BAP1 Loss Promotes Suppressive Tumor Immune Microenvironment via Upregulation of PROS1 in Class 2 Uveal Melanomas

Abstract

Uveal melanoma (UM) is the most common primary cancer of the eye and is associated with a high rate of metastatic death. UM can be stratified into two main classes based on metastatic risk, with class 1 UM having a low metastatic risk and class 2 UM having a high metastatic risk. Class 2 UM have a distinctive genomic, transcriptomic, histopathologic, and clinical phenotype characterized by biallelic inactivation of the BAP1 tumor-suppressor gene, an immune-suppressive microenvironment enriched for M2-polarized macrophages, and poor response to checkpoint-inhibitor immunotherapy. To identify potential mechanistic links between BAP1 loss and immune suppression in class 2 UM, we performed an integrated analysis of UM samples, as well as genetically engineered UM cell lines and uveal melanocytes (UMC). Using RNA sequencing (RNA-seq), we found that the most highly upregulated gene associated with BAP1 loss across these datasets was PROS1, which encodes a ligand that triggers phosphorylation and activation of the immunosuppressive macrophage receptor MERTK. The inverse association between BAP1 and PROS1 in class 2 UM was confirmed by single-cell RNA-seq, which also revealed that MERTK was upregulated in CD163+ macrophages in class 2 UM. Using ChIP-seq, BAP1 knockdown in UM cells resulted in an accumulation of H3K27ac at the PROS1 locus, suggesting epigenetic regulation of PROS1 by BAP1. Phosphorylation of MERTK in RAW 264.7 monocyte-macrophage cells was increased upon coculture with BAP1^-/- UMCs, and this phosphorylation was blocked by depletion of PROS1 in the UMCs. These findings were corroborated by multicolor immunohistochemistry, where class 2/BAP1-mutant UMs demonstrated increased PROS1 expression in tumor cells and increased MERTK phosphorylation in CD163+ macrophages compared with class 1/BAP1-wildtype UMs. Taken together, these findings provide a mechanistic link between BAP1 loss and the suppression of the tumor immune microenvironment in class 2 UMs, and they implicate the PROS1-MERTK pathway as a potential target for immunotherapy in UM.

Keywords: BAP1; MERTK; PROS1; macrophage; metastasis; tumor immune microenvironment; uveal melanoma.

Figure 1

PROS1 expression is regulated by BAP1 in uveal melanocytes and uveal melanoma cells. (A) Summary of RNA sequencing data from BAP1-wildtype Mel202, 92.1, and MP41 uveal melanoma cells, and UMC026 uveal melanocytes with or without shRNA-mediated knockdown of BAP1, and 80 UM samples from The Cancer Genome Atlas (TCGA) data repository with or without mutational inactivation of BAP1. Numbers in parenthesis indicate the number of genes upregulated by loss of BAP1. Numbers within the Venn diagram indicate the number of overlapping genes between indicated subsets of genes. PROS1 was one of only two genes that was upregulated across all datasets. (B) Immunoblots confirming upregulation of PROS1 protein following knockout of BAP1 in UMC026 cells, or knockdown of BAP1 in Mel202 cells. Conversely, ectopic expression of BAP1 in MP46 BAP1-mutant class 2 uveal melanoma cells resulted in downregulation of PROS1. PROS1/β-actin densitometry ratios (lanes 1–6): 0.29592, 1.2059, 0.61066, 1.1644, 0.34334, 0.10613 (C) ChIP-seq analysis of H3K27ac at the PROS1 locus in Mel202 cells with or without doxycycline-induced, shRNA-mediated knockdown of BAP1.

Figure 2

Single-cell RNA sequencing analysis of PROS1 and MERTK in uveal melanomas. (A) UMAP dimensionality reduction plot of 59,915 neoplastic and non-neoplastic cells from 11 uveal melanoma samples, with cell types indicated in the legend. (B) Left: UMAP plot of the 42,230 uveal melanoma cells in the dataset, with colors indicating gene expression profile (GEP) class 1 (blue) and class 2 (red). Right: corresponding UMAP plot demonstrating log normalized expression of PROS1, as indicated by heatmap. (C) Left: UMAP plot of the 5053 monocytes/macrophages in the dataset, with colors indicating GEP class 1 (blue) and class 2 (red) of the tumors from which the cells were derived. Right: corresponding UMAP plots demonstrating log normalized expression of CD68, CD163, and MERTK, as indicated by a heatmap. (D) Dot plot demonstrating MERTK expression in monocytes/macrophages with respect to GEP class status of the tumors from which the cells were derived. (E) Scatter plot demonstrating association between expression of MERTK and CD163 in monocytes/macrophages from class 2 tumors.

Figure 3

Multicolor immunohistochemistry of PROS1, MERTK, and CD163 in uveal melanomas. (A) Top, representative photomicrographs of class 1 and class 2 uveal melanomas analyzed with hematoxylin and eosin (H&E) and with immunostaining for BAP1, PROS1, phosphorylated MERTK (phospho-MERTK), and CD163. Scale bar (lower left), 100 µm. Bottom: donut plots summarizing percentage of cells that were positive for each biomarker with respect to GEP tumor class 1 versus class 2. (B) Donut plot demonstrating the proportion of CD163+/phospho-MERTK+ cells with respect to GEP tumor class 1 versus class 2. Total number of cells analyzed and number (and percentage) of cells staining positive for each indicated protein are summarized in Table 1.

Figure 4

Loss of BAP1 in uveal melanocytes activates MERTK in macrophages in a PROS1-dependent manner. (A) Soluble PROS1 protein levels measured by ELISA in media from cultured UMC026 cells with or without knockout of BAP1. (B) Confocal microscopy of UMC026 cells with or without knockout of BAP1 immunostained for PROS1. Scale bar (lower right), 10 µm. (C) Representative immunoblot of lysates from RAW 264.7 cells cocultured with UMC026 cells with or without knockout of BAP1, immunoprecipitated with total-MERTK antibody, and probed with either total-MERTK or phospho-MERTK antibody. Immunoprecipitated MERTK is undetectable when UMC026 cells are cultured independently of RAW 264.7 cells (Supplementary Figure S11). Phospho-MERTK/total-MERTK densitometry ratios (lanes 1–2): 0.11181, 0.46660. (D) Densitometric analysis of immunoblots from triplicate coculture experiments represented in panel C. (E) Representative immunoblot of lysates from RAW 264.7 cells cocultured with UMC026 cells knocked out for BAP1, with or without siRNA-mediated knockdown of PROS1, immunoprecipitated with total-MERTK antibody, and probed with phospho-MERTK antibody. Phospho-MERTK/total-MERTK densitometry ratios (lanes 1–2): 0.33620, 0.71485. (F) Densitometric analysis of immunoblots from triplicate coculture experiments represented in panel E.

Figure 5

Proposed model for how BAP1 loss leads to suppression of the tumor immune microenvironment. In class 1 BAP1-wildtype uveal melanomas, tumor cells express low levels of PROS1, and tumor-associated macrophages are mostly M1-polarized with low MERTK expression and low MERTK phosphorylation. In BAP1-mutant class 2 uveal melanomas, PROS1 is upregulated since it is no longer repressed by BAP1. Mostly, membrane-bound PROS1 on tumor cells interacts with MERTK on nearby macrophages, leading to phosphorylation of MERTK and activation of downstream signaling that promotes M2 polarization.