Loss of the Tumor Suppressor BAP1 Causes Myeloid Transformation

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Science  21 Sep 2012:
Vol. 337, Issue 6101, pp. 1541-1546
DOI: 10.1126/science.1221711

Identifying BAP1 Targets

Inactivating mutations in the deubiquitinating enzyme BAP1 have been associated with cancer. Dey et al. (p. 1541, published online 9 August; see the Perspective by White and Harper) reveal molecular targets of the enzyme and show evidence for a role in leukemia. Mice specifically lacking the target of BAP1, HCF-1, in the bone marrow developed myeloid leukemia. BAP1 appears to be part of a complex that regulates modification of histones and gene expression important for normal hematopoiesis and tumor suppression.


De-ubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with increased risk of mesothelioma and uveal melanoma. Somatic BAP1 mutations occur in various malignancies. We show that mouse Bap1 gene deletion is lethal during embryogenesis, but systemic or hematopoietic-restricted deletion in adults recapitulates features of human myelodysplastic syndrome (MDS). Knockin mice expressing BAP1 with a 3xFlag tag revealed that BAP1 interacts with host cell factor–1 (HCF-1), O-linked N-acetylglucosamine transferase (OGT), and the polycomb group proteins ASXL1 and ASXL2 in vivo. OGT and HCF-1 levels were decreased by Bap1 deletion, indicating a critical role for BAP1 in stabilizing these epigenetic regulators. Human ASXL1 is mutated frequently in chronic myelomonocytic leukemia (CMML) so an ASXL/BAP1 complex may suppress CMML. A BAP1 catalytic mutation found in a MDS patient implies that BAP1 loss of function has similar consequences in mice and humans.

Somatic inactivating BAP1 mutations occur in the majority of metastatic uveal melanomas and approximately one-quarter of malignant pleural mesotheliomas. Somatic mutations also have been identified in breast, lung, and renal cell cancers (15). Recently, germline BAP1 mutations were linked to a tumor predisposition syndrome characterized by melanocytic tumors, mesothelioma, and uveal melanoma (6, 7).

We investigated the normal physiological role of BAP1 using BAP1-deficient mice (fig. S1A). Bap1−/− embryos showed developmental retardation at embryonic day 8.5 (E8.5) and were not detected beyond E9.5, indicating that BAP1 is essential for embryo development (fig. S1, B and C). To bypass this embryonic lethality, we bred mice that expressed the tamoxifen-inducible recombinase creERT2 ubiquitously from the Rosa26 locus (8) and had Bap1 exons 4 and 5 flanked by lox sites (floxed) (fig. S1A). The floxed Bap1 exons were deleted from most adult mouse tissues at 1 week after daily tamoxifen injections for 5 days were completed, the brain being the expected exception (fig. S1D). Loss of Bap1 mRNA from hematopoietic lineages at 1 week after the final tamoxifen injection was confirmed by quantitative reverse transcription polymerase chain reaction (fig. S1E), and BAP1 protein was no longer detected in splenocytes by Western blotting (fig. S1F). Within 4 weeks of the last tamoxifen injection, 100% of the Bap1fl/fl creERT2+ mice [hereafter referred to as BAP1 knockout (BAP1 KO) mice] developed splenomegaly (n = 12 mice). This phenotype was never observed in Bap1+/+ creERT2+ control mice [hereafter referred to as wild-type (WT) mice] (Fig. 1, A and B). Histopathology, flow cytometry, and myeloperoxidase immunohistochemistry revealed that splenomegaly in the KO mice resulted from extramedullary hematopoiesis and expansion of the myeloid lineage (Fig. 1, C to E). Myeloid cells also were increased in lymph nodes (fig. S2) and bone marrow (Fig. 1F).

