Abstract
Targeting innate immunity holds promise in cancer immunotherapy, particularly in improving checkpoint inhibitors. However, the use of agonists of the promising innate receptors TLRs and STING1,2,3,4 is facing challenges. Here we examined the antitumour function of the α-kinase 1 (ALPK1) receptor for bacterial ADP-heptose (ADP-Hep)5,6,7. Treatment of mice with ADP-Hep induced multiple proinflammatory factors including CXCL10 and CCL2, and stimulated Alpk1-dependent antitumour immunity. Mice bearing a gain-of-function ALPK1(T237M) disease variant8 also rejected grafted tumours. Using medicinal chemistry, we identified a more potent analogue, UDSP-Hep. In contrast to ADP-Hep, UDSP-Hep distinguished Alpk1 polymorphism, which correlates with mouse susceptibility to bacteria-associated colitis9,10,11,12. UDSP-Hep exhibited a stronger Alpk1-mediated antitumour effect and synergized with checkpoint inhibitors. The effect required CD8+ T cells, dendritic cells (DCs) and macrophages, and was sensitive to antibodies that block CXCL10 or CCL2 function. ALPK1 agonists activated DCs for cross-presentation, promoting tumour-specific T cell expansion in the tumour-draining lymph nodes. ALPK1 has wider expression than STING in non-immune cells with a distinct inflammatory signature. UDSP-Hep is differentiated from STING agonists in stimulating tumour-cell antigen presentation, macrophage–DC cross-priming and protective memory T cell differentiation, and it does not induce T cell apoptosis. Our study elucidates the antitumour effect of ALPK1 agonism and suggests the potential of ALPK1 agonists in cancer immunotherapy.
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Data availability
The scRNA-seq datasets generated in this study are available under GEO accession numbers GSM8479167 and GSM8479168. Raw RNA-seq datasets are available under GEO accession numbers GSE308610 and GSE308482. All other data supporting the findings of this study are provided with the Article and its Supplementary Information. Source data are provided with this paper.
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Acknowledgements
We thank X. Zhang for providing MB49 cells; X. Xia for Batf3−/− mice; Z. Shen at BeiGene for MC38 cells and anti-PD-1; the staff at Pyrotech for Hepa 1-6 cells and ALPK1-humanized mice; F. Du at Adagene for anti-CTLA-4, anti-4-1BB and anti-PD-L1; L. Gao and L. Ye for assistance with immunological assays; Y. Xu and the members of the NIBS Laboratory Animal Resource Center for mouse breeding; the staff at the NIBS sequencing centre for RNA-seq experiments; the staff at the CIBR Laboratory Animal Resource Center for animal radiation; and W. Shi and Y. Wang for technical assistance. The work was supported by the Basic Science Center Project (82388201) of the National Natural Science Foundation of China (NSFC), the Strategic Priority Research Program of Chinese Academy of Sciences (XDB37030202 and XDB29020202), the National Key Research and Development Program of China (2022YFA1304700) and the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2019-I2M-5-084) to F.S. F.S. is also supported by the Tencent New Cornerstone Investigator Program.
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Contributions
J.L., X.T. and F.S. conceived the study. J.L. and X.T. performed the experiments. Y. Li synthesized ADP-Hep and it analogues, supervised by C.L.; Y. Luo analysed the scRNA-seq data. Y.M. helped with the LC–MS analyses. Y.W., H.H., Y.S. and J.D. provided technical assistance and valuable suggestions. P.Z. was involved in the early stage of the study and made some initial observations. J.L., X.T. and F.S. analysed the data and wrote the manuscript with input from all of the authors. All of the authors discussed the results and commented on the manuscript.
