Human Cancer Biology

NUP98 Is Fused to (DNA) IIB 18 0 kDa (TOP2B)ina Patient with Acute Myeloid Leukemia with a New t(3;11)(p24;p15) Karin Nebral,1Helmut H. Schmidt,2 Oskar A. Haas,1and Sabine Strehl1

Abstract Purpose: The nucleoporin 98 kDa (NUP98) has been reported to be fused to 17 different partner in various hematologic malignancies with11p15aberrations. Cytogenetic analysis of an adult de novo acute myelogenous leukemia (M5a) revealed a t(3;11)(p24;p15), suggesting rearrangement of NUP98 with a novel partner gene. Experimental Design: Fluorescence in situ hybridization (FISH) was used to confirm the involvement of NUP98 in the t(3;11)(p24;p15). Selection of possibleNUP98 partner genes was done by computer-aided analysis of the 3p24 region using the University of California Santa Cruz genome browser. Fusion gene ^ specific FISH and reverse -PCR analyses were done to verify the presence of the new NUP98 fusion. Results: FISH analysis using a NUP98 -specific clone showed a split signal, indicating that the NUP98 gene was affected by the translocation. Of the genes localized at 3p24, TOP2B was selected as a possible fusion partner candidate gene. Dual-color fusion gene ^ specific FISH and reverse transcription-PCR analysis verified that NUP98 was indeed fused to TOP2B.Inaddition to reciprocal NUP98-TOP2B and TOP2B-NUP98 in-frame fusion transcripts, an alternatively spliced out-of-frame TOP2B-NUP98 transcript that resulted in a premature stop codon was detected. Analysis of the genomic breakpoints revealed typical signs of nonhomologous end joining resulting from error-prone DNA repair. Conclusions: TOP2B encodes a type II topoisomerase, which is involved in DNA transcription, replication, recombination, and mitosis, and besides TOP1, represents the second NUP98 fusion partner gene that belongs to the topoisomerase gene family.This finding emphasizes the important role of in malignant transformation processes.

One of the most promiscuous fusion partner genes involved in (3–5). NUP98 consists of two NH2-terminal glycine-leucine- hematopoietic malignancy–associated chromosomal rear- phenylalanine-glycine (GLFG) repeat domains (usually called rangements is the recently identified NUP98 gene at 11p15, FG repeats) that flank a GLEBS-like motif, which is a RAE1 bind- which to date has been discovered to fuse to 17 different ing site, and a COOH-terminal ribonucleoprotein-binding partners (1). NUP98 fusions occur in de novo and therapy- motif (6). related myeloid as well as lymphoid malignancies (1, 2). The most frequently observed fusion partners of NUP98 NUP98 encodes a 98 kDa protein that is an important belong to the homeobox family of transcription factors and component of the nuclear pore complex, which mediates include the class I homeobox genes HOXA9, HOXA11, nucleocytoplasmic transport of proteins and RNA. It resides HOXA13, HOXC11, HOXC13, HOXD11, and HOXD13,as predominantly on the nucleoplasmic side of the nuclear well as the nonclustered class II homeobox genes PRRX1/PMX1 pore complex, but was also found at the cytoplasmic face as and PRRX2 (1, 7). The HOX family genes contain a conserved well as dispersed within the nucleus, moving between these helix-turn-helix motif, the homeodomain, which is fused to the different localizations in a transcription-dependent manner NH2-terminal FG-rich region of NUP98 in all NUP98-HOX chimeric proteins reported thus far (1). NUP98-HOX fusions may deregulate expression of HOX target genes by transcrip- tional transactivation via NUP98 GLFG repeat–mediated Authors’ Affiliations: 1Children’s Cancer Research Institute, Vienna, Austria and 2Division of Hematology, Department of Internal Medicine, University Medical recruitment of the CBP/p300 complex, and thus might Center, Graz, Austria influence HOX gene–regulated hematopoiesis (8, 9). The Received 1/20/05; revised 5/17/05; accepted 6/20/05. non-HOX NUP98 fusion partners compose a heterogeneous Grant support: ‘‘O« sterreichische Kinderkrebshilfe’’ and the research program group of genes that are associated with a wide range of ‘‘Genome Research for Health’’of the Austrian Ministry of Education, Science, and biological functions. In contrast to the HOX fusion partner Culture (GEN-AU Child, GZ 200.071/3-VI/2a/2002). The costs of publication of this article were defrayed in part by the payment of page genes, they have not been recognized to play any specific or charges. This article must therefore be hereby marked advertisement in accordance unique role in hematopoiesis. These fusion partners include with 18 U.S.C. Section 1734 solely to indicate this fact. DDX10, RAP1GDS1, TOP1, PSIP2/LEDGF, NSD1, WHSC1L1/ Requests for reprints: Sabine Strehl, Children’s Cancer Research Institute, NSD3, FN1, and ADD3 (1, 2, 10). An intriguing feature of Kinderspitalgasse 6, A-1090 Vienna, Austria. Phone: 43-1-40170-449; Fax: 43-1- 40170-437; E-mail: [email protected]. all non-HOX partners is their propensity to adopt a coiled-coil F 2005 American Association for Cancer Research. conformation (1). Most of the non-HOX NUP98 fusion doi:10.1158/1078-0432.CCR-05-0150 partners are either transcriptional coactivators or factors that

