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BAP1, PBRM1 and SETD2 in clear-cell renal cell carcinoma: molecular diagnostics and possible targets for personalized therapies Expert Rev. Mol. Diagn. Early online, 1–10 (2015)
Francesco Piva‡1, Matteo Santoni‡2, Marc R Matrana3, Suma Satti3, Matteo Giulietti1, Giulia Occhipinti1, Francesco Massari4, Liang Cheng5, AntonioLopez-Beltran6, Marina Scarpelli7, Giovanni Principato1, Stefano Cascinu2 and Rodolfo Montironi*7
Several novel recurrent mutations of histone modifying and chromatin remodeling genes have been identified in renal cell carcinoma. These mutations cause loss of function of several genes located in close proximity to VHL and include PBRM1, BAP1 and SETD2. PBRM1 encodes for BAF180, a component of the SWI/SNF chromatin remodeling complex, and is inactivated in, on average, 36% of clear cell renal cell carcinoma (ccRCC). Mutations of BAP1 encode for the histone deubiquitinase BRCA1 associated protein-1, and are present in 10% of ccRCCs. They are largely mutually exclusive with PBRM1 mutations. Mutations to SETD2, a histone methyltransferase, occur in 10% of ccRCC. BAP1- or SETD2-mutated ccRCCs have been associated with poor overall survival, while PBRM1 mutations seem to identify a favorable group of ccRCC tumors. This review describes the roles of PBRM1, BAP1 and SETD2 in the development and progression of ccRCC and their potential for future personalized approaches. KEYWORDS: BRCA1-associated protein-1 . clear cell renal cell carcinoma . molecular diagnostics . Polybromo-1 .
personalized therapies . SETD2
1
Department of Specialistic Clinical and Odontostomatological Sciences, Polytechnic University of Marche, Ancona, Italy 2 Medical Oncology, Polytechnic University of the Marche Region, School of Medicine, United Hospitals, Ancona, Italy, 3 Ochsner Cancer Institute, New Orleans, LA, USA, 4 Medical Oncology, Azienda Ospedaliera Universitaria Integrata, University of Verona, Verona, Italy, 5 Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA, 6 Department of Pathology and Surgery, Faculty of Medicine, Cordoba, Spain, 7 Section of Pathological Anatomy, Polytechnic University of the Marche Region, School of Medicine, United Hospitals, Ancona, Italy *Author for correspondence: [email protected] ‡
Authors equally contributed
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Renal cell carcinoma (RCC) includes sporadic or hereditary carcinomas arising from cells of the proximal renal tubular epithelium. Clear cell renal carcinomas (ccRCCs) represent the majority of kidney tumors, accounting for the 70% of RCC. They constitute a heterogeneous group that varies in histology, molecular alterations in tumor-suppressor gene and oncogenes, clinical courses and responses to treatment. In the last years, much research has focused on identifying clinical and pathological features associated with the prognosis of ccRCC patients, especially in the context of relatively new targeted therapies. These agents have led to major advances in the management of RCC patients and are directed toward VEGF [1,2] or its receptor [3–6] and mammalian target of rapamycin (mTOR) [7,8]. However, despite recent success, responses of primary ccRCC tumors and metastases to these agents are
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variable, and complete responses are relatively uncommon [9]. Indeed, the increasing availability of molecularly targeted drugs for patients with ccRCC stresses the need for reliable biomarkers to predict tumor behavior. The inactivation of the von Hippel–Lindau (VHL) tumor-suppressor gene constitutes a crucial step in ccRCC carcinogenesis; however, additional genetic and epigenetic alterations are required for the malignant transformation to ccRCC [10]. In ccRCC, one VHL allele is damaged from an intragenic mutation, and then the loss of the second allele due to a large deletion follows. This event causes a loss of heterozygosity, but it is not sufficient to cause cancer [11]. Therefore, VHL is thought to be a weak tumor-suppressor gene. In fact, it has been shown that VHL inactivation alone causes a senescent-like phenotype [11]. As deletions along VHL gene are usually large (several megabases) and located in 3p25 chromosomal
2015 Informa UK Ltd
ISSN 1473-7159
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region, additional near tumor-suppressor genes, such as BRCA1-associated protein-1 (BAP1), Polybromo-1 (PBRM1) and Set domain-containing 2 (SETD2), are lost (FIGURE 1). Subsequently, the loss of the remaining PBRM1 or BAP1 allele can be associated with different ccRCC grades and aggressiveness [12]. Patients with BAP1 or SETD2 mutant ccRCCs have been associated with poor overall survival, while PBRM1 mutations seem to identify a favorable group of ccRCC tumors [13,14]. The roles of these genes as therapeutic targets have not been elucidated yet. This review describes the functions and role of PBRM1, BAP1 and SETD2 in the development and progression of ccRCC, as well as their potential for future personalized approaches in the diagnosis and treatment of these patients. BAP1
The BAP1 gene is constituted by 17 exons. The protein is a deubiquitinating enzyme that binds to breast cancer type 1 susceptibility protein (BRCA1) and BRCA1-associated RING domain protein 1 (BARD1), inhibiting their capability to mediate ubiquitination and autoubiquitination and acting as a tumor suppressor [15]. The BAP1 region that interacts with BRCA1 is located between amino acids 596 and 721, while the region that interacts with BARD1 is from 182 to 365. However, a previous study did not detect the deubiquitylation of BRCA1 [16], maybe due to a different method to flag NH2 terminus of BAP1, the region where the catalytic domain should be. Thus, further data are necessary to support these properties. Moreover, BAP1 modulates histone H2A and HCFC1 deubiquitination and regulates transcription, cell cycle and growth, response to DNA damage and chromatin dynamics. In particular, BAP1 deubiquitinates H2A and makes E2F1 target gene promoters accessible. BAP1 is mainly localized in the nucleus, bound to chromatin and retained in the cytoplasm when monoubiquitinated from the E2/E3 hybrid ubiquitin-protein ligase (FIGURE 2). In the mouse, VHL is on a different chromosome than BAP1, PBRM1 or SETD2. While the loss of 3p in humans simultaneously deletes one copy of BAP1, loss of heterozygosity in the mouse corresponding VHL region does not affect BAP1 (FIGURE 1). These results explain why VHL heterozygous humans, but not mice, develop ccRCC and why a mouse model of ccRCC has been lacking [17]. BAP1 regulates G1/S phase transition and is highly expressed in testis, placenta and ovary, and present in breast tissue. Mutant BAP1 does not act as dominant negative, because the wild type allele compensates for the mutated allele. Therefore, BAP1 acts as a two-hit tumor suppressor in ccRCC. Although both copies of BAP1 can be lost, the inactivation of both BAP1 alleles through a point mutation has not been observed [12]. Although BAP1 is a tumor suppressor when overexpressed, and its mutants are associated with the development of metastases in uveal melanoma [18], its knockdown seems to have conflicting effects. In fact, its loss has been associated with the doi: 10.1586/14737159.2015.1068122
tumorigenesis of 92.1 uveal melanoma cells [19]. On the other hand, its knockdown down-regulated E2F1 target genes, resulting in the blockade of S phase initiation, cell growth and invasiveness in OCM1 and OM431 uveal melanoma cells [20]. Depletion of BAP1 expression in cutaneous melanoma cells reduced proliferation and induced apoptosis, inhibiting tumor growth in vivo, but its overexpression in melanocytes suppressed proliferation [21]. In mesothelioma cells, BAP1 knockdown has been shown to inhibit growth by the down-regulation of the E2F-responsive growth-related genes Cyclin A2, E2F1, p107 (RBL1) and CDC25C. This could be due to the post-translational inactivation of HCF-1. To explain this paradox, it has been hypothesized that BAP1 does not promote tumorigenesis by accelerating cell growth, but allows a slowed down but tolerant G1/S checkpoint, leading to slower but uncontrolled tumor growth [22]. Molecular effects of BAP1 mutations
The functionality of some BAP1 missense mutant proteins (Ser63Cys, Phe81Val, Cys91Trp and Ala95Asp) was assessed by in vitro enzymatic assay. Phe81Val, Cys91Trp and Ala95Asp showed decreased levels of ubiquitin cleavage activity, whereas Ser63Cys showed an increased level compared with wild-type BAP1. However, both over- and under-ubiquitination of target histones led to genomic instability [22]. The influence of mutated BAP1 was investigated on binding capacity with its most important interactors showing that HCF-1 levels did not differ between BAP1-deficient and reconstituted 769-P cells. Impairing the HCF-1 binding to BAP1 prevented the BAP1 inhibitory effect on cell proliferation but did not alter its ability to deubiquitinate H2A. The interaction of BAP1 with HCF-1 was analyzed in tumor grafts and the results were similar to those in cell lines. BAP1 bound to HCF-1 and BAP1 mutations did not correlate with ubiquitinated H2A (H2Aub1) levels. In other words, BAP1 binding to HCF-1, but not H2Aub1 deubiquitination, is involved in RCC tumor-suppression function [12]. PBRM1
The PBRM1 (also PB1 or BAF180) gene gives rise to at least nine splicing isoforms. It is a subunit of the SWI/SNF transcription-modulating chromatin remodeling complex that controls DNA accessibility for transcription [23]. The complex contains an ATPase subunit to supply energy for chromatin remodeling operations. PBRM1 protein harbors six bromodomains that bind acetylated residues on histone tails. The bromodomains are not equivalent in specificity as they have different target affinity. This suggests that they may ‘read and interpret’ the histone code. The bromodomains and bromoadjacent homology domains are frequently hit by mutations in ccRCC. In breast cancers where PBRM1 was found to be mutated, it was demonstrated that PBRM1 is a regulator of p21 gene promoter accessibility, since PBRM1 induces p21 expression that, in turn, inhibits cell proliferation [24]. PBRM1 seems to be required for cellular senescence, a Expert Rev. Mol. Diagn.
