Leukemia (2013) 27, 10–15 & 2013 Macmillan Publishers Limited All rights reserved 0887-6924/13 www.nature.com/leu

SPOTLIGHT REVIEW The ASXL–BAP1 axis: new factors in myelopoiesis, cancer and epigenetics

O Abdel-Wahab1 and A Dey2

The recent identification of germline and somatic in BAP1 as well as in multiple members of the ASXL (additional sex combs-like) family of has highlighted the role of these in a diverse array of biological functions. A diverse number of possible functions have previously been ascribed to ASXL1 in non-hematopoietic contexts, including physical co-operativity with HP1a and LSD1. Here we discuss new evidence for a BAP1-independent function of ASXL1 in regulating histone H3 lysine 27 methylation through interactions with the Polycomb-repressive complex 2 (PRC2). BAP1, a nuclear-localized deubiquitinase, has been shown to interact with a number of proteins, including ASXL1 and/or ASXL2, but the functional importance of this interaction has remained elusive. Here, we highlight recent work revealing the critical function of BAP1 in restricting myelopoiesis and in regulating hematopoietic stem cell function. These data provide evidence that BAP1 and ASXL1 function as a novel class of tumor suppressors in myeloid malignancies. BAP1 functions through effects on stability of host cell factor-1, and O-GlcNAcylation, and ASXL1 impacts histone post-translational modifications through interaction with PRC2. Future studies investigating the mechanism of transformation by loss of BAP1 and ASXL1 may result in new therapeutic approaches to treat hematological malignancies. SPOTLIGHT

Leukemia (2013) 27, 10–15; doi:10.1038/leu.2012.288 Keywords: ASXL1; ASXL2; BAP1; chronic myelomonocytic leukemia; myelodysplastic syndrome; Polycomb

INTRODUCTION phenotype characteristic of both Polycomb group (PcG; repressive 5 The dysregulation of factors has long been suspected complex) and Trithorax group (activating) deletions, which to have an important role in myeloid transformation based on the led to the hypothesis that Asx has dual functions in silencing and identification of recurrent translocations involving transcription activation of homeotic . In addition, functional factors in myeloid malignant disorders. More recently, recurrent studies in suggest that Asx encodes a - somatic mutations in transcription factors as well as epigenetic associated with similarities to PcG proteins (5). A series of regulators of transcription have been identified in a large in vitro studies in non-hematopoietic cells have suggested a proportion of patients with cytogenetically normal myelodysplastic variety of functions for ASXL1, including physical co-operativity syndrome (MDS) and . In addition to with HP1a and LSD1 to repress retinoic acid-receptor activity and continuing discovery efforts in these disorders, a great interaction with peroxisome proliferator-activated receptor-g to 6–8 deal of effort is also being placed in functionally understanding suppress lipogenesis. how these mutations contribute to myeloid transformation, and All three ASXL family members are characterized by an amino- identifying the critical target genes and downstream transcrip- terminal homology domain and a C-terminal plant homeodo- 8,9 tional programs affected by these alterations. Here we discuss two main. Recent bioinformatics analysis of the conserved domains recent studies that identify novel functions for BAP11 and ASXL1 of mammalian ASXL proteins has suggested that the N-terminal (additional sex combs-like)2 in myeloid transformation. BAP1 and domain of ASXL1 (amino acids 10–100) might represent a unique the ASXL family of proteins have been shown to interact in DNA binding motif, termed a HARE-HTH domain (HB1, ASXL1, 10 metazoan cells using multiple proteomic approaches.1,3,4 Although restriction endonuclease helix-turn helix domain). On the basis little was previously known about the function or importance of of comparative analysis with other plant homeodomain- these proteins, recent reports from cancer genome sequencing containing proteins, the plant homeodomain of ASXL proteins studies, biochemical characterization and novel mouse models appears unique and has been predicted to potentially recognize have identified overlapping as well as independent roles for these internal methylated lysines on histone H3 tails as opposed to two genes in hematopoiesis and myeloid malignancies. lysines on the N-terminal tail of histone H3. Further functional investigation will be needed to understand the role of these domains of the ASXL family members. ASXL1: A POLYCOMB GROUP PROTEIN FREQUENTLY Interestingly, frequent mutations in ASXL1 have recently been MUTATED IN MYELOID MALIGNANCIES identified in the entire spectrum of myeloid malignancies11–16 ASXL1, ASXL2 and ASXL3 are the human homologs of Drosophila (Table 1). In fact, mutations in ASXL1 appear to be novel additional sex combs (Asx). Asx deletion results in a homeotic biomarkers of adverse overall survival in patients with MDS and

