BAP1 loss defines a new class of renal cell carcinoma

Abstract

The molecular pathogenesis of renal cell carcinoma (RCC) is poorly understood. Whole-genome and exome sequencing followed by innovative tumorgraft analyses (to accurately determine mutant allele ratios) identified several putative two-hit tumor suppressor genes, including BAP1. The BAP1 protein, a nuclear deubiquitinase, is inactivated in 15% of clear cell RCCs. BAP1 cofractionates with and binds to HCF-1 in tumorgrafts. Mutations disrupting the HCF-1 binding motif impair BAP1-mediated suppression of cell proliferation but not deubiquitination of monoubiquitinated histone 2A lysine 119 (H2AK119ub1). BAP1 loss sensitizes RCC cells in vitro to genotoxic stress. Notably, mutations in BAP1 and PBRM1 anticorrelate in tumors (P = 3 × 10(-5)), [corrected] and combined loss of BAP1 and PBRM1 in a few RCCs was associated with rhabdoid features (q = 0.0007). BAP1 and PBRM1 regulate seemingly different gene expression programs, and BAP1 loss was associated with high tumor grade (q = 0.0005). Our results establish the foundation for an integrated pathological and molecular genetic classification of RCC, paving the way for subtype-specific treatments exploiting genetic vulnerabilities.

Fig. 1. Integrative mutation and DNA copy-number analyses in a tumor and tumorgraft from the index subject

(a) Representative capillary sequencing chromatograms of normal (N), patient’s tumor (T), and tumorgraft (TG) illustrating different examples of mutant allele enrichment in the tumorgraft. Arrowheads indicate mutations. (b) Allele specific (ASCN) and paired (PCN) copy number representation of high-density SNP array data incorporating the estimated position of mutated genes. Green, paired copy numbers. Red and blue, maximum and minimum copy numbers for each heterozygous SNP.

Fig. 2. BAP1 is a tumor suppressor in ccRCC

(a) Schematic of BAP1 with mutations (UCH, ubiquitin C-terminal hydrolase domain; HBM, HCF-1 binding motif; BRCA1, [putative] BRCA1 interacting domain; ULD, Uch37-like domain; NLS, nuclear localization signal; I, insertion; Δ, deletion; †, missense; *, non-sense; S, splice site; *L, stop codon loss. (b) Structural model of BAP1 UCH domain (purple) and ULD tail (green) superimposed on a template DUB (not shown) bound to ubiquitin (cyan); structural elements that alter upon ubiquitin binding are colored salmon. Left, cartoon of BAP1 model. Right, surface representation of BAP1 highlighting the positions of RCC alterations on interaction surfaces. Left inset, enlarged view of the DUB active site. Right inset, enlarged view of the Gly¹³, interaction with an aromatic residue in the ULD tail (dots indicate interaction radius). (c) Western blot of extracts from tumors with defined BAP1 mutation and chromosome 3p status. 769-P cells transfected with either an empty vector (EV) or wild-type (WT) BAP1 were used as controls. PPIB, cyclophilin B is shown as a loading control. Arrow indicates BAP1. (d) Representative IHC of tumors and tumorgrafts positive or negative for nuclear BAP1. Scale bar, 50 μm. Open arrow, tumor cells; simple arrow, endothelial cells and lymphocytes, which express BAP1 and serve as internal controls.

Fig. 3. HCF-1-dependent suppression of cell proliferation by BAP1

(a) Proliferation curves of empty vector (EV) and BAP1 reconstituted 769-P cells with inset showing BAP1 western blot. (b) Proliferation curves of 769-P cells stably expressing an shRNA targeting endogenous (mutant) BAP1 (shBAP1) or a vector control (shCtrl) and in addition wild-type BAP1 (BAP1), BAP1^Y33D (Y33D) or empty vector (EV). Western blot of cells transduced with shRNA targeting endogenous mutant BAP1 (A and B) or vector control (shCtrl) and transduced with expression vectors as indicated. (c) Western blot of partially purified histone fractions of 769-P cells reconstituted with an empty vector (EV) or wild-type BAP1 (BAP1). (d) Western blot of input (cytosolic [Cy] or nuclear [Nu] fractions) as well as immunoprecipitates (from nuclear fractions) and corresponding flow-through from empty vector (−) or wild-type BAP1 (+) expressing cells. Short and long exposures are indicated. Both ectopically expressed epitope tagged wild-type as well as endogenous mutant BAP1 bind HCF-1. (e) Proliferation curves of 769-P cells depleted of endogenous BAP1 shRNA and reconstituted with an empty vector (EV), wild-type BAP1 (WT) or HCF-1 binding motif mutant (HBM). Western blot from input as well as BAP1 immunoprecipitates. (f) Western blot of partially purified histone fractions or cell lysates from 769-P cells depleted of endogenous BAP1 and transduced with an empty vector (EV), wild-type BAP1 (WT), an HCF-1 binding motif mutant (HBM), or BAP1^Y33D (Y33D). Error bars represent SEM (n=3). *, p<0.05; **, p<0.01.

Fig. 4. BAP1 binds to and elutes with HCF-1 in tumorgrafts

(a) Western blot from inputs as well as BAP1 immunoprecipitates (BAP1-IP) from the indicated tumorgrafts. wt, wild type; M, mutant. (b) Western blot of TCA precipitated gel-filtration fractions of tumorgrafts either wild type for BAP1 (TG143 and TG144) or mutant (TG26). Ct, Control lysate from 769-P cells. (c) Western blot of partially purified histone fractions from tumorgrafts with the indicated BAP1 status.

Fig. 5. Loss of BAP1 and PBRM1 sets foundation for molecular genetic classification of ccRCC

(a) Representative H&E and immunohistochemistry (IHC) images of tumors with loss of BAP1, PBRM1, or both. Scale bar, 50 µm; 10 µm for inset. Open arrows, tumor cells; simple arrows, stroma/inflammatory cells; filled arrow, rhabdoid tumor cell. (b) Pie chart of the distribution of ccRCC subtypes. (c) Heatmap of statistically significant probes distinguishing BAP1- and PBRM1-deficient tumors/tumorgrafts vs. wild type. Expression of the same probes in renal cortex included as a reference. The full data set is provided in Supplementary Data 4. (d) Venn diagram illustrating the overlap in BAP1 and PBRM1 gene expression signatures with associated global pathway analyses.