Fig. 1

BAP1 deficiency results in MDS/CMML-like disease. (A) Bap1+/+ creERT2+ (WT) and Bap1fl/fl creERT2+ (KO) spleens at 4 weeks after tamoxifen treatment. (B) Total splenocytes at 4 weeks. (C) Hematoxylin and eosin staining of spleens at 4 weeks. (D) Leukocyte subsets in spleen at 4 weeks analyzed by flow cytometry. Identifying surface markers were B220 (B cells), CD3ε (T cells), CD11b (myeloid cells), and CD11b plus Gr1 (neutrophils). (E and F) Myeloperoxidase staining of spleen (E) and bone marrow (F) at 4 weeks. (G) Peripheral blood smears at 4 weeks. (H to L) Peripheral blood cell counts. All graphs show the mean ± SD for five mice of each genotype. Asterisks indicate statistically significant differences between WT and KO mice based on two-way analysis of variance (ANOVA), followed by Bonferroni post-test analysis. 10E3/ul, 1 × 103/μl; WBC, white blood cells. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Peripheral blood taken from 11 out of 12 BAP1 KO mice at 4 weeks after the final tamoxifen injection showed cytological features consistent with myelodysplasia and ineffective erythropoiesis (Fig. 1G). Total leukocyte numbers were elevated (Fig. 1H) because of monocytosis (Fig. 1I) and neutrophilia (Fig. 1J), which is consistent with chronic myelomonocytic leukemia (CMML)–like disease [classified as a myelodysplastic/myeloproliferative disease according to the new World Health Organization classification of myeloid neoplasms (9)]. Thrombocytopenia was detected as early as 1 week after the final tamoxifen injection (Fig. 1K), and all diseased mice developed severe progressive anemia (Fig. 1L). We noted morphologic features of erythroid dysplasia, including increased numbers of nucleated red blood cells, anisopoikilocytosis, and prominent basophilic stippling (Fig. 1L). Hypersegmented neutrophils (Fig. 1J), bilobed granulocytes (Fig. 1I), giant platelets (Fig. 1K), hyposegmented neutrophils consistent with pseudo-Pelger-Huët anomaly, and atypical immature cells with myelomonocytic features were also observed. Mitotic figures and apoptotic cells (fig. S3) were consistent with human myelodysplastic syndrome (MDS) (10), whereas blasts were rare. Collectively, these data show that Bap1 deletion produces a myeloproliferative/myelodysplastic disorder with features of human CMML. Consistent with what is seen in patients with end-organ damage from myeloid neoplasms, the BAP1 KO heart contained microthrombi with multifocal necrosis, neutrophilic inflammation, and infiltration of myeloblastic cells (fig. S4).

Given that chronic myeloid neoplasms originate in the phenotypic hematopoietic stem cell (HSC) compartment (11), we characterized the lineage-depleted hematopoietic progenitor cell population in the BAP1 KO mice. Lineage-negative ScaI c-Kit+ myeloid progenitor cells and HSC-enriched lineage-negative ScaI+ c-Kit+ (LSK) cells were increased in BAP1 KO spleen and bone marrow as early as 2 weeks after the final tamoxifen injection (fig. S5A). Given that BAP1 KO mice develop monocytosis and neutrophilia, BAP1 KO LSK cells harvested 1 month after tamoxifen treatment expressed higher levels of a subset of genes involved in myelopoiesis [fig. S5, B and C (12)]. In methylcellulose colony-forming assays, BAP1 KO LSK cells yielded fewer colonies than WT LSK cells (fig. S6, A and B). In addition, unlike cells from WT colonies, which could be replated after 10 days in culture to form new colonies, replated BAP1 KO cells did not produce well-formed colonies, and many exhibited cytoplasmic blebbing characteristic of apoptosis (fig. S6, C and D). These in vitro data suggest that BAP1 deficiency impairs HSC survival and/or self-renewal, but this may be context-dependent, in such a way that sufficient BAP1 KO HSCs survive in vivo to reveal the skewing of differentiation toward the myeloid lineage (Fig. 1 and fig. S5)

Next, we performed bone marrow transplantation studies to determine whether CMML-like disease is intrinsic to the BAP1 KO hematopoietic compartment. BAP1 KO CD45.2+ lineage-negative bone marrow cells harvested either 1 week (fig. S7A) or 1 month (fig. S7B) after the final tamoxifen injection were unable to reconstitute the bone marrow of lethally irradiated congenic CD45.1+ B6.SJL recipient mice like their WT counterparts. This finding was confirmed in competitive repopulation assays in which recipient mice received equal numbers of WT B6.SJL and BAP1 KO C57BL/6 bone marrow cells (fig. S8). Failure of the BAP1 KO cells to engraft might reflect an inability to home to the appropriate stem cell niche. However, Bap1 deletion after the bone marrow of B6.SJL recipient mice was reconstituted with Bap1fl/fl creERT2+ bone marrow cells (Fig. 2A and fig. S9) produced features of CMML, including thrombocytopenia (Fig. 2B), neutrophilia (Fig. 2C), monocytosis (Fig. 2D), and anemia (Fig. 2, E and F) at 4 weeks. Myeloid cells were increased in the spleen (Fig. 2G), and lineage-negative ScaI c-Kit+ myeloid progenitor cells and LSK cells were increased in the bone marrow (Fig. 2, H to J). These data indicate that BAP1 deficiency restricted to the hematopoietic compartment is sufficient for the development of myeloid leukemia, and there is no requirement for BAP1 deficiency in the bone marrow stoma.

