Emerging Roles of the Spliceosomal Machinery in Myelodysplastic Syndromes and Other Hematological Disorders

Home , PRPF40B, PRPF8, SF3B1, SF3B2, Spliceosome

REVIEW Emerging roles of the spliceosomal machinery in myelodysplastic syndromes and other hematological disorders

V Visconte1, H Makishima1, JP Maciejewski1,2 and RV Tiu1,2

In humans, the majority of all protein-coding transcripts contain introns that are removed by mRNA splicing carried out by spliceosomes. Mutations in the spliceosome machinery have recently been identified using whole-exome/genome technologies in myelodysplastic syndromes (MDS) and in other hematological disorders. Alterations in splicing factor 3 subunit b1 (SF3b1) were the first spliceosomal mutations described, immediately followed by identification of other splicing factor mutations, including U2 small nuclear RNA auxillary factor 1 (U2AF1) and serine arginine-rich splicing factor 2 (SRSF2). SF3b1/U2AF1/SRSF2 mutations occur at varying frequencies in different disease subtypes, each contributing to differences in survival outcomes. However, the exact functional consequences of these spliceosomal mutations in the pathogenesis of MDS and other hematological malignancies remain largely unknown and subject to intense investigation. For SF3b1, a gain of function mutation may offer the promise of new targeted therapies for diseases that carry this molecular abnormality that can potentially lead to cure. This review aims to provide a comprehensive overview of the emerging role of the spliceosome machinery in the biology of MDS/hematological disorders with an emphasis on the functional consequences of mutations, their clinical significance, and perspectives on how they may influence our understanding and management of diseases affected by these mutations.

Leukemia (2012) 26, 2447–2454; doi:10.1038/leu.2012.130 Keywords: spliceosome; mutations; MDS

INTRODUCTION myelodysplastic syndromes (MDS), myeloproliferative neoplasms Protein synthesis is a carefully regulated mechanism that begins (MPN), MDS/MPN overlap, and finally in chronic lymphocytic with replication, followed by transcription, and culminating with leukemia (CLL) and other hematological disorders. Subsequently, translation of the protein. Post-transcriptional modifications, a mutations in other spliceosomal genes such as U2 small nuclear myriad of intermediary steps that occur mainly between the RNA auxillary factor 1 (U2AF1) and serine arginine-rich splicing transcription and translation process, ensure the integrity, factor 2 (SRSF2) were identified (Table 1). Within individual disease diversity and fidelity of the final protein product. One of the subtypes, mutations of specific genes confer varying functional crucial post-transcriptional processes is RNA splicing, whereby and clinical consequences. The discovery of this new class of noncoding sequences called introns are removed from the molecular lesions represents a leap in our understanding of the primary transcript (pre-mRNA). This process is carried out by a biology of hematological diseases and their pathogenesis. series of proteins consisting of small nuclear ribonucleoproteins (snRNPs) and small nuclear RNAs that form spliceosomes.1 Alternative splicing is a process that increases genomic diversity OVERVIEW OF THE BIOLOGY OF SPLICEOSOMES through alterations in the composition of the exons by using The dynamic process of intron splicing is made possible through alternative 50 and 30 splice sites, retained introns and unconven- the active participation of two types of spliceosomal complexes, tional exons.2 As RNA splicing is a ubiquitous process in eukaryotic major and minor spliceosomes. Major spliceosomes, which consist cells, it is not surprising that dysfunction of this pathway can of U1, U2, U4/U6 and U5snRNPs, catalyze most of the splicing lead to disease. Polymorphisms or mutations altering regulatory processes. Minor spliceosomes, consisting of U11 and U12snRNPs sequences or producing different splice variants ultimately lead and U4atac and U6atac small nuclear RNAs, carry out the splicing to hereditary diseases and cancer. This phenomenon has been of minor class introns.5 This process is responsible for the excision observed in neuromuscular, psychiatric, X-linked and neoplastic of B1 in 300 introns from human pre-mRNA. Although generally disorders.3 perceived as a post-transcriptional process, splicing can also occur Recently, whole-genome sequencing as a genetic tool has been as a co-transcriptional process in the nucleus.6 Most of the instrumental in the discovery of novel mutations in genes such as individual components of the major spliceosomes are partly DNMT3A in hematological malignancies, including acute myeloid synthesized in the cytoplasm but mature forms are mainly leukemia (AML).4 A similar approach was utilized to further localized in the nucleus. elucidate the pathogenesis of other hematological malignancies, Spliceosomes, which catalyze the essential process of RNA which led to the identification of mutations in spliceosomal splicing and ligation of flanking exons, rely on specific recognition genes, in particular splicing factor 3 subunit b1 (SF3b1), first in sites in the target pre-mRNA transcript for appropriate binding

1Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA and 2Department of Hematologic Oncology and Blood Disorders, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA. Correspondence: Dr RV Tiu, Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, 9500 Euclid Avenue R40, Cleveland, OH 44195, USA E-mail: [email protected] Received 25 January 2012; revised 4 May 2012; accepted 7 May 2012; accepted article preview online 15 May 2012; advance online publication, 8 June 2012 Role of spliceosomal machinery in MDSs V Visconte et al 2448 processing (cis-acting elements) or in components implicated in Table 1. Spliceosomal genes the regulation (trans-acting elements) of splicing. Splicing requires Gene name Reference accuracy in the recognition of the exon–intron boundaries and insertion or removal of nucleotides by mutations leading to a shift SF3b1 Visconte et al.11 in the open reading frame of the future protein. Mutations Yoshida et al.12 affecting alternative splicing, intronic regions, recognition sites, Papaemmanuil et al.13 exonic regions and changes in the abundance and ratio of Malcovati et al.28 different splicing isoforms have been previously described and 34 Lasho et al. implicated in the biogenesis of several human diseases.10 Damm et al.29 30 Recently, SF3b1 has been found to be highly mutated in MDS Patnaik et al. 11–13 12 Rossi et al.36 and related disorders. Yoshida et al. also described Wang et al.35 mutations in SF3a1, albeit at lower frequency. Quesada et al.37 U2AF1 Yoshida et al.12 Graubert et al.17 MECHANISMS OF SPLICEOSOME REGULATION Makishima et al. The process of spliceosome assembly has been associated with U2AF2 Yoshida et al.12 kinases and phosphatases. Thus, mechanisms of sequential 12 40 SRSF2 Yoshida et al. Makishima et al. phosphorylation and dephoshorylation regulate components of ZRSR2 Yoshida et al.12 40 the spliceosomes and dictate the steps in spliceosome assembly. Makishima et al. Serine/arginine (SR) proteins are factors essential in both the SF3a1 Yoshida et al.12 PRPF40B Yoshida et al.12 constitutive and alternative RNA splicing. The phosphorylation SF1 Yoshida et al.12 usually occurs at the serine residues in the RS domain promoting 14 PRPF8 Makishima et al.40 protein interactions that lead to spliceosome assembly. LUC7L2 Makishima et al.40 Subsequently, SR proteins must be dephosphorylated to allow the step of transesterification to proceed. Several kinases have Abbreviations: LUC7L2, putative RNA-binding protein Luc7-like 2; PRPF8, been described to phosphorylate SR proteins, such as SRPK, PRP8 pre-mRNA processing factor 8 homolog; PRPF40B, PRP40 pre-mRNA processing factor 40 homolog B; SF1, splicing factor 1; SRSF2, serine PRP4, Clk1–4, Cdc2 kinase and topoisomerase 1. All these argine-rich splicing factor 2; SF3a1, splicing factor 3a, subunit 1; SF3b1, kinases target serine/threonine residues and are localized in splicing factor 3b subunit 1; U2AF1/2, U2 small nuclear RNA auxillary the cytosol and nuclear speckles. Several kinase inhibitors have factor 1/2; ZRSRS2, zinc finger (CCCH type), RNA-binding motif and been developed including TG003, a derivate of benzothiazol, serine/arginine-rich 2. which is a selective pharmacological inhibitor of Clk1 activity suppressing the SR-rich domains and SR phosphorylation. Different phosphatases, such as PP2Cg, PP1 and PP2A have also 0 0 and assembly, namely the 5 end and the 3 end splice sites. been implicated in the early step and in the second catalytic 0 The 5 end splice site marks the exon–intron junction at the step of spliceosome assembly.15 50 end of the intron. The 30 end splice site marks the exon–intron junction site at the 30 of the intron and consists of the branch point sequence, a polypyrimidine tract. Dinucleotides that are CURRENT EVIDENCE AND PERSPECTIVES ON SPLICEOSOME located at 50 and 30 sites are donor and acceptor splice sites COMPONENT MUTATIONS: BIOLOGICAL AND CLINICAL for intron removal. Major spliceosomes remove introns with the SIGNIFICANCE canonical dinucleotide, GU–AG, and rarely AT–AC and GC–AG Splicing factor 3 subunit b1 dinucleotides. Minor spliceosomes carry out the removal of AT–AC 5,7,8 Splicing mutations have been found to lead to hereditary diseases, and GT–AC introns. The process of spliceosome assembly is such as X-linked disorders of copper metabolism and retinitis a systematic process that begins with U1snRNP binding at the pigmentosa.16 Somatic mutations of vital components of the 50 end splice site of the pre-mRNA to form an early or E complex. spliceosome machinery were recently discovered by several This E complex requires ATP and commits the pre-mRNA to investigators using whole-exome and genome sequencing in splicing. U2snRNP is subsequently recruited to the branch region hematological malignancies, first in MDS and MDS/MPN overlap and becomes tightly associated with the branch point sequence syndromes, particularly refractory anemia with ring sideroblasts through an ATP-dependent process to form complex A. The (RARS) and RARS with thrombocytosis (RARS-T) and then in other duplex formed between U2snRNP and the pre-mRNA branch hematological diseases. Mutations in SF3b1 were first reported by region forces out the branch adenosine and commits it for the first three groups.11–13 Beside SF3b1, mutations were found in other transesterification process. A pseudouridine residue in U2 small genes involved in RNA splicing machinery, such as U2AF1, SRSF2, nuclear RNA changes the duplex conformation, making it more ZRSR2, SF3a1, PRPF40B, U2AF65 and SF1.12,17 accessible for the first step of splicing. The tri-snRNP U4/U5/U6 are Mutational frequencies for SF3b1 ranging from 68–75% were recruited to the spliceosome to form complex B, which ultimately reported for RARS and 81% in RARS-T. In our laboratory, SF3b1 becomes rearranged and forms complex C, characterized by the mutations were always associated with acquired rather than with loss of the U1 and U4 snRNPs from the complex and replacement hereditary cases of refractory anemia, as no mutation was of U1snRNP at the 5-splice site with U6 snRNP (Figure 1). observed in a small group of patients with congenital sideroblastic U2snRNP is formed by a complex of proteins including SF3a, anemia. Lower mutational frequencies of SF3b1 (0–7%) were SF3b1 and the 12s RNA unit. SF3b is a 450-kDa multiprotein noted in MDS and MDS/MPN with o15% RS.11,12 The notable complex composed of SF3b1, SF3b2, SF3b3, SF3b4, SF3b5, SF3b14 difference in the prevalence of SF3b1 mutations in MDS and MDS/ and PHF5A.9 SF3b1 allows for the binding of the spliceosomal MPN with 415% RS led some investigators to believe that this U2snRNP to the branch point near the 30 splicing site. The main mutation is important in the pathogenesis of RARS/RARS-T. function of this complex is to prevent an inappropriate The clinical entity RARS was first defined in 1982, a time when nucleophilic attack by additional components of the spliceo- a set of new diagnostic criteria was created for the classification some before the initial transesterification reaction that must of MDS.18 The traditional distinction of X15% RS in erythroid occur in order to achieve RNA splicing. Splicing can be affected precursors was an arbitrary distinction without a biologic basis; by mutations in the components important for pre-mRNA however, it now appears that this cutoff does have some