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F.S. is the scientific founder and chair of the scientific advisory board of Pyrotech Therapeutics. This relationship did not influence this study. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Activation of ALPK1 by ADP-Hep stimulates innate and adaptive immune responses as well as antitumour immunity.
a, BMDMs from WT or Alpk1−/− C57BL/6 mice were treated with PBS or 100 μM ADP-Hep for 4 h. Transcript levels of the indicated genes are shown (mean ± s.e.m., n = 3). b, Cytokine concentrations in the supernatants of ADP-Hep-treated human PBMCs (mean ± s.d.); see Fig. 2d and Supplementary Fig. 1b for more comprehensive data. c, Heatmap of cytokine concentrations in the sera of WT (n = 6), Alpk1−/− (n = 6), or Tifa−/− (n = 5) C57BL/6 mice injected (i.v.) with PBS or ADP-Hep (500 μg per mouse). d, Heatmap of cytokine concentrations in the sera of C57BL/6 mice injected with PBS (n = 6) or ADP-Hep (500 μg per mouse; n = 6 for i.p. and 7 for the other groups) intraperitoneally (i.p.), subcutaneously (s.c.), intramuscularly (i.m.), or intravenously (i.v.). e, WT C57BL/6 mice were immunized with OVA (100 μg per mouse) alone or in combination with aluminium (Alum, 2 mg per mouse) or ADP-Hep (500 μg per mouse). Anti-OVA IgG production was measured by ELISA on day 14 after immunization, and the absorbance values are shown as mean ± s.e.m. (n = 6); P values were calculated using one-way ANOVA. f, WT or Alpk1−/− C57BL/6 mice bearing B16F10-OVA tumours were treated with PBS or ADP-Hep (500 μg per mouse) (n = 8–10 mice as shown in the tumour photographs taken on day 22 after tumour challenge). g, MC38 tumours were grafted (s.c.) into WT (n = 6), Alpk1T237M/+ (n = 8), or Alpk1T237M/T237M (n = 6) C57BL/6 mice. Left, average tumour growth curves for the time duration when all mice within the group remained on study (mean ± s.e.m., two-way ANOVA, NS, not significant); middle, tumour growth curves in individual animals; right, mouse survival (log-rank (Mantel–Cox) test). All data are representative of three independent experiments.
Extended Data Fig. 2 Chemical modifications of ADP-Hep identifies UDSP-Hep as a more potent and stable ALPK1 agonist.
a, Chemical structures and EC50 of ADP-Hep analogues. Synthesis and preparation of the analogues are in Supplementary Data 1. EC50 was determined using the NF-κB luciferase reporter assay in HEK293 T cells (see Supplementary Fig. 1a). The ADP-Hep and UDSP-Hep data are the same as shown in Fig. 2b. b, e, HEK293 T cells with eGFP-TIFA integrated into the genome were treated with ADP-Hep or an indicated analogue. c, LC-MS analysis of the stability of ADP-Hep, UDP-Hep, ADSP-Hep, and UDSP-Hep in PBS in the presence or absence of 20% FBS (v/v). d, HEK293 T cells, cultured in serum-free or 10% FBS-supplemented DMEM, were stimulated extracellularly with ADP-Hep (n = 6) or UDSP-Hep (n = 3). f, HEK293 T cells were electroporated with ADP-Hep or UDSP-Hep (n = 3 for each group). g, Activation of recombinant human ALPK1 by ADP-Hep and UDSP-Hep in vitro, quantified via the ADP-GloTM luminescent kinase assay (mean ± s.d.). b, e, Anti-pT9-TIFA immunoblotting. d, f, NF-κB luciferase reporter assay data (mean ± s.d.). d, f, g, Curves were fitted to calculate EC50 values. All data are representative of three independent experiments.
Extended Data Fig. 3 UDSP-Hep has much stronger immunostimulant activity than ADP-Hep and distinguishes Alpk1 alleles in mice.