BAP1, PBRM1 & SETD2 in ccRCC
Human chromosomes 1 2 3
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4
Review
Mouse chromosomes 1
VHL (10, 141, 635–10, 153, 670; 3p25.3) BAP1 (52, 401, 004-52, 410, 105; 3p21.1) PBRM1 (52, 545, 352-52, 685, 850; 3p21.1) SETD2 (47, 016, 408-47, 163, 977; 3p21.31)
2 3 4
5
5
6
6
7
7
8
8
9
9
10
10
11
11
12
12
13
13
14
14
15
15
16
16
17
17
18
18
19
19
20
X
21
Y
VHL (113, 624, 021-113, 631, 633; 6qE3)
SETD2 (110, 532, 597-110, 618, 633; 9qF2)
BAP1 (31, 251, 489-31, 259, 929; 14qB) PBRM1 (31, 019, 138-31, 121, 592; 14qB)
22 X Y
Figure 1. Different chromosome location of VHL, BAP1, PBRM1 and SETD2 between human and mouse.
tumor-suppressor mechanism, by regulating p53-mediated transcription of specific genes [25]. PBRM1 is important for transcriptional silencing induced by DNA double-strand breaks (DSBs) and promotes repair at damaged sites, and since PBRM1 mutants failed to perform these functions, it supports its tumor-suppressor activity [26]. Besides the role in transcriptional regulation, PBRM1 prevents tumorigenesis by promoting centromeric cohesion and genome stability [27]. Moreover, genes involved in chromosomal instability and cellular proliferation result in up-regulation following PBRM1 knockdown. In particular, RNAi-mediated depletion of PBRM1 in ccRCC cell lines has been shown to increase proliferation, colony formation and cell migration, consistent with a role as a tumor suppressor [28]. This role is also supported by the evidence that PBRM1 is involved in other malignancies, such as pancreatic cancer [29]. SETD2
SETD2 produces at least three alternative splicing transcripts and its products are histone methyltransferases. SETD2 has been found to act as tumor-suppressor gene in patients with breast cancer and leukemias [30,31]. Mutations in SETD2 are associated with increased loss of DNA methylation at nonpromoter regions. This is probably due to reductions of histone
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H3 (H3K36) trimethylation (H3K36me3) that is involved in the maintenance of a heterochromatic state [32]. A study that compared 42 primary ccRCC tumors versus normal kidney samples identified thousands of genomic regions with differences in chromatin accessibility. In particular, SETD2-mutated tumors were associated with nucleosome depletion, that is, an increased chromatin accessibility, and, most likely, this was due to H3K36me3 deficiency. Genes harboring alterations in nucleosome positioning and histone modifications may show aberrancies in splicing (mainly including exon skipping and intron retention) and alternative transcription start and termination sites [33]. SETD2 plays an important role also in DNA damage repair. In fact, cells lacking SETD2 show microsatellite instability and an elevated spontaneous mutation rate, since H3K36me3 is required to recruit the mismatch recognition protein hMutSa onto chromatin [34]. Moreover, H3K36me3 is required for ATM and p53-mediated checkpoint activations upon DNA DSBs [35] and homologous recombination repair of DSBs, as it facilitates the recruitment of repairing proteins [35,36]. Frequency of BAP1, PBRM1 & SETD2 mutations
Several studies have estimated the prevalence of VHL, BAP1, PBRM1 and SETD2 mutations. We have collected these data
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samples expressing PBRM1 had the wildtype gene [12]. As expected, most of the truncating mutations affected PBRM1 production. In the same samples, only in 15 out of 348 ccRCCs SETD2 was mutated. Interestingly, the inactivation of PBRM1 and BAP1 is less common in papillary RCC, chromophobe RCC (chRCC) and oncocytoma than in ccRCC tumors. Ub In particular, loss of PBRM1 protein Ub BAP1 expression occurred in only 3, 6 and 0% BRCA1 of papillary RCC, chRCC and oncocytoUb BARD1 Ub mas, whereas BAP1 loss was not observed BAP1 in any of these three tumors [41]. A recent Cancer study on a cohort of papillary RCCs and their matched normal samples highlighted BRCA1 BAP1 that SETD2 was significantly mutated [42]. UBE2O Interestingly, Gerlinger et al. analyzed BARD1 distinct subclones within primary tumors Nucleus Tumor suppression Intracellular or their metastases of 10 sporadic ccRCCs to assess the intratumor heteroFigure 2. BAP1 activity in ccRCC. BAP1 protein is a deubiquitinating enzyme that geneity. They showed that ccRCCs binds to breast cancer type 1 susceptibility protein (BRCA1) and BRCA1-associated RING adopted a branched rather than linear domain protein 1 (BARD1), acting as a tumor suppressor. BAP1 is mainly localized in the evolution. Moreover, VHL and PBRM1 nucleus and secondly retained in the cytoplasm when monoubiquitinated from E2/E3 hybrid ubiquitin-protein ligase (UBE2O). mutations were on the trunks of the phylogenetic trees, supporting that the in TABLE 1. However, it is important to assess how severe a muta- VHL and PBRM1 inactivating events occur early in tumor tion is in terms of alteration of protein function or protein development [43]. loss. Not only can destruction of the classical 5’ and 3’ splice sites alter pre-mRNA splicing and give rise to partly or totally Correlations among BAP1, PBRM1 & SETD2 mutated exon skipping or intron retention, but all mutations lying in genes exons or introns, both synonymous and missense, can cause Several studies have searched for favored or disfavored correlasplicing alterations [37]. Splicing alterations can cause severe tions between mutated genes. Surprisingly, although loss of effects, provoking frameshift errors and, consequently, the crea- BAP1 or PBRM1 was observed in 45% of ccRCC, concomition of premature termination codons. This event triggers a tant BAP1 and PBRM1 loss was reported only in 3% of surveillance mechanism called ‘nonsense mediated decay’ caus- ccRCC cases [32]. In particular, few tumors had both ing transcript degradation and protein loss. Although some BAP1 and PBRM1 loss, and this simultaneous loss in ccRCC softwares are able to predict if a mutation can alter splicing [38], may be disadvantageous, although the reasons are such softwares are unable to show the resulting splicing pattern unclear [12,44]. These results, confirmed in other investigain terms of sequences lost or retained. tions [45], suggest that contemporary mutations in BAP1 and In VHL, BAP1, PBRM1 and SETD2, there are base substitu- PBRM1 are negatively selected in ccRCC; in other words tions, insertions or deletions that could cause frameshift and tumors may segregate into BAP1 or PBRM1-deficient subnonsense mutations. The latter two are truncating mutations types. Instead, in a study on 185 ccRCC tumors, mutual that can directly cause loss of the protein or its function. exclusivity between PBRM1 and BAP1 mutations was not A study that found 49.2, 29.2, 7.6 and 5.9% of tumors har- statistically significant [39]; according to another study, loss of bored mutated VHL, PBRM1, SETD2 and BAP1 and also PBRM1 expression did not correlate with VHL mutations [40]. highlighted that truncating mutations represent 67, 82.5, However, it is important to note that studies with small 78.50 and 36% of mutations in VHL, PBRM1, SETD2 and sample size might not have enough power to detect an BAP1 respectively [39]. association. Furthermore, some authors showed that PBRM1 protein Concerning PBRM1 and SETD2 genes, a meta-analysis across expression is present only in about 30% of ccRCC [40]; corre- ccRCC studies confirmed that the frequency of tumors lating sequencing with immunohistochemistry data showed that with both PBRM1 and SETD2 mutated exceeded the frequency about 90% of samples that did not express PBRM1 had a expected by chance alone [13]. Therefore, PBRM1 and mutation in the corresponding gene, and about 90% of the SETD2 can cooperate in renal tumorigenesis [44].