Leukemia (2012) 2447 – 2454 & 2012 Macmillan Publishers Limited Role of spliceosomal machinery in MDSs V Visconte et al 2449

Figure 1. Alternative splicing pattern and disease causation. Left panel: spliceosomes catalyze RNA splicing, which leads to the ligation of two flanking exonic regions. The GU dinucleotide at the 50 end, branch point sequence and terminal AG dinucleotide at the 30 end serve as specific recognition sites. Three different complexes (E, A and B) are subsequently formed by the spliceosome assembly. In order, U1, U2 and U4/U5/ U6snRNP all cooperate in the formation of each complex. SF3b1 encodes a protein responsible for the binding of the U2snRNP to the branch point at the 30 splicing site. Right panel: alternative splicing is the process that generates variability in transcripts, ultimately leading to biological differences in protein function and structure through different mechanisms (A–G). The first type is called (A) exon skipping or sometimes called exon cassette, which involves either retention or splicing out of the involved exon from the primary transcript. It is the most common mode of alternative splicing in mammalian pre-mRNA. (B) Mutually exclusive exons, one of two exons are retained in the primary transcript after splicing. (C) Competitive 50 splice site, a new 50 splice junction is used. (D) Competive 30 splice site, a new 30 splice junction is used. (E) Retained intron, a sequence is spliced out or kept. (F) Multiple promoters, a transcriptional process where different starting points in the transcription can lead to transcripts with different 50 sites. (G) Multiple Poly-A sites, is the process that generates different 30 ends. Several hypotheses are illustrated to explain the disease causation. Illustrated by Ramon V Tiu, MD. biological significance, as evidenced by the higher frequency of such as copper deficiency, chronic neoplastic diseases and SF3b1 mutations. RS are an abnormal localization of ferritin iron in exposures to ethanol, acetaldehyde and other toxins.22–24 Few the mitochondria of erythroid precursors and are usually an index reports have described the presence of rare RS in the BM of of altered erythropoiesis.19 Although clearly important in the patients with copper deficiency and in patients with chronic subsequent morphological classification of RARS and RARS-T, the alcohol abuse. Very little is known regarding the mechanism/s presence of RS is not in itself pathognomonic for MDS. As the leading to the RS formation in this context. Although, in general, World Health Organization (WHO) has clearly defined, definitive trancriptional and post-transcriptional regulation have been evidence of dysplasia, cytopenias and certain types of cytogenetic characterized in the iron utilization through iron-regulatory abnormalities remain crucial in the diagnosis of MDS and related proteins.22,25 diseases such as MDS/MPN overlap neoplasms. The high frequency of SF3b1 mutations in RARS/RARS-T makes As mentioned, these iron deposits are not unique to RARS and this gene a very strong candidate for the pathogenesis of these RARS-T but sometimes are observed in other disease entities, diseases. Indeed, we are currently investigating this particular especially congenital sideroblastic anemias. The molecular patho- association using specific pharmacological inhibitors of the genesis of certain types of congenital sideroblastic anemias are spliceosome machinery, such as meayamycin, which specifically more clearly defined. For example, germ-line mutations in targets the SF3b complex. We also hypothesized that structural aminolevulinate delta-synthase (ALAS2) ultimately result in X-linked differences in iron distribution may be observed between SF3b1 sideroblastic anemia.20 It is imperative therefore to assume mutant and wild-type cases. This is being investigated using that certain types of mitochondrial genes may be at fault for transmission electron microscopy. We are also performing RARS and RARS-T. Several genes, included ATP-binding cassette, experiments in Sf3b1 heterozygous mice26 to further clarify the subfamily B, member 7 (ABCB7), Pseudouridine synthase-1 (PUS-1) association between SF3b1 mutations and RS formation. Part of and Ferrochelatase (FECH) are potential candidates, but to date no the data were presented in the recent 53rd ASH meeting.27 mutations in these genes have been associated with acquired Furthermore, we found two cases of rare diseases with SF3b1 cases of RARS and are confined solely to hereditary cases of mutations. The two informative cases were: a patient with post- sideroblastic anemia.21 RS can appear in other clinical conditions polycythemia vera myelofibrosis in a cohort of 30 patients with