a, RT-qPCR analysis of CXCL10, IL1B, CXCL8, CCL2, TNF, and ALPK1 expression in PMA-differentiated THP-1 cells treated with ADP-Hep, UDP-Hep, or UDSP-Hep for 12 h. Levels of mRNA were normalized to that of GAPDH (mean ± s.d., n = 3). b, Heatmap of cytokine concentrations in the sera of WT or Alpk1−/− C57BL/6 mice injected (i.v.) with PBS or an indicated dose of ADP-Hep or UDSP-Hep (n = 7 for untreated WT group, 5 for 5-µg ADP-Hep- and 500-µg UDSP-Hep-treated WT group, and 6 for other groups). c, e, WT or Alpk1−/− C57BL/6 mice or indicated strains of mice were immunized with OVA (100 μg per mouse) alone or in combination with aluminium (Alum, 2 mg per mouse) or an indicated dose of ADP-Hep or UDSP-Hep (c) or ADP-Hep (500 µg per mouse), or UDSP-Hep (20 µg per mouse) (e). Anti-OVA IgG production was measured by ELISA on day 21 after immunization, and the absorbance values are shown as mean ± s.e.m. (c, n = 6 for WT and 7 for Alpk1−/−; e, n = 6). Two-way ANOVA. d, Heatmap of cytokine concentrations in the sera of C57BL/6, 129, or BALB/c mice injected (i.v.) with PBS, ADP-Hep or UDSP-Hep at the indicated doses. Each lane indicates the average cytokine expression (n = 5–6 mice per group). f, RT-qPCR analyses of Cxcl10, Tnf, and Ifi205 expression in C57BL/6, 129, or BALB/c strain-derived BMDMs treated with ADP-Hep, UDP-Hep, or UDSP-Hep for 6 h. Levels of mRNA were normalized to that of Gapdh (mean ± s.d., n = 3). g, ALPK1−/− HEK293 T cells expressing eGFP-TIFA and Flag-ALPK1 derived from the C57BL/6 or 129 mice or humans were treated with ADP-Hep, UDP-Hep, or UDSP-Hep. TIFA phosphorylation was assessed by anti-pT9-TIFA immunoblotting. h, Serum concentrations of UDSP-Hep over time after a single s.c. or i.v. injection (2.5 mg kg−1) into C57BL/6 mice. Blood was collected at 0, 0.083, 0.25, 0.5, 1, 2, 4, and 7 h after the UDSP-Hep injection; its concentrations were quantified by LC-MS/MS (mean ± s.e.m., n = 3). i, Local biodistribution after peri-tumoural injection of UDSP-Hep. The concentrations of UDSP-Hep in tumour tissues and tdLNs 15 min after the injection (2.5 mg kg−1) are shown (mean ± s.e.m., n = 4). Data are representative of two (h, i) or three (a–g) independent experiments.
Extended Data Fig. 4 UDSP-Hep-induced Alpk1-dependent antitumour immunity in mice and can enhance the effects of various checkpoint inhibitors.
a, b, Growth curves of B16F10-OVA (a) and MC38 tumours (b) in WT and Alpk1−/− C57BL/6 mice treated with PBS or UDSP-Hep (a, n = 11 for PBS-treated WT group and 8 for other groups; b, n = 11 for WT and 9 for Alpk1−/−). c, d, Growth curves (left) and survival analysis (right) of B16F10-OVA (c) and MC38 tumours (d) treated with PBS or UDSP-Hep in NSG mice (n = 7 for PBS-treated B16F10-OVA and 8 for other groups). e, f, Hepa 1-6 (e) and B16F10-OVA (f) were grafted into both sides of the back of C57BL/6 mice. Tumours on the right flank were treated with PBS (e, n = 8; f, n = 10) or UDSP-Hep (e, n = 11; f, n = 9). g, h, Growth curves (left) and survival analysis (right) of orthotopic 4T1-OVA (g) or E0771 (h) mammary carcinoma in BALB/c (n = 7) or C57BL/6 mice (n = 8), respectively, subjected to intratumour injection of PBS, ADP-Hep, or UDSP-Hep. i, j, Growth curves of MC38 tumours treated with PBS, UDSP-Hep (50 μg per mouse), anti-CTLA-4 antibody (0.5 mg kg−1), anti-PD-1 antibody (high dose, 5 mg kg−1; low dose, 0.25 mg kg−1), or UDSP-Hep combined with either antibody (i, n = 12 for PBS and 11 for other groups; j, Left, n = 11 for PBS and UDSP-Hep groups, 21 for anti-PD-1, and 23 for UDSP-Hep and anti-PD-1 co-treatment group; Right, n = 10). k, Average and individual-animal tumour growth curves and mouse survival of advanced MC38 tumours (nearly 400 mm3) treated with PBS (n = 9), UDSP-Hep (50 μg per mouse; n = 8), anti-PD-1 antibody (10 mg kg−1; n = 9), or UDSP-Hep combined with anti-PD-1 antibody (n = 8). l, m, C57BL/6 mice bearing MC38 tumours were treated with PBS (n = 11), UDSP-Hep (50 μg per mouse; n = 12), anti-4-1BB antibody (5 mg kg−1; n = 12), anti-PD-L1 antibody (5 mg kg−1; n = 12), or UDSP-Hep combined with either antibody (n = 12). The PBS and UDSP-Hep-alone groups in l and m are the same experiment. a–m, Data are shown as mean ± s.e.m., and two-way ANOVA and log-rank (Mantel–Cox) tests were used for statistical comparisons of tumour volume and mouse survival data, respectively (NS, not significant). Data are representative of two independent experiments.