1Human Oncology and Pathogenesis Program and Leukemia Service, Memorial Sloan-Kettering Cancer Center, New York, NY, USA and 2Department of Physiological Chemistry, Genentech, South San Francisco, CA, USA. Correspondence: Dr O Abdel-Wahab, Human Oncology and Pathogenesis Program and Leukemia Service, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10065, USA E-mail: [email protected] or Dr A Dey, Department of Physiological Chemistry, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA. E-mail: [email protected] Received 3 September 2012; revised 2 October 2012; accepted 3 October 2012; accepted article preview online 9 October 2012; advance online publication, 13 November 2012 New factors in myelopoiesis, cancer and epigenetics O Abdel-Wahab and A Dey 11 Table 1. The mutational spectrum of BAP1, ASXL1 and ASXL2 in human disordersa

Gene Disease Frequency of Comments mutations

Myeloid malignancies ASXL1 De novo MDS13,15–17 14–25% CMML12,17 27–49% Chronic MPNs12,42,43 2–23% Most common in primary myelofibrosis and post-polycythemia vera or essential thrombocytosis myelofibrosis De novo AML11,14 5–20% AML from an antecedent hematological 15–20% disorder44 BAP1 MDS, CMML1 B3% (1/32 samples) BAP1 also downregulated in expression in CD34 þ cells from MDS patients compared with age-matched controls1

Other malignancies BAP1 Atypical Spitz Nevi28 11.1% Cutaneous melanoma28 5.0% Uveal melanoma24,28 39–47% Frequency of BAP1 mutations even higher in metastatic (84%). Clear cell renal cell carcinoma29,30 15% BAP1 mutations associated with higher tumor grades and anti-correlated with PBRM1 mutations Malignant pleural mesothelioma26,27 19.8–23% Mutated in germline in patients with hereditary in conjunction with uveal melanoma Breast cancer45 0.6% Lung carcinoma46 0.4% ASXL1 Breast cancer45 15% (2/13 samples) Metastatic castration resistant prostate 1–5% cancer34 ASXL2 Metastatic castration resistant prostate 1–5% cancer34

Developmental disorders ASXL1 Bohring–Opitz syndrome33 Present Germline mutations Abbreviations: AML, acute myeloid leukemia; ASXL, additional sex combs-like; CMML, chronic myelomonocytic leukemia; MDS, myelodysplastic syndrome; MPN, myeloproliferative neoplasms. aThis table reports only somatic mutations in BAP1, ASXL1 and ASXL2. Copy-number alterations and changes in expression of each of these genes have been reported, but have not been systematically performed or reported across studies or tumor types. Rare somatic ASXL3 mutations have been described in ovarian and pancreatic adenocarcinoma, but recurrent mutations (for example, with a frequency of 45%) in ASXL3 have not been identified in any single human disorder. acute myeloid leukemia,11–16 independent from other clinically histone H3 lysine 4 trimethyl (H3K4me3), respectively). Western utilized prognosticators. blots of purified histones from isogenic myeloid hematopoietic

