DAPK3(WT) unstimulated or stimulated with 2′,3′-cGAMP (50 μg/ml) for 3 h. Localization of GFP-DAPK3, STING, and TBK1. (Lower) Co-localization of GFP-DAPK3/TBK1, GFP-DAPK3/STING, and TBK1/STING was analyzed using Image J software. Data are pooled from three independent experiments (n > 1,500 cells for unstimulated and cGAMP-stimulated 70 images). Scale bars, 15 μm. d, Schematic representation of human STING mutants. e, (Upper) Immunoprecipitation and immunoblot of HEK293T transfected with plasmid encoding HA-tagged human STING (WT, 1-379), phospho-deficient mutant (3S-3A), or C-terminal deletion mutant (aa 1-340) unstimulated or stimulated with 2′,3′-cGAMP (5 μg/ml) for 2 h, and (lower) immunoblot of whole cell lysates (WCL). Values represented as mean ± s.d. Data in (a, c, e) are representative of three independent experiments.

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Extended Data Fig. 7 Phosphorylation of TRIP12 on S312 or TRIM56 on T442 are not involved in STING K63-linked poly ubiquitination.

a, Primary RNAi screen of E3 ligases in THP1-Blue ISG transfected with indicated siRNA. SEAP activity was measured after normalization with CellTiter-Glo. Black; siControl, Blue; previously reported E3 ligases for K63-linked poly-ubiquitination of STING, Red; positive control (for example siSTING1 and siTBK1). siTRIM56 value was used for determining cut-off. b, Secondary RNAi screen of E3 ligases in THP1-Blue ISG transfected with indicated siRNA. qRT-PCR of IFNB1 was performed. #; candidates for subsequent analysis. c, d, In vitro kinase assay of (c) GST-tagged human TRIP12 peptide (aa 260-360) and (d) GST-tagged human TRIM56 peptide (aa 400-500). Peptides were incubated with GST-tagged DAPK3 or TBK1 in the presence of [γ-32P] ATP. e, Schematic representation of human TRIP12 mutants. f, (Upper) Immunoprecipitation and immunoblot of HEK293T transfected with plasmids encoding HA-tagged human STING and V5-tagged human TRIP12 (WT) or phospho-deficient TRIP12 (S312A), and (lower) immunoblot of whole cell lysates (WCL). g, (Upper) Immunoprecipitation and immunoblot of HEK293T transfected with plasmids encoding 3×Flag-tagged human STING, HA-tagged Ub(K63O), and V5-tagged human TRIP12(WT), phospho-deficient TRIP12(S312A), or HECT domain-deficient TRIP12(ΔHECT), and (lower) immunoblot of WCL. h, (Upper) Immunoprecipitation and immunoblot of HEK293T transfected with plasmids encoding 3×Flag-tagged human STING, HA-tagged Ub(K63O), and V5-tagged human TRIM56(WT), phospho-deficient TRIM56(T442A), or enzyme-inactive TRIM56(C24S), and (lower) immunoblot of WCL. Data in (c, d, f-h) are representative of three independent experiments. Values represent mean ± s.d. *P < 0.05, **P < 0.01, and ***P < 0.001 (compared to siControl) (a, b). Statistical comparisons were conducted using two-tailed t-test (a, b).

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Extended Data Fig. 8 DAPK3, LMO7, and TRIP12 are highly mutated in human cancers.

a-c, Genomic alterations of (a) DAPK3, (b) LMO7 and (c) TRIP12 in human cancers from cBioportal.

Extended Data Fig. 9 LMO7 and TRIP12 are positive regulators of STING-IFNβ signaling in THP1 and HUVEC.

a, In vitro kinase assay of GST-tagged human LMO7 (aa 360-460). Peptides were incubated with GST-tagged DAPK3 or TBK1 in the presence of [γ-32P] ATP. b, (Upper) Immunoprecipitation and immunoblot of HEK293T transduced with indicated shRNA prior to transfection with plasmids encoding 3×Flag-tagged human STING, HA-tagged Ub(K63O), and V5-tagged human LMO7(WT) and (lower) immunoblot of whole cell lysates (WCL). c, (Upper) Immunoblot of THP1-Blue ISG transduced with two distinct shLMO7 or (lower) shTRIP12 sequences. d, (Upper) Immunoprecipitation and immunoblot of THP1-Blue ISG transduced with indicated shRNA and stimulated with 2′,3′-cGAMP (10 μg/ml) for 3 h and 6 h, and (lower) immunoblot of WCL. e, f, Immunoblot of THP1-Blue ISG transduced with two distinct (e) shLMO7 or (f) shTRIP12 sequences and stimulated with 2′,3′-cGAMP (10 μg/ml) for 3 h and 6 h. g, h, Immunoblot of (g) THP1-Blue ISG and (h) HUVEC transfected with indicated siRNA. i, j, qRT-PCR of IFNB1 and CXCL10 in (i) THP1-Blue ISG and (j) HUVEC transfected with indicated siRNA stimulated with VACV70 (2 μg/ml), 2′,3′-cGAMP (10 μg/ml), and c-di-GMP (10 μg/ml). Data in (a-h) are representative or (i, j) mean of three independent experiments. Values represent mean ± s.d. *P < 0.05, **P < 0.01, and ***P < 0.001. Statistical comparisons were conducted using two-tailed t-test (i, j).

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Extended Data Fig. 10 Schematic model of the DAPK3-STING axis.

In unstimulated cells (L929 and MCA205), DAPK3 maintains steady-state STING levels by inhibiting STING K48-linked poly-ubiquitination and proteasome-mediated degradation. In DNA-stimulated cells (THP1), DAPK3 promotes STING activation by phosphorylating the E3 ligase LMO7 at S863, enabling LMO7-STING interaction, STING K63-linked poly-ubiquitination, and recruitment of TBK1.

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Takahashi, M., Lio, CW.J., Campeau, A. et al. The tumor suppressor kinase DAPK3 drives tumor-intrinsic immunity through the STING–IFN-β pathway. Nat Immunol 22, 485–496 (2021). https://doi.org/10.1038/s41590-021-00896-3

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