Extended Data Fig. 5 UDSP-Hep inflames the tumours and the tumour control requires chemokines and bone marrow-derived cells.
a, b, Quantification of cytokines within Hepa 1-6 tumours upon UDSP-Hep treatment (n = 6 mice per group). c, Growth curves (left) and mouse survival (right) of B16F10-OVA tumours treated with PBS (n = 8), UDSP-Hep (50 μg per mouse; n = 9), PTx (400 ng per mouse; n = 9), or UDSP-Hep combined with PTx (n = 9). d, Growth curves of B16F10-OVA tumours treated with PBS (n = 9), UDSP-Hep (n = 10), CXCR3-blocking antibody (n = 9), CCL2-neutralizing antibody (n = 7), or UDSP-Hep combined with either antibody (n = 9 for anti-CXCR3 plus UDSP-Hep and 7 for anti-CCL2 plus UDSP-Hep). e, Growth curves of Hepa 1-6 tumours treated with UDSP-Hep alone or in combination with control or clodronate liposomes (n = 7 for PBS and 6 for other groups). f, Tumour growth curves in bone marrow (BM) chimera mice. C57BL/6 WT mice reconstituted with WT BM (WT → WT, left) or Alpk1−/− BM (Alpk1−/−→WT, middle) or Alpk1−/− mice reconstituted with WT BM (WT→Alpk1−/−, right) were grafted with B16F10-OVA tumours and then treated with PBS (n = 8 for WT → KO and 6 for the other groups) or UDSP-Hep (50 μg per mouse; n = 6 for each group). g, scRNA-seq analysis of Alpk1 and Tifa expression in intact B16F10-OVA tumours. Violin plots showing normalized Alpk1 and Tifa expression across annotated cell clusters. h, Flow cytometry quantification of IFNγhi or GZMBhi NK cells among CD45+ cells in TILs of PBS or UDSP-Hep-treated MC38 tumours (n = 8 mice per group). i, Flow cytometry quantification of CD3+ T cells and NK cells among CD45+ cells in TILs of PBS or UDSP-Hep-treated B16F10-OVA tumours (n = 8 mice per group). a–f, h, i, Data are shown as mean ± s.e.m. Two-tailed unpaired Student’s t-test or Welch’s t-test (h, i), one-way ANOVA (a, b), and two-way ANOVA (c–f) were used for statistical comparisons. Data are representative of three (c–f) or two (a, b, h, i) independent experiments.
Extended Data Fig. 6 UDSP-Hep treatment activates DCs and T cells in the tdLN to control tumour growth.