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Extracellular
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45 N/A 11 N/A N/A 2 The percentage is the sum of synonymous and non-synonymous mutations. N/A: Not assessed data.
52 N/A 14 Selected case cohort 176
81
[69]
41 10 10 5 1 1 41 11 10 Selected case cohort 240
82
[69]
11 3 4 4 0 0 26 11 8 Selected case cohort 106
41
[68]
7 1 2 3 0 0 42 8 17 55 Selected case cohort 98
[39]
19 6 3 3 1 1 29 8 6 Selected case cohort 185
49
[13]
20 6 4 4 1 1 30 7 6 Selected case cohort 188
50
[32]
22 6 1 6 1 1 34 13 11 Selected case cohort 417
56
Ref VHL (%) Study design
The promoter hypermethylation has been hypothesized to explain why about 10% of ccRCC are negative for PBRM1 or BAP1 proteins, according to immunohistochemistry, but they do not show evidence of point mutations. In fact, CpG hypermethylation represents another way to inactivate one allele of a gene, alternative to mutations. To sustain this hypothesis, the methylation status of CpG loci near the transcription start site of BAP1, PBRM1 and SETD2 was assessed in 50 primary ccRCCs. These genes were not or rarely methylated in both tumor and normal specimens [55]. Instead, in clinical samples of ccRCC, the identification of chromatin accessibility decreases in PBRM1 and SETD2 genes, suggesting the importance of epigenetic changes in this tumor and supporting the manipulation of the epigenome as a potential therapeutic strategy [56]. Thus, further efforts are required to identify the mechanisms underlying such events.
Cohort
Epigenetics
BAP1 (%)
SETD2 (%)
PBRM1 (%)
BAP1 + PBRM1 (%)
BAP1 + SETD2 (%)
SETD2 + PBRM1 (%)
VHL + BAP1 (%)
VHL + SETD2 (%)
VHL + PBRM1 (%)
A germline BAP1 mutation G121A (Gly41Ser) was identified in one individual, not having a germline VHL mutation, who had two and one first and second-degree relatives, respectively, with RCC [12]. The mutation A277G (Thr93Ala) in BAP1 has been shown to alter the RNA splicing pattern, which gives rise to 80% of aberrant mature transcripts that lead to a frameshift, whereas approximately 20% corresponded to the missense mutation. In TABLE 1, we summarize the BAP1 mutations identified by the same authors and associated with a tumor predisposition syndrome, including uveal melanoma, malignant pleural mesothelioma and cutaneous melanoma [46]. BAP1 sequencing in 83 unrelated probands with familial RCC revealed that a new germline mutation, T41A (L14H), predisposed to early onset of ccRCC. This was supported by several findings, including: this variant co-segregates with the RCC phenotype; it targets the catalytic domain of BAP1; the wild type amino acid L14 is highly conserved across species suggesting its irreplaceableness; the mutation is associated with LOH [47]. In the same study, other two mutations were discovered, although they have been likely considered as benign (TABLE 2). Germline mutations leading to BAP1 hereditary cancer predisposition syndrome can also cause uveal and cutaneous melanoma, mesothelioma [48–51] and basal cell carcinoma [52]. Notably, patients with the same BAP1 mutational status may develop different cancer types depending on the type of carcinogen exposure and, naturally, on the presence of other gene mutations [53]. BAP1 germline mutations have been found in several tissues. However, not all these tissues harboring such mutations develop cancer, suggesting that the mechanisms underlying such tolerance to mutations are still unrevealed. Moreover, Wang et al. have presented the preliminary The Cancer Genome Atlas exome sequencing data from 499 RCC cases [54]. The final result from this study on the loss of heterozygosity patterns to assess the interactions between germline and somatic mutations are awaited.
Table 1. ccRCC marker frequencies.
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Are germinal BAP1 mutations associated with RCC predisposition?
[12]
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BAP1, PBRM1 & SETD2 in ccRCC
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Table 2. Germline mutations in BAP1 gene causing renal cell carcinoma. Gene
Protein
Predicted effects
Ref.
37+1 delG
N-A
Probably affecting splicing
[46]
T41A
Leu14His
Missense (or splice) exon 1
[47]
C45G
N-A
3’UTR
[48]
78_79 delGG
Val27Ala
Truncating
[46]
G121A
Gly41Ser
Probably benign
[47]
256_277 and A277G
Ile87Met and Thr93Ala
In/del exon 5
[46]
A277G
Thr93Ala
Affecting splicing and truncating
[46]
437+1 G>A
N-A
Truncating
[46]
629 dupT
Met211His
Truncating
[46]
660-11 T>A
N-A
Probably affecting splicing
[46]
A869G
Asn290Ser
probably benign
[47]
932-58_59 delTG
N-A
Probably affecting splicing
[48]
C1182G
Tyr394*
Unknown
[48]
1654 delG
Asp552Ile
Truncating
[46]
1882_1885 del TCAC
Ser628Pro
Unknown
[48]
C2050T
Gln684*
Unknown
[48]
2057-22A>C
N-A
Probably affecting splicing
[48]
2057-4G>T
N-A
Probably affecting splicing
[48]
Prognostic significance of BAP1, PBRM1 & SETD2 mutations
Several studies focused on the prognostic significance of BAP1, PBRM1 and SETD2 mutational status. An analysis on 176 tumors showed that BAP1 loss correlated with high Fuhrman nuclear grade and mTORC1 activation [12]. Other authors confirmed a strong correlation between BAP1 mutations and higher Fuhrman nuclear grades [39]. Moreover, BAP1 loss in ccRCC may be associated with poor prognosis [14,32,39,45]. In particular, a retrospective analysis of 145 patients validated using a further independent cohort of 327 patients confirmed that tumors with mutations of either PBRM1 or BAP1 were more likely to present with higher tumor stage 3–4. They found that BAP1 mutations, but no VHL or PBRM1 mutations, were significantly associated with worse cancer-specific survival. They also reported a poor median overall survival (4.6 years) in patients with BAP1-mutated ccRCC compared to patients with PBRM1-mutated tumors (10.6 years) [14]. A study of BAP1 protein expression in 1479 patients with clinically localized ccRCC showed that BAP1 loss correlated with the most aggressive forms of ccRCC, even among low-risk patients. However, in this study, not all high-grade tumors showed loss of BAP1 expression [57]. A potential explanation of its negative prognostic role may be represented by mTORC1 activation following BAP1 inactivation, suggesting doi: 10.1586/14737159.2015.1068122
that these tumors could be more responsive to mTORC1 inhibitors. Interestingly, BAP1 truncating, compared with missense mutations, presented worse prognosis, but this was not maintained for BAP1 and SETD2 in a different cohort. Moreover, SETD2 mutations were related to the time to recurrence [13]. BAP1- and PBRM1-mutant tumors were also associated with different gene expression signatures. The former had alterations of genes implicated in growth-factor signaling and the latter involved genes implicated in the cytoskeleton and tissue architecture. This suggests that ccRCC can be classified into at least two distinct entities, biologically and clinically [14]. SETD2 mutations were related with little worse prognosis [13], whereas SETD2 frameshift mutations were associated with advanced tumors stages [39]. A retrospective study that investigated the associations between computed tomography diagnostic imaging features and RCC mutations highlighted that VHL-mutated tumors were associated with well-defined tumor margins, nodular tumor enhancement and gross appearance of intratumor vascularity. BAP1-mutated tumors were associated with renal vein invasion, whereas SETD2-mutated tumors were not associated with any computed tomography feature studied. Moreover, multicystic ccRCC did not harbor mutations of SETD2 and BAP1 [58]. Expert Rev. Mol. Diagn.
