Leukemia (2001) 15, 1689–1695  2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu REVIEW

NUP98 fusions in hematologic malignancies DH Lam1,2 and PD Aplan2

1Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY; and 2Genetics Department, Medicine Branch, Division of Clinical Sciences, National Cancer Institute, Bethesda, MD, USA

Acute leukemia is associated with a wide spectrum of recur- the malignant cells ofpatients with both myeloid and rent, non-random chromosomal translocations. Molecular lymphoid malignancies, including AML, CML, myelodysplas- analysis of the involved in these translocations has led to a better understanding of both the causes of chromosomal tic syndrome (MDS), and T acute lymphoblastic leukemia rearrangements as well as the mechanisms of leukemic trans- (T-ALL). This review will summarize the clinical and formation. Recently, a number of laboratories have cloned biological features of this emerging class of chromosomal translocations involving the NUP98 gene on rearrangements. 11p15.5, from patients with acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS), chronic myelogen- ous leukemia (CML), and T cell acute lymphoblastic leukemia (T-ALL). To date, at least eight different chromosomal Structure and function of NUP98 rearrangements involving NUP98 have been identified. The resultant chimeric transcripts encode fusion that The NUP98 is a component ofthe com- juxtapose the N-terminal GLFG repeats of NUP98 to the C-ter- plex (NPC), which regulates nucleocytoplasmic transport of minus of the partner gene. Of note, several of these protein and RNA (Figure 1a).17–22 The yeast NPC consists of translocations have been found in patients with therapy-related approximately 30 ;23 the mammalian NPC has acute myelogenous leukemia (t-AML) or myelodysplastic syn- 20,22,24 drome (t-MDS), suggesting that genotoxic chemotherapeutic been estimated to contain 50–100 different proteins. agents may play an important role in generating chromosomal NUP98 resides asymmetrically at the nucleoplasmic side of rearrangements involving NUP98. Leukemia (2001) 15, 1689– the NPC and colocalizes with TPR (translocated promoter 1695. region) to intranuclear sites, extending to and perhaps within Keywords: NUP98; acute leukemia; myelodysplastic syndrome; the .17,20,25 NUP98 belongs to a subgroup ofnucleo- 11p15.5 translocations porins, which contains FXFG or FG repeats (Figure 1b). Unlike the other members ofthis subgroup, NUP98 also contains GLFG repeats. The 37 FG repeats are located in the N-ter- Introduction minus and comprise the first and third functional domains of

Similar to other malignancies, leukemia is a heterogeneous disease. This observation is presumably a direct manifestation ofthe vast array ofgenomic changes that occur in leukemic cells. However, certain types ofleukemia are oftenassociated with very specific, recurrent chromosomal abnormalities, most commonly chromosomal inversions or translocations.1–4 Correlation ofrecurrent chromosomal abnormalities with clinical features allows the establishment of new disease classifications and therapeutic approaches. Importantly, these chromosomal abnormalities have begun to serve as bio- markers useful in identifying distinct clinical subgroups with predictable clinical features and therapeutic responses.5,6 The cloning ofchromosomal breakpoints and genes involved in chromosomal rearrangements has also provided considerable insight into the mechanisms ofchromosomal rearrangement and leukemic transformation. The first case reports ofa t(7;11)(p15;p15.5) associated with acute myelogenous leukemia (AML) or chronic myelogenous leukemia (CML) were published in 1982.7 Subsequently, two Figure 1 Representation ofthe nuclear pore complex (NPC) and laboratories identified 98 kD (NUP98) as one of the functional domains of the nucleoporin 98kD protein (NUP98). (a) the genes involved in the t(7;11)(p15;p15.5).8,9 This obser- The nuclear localization ofNUP98, subsequent to autoproteolysis in the , is indicated by this schematic ofthe NPC. TAP and vation has led to the identification and cloning ofat least eight RAE1 have been localized to the cytoplasm and nucleoplasm as well different fusion transcripts encoding NUP98 fusion pro- as on both sides ofthe NPC. NUP98 colocalizes with TPR at the teins.10–16 These chromosomal abnormalities are present in nuclear basket ofthe NPC and also within the nucleoplasm. CC, cen- tral core; CF, cytoplasmic filaments; CR, cytoplasmic ring; NR, nuclear ring; NB, nuclear basket; NE, ; NO, Correspondence: PD Aplan, National Cancer Institute, Division of nucleolus. (b) The Phe-Gly (FG) repeats ofNUP98 are indicated, as Clinical Sciences, Advanced Technology Center, 8717 Grovemont are the RAE1 (GLEBS) and RNA binding domains (bd). An asterisk Circle, Gaithersburg, MD 20877, USA; Fax: 301-402-3134 marks residue 863 where autoproteolysis truncates the NUP98 Received 12 April 2001; accepted 11 July 2001 protein. Review DH Lam and PD Aplan 1690 the NUP98 protein. These FG repeats have been shown to be docking sites for transport receptors such as karyopherin ␤1, which interacts with karyopherin ␣ to form a heterodimer transport complex. Karyopherin ␣ recognizes the nuclear localization signals (NLS) and nuclear export signals (NES) of substrates that are being transported, but requires karyopherin ␤1 to dock on to nucleoporins.17,26–28 Once docked, the small GTPase Ran and p10 (NTF2) facilitate entry into or out of the nucleus. Although karyopherin ␤1 binds the FG repeats ofNUP98, whether NUP98 actually plays a direct role in protein trans- port is unclear since depletion ofNUP98 does not seem to disrupt protein transport in reconstituted nuclei.29 NUP98- specific antibodies inhibit the transport ofsnRNA, 5S RNA, large rRNA, and mRNA without perturbing other functions,19 but NUP98-deficient embryonic stem (ES) cells remain viable, suggesting that NUP98 is not required for mRNA transport.17– 22 Other studies suggest that the