a, Growth curves of MC38 or B16F10-OVA tumours treated with PBS, UDSP-Hep, anti-CD4 depletion antibody, or UDSP-Hep combined with the depletion antibody (n = 8 for the PBS group of B16F10-OVA tumours and 9 for the other groups). b, Growth curves of MC38 or B16F10-OVA tumours treated with PBS, UDSP-Hep, anti-NK1.1 depletion antibody, or UDSP-Hep combined with the depletion antibody (n = 9 for MC38 tumour groups and 7 for B16F10-OVA tumour groups). The PBS and UDSP-Hep-alone groups in the left panel of a and b are the same as those in Fig. 4e. c, d, C57BL/6 mice bearing B16F10-OVA tumours were subjected to lymphadenectomy operation prior to PBS or UDSP-Hep treatment. c, Schematic diagram of the experiment. d, Tumour growth curves (n = 5). e, f, CD8α−/− mice receiving CD45.1+ CD8+ T cells adoptively transferred from tdLNs of B16F10-OVA-bearing donors treated with PBS or UDSP-Hep. e, Schematic diagram of the experiment. f, Tumour growth curves (female, n = 4 for PBS and 3 for other groups; male, n = 4 for UDSP-Hep and 5 for other groups). g, Flow cytometry quantification of PD-1+ CD8+ T cells among total CD8+ T cells in PBS or UDSP-Hep-treated B16F10-OVA tumours (n = 8 mice per group). h, i, Analyses of CD8+ Tpex and Ttex in the TILs of B16F10-OVA tumour-bearing mice treated with PBS or UDSP-Hep. h, Representative flow cytometry plots of anti-TCF1 and anti-TIM-3 staining. i, Quantification of the flow cytometry analyses (n = 8 mice per group). j, Quantification of the mean fluorescence intensity of anti-CD80, CD86, and CD40 staining of WT and Alpk1−/− iCD103-DCs treated with PBS, LPS, ADP-Hep, UDSP-Hep, or R848 (mean ± s.d., n = 3). k, Examination of cDC1s in tdLNs of PBS or UDSP-Hep-treated B16F10-OVA tumours. Shown are histograms of anti-CD80 and CD86 staining of the cDC1s. l, Purified iCD103-DCs were treated with 1 μg mL−1 OVA plus PBS, 100 nM UDSP-Hep, or 100 nM ADU-S100 for 8 h and then injected (1×105 per mouse) adjacent to the tdLN in B16F10-OVA-bearing mice on day 7, 10 and 13 after tumour challenge. Shown are tumour growth curves (n = 9 mice per group). m, n, Anti-CD69 staining of CD4+ and CD8+ T cells in tdLNs of PBS (n = 7) or UDSP-Hep (n = 8)-treated B16F10-OVA tumours. m, Representative flow cytometry plots. n, Quantification of CD69+ populations among the T cells. a, b, d, f, g, i, l, n, Data are shown as mean ± s.e.m.; two-way ANOVA (a, b, d, f, l) and two-tailed unpaired Student’s t-test (g, i, n), were used for statistical comparisons (NS, not significant). All data are representative of three independent experiments.
Extended Data Fig. 7 Expression patterns of ALPK1, TIFA, STING, TLR7, TLR8, and TLR9 in NCI-60 plus 13 additionally selected cell lines.
Cell lysates were blotted with indicated antibodies. Immune-relevant cells are marked in red. Data are representative of three independent experiments.
Extended Data Fig. 8 Distinct transcriptional and cytokine signatures elicited by ALPK1, STING, and TLR agonists in immune-relevant cells and mice.
a, b, Heatmap of gene expression in THP-1 monocytes (a) or RPMI-8226 B lymphocytes (b) treated with ALPK1 agonist (ADP-Hep or UDSP-Hep), TLR7/8 agonist (R848), or STING agonist (ADU-S100) at the indicated doses. Cells were treated for 4 h; mRNAs were isolated for RNA-seq analysis. c, Heatmap of cytokine concentrations in the sera of C57BL/6 WT mice injected (i.v.) with PBS, UDSP-Hep, R848, or ADU-S100 at the indicated doses (n = 5 for UDSP-Hep and R848 and 6 for ADU-S100). See Supplementary Fig. 8 for more comprehensive data. All data are representative of two independent experiments.