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BAP1, PBRM1 & SETD2 in ccRCC
As for PBRM1 loss, it was associated neither with high-grade nor with low-grade tumor, but there was a correlation between tumors that had exclusively lost PBRM1 and low tumor grade [12]. Another study showed that loss of PBRM1 expression correlated with tumor T stage. Indeed, in this study, approximately half of the pT1/pT2 and only about 20% of the pT3/ pT4 tumors expressed PBRM1. Late-stage, high-grade and poorly differentiated tumors were mainly PBRM1-negative and independent from VHL mutation status. PBRM1-positive tumors correlated with better prognosis than patients with PBRM1 absent tumors [40]. In addition, PBRM1 and SETD2 mutations were not associated with differences in overall survival between patients with one or both mutated genes [44]. Finally, ccRCC with mutations either in PBRM1 or BAP1 or SETD2 correlated with higher stage but not with tumor size, supporting the idea that chromatin alteration are involved in disease progression. Moreover, specific PBRM1, SETD2, BAP1 mutations are associated advanced tumor stage and grade [39]. BAP1 & treatment sensitivity
From a therapeutic point of view, it is important to assess if distinct tumor subtypes differ in treatment response to different therapies, for example, ionizing radiation or drugs. It is known that stresses causing DNA DSBs, such as ionizing radiation, trigger BAP1 phosphorylation, assembly of the homologous recombination factors BRCA1 and RAD51, recruiting chromatin in the proximity of damaged sites [59,60]. It has been shown that BAP1-deficient cells were more vulnerable to g-radiation [12], suggesting greater sensitivity to radiotherapy in BAP1-deficient RCC than RCC wild-type for BAP1. In a previous study, mesothelioma cell lines with both wild-type and mutated or deleted BAP1 were exposed to 10 Gy of ionizing radiation, and the formation of RAD51 or BRCA1 complexes was assessed. No differences were found between BAP1 wild-type and deficient cell lines, highlighting that BAP1 may not have an important role in the formation of DNA repair complexes [22]. Regarding drugs, since BAP1 loss determines ubiquitinated H2A accumulation, masking various gene promoter transcripts, histone deacetylase (HDAC) inhibitors may reverse this effect. Indeed, valproic acid decreased H2A-ub levels and reduced the fraction of viable 92.1 human uveal melanoma cells, showing that BAP1 made these cells vulnerable to HDAC inhibitors [19]. At present, several HDAC inhibitors are under evaluation in patients with metastatic RCC. LBH589 (panobinostat), a panDAC inhibitor, was tested in a Phase II trial enrolling patients previously treated with tyrosine kinase inhibitors and mTOR inhibitors. They did not report objective responses, as all patients progressed or stopped treatment prior to the 16-week revaluation [61]. Similar results in terms of efficacy were obtained in another Phase II trial of FK228 (depsipeptide), another HDAC inhibitor [62]. The list of HDAC inhibitors also includes vorinostat, MS-275, belinostat and entinostat (in combination with IL-2), which are currently under study in mRCC patients [63–66], respectively. informahealthcare.com
Review
Furthermore, BAP1 loss sensitized cells to the PARP inhibitor olaparib [12]. Currently, a Phase I study is evaluating the combination of olaparib and AZD5363, an AKT inhibitor, in patients with advanced solid tumors including RCC refractory to standard therapy [67]. Expert commentary
Greater understanding of the molecular drivers of ccRCC has led to revolutionary new targeted therapies for patients with metastatic or otherwise inoperable ccRCC over the last decade. These newer targeted therapies, including VEGF-targeted tyrosine kinase inhibitors mTOR inhibitors and a monoclonal antibody, have virtually supplanted the older immunotherapies for ccRCC in most patients, offering a higher response rate and much less toxicity. But, despite the therapy advances made, there are virtually no complete responses in metastatic ccRCC to targeted therapy, and nearly all patients with metastatic ccRCC treated with targeted therapy alone eventually succumb to this disease [9]. Thus, even greater understanding of the molecular and genetic drivers of ccRCC is necessary to identify novel therapeutic targets in hope of eliciting cures. Likewise, identifying biomarkers in ccRCC is critical for prognostication and stratifying patients in order to properly pair them with the most appropriate therapy. BAP1, PBRM1 and SETD2 represent three candidate genes that have strong potential implications as possible biomarkers for prognostication in ccRCC and may represent possible therapeutic targets. Several studies have shown that BAP1 mutations are associated with more aggressive tumors including higher Fuhrman grade, later stage at presentation and upregulation of the mTOR pathway [12–14,39,45]. This also means that BAP1 presents itself as a potential therapeutic target, and as discussed above, trials of HDAC inhibitors in ccRCC are ongoing. Likewise, there is evidence to suggest that BAP1 mutation may be predictive of mTOR inhibitor sensitivity, but there is no data to support this either way [12]. BAP1 mutations may be associated with great sensitivity to radiotherapy, which may be an important predictive implication in ccRCC, which is a relatively radio-insensitive tumor, but more clinical research is necessary to confirm this [12]. PBRM1 also has potential prognostic implications as mutations in this gene have been associated with lower tumor grade and earlier stage disease at presentation. And, while there has been less translational and clinical research into the implications of SETD2 mutations, these histone-modifying mutations may also hold important clinical implications. Obviously, much more research is needed to further define the roles of these three genes as they relate to defining prognosis in ccRCC and as potential therapeutic targets. Five-year view
In the next years, the assessment of BAP1, PBRM1 and SETD2 mutations should be integrated with the clinicopathological features of ccRCC patients, such as prognostic groups. Furthermore, the predictive role of these mutations should be doi: 10.1586/14737159.2015.1068122
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Piva, Santoni, Matrana, et al.
investigated in patients treated with targeted agents. Moreover, research should be focused on the potential correlation between the risk of relapsing after partial or complete nephrectomy and BAP1, PBRM1 and SETD2 mutations. New targeted immunotherapy, including PD-1 and PD-L1 targeted agents, are being developed and are expected to be approved in ccRCC in the near future. Appropriately sequencing and/or combining these new drugs with current targeted therapies or other agents will be essential in maximizing patient outcomes. Potential biomarkers, such as BAP1, PBRM1 and SETD2, and others will be all the more important in order to personalize therapies for patients as our understanding of the molecular heterogeneity of ccRCC expands and the number of available treatment options for
this disease continues to grow. And, it is likely that diseasespecific panels of important genes such as VHL, BAP1, PBRM1 and SETD2 will one day help clinicians to better define prognosis and personalize targeted treatments plans for patients with ccRCC. Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Key issues .
Mutations to BAP1, PBRM1 and SETD2, in close proximity to VHL gene, are frequent in ccRCC patients.
.
In the mouse, VHL is on a different chromosome than BAP1, PBRM1 or SETD2, thus explaining why VHL heterozygous humans, but not mice, develop.
.
The inactivation of PBRM1 and BAP1 is less common in papillary, chromophobe RCC and oncocytoma than in ccRCC.
.
BAP1 mutation may be predictive of the sensitivity to mTOR inhibitors and radiotherapy.
.
Patients with BAP1- or SETD2-mutant ccRCC have been associated with poor overall survival, while PBRM1 mutations seem to identify a favorable group of ccRCC tumors.
.
Potential biomarkers, such as BAP1, PBRM1 and SETD2, and others will be all the more important in order to personalize therapies for ccRCC patients.
.
The roles of these genes as therapeutic targets in ccRCC patients have not been elucidated yet.
.
Research should be also focused on the potential correlation between the risk of relapsing after partial or complete nephrectomy and BAP1, PBRM1 and SETD2 mutations.
