Figures and data
MRGPRX4 is highly expressed in human melanoma
(A,B) MRGPRX4 mRNA expression in (A) GTEx normal skin and TCGA melanoma (SKCM) and (B) in uveal melanoma compared to other TCGA cancers. Data is analysed using Mann-Whitney U test or one-way ANOVA followed by post-hoc test. (C-E) MRGPRX4 and canonical gene expression in normal human skin (C) melanocytes, (D) keratinocytes and (E) fibroblasts. (F) Cancer type-specific enrichment of MRGPRX4 across TCGA PanCancer Atlas malignancies. Each point represents one TCGA cancer type. The x-axis shows the median MRGPRX4 expression difference between that cancer type and all remaining TCGA cancers, and the y-axis shows the −log10 Wilcoxon p-value for the same one-vs-rest comparison. Cutaneous melanoma is highlighted in red and represents SKCM compared with all non-SKCM cancers. (G) Cell type-specific enrichment of MRGPRX4 in DepMap cancer cell line data (H) MRGPRX4 expression in DepMap skin cancer cell lines subdivided by cancer type. Data is analysed by one-way ANOVA followed by post-hoc test. (I) Relationship between MRGPRX4 copy number and expression in TCGA SKCM patients (J) Relationship between MRGPRX4 methylation and expression in TCGA SKCM patients. Red dotted line indicates best-fit linear regression (p<0.001). (K) Confocal images of human cutaneous melanoma sample stained for MRGPRX4 (red), SOX10 (magenta), HMB-45 (green) and Hoechst (cyan) nuclear counterstain. 20x magnification, scale bar 50um. (L) UMAP of integrated melanoma patient scRNA-seq datasets annotated into major melanoma transcriptional states. (M) MITF program scores across melanoma transcriptional states, showing highest activity in melanocytic cells and reduced activity in dedifferentiated lineages. (N) Heatmap of top cluster genes enriched in melanoma transcriptional states (O) MRGPRX4 expression in melanoma transcriptional states
MRGPRX4 drives metastatic melanoma in mouse model
(A-G) (A) Representative image of melanoma on the ears and tails of tamoxifen-treated TyrCreER+;MRGPRX4LSL+/- mice (11 weeks old). (B) Cutaneous melanoma visualized following depilation. (C) Gross and H&E images of uveal melanoma from control TyrCreER-;MRGPRX4LSL+/-and melanoma TyrCreER+;MRGPRX4LSL+/- animals. (D) Meningeal melanoma visualized in TyrCreER+;MRGPRX4LSL+/- mouse after removal of skull and brain. TyrCreER+;MRGPRX4LSL+/-melanoma animals showing (E) lymph node metastasis (white arrows), (F) lung metastasis (white arrows) and (G) melanoma cells in brain parenchyma. (H) Tile-scan confocal images of MRGPRX4 staining in tamoxifen-treated experimental (TyrCreER+;MRGPRX4LSL+/-) and control (TyrCreER-;MRGPRX4LSL+/-) littermate mice at 20x magnification. Data is representative of 2-3 independent experiments. Scale bar, 50um (I-J) Proteomics analysis comparing tumor (TyrCreER+;MRGPRX4LSL+/) vs control (TyrCreER-;MRGPRX4LSL+/-) skin (I) Volcano plot where red dots are proteins significantly enriched in melanoma, and blue dots indicate significantly decreased proteins. (J) Top pathways enriched in melanoma skin samples (GSEA). Bubble plot shows normalized enrichment scores (NES), where bubble diameter represents statistical significance (-log10 p-value) and color indicates pathway database (GO biological process, KEGG, Reactome). (K) Cross-species conservation between MRGPRX4-driven melanoma proteome and human SKCM transcriptomics. Mouse protein log2 fold-change values (melanoma/control) aligned to orthologous human RNA expression (TCGA SKCM; z-scored). Genes enriched in mouse tumors also show elevated expression in human melanoma (p = 2.9 × 10⁻¹², linear regression) (L-O) Immune microenvironment in MRGPRX4-driven melanoma. (L) Gating scheme for the identification of tumor myeloid celltypes. Frequencies of (M) myeloid cells and (N) PD-L1+ myeloid cells and (O) PD-L1 MFI of CD45neg population in skin samples of control (TyrCreER-;MRGPRX4LSL+/-) vs melanoma mice (TyrCreER+;MRGPRX4LSL+/-). Data are mean+SEM and each dot represent an animal (n = 4-5), and are representative of 2-3 independent experiments. For all experiments both male and female mice were used and data are analyzed by Student’s t test or one-way ANOVA followed by post hoc test. *p < 0.05, **p < 0.01
MRGPRX4 tumors display an enhanced NC/EMT signature
