ZEB1 transcription factor promotes immune escape in melanoma

Abstract

Background: The efficacy of immunotherapies in metastatic melanoma depends on a robust T cell infiltration. Oncogenic alterations of tumor cells have been associated to T cell exclusion. Identifying novel cancer cell-intrinsic non-genetic mechanisms of immune escape, the targeting of which would reinstate T cell recruitment, would allow to restore the response to anti-programmed cell death protein 1 (PD-1) antibody therapy. The epithelial-to-mesenchymal transition (EMT)-inducing transcription factor ZEB1 is a major regulator of melanoma cell plasticity, driving resistance to mitogen-activated protein kinase (MAPK) targeted therapies. We thus wondered whether ZEB1 signaling in melanoma cells may promote immune evasion and resistance to immunotherapy.

Methods: We evaluated the putative correlation between ZEB1 expression in melanoma cells and the composition of the immune infiltrate in a cohort of 60 human melanoma samples by combining transcriptomic (RNA-sequencing) and seven-color spatial multi-immunofluorescence analyses. Algorithm-based spatial reconstitution of tumors allowed the quantification of CD8+, CD4+ T cells number and their activation state (PD-1, Ki67). ZEB1 gain-of-function or loss-of-function approaches were then implemented in syngeneic melanoma mouse models, followed by monitoring of tumor growth, quantification of immune cell populations frequency and function by flow cytometry, cytokines secretion by multiplex analyses. Chromatin-immunoprecipitation was used to demonstrate the direct binding of this transcription factor on the promoters of cytokine-encoding genes. Finally, the sensitivity to anti-PD-1 antibody therapy upon ZEB1 gain-of-function or loss-of-function was evaluated.

Results: Combined spatial and transcriptomic analyses of the immune infiltrates in human melanoma samples demonstrated that ZEB1 expression in melanoma cells is associated with decreased CD8+ T cell infiltration, independently of β-catenin pathway activation. ZEB1 ectopic expression in melanoma cells impairs CD8+ T cell recruitment in syngeneic mouse models, resulting in tumor immune evasion and resistance to immune checkpoint blockade. Mechanistically, we demonstrate that ZEB1 directly represses the secretion of T cell-attracting chemokines, including CXCL10. Finally, Zeb1 knock-out, by promoting CD8+ T cell infiltration, synergizes with anti-PD-1 antibody therapy in promoting tumor regression.

Conclusions: We identify the ZEB1 transcription factor as a key determinant of melanoma immune escape, highlighting a previously unknown therapeutic target to increase efficacy of immunotherapy in melanoma.

Trial registration number: NCT02828202.

Keywords: immunotherapy; lymphocytes; melanoma; tumor escape; tumor microenvironment; tumor-infiltrating.

Conflict of interest statement

Competing interests: None declared.

© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

High ZEB1 expression in tumor cells is associated with decreased CD8+ T cell infiltration in a cohort of human melanoma samples. (A) Schematic diagram of the RNA-seq and spatial multi-immunofluorescence (multi-IF) analyses of human cutaneous melanoma samples. (B) Representative pictures of multi-immunofluorescence opal staining for ZEB1 (red), SOX10 (white), CD8 (green), CD4 (orange), Ki67 (magenta) and PD-1 (yellow). Blue: DAPI. Scale bar=100 µm. ZEB1-expressing CD8, CD4, and stromal cells are indicated with green, orange and red arrows, respectively. (C) Schematic representation of digital image analyses pipeline and tumor reconstruction. The entire tumor sections were selected for the analysis. Each cell was detected based on the nucleus and associated with a phenotype based on the expression of SOX10 (melanoma cell), CD8 (CD8+ T cells), and CD4 (CD4+ T cells). For each cell, both the location and immunofluorescence intensities of each markers were stored in a so-called phenotype matrix. Tumors were then reconstructed, and quantification was performed using the R software. (D) Whole tumor regions were spatially reconstituted using the R software. Melanoma cells are represented using a color gradient (green, orange, red, and black) corresponding to ZEB1 nuclear intensity in melanoma cells (respectively <1; (1;3); (3;10); >10). Spatial reconstitution of CD8 localization is represented with blue dots. The black insets correspond to representative pictures of multi-IF opal staining for ZEB1 (red), CD8 (green), and SOX10 (white) of a ZEB1 low/CD8 infiltrated (upper panel) and a ZEB1 high/CD8 excluded (lower panel) tumor. Blue: DAPI. Note that in the ZEB1 low tumor, ZEB1 high cells are not melanoma SOX10-positive cells. Scale bars=100 µm. (E) Stacked bar representing the percentage of CD8 excluded (light blue) and CD8 infiltrated (dark blue) tumors within ZEB1 low and ZEB1 high tumors. (F) Percentage of CD8 T cells as quantified by IF in ZEB1 low (green: n=28) and ZEB1 high (red: n=10) tumors (mean with SD, Mann-Whitney test).