www.aacrjournals.org 6489 Clin Cancer Res 2005;11(18) September 15, 2005 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Human Cancer Biology indirectly contribute to transcriptional control. Thus, oligomer- which spans the TOP2B gene. Probes were differentially labeled by nick ization via the coiled-coil domain and/or the transactivation translation: the NUP98 probe with digoxigenin-11-dUTP and the potential of the NUP98 GLFG repeats might confer aberrant TOP2B clone with biotin-16-dUTP (Roche Diagnostics, Vienna, transcriptional properties to the NUP98 fusions. This hypoth- Austria). Slides for fluorescence in situ hybridization (FISH) were esis is supported by the nuclear localization of the NUP98 prepared from the methanol/acetic acid–fixed cell suspension used for cytogenetic analysis and FISH was done as previously described (14). fusion proteins and oligonucleotide array expression analysis All samples were evaluated using an Axioplan fluorescence microscope (9, 11). Furthermore, the NUP98 fusions might contribute to (Zeiss, Vienna, Austria) equipped with the appropriate filter sets for leukemogenesis by triggering aberrant nucleocytoplasmic trans- FITC, Cy3, and 4V,6-diamidino-2-phenylindole, and images were taken port (8). with a CCD camera (Photometrix, Tucson, AZ) using the IPLab software In this study, we describe the identification of the novel (Vysis, Inc., Stuttgart, Germany). NUP98 partner gene TOP2B in a case of an adult de novo acute Reverse transcription-PCR analysis. Total RNA was extracted from myelogenous leukemia with a t(3;11)(p24;p15). TOP2B is the peripheral blood cells obtained at diagnosis of the patient and from second topoisomerase that fuses to NUP98, and with all non- peripheral blood cells of a healthy individual using the Qiagen RNeasy HOX partners shares the high propensity to adopt a coiled-coil Mini Kit (Qiagen, Inc., Vienna, Austria) according to the recommen- conformation. dations of the manufacturer. RNA was reverse transcribed using ran- dom hexamers and 200 units of Moloney murine leukemia virus re- verse transcriptase (Invitrogen, Lofer, Austria) at 42jC for 60 minutes. Materials and Methods The NUP98-TOP2B fusion transcript was detected using NUP98ex12 and TOP2Bex28 primers, and the reciprocal TOP2B-NUP98 using Case history. A 63-year-old male suffered from thrombocytopenia TOP2Bex24 and NUP98ex14 primers (Table 1). Expression of the nor- of unclear genesis and continuous difficulty swallowing. After biopsy of mal TOP2B allele was detected using TOP2Bex24 and TOP2Bex28 the pharynx, immunohistochemistry revealed an extramedullary primers. All reverse transcription-PCR reactions were done using Hot myeloid cell infiltrate with a French-American-British acute myeloge- Start Taq Polymerase (Qiagen) and an initial activation step at 95jC nous leukemia M5a phenotype. The blast cells were negative for the for 14 minutes. Cycling conditions for all reverse transcription-PCR B-cell markers CD20 and CD21, immunoglobulin n and E, as well as reactions were as follows: denaturation at 95jC for 30 seconds, anneal- for the T-cell markers CD45RO and CD3, and CD15, CD30, and S-100; ing at 63jC for 30 seconds, and elongation at 72jC for 60 seconds but positive for CD43 and CD68. Bone marrow and peripheral blood carried out for 40 cycles, followed by a final extension at 72jC for showed 80% and 99% blast cells, respectively, positive for the myeloid 7 minutes. Sequencing of the PCR products was done by MWG Biotech markers HLA-DR, CD13, and CD33, as well as CD68. Cytogenetic (Ebersberg, Germany) and VBC-Genomics (Vienna, Austria). analysis revealed a 46,XY,t(3;11)(p24;p15) karyotype. Induction Long-range PCR. The genomic breakpoints of the der(11) and therapy with Ara-C (Cytosar-U), amsacrine, and thioguanine failed to der(3) were identified by long-range PCR of genomic significantly reduce the tumor load. After additional high-dose DNA using primers NUP98ex13 and TOP2Bex26, and TOP2Bin25-5 chemotherapy with Ara-C and novantrone, the patient achieved and NUP98in13-4, respectively (extension at 68jC for 5 minutes). complete histologic remission but relapsed 15 months after diagnosis Amplification of NUP98 and TOP2B normal alleles was done using and showed a 90% blast cell infiltration in the bone marrow. The blast NUP98in13 and NUP98in13-4, and TOP2Bin25-5 and TOP2Bin25-4 cells were resistant to Ara-C and topotecan (Hycamtin) and the patient primers, respectively (Table 1). died 20 months after initial diagnosis. Conventional and molecular cytogenetics. Cytogenetic analysis was done on GTG-banded metaphases and karyotypes were described Results and Discussion according to the International System for Human Cytogenetic Nomenclature (ISCN, 1995; ref. 12). Detection of the NUP98-TOP2B Cytogenetic analysis revealed a t(3;11)(p24;p15) transloca- rearrangement was done using PAC 1173K1 (13) in combination with tion in a patient with acute myelogenous leukemia M5a. the TOP2B-specific RP11-659P16 clone (obtained from Dr. Mariano Subsequent FISH analysis with the NUP98-specific clone Rocchi, Department of Cytogenetics, University of Bari, Bari, Italy) 1173K1 showed a split signal, suggesting that NUP98 was

