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c-Rel is a myeloid checkpoint for cancer immunotherapy

Nature Cancer volume 1, pages 507–517 (2020)Cite this article

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Abstract

Immunotherapy that targets lymphoid cell checkpoints holds great promise for curing cancer. However, many cancer patients do not respond to this form of therapy. In addition to lymphoid cells, myeloid cells play essential roles in controlling immunity to cancer. Whether myeloid checkpoints exist that can be targeted to treat cancer is not well established. Here we show that c-Rel, a member of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) family, specified the generation of myeloid-derived suppressor cells by selectively turning on protumoral genes while switching off antitumoral genes through a c-Rel enhanceosome. c-Rel deficiency in myeloid cells markedly inhibited cancer growth in mice and pharmaceutical inhibition of c-Rel had the same effect. Combination therapy that blocked both c-Rel and the lymphoid checkpoint protein programmed cell death protein 1 was more effective in treating cancer than blocking either alone. Thus, c-Rel is a myeloid checkpoint that can be targeted for treating cancer.

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Fig. 1: Global and myeloid Rel gene deletion blocks tumor growth and reduces MDSCs in mice.
Fig. 2: Reduced suppressive function and altered metabolism of Rel−/− Gr-1+ myeloid cells.
Fig. 3: Loss of MDSC gene signatures from Rel−/− myeloid cells.
Fig. 4: c-Rel activates the MDSC signature genes Cebpb and Arg1.
Fig. 5: c-Rel regulates MDSC gene expression, metabolism and suppressive function via Cebpb.
Fig. 6: c-Rel inhibitor blocks tumor growth and MDSC development, and enhances the effect of anti-PD1 therapy.
Fig. 7: c-Rel inhibitor blocks human MDSCs.

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Data availability

Microarray and RNA-seq data that support the findings of this study have been deposited in the ArrayExpress under accession nos. E-MTAB-8674 and E-MTAB-8714. Source data for Fig. 1 and Figs. 4–7 are provided with this paper and are available online. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank H.-C. Liou (Cornell University), W. Weinberg (US FDA) and J. Zakrzewski (Cornell University) for providing the breeding pair of the Rel−/− mice. We thank D. Gabrilovich, J. Goldsmith, P. Fang, L. Wan, M. Lin, D. Zhang, L. Guan, J. Devergiilis and J. Sun for valuable discussions, technical support and reagents. We thank the University of Pennsylvania Pancreatic Islet Cell Biology Core and D.P. Beiting from the University of Pennsylvania School of Veterinary Medicine for technical support. This work was supported in part by grants from the National Institutes of Health (nos. R01-AI152195, R01-AI099216, R01-AI121166, R01-AI143676 and R01-AI136945 to Y.H.C.); X.L. was partially supported by grant no. NIH-T32-DK007780.

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Author notes

  1. These authors contributed equally: Ting Li, Xinyuan Li.

Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Ting Li, Xinyuan Li, Ali Zamani, Wei Wang, Chin-Nien Lee, Mingyue Li, George Luo, Emily Eiler, Honghong Sun & Youhai H. Chen

  2. Department of Microbiology and Immunology, College of Physicians & Surgeons, Columbia University, New York, NY, USA

    Sankar Ghosh

  3. Mount Sinai Center for Therapeutics Discovery, Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Jian Jin

  4. Department of Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Jian Jin

  5. Department of Biomedical Sciences, Research Division of Immunology, Cedars-Sinai Medical Center, Los Angeles, CA, USA

    Ramachandran Murali

  6. Shandong Eye Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, China

    Qingguo Ruan & Weiyun Shi

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Contributions

T.L. and X.L. designed and executed the experiments and wrote the manuscript. A.Z. designed and performed some of the MDSC suppression experiments. H.S., M.L., W.W., C.-N.L., G.L., E.E., Q.R. and W.S. helped complete certain molecular, cellular or animal experiments. S.G. provided the Rel gene floxed mice. J.J. and R.M. designed and optimized the c-Rel inhibitor. Y.H.C. conceived and supervised the study and wrote the manuscript.

Corresponding author

Correspondence to Youhai H. Chen.

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Competing interests

Y.H.C. and R.M. are inventors of the following patent that describes the c-Rel inhibitor used in this study: Chen Y.H., Murali R. & Sun J. Rel inhibitors and methods of use thereof (USA patent no. US8609730B2). Y.H.C. is a member of the advisory board of Amshenn Pharmaceutical Company and Binde Company.