Figure 2

ZEB1-mediated T cell recruitment defect is independent of β-catenin pathway activation. (A) β-catenin ssGSEA score calculated from the RNA-seq data, using the previously described score from Spranger et al based on seven genes: EFNB3, APC2, TCF1, C-MYC, TCF12, VEGFA and CTNNB1. Tumors were classified as ZEB1 low (n=38) and ZEB1 high (n=12) following the IF analysis (mean with SD, Mann-Whitney test). (B) Single cell RNA-seq from Jerby-Arnon et al. X-axis: β-catenin single-sample gene set enrichment analysis (ssGSEA) score calculated on melanoma cells. Y-axis: ZEB1 expression (TPM) in melanoma cells. R²=0.0005748, p value=0.2817. (C) Pie charts representing the proportion of nuclear β-catenin (dark orange) and cytoplasmic β-catenin (light orange) tumors within ZEB1high melanoma (n=12). Representative pictures of cytoplasmic (on the left) and nuclear (on the right) β-catenin staining (in red). DAPI in blue. Scale bars=100 µm and 200 µm, respectively. (D) Representative picture of cytoplasmic β-catenin staining in a ZEB1high tumor: β-catenin (red), ZEB1 (yellow) and SOX10 (cyan). DAPI in blue. Scale bar=50 µm. RNA-seq, RNA-sequencing.

Figure 3

ZEB1 ectopic expression in melanoma cells increases tumor growth in immunocompetent mice. (A) Br16M3 and NR6.1 murine melanoma cells were infected with retroviruses expressing ZEB1. Western blot analyses of ZEB1. GAPDH was used as a loading control. (B) 3×106 Br16M3 (left) or NR6.1 (right) control (green) or ZEB1-overexpressing (red) cells were injected subcutaneously into C57BL6 immunocompetent mice (upper panel) or into immunodeficient RAG2 KO mice (lower panel). The mean tumor volume is represented (±SEM, Mann-Whitney test). Br16M3: n=17–19 and n=4 for immunocompetent and RAG2 knock-out (KO) mice respectively; NR6.1: n=5 for both immunocompetent and RAG2 KO mice.

Figure 4

ZEB1 ectopic expression in melanoma cells impairs the recruitment of CD8+ T lymphocytes. (A) FACS analyses of CD45 infiltration 2 weeks after injection into immunocompetent C57BL6 mice of Br16M3 (left) or NR6.1 (right) models: control (blue); ZEB1 ectopic expression (red), n=4–5 per group. Bar chart representing the number of CD45+ cells/g of tumor (mean with SD, Mann-Whitney test). (B and C) FACS analyses of CD8+ and CD4+ infiltration in Br16M3 and NR6.1 control and ZEB1-overexpressing tumors. Bar charts representing the number of CD8+ (B) or CD4+ (C) cells per gram of tumor. (D) Representative pictures of H&E coloration, ZEB1 or CD8 immunostaining in Br16M3 (left) and NR6.1 (right) control and ZEB1-overexpressing tumors collected 2 weeks after injection into C57BL6 mice. Purple chromogen was used for IHC visualization in pigmented NR6.1 tumors instead of brown DAB for non-pigmented Br16M3 tumors. Scale bars=50 µm. Arrows indicate purple positive CD8+ T cells. (E) Quantification of CD8+ T cell infiltration in Br16M3 and NR6.1 control versus ZEB1-overexpressing tumors. Bar chart representing the number of CD8+ T cells per mm² of tumor (n=5–6, mean with SD, Mann-Whitney test). (F) FACS analyses of FOXP3+ CD4+ regulatory T cells (Treg) infiltration in Br16M3 and NR6.1 control and ZEB1-overexpressing tumors. Bar charts representing the percentage of Tregs among CD4+ T cells in Br16M3 control (blue) and ZEB1-overexpressing (red) tumors, 14 (left) and 21 (right) days after injection (mean with SD, Mann-Whitney test). (G) Bar chart representing the ratio between the number of CD8+ T cells and the number of Tregs in Br16M3 control and ZEB1-overexpressing tumors (mean with SD, Mann-Whitney test).

