Abstract
Targeting tumor-infiltrating regulatory T (TI-Treg) cells is a potential strategy for cancer therapy. The ATPase p97 in complex with cofactors (such as Npl4) has been investigated as an antitumor drug target; however, it is unclear whether p97 has a function in immune cells or immunotherapy. Here we show that thonzonium bromide is an inhibitor of the interaction of p97 and Npl4 and that this p97–Npl4 complex has a critical function in TI-Treg cells. Thonzonium bromide boosts antitumor immunity without affecting peripheral Treg cell homeostasis. The p97–Npl4 complex bridges Stat3 with E3 ligases PDLIM2 and PDLIM5, thereby promoting Stat3 degradation and enabling TI-Treg cell development. Collectively, this work shows an important role for the p97–Npl4 complex in controlling Treg–TH17 cell balance in tumors and identifies possible targets for immunotherapy.
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Data availability
All high-throughput sequencing data have been deposited in the Gene Expression Omnibus database under the accession code GSE240681. Source data are provided with this paper.
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Acknowledgements
We thank Y. Shi from Soochow University, B. Li from Shanghai Jiao Tong University, and Y. Wang and X. Liu from University of Chinese Academy of Sciences for their helpful discussion. This work was supported by the National Key R&D Program of China (2020YFA0803200), National Natural Science Foundation of China grants (82150112, 82303148, 92168116, 82360502, 82060542, 31930026, 82361168638, 82372613, 82373251 and 82222052), China Postdoctoral Science Foundation (2023M742656), Yunnan Province’s Ten Thousand Talents Plan for Top Young Talents (YNWR-QNBJ-2019-275), Xingdian Talents and Famous Doctors Special Project (XDYC-YLWS-2023-0029) and Shanghai Super Postdoctoral Incentive Program.
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Contributions
P.N. and R.Y. performed functional experiments. Z.C. performed biochemical experiments. Y.M. conducted scRNA-seq analysis. X.J. and S.W. performed genotyping of mice. B.L., C.D., L.T., C.L., Y.X., L.A., Y.P., Y.Z., W.Z., H.Z., Z.T., M.Z., Y.H. and W.W. discussed and analyzed the results. P.N., S.J. and Z.Z. designed the experiments and wrote the manuscript. S.J. and Z.Z. supervised the project.
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Z.Z. (CN201510362643.5, CN201510359075.3, CN201910753411.0, CN2024108876845), S.J. (CN201510362643.5, CN201510359075.3, CN201910753411.0, CN2024108876845), P.N. (CN201910753411.0) and W.W. (CN201910753411.0) have filed patents regarding the potential application of TB in cancer therapy. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 TB displayed a broader antitumor therapeutic window than CB-5083.
a, Summary of p97-related inhibitors. b, Strep pulldown assay to assess the interaction between p97 and Npl4. Data shown are representative results of two independent experiments. c, Co-IP assay to detect the interaction of p97 with Npl4. Data shown are representative results of two independent experiments. d, Inhibition of cell viability in HGC-27 cells. Data shown are representative results of two independent experiments. e, Inhibition of cell viability in GES-1 cells. Data shown are representative results of two independent experiments. f, mRNA levels of CHOP in the indicated cells that had been treated with TB (upper) or CB-5083 (lower). Data shown are representative results of two independent experiments. g, Schematic illustration of the tumor graft model and treatment with compounds. h, Experimental flow chart of a mouse model for MNNG/H. pylori-induced GC and the treatment with TB, CB-5083 or 5-FU. i, Therapeutic windows and tumor growth inhibition abilities of TB and CB-5083. IC50 values of the compounds are shown. j, Tumor weights of tumor graft models after treatment with TB (intragastrically administered). n = 6 biologically independent samples examined over three independent experiments. k, Tumor weights for MC38-bearing mice that had been treated with TB (intravenously injected, i.v.). n = 4 biologically independent samples examined over two independent experiments. l, Body weights of TB-treated (intraperitoneal administration) mice and control group. n = 4 biologically independent samples examined over two independent experiments. m, Body weights of TB-treated (i.v.) mice and control group. n = 4 biologically independent samples examined over two independent experiments. n, Body weights of TB-treated (25mg/kg, i.v.) mice and control group. n = 6 biologically independent samples examined over two independent experiments. o, Whole blood cell routine test of TB-treated mice and control group from panel n. n = 6 biologically independent samples examined over two independent experiments. Data shown as means ± s.d. (d-f) or means ± s.e.m. (j,k,l,m and n). Data were analyzed for significant differences by performing a one-way ANOVA with Dunnett’s post hoc analysis (f, j and k), or two-way ANOVA with Dunnett’s post hoc analysis (l,m), or two-tailed, unpaired Student’s t-tests (n).