Papers of special note have been highlighted as: . of interest .. of considerable interest 1.
2.
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Expert Rev. Mol. Diagn.
Review
BAP1, PBRM1 and SETD2 in clear-cell renal cell carcinoma: molecular diagnostics and possible targets for personalized therapies Expert Rev. Mol. Diagn. Early online, 1–10 (2015)
Francesco Piva‡1, Matteo Santoni‡2, Marc R Matrana3, Suma Satti3, Matteo Giulietti1, Giulia Occhipinti1, Francesco Massari4, Liang Cheng5, AntonioLopez-Beltran6, Marina Scarpelli7, Giovanni Principato1, Stefano Cascinu2 and Rodolfo Montironi*7
Several novel recurrent mutations of histone modifying and chromatin remodeling genes have been identified in renal cell carcinoma. These mutations cause loss of function of several genes located in close proximity to VHL and include PBRM1, BAP1 and SETD2. PBRM1 encodes for BAF180, a component of the SWI/SNF chromatin remodeling complex, and is inactivated in, on average, 36% of clear cell renal cell carcinoma (ccRCC). Mutations of BAP1 encode for the histone deubiquitinase BRCA1 associated protein-1, and are present in 10% of ccRCCs. They are largely mutually exclusive with PBRM1 mutations. Mutations to SETD2, a histone methyltransferase, occur in 10% of ccRCC. BAP1- or SETD2-mutated ccRCCs have been associated with poor overall survival, while PBRM1 mutations seem to identify a favorable group of ccRCC tumors. This review describes the roles of PBRM1, BAP1 and SETD2 in the development and progression of ccRCC and their potential for future personalized approaches. KEYWORDS: BRCA1-associated protein-1 . clear cell renal cell carcinoma . molecular diagnostics . Polybromo-1 .
personalized therapies . SETD2
1
Department of Specialistic Clinical and Odontostomatological Sciences, Polytechnic University of Marche, Ancona, Italy 2 Medical Oncology, Polytechnic University of the Marche Region, School of Medicine, United Hospitals, Ancona, Italy, 3 Ochsner Cancer Institute, New Orleans, LA, USA, 4 Medical Oncology, Azienda Ospedaliera Universitaria Integrata, University of Verona, Verona, Italy, 5 Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA, 6 Department of Pathology and Surgery, Faculty of Medicine, Cordoba, Spain, 7 Section of Pathological Anatomy, Polytechnic University of the Marche Region, School of Medicine, United Hospitals, Ancona, Italy *Author for correspondence: [email protected] ‡
Authors equally contributed
informahealthcare.com
Renal cell carcinoma (RCC) includes sporadic or hereditary carcinomas arising from cells of the proximal renal tubular epithelium. Clear cell renal carcinomas (ccRCCs) represent the majority of kidney tumors, accounting for the 70% of RCC. They constitute a heterogeneous group that varies in histology, molecular alterations in tumor-suppressor gene and oncogenes, clinical courses and responses to treatment. In the last years, much research has focused on identifying clinical and pathological features associated with the prognosis of ccRCC patients, especially in the context of relatively new targeted therapies. These agents have led to major advances in the management of RCC patients and are directed toward VEGF [1,2] or its receptor [3–6] and mammalian target of rapamycin (mTOR) [7,8]. However, despite recent success, responses of primary ccRCC tumors and metastases to these agents are
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variable, and complete responses are relatively uncommon [9]. Indeed, the increasing availability of molecularly targeted drugs for patients with ccRCC stresses the need for reliable biomarkers to predict tumor behavior. The inactivation of the von Hippel–Lindau (VHL) tumor-suppressor gene constitutes a crucial step in ccRCC carcinogenesis; however, additional genetic and epigenetic alterations are required for the malignant transformation to ccRCC [10]. In ccRCC, one VHL allele is damaged from an intragenic mutation, and then the loss of the second allele due to a large deletion follows. This event causes a loss of heterozygosity, but it is not sufficient to cause cancer [11]. Therefore, VHL is thought to be a weak tumor-suppressor gene. In fact, it has been shown that VHL inactivation alone causes a senescent-like phenotype [11]. As deletions along VHL gene are usually large (several megabases) and located in 3p25 chromosomal
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Piva, Santoni, Matrana, et al.
region, additional near tumor-suppressor genes, such as BRCA1-associated protein-1 (BAP1), Polybromo-1 (PBRM1) and Set domain-containing 2 (SETD2), are lost (FIGURE 1). Subsequently, the loss of the remaining PBRM1 or BAP1 allele can be associated with different ccRCC grades and aggressiveness [12]. Patients with BAP1 or SETD2 mutant ccRCCs have been associated with poor overall survival, while PBRM1 mutations seem to identify a favorable group of ccRCC tumors [13,14]. The roles of these genes as therapeutic targets have not been elucidated yet. This review describes the functions and role of PBRM1, BAP1 and SETD2 in the development and progression of ccRCC, as well as their potential for future personalized approaches in the diagnosis and treatment of these patients. BAP1
The BAP1 gene is constituted by 17 exons. The protein is a deubiquitinating enzyme that binds to breast cancer type 1 susceptibility protein (BRCA1) and BRCA1-associated RING domain protein 1 (BARD1), inhibiting their capability to mediate ubiquitination and autoubiquitination and acting as a tumor suppressor [15]. The BAP1 region that interacts with BRCA1 is located between amino acids 596 and 721, while the region that interacts with BARD1 is from 182 to 365. However, a previous study did not detect the deubiquitylation of BRCA1 [16], maybe due to a different method to flag NH2 terminus of BAP1, the region where the catalytic domain should be. Thus, further data are necessary to support these properties. Moreover, BAP1 modulates histone H2A and HCFC1 deubiquitination and regulates transcription, cell cycle and growth, response to DNA damage and chromatin dynamics. In particular, BAP1 deubiquitinates H2A and makes E2F1 target gene promoters accessible. BAP1 is mainly localized in the nucleus, bound to chromatin and retained in the cytoplasm when monoubiquitinated from the E2/E3 hybrid ubiquitin-protein ligase (FIGURE 2). In the mouse, VHL is on a different chromosome than BAP1, PBRM1 or SETD2. While the loss of 3p in humans simultaneously deletes one copy of BAP1, loss of heterozygosity in the mouse corresponding VHL region does not affect BAP1 (FIGURE 1). These results explain why VHL heterozygous humans, but not mice, develop ccRCC and why a mouse model of ccRCC has been lacking [17]. BAP1 regulates G1/S phase transition and is highly expressed in testis, placenta and ovary, and present in breast tissue. Mutant BAP1 does not act as dominant negative, because the wild type allele compensates for the mutated allele. Therefore, BAP1 acts as a two-hit tumor suppressor in ccRCC. Although both copies of BAP1 can be lost, the inactivation of both BAP1 alleles through a point mutation has not been observed [12]. Although BAP1 is a tumor suppressor when overexpressed, and its mutants are associated with the development of metastases in uveal melanoma [18], its knockdown seems to have conflicting effects. In fact, its loss has been associated with the doi: 10.1586/14737159.2015.1068122
tumorigenesis of 92.1 uveal melanoma cells [19]. On the other hand, its knockdown down-regulated E2F1 target genes, resulting in the blockade of S phase initiation, cell growth and invasiveness in OCM1 and OM431 uveal melanoma cells [20]. Depletion of BAP1 expression in cutaneous melanoma cells reduced proliferation and induced apoptosis, inhibiting tumor growth in vivo, but its overexpression in melanocytes suppressed proliferation [21]. In mesothelioma cells, BAP1 knockdown has been shown to inhibit growth by the down-regulation of the E2F-responsive growth-related genes Cyclin A2, E2F1, p107 (RBL1) and CDC25C. This could be due to the post-translational inactivation of HCF-1. To explain this paradox, it has been hypothesized that BAP1 does not promote tumorigenesis by accelerating cell growth, but allows a slowed down but tolerant G1/S checkpoint, leading to slower but uncontrolled tumor growth [22]. Molecular effects of BAP1 mutations
The functionality of some BAP1 missense mutant proteins (Ser63Cys, Phe81Val, Cys91Trp and Ala95Asp) was assessed by in vitro enzymatic assay. Phe81Val, Cys91Trp and Ala95Asp showed decreased levels of ubiquitin cleavage activity, whereas Ser63Cys showed an increased level compared with wild-type BAP1. However, both over- and under-ubiquitination of target histones led to genomic instability [22]. The influence of mutated BAP1 was investigated on binding capacity with its most important interactors showing that HCF-1 levels did not differ between BAP1-deficient and reconstituted 769-P cells. Impairing the HCF-1 binding to BAP1 prevented the BAP1 inhibitory effect on cell proliferation but did not alter its ability to deubiquitinate H2A. The interaction of BAP1 with HCF-1 was analyzed in tumor grafts and the results were similar to those in cell lines. BAP1 bound to HCF-1 and BAP1 mutations did not correlate with ubiquitinated H2A (H2Aub1) levels. In other words, BAP1 binding to HCF-1, but not H2Aub1 deubiquitination, is involved in RCC tumor-suppression function [12]. PBRM1
The PBRM1 (also PB1 or BAF180) gene gives rise to at least nine splicing isoforms. It is a subunit of the SWI/SNF transcription-modulating chromatin remodeling complex that controls DNA accessibility for transcription [23]. The complex contains an ATPase subunit to supply energy for chromatin remodeling operations. PBRM1 protein harbors six bromodomains that bind acetylated residues on histone tails. The bromodomains are not equivalent in specificity as they have different target affinity. This suggests that they may ‘read and interpret’ the histone code. The bromodomains and bromoadjacent homology domains are frequently hit by mutations in ccRCC. In breast cancers where PBRM1 was found to be mutated, it was demonstrated that PBRM1 is a regulator of p21 gene promoter accessibility, since PBRM1 induces p21 expression that, in turn, inhibits cell proliferation [24]. PBRM1 seems to be required for cellular senescence, a Expert Rev. Mol. Diagn.
