BAP1 Loss Is Associated with DNA Methylomic Repatterning in Highly Aggressive Class 2 Uveal Melanomas.

Abstract

The strong association between <i>BAP1</i> 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 <i>BAP1</i> loss on the DNA methylome in UM.<b>Experimental Design:</b> 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 <i>BAP1</i> 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 <i>BAP1</i> locus was found in all Class 2 tumors, suggesting that <i>BAP1</i> 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, <i>BAP1</i> 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 <i>BAP1</i> on the remaining copy of chromosome 3, leading to the methylomic repatterning profile characteristic of Class 2 UMs.