Table 1. Oligonucleotide primer sequences

Primer Orientation Oligonucleotide sequence (5V-3V) Gene/exon-intron* Nucleotides*

NUP98 ex12 Sense GACAGCCACTTTGGGCTTTG NUP98 /exon12 1,544-1,563 NUP98 ex14 Antisense TGACTTGGCTGTGCCTGTTG NUP98 /exon14 1,844-1,825 TOP2B ex28 Antisense TGAGGGCATTGTCTCTTCCAA TOP2B/exon 28 3,708-3,688 TOP2B ex24 Sense AGGAGTGGCTTGTGGGAATG TOP2B/exon 24 3,164-3,183 NUP98 ex13 Sense TTCTCCAGCAGCACATCAATAG NUP98 /exon13 1,624-1,645 TOP2B ex26 Antisense CCTGAATCGGAGGAACTATCATC TOP2B/exon 26 3,407-3,385 TOP2Bin25-5 Sense GTTGCCCATTGCAGATCAC TOP2B/intron 25 562-580 NUP98 in13-4 Antisense TGCCTTGCACATATTCAGCTAC NUP98 /intron13 1,305-1,284 NUP98 in13 Sense TCTGTGAGACATTGGAAAATGAG NUP98 /intron 13 627-649 TOP2Bin25-4 Antisense GCGCCCATCTTGTCCTACC TOP2B/intron 25 1,060-1,042

NOTE: Gene IDs ENSG00000110713(NUP98) and ENSG00000077097 (TOP2B ) for intronic sequences; nucleotide positions relative to the respective introns. Tran- script IDs ENST00000324932 (NUP98) and ENST00000264331 (TOP2B ) for nucleotide positions of cDNA sequences. *Nucleotide positions and exon nomenclature according to the Ensembl genome browser of the Sanger Institute (http://www.ensembl.org/).

