Fig 2: The ectopic expression of TWIST1 or ZEB1 in the ZEB1low 501MEL cell line promotes BRAFi resistance 501MEL cells were infected with retroviruses expressing either TWIST1 or ZEB1. Western blot analyses of ZEB1 and TWIST1. GAPDH was used as a loading control.Quantitative PCR analyses of MITF expression upon ZEB1 ectopic expression (mean ± SD, n = 3). FACS analyses of p75 expression upon ZEB1 ectopic expression, after 10 days with or without 100 nM PLX4032 treatment, showing that p75 is not induced in either of these conditions.Soft agar colony formation assay in the presence or absence of PLX4032 (100 nM). Scale bar = 500 μm. The graph represents the mean number of colonies (± SD) in three independent experiments (Student's t‐test). Source data are available online for this figure.

Fig 3: 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).

Fig 4: High ZEB1 and low MITF levels are associated with inherent resistance to MAPKi in BRAFV 600‐mutated melanoma tumors MITF mRNA expression levels according to ZEB1 expression in 467 melanoma tumors from the TCGA data set (Pearson correlation test). Representative pictures of ZEB1 and MITF immunostaining in primary melanomas. Scale bar = 40 μm. The aberrant activation of ZEB1 in melanomas is correlated with a MITFlow phenotype.Representative pictures of ZEB1 immunostaining in BRAF V600 tumors from patients, classified into ZEB1high, ZEB1int, and ZEB1low subgroups based on the intensity of ZEB1 staining and on the percentage of cells positive for ZEB1. Scale bar = 80 μm.Pie charts representing the distribution of ZEB1 alone (upper part), or ZEB1 and TWIST1 (lower part) immunohistochemical staining in tumors according to their initial response to vemurafenib ± cobimetinib treatment. ZEB1 ± TWIST1 levels are higher in MAPKi primary resistant melanomas (initial non‐responders) compared to tumors that initially respond to treatment (n = 70, Fisher's exact test).Representative pictures of ZEB1 and MITF immunostainings, before and after vemurafenib treatment, in the tumor from patient 1, exhibiting primary resistance to BRAFi. Scale bar = 80 μm. Right: Magnification of MITFhigh and MITFlow clones in the resistant tumor under treatment. Arrows indicate endothelial and stromal cells that also show positive staining for ZEB1, besides tumor cells.

Fig 5: 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.