TCGA_Methyl_DMP: All significantly methylated probes (FDR < 0.05) in Class 2 uveal melanomas compared to Class 1 uveal melanomas from the 80 TCGA cases. JWH_Methyl_DMP: All significantly methylated probes (p < 0.05) in Class 2 uveal melanomas compared to Class 1 uveal melanomas from 12 cases of the senior author (JWH). TCGA_D12_PC1_PC2_Methyl: The 67,261 probes shared between the top 20% most variable methylated probes from the TCGA and JWH datasets used in the principal component analyses. The percentage contribution to PC1 and PC2 are displayed for all probes. TCGA_D12_Probes_Top5000PC1: The 204 probes shared between the TCGA and JWH datasets out of the Top 5000 shared most variable methylated probes contributing to PC1 in the principal component analyses.
ARTICLE ABSTRACT
The strong association between BAP1 mutations and metastasizing Class 2 uveal melanoma (UM) suggests that epigenetic alterations may play a significant role in tumor progression. Thus, we characterized the impact of BAP1 loss on the DNA methylome in UM.Experimental Design: Global DNA methylation was analyzed in 47 Class 1 and 45 Class 2 primary UMs and in UM cells engineered to inducibly deplete BAP1. RNA-Seq was analyzed in 80 UM samples and engineered UM cells.
Hypermethylation on chromosome 3 correlated with downregulated gene expression at several loci, including 3p21, where BAP1 is located. Gene set analysis of hypermethylated and downregulated genes identified axon guidance and melanogenesis as deregulated pathways, with several of these genes located on chromosome 3. A novel hypermethylated site within the BAP1 locus was found in all Class 2 tumors, suggesting that BAP1 itself is epigenetically regulated. Highly differentially methylated probes were orthogonally validated using bisulfite sequencing, and they successfully distinguished Class 1 and Class 2 tumors in 100% of cases. In functional validation experiments, BAP1 knockdown in UM cells induced methylomic repatterning similar to UM tumors, enriched for genes involved in axon guidance, melanogenesis, and development.
This study, coupled with previous work, suggests that the initial event in the divergence of Class 2 UM from Class 1 UM is loss of one copy of chromosome 3, followed by mutation of BAP1 on the remaining copy of chromosome 3, leading to the methylomic repatterning profile characteristic of Class 2 UMs.