role ofNUP98 in RNA trans- port may be mediated by TAP (TIP associated protein), a novel cellular factor first identified for its interaction with TIP (tyrosine kinase-interacting protein) (Figure 1a).30–32 TAP is required for export of cellular mRNA substrates. The gluta- mine-rich C-terminal domain ofTAP interacts with the FG repeats ofNUP98. Thus, in conjunction with other nuclear proteins, TAP appears to mediate mRNA export through its interaction with NUP98. The second and fourth functional domains of NUP98 con- tain a GLEBS-like motifand a RNP binding motif,respectively, surrounded by charged residues (Figure 1b). In a recent report, Figure 2 Genomic structure ofthe human NUP98 gene and its the NUP98 C-terminus has also been shown to function as a alternatively spliced transcripts. (a) NUP98 exons 1–20 are indicated nuclear localization signal (NLS).25 GLEBS motifs serve as by black boxes. Broken lines indicate gaps ofunknown size between binding sites for RAE1—a molecule purported to be a mRNA- exons 1 and 2 and 2 and 3. The approximate location oftranslocation specific carrier containing the nuclear export signal (NES) breakpoints, based on fusion mRNA sequences, is shown. (b) Five 33 alternatively spliced NUP98 transcripts are shown. Numbers above (Figure 1a). RAE1 and TAP both bind to NUP98 and, at the opened boxes refer to NUP98 or NUP96 exons. The bold, gray same time, can bind to mRNA-associated proteins. Since vertical lines indicate splicing within an exon; bold, dash vertical line mRNA traverses the NPC as part ofa RNP complex, it seems indicates the autoproteolytic site in exon 20 of NUP98. likely that RAE1 and TAP act as nuclear transport receptors for mRNA, much like karyopherins for proteins. Although the function of the putative RNP binding domain is unknown, of186 kDa. The precursor is then proteolytically cleaved to given the data on NUP98’s role in mRNA transport, this produce NUP98 and NUP96 proteins. In order for NUP98 to domain may also participate in mRNA transport. be properly targeted to the nuclear basket, both the NUP98- The NUP98 gene is located on chromosome 11p15.5 near NUP96 precursor and the immature NUP98 protein must be an imprinted region that includes IPL, IGF2 and H19;34 how- cleaved to produce an 863 aa NUP98 protein.20 Upon cleav- ever, NUP98 is not imprinted in the mouse (DHL and PDA, age ofthe immature NUP98 protein, a 6 kDa oligopeptide unpublished data). Two major NUP98 transcripts of4.0 and containing an NLS is produced from the C-terminus; this 7.0 kb can be detected. The 4.0 kb transcript consists of20 6 kDa product remains associated with NUP98.25 Post- exons35 (Figure 2a) and encodes a 920 amino acid (aa) pro- translational modification occurs in the nucleoplasm through tein.20 Several minor NUP98 transcripts are produced through autoproteolysis, not requiring additional proteases.39 This alternative splicing (Figure 2b). One alternatively spliced form remarkable biogenesis pathway is evolutionarily conserved deletes 570 bp from the full-length transcript and results in the between Homo sapiens and Saccharomyces cerevisiae. loss ofresidues 254 to 444. 36 Two other rare transcripts result from either a 51 bp extension of the 3Ј end ofexon 10 37 or from splicing around exon 12.38 Interestingly, these alternate NUP98 partner genes I:the HOX family splice forms can be detected in both human and mouse NUP98 transcripts (DHL and PDA, unpublished data). The nucleoporins were first studied in Saccharomyces cerevis- Recently, it was reported that another rare transcript is pro- iae and Xenopus laevis.17,40,41 These studies led to the cloning duced by splicing around exons 3 and 4.22 In all ofthese alter- and characterization ofthe rat NUP98.18 Subsequently, the natively spliced transcripts, the reading frame is preserved, human NUP98 gene was identified at the site ofa encoding a truncated NUP98 protein. t(7;11)(p15;p15.5) chromosomal translocation.8,9 The partner The 7.0 kb transcript is unique in that it encodes a NUP98- gene in this translocation was the homeobox gene HOXA9. NUP96 precursor protein (Figure 2b).20 This 7.0 kb transcript Since then, two other chromosomal translocations have been is created by splicing NUP98 exon 20 sequences shown to result in NUP98–homeobox fusions, the (corresponding to residue 914) to downstream exons encoding t(2;11)(q31;p15.5)11 and t(1;11)(q23;p15.5),12 involving, the NUP96 protein. This alternatively spliced transcript enco- HOXD13 and PMX1, respectively (Table 1). des a precursor protein of1712 aa, with a molecular mass The HOX or homeobox genes are a superfamily of tran-

Leukemia Review DH Lam and PD Aplan 1691 Table 1 Recurrent NUP98 chromosomal rearrangements iesis.46 Studies with leukemia cell lines suggested that there may be some lineage specificity for these three clusters. For Rearrangement NUP98 Homeodomain Disease example, clusters B and C are expressed mainly in erythroid partner ? cell lines whereas cluster A is predominantly expressed in gene myeloid cell lines.47–50 Expression ofvirtually all the HOX genes ofthe A, B and C clusters is detected in normal primitive t(7;11)(p15;p15.5) HOXA9 Yes AML, MDS, t- 46 MDS/AML, CML hematopoietic cells. Expression ofHOX cluster genes tends t(2;11)(q31;p15.5) HOXD13 Yes AML, t-MDS/AML to decline in a pattern that follows their chromosomal pos- t(1;11)(q23;p15.5) PMX1 Yes t-MDS/AML ition; genes in the 3Ј region are downregulated earlier than inv11(p15.5q22) DDX10 No AML, t-MDS/AML genes in the 5Ј region ofeach cluster. As hematopoietic stem t(11;20)(p15.5;q11) TOP1 No AML, t-MDS/AML cells become increasingly committed to a specific lineage, t(9;11)(p22;p15.5) LEDGF No AML there is a loss of HOX .51 Some ‘orphan’ t(5;11)(q35;p15.5) NSD1 No AML t(4;11)(q21;p15.5) RAP1GDS1 No T-ALL homeobox genes are expressed during normal hematopoietic development as well. However, the entire D cluster may be non-essential for normal blood cell development. Expression ofHOXD cluster genes