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caused by topoisomerase II as described by Ahuja et al. (20) in two cases with NUP98-TOP1 and t-MDS. Of note, our patient had primary leukemia and did not receive any topoisomerase II poisons. DNA topoisomerase II (TOP2) is a nuclear phosphoprotein that controls and alters DNA topology. TOP2 is involved in condensation, chromatid separation, and in the relief of DNA torsional stress that occurs during transcription and replication (21). More specifically, TOP2 catalyzes the transient breakage and the subsequent rejoining of double- strand DNA and acts as an ATP-driven clamp that captures one DNA segment and transports it through the -bridged break in the second DNA duplex (22–24). Two closely related isoforms, TOP2A (a; 170 kDa) and TOP2B (h; 180 kDa), have Fig. 1. FISH analysis of the patient using clones 1173K1specific for NUP98 (green) been identified in human cells (25). Although sharing about and RP11-659P16 encompassing the whole TOP2B gene (red). Metaphase (A) 70% amino acid identity, TOP2A and TOP2B are encoded and interphase (B) nuclei displaying two fusion signals (A, arrows). by separate genes on 17q21 and 3p24, respectively, which are likely to have evolved through a gene duplication event disrupted as a result of the translocation (15). To identify (25–27). Although the catalytical mechanisms of TOP2A and candidate partner genes, computer-aided analysis of the 3p24 TOP2B seem to be the same, considerable evidence suggests region using the University of California Santa Cruz genome that the physiologic functions of the two isoforms differ from 3 browser was done and this analysis determined that roughly each other. In this regard, TOP2A and TOP2B are differentially 35 reference sequence derived genes are localized at 3p24. Of expressed throughout the cell cycle, vary in their predominant these genes, TOP2B seemed to be an excellent candidate gene because TOP1 has already been identified as a NUP98 fusion partner (16). Dual-color FISH analysis with the NUP98 clone and a TOP2B-specific BAC resulted in two fusion signals in metaphase and interphase cells, providing compelling evidence that NUP98 was indeed fused to TOP2B (Fig. 1). Fusion gene– specific reverse transcription-PCR experiments using primers localized in exon 12 and exon 28 of NUP98 and TOP2B, respectively, led to the identification of an in-frame fusion of NUP98 exon 13 to TOP2B exon 26 (Fig. 2A and B). Analysis of the reciprocal TOP2B-NUP98 transcript identified two chimeric mRNA species fusing TOP2B exon 25 in-frame to NUP98 exon 14, and an alternatively spliced smaller transcript that fused TOP2B exon 24 out-of-frame to NUP98 exon 14, thereby generating a premature stop codon (Fig. 2A and B). According to the FISH data, the second alleles of both genes involved in the translocation were retained and expression of normal TOP2B (Fig. 2A) and NUP98 (data not shown) transcripts was verified by reverse transcription-PCR. Analysis of the genomic breakpoints of the der(11) and der(3) chromosomes and comparison with the sequences of the corresponding non-rearranged alleles revealed a 2-bp deletion in NUP98 (GA) and a 4-bp microduplication of a short TOP2B sequence (AGTA) that was present on both derivative chromosomes (Fig. 3). The simplest interpretation of such a configuration is two double-strand DNA breaks within NUP98 (nt 1197 and 1199 of intron 13) and a 4-bp staggered break within TOP2B (nt 683-687 of intron 25), followed by nonhomologous end joining and gap repair. As previously shown, such deletions and duplications of base pairs are typical Fig. 2. A, reverse transcription-PCR analysis of the NUP98/TOP2B and the signs of nonhomologous end joining resulting from an error- reciprocalTOP2B/NUP98 fusion transcripts (left), and of the normal TOP2B allele (right). M, molecular weight marker,100 bp ladder; lanes1and 4, patient; lanes 2 and prone DNA repair mechanism (17, 18). The genomic break- 5, normal peripheral blood; lanes 3 and 6, negative control (same order on both points described herein seem to differ from those detected for gels). B, partial nucleotide and amino acid sequences of the NUP98/TOP2B and NUP98-TOP1 (19, 20). The fine structure of the breakpoints is TOP2B/NUP98 in-frame fusions. C, schematic representation of NUP98 and TOP2B wild-type proteins, the putative chimeric NUP98/TOP2B andTOP2B/ also not compatible with the typical 4-bp staggered breaks NUP98a proteins, and theTOP2B/NUP98b truncated protein. ATPase, ATPase domain; C-Term, COOH-terminal domain; DNA Breakage, DNA breakage-rejoining domain; GLEBS, GLEBS-like motif; GLFG, glycine-leucine-phenylalanine-glycine repeats; NES, functional nuclear export signal; NLS, functional nuclear localization 3 http://genome.ucsc.edu/. signal; RNP, ribonucleoprotein-binding domain.