& 2012 Macmillan Publishers Limited Leukemia (2012) 2447 – 2454 Role of spliceosomal machinery in MDSs V Visconte et al 2450 MPN11 and a patient with paroxysmal nocturnal hemoglobinuria thrombosis, mainly arterial.27 How thrombosis occurs in the in a group of aplastic anemia (AA, N ¼ 22), T-cell large granular setting of SF3b1 mutations is unclear and will be the subject lymphocyte leukemia (T-LGL, N ¼ 17) and paroxysmal nocturnal of further investigation. hemoglobinuria (PNH, N ¼ 24).27 Interestingly, both cases Another study screened for the presence of SF3b1 mutations in presented RS with no other morphological or cytogenetic 155 patients with primary myelofibrosis and also found a low features suggesting concomitant MDS, further supporting the frequency of mutations in this group of patients (10/155; 6.5%), a role of SF3b1 in RS biogenesis. majority of which (60%) carried a concomitant Janus kinase In terms of clinical significance, the two studies that initially 2V617F mutation. In six patients with bone marrow available for explored the effects of SF3b1 mutation on overall survival had Prussian blue staining, they also found that all patients with SF3b1 slightly different results. The study by Visconte et al. did not mutations had RS. Neither clinicopathological nor overall survival initially find any difference in overall survival. In contrast, a larger differences were observed between patients with SF3b1 mutations study by Papaemmanuil et al. showed better overall survival and their wild-type counterparts.34 Furthermore, no mutations (P ¼ 0.01), leukemia-free survival (P ¼ 0.05) and event-free survival have been found so far in our hands in patients with essential (P ¼ 0.008) in patients with SF3b1 mutations compared with thrombocythemia (N ¼ 15), polycythemia vera (N ¼ 24) and wild-type patients. Papaemmanuil et al.13 and Malcovati et al.28 in very few cases of chronic myelogenous leukemia (N ¼ 7). also noted that SF3b1 mutations are associated with lower risk of Mutations in SF3b1 were also found to be rare in much larger evolution to AML. Several other studies subsequently followed cohort of MPN as reported by Yoshida et al. (0%), and exploring the clinical correlations of SF3b1. Damm et al.29 studied Papaemmanuil et al. (primary myelofibrosis, 4.4%, polycythemia 317 patients with MDS, although clinical data were only analyzed vera, 0% and essential thrombocythemia, 3.1%, chronic in 253 patients, and found no difference in overall survival and myelogenous leukemia, 4.7%). Interestingly, we found 2/32 rate of AML transformation. The multivariate analysis performed (6.2%) patients with mastocytosis with SF3b1 mutations by Patnaik et al.30 determined that the prognostic value of SF3b1 (Visconte et al., unpublished data). The low frequency of SF3b1 mutations was completely accounted for the WHO morphological mutations in MPN further supports the selective specificity of the grouping. In opposition to these negative findings and our own presence of SF3b1 mutations for RARS and RARS-T. The majority of original negative findings in a small cohort of patients, we recently the studies utilized whole-exome sequencing as their initial presented data on 511 cases of MDS, MDS/MPN and other mutation screening tool. Few studies performed whole-genome hematological disorders (paroxysmal nocturnal hemoglobinuria, sequencing.13,17,35 In the vast majority of the studies, SF3B1 was AA, T-cell large granular lymphocyte leukemia, Mast cell diseases sequenced for all the exons in a small target cohort preliminarily and MPN), finding better overall survival in MDS and MDS/MPN then a much more focusing sequencing of the most frequently cases,27 further supporting the results of Papaemmanuil et al. mutated exons were performed. More recently, it has been We also observed that the rate of overall survival is better in reported that aside from RARS and RARS-T, the disease with the SF3b1 mutants versus wild-type patients within the subset of most frequent mutations in SF3b1 is CLL.35–37 Papaemmanuil RARS/RARS-T. et al.13 reported SF3b1 mutations in 2/40 CLL patients, although It is possible that the good prognosis seen in SF3b1 mutants only in one patient was the mutation somatically confirmed. A is