Figure 5

Melanoma cell-intrinsic ZEB1 signaling decreases the production of T cell-attracting chemokines. (A) Heatmap representing the relative quantity of 35 analytes measured using the Meso Scale Diagnostics (MSD) technology in supernatants of dilacerated control (n=5) or ZEB1-overexpressing (n=8) Br16M3 tumors. TGF-β isoforms were also quantified with the same technology in n=6 and n=8 control or ZEB1-overexpressing tumors, respectively. Mann-Whitney test. (B) Bar charts representing the concentration of CXCL10, CCL4, TGF-β1 and 2 (pg/mL), in the supernatant of dilacerated Br16M3 control (n=5 or 6) and ZEB1-overexpressing (n=8) tumors (mean with SD, Mann-Whitney test). (C) Chromatin immunoprecipitation assay of ZEB1 showing binding to the promoter of Cxcl10 and Ccl4 in Br16M3 cells. The fraction of chromatin bound to the promoter, with IgG control or anti-ZEB1 antibody is represented as a percentage of input (n>=3) (mean with SD, t-test). (D) NR6.1 control and ZEB1-overexpressing murine melanoma cells were infected with control or Cxcl10-expressing lentiviruses. qPCR analyses of CXCL10 mRNA expression in NR6.1 control (CT), ZEB1 control (Z1 CT) and ZEB1-overexpressing CXCL10 (Z1 CXCL10) (n=1). (E) 2.5×106 NR6.1 ZEB1 control (Z1 CT) and ZEB1 CXCL10 (Z1 CXCL10) cells were injected subcutaneously into C57BL6 immunocompetent mice (n=5 per group). The mean tumor volume is represented (±SEM, Mann-Whitney test). (F) FACS analyses of CD45 and CD8 infiltration 17 days after injection into immunocompetent C57BL6 mice of NR6.1 ZEB1 control (n= 4) and ZEB1 CXCL10 (n= 5). Bar chart representing the number of CD45+ cells and CD8+ T cells/g of tumor (mean with SD, Mann-Whitney test). (G) Quantification of CD8+ T cell infiltration in NR6.1 ZEB1 control and ZEB1 CXCL10 tumors. Bar chart representing the number of CD8+ T cells per mm² of tumor (n=5 per group, mean with SD, Mann-Whitney test).

Figure 6

Zeb1 knock-out in melanoma cells strongly reduces tumor growth and favors CD8+ T cell infiltration. (A) Br42M6 murine melanoma cells were infected with lentiviruses expressing the Cas9 and guide RNA (sgRNA) targeting ZEB1 or scramble sgRNA. 42C1 is a scramble clone, and 42Z6 is a Zeb1 knock-out clone. Western blot analyses of ZEB1. GAPDH was used as a loading control. (B) 2.5×106 42C1 (orange) or 42Z6 (blue) cells were injected subcutaneously into C57BL6 immunocompetent mice (left, n=6–10 per group) or into RAG KO mice (right, n=3 per group). The mean tumor volume is represented (±SD, Mann-Whitney test). (C) Representative pictures of ZEB1 and CD8 immunostaining (brown, DAB) in 42C1 (scramble) and 42Z6 (ZEB1 KO) tumors. Scale bars=50 µm. Quantification of CD8 infiltration in 42C1 (scramble) and 42Z6 (ZEB1 KO) tumors. Bar chart representing the number of CD8+ T cell per mm² of tumor (mean with SD, Mann-Whitney test).

Figure 7

ZEB1 overexpression in melanoma cells triggers resistance while knock-out of Zeb1 improves the response to immune checkpoint blockade. (A) NR6.1 murine melanoma cells were infected with retroviruses expressing ZEB1. 2×106 NR6.1 control (red) or ZEB1-overexpressing (blue) cells were injected subcutaneously into C57BL6 immunocompetent mice (five per group) and treated with either anti-PD-1 (dashed lines) or control isotype (iso, solid lines) 5, 7, and 9 days after injection. The mean tumor volume for 5 mice is represented (±SD, Student’s t-test). (B) NR6.1 murine melanoma Kaplan-Meier survival curves with log-rank test, mice were euthanized when tumor volume reached 1500mm3 or diameter >15mm. (C) 2×106 control 25C19 (orange) or Zeb1 KO 25Z19 (blue) cells were injected subcutaneously into C57BL6 immunocompetent mice (five per group) and treated with either anti-PD-1 (dashed lines) or control isotype (iso, solid lines) 5, 7, and 9 days after injection. The mean tumor volume for 5 mice is represented (±SD, Student’s t-test). (D) Br25F4 murine melanoma Kaplan-Meier survival curves with log-rank test, mice were euthanized when tumor volume reached 1500 mm3 or diameter >15mm. (*P<0.05, **p<0.01, ***p<0.001, ****p<0.0001).