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Extended data

Extended Data Fig. 1 Global and myeloid Rel gene deletion blocks tumor growth and reduces MDSCs in mice.

a, Tumor growth in WT and Rel−/− mice (n = 5 mice/group) injected s.c. with B16F0 tumor cells (***, P < 0.0001). b, Tumor weight of WT and Rel−/− mice (n = 10 mice/group) treated in Fig. 1a. (*, P = 0.0188). c, Percentages of CD11b+Gr-1+ cells in the blood of WT and Rel−/− mice treated with anti-Gr-1 or IgG isotype control (n = 3 mice/group), 7 days post B16F10 tumor cell inoculation (**, P = 0.0037; ***, P < 0.0001). d, Percentages of CD11b+Gr-1+ cells in the tumor of WT mice treated with anti-Gr-1 or IgG isotype control (n = 3 mice/group), 7 days post B16F10 tumor cell inoculation (***, P = 0.0008). e, Percentages of CD4+CD25+ cells in the spleen of WT and Rel−/− mice treated with anti-CD25 or IgG isotype control (n = 3 mice/group), 14 days post B16F10 tumor cell inoculation (***, P < 0.0001). f, Tumor size on Day 14 of WT and Rel−/− mice treated as in Fig. 1c–e. n = 16 for the WT + IgG group, n = 15 for the Rel−/−+IgG group, n = 11 for the WT + anti-Gr1 group, n = 10 for the Rel−/−+anti-Gr1 group, n = 9 for the WT + anti-CD25 group, and n = 9 for the Rel−/−+anti-CD25 group (*, P = 0.0303; **, P < 0.01; ***, P = 0.0001). g, c-Rel expression in Gr-1+ and Gr-1- splenocytes of LyzM-Cre (Cre) and LyzM-Cre RelF/F (RelF/F) tumor-bearing mice as determined by Western blot. Representative blots from biologically independent experiments were shown and the bar graph shows the relative quantities of the c-Rel protein in the corresponding group shown below (n = 3 mice for each group; *, P = 0.0101). h,i, Percentages of CD11b+Ly6G+ (h) and CD11b+Ly6C+ (i) leukocytes in the tumor of WT and Rel−/− mice treated in Fig. 1a (n = 5 mice/group). **, P = 0.0011 for panel h; **, P = 0.0087 for panel i. j, Percentages of CD8+CD25+ leukocytes in the tumor of LyzM-Cre and LyzM-Cre RelF/F mice treated in Fig. 1f (n = 8 mice/group; ***, P = 0.0005). k-n, Percentages of the indicated leukocyte subsets in the spleen of WT and Rel−/− mice treated in Fig. 1a. (n = 4 mice/group in the panels k and l; n = 5 mice/group in the panels m and n; *, P = 0.037). Statistical significance was determined by two-tailed Mann-Whitney U-test (a), two-tailed unpaired t-test (b-f, h-j, m), or one-way ANOVA with Tukey post-hoc test (g). For all panels, data are presented as means ± s.e.m.

Source data

Extended Data Fig. 2 Percentages of immune cell subsets in tumor-bearing and naïve LyzM-Cre and LyzM-Cre RelF/F mice.

a, Percentages of CD11b+Gr-1+ cells in the spleen of mice treated in Fig. 1f. n = 6 mice/group. b-d, Percentages of CD4+ cells in the spleen (b, n = 6/group), blood (c, n = 6/group), and tumor (d, n = 8/group) of mice treated in Fig. 1f. e-g, Percentages of CD4+CD25+ cells in the spleen (e, n = 6/group), blood (f, n = 6 for the LyzM-Cre group and n = 5 for the LyzM-Cre RelF/F group), and tumor (g, n = 3 for the LyzM-Cre group and n = 6 for the LyzM-Cre RelF/F group) of mice treated in Fig. 1f. h-j, Percentages of CD8+ cells in the spleen (h, n = 6/group), blood (i, n = 6/group), and tumor (j, n = 8/group) of mice treated in Fig. 1f. k-n, Percentages of the indicated leukocyte subsets in the tumor (n = 3 for the LyzM-Cre group and n = 6 for the LyzM-Cre RelF/F group) of mice treated in Fig. 1f (k, l), and the spleen (n = 3 for the LyzM-Cre group and n = 5 for the LyzM-Cre RelF/F group) of naïve mice (m, n). (***, P = 0.0002) Statistical significance was determined by two-tailed unpaired t-test (k). For all panels, data are presented as means ± s.e.m.