cells with and without ASXL1, as well as chromatin immunopre- SPOTLIGHT cipitation sequencing revealed a striking global loss of H3K27me3 ASXL1 REGULATES HISTONE H3 LYSINE 27 METHYLATION upon ASXL1 loss. Moreover, this loss of H3K27me3 was seen To investigate the functional implication of ASXL1 mutations, we despite no change in the levels of the core Polycomb- recently characterized the effects of these mutations on ASXL1 repressive complex 2 (PRC2) members (EZH2, EED and SUZ12) protein expression in vitro.2 Mutations in ASXL1, although present responsible for H3K27 methylation.2 Interestingly, chromatin throughout the coding region, occur most frequently as nonsense immunoprecipitation for EZH2 in the presence and absence of as well as frameshift mutations in the 50-end of the last exon, ASXL1 suggested that ASXL1 is important for recruitment and/or suggesting that these mutations might result in a truncated stabilization of the PRC2 complex at key locations in the genome. protein product lacking the plant homeodomain.12,17 However, In accordance with this, ASXL1 was also found to physically characterization of ASXL1 protein expression in cell types bearing interact with core PRC2 members. Conversely, re-expression of endogenous expression of these mutant forms of ASXL1 revealed ASXL1 in myeloid leukemia cells with complete loss of ASXL1 due downregulation in ASXL1 protein expression with no truncated to homozygous mutation revealed an increase in H3K27me1/2/3 protein product in these cell lines.2 Indeed, leukemia-associated levels by chromatin immunoprecipitation and mass-spectrometric mutations in ASXL1 appear to result in reduced stability of the analysis of histones. Though the BAP1–ASXL1 Polycomb-repres- mutant forms of ASXL1 relative to wild type. The mutant forms of sive deubiquitinase complex in has ASXL1 undergo more rapid degradation following cycloheximide been suggested to be a H2A deubiquitinase (described below),18 exposure in cells.2 the effects of ASXL1 loss on lysine 119 Given that Asx mutations in Drosophila appear to affect both (H2AK119Ub) levels were minimal compared with the effects PcG and Trithorax group function, we characterized the effects of on H3K27me3.2 Given the epigenomic and physical interaction ASXL1 loss on canonical marks of the PcG and Trithorax group between ASXL1- and the EZH2-containing PRC2 complex gene function (histone H3 lysine 27 trimethyl (H3K27me3) and members, it will be important to identify whether ASXL1

& 2013 Macmillan Publishers Limited Leukemia (2013) 10 – 15 New factors in myelopoiesis, cancer and epigenetics O Abdel-Wahab and A Dey 12 similarly interacts with EZH1, the homolog of EZH2 with known In addition to the above, we recently identified a somatic ability to methylate H3K27 in vitro and in vivo.19,20 heterozygous BAP1 mutation in a patient with de novo MDS and To identify potential target genes affected by ASXL1 loss, gene deletion of 20q as the sole additional somatic abnormality.1 expression data from isogenic cells with ASXL1 knockdown or Although only a small cohort of patients (32 total) was sequenced control was intersected with H3K27me3 and H3K4me3 chromatin for BAP1 mutations, we identified more frequent downregulation immunoprecipitation sequencing data from the same cell type. in expression of BAP1 in CD34 þ cells from patients with de novo This analysis revealed that the HOXA cluster of genes had a MDS compared with age-matched counterparts.31 Given the distinct H3K27me3 loss and a concomitant upregulation in striking phenotype resulting from systemic deletion of Bap1 in expression upon ASXL1 loss. Despite the striking effects of BAP1 mice (described below), further work to identify the frequency of loss on myeloproliferation (described below), there appears to be somatic BAP1 mutations in myeloid malignancies, clinical minimal effect of conditional deletion of Bap1 in mice or correlates of BAP1 mutations and downregulated expression, knockdown of BAP1 in vitro on upregulation of HOXA gene and mutations co-occurring with alterations in BAP1 would aid in members,1,2 suggesting some divergence in the function of BAP1 our understanding of the role of BAP1 in the pathogenesis of and ASXL1. myeloid malignancies. Although upregulation of HOXA gene members was prominent from in vitro analyses following ASXL1 knockdown in primary hematopoietic cell types and transformed myeloid leukemia cell A NOVEL ROLE FOR BAP1 IN HEMATOPOIESIS lines, prior work characterizing a mouse model with constitutive To investigate the biological function of Bap1, we recently created loss of Asxl1 revealed no overt hematopoietic malignancy upon a murine model with deletion of Bap1.1 Given that constitutive Asxl1 loss.9 Analysis of the effects of Asxl1 loss from this previous loss of Bap1 resulted in embryonic lethality, we generated mice mouse model was possibly obscured due to high perinatal with inducible deletion of Bap1 using creERT2 technology. lethality seen in mice with germline loss of Asxl1. Moreover, loss of Strikingly, within 4 weeks following tamoxifen injection to Asxl1 using in vivo RNA-interference methodology in the presence induce deletion of Bap1, 100% of Bap1 knockout (KO) mice of oncogenic forms of NRasG12D revealed a clear collaborative developed splenomegaly due to extramedullary hematopoiesis.