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Fig. 3. Genomic sequences of germ line chromosomes 11(NUP98)and3(TOP2B ), and derivative chromosomes 11and 3. All sequences are shown in 5V-3Vorientation with respect to the NUP98 and TOP2B coding sequences. Nucleotides at the breakpoint junctions that are present on both derivative chromosomes are in boldface, and the 2-bp deletion in the NUP98 sequence is boxed.The breakpoints in NUP98 occurred at nucleotides 1,197 and 1,199 of intron 13, generating a deletion of nucleotides GA, and those inTOP2B at nucleotides 683 and 687 of intron 25. subcellular distribution, and the tissue-specific expression contributes to oncogenic transformation, and if the putative pattern differs during fetal development and in adult verte- reciprocal TOP2B-NUP98 and/or the truncated TOP2B isoform brates (28–33). plays an essential role in this process. In a significant number of Several lines of evidence suggest that topoisomerase expres- cases with NUP98 fusions, the reciprocal partner-NUP98 sion is associated with resistance to anticancer drugs in that transcript is absent, which indicates that the NUP98-partner topoisomerase-targeting drugs stabilize the TOP2B enzyme/ fusion protein is critical for the leukemic transformation DNA complex, resulting in the accumulation of DNA lesions process (1, 2). and cell death via apoptosis (34). Chemotherapy resistance The unifying feature of all NUP98 fusion proteins is that might thus be related to altered or reduced topoisomerase the NH2-terminal FG repeats, which have a strong transcrip- expression. However, in particular in leukemia, no obvious tional transactivation potential through direct interaction with correlation between the levels of TOP2A and/or TOP2B mRNA the CBP/p300, are always fused to different partner proteins and protein expression and chemotherapy failure could be (1). In particular, malignant transformation induced by determined (34). expression of NUP98-HOX chimeric proteins seems to be Protein analyses revealed that type II DNA topoisomerases caused by aberrant transcriptional regulation of target genes exist as homodimers, with each subunit consisting of three (8, 11, 38). In contrast, the oncogenic mechanism of NUP98 functional domains: an NH2-terminal ATPase domain fusions that involve non-HOX partner genes seems to differ (encoded by TOP2B exons 1-11), a central DNA breakage- from that described for HOX partners because most of these rejoining domain (TOP2B exons 12-28), which contains a fusion partners have neither direct DNA binding properties nucleotide-binding motif and the catalytic tyrosine (TOP2B nor are they implicated in hematopoietic development. All exon 20), and a relatively poorly conserved COOH-terminal proteins encoded by non-HOX NUP98 fusion partners domain (TOP2B exons 29-36; refs. 22, 35). The COOH termini described to date contain regions with a significant probabil- of the two TOP2 isoforms seem to be important for subcellular ity to adopt a coiled-coil conformation. These domains, localization, and functional bipartite nuclear localization signal which are present in f3% to 5% of all human proteins sequences as well as nuclear export signals are localized in these and are thought to be involved in homo- and heterodime- domains (36, 37). rization (39), are retained in the chimeric proteins and fused The NUP98-TOP2B fusion transcript fuses the NH2-terminal to the transactivating FG repeat–rich NH2 terminus of FG repeat and GLEBS motifs of NUP98 with the COOH- NUP98 (1, 40). Protein analysis with the COILS 2.2 (39)4 terminal domain of TOP2B, thereby retaining the functional and the MULTICOIL (41)5 programs revealed this intriguing nuclear localization signal but eliminating the nuclear export feature also in the COOH-terminal region of TOP2B between signals (Fig. 2C). Consequently, the putative reciprocal TOP2B- amino acids 1,150 to 1,175 (P = 1.0) and 1,148 to 1,174 (P = NUP98 chimeric protein retains the ATPase, the DNA breakage- 0.55), respectively. rejoining, and the nuclear export signal domains of TOP2B that Regarding the other NUP98 topoisomerase fusion partner, are fused to the ribonucleoprotein-binding and nuclear TOP1, recent findings have shown that engineered expression of localization signal domains of NUP98 (Fig. 2C). The shorter NUP98-TOP1 results in rapid perturbations of hematopoiesis out-of-frame TOP2Bexon24-NUP98exon14 fusion transcript (9). Strikingly, these perturbations seemed to be independent might encode a truncated TOP2B isoform that consists of the ATPase, the DNA breakage-rejoining, and nuclear export signal domains fused to 18 fusion partner unrelated amino acids 4 http://www.ch.embnet.org/software/COILS___form.html. (Fig. 2C). It is unclear in which way the NUP98-TOP2B protein 5 http://multicoil.lcs.mit.edu/cgi-bin/multicoil.