related to its close association with a clinic-pathologic entity second independent study initially looked at a limited series of 11 that is known to have good outcomes, but recent data from our fludarabine-refractory CLL by whole-exome sequencing and found group also demonstrate that it may be independent of disease mutations in 3/11 cases.36 This lead to the screening of a larger classification. Biologically, it is possible that SF3b1 mutations result cohort of 393 CLL patients (fludarabine refractory (N ¼ 59); newly in aberrations in alternative splicing, leading to a loss of activity of diagnosed and previously untreated (N ¼ 301) and clonally related protein transcripts from pathway signals that typically portend Richter’s syndrome (N ¼ 33)), which found a statistically significant poor outcomes in AML. It is also possible that alternative splicing higher frequency of SF3b1 mutations in patients with fludarabine results in activation of tumor suppressor gene pathways, making refractory CLL (10/59; 17%) compared with just 5% (17/301) in these cells less likely to acquire poor prognostic chromosomal and newly diagnosed, previously untreated CLL (P ¼ 0.002). A majority genetic mutations, further supported by the absence of SF3b1 of the mutations were missense (9/10; 90%). Patients with clonally mutations at the time of AML transformation in serial studies. related Richter’s syndrome have a mutational frequency of 6% In vivo studies by Isono et al. have demonstrated that Sf3b1 is (2/33). In this same study, multivariate analysis revealed that important in Polycomb (PcG)-mediated repression of Hox genes the presence of SF3b1 mutations independently predicted for in mice. Sf3b1 heterozygous mice develop abnormal skeletal increased risk of death (hazard ratio: 3.02 (confidence interval: phenotypes similar to what is observed in PcG complex mutant 1.24–7.35), P ¼ 0.015). The group also studied 163 cases of mature cases. Several inactivating mutations in genes of the PcG complex B-cell neoplasms, including mantle cell lymphoma, follicular cell have already been associated with myeloid malignancies, such as lymphoma, diffuse large B-cell lymphoma, splenic marginal zone ASXL1 in chronic myelomonocytic leukemia (CMML) and EZH2 in a lymphoma, extranodal marginal zone lymphoma, hairy cell variety of diseases mostly associated with increased platelets.31,32 lymphoma and multiple myeloma, and found no SF3b1 Other components of the polycomb-repressor complex have also mutations in these cases, supporting its specificity in CLL within been found to be infrequently mutated in myeloid malignancies.33 lymphoid neoplasms.36 Another group using whole-exome/ As the function of the PcG complex seems to be dependent genome and targeted sequencing of a smaller cohort of CLL on SF3B as demonstrated in the mouse studies, it is possible patients (N ¼ 91) found SF3b1 mutations in 15% (14/91) of cases.35 that this is what leads to the subsequent development of MDS Several clinical associations were observed in this study, including in SF3b1 mutated cases. However, ASXL1 and EZH2 mutations are a higher frequency of mutated patients having a concomitant almost always associated with more aggressive MDS phenotypes deletion in chromosome 11q, and K700E mutants were associated that tend to progress to AML, which is contrary to the phenotype with unmutated immunoglobulin heavy chain gene status (P ¼ 0.048). where SF3b1 mutations is frequently found, such as low-risk Moreover, Cox multivariate regression model found that the MDS (RARS/RARS-T). This is further supported but the very presence of SF3b1 mutations in these patients was predictive of an low frequency of SF3b1 mutations in AML (de novo ¼ 2.6%; earlier need for treatment (hazard ratio: 2.2; P ¼ 0.03) independent secondary ¼ 4.8%). of other factors including immunoglobulin heavy chain gene Initial studies from our group also suggest its potential link mutation status, del17p or ataxia telangiectasia mutated gene to increased risk of thrombosis within these patients. Further mutation. Using intron retention of two endogenous genes (BRD2 analysis has clarified that B40% of mutant patients developed and RIOK3), they demonstrated that the spliceosome inhibitor