BAP1, PBRM1 & SETD2 in ccRCC
Human chromosomes 1 2 3
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Mouse chromosomes 1
VHL (10, 141, 635–10, 153, 670; 3p25.3) BAP1 (52, 401, 004-52, 410, 105; 3p21.1) PBRM1 (52, 545, 352-52, 685, 850; 3p21.1) SETD2 (47, 016, 408-47, 163, 977; 3p21.31)
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BAP1 (31, 251, 489-31, 259, 929; 14qB) PBRM1 (31, 019, 138-31, 121, 592; 14qB)
22 X Y
Figure 1. Different chromosome location of VHL, BAP1, PBRM1 and SETD2 between human and mouse.
tumor-suppressor mechanism, by regulating p53-mediated transcription of specific genes [25]. PBRM1 is important for transcriptional silencing induced by DNA double-strand breaks (DSBs) and promotes repair at damaged sites, and since PBRM1 mutants failed to perform these functions, it supports its tumor-suppressor activity [26]. Besides the role in transcriptional regulation, PBRM1 prevents tumorigenesis by promoting centromeric cohesion and genome stability [27]. Moreover, genes involved in chromosomal instability and cellular proliferation result in up-regulation following PBRM1 knockdown. In particular, RNAi-mediated depletion of PBRM1 in ccRCC cell lines has been shown to increase proliferation, colony formation and cell migration, consistent with a role as a tumor suppressor [28]. This role is also supported by the evidence that PBRM1 is involved in other malignancies, such as pancreatic cancer [29]. SETD2
SETD2 produces at least three alternative splicing transcripts and its products are histone methyltransferases. SETD2 has been found to act as tumor-suppressor gene in patients with breast cancer and leukemias [30,31]. Mutations in SETD2 are associated with increased loss of DNA methylation at nonpromoter regions. This is probably due to reductions of histone
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H3 (H3K36) trimethylation (H3K36me3) that is involved in the maintenance of a heterochromatic state [32]. A study that compared 42 primary ccRCC tumors versus normal kidney samples identified thousands of genomic regions with differences in chromatin accessibility. In particular, SETD2-mutated tumors were associated with nucleosome depletion, that is, an increased chromatin accessibility, and, most likely, this was due to H3K36me3 deficiency. Genes harboring alterations in nucleosome positioning and histone modifications may show aberrancies in splicing (mainly including exon skipping and intron retention) and alternative transcription start and termination sites [33]. SETD2 plays an important role also in DNA damage repair. In fact, cells lacking SETD2 show microsatellite instability and an elevated spontaneous mutation rate, since H3K36me3 is required to recruit the mismatch recognition protein hMutSa onto chromatin [34]. Moreover, H3K36me3 is required for ATM and p53-mediated checkpoint activations upon DNA DSBs [35] and homologous recombination repair of DSBs, as it facilitates the recruitment of repairing proteins [35,36]. Frequency of BAP1, PBRM1 & SETD2 mutations
Several studies have estimated the prevalence of VHL, BAP1, PBRM1 and SETD2 mutations. We have collected these data
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samples expressing PBRM1 had the wildtype gene [12]. As expected, most of the truncating mutations affected PBRM1 production. In the same samples, only in 15 out of 348 ccRCCs SETD2 was mutated. Interestingly, the inactivation of PBRM1 and BAP1 is less common in papillary RCC, chromophobe RCC (chRCC) and oncocytoma than in ccRCC tumors. Ub In particular, loss of PBRM1 protein Ub BAP1 expression occurred in only 3, 6 and 0% BRCA1 of papillary RCC, chRCC and oncocytoUb BARD1 Ub mas, whereas BAP1 loss was not observed BAP1 in any of these three tumors [41]. A recent Cancer study on a cohort of papillary RCCs and their matched normal samples highlighted BRCA1 BAP1 that SETD2 was significantly mutated [42]. UBE2O Interestingly, Gerlinger et al. analyzed BARD1 distinct subclones within primary tumors Nucleus Tumor suppression Intracellular or their metastases of 10 sporadic ccRCCs to assess the intratumor heteroFigure 2. BAP1 activity in ccRCC. BAP1 protein is a deubiquitinating enzyme that geneity. They showed that ccRCCs binds to breast cancer type 1 susceptibility protein (BRCA1) and BRCA1-associated RING adopted a branched rather than linear domain protein 1 (BARD1), acting as a tumor suppressor. BAP1 is mainly localized in the evolution. Moreover, VHL and PBRM1 nucleus and secondly retained in the cytoplasm when monoubiquitinated from E2/E3 hybrid ubiquitin-protein ligase (UBE2O). mutations were on the trunks of the phylogenetic trees, supporting that the in TABLE 1. However, it is important to assess how severe a muta- VHL and PBRM1 inactivating events occur early in tumor tion is in terms of alteration of protein function or protein development [43]. loss. Not only can destruction of the classical 5’ and 3’ splice sites alter pre-mRNA splicing and give rise to partly or totally Correlations among BAP1, PBRM1 & SETD2 mutated exon skipping or intron retention, but all mutations lying in genes exons or introns, both synonymous and missense, can cause Several studies have searched for favored or disfavored correlasplicing alterations [37]. Splicing alterations can cause severe tions between mutated genes. Surprisingly, although loss of effects, provoking frameshift errors and, consequently, the crea- BAP1 or PBRM1 was observed in 45% of ccRCC, concomition of premature termination codons. This event triggers a tant BAP1 and PBRM1 loss was reported only in 3% of surveillance mechanism called ‘nonsense mediated decay’ caus- ccRCC cases [32]. In particular, few tumors had both ing transcript degradation and protein loss. Although some BAP1 and PBRM1 loss, and this simultaneous loss in ccRCC softwares are able to predict if a mutation can alter splicing [38], may be disadvantageous, although the reasons are such softwares are unable to show the resulting splicing pattern unclear [12,44]. These results, confirmed in other investigain terms of sequences lost or retained. tions [45], suggest that contemporary mutations in BAP1 and In VHL, BAP1, PBRM1 and SETD2, there are base substitu- PBRM1 are negatively selected in ccRCC; in other words tions, insertions or deletions that could cause frameshift and tumors may segregate into BAP1 or PBRM1-deficient subnonsense mutations. The latter two are truncating mutations types. Instead, in a study on 185 ccRCC tumors, mutual that can directly cause loss of the protein or its function. exclusivity between PBRM1 and BAP1 mutations was not A study that found 49.2, 29.2, 7.6 and 5.9% of tumors har- statistically significant [39]; according to another study, loss of bored mutated VHL, PBRM1, SETD2 and BAP1 and also PBRM1 expression did not correlate with VHL mutations [40]. highlighted that truncating mutations represent 67, 82.5, However, it is important to note that studies with small 78.50 and 36% of mutations in VHL, PBRM1, SETD2 and sample size might not have enough power to detect an BAP1 respectively [39]. association. Furthermore, some authors showed that PBRM1 protein Concerning PBRM1 and SETD2 genes, a meta-analysis across expression is present only in about 30% of ccRCC [40]; corre- ccRCC studies confirmed that the frequency of tumors lating sequencing with immunohistochemistry data showed that with both PBRM1 and SETD2 mutated exceeded the frequency about 90% of samples that did not express PBRM1 had a expected by chance alone [13]. Therefore, PBRM1 and mutation in the corresponding gene, and about 90% of the SETD2 can cooperate in renal tumorigenesis [44].