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of TOP1 catalytic activity because a mutation known to lized in the nucleus. Whether disturbances of the nucleocyto- abrogate TOP1 DNA isomerase activity essentially did not alter plasmic transport are caused by NUP98 fusions has not been the NUP98-TOP1 proliferative and oncogenic potential. These investigated to date. Intriguingly, recent investigations of a data suggest that leukemogenic transformation does not occur nucleoporin NUP214 rearrangement suggest that the SET- through up-regulated TOP1 DNA unwinding activity. In NUP214 fusion protein, which also resides in the nucleus, leads addition, no genome instability was observed, and thus to disorganization of nuclear export by causing aberrant NUP98-TOP1 expression does not seem to interfere with localization of CRM1 (XPO1, exportin 1; ref. 44). In this normal TOP1 activity. Therefore, the transcriptional coactivator context, in vitro studies of NUP98 showed interactions with property of TOP1 provides a possible functional similarity to various export factors such as RaeI/Gle2, Mex67p/Mtr2p, TAP, HOX genes in that the NUP98-TOP1 chimeric protein acts as an and also CRM1 (1). Thus, it cannot be entirely ruled out that at aberrant transcription factor (9). However, for TOP2B,no least some of the NUP98 chimeric proteins interfere with involvement in transcriptional regulation has been determined. appropriate nucleocytoplasmic shuttling. In addition, TOP1 belongs to the type IB subfamily of In conclusion, the identification of TOP2B as the second topoisomerases I that share no sequence or structural homology NUP98 topoisomerase fusion partner confirms and extends the with other known topoisomerases, and are thus functionally previously recognized important role of topoisomerases in distinct (42). Moreover, the NUP98-TOP1 and the NUP98- malignant transformation processes as well as chemotherapy TOP2B fusion proteins differ significantly from each other in resistance mechanisms. In line with the vast majority of NUP98 that virtually all of the TOP1 proteins (amino acids 170-514) gene rearrangements, the fusion with TOP2B was detected in an containing the core, the linker, and the COOH-terminal acute leukemia derived from the myeloid lineage. Further domains but only the TOP2B COOH terminus, respectively, extensive functional analyses of NUP98 fusion proteins are are fused to the FG repeat motif of NUP98. needed to elucidate the complex pathways of NUP98-associat- Interestingly, complete disruption of the nucleoplasmically ed leukemogenesis. oriented NUP98 protein triggers dramatic changes at the cytoplasmic face of the nuclear pore complex and causes Acknowledgments distinct protein import pathways to operate with reduced efficiency (43). However, the NUP98 chimeric proteins We thank Rolf Marschalek for his expertise in the analysis of the genomic break- analyzed (HOXA9, PMX1, RAP1GDS1, and TOP1) were loca- points of the translocation.

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Karin Nebral, Helmut H. Schmidt, Oskar A. Haas, et al.

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