Leukemia (2012) 2447 – 2454 & 2012 Macmillan Publishers Limited Role of spliceosomal machinery in MDSs V Visconte et al 2451 E7107 leads to disruption of splicing of BRD2 and RIOK3 in both Nagata et al.42 further confirmed these results. The most common normal and CLL cells. SF3b1 mutated patients have aberrant mutation occurs at amino-acid position P95 between the RNA endogenous splicing activity in tumor samples compared with the recognition motif and the RS domain. Lower mutations frequen- wild-type cases and the ratio of unspliced to spliced mRNA forms cies were reported in other malignancies (Table 2). A presentation of BRD2 and RIOK3 was significantly higher in SF3b1 mutation at the 2011 annual ASH meeting reported a frequency of 47.2% in carriers versus wild-type cases (median ratio: 2:1 vs 0.5:1, Pp.001 their cohort of patients with CMML.43 More recently it has been and 4.5:1 vs 2.1:1, P ¼ 0.006).35 Finally, a recent paper was emphasized that DNA hypermutability might explain the co- published examining 105 CLL patients in whom whole-exome existence of SRSF2 with other mutations.42 Depletion of SRSF2 sequencing was performed, and found SF3b1 to be mutated in leads to genomic instability and might possibly explain the worse 9.7%. Similar to earlier findings, the presence of SF3b1 mutations outcome in patients with SRSF2 mutations.44,45 There are some was associated with worse 5 year time to progression in Binet clinical associations linking this mutation to a clinical phenotype stage A patients (34% vs 73%, P ¼ 0.002) and 10-year overall associated with increased age, higher hemoglobin levels and survival (30% vs 77%, P ¼ 0.0002).37 Overall, all three studies coincidental TET2 and RUNX1 mutations. It was also noted that it reported SF3b1 mutations in codons, which have been described seems to be mutually exclusive from mutations in EZH2, a poor to be mutated also in MDS and other disorders. prognostic marker. SRSF2 mutations showed better outcomes in CMML patients with concomitant RUNX1 mutations.43 U2 Small Nuclear RNA Auxillary Factor 1 (U2AF1) Mammalian U2AF is a heterodimer composed of a 65-kDa subunit Other spliceosome genes 65 35 65 (U2AF ; U2AF2) and a 35-kDa subunit (U2AF ; U2AF1). U2AF Alterations of components of the spliceosome machinery might 35 contacts the pyrimidine tract while U2AF interacts with the AG be a key biological process in the pathogenesis of many splice acceptor dinucleotide of the target intron at the 30 splice hematological diseases, where each gene might be determinant site. Recently, whole-exome/genome sequencing has helped in for different clinical phenotypes. Yoshida et al. published the the discovery of somatic mutations of U2AF in MDS. Mutations largest comprehensive map of splicing factors genes found were found mostly in U2AF1 with frequencies of 8.7–11.6% in to be mutated in 29 patients with myelodysplasia by whole- 12,17 de novo MDS. Yoshida et al. also reported four patients with exome sequencing. Other spliceosomal-related genes were found mutations in U2AF2. Mutations of this gene are distributed among mutated in cases of MDS in addition to the well-known SF3b1, a myriad of myeloid malignancies and mainly involve two U2AF1 and SRSF2 and include U2AF65, ZRSR2, SF1, SRSF1, SF3a1 conserved amino-acid positions (S34 and Q157) located within and PRF40B. More recently, Makishima et al.40 added PRPF8 and 12,17 zinc finger domains. Mutational frequencies were found LUC7L2 to the list of somatic mutations in spliceosomal genes to be 12% in MDS without RS, 8% in CMML and 10% in cases of (Table 1). MDS-derived secondary AML. A lower frequency was observed in primary AML and MPNs. Recent evidence shows that a p.Ser34Phe or p.Ser34Tyr mutation occurs in 8.7% of de novo MDS.17 38 SPLICEOSOMAL MUTATIONS AND POTENTIAL MECHANISMS Subsequently, Makishima et al. extended the study to juvenile OF DISEASE PATHOGENESIS myelomonocytic leukemia and pediatric AML, finding no Beside the strong correlation between spliceosomal mutations mutations in these disease entities. Yoshida et al.12 first reported and certain hematological diseases, the mechanism through that mutant U2AF1 transduced TF-1 and HeLa cells present with a which these mutations perturb the process of RNA splicing and decrease in cell proliferation rather than a growth advantage, subsequently lead to disease is still unknown. Physiologically, suggesting a loss of function mutation. Cells transiently expressing multiple protein isoforms are generated by the process of mutant U2AF1 also displayed lower reconstitution capacity by differential splicing of RNA transcripts, also known as alternative competitive reconstitution assay in mice. Graubert et al.17 splicing. Alternative splicing determines the final set of coding described a significant increase in exon skipping when the sequences that will ultimately be translated and contributes to the mutant p.Ser34Phe cDNA was transiently expressed in vitro, differential biological and chemical function of the final protein suggesting a gain of function. Clinically, no correlation with product. Different patterns of alternative splicing can occur and response to hypomethylating agents was found in a group of 92 include: (1) exons that are not always consistently included in the patients.39 Makishima et al.40 reported a significant association final mRNA transcript, called cassette exons, may be involved, (2) with worse overall survival in patients with MDS/MPN. In the certain cassette exons may be mutually exclusive, thus if two or context of spliceosome regulation and interaction, U2AF65 usually more cassette exons are present, only one will be incorporated in binds to SF1 factor, increasing the affinity of SF1 factor for the pre- the mRNA, (3) alternative 50 splice sites, (4) alternative 30 splice mRNA branch point sequence. Later during splicing, SF1 is usually sites, (5) intron retention, (6) the presence of multiple promoters displaced from U2AF65 and replaced by the U2snRNP protein, and (7) multiple polyadenylation sites.46 It has been reported that SF3b1. It is of great interest to understand how genes that share almost 50% of mutations in spliceosomal genes affect splicing and almost the same mechanism are pathognomonic of different regulation of alternative splicing leading to causative defects and diseases. contributing to increased susceptibility to diseases (Figure 1).3,47 Diseases that result from aberrant alternative splicing patterns Serine argine-rich splicing factor 2 include neuropsychiatric diseases like frontotemporal dementia SRSF2 encodes a member of the SR-rich family of pre-mRNA with Parkinsonism-17 characterized by misregulation of tau exon splicing factors. A majority of these factors contains an RNA 10 alternative splicing,48 amyotrophic lateral sclerosis where recognition motif and a RS domain. The presence of the RS abnormal patterns of intron retention and exon skipping in domain helps the interaction between different SR splicing factors. astrocytes were observed49 and schizophrenia that has an SRSF2 has been reported to be critical for constitutive and abnormally higher frequency of exon selection and skipping.50,51 alternative mRNA splicing. Interestingly, SRSF2 is believed to be a This mechanism has also been observed in solid cancer like Wilm’s key element in the acetylation/phosphorylation network and an tumor, where misregulation of alternative splicing of WT1 occurs important regulator of the DNA stability.41 Yoshida et al.12 were and glioblastoma where reduced splicing of FGF-R1 alpha exon is first to report a mutation involving this gene in hematological seen.52,53 It is therefore conceivable that dysfunction or changes in malignancies, particularly in MDS and related diseases. It is most alternative splicing patterns can be caused by mutations in the frequent in CMML, with frequencies ranging between 28.4–30%. spliceosome machinery (SF3b1, U2AF1, SRSF2 and others) and can