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Extracellular
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45 N/A 11 N/A N/A 2 The percentage is the sum of synonymous and non-synonymous mutations. N/A: Not assessed data.
52 N/A 14 Selected case cohort 176
81
[69]
41 10 10 5 1 1 41 11 10 Selected case cohort 240
82
[69]
11 3 4 4 0 0 26 11 8 Selected case cohort 106
41
[68]
7 1 2 3 0 0 42 8 17 55 Selected case cohort 98
[39]
19 6 3 3 1 1 29 8 6 Selected case cohort 185
49
[13]
20 6 4 4 1 1 30 7 6 Selected case cohort 188
50
[32]
22 6 1 6 1 1 34 13 11 Selected case cohort 417
56
Ref VHL (%) Study design
The promoter hypermethylation has been hypothesized to explain why about 10% of ccRCC are negative for PBRM1 or BAP1 proteins, according to immunohistochemistry, but they do not show evidence of point mutations. In fact, CpG hypermethylation represents another way to inactivate one allele of a gene, alternative to mutations. To sustain this hypothesis, the methylation status of CpG loci near the transcription start site of BAP1, PBRM1 and SETD2 was assessed in 50 primary ccRCCs. These genes were not or rarely methylated in both tumor and normal specimens [55]. Instead, in clinical samples of ccRCC, the identification of chromatin accessibility decreases in PBRM1 and SETD2 genes, suggesting the importance of epigenetic changes in this tumor and supporting the manipulation of the epigenome as a potential therapeutic strategy [56]. Thus, further efforts are required to identify the mechanisms underlying such events.
Cohort
Epigenetics
BAP1 (%)
SETD2 (%)
PBRM1 (%)
BAP1 + PBRM1 (%)
BAP1 + SETD2 (%)
SETD2 + PBRM1 (%)
VHL + BAP1 (%)
VHL + SETD2 (%)
VHL + PBRM1 (%)
A germline BAP1 mutation G121A (Gly41Ser) was identified in one individual, not having a germline VHL mutation, who had two and one first and second-degree relatives, respectively, with RCC [12]. The mutation A277G (Thr93Ala) in BAP1 has been shown to alter the RNA splicing pattern, which gives rise to 80% of aberrant mature transcripts that lead to a frameshift, whereas approximately 20% corresponded to the missense mutation. In TABLE 1, we summarize the BAP1 mutations identified by the same authors and associated with a tumor predisposition syndrome, including uveal melanoma, malignant pleural mesothelioma and cutaneous melanoma [46]. BAP1 sequencing in 83 unrelated probands with familial RCC revealed that a new germline mutation, T41A (L14H), predisposed to early onset of ccRCC. This was supported by several findings, including: this variant co-segregates with the RCC phenotype; it targets the catalytic domain of BAP1; the wild type amino acid L14 is highly conserved across species suggesting its irreplaceableness; the mutation is associated with LOH [47]. In the same study, other two mutations were discovered, although they have been likely considered as benign (TABLE 2). Germline mutations leading to BAP1 hereditary cancer predisposition syndrome can also cause uveal and cutaneous melanoma, mesothelioma [48–51] and basal cell carcinoma [52]. Notably, patients with the same BAP1 mutational status may develop different cancer types depending on the type of carcinogen exposure and, naturally, on the presence of other gene mutations [53]. BAP1 germline mutations have been found in several tissues. However, not all these tissues harboring such mutations develop cancer, suggesting that the mechanisms underlying such tolerance to mutations are still unrevealed. Moreover, Wang et al. have presented the preliminary The Cancer Genome Atlas exome sequencing data from 499 RCC cases [54]. The final result from this study on the loss of heterozygosity patterns to assess the interactions between germline and somatic mutations are awaited.
Table 1. ccRCC marker frequencies.
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Are germinal BAP1 mutations associated with RCC predisposition?
[12]
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BAP1, PBRM1 & SETD2 in ccRCC
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Table 2. Germline mutations in BAP1 gene causing renal cell carcinoma. Gene
Protein
Predicted effects
Ref.
37+1 delG
N-A
Probably affecting splicing
[46]
T41A
Leu14His
Missense (or splice) exon 1
[47]
C45G
N-A
3’UTR
[48]
78_79 delGG
Val27Ala
Truncating
[46]
G121A
Gly41Ser
Probably benign
[47]
256_277 and A277G
Ile87Met and Thr93Ala
In/del exon 5
[46]
A277G
Thr93Ala
Affecting splicing and truncating
[46]
437+1 G>A
N-A
Truncating
[46]
629 dupT
Met211His
Truncating
[46]
660-11 T>A
N-A
Probably affecting splicing
[46]
A869G
Asn290Ser
probably benign
[47]
932-58_59 delTG
N-A
Probably affecting splicing
[48]
C1182G
Tyr394*
Unknown
[48]
1654 delG
Asp552Ile
Truncating
[46]
1882_1885 del TCAC
Ser628Pro
Unknown
[48]
C2050T
Gln684*
Unknown
[48]
2057-22A>C
N-A
Probably affecting splicing
[48]
2057-4G>T
N-A
Probably affecting splicing
[48]
Prognostic significance of BAP1, PBRM1 & SETD2 mutations
Several studies focused on the prognostic significance of BAP1, PBRM1 and SETD2 mutational status. An analysis on 176 tumors showed that BAP1 loss correlated with high Fuhrman nuclear grade and mTORC1 activation [12]. Other authors confirmed a strong correlation between BAP1 mutations and higher Fuhrman nuclear grades [39]. Moreover, BAP1 loss in ccRCC may be associated with poor prognosis [14,32,39,45]. In particular, a retrospective analysis of 145 patients validated using a further independent cohort of 327 patients confirmed that tumors with mutations of either PBRM1 or BAP1 were more likely to present with higher tumor stage 3–4. They found that BAP1 mutations, but no VHL or PBRM1 mutations, were significantly associated with worse cancer-specific survival. They also reported a poor median overall survival (4.6 years) in patients with BAP1-mutated ccRCC compared to patients with PBRM1-mutated tumors (10.6 years) [14]. A study of BAP1 protein expression in 1479 patients with clinically localized ccRCC showed that BAP1 loss correlated with the most aggressive forms of ccRCC, even among low-risk patients. However, in this study, not all high-grade tumors showed loss of BAP1 expression [57]. A potential explanation of its negative prognostic role may be represented by mTORC1 activation following BAP1 inactivation, suggesting doi: 10.1586/14737159.2015.1068122
that these tumors could be more responsive to mTORC1 inhibitors. Interestingly, BAP1 truncating, compared with missense mutations, presented worse prognosis, but this was not maintained for BAP1 and SETD2 in a different cohort. Moreover, SETD2 mutations were related to the time to recurrence [13]. BAP1- and PBRM1-mutant tumors were also associated with different gene expression signatures. The former had alterations of genes implicated in growth-factor signaling and the latter involved genes implicated in the cytoskeleton and tissue architecture. This suggests that ccRCC can be classified into at least two distinct entities, biologically and clinically [14]. SETD2 mutations were related with little worse prognosis [13], whereas SETD2 frameshift mutations were associated with advanced tumors stages [39]. A retrospective study that investigated the associations between computed tomography diagnostic imaging features and RCC mutations highlighted that VHL-mutated tumors were associated with well-defined tumor margins, nodular tumor enhancement and gross appearance of intratumor vascularity. BAP1-mutated tumors were associated with renal vein invasion, whereas SETD2-mutated tumors were not associated with any computed tomography feature studied. Moreover, multicystic ccRCC did not harbor mutations of SETD2 and BAP1 [58]. Expert Rev. Mol. Diagn.