has not been detected in normal hem- atopoietic cells,46 and neither Hoxd-13 nor Hoxd-11–Hoxd- scription factors that play a role in early embryonic develop- 13 knock-out mice show defects in hematopoiesis.52 ment.42,43 Members ofthe HOX family share a 60 aa A regulatory role for HOX genes in normal and malignant sequence, which constitutes the DNA binding homeodomain hematopoiesis can be inferred from experiments in which ofthese proteins (Figure 3a). Mammalian HOX genes are expression ofspecific genes have been blocked or amplified organized into four clusters (A, B, C and D) located on human in cell lines or animal models.46 Overexpression of HOXA10 7, 17, 12, and 2, respectively.44,45 The 39 genes in bone marrow cells results in AML when these cells are in these four clusters are referred to as class-I homeobox transplanted into sub-lethally irradiated mice.53 Insertional genes. There are also numerous class-II or ‘orphan’ homeobox activation of Hoxa-7 or Hoxa-9, in collaboration with acti- genes, which contain a divergent homeodomain, dispersed vation of Meis1 (a Pbx-related ‘orphan’ homebox gene) also throughout the genome. leads to AML in mice,54 and mRNA expression profile experi- Notably, three ofthe fourclusters (clusters A, B and C) have ments have demonstrated that HOXA9 is frequently expressed been shown to regulate normal and leukemic hematopo- in AML cells.55 Intriguingly, the t(1;19)(q23;p13) in human ALL creates a chimeric E2A-PBX1 protein; this protein has been shown to be oncogenic in a transgenic mouse model.56 Prior to the discovery ofthe NUP98 gene fusions, HOX1157 and the aforementioned PBX158 were the only homeobox genes implicated in human leukemia. The first class-I HOX genes to be linked to human leukemias are HOXA98,9 and HOXD13,11 as partners in the NUP98 gene fusions. The NUP98 translocation involving the HOXA9 gene, t(7;11)(p15;p15.5), is associated with AML, MDS, and CML. Although it has been suggested that the t(7;11) is most com- monly found in older Asian men, it remains unclear whether the racial or geographical predisposition is coincidental.38,59– 64 HOXA9 consists ofthree exons: 1A, 1B and 2. The translocation breakpoint occurs between exons 1A and 1B, effectively separating the homeodomain in exon 2 from the regulatory elements in exon 1A (Figure 3a). The NUP98 break- point in these translocations lies in either intron 11 or 12 (Figure 2a). The t(2;11)(q31;p15.5) has been found in patients with AML and t-MDS/AML, and fuses NUP98 to HOXD13.11,65 Similar to HOXA9, the second exon of HOXD13 contains the highly conserved homeodomain, which is separated from its regulat- ory elements in the gene fusion since the breakpoint lies between the two exons (Figure 3a). The breakpoint in NUP98 lies in intron 12, the most common breakpoint region (Figure 2a). A variant ofthis translocation has a breakpoint upstream ofthe HOXD13 gene, adjacent to a putative gene called FN1.37 In this case, both NUP98-FN1 and NUP98-HOXD13 fusion transcripts can be detected in the same patient. A third HOX gene implicated in NUP98 translocations is PMX1. The t(1;11)(q23;p15.5) is associated with t-AML and Figure 3 Functional domains ofNUP98 and fusionpartner pro- produces a NUP98-PMX1 fusion gene.12,66 PMX1 is a class-ll teins. (a) A representative chimeric protein (der(11)) is shown which homeobox gene that has a genomic structure similar to fuses the N-terminus of NUP98 to the C-terminal homeodomain (HD) ofa HOX protein. Arrows indicate points ofgene fusion.(b) The func- HOXA9 in that the homeodomain of PMX1 is located in exon tional domains ofnon-HOX, variant partner proteins and fusionpoints 2. The breakpoint in PMX1 lies between exons 1A and 1B are shown. (Figure 3a). Alternative splicing fuses NUP98 to both exons

Leukemia Review DH Lam and PD Aplan 1692 1B and 2 of PMX1, producing two chimeric transcripts. is expressed in hematological tissues, as shown by Northern Although the fusion of NUP98 to PMX1 exon 2 encodes a analysis.16 The chromosomal translocation breakpoint has not NUP98-homeodomain similar to NUP98-HOXA9 and yet been isolated and further characterization of this novel NUP98-HOXD13, PMX1 exon 1B is non-coding and would human gene in the translocation is anticipated. result in the production ofa truncated NUP98-PMX1 fusion The t(4;11)(q21;p15) has the distinction ofbeing the only protein. NUP98 gene rearrangement associated with T-ALL (Table Two other translocations found in t-MDS/AML patients, 1).13,71,72 The RAP1GDS1 gene on chromosome 4q21 codes t(11p15.5;17q21) and t(11p15.5;12q13),62 may also generate for a ubiquitously expressed guanine nucleotide exchange fac- NUP98-HOX fusions. The 17q21 and 12q13 breakpoints in tor called smgGDS (Figure 3b). SmgGDS mediates conversion these two translocations map to the HOXB and HOXC clus- ofinactive GDP-bound GTPases to the active GTP-bound ters, respectively, raising the possibility that these translo- form. However, it has been speculated that smgGDS might cations produce NUP98-HOXB and NUP98-HOXC fusions. also play a role in nucleocytoplasmic transport, as smgGDS However, proofthat these rearrangements involve HOX genes is composed ofmultiple armadillo repeats that are thought to awaits the molecular characterization ofthese translocations. mediate protein–protein interactions. NUP98 is fused to nucleotide 5 ofthe RAP1GDS1 ORF, preserving the entire protein except for the first methionine. One of the NUP98 NUP98 partner genes II:the variants breakpoints in these translocations lies in intron 12, whereas the other lies in intron 10 (Figure 2a). Regardless ofwhich In addition to the HOX genes described above, five other NUP98 exon (10 or 12) is fused to RAP1GDS1, the reading NUP98 fusion partners have been identified at the molecular frame of the fusion transcript is preserved. As with the NUP98- level: DDX10,10 TOP1,14 LEDGF,15 NSD1,16 and DDX10 translocations, a reciprocal RAP1GDS1-NUP98 RAP1GDS113 (Table 1). Presently, there does