& 2012 Macmillan Publishers Limited Leukemia (2012) 2447 – 2454 Role of spliceosomal machinery in MDSs V Visconte et al 2452 Table 2. Mutations of splicing factor genes in MDSs and other hematological disorders

Spliceosome Studies Discovery % Mutations within disease groups Effects on Effects Other clinical genes tool overall on AML associations survival evolution/ any progression

SF3b1 Visconte et al.11 Whole MDS: RARS (68%), RAEB1/2: (2.9%) No No difference Higher risk of (N ¼ 56) exome RCMD/RCUD (4.3%) MDS/MPN: difference thrombosis very RARS-T (81%), CMML (6.8%), MDS/ uncommon in MPN-U (0) AML: AML (4.7%), 20 AML o15% RS (5.9%) BMF (1.6%) MPN (1.4%) Yoshida et al.12 Whole MDS: RARS (82.6%), RCMD-RS (76%), NA NA NA (N ¼ 582) exome MDS without RS (6.5%) MDS/MPN: CMML (4.5%) AML: 10 AML (2.6%) 20 AML/MDS (4.8%) MPN (0%) Papaemmanuil Whole Better Lower risk of NA et al.13 genome progression (N ¼ 354) Malcovati et al.28 Whole Better Lower risk of NA (N ¼ 564) genome progression Better Lower risk of progression Damm et al.29 Direct No No difference NA (N ¼ 317) sequencing difference Lasho et al.34 Direct No NA NA (N ¼ 155) sequencing difference Rossi et al.36 Whole Fludarabine-refractoryCLL (15%) Poor NA NA (N ¼ 59/301/33)* exome CLL at diagnosis (5%) Wang et al.35 Whole CLL (15%) NA NA Association with (N ¼ 91) exome/ del(11q) and genome unmutated IGHV status. Predictor factor for an earlier treatment 37 Quesada et al. Whole CLL (9.7%): unmutated IGHV (7.9%) Poor Short time to High b2- (N ¼ 279) exome mutated (20.5%) progression microglobulin, unmutated IGHV

U2AF1 Yoshida et al.12 Whole MDS no RS (11.6%) MDS/MPN: NA NA NA (N ¼ 582) exome CMML (8%) AML: 10 AML (1.3%), 20 AML (9.7%) MPN: (1.9%) Makishima Whole Low-risk MDS (6%) high-risk MDS Worse NA NA et al.40 exome (11%) MDS/MPN: CMML 17% AML: (N ¼ 310) 9% JMML/pediatric AML: (0%)\ Graubert et al.17 Whole MDS de novo (8.7%) MDS/sAML No Increased More frequent in del (N ¼ 150) genome (15.2%) difference probability to 20q and monosomy progress to 20 patients sAML

SRSF2 Yoshida et al.12 Whole MDS no RS (11.6%) RCMD/RCMD-RS NA NA NA (N ¼ 582) exome (5.5%) MDS/MPN: CMML (28.4%) AML: 10 AML (0.7%), 20 AML (6.5%) MPN (1.9%) Abbreviations: AML, acute myeloid leukemia; BMF, bone marrow failure; CLL, chronic lymphocytic leukemia; CMML, chronic myelomonocytic leukemia; IGHV, immunoglobulin heavy chain gene; JMML, juvenile myelomonocytic leukemia; MDS, myelodysplastic syndrome; MPN, myeloproliferative neoplasm; NA, not available; RARS, refractory anemia with ring sideroblasts; RS, ring sideroblasts. *The numbers represent three different cohorts utilized by Rossi et al.36

lead to disease manifestations observed in hematological have oncogenic potential and have the capacity of evading the neoplasms such as MDS, CLL and others. normal process of nonsense-mediated decay. It is also plausible In support of this hypothesis, experimental studies in the that these abnormal protein isoforms can lead to upregulation of nervous system demonstrated that alternative splicing can lead certain pathways already known to be abnormal in MDS like the to exacerbation or inhibition of certain apoptotic pathways.54 high aberrant pSTAT5 activation seen in some Janus kinase 2/MPL- Increased apoptosis has been shown to be important in the negative RARS-T. Conversely, these mutations can possibly lead to pathogenesis of MDS, supported by data showing overexpression repression of certain metabolic pathways (decreased conversion of proapoptotic proteins like Minichromosome Maintenance of 5-MC to 5-HMC in some cases of TET2 wild-type MDS) relevant Protein 2.55 It is possible that spliceosomal mutations leading in MDS as was recently reported by Jankowska et al.56 A similar to aberrant alternative splicing may be responsible for the mechanism may be responsible for the altered iron deposition dysregulation in apoptotic pathways seen in MDS. Similarly, it is seen in patients with RS. Mitochondria with their supporting possible that mutations of the spliceosomal pathway can result in proteins have a crucial role in iron passage, leading to the variable effects on splicing enhancers and splicing repressors, ﬁnal incorporation of iron in hemoglobin during normal heme leading to differential expression of protein isoforms that may biosynthesis. However, mutations of mitochondrial genes are