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BAP1, PBRM1 & SETD2 in ccRCC
As for PBRM1 loss, it was associated neither with high-grade nor with low-grade tumor, but there was a correlation between tumors that had exclusively lost PBRM1 and low tumor grade [12]. Another study showed that loss of PBRM1 expression correlated with tumor T stage. Indeed, in this study, approximately half of the pT1/pT2 and only about 20% of the pT3/ pT4 tumors expressed PBRM1. Late-stage, high-grade and poorly differentiated tumors were mainly PBRM1-negative and independent from VHL mutation status. PBRM1-positive tumors correlated with better prognosis than patients with PBRM1 absent tumors [40]. In addition, PBRM1 and SETD2 mutations were not associated with differences in overall survival between patients with one or both mutated genes [44]. Finally, ccRCC with mutations either in PBRM1 or BAP1 or SETD2 correlated with higher stage but not with tumor size, supporting the idea that chromatin alteration are involved in disease progression. Moreover, specific PBRM1, SETD2, BAP1 mutations are associated advanced tumor stage and grade [39]. BAP1 & treatment sensitivity
From a therapeutic point of view, it is important to assess if distinct tumor subtypes differ in treatment response to different therapies, for example, ionizing radiation or drugs. It is known that stresses causing DNA DSBs, such as ionizing radiation, trigger BAP1 phosphorylation, assembly of the homologous recombination factors BRCA1 and RAD51, recruiting chromatin in the proximity of damaged sites [59,60]. It has been shown that BAP1-deficient cells were more vulnerable to g-radiation [12], suggesting greater sensitivity to radiotherapy in BAP1-deficient RCC than RCC wild-type for BAP1. In a previous study, mesothelioma cell lines with both wild-type and mutated or deleted BAP1 were exposed to 10 Gy of ionizing radiation, and the formation of RAD51 or BRCA1 complexes was assessed. No differences were found between BAP1 wild-type and deficient cell lines, highlighting that BAP1 may not have an important role in the formation of DNA repair complexes [22]. Regarding drugs, since BAP1 loss determines ubiquitinated H2A accumulation, masking various gene promoter transcripts, histone deacetylase (HDAC) inhibitors may reverse this effect. Indeed, valproic acid decreased H2A-ub levels and reduced the fraction of viable 92.1 human uveal melanoma cells, showing that BAP1 made these cells vulnerable to HDAC inhibitors [19]. At present, several HDAC inhibitors are under evaluation in patients with metastatic RCC. LBH589 (panobinostat), a panDAC inhibitor, was tested in a Phase II trial enrolling patients previously treated with tyrosine kinase inhibitors and mTOR inhibitors. They did not report objective responses, as all patients progressed or stopped treatment prior to the 16-week revaluation [61]. Similar results in terms of efficacy were obtained in another Phase II trial of FK228 (depsipeptide), another HDAC inhibitor [62]. The list of HDAC inhibitors also includes vorinostat, MS-275, belinostat and entinostat (in combination with IL-2), which are currently under study in mRCC patients [63–66], respectively. informahealthcare.com
Review
Furthermore, BAP1 loss sensitized cells to the PARP inhibitor olaparib [12]. Currently, a Phase I study is evaluating the combination of olaparib and AZD5363, an AKT inhibitor, in patients with advanced solid tumors including RCC refractory to standard therapy [67]. Expert commentary
Greater understanding of the molecular drivers of ccRCC has led to revolutionary new targeted therapies for patients with metastatic or otherwise inoperable ccRCC over the last decade. These newer targeted therapies, including VEGF-targeted tyrosine kinase inhibitors mTOR inhibitors and a monoclonal antibody, have virtually supplanted the older immunotherapies for ccRCC in most patients, offering a higher response rate and much less toxicity. But, despite the therapy advances made, there are virtually no complete responses in metastatic ccRCC to targeted therapy, and nearly all patients with metastatic ccRCC treated with targeted therapy alone eventually succumb to this disease [9]. Thus, even greater understanding of the molecular and genetic drivers of ccRCC is necessary to identify novel therapeutic targets in hope of eliciting cures. Likewise, identifying biomarkers in ccRCC is critical for prognostication and stratifying patients in order to properly pair them with the most appropriate therapy. BAP1, PBRM1 and SETD2 represent three candidate genes that have strong potential implications as possible biomarkers for prognostication in ccRCC and may represent possible therapeutic targets. Several studies have shown that BAP1 mutations are associated with more aggressive tumors including higher Fuhrman grade, later stage at presentation and upregulation of the mTOR pathway [12–14,39,45]. This also means that BAP1 presents itself as a potential therapeutic target, and as discussed above, trials of HDAC inhibitors in ccRCC are ongoing. Likewise, there is evidence to suggest that BAP1 mutation may be predictive of mTOR inhibitor sensitivity, but there is no data to support this either way [12]. BAP1 mutations may be associated with great sensitivity to radiotherapy, which may be an important predictive implication in ccRCC, which is a relatively radio-insensitive tumor, but more clinical research is necessary to confirm this [12]. PBRM1 also has potential prognostic implications as mutations in this gene have been associated with lower tumor grade and earlier stage disease at presentation. And, while there has been less translational and clinical research into the implications of SETD2 mutations, these histone-modifying mutations may also hold important clinical implications. Obviously, much more research is needed to further define the roles of these three genes as they relate to defining prognosis in ccRCC and as potential therapeutic targets. Five-year view
In the next years, the assessment of BAP1, PBRM1 and SETD2 mutations should be integrated with the clinicopathological features of ccRCC patients, such as prognostic groups. Furthermore, the predictive role of these mutations should be doi: 10.1586/14737159.2015.1068122
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Piva, Santoni, Matrana, et al.
investigated in patients treated with targeted agents. Moreover, research should be focused on the potential correlation between the risk of relapsing after partial or complete nephrectomy and BAP1, PBRM1 and SETD2 mutations. New targeted immunotherapy, including PD-1 and PD-L1 targeted agents, are being developed and are expected to be approved in ccRCC in the near future. Appropriately sequencing and/or combining these new drugs with current targeted therapies or other agents will be essential in maximizing patient outcomes. Potential biomarkers, such as BAP1, PBRM1 and SETD2, and others will be all the more important in order to personalize therapies for patients as our understanding of the molecular heterogeneity of ccRCC expands and the number of available treatment options for
this disease continues to grow. And, it is likely that diseasespecific panels of important genes such as VHL, BAP1, PBRM1 and SETD2 will one day help clinicians to better define prognosis and personalize targeted treatments plans for patients with ccRCC. Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Key issues .
Mutations to BAP1, PBRM1 and SETD2, in close proximity to VHL gene, are frequent in ccRCC patients.
.
In the mouse, VHL is on a different chromosome than BAP1, PBRM1 or SETD2, thus explaining why VHL heterozygous humans, but not mice, develop.
.
The inactivation of PBRM1 and BAP1 is less common in papillary, chromophobe RCC and oncocytoma than in ccRCC.
.
BAP1 mutation may be predictive of the sensitivity to mTOR inhibitors and radiotherapy.
.
Patients with BAP1- or SETD2-mutant ccRCC have been associated with poor overall survival, while PBRM1 mutations seem to identify a favorable group of ccRCC tumors.
.
Potential biomarkers, such as BAP1, PBRM1 and SETD2, and others will be all the more important in order to personalize therapies for ccRCC patients.
.
The roles of these genes as therapeutic targets in ccRCC patients have not been elucidated yet.
.
Research should be also focused on the potential correlation between the risk of relapsing after partial or complete nephrectomy and BAP1, PBRM1 and SETD2 mutations.
Papers of special note have been highlighted as: . of interest .. of considerable interest 1.
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3.
4.
5.
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