Extended Data Fig. 2 TB treatment promoted the effector function of CD8+ T cells.
a, Apoptosis scores of CD8+ (left) and CD4+ T cells (right) based on the expression of T cell apoptosis-correlated genes from the indicated group. b, Violin plot showing the expression of genes correlated to CD8+ T cell effector function in the indicated group. c, Heatmap showing the expression of genes correlated to CD4+ T cell effector function in the indicated group. d, Flow cytometry analysis showing the proportions of CD4+ and CD8+ T cells among the tumor-infiltrated CD45+ cells from Ctrl and TB-treated mice. e, Proportions (left) and absolute numbers (right) of tumor-infiltrated CD4+ and CD8+ T cells per milligram tumor from Ctrl and TB-treated mice. n = 4 biologically independent samples examined over two independent experiments. f, Gating strategy for tumor-infiltrated CD8+ T cells and representative flow cytometry plots of unstimulated samples of the indicated cytokine-producing CD8+ T cells and samples of these cells stimulated with PMA and ionomycin. g, Proportions of tumor-infiltrated IFNγ+ CD8+ T cells per milligram tumor from Ctrl and TB-treated mice. n = 6 biologically independent samples examined over two independent experiments. Data shown as means ± s.e.m. (e,g). Data were analyzed for significant differences by performing two-tailed, unpaired Student’s t-tests (a), or one-way ANOVA with Dunnett’s post hoc analysis (b,e,g).
Extended Data Fig. 3 Mutational analysis of the p97-Npl4 complex in T cells.
a, Flow cytometry analysis of CD4+ and CD8+ T cell subpopulations in thymuses and spleens from Vcp3A/3A and Vcp3A/3A CD4Cre mice. b, Flow cytometry analysis showing the expression levels of GzmB and IFNγ in CD8+ T cells isolated from spleens of Vcp3A/3A or Vcp3A/3A CD4Cre mice. CD8+ T cells were sorted from spleens stimulated with anti-CD3 (2 μg/mL) and anti-CD28 (1 μg/mL) for the indicated times, and cytokine expression was determined by performing flow cytometry. c, Tumor growth curves of MC38-derived subcutaneous tumors from, respectively, Vcp3A/3A and Vcp3A/3A CD4Cre mice. n = 6 biologically independent samples examined over two independent experiments. d, Percentages of CD4+ and CD8+ T cells among the tumor-infiltrated CD45+ cells for the indicated mice. n = 4 biologically independent samples examined over two independent experiments. e, Frequencies of CD8+GzmB+, CD8+IFNγ+ and CD8+TNF+ cells among tumor-infiltrated CD8+ T cells. n = 4 biologically independent samples examined over two independent experiments. f, Number of tumor-infiltrated CD45.2+ Treg, Th17, Th1 and Th2 cells per milligram tumor from CD45.1 mice that were adoptively transferred with vector (Ctrl) or Flag-Vcp 3A lentivirus-infected (Vcp3A) CD45.2+ CD3+ T cells. n = 4 biologically independent samples examined over two independent experiments. Data shown as means ± s.e.m. (c-f). Data were analyzed for significant differences by performing two-way ANOVA with Dunnett’s post hoc analysis (c), or two-tailed, unpaired Student’s t-test (d-f).
Extended Data Fig. 4 Targeting the p97-Npl4 interaction skewed Treg-Th17 cell balance.