SPOTLIGHT effect of Asxl1 loss in promoting myeloproliferation induced by The peripheral blood of these mice was characterized by severe, NRasG12D, and this was associated with upregulation of 50-HOXA progressive monocytosis, neutrophilia, anemia and thrombo- genes.2 Currently, there is no published description of the effects cytopenia. Morphological examination revealed dysplastic of conditional deletion of Asxl1 from the hematopoietic features in the myeloid cells. The proliferative features combi- compartment and further work to characterize this will be ned with features of ineffective hematopoiesis following Bap1 important to clarify the in vivo effects of Asxl1 on hematopoiesis. deletion most resembled an MDS/Myeloproliferative neoplasm overlap disorder such as human chronic myelomonocytic leukemia. Deletion of only a single copy of Bap1 resulted in a BAP1: A MUTATED IN HUMAN significantly milder phenotype than deletion of both copies of MALIGNANCY Bap1. BAP1 is a nuclear-localized deubiquitinating enzyme and a At the hematopoietic stem cell level, systemic Bap1 deletion member of the ubiquitin C-terminal hydrolase family. It was the resulted in an increase in the number of cells containing the first nuclear-localized ubiquitin C-terminal hydrolase enzyme to be hematopoietic stem cells (the lineage-negative, Sca-1 þ , c-KIT þ identified and was originally discovered as a protein interacting (LSK) cells and lineage-negative, Sca-1 À , c-KIT þ (myeloid with the RING finger domain of BRCA1.21 BAP1 was shown to progenitor) cells) in the bone marrow. Intriguingly, the Bap1- enhance the growth-suppressive effect of BRCA1 and, thus, it was deficient bone marrow progenitors exhibited decreased self- hypothesized that BAP1 may deubiquitinate BRCA1.21 It was renewal as assayed by in vitro colony-forming assays and subsequently found that BAP1 does not deubiquitinate BRCA1, competitive transplantation assays using whole bone marrow and BAP1 can inhibit malignant cell proliferation in a BRCA1- mononuclear cells depleted of Bap1 before transplantation.1 One independent manner, suggesting that BAP1 likely functions in possible explanation for this apparent conundrum is that Bap1 processes not related to BRCA1.22 loss effects hematopoietic stem cell engraftment and/or homing, Although the function of BAP1 remained elusive for some time, although further work will be needed to ascertain this. Finally, several pieces of evidence suggested that BAP1 was a likely tumor Bap1 deficiency resulted in a chronic myelomonocytic leukemia- suppressor shortly after its discovery. First, the BAP1 (3p21.3) like phenotype in a cell-autonomous manner, as Bap1 deletion undergoes frequent copy-number loss and loss-of-heterozygosity in after reconstitution of lethally irradiated recipient mice resulted in several cancers. For instance, deletions in 3p are seen in the vast the same features as the primary Bap1 conditional KO mice. majority of small-cell and non-small cell lung cancer cell lines,21 as well as renal cell carcinoma tumors.23 Second, BAP1 overexpression was shown to suppress tumorigenicity of many transformed cell THE BAP1–ASXL CONNECTION types that have homozygous loss of the locus containing BAP1.22 In As a member of the ubiquitin C-terminal hydrolase family, BAP1 line with this, loss-of-function BAP1 mutations have recently been possesses a conserved catalytic domain.21 In addition, BAP1 identified in a diverse array of solid tumor types24–30 (Table 1). This possesses a large C-terminal domain not present in other ubiquitin includes frequent somatic mutations in malignant pleural C-terminal hydrolase members, which was previously predicted to mesothelioma,26 uveal melanoma24 and clear cell renal have an important role in regulating and coordinating its carcinoma.29,30 The BAP1 mutations have been reported to occur deubiquitinating enzyme activity through selective association as missense, nonsense and out-of-frame insertions/deletions with potential substrates or regulatory components. In order to throughout the open-reading frame. Furthermore, germline BAP1 identify BAP1 interacting proteins, we utilized a novel mutations have been prominently described in patients with uveal methodology of immunoprecipitation and mass-spectrometric melanoma,25,28 mesothelioma,25,28 as well as patients with both detection coupled with in vivo stable isotope labeling of amino uveal melanoma and mesothelioma.25,27,28 Given the rarity of acids.1 We created a Bap1.3 Â Flag knock-in mouse and then spontaneous cases of either uveal melanoma or mesothelioma, it performed an immunoprecipitation for FLAG epitope in lysates appears that germline mutations in BAP1 define a new hereditary from spleen and brain of knock-in mice and littermate wild-type 13 cancer syndrome characterized by uveal melanoma and mice fed a diet of C6-lysine in their chow. By discriminating mesothelioma, and possibly other malignancies. peptides identified from wild-type animals and Flag knock-in

