A Multi-Step Immune-Competent Genetically Engineered Mouse Model Reveals Phenotypic Plasticity in Uveal Melanoma

Abstract

Uveal melanoma (UM) is a highly aggressive intraocular malignancy with limited therapeutic options for metastatic disease. Existing transgenic UM mouse models inadequately recapitulate human disease progression, while transplant models lack immune competence for studying the tumor immune microenvironment and therapeutic interventions. To address these limitations, we developed a genetically engineered mouse model incorporating stepwise genetic alterations implicated in human UM progression. Spatiotemporally controlled expression of mutant GNAQQ209L from the endogenous locus induced choroidal nevi with limited penetrance. Concomitant BAP1 deletion enhanced nevus formation, while further MYC activation led to fully penetrant intraocular tumors with the potential to disseminate. Single-cell RNA sequencing revealed malignant cells segregated into melanocytic and neural crest-like subpopulations characterized by distinct transcriptional and biosynthetic programs. Trajectory analyses inferred dedifferentiation from the melanocytic toward the neural crest-like state during tumor progression. Comparison to human UM revealed commonalities with highly aggressive class 2 UM, including gene expression signatures and copy number gains affecting genes that map to human chromosome 8q beyond the activated MYC allele, suggesting cooperative effects of multiple drivers in this chromosomal region. The tumor microenvironment featured immunosuppressive macrophage populations and exhausted T cells, closely resembling human UM. This physiologically relevant, immune-competent model provides a platform for investigating UM biology, functionally characterizing candidate driver genes, and developing immune-based therapeutic strategies.

Figure 1.. Generation of a genetically engineered mouse model of choroidal nevi driven by Gnaq mutation and Bap1 loss

A, Schematic diagram of the Gnaq conditional activation (Gnaq^CA) allele (Created in BioRender. Xu, X. (2026) https://BioRender.com/ccwj1ps). B, Mouse embryonic fibroblasts (MEFs) isolated from Gnaq^CA mice were infected by lentiviral Cre. PCR amplification and Sanger sequencing of Gnaq exon 5 from cDNA confirmed expression of the Q209L mutation. C, Western blot of Cre-infected Gnaq^CA MEFs revealed changes in Gαq downstream signaling but physiological Gnaq expression. D, Schematic diagram of Gnaq conditional activation (Gnaq^CA) in the uvea by in situ lentiviral delivery of Cre and Luciferase (pL-CL) (Created in BioRender. Xu, X. (2026) https://BioRender.com/ccwj1ps). E, Representative in vivo bioluminescence imaging of a Gnaq^CA mouse infected with pL-CL. F, Quantification of frequency of choroidal hyperplasia in Gnaq^CA mice at the indicated timepoints. G, Representative image of H&E staining showing localized choroidal hyperplasia in a Gnaq^CA mouse. H, Representative fluorescent IHC showing Sox10 and Rpe65 expression in uveal tracts of wildtype and Gnaq^CA mice. I, Schematic diagram of Gnaq conditional activation and Bap1 conditional CRISPR knockout (Gnaq^CA; Bap1^CKO) in the uvea by in situ lentiviral delivery of Cre, Luciferase, and sgBap1 (pL-CLB) (Created in BioRender. Xu, X. (2026) https://BioRender.com/ccwj1ps). J, Quantification of choroidal hyperplasia and tumors in Gnaq^CA; Bap1^CKO mice at 7 months. K and L, Representative images of H&E staining of choroidal hyperplasia (K) and micro-tumor (L) in Gnaq^CA; Bap1^CKO mice. M, Representative fluorescent IHC showing Sox10 and Rpe65 expression in the uveal tract of a Gnaq^CA; Bap1^CKO mouse.