a, Gating strategy for TI-Treg and Th17 cells gated from tumor-infiltrated CD4+ T cells, with a fluorescence minus one (FMO) sample used as negative control. b, Absolute numbers of intratumoral Treg (TI-Treg, Foxp3+) and Th17 cells (IL17a+) per milligram tumor from tumor-bearing control (Ctrl) and TB-intraperitoneally (i.p.)-injected mice. n = 4 biologically independent samples examined over two independent experiments. c, Absolute numbers of TI-Treg and Th17 cells per milligram tumor from tumor-bearing Ctrl and TB-intravenously (i.v.)-injected mice. n = 4 biologically independent samples examined over two independent experiments. d, Representative flow cytometry plots and percentages of TI-Treg cells from tumor-bearing Ctrl or TB-intratumorally (i.t.)-injected mice. n = 4 biologically independent samples examined over two independent experiments. e, Absolute number of TI-Treg cells per milligram tumor in each of the indicated mice. n = 4 biologically independent samples examined over two independent experiments. f, Representative plots and percentages of tumor-infiltrated Th17 cells from tumor-bearing Ctrl or TB-intratumorally (i.t.)-injected mice. n = 4 biologically independent samples examined over two independent experiments. g, Absolute number of tumor-infiltrated Th17 cells per milligram tumor in each of the indicated mice. n = 4 biologically independent samples examined over two independent experiments. h, Schematic illustration of the iTreg-cell-transfer assay procedure. CD45.1 tumor-bearing mice were intravenously injected with Vcp3A/3A iTreg (WT) or Vcp3A/3A Foxp3Cre (3A) iTreg cells. i, Flow cytometry analysis of the purity of iTreg cells derived from WT and 3A mice for adoptive transfer assay. j, Absolute numbers of TI-Treg cells per milligram tumor from tumor-bearing Vcp3A/3A, Foxp3Cre and Vcp3A/3A Foxp3Cre mice. n = 10 biologically independent samples examined over four independent experiments. k, Absolute numbers of intratumoral Th17 cells per milligram tumor from tumor-bearing Vcp3A/3A, Foxp3Cre and Vcp3A/3AFoxp3Cre mice. n = 10 biologically independent samples examined over four independent experiments. l, Absolute numbers of tumor-infiltrated IFNγ+CD8+ T cells per milligram tumor from tumor-bearing Vcp3A/3A, Foxp3Cre and Vcp3A/3AFoxp3Cre mice. n = 10 biologically independent samples examined over four independent experiments. Data shown as means ± s.e.m. (b-g,j-l). Data were analyzed for significant differences by performing one-way ANOVA with Dunnett’s post hoc analysis (b–g) or two-tailed, unpaired Student’s t-test (j–l).
Extended Data Fig. 5 Targeting p97-Npl4 interaction did not disrupt immune homeostasis.
a, Flow cytometry analysis showing the proportions of Treg cells (Foxp3+) and Th17 cells (IL17a+RORγt+) among CD4+ T cells in lungs from Vcp3A/3A, Foxp3Cre and Vcp3A/3A Foxp3Cre mice. b, Flow cytometry analysis showing the proportions of Treg cells (Foxp3+) and Th17 cells (IL17a+RORγt+) among CD4+ T cells in the colon from Vcp3A/3A, Foxp3Cre and Vcp3A/3A Foxp3Cre mice. c, The proportions of CD44hiCD62Llow subpopulation among the CD4+ or CD8+ T cells from spleens and lymph nodes of Vcp3A/3A, Foxp3Cre and Vcp3A/3A Foxp3Cre mice. n = 5 biologically independent samples examined over two independent experiments. d, Absolute numbers of Treg (Foxp3+) and Th17 cells (IL17a+) in draining lymph nodes (dLN) from Ctrl and TB-treated mice. n = 4 biologically independent samples examined over two independent experiments. e, Absolute numbers of Treg (Foxp3+) and Th17 cells (IL17a+) in lungs from Ctrl and TB-treated mice. n = 4 biologically independent samples examined over two independent experiments. f, Absolute numbers of Treg (Foxp3+) and Th17 cells (IL17a+) in colons from Ctrl and TB-treated mice. n = 4 biologically independent samples examined over two independent experiments. Data shown as means ± s.e.m. (c-f). Data were analyzed for significant differences by performing one-way ANOVA with Dunnett’s post hoc analysis (c–f).
