Monosomy 3 Is Linked to Resistance to MEK Inhibitors in Uveal Melanoma

Abstract

The use of MEK inhibitors in the therapy of uveal melanoma (UM) has been investigated widely but has failed to show benefits in clinical trials due to fast acquisition of resistance. In this study, we investigated a variety of therapeutic compounds in primary-derived uveal melanoma cell lines and found monosomy of chromosome 3 (M3) and mutations in BAP1 to be associated with higher resistance to MEK inhibition. However, reconstitution of BAP1 in a BAP1-deficient UM cell line was unable to restore sensitivity to MEK inhibition. We then compared UM tumors from The Cancer Genome Atlas (TCGA) with mutations in BAP1 with tumors with wild-type BAP1. Principal component analysis (PCA) clearly differentiated both groups of tumors, which displayed disparate overall and progression-free survival data. Further analysis provided insight into differential expression of genes involved in signaling pathways, suggesting that the downregulation of the eukaryotic translation initiation factor 2A (EIF2A) observed in UM tumors with BAP1 mutations and M3 UM cell lines might lead to a decrease in ribosome biogenesis while inducing an adaptive response to stress. Taken together, our study links loss of chromosome 3 with decreased sensitivity to MEK inhibition and gives insight into possible related mechanisms, whose understanding is fundamental to overcome resistance in this aggressive tumor.

Keywords: BRCA1-associated protein 1; MAPK; eIF2 signaling; eIF2alpha; monosomy 3; ocular melanoma; targeted therapy.

Figure 1

Uveal melanoma cell lines with monosomy 3 (M3) are resistant to MEK inhibition compared to cell lines with disomy 3 (D3). Dose-response curves of uveal melanoma cell lines with M3 (blue symbols) and D3 (red symbols) that were treated with trametinib (A) or refametinib (B) at different concentrations (from 5 nM to 25 µM) for 5 days and minimum cell viability of each cell line after treatment (C,D). Cell viability is expressed as normalized values in percentage to DMSO controls. Summarized results of three independent experiments are shown. Data are average ± SD. ****, p < 0.0001.

Figure 2

Uveal melanoma cell lines with monosomy 3 (M3) show BAP1 loss. BAP1 protein expression was assessed in the indicated uveal melanoma cell lines by Western blot. A representative result of three independent experiments is shown. Uncropped blots are displayed in Supplementary Figure S2.

Figure 3

Establishment of a BAP1-reconstituted UM cell line. M3 cell line UPMM2 was reconstituted with an empty vector (EV), wild-type BAP1 or an inactive p.C91S mutant BAP1 variant and further characterized. (A) Western blot confirms protein expression of BAP1 in BAP1-deficient cell line UPMM2 after wild-type or mutant BAP1 reconstitution. (B) Reconstitution of wild-type BAP1 in cell line UPMM2 leads to a significant decrease in proliferation rate. A representative result of three independent experiments is shown. (C) Reconstitution of wild-type BAP1 in cell line UPMM2 shows a tendency of less colony formation ability when seeded at low density. A representative result of three independent experiments is shown. Data are average ± SD. *, p < 0.05. **, p < 0.01. Uncropped blots are displayed in Supplementary Figure S3.

Figure 4

Reconstitution of wild-type BAP1 does not sensitize UPMM2 cell line to MEK inhibition. UPMM2 cells were treated with trametinib (A) or refametinib (B) at different concentrations (from 5 nM to 25 µM) for 5 days and the minimum cell viability for each cell line after treatment was quantified (C,D). Cell viability is expressed as normalized values in percentage to DMSO controls. Summarized results of three independent experiments are shown. Data are average ± SD. *, p < 0.05. n.s., not significant.

Figure 5

Expression levels of phosphorylated MEK and ERK in uveal melanoma cell lines in basal conditions and after treatment with trametinib. Western blot of phosphorylated MEK1/2 (Ser221), phosphorylated ERK1/2 (Thr202/Tyr204) and total ERK1/2 in basal conditions (A) or after treatment with 15 nM trametinib (or DMSO control) for 24 h (B). Total ERK1/2 and Vinculin were detected on an additional membrane using the same protein lysates. A representative result of two independent experiments is shown. Uncropped blots are displayed in Supplementary Figures S4 and S5.

Figure 6

Uveal melanoma tumors from TCGA with BAP1 mutations show deletion of chromosome 3, but not tumors with wild-type BAP1. Paired DNA copy numbers from UVM-TCGA tumors in chromosome 3 were sorted by their average segmented copy number (n = 74). The chromosomal position of the BAP1 gene is indicated.

Figure 7

Uveal melanoma patients from TCGA with BAP1 mutations display poor overall (A) and progression-free survival (B) compared to patients without mutations in BAP1. HR = hazard ratio.

Figure 8

Transcriptome analysis of UVM-TCGA confidentially separates tumors with and without BAP1 mutations. Gene expression profiling of uveal melanoma TCGA dataset (n = 74) followed by principal component analysis (PCA) using Partek Genomics Suite reveals distinct clustering of samples in two subgroups.

Figure 9

Top differentially regulated genes in BAP1-mutant versus BAP1-wild type UM tumor samples. Uveal melanoma TCGA data (n = 74) were analyzed and grouped in mutant BAP1 and wild-type BAP1 tumor samples. The top significantly differentially regulated genes after a false discovery rate (FDR) corrected p value are shown. FC, fold change.

Figure 10

eIF2 signaling pathway. Ingenuity pathway analysis shows upregulated genes in shades of red and downregulated genes in shades of green in BAP1-mutated UVM-TCGA tumors.

Figure 11

eIF2 signaling pathway genes in BAP1-mutant and BAP1-wild type UM tumor samples. Uveal melanoma TCGA data (n = 74) were analyzed and grouped in BAP1-mutant and BAP1-wild type tumor samples. The genes of the eIF2 signaling pathway that were differentially expressed after a false discovery rate (FDR) corrected p value below 0.001 and a fold change (FC) higher than 1.5 are shown. Genes further assayed by qPCR are in bold.

Figure 12

Gene expression of eIF2 pathway-related genes in UM cell lines. Expression of five eIF2 pathway-related genes was assessed by qPCR in UM cell lines (A) and BAP1-reconstituted UPMM2 cell lines (B). Gene expression was normalized to PPIB levels relative to the average expression levels in D3 cell lines or the empty vector (EV) control. Summarized results of two biological replicates are shown (cells in B were from the same passage). Data are average ± SD. *, p < 0.05. **, p < 0.01. ***, p < 0.001. ****, p < 0.0001. n.s., not significant.