Fig. 2

BAP1 deficiency in hematopoietic cells is sufficient for MDS/CMML-like disease. (A) Bone marrow cells from CD45.2+ Bap1+/+ creERT2+ or Bap1fl/fl creERT2+ mice were transplanted into CD45.1+ lethally irradiated WT recipients. Tamoxifen was given to recipients at 5 weeks after transplantation to induce Bap1 deletion. (B to F) Peripheral blood cell counts [(B) to (E)] and hemoglobin (HGB) levels (F) of reconstituted mice. (G) Splenic subsets at 4 weeks after tamoxifen treatment. Identifying surface markers were B220 (B cells), CD3ε (T cells), and CD11b (myeloid cells). (H) Flow cytometric analysis of lineage-negative bone marrow cell populations at 4 weeks after tamoxifen treatment. (I and J) Absolute numbers of lineage-negative ScaI c-Kit+ myeloid progenitors (I) and lineage-negative ScaI+ c-Kit+ (LSK) cells (J) in bone marrow at 4 weeks after tamoxifen treatment. All graphs show the mean ± SD for three to five mice of each genotype. Asterisks indicate statistically significant differences between WT and KO mice based on two-way ANOVA, followed by Bonferroni post-test analysis. *P < 0.05, ***P < 0.001, ****P < 0.0001.

To investigate the mechanism for tumor suppression by BAP1, we characterized by mass spectrometry endogenous BAP1-interacting proteins, which we affinity-purified from BAP1.3xFlag knockin mouse spleen and brain (Fig. 3 and fig. S10A). These organs were chosen because they express BAP1 (fig. S10B), and their size means that protein yield is not a limiting factor. It is worth noting, however, that Bap1 mRNA was detected in LSK cells (fig. S1E), and BAP1.3xFlag protein was immunoblotted in lineage-negative bone marrow cells containing the progenitor and stem cell populations (fig. S11). Because antibodies to Flag also recognize several proteins in WT tissues (fig. S10B), we used a subtraction strategy employing stable isotope labeling. Specifically, BAP1.3xFlag tissue lysates were mixed, before immunoprecipitation, in a 1:1 ratio with WT lysates prepared from animals fed 13C6-lysine in their chow (13). If the light version of a peptide deriving from the BAP1.3xFlag knockin was not more abundant than its heavy WT counterpart when analyzed by mass spectrometry, then the protein was excluded as a BAP1-interacting protein. Immunoprecipitations with tissues from an unrelated knockin strain expressing 3xFlag.ARMC8 (fig. S10C), which is a component of the CTLH (C-terminal to LisH motif) complex (14), provided a further control for specificity.

Fig. 3

Identification and characterization of BAP1-associated proteins in mouse brain and spleen. (A) BAP1.3xFlag knockin (KI) mice fed regular chow and WT mice fed “heavy” 13C6-lysine in their chow were used to make tissue lysates for anti-Flag immunoprecipitation (IP) and mass spectrometry (MS). Top IP hits that were represented by multiple unique peptides deriving primarily from the “light” BAP1.3xFlag KI are listed. (B) The mouse BAP1 interactome. Large nodes represent BAP1-interacting proteins identified in the brain (blue lines) or spleen (red lines). Gray lines indicate known interactions from the STRING database. Smaller gray nodes represent proteins from the STRING database that are connected to two or more proteins from our data set and include manually crated annotations. (C) Anti-Flag immunoprecipitations from WT and BAP1.3xFlag splenocytes. (D) Western blot analysis of WT and BAP1 KO splenocytes at 3 weeks after tamoxifen treatment. (E) Myc-tagged OGT was affinity-purified from HEK293T cells cotransfected with hemagglutinin (HA)–tagged ubiquitin and then incubated with BAP1/ASXL1 purified from Sf9 cells. Where indicated, BAP1 protease activity was inactivated with NEM before OGT was added.