not appear to be fusion transcript has been detected. an obvious theme among these genes. The inv(11)(p15.5q22) In all ofthe NUP98 translocations described thus far, a is associated with AML and MDS, and generates two different chimeric transcript consisting ofthe 5 Ј portion of NUP98 in-frame fusion transcripts, with NUP98 exon 12 fused to fused in-frame to the 3Ј portion ofthe partner genes is gener- DDX10 exon 6 or NUP98 exon 14 fused to DDX10 exon ated. Despite the fact that the NUP98 breakpoints in these 7.10,67,68 The DDX10 gene encodes a DEAD-box protein with translocations are located between introns 9 to 14 (Figure 2a), a putative RNA motif(Figure 3b). DEAD box RNA the FG repeats in the N-terminus are always retained (Figure are implicated in a number ofRNA metabolic path- 3a). Interestingly, FG repeats are also retained in translo- ways including splicing, translation, and ribosome assembly. cations involving NUP214.73,74 In the t(6;9)(p23;q34), the FG In contrast to the HOX genes, DDX10 is ubiquitously repeats in the C-terminus ofNUP214 are fusedto the N-ter- expressed, but the reciprocal DDX10-NUP98 gene fusion is minus ofDEK. Similarly, in the interstitial deletion of9q34, only detected sporadically. the N-terminus ofSET is fusedto the FG repeats ofNUP214. I (TOP1) is the NUP98 fusion partner in the t(11;20)(p15.5;q11).14,35 This translocation is also associated with MDS and AML. TOP1 is functionally organized into four Possible mechanisms of translocation domains (Figure 3b) and normally carries out a cycle of transesterification reactions, during which it creates transient Although initial studies indicated an association between single-stranded DNA nicks, resulting in topological NUP98 fusions and de novo AML, recent reports demonstrate transformations of DNA. The TOP1 breakpoint leads to a sep- that several ofthe reported NUP98 chromosomal rearrange- aration ofthe TOP1 NLS and catalytic domains, and results ments could be identified in patients with therapy-related in a fusion between the amino terminal portion of NUP98 AML or MDS (t-AML, t-MDS) (Table 1).10–12,14,35,62,75 Addition- and the TOP1 catalytic domain. Although a reciprocal fusion ally, 7% ofall pediatric t-MDS/AML cases were associated transcript is predicted, none have been isolated. with 11p15.5 translocations in one series.75 The association The t(9;11)(p22;p15.5), is associated with AML and leads of NUP98 gene rearrangements with t-AML/MDS suggests that to production ofa NUP98-LEDGF fusion.15 The LEDGF gene the NUP98 may be susceptible to gross chromosomal encodes the transcriptional coactivators p52 and p75 (also rearrangements induced by genotoxic agents. known as lens epithelium-derived growth factor) (Figure 3b). Thus far, there are only two reports in which the genomic The p52 and p75 isoforms are produced as a result of alterna- translocation breakpoints have been characterized. In two tive splicing and differ only in their C-termini.69 The N-ter- cases oft-MDS and a t(11;20)(p15.5;q11), the genomic break- minus ofp52 and p75 contains a HATH domain, followedby points showed 4 bp microduplications which the authors a linker region, and then an HMG-1 like C-terminal domain speculated might have been generated by a 4 bp staggered with highly charged residues. p52 is a potent activator oftran- double-strand DNA break followed by inter-chromosomal scription and serves to bridge sequence-specific transcription strand exchange.35 This observation is intriguing, given that activation proteins.70 The LEDGF breakpoint is in the linker topo II normally generates 4 bp staggered breaks and the region, which would produce a fusion that contains the patients had received topo II inhibitors. Moreover, a very simi- charged residues ofLEDGF at the C-terminus. The 3 Ј end of lar translocation has been produced in vitro by treating cells both the p52 and the p75 transcript is fused to exon 9 of with topo II inhibitors.76,77 NUP98 (Figure 2a). Again, a reciprocal fusion transcript is The breakpoint junctions in a case ofAML with a presumed to exist, but has not been detected. t(2;11)(q31;p15.5) were within intron 12 of NUP98 and The t(5;11)(q35;p15.5), recently identified in several cases upstream ofboth HOXD13 exon 1 and the putative FN1 gene ofchildhood AML, fuses NUP98 exon 12 to the NSD1 gene.16 on chromosome 2q31.37 Sequence comparisons demonstrated In the mouse, nsd1 has been shown to exhibit the properties that the translocation was not balanced at the molecular level ofboth corepressors and coactivators oftranscription, but the and that 49 bp from and 110 bp from chro- function of its human counterpart is unknown. Human NSD1 mosome 2 were duplicated and present on both the der(2)

Leukemia Review DH Lam and PD Aplan 1693 and der(11) chromosomes. Interestingly, the breakpoint in expressed suggesting that the FG repeats possess an important intron 12 of NUP98 lies within a LINE repetitive sequence. function for leukemic transformation. Although it has been Similar to the t(11;20) described above, a potential mech- suggested that NUP98 translocations may be over-represented anism for this translocation is again a staggered double-strand in the Asian population, additional surveys will be needed to DNA break, followed by chromosomal interchange and determine the incidence and prevalence ofthese translo- repair. cations, and to clarify the issue of racial predisposition. Additionally, the mechanisms leading to chromosomal rearrangement and leukemic transformation are proposed Potential mechanisms of leukemic transformation only tentatively and many important questions remain. For instance, do different mechanisms exist for different fusion The association ofchromosomal rearrangements that produce proteins, or is there a unifying theme for all NUP98 fusion NUP98 fusion proteins with acute leukemia suggests that proteins? Finally, the clinical course ofpatients with NUP98 expression ofNUP98 fusionproteins may be a causal event