Extended Data Fig. 6 Target the p97-Npl4 interaction did not affect Treg cell stability and suppressive function.
a, Apoptosis scores of TI-Treg cells from Ctrl (grey) and TB (red)-treated groups. In each case, the apoptosis score was calculated based on the expression profile of the apoptosis-correlated transcriptomic signature. b, CTV proliferation analysis of iTreg (upper) and Th17 (lower) cells upon TB treatment at the indicated concentrations. Data shown are representative results from two independent experiments. c, Representative plots (left) and percentages (right) of Foxp3+ cells, IL17a+ cells and Foxp3+ IL17a+ cells among total tumor-infiltrating CD4+ T cells from Vcp3A/3A and Vcp3A/3AFoxp3Cre mice. n = 4 biologically independent samples examined over two independent experiments. d, Ex vivo suppression of carboxyfluorescein succinimidyl ester (CFSE)-labeled WT naïve CD8+ T cell proliferation by iTreg cells derived from Vcp3A/3A and Vcp3A/3AFoxp3Cre mice with the indicated ratios of Treg to Teff cells. Teff, CD8+ T effector cells (n = 3 repeats/group). Data shown are representative results from two independent experiments. e, Heatmap of downregulated regulon activities in TI-Treg cells from Ctrl and TB-treated tumor bearing mice. f, Heatmap showing the transcription levels of Stat3 target genes in the TI-Treg cells from Ctrl and TB-treated tumor bearing mice. g, Flow cytometry analysis of induced Treg (iTreg, CD25+Foxp3+, upper) cells and Th17 (IL17a+, lower) cells in the CD4+ T cells after treatment with TB or a mixture of TB with the Stat3 inhibitor Stattic. Naïve CD4+ T cells were sorted from spleen tissues and then stimulated with anti-CD3 (10 μg/mL) and anti-CD28 (10 μg/mL/) under Treg or Th17 differentiation conditions together with TB (1 μM) or a mixture of TB (1 μM) with Stattic (1, 2 μM). Data shown as means ± s.e.m. (c). Data were analyzed for significant differences by performing two-tailed, unpaired Student’s t-test (a), or one-way ANOVA with Dunnett’s post hoc analysis (c).
Extended Data Fig. 7 Targeting the p97-Npl4 interaction inhibited ubiquitination of Stat3.
a, Western blot analysis of Stat3 in wildtype (WT) cells and that in p97 KO 293FT cells, each transfected with Flag-UB, Myc-Stat3, wildtype or its 3A mutant HA-p97. Data shown are representative results from two independent experiments. b, Immunoblotting of the indicated proteins in CD4+ T cells at different activation and differentiation stages, and in CD8+ T cells upon TCR stimulation. Data shown are representative results from two independent experiments. c, Schematic illustration of the knocking out of p97, Npl4 or PDLIM2/5 using the CRISPR-Cas9 system. d, Immunoblotting analysis showing the efficiency of the knockout of each of p97, Npl4 and PDLIM2/5 in CD4+ T cells. Data shown are representative results from two independent experiments. e, Proportion of the iTreg (Foxp3+) cells among the CD4+ T cells after p97, Npl4 or PDLIM2/5 deficiency (n = 3 repeats/group). Data shown are representative results from two independent experiments. f, Proportion of the Th17 (IL17a+RORγt+) cells among the CD4+ T cells after p97, Npl4 or PDLIM2/5 deficiency (n = 3 repeats/group). Data shown are representative results from two independent experiments. g, Co-IP analysis to detect the interaction between p97 and PDLIM5 in 293FT cells. Data shown are representative results from two independent experiments. h, Co-IP analysis to detect the interaction between p97 and PDLIM2 in 293FT cells. Data shown are representative results from two independent experiments. i, qPCR analysis to determine the knockdown efficiency of PDLIM2/5 siRNAs (n = 3 repeat/group). Data shown are representative results from two independent experiments. j, Flow cytometry analysis showing the proportions of iTreg cells (Foxp3+) and Th17 cells (IL17a+RORγt+) among the indicated CD4+ T cells that had been subjected to knockdown of PDLIM2 or PDLIM5 with their specific siRNAs (n = 3 repeats/group). Data shown are representative results from two independent experiments. Data shown as means ± s.d. (e,f,I and j). Data were analyzed for significant differences by performing two-tailed, unpaired Student’s t-test (e,f,I and j).
Supplementary information
Supplementary Information
Supplementary Tables 1–3. Supplementary Table 1: List of primers for mouse genotyping. Supplementary Table 2: List of key resources. Supplementary Table 3: List of primers for qPCR.
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Nie, P., Cao, Z., Yu, R. et al. Targeting p97–Npl4 interaction inhibits tumor Treg cell development to enhance tumor immunity. Nat Immunol 25, 1623–1636 (2024). https://doi.org/10.1038/s41590-024-01912-y
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DOI: https://doi.org/10.1038/s41590-024-01912-y
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