Figure 2.. MYC promotes UM formation

A, Schematic diagram of Gnaq conditional activation, Bap1 conditional CRISPR knockout, and Myc conditional knock-in (Gnaq^CA; Bap1^CKO; Myc^CKI) in the uvea by in situ delivery of pL-CLB (Created in BioRender. Xu, X. (2026) https://BioRender.com/ccwj1ps). B, Kaplan-Meier overall survival curve of Gnaq^CA; Bap1^CKO; Myc^CKI mice after pL-CLB administration. C, Representative in vivo bioluminescence imaging showing strong signal from a primary intraocular tumor. D and E, Representative images of H&E staining of intraocular tumors from Gnaq^CA; Bap1^CKO; Myc^CKI mice. F, Representative fluorescent IHC showing Sox10 and Rpe65 expression in a tumor from a Gnaq^CA; Bap1^CKO; Myc^CKI mouse. Tumors are clonally positive for Sox10 and negative for RPE65. G, Representative image of Periodic acid–Schiff (PAS) staining of a tumor from a Gnaq^CA; Bap1^CKO; Myc^CKI mouse. Tumors displayed minimal PAS positivity. H, Representative image of multiplex IHC (mIHC) showing Gli2, Melan-A, and Tyrp1 expression in an unpigmented tumor from Gnaq^CA; Bap1^CKO; Myc^CKI mice. I and J, Representative images of H&E staining of a pigmented tumor from Gnaq^CA; Bap1^CKO; Myc^CKI mice. K, Representative image of multiplex IHC (mIHC) showing Gli2, Melan-A, and Tyrp1 expression in a pigmented tumor from Gnaq^CA; Bap1^CKO; Myc^CKI mice. L, Representative images of IHC using a melanoma cocktail (Tyrosinase + Melan-A) to detect disseminated UM cells in the livers of Gnaq^CA; Bap1^CKO; Myc^CKI mice. M, Representative images of IHC using GFP and Myc to detect disseminated UM cells in the livers of Gnaq^CA; Bap1^CKO; Myc^CKI mice.

Figure 3.. Transplant models for UM liver metastasis

A, Schematic diagram of orthotopic transplant of Gnaq^CA; Bap1^CKO; Myc^CKI primary tumor cells in immunocompetent and immunocompromised mice (Created in BioRender. Xu, X. (2026) https://BioRender.com/g7sncqs). B, Representative images of H&E staining of orthotopic transplant of Gnaq^CA; Bap1^CKO; Myc^CKI primary tumor cells (QBM12) in immunocompetent mice. C, Representative image of multiplex IHC (mIHC) showing Gli2, Melan-A, and Tyrp1 expression in a QBM12 orthotopic transplant tumor. D, Schematic diagram of metastatic transplant of Gnaq^CA; Bap1^CKO; Myc^CKI primary tumor cells in NSG mice via tail vein injection (Created in BioRender. Xu, X. (2026) https://BioRender.com/g7sncqs). E, Representative in vivo bioluminescence imaging showing signal from QBM12 cells 75 days after tail vein inoculation. F-H, Representative images of H&E staining of metastasis in lung (F), kidney (G), and liver (H). I, Schematic diagram of liver metastatic transplant of Gnaq^CA; Bap1^CKO; Myc^CKI primary tumor cells in NSG mice via intrasplenic injection followed by splenectomy (Created in BioRender. Xu, X. (2026) https://BioRender.com/g7sncqs). J, Representative in vivo bioluminescence imaging showing signal from QBM40 cells 30 days after tail vein inoculation. K, Representative images of H&E staining of metastasis in liver. L and M, Representative image of multiplex IHC (mIHC) showing Gli2, Melan-A, and Tyrp1 expression in liver metastasis formed by QBM12 (L) and QBM40 (M).