rearrangements seems quite aggressive and the outcome of for leukemic transformation. Of note, a chimeric transcript treatment is disappointing. It is our hope that a better under- that encodes the NUP98 N-terminal FG repeats fused to the standing ofthe disease process will help identifynew treat- C-terminus ofthe partner protein, is expressed in all NUP98 ment strategies to contend with this poor clinical outcome. fusions reported, suggesting that the NUP98 N-terminus may be important for leukemogenicity. These FG repeats have been shown to activate transcription and interact with both References CREB binding protein and p300.12,36 Expression ofa NUP98- HOXA9 fusion protein induces transformation of NIH 3T3 1 Rabbitts TH. Translocations, master genes, and differences 36 fibroblasts in vitro. Recently, two groups have reported that between the origins ofacute and chronic leukemias. Cell 1991; NUP98 fusion proteins are leukemogenic in murine bone 67: 641–644. marrow transplantation experiments. Ectopic expression ofa 2 Rabbitts TH. Chromosomal translocations in human cancer. Nat- NUP98-HOXA9 fusion in mouse bone marrow cells was ure 1994; 372: 143–149. shown to predispose mice to AML; this effect was accentuated 3 Cline MJ. The molecular basis ofleukemia. N Engl J Med 1994; 78 330: 328–336. by co-expression ofMeis 1. When expressed in differen- 4 Thandla S, Aplan PD. Molecular biology ofacute lymphocytic leu- tiating murine embryonic stem cells, NUP98-HOXD13 fusion kemia. Semin Oncol 1997; 24: 45–56. protein leads to an increase in myeloproliferation and 5 Look AT. Oncogenic transcription factors in the human acute leu- decrease in erythropoiesis.79 Furthermore, NUP98-HOXD13 kemias. Science 1997; 278: 1059–1064. induces myeloproliferation and, again in collaboration with 6 Rowley JD. The critical role ofchromosome translocations in Meis1, acute myeloid leukemia in murine bone marrow trans- human leukemias. Annu Rev Genet 1998; 32: 495–519. 80 7 Tomiyasu T, Sasaki M, Kondo K, Okada M. Chromosome banding plantation experiments. studies in 106 cases ofchronic myelogenous leukemia. Jinrui Iden- Reports that NUP98 fusion proteins function as robust tran- gaku Zasshi 1982; 27: 243–258. scription factors imply that these fusion proteins localize to 8 Nakamura T, Largaespada DA, Lee MP, Johnson LA, Ohyashiki K, the nucleus. The NUP98-HOXA9,36 NUP98-PMX1,12 NUP98- Toyama K, Chen SJ, Willman CL, Chen IM, Feinberg AP, Jenkins RAP1GDS1,72 and DEK-NUP21481 fusion proteins do show NA, Copeland NG, Shaughnessy JD. Fusion ofthe nucleoporin nuclear localization, although NUP98-RAP1GDS1 can also gene NUP98 to HOXA9 by the chromosome translocation t(7;11)(p15;p15) in human myeloid leukaemia. Nat Genet 1996; be found in the cytoplasm. If NUP98 fusion proteins serve as 12: 154–158. transcription factors, then what are the downstream targets? 9 Borrow J, Shearman AM, Stanton VP, Becher R, Collins T, Willi- Presumably, the fusion partners provide binding specificity ams AJ, Dube I, Katz F, Kwong YL, Morris C, Ohyashiki K, Toyama and therefore can determine these downstream target genes. K, Rowley J, Housman DE. The t(7;11)(p15;p15) translocation in In the case ofthe HOX partners, such a mechanism is plaus- acute myeloid leukaemia fuses the genes for nucleoporin NUP98 ible since HOX genes play a role in hematopoietic develop- and class I homeoprotein HOXA9. Nat Genet 1996; 12: 159–167. 10 Arai Y, Hosoda F, Kobayashi H, Arai K, Hayashi Y, Kamada N, ment. It is more difficult to propose this mechanism for Kaneko Y, Ohki M. The inv(11)(p15q22) chromosome translo- DDX10, LEDGF, NSD1, and RAP1GDS1 since these proteins cation of de novo and therapy-related myeloid malignancies are not known to have direct DNA binding capabilities nor results in fusion of the nucleoporin gene, NUP98, with the puta- are they implicated in blood cell development. tive RNA helicase gene, DDX10. Blood 1997; 89: 3936–3944. Alternatively, the FG domain ofNUP98 may be the critical 11 Raza-Egilmez SZ, Jani-Sait SN, Grossi M, Higgins MJ, Shows TB, component, with the partners providing a non-specific func- Aplan PD. NUP98-HOXD13 gene fusion in therapy-related acute myelogenous leukemia. Cancer Res 1998; 58: 4269–4273. tion. There is precedence for such a mechanism. For instance, 12 Nakamura T, Yamazaki Y, Hatano Y, Miura I. NUP98 is fused to in MLL gene fusions, it has been suggested that the importance PMX1 homeobox gene in human acute myelogenous leukemia ofpartner genes lies principally in their capacity to facilitate with chromosome translocation t(1;11)(q23;p15). Blood 1999; 94: oligomerization ofthe MLL fusionproteins. 82 Reviewing the 741–747. function of the NUP98 partner genes described, it is tempting 13 Hussey DJ, Nicola M, Moore S, Peters GB, Dobrovic A. The to invoke such a mechanism since most ofthe NUP98 partner (4;11)(q21;p15) translocation fuses the NUP98 and RAP1GDS1 genes and is recurrent in T-cell acute lymphocytic leukemia. genes encode products capable ofprotein–protein interac- Blood 1999; 94: 2072–2079. tions. It is possible that NUP98 may indirectly control gene 14 Ahuja HG, Felix CA, Aplan PD. The t(11;20)(p15;q11) chromo- expression by a dominant-negative mechanism or by altering somal translocation associated with therapy-related myelodysplas- nucleocytoplasmic transport ofmRNA and/or protein. tic syndrome results in an NUP98-TOP1 fusion. Blood 1999; 94: The studies of NUP98 gene fusions and leukemia reported 3258–3261. thus far form only a framework from which to proceed. At 15 Ahuja HG, Hong J, Aplan PD, Tcheurekdjian L, Forman SJ, Slovak ML. t(9;11)(p22;p15) in acute myeloid leukemia results in a fusion least eight different genes are involved in NUP98 fusions and between NUP98 and the gene encoding transcriptional coacti- there seems to be a predilection for homeobox genes. A fusion vators p52 and p75-lens epithelium-derived growth factor transcript encoding the NUP98 FG repeats is consistently (LEDGF). Cancer Res 2000; 60: 6227–6229.