Leukemia (2013) 10 – 15 & 2013 Macmillan Publishers Limited New factors in myelopoiesis, cancer and epigenetics O Abdel-Wahab and A Dey 13 animals, we were able to identify peptides specifically enriched in been fully appreciated, given the lack of published candidate the Bap1.3 Â Flag immunoprecipitations. This revealed host cell gene-sequencing studies of BAP1 in patients with hematopoietic factor-1 (HCF-1), O-linked N-acetylglucosamine transferase (OGT), disorders. ASXL1, ASXL2 and a lysine demethylase KDM1B as the most specific, quantitative and endogenous interactors of BAP1.3,4,32 Recent studies from D. melanogaster suggested that BAP1 and BAP1 REGULATES HCF-1 AND OGT STABILITY ASXL1 associate to a new PcG protein complex(Polycomb- Previous work by Misaghi et al.,35 Machida et al.3 and Sowa et al.4 repressive deubiquitinase complex), which serves to remove identified that BAP1 interacts with HCF-1, a pleiotropic mono-ubiquitin from H2AK119.18 This interaction is critical for transcriptional regulator that has been extensively studied as a the enzymatic activity of BAP1. Moreover, there appears to be regulator of cell proliferation (reviewed in Eletr and Wilkinson36). reduced co-immunoprecipitation of BAP1 with ASXL1 in cells BAP1 deubiquitylates HCF-1 to prevent its proteasomal bearing mutant forms of ASXL1.2 It is worth noting that this could degradation3,4,35 (Figure 1). Confirming the functional interaction be due to the decreased stability of the ASXL1 mutants. Although of BAP1 with HCF-1, we found that Bap1 KO mouse cells exhibit the intersection of ASXL1 and BAP1 in H2AUb deubiquitination strikingly decreased HCF-1 levels.1 Moreover, Bap1 KO cells and thereby in the regulation of myelopoiesis seems attractive, a showed a decrease in OGT levels and O-GlcNAc (O-linked b-D-N- number of findings suggest that BAP1 and ASXL1 may function acetylglucosamine) levels,1 suggesting that Bap1 regulates the independently. First, although germline33 and somatic mutations stability of OGT and HCF-1. This data further suggests that BAP1 in ASXL112,17 as well as somatic mutations in ASXL234 have now HCF-1, ASXL1 and/or ASXL2, and OGT might form a BAP1 core been described in a number of disorders, there appears to be very complex, and that ASXL1 may also serve in BAP1-independent little overlap in the disorders where BAP1 and ASXL1 members are complexes (Figure 1). mutated (Table 1). For instance, as mentioned earlier BAP1 appears On the basis of the discovery of downregulation of BAP1 to be mutated most commonly in mesothelioma, uveal melanoma expression in MDS patients, the discovery of an MDS patient with and clear cell renal cell carcinoma, whereas ASXL1 is commonly a somatic BAP1 mutation and the murine model clearly linking mutated in myeloid malignancies and ASXL2 is mutated in Bap1 loss to a chronic myelomonocytic leukemia-like phenotype, metastatic prostate carcinoma (Table 1). Moreover, germline it will be fascinating to investigate the role of HCF-1 and/or OGT in mutations in BAP1 are associated with the hereditary cases of hematopoiesis and myeloid malignancies. HCF-1 is thought to uveal melanoma and/or mesothelioma, whereas germline promote progression at the G1/S boundary by recruiting mutations in ASXL1 are associated with the severe H3K4 methyltransferases to transcription factors, to transcribe developmental disorder Bohring–Opitz Syndrome.33 Finally, a genes needed to enter S-phase.37–39 Effect of Bap1 loss on clear relationship between BAP1 or ASXL1 mutation/loss and quiescence, proliferation and cycling of hematopoietic stem cells global H2AK119Ub levels in mammalian cells has not been readily would be enlightening. apparent.1,2,30 These differences may, however, be context- OGT is a single, highly conserved enzyme, which catalyzes the dependent and warrant further investigation. At the same time, addition of the sugar O-GlcNAc to proteins.40 O-GlcNAcylation is a we cannot exclude the possibility that the true incidence of BAP1 nutrient-sensitive sugar modification that participates in a mutations/downregulation in hematopoietic malignancies has not bewildering array of cellular processes, and is thought to link SPOTLIGHT