Figure 4.. Characterization of phenotypic diversity in murine UM

A, UMAP plot showing malignant and microenvironmental cell types in UMs from Gnaq^CA; Bap1^CKO; Myc^CKI mice analyzed by scRNA-seq. Cell types are color-coded. B, UMAP plot showing the two major subtypes in UMs from Gnaq^CA; Bap1^CKO; Myc^CKI mice, Melanocytic and Neural Crest-like (color-coded). C, Violin plot showing the expression of Melanocytic and Neural Crest-like subtype markers. D, UMAP plot showing phenotypic clusters among the malignant UM cells from Gq^CA; Myc^CKI; Bap1^CKO tumors. Clusters are color-coded. E, Dot plot showing the expression of cluster markers, with dot color indicating the average expression levels and dot size indicating the percentage of cells in each cluster expressing the respective marker. F, Heatmap showing the relationship of murine UM cell states to murine cutaneous melanoma phenotypic states based on the relative expression of the indicated gene signatures.

Figure 5.. MYC activity across the UM progression continuum

A, UMAP plot showing Myc expression in malignant UM cells from Gnaq^CA; Bap1^CKO; Myc^CKI tumors. B, Violin plots comparing endogenous Myc expression between the Melanocytic and Neural Crest-like subtypes. C, UMAP plot showing expression of Myc targets in murine UM cells, color coded by average expression of canonical Myc targets from “GSEA_Hallmark_MYC_targets_v1/v2”. D and E, Gene set enrichment analysis (GSEA) of Hallmark signature (D) and gene ontology (GO) (E) in Melanocytic and Neural Crest-like UM cells. F, Spearman correlation between Myc expression and the indicated genes in murine UM from the scRNA-seq analysis and human UM from the UM dataset form TCGA (TCGA-UVM). G, Scatter plot showing the expression changes of 90 ribosome genes and 78 mitochondrial ribosome genes between Melanocytic and Neural Crest-like cells. H, UMAP of human UM scRNA-seq dataset, color coded by Class 1 and Class 2 primary tumors. I and J, Violin plots showing expression of MYC (I) and MYC targets (J) in Class 1 and Class 2 human UM. K, Percentage of Class 1 and Class 2 human UM cells positive for the Melanocytic or Neural Crest-like signatures. L, Violin plot showing MYC expression in Class 2 human UM cells positive for the Melanocytic or Neural Crest-like signatures.

Figure 6.. The transcription factor landscape across murine UM subtypes

A, Heatmap showing Z-score normalized transcription factor (TF) motif activity across mouse UM single cells from Gnaq^CA; Bap1^CKO; Myc^CKI tumors, as inferred by SCENIC analysis of the scRNA-seq data. Hierarchical clustering of both cells and motifs reveals distinct regulatory programs associated with Melanocytic and Neural Crest-like UM states. B-I, UMAP showing the expression of Jun (B), Jund (C), Fosb (D), Fos (E), Yap1 (F), Tead1 (G), Runx1 (H), and Fosl1 (I) in mouse UM cells from Gnaq^CA; Bap1^CKO; Myc^CKI tumors.

Figure 7.. Progression trajectory analysis of murine UM

A, RNA velocity analysis based on spliced and unspliced transcript abundances revealed directional transitions across mouse UM cell states from Gnaq^CA; Bap1^CKO; Myc^CKI tumors. Velocity vector fields projected onto the UMAP embedding indicate major evolutionary paths. B, Latent time analysis shows the latency of single UM cells, color coded by pseudotime. C, Expression dynamics of representative genes plotted along latent pseudotime. D-F, UMAP plots showing the expression of Foxo3 (D), Cd44 (E), and Bach2 (F) in murine UM cells.

Figure 8.. Copy number alterations correlate with UM subtypes

A, Inferred copy number alteration (CNA) profiles of individual malignant cells from murine UM tumors using inferCNV, using immune cells as diploid references. Heatmap shows relative genomic alteration levels across chromosomes, and hierarchical clustering of UM cells revealed two distinct CNA-defined UM branches. B, UMAP plot of mouse UM cells colored by CNA branch assignment. C and D, Proportional composition of CNA branch within each major subtypes (C) and phenotypic clusters (D). E and F, Heatmap of genes frequently amplified (E) or deleted (F) in both murine and human UM, color coded by frequency of chromosomal gain (E) or loss (F) in mouse UM cells or TCGA-UVM samples.