Leukemia Review DH Lam and PD Aplan 1694 16 Jaju RJ, Fidler C, Haas OA, Strickson AJ, Watkins F, Clark K, Aplan 37 Arai Y, Kyo T, Miwa H, Arai K, Kamada N, Kita K, Ohki M. Heter- PD, Kearney L, Boultwood J, Wainscoat JS. A novel gene is fused ogenous fusion transcripts involving the NUP98 gene and to NUP98 in the t(5;11)(q35;p15.5) in de novo childhood AML. HOXD13 gene activation in a case ofacute myeloid leukemia Blood 2000; 96 (Suppl. A): 692a. with the t(2;11)(q31;p15) translocation. Leukemia 2000; 14: 17 Radu A, Blobel G, Moore MS. Identification ofa protein complex 1621–1629. that is required for import and mediates docking 38 Hatano Y, Miura I, Nakamura T, Yamazaki Y, Takahashi N, Miura ofimport substrate to distinct nucleoporins. Proc Natl Acad Sci AB. Molecular heterogeneity ofthe NUP98/HOXA9 fusiontran- USA 1995; 92: 1769–1773. script in myelodysplastic syndromes associated with 18 Radu A, Moore MS, Blobel G. The peptide repeat domain of t(7;11)(p15;p15). Br J Haematol 1999; 107: 600–604. nucleoporin Nup98 functions as a docking site in transport across 39 Rosenblum JS, Blobel G. Autoproteolysis in nucleoporin biogen- the nuclear pore complex. Cell 1995; 81: 215–222. esis. Proc Natl Acad Sci USA 1999; 96: 11370–11375. 19 Powers MA, Forbes DJ, Dahlberg JE, Lund E. The vertebrate GLFG 40 Wente SR, Rout MP, Blobel G. A new family of yeast nuclear pore nucleoporin, Nup98, is an essential component ofmultiple RNA complex proteins. J Cell Biol 1992; 119: 705–723. export pathways. J Cell Biol 1997; 136: 241–250. 41 Wimmer C, Doye V, Grandi P, Nehrbass U, Hurt EC. A new sub- 20 Fontoura BM, Blobel G, Matunis MJ. A conserved biogenesis path- class ofnucleoporins that functionallyinteract with nuclear pore way for nucleoporins: proteolytic processing of a 186-kilodalton protein NSP1. EMBO J 1992; 11: 5051–5061. precursor generates Nup98 and the novel nucleoporin, Nup96. J 42 KrumlaufR. Hox genes in vertebrate development. Cell 1994; 78: Cell Biol 1999; 144: 1097–1112. 191–201. 21 Fontoura BM, Blobel G, Yaseen NR. The nucleoporin is a 43 Maconochie M, Nonchev S, Morrison A, KrumlaufR. Paralogous site for GDP/GTP exchange on ran and termination of karyopherin Hox genes: function and regulation. Annu Rev Genet 1996; 30: beta 2-mediated nuclear import. J Biol Chem 2000; 275: 529–556. 31289–31296. 44 Scott MP. Vertebrate homeobox (letter). Cell 22 Wu X, Kasper LH, Mantcheva RT, Mantchev GT, Springett MJ, 1992; 71: 551–553. van Deursen JM. Disruption ofthe FG nucleoporin NUP98 causes 45 Scott MP. A rational nomenclature for vertebrate homeobox selective changes in nuclear pore complex stoichiometry and (HOX) genes. Nucleic Acids Res 1993; 21: 1687–1688. function. Proc Natl Acad Sci USA 2001; 98: 3191–3196. 46 Thorsteinsdottir U, Sauvageau G, Humphries RK. Hox homeobox 23 Rout MP, Aitchison JD, Suprapto A, Hjertaas K, Zhao Y, Chait BT. genes as regulators ofnormal and leukemic hematopoiesis. Hema- The yeast nuclear pore complex: composition, architecture, and tol Oncol Clin NorthAm 1997; 11: 1221–1237. transport mechanism. J Cell Biol 2000; 148: 635–651. 47 Lawrence HJ, Sauvageau G, Humphries RK, Largman C. The role 24 Gorlich D, Kutay U. Transport between the and the ofHOX homeobox genes in normal and leukemic hematopoiesis. cytoplasm. Annu Rev Cell Dev Biol 1999; 15: 607–660. Stem Cells 1996; 14: 281–291. 25 Fontoura BM, Dales S, Blobel G, Zhong H. The nucleoporin 48 Lawrence HJ, Stage KM, Mathews CH, Detmer K, Scibienski R, Nup98 associates with the intranuclear filamentous protein net- MacKenzie M, Migliaccio E, Boncinelli E, Largman C. Expression work ofTPR. Proc Natl Acad Sci USA 2001; 98: 3208–3213. ofHOX C homeobox genes in lymphoid cells. Cell GrowthDiffer 26 Moroianu J, Hijikata M, Blobel G, Radu A. Mammalian kary- 1993; 4: 665–669. opherin alpha 1 beta and alpha 2 beta heterodimers: alpha 1 or 49 Mathews CH, Detmer K, Boncinelli E, Lawrence HJ, Largman C. alpha 2 subunit binds nuclear localization signal and beta subunit Erythroid-restricted expression ofhomeobox genes ofthe human interacts with peptide repeat-containing nucleoporins. Proc Natl HOX 2 locus. Blood 1991; 78: 2248–2252. Acad Sci USA 1995; 92: 6532–6536. 