Extended Data Fig. 9 UDSP-Hep can act together with STING or TLR agonist to render enhanced antitumour activity.
a, Growth curves (left) and mouse survival (right) of B16F10 tumours treated with PBS or DMXAA (n = 7 mice per group). b, Growth curves of B16F10 tumours treated with PBS or high-dose UDSP-Hep (n = 9 mice per group). This is the same experiment as the PBS and UDSP-Hep treatment groups in Extended Data Fig. 9d (left). c, Growth curves of B16F10 tumours treated with PBS, UDSP-Hep (50 μg per mouse), DMXAA (50 μg per mouse), or UDSP-Hep combined with DMXAA (n = 9 for PBS and 8 for other groups). d, e, Growth curves (left) and mouse survival (right) of B16F10 (d) and MC38 (e) tumours treated with PBS, UDSP-Hep (100 μg per mouse for B16F10; 50 μg per mouse for MC38), R848 (100 μg per mouse for B16F10; 50 μg per mouse for MC38), or UDSP-Hep combined with R848. d, n = 9 mice for each group. e, n = 11 for PBS, 8 for UDSP-Hep, 9 for R848, and 7 for UDSP-Hep combined with R848. f, g, Growth curves of MC38 tumours treated with R848 (50 μg per mouse, f) or DMXAA (50 μg per mouse, g) alone or in combination with CXCR3 blocking antibody (left) or CCL2 neutralizing antibody (right). f, n = 12 for PBS, 7 for R848 and anti-CXCR3 blocking antibody alone, 8 for CCL2 neutralizing antibody alone and R848 plus anti-CXCR3 blocking antibody, and 7 for R848 plus CCL2 neutralizing antibody. g, n = 11 for PBS, and 8 for all other groups. h, i, Measurements of the Ttsm population on day 60 in Hepa 1-6 tumour-inoculated mice that already achieved complete tumour clearance by UDSP-Hep. Scheme of the experiments and the Day-23 data are in Fig. 5e–g. h, Representative flow cytometry plots gated on Ttsm. i, Quantification of the numbers of Ttsm at the inoculation site (Skin in situ), tdLNs, spleen, and non-tdLN (n = 7 for skin and 8 for other groups). a–g, i, Data are shown as mean ± s.e.m. Two-way ANOVA (a–g) and log-rank (Mantel–Cox) test (a, d, e) were used for statistical comparisons. All data are representative of three independent experiments.
Extended Data Fig. 10 Comparisons of immune activation properties of ALPK1, STING, and TLR7/8 agonists.
a, Induction of CXCL10, IL-6, and TNF by increasing doses of UDSP-Hep, the TLR7 agonist 41c-A from Roche, and ADU-S100 in human PBMCs (three donors). Shown are concentrations of the cytokines in the supernatants. b, Quantification (n = 3) of anti-CD86, CD80, and CD40 staining of purified iCD103-DCs treated with PBS, LPS, UDSP-Hep or ADU-S100. The histograms are shown in Fig. 5c. c, d, WT and Alpk1−/− BMDMs were treated with PBS, UDSP-Hep, R848, or ADU-S100. Shown are histograms (c) and quantification (d, n = 3) of anti-CD86 staining of the cells. e, f, OT-I CD8+ T cells were co-cultured for 72 h with WT and Alpk1−/− BMDMs pre-stimulated with OVA alone or in combination with an indicated immune agonist. Shown are flow cytometry histograms (e) and quantification (f, n = 3) of total T-cell numbers. g, Viability of anti-CD3ε/CD28-activated mouse CD3+ T cells after indicated treatments (n = 3). h, Growth curves of Hepa 1-6 tumours treated with PBS, UDSP-Hep (50 μg per mouse), or DMXAA (50 μg per mouse) (mean ± s.e.m., n = 8 for PBS and 9 for other two groups). Two-way ANOVA was used for statistical comparisons. a, b, d, f, g, mean ± s.d. All data are representative of three independent experiments.
Supplementary information
Supplementary Figures
Supplementary Figs. 1–8.
Supplementary Data 1
Detailed methods and procedures for chemical synthesis and characterization of ADP-Hep and its analogues listed in Extended Data Fig. 2.
Supplementary Data 2
The uncropped immunoblots for key data presented in the main text and Extended Data.
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Tian, X., Liu, J., Li, Y. et al. Agonists for cytosolic bacterial receptor ALPK1 induce antitumour immunity. Nature (2025). https://doi.org/10.1038/s41586-025-09828-9
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DOI: https://doi.org/10.1038/s41586-025-09828-9
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