Figure 1. BAP1 and ASXL1 regulate gene expression through effects on a number of epigenetic modifications. BAP1 and ASXL1 physically interact, and ASXL1 is important for the enzymatic activity of BAP1. BAP1 deubiquitinates the cell cycle regulating protein host cell factor-1 (HCF-1) and the enzyme OGT (O-GlcNAc transferase) (a). HCF-1 is a heterodimer produced by proteolytic cleavage of a precursor protein consisting of HCF-1N and HCF-1C. Cleavage of HCF-1 precursor requires O-GlcNAcylation by OGT, the sole enzyme responsible for addition of the sugar modification O-linked b-D-N-acetylglucosamine to proteins. Loss of BAP1 results in decreased levels of both HCF-1 and OGT due to destabilization of both proteins. It is not yet clear whether ASXL1 loss effects HCF-1 and/or OGT levels in a similar manner. Ub, ubiquitination; G, O-GlcNac modification by OGT). (b) Expression of ASXL1 was recently shown to be strongly associated with the abundance of the transcriptionally repressive histone modification H3K27me3. Loss of ASXL1 is associated with global loss of this modification despite presence of the core members of the PRC2, the enzymatic complex necessary for methylation of H3K27. ASXL1 is responsible for recruitment and/or stabilization of the PRC2 complex at various genomic locations.