50 Vieille-Grosjean I, Roullot V, Courtois G. Lineage and stage spe- 27 Moroianu J, Blobel G, Radu A. RanGTP-mediated nuclear export cific expression ofHOX 1 genes in the human hematopoietic sys- ofkaryopherin alpha involves its interaction with the nucleoporin tem. Biochem Biophys Res Commun 1992; 183: 1124–1130. Nup153. Proc Natl Acad Sci USA 1997; 94: 9699–9704. 51 Sauvageau G, Lansdrop PM, Eaves CJ, Hogge DE, Dragowska WH, 28 Bonifaci N, Moroianu J, Radu A, Blobel G. Karyopherin beta2 Reid DS, Largman C, Lawrence HJ, Humphries RK. Differential mediates nuclear import ofa mRNA binding protein. Proc Natl expression ofhomeobox genes in functionallydistinct CD34 + sub- Acad Sci USA 1997; 94: 5055–5060. populations ofhuman bone marrow cells. Proc Natl Acad Sci USA 29 Powers MA, Macaulay C, Masiarz FR, Forbes DJ. Reconstituted 1994; 91: 12223–12227. nuclei depleted ofa vertebrate GLFG nuclear pore protein, p97, 52 Zakany J, Duboule D. Synpolydactyly in mice with a targeted import but are defective in nuclear growth and replication. J Cell deficiency in the HoxD complex. Nature 1996; 384: 69–71. Biol 1995; 128: 721–736. 53 Lawrence HJ, Sauvageau G, Ahmadi N, Lopez AR, LeBeau MM, 30 Gruter P, Tabernero C, von Kobbe C, Schmitt C, Saavedra C, Bachi Link M, Humphries K, Largman C. Stage-and lineage-specific A, Wilm M, Felber BK, Izaurralde E. TAP, the human homolog of expression ofthe HOXA10 homeobox gene in normal and leu- Mex67p, mediates CTE-dependent RNA export from the nucleus. kemic hematopoietic cells. Exp Hematol 1995; 23: 1160–1166. Mol Cell 1998; 1: 649–659. 54 Nakamura T, Largaespada DA, Shaughnessy JD Jr, Jenkins NA, 31 Bachi A, Braun IC, Rodrigues JP, Pante N, Ribbeck K, von Kobbe Copeland NG. Cooperative activation ofHoxa and Pbx1-related C, Kutay U, Wilm M, Gorlich D, Carmo-Fonseca M, Izaurralde genes in murine myeloid leukaemias (see comments). Nat Genet E. The C-terminal domain ofTAP interacts with the nuclear pore 1996; 12: 149–153. complex and promotes export ofspecific CTE-bearing RNA sub- 55 Golub TR, Slonim DK, Tamayo P, Huard C, Gaasenbeek M, Mesi- strates. RNA 2000; 6: 136–158. rov JP, Coller H, Loh ML, Downing JR, Caligiuri MA, Bloomfield 32 Tan W, Zolotukhin AS, Bear J, Patenaude DJ, Felber BK. The CD, Lander ES. Molecular classification ofcancer: class discovery mRNA export in Caenorhabditis elegans is mediated by Ce-NXF- and class prediction by gene expression monitoring. Science 1, an ortholog ofhuman TAP/NXF and Saccharomyces cerevisiae 1999; 286: 531–537. Mex67p. RNA 2000; 6: 1762–1772. 56 Monica K, LeBrun DP, Dedera DA, Brown R, Cleary ML. Trans- 33 Pritchard CE, Fornerod M, Kasper LH, van Deursen JM. RAE1 is formation properties of the E2a-Pbx1 chimeric oncoprotein: fusion a shuttling mRNA export factor that binds to a GLEBS-like NUP98 with E2a is essential, but the Pbx1 homeodomain is dispensable. motifat the nuclear pore complex through multiple domains. J Mol Cell Biol 1994; 14: 8304–8314. Cell Biol 1999; 145: 237–254. 57 Hatano M, Roberts CW, Minden M, Crist WM, Korsmeyer SJ. 34 Joyce JA, Schofield PN. Genomic imprinting and cancer. Mol Deregulation ofa homeobox gene, HOX11, by the t(10;14) in T Pathol 1998; 51: 185–190. cell leukemia. Science 1991; 253: 79–82. 35 Ahuja HG, Felix CA, Aplan PD. Potential role for DNA topoiso- 58 Kamps MP, Murre C, Sun XH, Baltimore D. A new homeobox merase II poisons in the generation oft(11;20)(p15;q11) translo- gene contributes the DNA binding domain ofthe t(1;19) translo- cations. Genes Chromosomes Cancer 2000; 29: 96–105. cation protein in pre-B ALL. Cell 1990; 60: 547–555. 36 Kasper LH, Brindle PK, Schnabel CA, Pritchard CE, Cleary ML, 59 Huang SY, Tang JL, Liang YJ, Wang CH, Chen YC, Tien HF. Clini- van Deursen JM. CREB binding protein interacts with nucleoporin- cal, haematological and molecular studies in patients with chro- specific FG repeats that activate transcription and mediate mosome translocation t(7;11): a study offourChinese patients in NUP98-HOXA9 oncogenicity. Mol Cell Biol 1999; 19: 764–776. Taiwan. Br J Haematol 1997; 96: 682–687.