Captured proteins were washed briefly under low-stringency conditions, so that silver staining revealed no differential bands between WT versus BAP1.3xFlag spleen (fig. S10D). After the WT background was subtracted out, however, known BAP1-interacting proteins were revealed (15, 16), including host cell factor–1 (HCF-1), O-linked N-acetylglucosamine transferase (OGT), the polycomb group proteins ASXL1 and ASXL2 (Fig. 3A), the lysine demethylase KDM1B, and the transcriptional regulator FOXK1. The BAP1 network defined by these experiments is represented in Fig. 3B, and it also includes select interactors from the STRING database or manually curated annotations if they connected to two or more proteins within our experimental data set. Immunoprecipitation and Western blotting of BAP1.3xFlag mouse spleen validated the interaction of OGT and HCF-1 with BAP1 (Fig. 3C), but we lacked quality antibodies to assess the other endogenous interactions in this manner.ASXL1 is a cofactor that is essential for BAP1 enzymatic activity (17) and is mutated frequently in MDS and CMML (18, 19). Given the development of MDS in our BAP1 KO mice, it is tempting to speculate that the BAP1-ASXL1 axis has a critical role in suppressing CMML.

De-ubiquitylation of the conserved epigenetic regulator HCF-1 by BAP1 prevents HCF-1 proteasomal degradation (20) and, consistent with this notion, BAP1 KO splenocytes contained far less HCF-1 than their WT counterparts (Fig. 3D). OGT also was decreased in BAP1 KO splenocytes (Fig. 3D), and, being the sole cellular enzyme responsible for O-GlcNAcylation, the cells showed a corresponding decrease in O-GlcNAcylated proteins. To investigate whether BAP1 can de-ubiquitylate and stabilize OGT directly, rather than decreased OGT being secondary to a reduction in HCF-1, we affinity-purified ubiquitylated and Myc-tagged human OGT from HEK293T cells and then incubated it with human BAP1 and human ASXL1 residues 2 to 365, copurified from Sf9 cells. Consistent with OGT being a BAP1 substrate, ubiquitylation on OGT was reduced by the BAP1/ASXL1 complex, and this de-ubiquitylation was blocked when purified BAP1/ASXL1 was pretreated with the cysteine protease inhibitor N-ethylmaleimide (NEM) (Fig. 3E). NEM-mediated inactivation of BAP1 catalytic activity was confirmed in a ubiquitin-7-amido-4-methylcoumarin (Ub-AMC) cleavage assay (fig. S12A). In contrast to the situation in splenocytes, BAP1 deficiency in mouse embryo fibroblasts did not alter steady-state OGT protein abundance, but, in keeping with OGT being a BAP1 substrate, increased OGT protein turnover was revealed when translation was inhibited with cycloheximide (fig. S12B). Because O-GlcNAcylation of HCF-1 by OGT is necessary for the proteolytic maturation of HCF-1 (21, 22), our data indicate that BAP1 not only stabilizes HCF-1 but also is necessary for HCF-1 activation via OGT stabilization.

HCF-1 is a pleiotropic transcriptional coregulator (23), so BAP1 probably regulates gene expression via HCF-1 stabilization. To identify potentially critical BAP1-regulated genes, we determined what genes are (i) dysregulated in BAP1 KO cells by microarray analyses, and (ii) normally have BAP1 bound indirectly to their promoters (via yet-to-be-characterized DNA binding proteins), as judged by chromatin immunoprecipitation and DNA sequencing (ChIP-seq). Low chromatin yields from hematopoietic stem/progenitor cell populations precluded ChIP-seq studies, so we used BAP1.3xFlag knockin bone marrow–derived macrophages (BMDMs) as a surrogate to identify genome-wide BAP1 binding sites. A total of 9128 significant BAP1 peaks were identified after ChIP with antibodies to Flag (assessed with MACS analysis software with stringent criteria: P value < 1 × 10−10 and fold enrichment >5). Of these sites, 5926 (65%) were located near the transcription start sites of 5731 genes (Fig. 4A). Motif enrichment analysis of the BAP1-binding sites revealed that the top two occurring motifs are most similar to known binding sites of the Ets (CGGAAG) family of transcription factors and of SP1 (GGGCGGGG) (Fig. 4B). Gene expression data sets were generated with WT and BAP1 KO lineage-negative ScaI c-Kit+ myeloid progenitor cells, as well as LSK cells. Among the 5731 genes with BAP1 peaks by ChIP-seq, 32 were down-regulated significantly in BAP1 KO cells (Fig. 4C), whereas 18 were up-regulated. We validated BAP1 localization to 30 out of 32 of the down-regulated genes by ChIP–qualitative polymerase chain reaction (qPCR) (Fig. 4D). Several of the genes identified affect the immune system, including Il7r, which is particularly interesting as a candidate BAP1 target gene because it is a known regulator of hematopoietic cell survival, and IL7R expression is decreased in MDS patients (24).