Leukemia (2012) 2447 – 2454 & 2012 Macmillan Publishers Limited Role of spliceosomal machinery in MDSs V Visconte et al 2453 not frequent in RARS and RARS-T and therefore it is intuitive to well as alternative splicing.59 The low toxic effect of meayamycin assume that an alternative mechanism to the altered iron against IMR-90 human lung fibroblasts and the selectivity against deposition may be at play.13 Furthermore, Yoshida et al.12 transformed cells might be due to the splicing inhibition. studied the impact of the spliceosomal mutations transiently Pharmacological analogs called sudemycins were also developed expressing mutant and wild-type U2AF1 in Hela cells and found that target SF3b and are capable of modulating alternative that mutants cells display an overrepresentation of nonsense splicing in human tumor xenografts.60 Drugs such as Pladienolide mediated mRNA decay genes by gene expression analysis. and FR901464 represent novel ways of targeting the spliceosome Graubert et al.17 proposed a mechanism of exon-skipping in with potent cytotoxic effects at lower IC50. FR901464 is also cells expressing mutant U2AF1. Exon skipping is a process that capable of arresting the cell cycle at G1/G2/M phases. results in the loss of an exon or in a lengthened/shortened (alternative 50 or 30 splicing) alternatively spliced mRNA. Cells transiently expressing mutant U2AF1 exhibited a high proportion FUTURE DIRECTIONS/SUMMARY of transcripts with exon skipping compared with wild types. More Aberrant splicing has been described in many cancers. The discovery comprehensive analyses will help clarify these possibilities. In of mutations in the spliceosome pathway and their specificity in addition, introns might not be spliced out from the RNA transcript RARS, RARS-T and CLL offers a new area of investigation for and retained in the mRNA as part of an exon. Up to 15% of human explaining disease biology, diagnostics and possibly therapeutic diseases are due to mis-splicing. Mutations in certain genes can interventions for these conditions. New information regarding the change the DNA sequences and results in inefficient recognition crucial targets may be helpful to clarify the exact mechanism that of canonical sites by the spliceosome generating a mutated pre- leads to the pathophysiology of many disease subtypes. mRNA. Genetic therapies have actually been used to targeted mis- Our group and others are actively searching for viable targets spliced transcripts in certain diseases.57 of the SF3b1 gene. In vitro SF3b1 knockdown in cell lines have addressed some important issues related to U2-dependent introns. In fact, downregulation of SF3b1 mRNA leads to a lower DRUGS THAT TARGET THE SPLICEOSOME MACHINERY splicing activity of U2 introns as compared with U12 introns.27 Identification of a novel gene typically altered in a specific disease Global analysis of the transcriptome by RNA sequencing is group offers the promise of potential targeted therapy. We now being performed in our laboratory to map the targets of know that SF3b1 mutations are mainly heterozygous in MDS, CLL SF3b1 mutants. Functionally, spliceosome assembly and splicing and other hematological neoplasms. Disruption of the remaining catalysis mechanisms may be key biological processes in SF3b1 allele using a pharmacological inhibitor may induce cell killing regulation. SF3b1 contains threonine–proline dipeptides at specific for carriers of the mutations and can hypothetically spare the N-terminal chain, which are ‘hotspots’ of phosphorylation. normal cells because of the presence of two normal SF3b1 alleles. In splicing catalysis, phosphorylation of SF3b1 is functionally Alternatively, if the disease seen in SF3b1 mutants is related to important. An orchestra of substrates, proline kinases, serine– downstream abnormalities in splicing and restoration of normal threonine kinases, cyclin E and phosphatase may work in the splicing is necessary to improve disease, then the fundamental regulation of SF3b1. In fact, we also sequenced DYRK1A, a protein knowledge of whether this mutation is a gain of function or a loss discovered to phosphorylate SF3b1 in vitro without finding of function mutation is paramount. If SF3b1 mutations are gain of any mutations, leading us to theorize that regulation of SF3b1 function mutations and result in the aberrant alternative splicing occurs at the protein rather than at the genetic level. Therefore, seen, one can hypothesize that pharmacological therapy can our laboratory is currently investigating whether specific kinases restore the normal splicing patterns of these individuals by inhibitors, including TG003. inhibiting the gain of function effects of the mutation. Conversely, if this is a loss of function mutation, then the addition of a pharmacological inhibitor that disrupts the function of the CONFLICT OF INTEREST remaining normal allele can worsen disease manifestations. The authors declare no conflict of interest. However, the process and degree of alternative splicing as a measure of function of SF3b1 is complex. There are multiple regulators of alternative splicing aside from SF3b1, ranging from ACKNOWLEDGEMENTS other snRNPs but more importantly the pre-mRNP itself, and the We thank Dr K Koide from the University of Pittsburgh, PA for providing meayamycin type of alternative splicing observed are tissue specific as can be and for sharing his expertise. We also thank Dr H Koseki from Riken, Japan for giving seen in neural and muscle tissues. Knowing the crucial role had by us the access to the SF3b1 heterozygous mice. 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