& 2013 Macmillan Publishers Limited Leukemia (2013) 10 – 15 New factors in myelopoiesis, cancer and epigenetics O Abdel-Wahab and A Dey 14 nutrient availability to chromatin structure. How exactly BAP1 loss 2 Abdel-Wahab O, Adli M, Lafave LM, Gao J, Hricik T, Shih AH et al. ASXL1 mutations may affect the pattern of O-GlcNAcylated proteins and the promote myeloid transformation through loss of PRC2-mediated gene repression. ensuing effects on chromatin structure remain to be identified. Cancer Cell 2012; 22: 180–193. Although Asx in D. melanogaster is required for the 3 Machida YJ, Machida Y, Vashisht AA, Wohlschlegel JA, Dutta A. The deubiquiti- enzymatic activity of BAP1 (Calypso),18 it is not clear if loss of nating enzyme BAP1 regulates cell growth via interaction with HCF-1. J Biol Chem ASXL proteins affects the levels of HCF-1 or OGT in the 2009; 284: 34179–34188. manner that BAP1 loss affects the abundance of these proteins. 4 Sowa ME, Bennett EJ, Gygi SP, Harper JW. Defining the human deubiquitinating One possibility is that ASXL1 and ASXL2 may have redundant enzyme interaction landscape. Cell 2009; 138: 389–403. 5 Sinclair DA, Milne TA, Hodgson JW, Shellard J, Salinas CA, Kyba M et al. The functions in the interactions with BAP1, thus limiting the additional sex combs gene of Drosophila encodes a chromatin protein that binds deleterious effects of either ASXL1 or ASXL2 alone. Interestingly, to shared and unique Polycomb group sites on polytene . Devel- a prior gene trap KO model of Asxl2 revealed global effects opment 1998; 125: 1207–1216. of downregulated H3K27 methylation as has been recently 6 Park UH, Yoon SK, Park T, Kim EJ, Um SJ. Additional sex comb-like (ASXL) proteins identified with Asxl1.41 Further work to understand the effects 1 and 2 play opposite roles in adipogenesis via reciprocal regulation of peroxi- of Asxl2 loss using conditional deletion of Asxl2 may be fruitful some proliferator-activated receptor {gamma}. J Biol Chem 2011; 286: 1354–1363. given the recently identified presence of ASXL2 mutations in 7 Lee SW, Cho YS, Na JM, Park UH, Kang M, Kim EJ et al. ASXL1 represses retinoic human malignancies (Table 1). acid receptor-mediated transcription through associating with HP1 and LSD1. J Biol Chem 2010; 285: 18–29. 8 Cho YS, Kim EJ, Park UH, Sin HS, Um SJ. Additional sex comb-like 1 (ASXL1), in cooperation with SRC-1, acts as a ligand-dependent coactivator for retinoic acid CONCLUDING REMARKS receptor. J Biol Chem 2006; 281: 17588–17598. Mutations in BAP1 and ASXL gene members are present in a 9 Fisher CL, Pineault N, Brookes C, Helgason CD, Ohta H, Bodner C et al. Loss-of- diverse number of human disorders highlighting the importance function additional sex combs like 1 mutations disrupt hematopoiesis but do not of these genes in a number of biological processes. Investigations cause severe myelodysplasia or leukemia. Blood 2010; 115: 38–46. into the function of BAP1 and ASXL1 in the context of 10 Aravind L, Iyer LM. The HARE-HTH and associated domains: novel modules in the hematopoietic cells have uncovered a number of novel insights, coordination of epigenetic DNA and protein modifications. Cell Cycle 2012; 11: 119–131. SPOTLIGHT including the role of deubiquitylation of nuclear proteins in 11 Metzeler KH, Becker H, Maharry K, Radmacher MD, Kohlschmidt J, Mrozek K et al. myeloid malignancies, a possible role for HCF-1 and OGT in ASXL1 mutations identify a high-risk subgroup of older patients with primary myeloid transformation, and new insights into PRC2-mediated cytogenetically normal AML within the ELN Favorable genetic category. Blood gene repression in mammals. BAP1 and ASXL1 appear to be 2011; 118: 6920–6929. involved in a large number of protein–protein interactions. Future 12 Abdel-Wahab O, Pardanani A, Patel J, Wadleigh M, Lasho T, Heguy A et al. Con- work dissecting the overlapping and independent complexes comitant analysis of EZH2 and ASXL1 mutations in myelofibrosis, chronic mye- where BAP1 and ASXL1 are involved will undoubtedly shed light lomonocytic leukemia and blast-phase myeloproliferative neoplasms. Leukemia on the role of these proteins in malignancies. Also, given the 2011; 25: 1200–1202. frequency of BAP1 mutations in many epithelial malignancies, it 13 Bejar R, Stevenson K, Abdel-Wahab O, Galili N, Nilsson B, Garcia-Manero G et al. will be interesting to ascertain the effect of Bap1 loss on non- Clinical effect of point mutations in myelodysplastic syndromes. N Engl J Med hematopoietic tissues. 2011; 364: 2496–2506. 14 Patel JP, Gonen M, Figueroa ME, Fernandez H, Sun Z, Racevskis J et al. Prognostic On the basis of the frequent mutations of ASXL1 in myeloid relevance of integrated genetic profiling in acute myeloid leukemia. 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