Fig. 4

Identification of BAP1-regulated genes. (A) Characterization of BAP1 binding sites identified by anti-Flag ChIP-seq analysis of BAP1.3xFlag BMDMs. TSS, transcription start site. (B) De novo motif enrichment analysis of BAP1 binding sites identified similarities to Ets (CGGAAG) and SP1 (GGGCGGGG) transcription factor binding sites. (C) Venn diagram of genes with BAP1 recruited to their promoter in BAP1.3xFlag BMDMs (blue circle), and genes with reduced expression in BAP1 KO lineage-negative ScaI c-Kit+ myeloid progenitors (MP; green circle) or BAP1 KO LSK cells (red circle). (D) Putative BAP1 target genes identified in (C) were validated by ChIP-qPCR on BAP1.3xFlag BMDMs (top panel). Negative control (Neg. Ctrl) primers amplify a region in a gene desert, and therefore binding of transcription factors is not expected. Bars show the mean ± SD of triplicate wells, the chromatin deriving from BMDMs pooled from multiple KI mice. The lower panel shows microarray expression data for these genes in WT versus BAP1 KO MP or LSK cells. (E) Venn diagram showing the overlap between promoters occupied by BAP1.3xFlag, HCF-1, or OGT, based on ChIP-seq analyses. (F) Distances separating BAP1 peaks and OGT peaks from HCF-1 peaks by ChIP-seq.

Next we performed additional ChIP-seq studies for HCF-1 and OGT using WT BMDMs, the goal being to determine which promoters occupied by BAP1 also contain HCF1 and/or OGT. Most HCF-1 and OGT peaks (57.3 and 52.4%, respectively) were located near transcriptional start sites (fig. S13). Eighty-five percent of promoters occupied by BAP1 also contained HCF-1 (Fig. 4E). Fewer OGT sites were identified, but of these, most were found in genes containing BAP1 and/or HCF-1 sites (Fig. 4E). Within the 1827 promoters occupied by BAP, OGT, and HCF-1, more than 65% of BAP1 peaks are within 200 base pairs (bp) of an HCF-1 peak, whereas 70% of OGT peaks are within 400 bp of an HCF1 peak (Fig. 4F). The close proximity of the BAP1, OGT, and HCF-1 peaks (Fig. 4F) fits with their being recruited to promoters as a complex.

Given that BAP1 KO mice develop MDS, we investigated whether BAP1 mutations occur in human MDS by full-length resequencing of BAP1 in 32 paired tumor and normal samples from patients with de novo MDS. We identified a patient with a frameshift mutation that causes premature termination within the UCH catalytic domain of BAP1. Analysis of matched normal DNA did not identify the frameshift allele, which is consistent with somatic acquisition of the frameshift BAP1 mutation by the MDS clone (fig. S14). The patient with the somatic BAP1 mutation presented with refractory cytopenias and multilineage dysplasia, similar to the multilineage dysplasia and cytopenias seen in our murine model. Mutational profiling revealed that this patient was WT for known MDS mutations, including TET2, ASXL1, EZH2, NRAS, C-KIT, FLT3, IDH1, and IDH2, and had del(20)(q11.2q13.3) as the sole cytogenetic abnormality on metaphase chromosome analysis. In a separate microarray data set (25), BAP1 mRNA expression was reduced significantly in CD34+ cells from MDS patients as compared to healthy controls (fig. S15), which is in keeping with BAP1 being a tumor suppressor. To mimic BAP1 haploinsufficiency, we characterized heterozygous Bap1fl/+ creERT2+ mice after tamoxifen treatment. Loss of one copy of Bap1 caused very mild, but progressive, hematological defects (fig. S16), which is important because the frameshift mutation identified in the patient is heterozygous.

Our results identify a previously unknown and potent tumor suppressor function for BAP1 in myeloid neoplasia. The BAP1 ortholog in Drosophila, called Calypso, suppresses hox gene expression (17), but we did not see increased Hox gene expression in BAP1 KO cells (fig. S17). Divergent epigenetic functions for fly and vertebrate BAP1 might reflect the fact that the HCF-1 binding motif conserved in vertebrate BAP1 (16, 20) is absent from Calypso. We propose that BAP1 forms a core complex with HCF-1 and OGT that can differentially recruit additional histone-modifying enzymes to regulate gene expression and thereby preserve normal hematopoiesis. It will be interesting to determine whether Bap1 deficiency restricted to nonhematopoietic mouse tissues also promotes tumor development.

Supplementary Materials

Materials and Methods

Figs. S1 to S17


References and Notes

  1. Acknowledgments: We thank members of the Dixit and Martin laboratories for advice and discussions and core laboratories for technical assistance.
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