(A-H) Spatial transcriptomic of MRGPRX4-driven melanoma (TyrCreER+;MRGPRX4LSL+/-). (A) Representative H&E-stained cross-section illustrating tumor, dermis, epidermis, stromal fascia, dWAT and muscle layers. Scale bar, 2 mm. (B) Spatial segmentation of Visium spots showing tissue compartment assignment based on canonical gene enrichment. (C) Spatial delineation of tumor-positive (blue) and tumor-negative (orange) regions (D–H) Spatial refinement using BayesSpace followed by AUCell scoring of predefined melanoma transcriptional programs. Shown are per-spot enrichment scores for (D) melanocytic, (E) mesenchymal-like, (F) RNA-processing, (G) antigen-presentation, and (H) hypoxia-associated states. Color intensity corresponds to AUCell score per spatial location. (I,J) (I) UMAP projection of dissociated melanoma cells colored by cell state, including melanocytic (orange), mesenchymal-like (hypoxia-associated; blue-green), IFN-stress-like (green), mesenchymal-like invasive (yellow), and cycling (dark blue) populations. (J) UCell-based scoring of melanoma-intrinsic programs shown at single-cell resolution of melanocytic, mesenchymal-like state, NCSC-like, and IFN-stress modules. Dot intensity reflects AUC score per cell. (K–M) (K) UMAP embedding of tumor-derived malignant cells colored by genotype, including TyrCreER+;MRGPRX4LSL (red), Tyr::NRASQ61K/°;Ink4a−/− (green), and TyrCreER;Ptenfl/fl;Braf V600E (blue) models. (L) UMAP colored by transcriptional states as indicated. (M) Heatmap of canonical state-defining genes across malignant clusters. (N, O) (O) Representative multiplex confocal image showing melanoma cells expressing MRGPRX4 (green) and PRRX1 (red) positioned directly adjacent to CD31⁺ blood vessels (magenta) and PROX1⁺ lymphatic endothelium (cyan). Nuclei are stained with Hoechst (white). Scale bar, 50 µm. (O) Quantification of phenotypically defined tumor subpopulations in blood vessel (BV) and lymphatic vessel (LV) positive areas (BV/LV+) versus BV/LV⁻ fields. BV/LV⁺ regions show significant enrichment of dual-positive MRGPRX4⁺PRRX1⁺ invasive cells as compared to BV/LV- areas (paired Wilcoxon test, p=0.0059). Dots represent independent imaged fields. (P, Q) (P) Volcano plot of differential expression between A2058 MRGPRX4 ⁺/⁺ and MRGPRX4 ⁻/⁻ cells highlighting down-regulation of melanocytic lineage markers (e.g., TYR, TYRP1, PMEL) and up-regulation of mesenchymal and extracellular-matrix genes (e.g., COL1A1, COL5A1, RARRES2, SERPINA3). (Q) GSEA enrichment analysis. Circle size denotes -log₁₀(p-value); color indicates database (Hallmark, KEGG, Reactome).
MRGPRX4 promotes an invasive phenotype dominated by an aberrant PI3K- AKT pathway
(A) Cell numbers in control and MRGPRX4 shRNA knockdown in A2058 cells. Data are mean+SEM and representative of 2 independent experiments. (B) MRGPRX4+/+ and MRGPRX4-/- cells were cultured and ATP measurements were taken every 24 hours. Data are mean+SEM and representative of 2-3 independent experiments. (C) Tumor volumes in NSG mice (n=5-7) injected with MRGPRX4+/+ and MRGPRX4-/- cells. Data are mean+SEM and representative of 2 independent experiments. (D-E) In vitro invasion assay. (D) Representative images (scale bar 250 um) of spheroids on days 0, 3 and (E) invasion of MRGPRX4+/+ and MRGPRX4-/- spheroids relative to wildtype on day 3. Data are mean+SEM and pooled from 2 independent experiments. (F-J) In vivo invasion assay in NSG mice injected i.v. with GFP+ MRGPRX4+/+ or GFP+MRGPRX4-/- cells and assessed 6-8 weeks later. (F) Representative flow plots, and (G) numbers of GFP+ cells in the lungs. (H) Representative images of the liver, (I) representative flow plots, and (G) numbers of GFP+ cells in the liver. Data are mean+SEM and pooled from 2 independent experiments. (K-M) Basal activity measured using PRESTO-Tango assay in HTLA cells transiently transfected with increasing doses of (K) MRGPRX1, (L) MRGPRX2 and (M) MRGPRX4. Data are mean+SEM and representative of 2-3 independent experiments. (N-O) Phosphosite enrichment in MRGPRX4-driven melanoma. (N) Heatmap of differentially regulated phosphosites in control (TyrCreER-;MRGPRX4LSL+/-) or melanoma skin samples (TyrCreER+MRGPRX4LSL+/-) or (O) Kinase enrichment analysis (KEA3) in tumor samples based on phosphosite signatures. (N) heatmap nomenclature for control vs melanoma is different (P) Western blot for pAKT, pS6K and pERK1/2 in MRGPRX4+/+ and MRGPRX4-/- A2058 cells or A2058 cells subjected to control (shLuc) or shRNA-mediated MRGPRX4 knockdown, cultured in decreasing serum conditions. Data are representative of 2-3 independent experiments. (Q,R) Cultured HTLA cells were transiently transfected with indicated MRGPRX-Tango constructs and treated with vehicle or MRGPRX4 antagonist. (Q) MRGPRX4 and (R) MRGPRX1 and MRGPRX2 basal activity was assessed. Data are mean+SEM and representative of 2 independent experiments. (S) A2058 cells were treated with vehicle or MRGPRX4 antagonist and cellular proliferation using ATP assay was measured on Day 6. Data are mean+SEM and representative of 2 independent experiments. (T,U) A2058 cells were treated with vehicle or MRGPRX4 antagonist and invasion using the spheroid assay was assessed 48h later for (T) MRGPRX4+/+ A2058 cells and (U) MRGPRX4-/-A2058 cells. Data are mean+SEM and pooled from 2 independent experiments. For all experiments both male and female mice were used and data are analyzed by Student’s t test or one-way ANOVA followed by post hoc test. *p < 0.05, **p < 0.01,
