The nuclear deubiquitinase BAP1 is commonly inactivated by somatic mutations and 3p21.1 losses in malignant pleural mesothelioma

Abstract

Malignant pleural mesotheliomas (MPMs) often show CDKN2A and NF2 inactivation, but other highly recurrent mutations have not been described. To identify additional driver genes, we used an integrated genomic analysis of 53 MPM tumor samples to guide a focused sequencing effort that uncovered somatic inactivating mutations in BAP1 in 23% of MPMs. The BAP1 nuclear deubiquitinase is known to target histones (together with ASXL1 as a Polycomb repressor subunit) and the HCF1 transcriptional co-factor, and we show that BAP1 knockdown in MPM cell lines affects E2F and Polycomb target genes. These findings implicate transcriptional deregulation in the pathogenesis of MPM.

Figure 1

RAE analysis of genomic gains and losses in 53 MPM tumors. The effective frequency of gain (red) or loss (blue) and corresponding statistical significance (false discovery rate (FDR), q values) of aberrations are indicated (y axis, right and left, respectively), as is their chromosomal position (center, gray; centromeres in red). Green lines adjacent to the axis indicate the cutoff for statistical significance (q value = 0.1, FDR = 10%). Genes selected for sequencing are identified near their relevant peaks (CDKN2A, the target of the 9p21 deletion, was not sequenced in the current study).

Figure 2

Heat map of chromosome 3p in MPM tumors. Genomic loss is indicated in blue. A subset of cases harbor more focal alterations centered on BAP1. Overall, 16 out of the 53 tumors (30%) have at least single copy genomic loss of the BAP1 locus. In the insets (scale bars, 50 µm), BAP1 immunohistochemistry shows loss of nuclear staining for BAP1 protein in tumors with a BAP1 mutation (for example, tumor A102 with a 1-bp insertion in exon 15) compared to those with wild-type BAP1 (for example, tumor A36). Also shown is the BAP1 FISH analysis (red signals, BAP1 probe; green signals, chromosome 3 centromere) confirming single copy loss in tumor A17 and normal diploid BAP1 status in tumor A97, validating array CGH results.

Figure 3

Distribution of BAP1 mutations relative to functional domains. Shown are the N-terminal ubiquitin hydrolase domain (blue), the HCF1-binding domain (HBM) and the C-terminal protein interaction domain (green) containing two nuclear localization signals (black boxes).

Figure 4

Integrated gene map showing BAP1 losses and mutations in relation to other identified genomic events in all 53 samples. Three samples had no alterations in the genes listed.

Figure 5

Relationship between the BAP1 knockdown expression signature in MPM and three published Polycomb target gene sets. The Bracken signature was derived by siRNA depletion of four PRC complex proteins (EZH2, EED, SUZ12 and BMI1) in embryonic fibroblasts. The Hassan signature reflects PRC targets identified by previous ChIP-on-chip experiments using SUZ12 in human embryonic stem cells. We generated the Douglas list using siRNA targeting BMI1 in A4573 Ewing Sarcoma cells. The significance of enrichment (overrepresentation) was assessed using the hypergeometric distribution. We noted significant overlap between genes in the BAP1 list and genes in all three PRC sets, with each pairwise overlap with the BAP1 signature showing P < 10⁻¹⁹. The 77 genes that appear in the BAP1 profile and at least two other Polycomb signatures are listed in Supplementary Table 8.