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Extended Data Fig. 3 Reduced suppressive function, reactive oxygen species (ROS) production, and cell growth in Rel−/− MDSCs.

a,b, Representative flow cytometry plots from the MDSC-T cell suppression assay for Fig. 2a (a) and Fig. 2b (b). c, Growth of bone marrow-derived MDSCs from WT (n = 4 mice/group) and Rel−/− (n = 9 mice/group) mice. (*, P = 0.03299). d, ROS production by bone marrow-derived MDSCs from WT and Rel−/− mice (n = 6 mice/group; ***, P < 0.0001). e, Percentages of CD11b+Ly6G+ and CD11b+Ly6C+ cells in bone marrow-derived MDSCs from WT and Rel−/− mice. Data representative of three independent experiments. f, Tumor growth in WT mice injected s.c. with B16F10 tumor cells plus Rel−/− MDSCs infected with control (n = 5 mice) or Rel-expressing retroviruses (n = 6 mice) (**, P = 0.0041). g, Percentages of CD44high cells in intratumor CD8+ cells of WT mice treated as in panel f. (n = 4 mice/group; *, P = 0.0187). h, The degree of suppression, at the indicated Effector:T cell ratio, of CD8+ T cell proliferation by bone marrow-derived MDSCs and BMDMs from naïve mice (n = 3 mice/group). The concentrations of anti-CD3 and anti-CD28 used (125 ng/mL each) were half of those in Fig. 2b (***, P < 0.0001). i, Representative flow cytometry plots for the MDSC-T cell suppression assay for Panel h. Statistical significance was determined by two-tailed unpaired t-test (c-e, g), two-tailed Mann-Whitney U-test (f), or two-way ANOVA with Tukey post-hoc test (h). Data are presented as means ± s.e.m. (c,d,f,h).

Source data

Extended Data Fig. 4 Rel gene deletion in MDSCs leads to upregulated expression of inflammatory genes and decreased Cebpb downstream genes.

a,b, Results from Ingenuity Pathway analysis of the RNA-seq data of bone marrow-derived WT and Rel−/− MDSCs, showing upregulated ‘inflammatory response’ genes (a, red) and downregulated Cebpb downstream genes (b, green) in Rel−/− MDSCs. Statistical significance was determined by calculated a right-tailed Fisher’s Exact Test. All data are pooled from 3 independent experiments.

Extended Data Fig. 5 Rel gene deletion in macrophages results in decreased expression of inflammatory genes.

BMDMs from WT and Rel−/− mice were treated with vehicle or LPS (100 ng/mL) for 1 hour and gene microarray analysis was performed for ~30,000 murine genes. Ingenuity Pathway Analysis was performed to identify c-Rel-regulated genes that were downstream of LPS response. Blue genes were decreased, and red genes were increased in Rel−/− cells as compared to WT cells, after LPS treatment.

Extended Data Fig. 6 The inhibitor is c-Rel-specific and blocks MDSC suppressive functions.

a, Relative numbers of WT and Rel−/− human Jurkat T cells treated with the c-Rel inhibitor (2.5 μM) or vehicle for the indicated times (n = 3 biologically independent cultures/group). b,c, Relative numbers of EL4 (b, n = 3 biologically independent cultures/group) and B16F10 (c, n = 3 biologically independent cultures/group) cells treated with or without the c-Rel inhibitor (5 μM) (**, P = 0.00767). d,e, Relative mRNA levels of IL-2 in WT, Rel−/− human Jurkat T cells (d, n = 3 biologically independent cultures/group; **, P = 0.0047), and normal human PBMCs (e, n = 3 biologically independent cultures/group; *, P = 0.0241) that were treated with or without c-Rel inhibitor (1 μM). For stimulation, plate-coated anti-mouse CD3 (250 ng/ml) plus soluble anti-mouse CD28 (250 ng/ml), or PMA (10 ng/ml) plus ionomycin (1 µM) were added to the culture for 4 hours, as indicated. f, Preferential inhibition of c-Rel binding to DNA by the c-Rel inhibitor. Western blot for the indicated NF-κB proteins after the NF-κB oligonucleotide pull-down of the nuclear extracts of WT and Rel−/− splenocytes treated with the c-Rel inhibitor (5 µM) or vehicle control (Ctr). g, Body weight change of mice that were injected i.p. with vehicle control only (n = 7 mice), or injected s.c. with B16F10 cells and i.p. with the c-Rel inhibitor (n = 3 mice) or vehicle control (n = 4 mice) as indicated. h,i, Representative flow cytometry plots of the MDSC-T cell suppression assay for Fig. 6e (h) and Fig. 6f (i). Data are presented as means ± s.e.m (a-e, g, and i). n = 3 mice/group (d,e,j). Statistical significance was determined by two-tailed unpaired t-test (b-e).

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Li, T., Li, X., Zamani, A. et al. c-Rel is a myeloid checkpoint for cancer immunotherapy. Nat Cancer 1, 507–517 (2020). https://doi.org/10.1038/s43018-020-0061-3

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