Leukemia Review DH Lam and PD Aplan 1695 60 Wong KF, So CC, Kwong YL. Chronic myelomonocytic leukemia 72 Hussey DJ, Albanese NO, Dobrovic A. Nuclear and cytoplasmic with t(7;11)(p15;p15) and NUP98/HOXA9 fusion. Cancer Genet localisation ofthe leukemia associated NUP98-RAP1GDS1 fusion Cytogenet 1999; 115: 70–72. protein. Blood 2000; 96 (Suppl. A): 85a. 61 Kwong YL, Pang A. Low frequency of rearrangements of the 73 von Lindern M, van Baal S, Wiegant J, Raap A, Hagemeijer A, homeobox gene HOXA9/t(7;11) in adult acute myeloid leukemia. Grosveld G. Can, a putative oncogene associated with myeloid Genes Chromosomes Cancer 1999; 25: 70–74. leukemogenesis, be activated by fusion of its 3Ј half to different 62 Nishiyama M, Arai Y, Tsunematsu Y, Kobayashi H, Asami K, Yabe genes: characterization ofthe set gene. Mol Cell Biol 1992; 12: M, Kato S, Oda M, Eguchi H, Ohki M, Kaneko Y. 11p15 translo- 3346–3355. cations involving the NUP98 gene in childhood therapy-related 74 Kraemer D, Wozniak RW, Blobel G, Radu A. The human CAN acute myeloid leukemia/myelodysplastic syndrome. Genes Chro- protein, a putative oncogene product associated with myeloid leu- mosomes Cancer 1999; 26: 215–220. kemogenesis, is a nuclear pore complex protein that faces the 63 Yamamoto K, Nakamura Y, Saito K, Furusawa S. Expression ofthe cytoplasm. Proc Natl Acad Sci USA 1994; 91: 1519–1523. NUP98/HOXA9 fusion transcript in the blast crisis of Philadelphia 75 Stark B, Jeison M, Shohat M, Goshen Y, Vogel R, Cohen IJ, Yaniv chromosome-positive chronic myelogenous leukaemia with I, Kaplinsky C, Zaizov R. Involvement of11p15 and 3q21q26 in t(7;11)(p15;p15). Br J Haematol 2000; 109: 423–426. therapy-related myeloid leukemia (t-ML) in children. Case reports 64 Ohnishi H, Taki T, Tabuchi K, Kobayashi M, Bessho F, Hayashi and review ofthe literature. Cancer Genet Cytogenet 1994; 75: Y. Down’s syndrome with myelodysplastic syndrome showing 11–22. t(7;11)(p13;p14). Am J Hematol 2000; 65: 62–65. 76 Zhou RH, Wang P, Zou Y, Jackson-Cook CK, Povirk LF. A precise 65 Shimada H, Arai Y, Sekiguchi S, Ishii T, Tanitsu S, Sasaki M. Gen- interchromosomal reciprocal exchange between hot spots for eration ofthe NUP98-HOXD13 fusiontranscript by a rare translo- cleavable complex formation by topoisomerase II in amsacrine- cation, t(2;11)(q31;p15), in a case ofinfantleukaemia. Br J treated Chinese hamster ovary cells. Cancer Res 1997; 57: Haematol 2000; 110: 210–213. 4699–4702. 66 Hatano Y, Miura I, Kume M, Miura AB. Translocation 77 Ripley LS. Deletion and duplication sequences induced in CHO (1;11)(q23;p15), a novel simple variant oftranslocation cells by teniposide (VM-26), a topoisomerase II targeting drug, can (7;11)(p15;p15), in a patient with AML (M2) accompanied by non- be explained by the processing ofDNA nicks produced by the Hodgkin lymphoma and gastric cancer. Cancer Genet Cytogenet drug-topoisomerase interaction. Mutat Res 1994; 312: 67–78. 2000; 117: 19–23. 78 Kroon E, Thorsteinsdottir U, Mayotte N, Nakamura T, Sauvageau 67 Ikeda T, Ikeda K, Sasaki K, Kawakami K, Takahara J. The G. NUP98-HOXA9 expression in hemopoietic stem cells induces inv(11)(p15q22) chromosome translocation oftherapy-related chronic and acute myeloid leukemias in mice. EMBO J 2001; 20: myelodysplasia with NUP98-DDX10 and DDX10-NUP98 fusion 350–361. transcripts. Int J Hematol 1999; 69: 160–164. 79 Pineault N, Buske C, Faulkes S, Helgason CD, Aplan PD, Humphr- 68 Nakao K, Nishino M, Takeuchi K, Iwata M, Kawano A, Arai Y, ies RK. The AML associated fusion gene NUP98-HOXD13 leads Ohki M. Fusion ofthe nucleoporin gene, NUP98, and the putative to increased myeloproliferation and decreased erythropoieisis in RNA helicase gene, DDX10, by inversion 11 (p15q22) chromo- differentiating embryonic stem cells. Blood 2000; 96: (Suppl. A): some translocation in a patient with etoposide-related myelodyspl- 458a. astic syndrome. Intern Med 2000; 39: 412–415. 80 Buske C, Pineault N, Feuring-Buske M, Aplan P, Humphries RK. 69 Singh DP, Kimura A, Chylack LT Jr, Shinohara T. Lens epithelium- Collaboration ofMEIS1 with the human leukemia-specific fusion derived growth factor (LEDGF/p75) and p52 are derived from a gene NUP98-HOXD13 causes acute myeloid leukemia (AML) in single gene by alternative splicing. Gene 2000; 242: 265–273. mice: a model ofNUP98-associated human leukemia. Blood 70 Ge H, Si Y, Roeder RG. Isolation ofcDNAs encoding novel tran- 2000; 96 (Suppl. A): 573a. scription coactivators p52 and p75 reveals an alternate regulatory 81 Fornerod M, Boer J, van Baal S, Jaegle M, von Lindern M, Murti mechanism oftranscriptional activation. EMBO J 1998; 17: KG, Davis D, Bonten J, Buijs A, Grosveld G. Relocation ofthe 6723–6729. carboxyterminal part ofCAN fromthe nuclear envelope to the 71 Mecucci C, La Starza R, Negrini M, Sabbioni S, Crescenzi B, Leoni nucleus as a result ofleukemia-specific chromosome rearrange- P, Di Raimondo F, Krampera M, Cimino G, Tafuri A, Cuneo A, ments. Oncogene 1995; 10: 1739–1748. Vitale A, Foa R. t(4;11)(q21;p15) translocation involving NUP98 82 Dobson CL, Warren AJ, Pannell R, Forster A, Rabbitts TH. Tumo- and RAP1GDS1 genes: characterization ofa new subset ofT acute rigenesis in mice with a fusion of the leukaemia oncogene Mll lymphoblastic leukaemia. Br J Haematol 2000; 109: 788–793. and the bacterial lacZ gene. EMBO J 2000; 19: 843–851.

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