Leukemia (2003) 17, 1858–1864 & 2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00 www.nature.com/leu Major form of NUP98/HOXC11 fusion in adult AML with t(11;12)(p15;q13) translocation exhibits aberrant trans-regulatory activity B-W Gu1, Q Wang1, J-M Wang2, Y-Q Xue3, J Fang1, KF Wong4, B Chen1, Z-Z Shi1, J-Y Shi1, X-T Bai1, D-H Wu1, Z Chen1 and S-J Chen1 1State Key Lab for Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital Affiliated to Shanghai Second Medical University, Shanghai, People’s Republic of China; 2ChangHai Hospital Affiliated to Second Military Medical University, Shanghai, People’s Republic of China; 3Leukemia Research Unit, JiangSu Institute of Hematology, First Hospital Affiliated to Suzhou University, Suzhou, People’s Republic of China; and 4Department of Pathology, Queen Elizabeth Hospital, Kowloon and HongKong SAR, People’s Republic of China

Three adult patients with de novo acute myeloid leukemia of (HD) with helix-turn-helix tertiary structure. A large body of distinct subtypes harboring t(11;12)(p15;q13) have been inves- evidence suggests that HOX family heterodimerize, tigated to characterize the involved in that translocation. Through molecular cytogenetics, a break was through HD, with other HD proteins to bind sequence-specific detected at the 30 part of nucleoporin 98 (NUP98) at 11p15. DNA, resulting in activation or repression of target genes. This Using rapid amplification of cDNA end, we identified the partner phenomenon is consistent with the fact that different organs gene at 12q13, HOXC11. Molecular analysis showed that exon display a specific combination of expression.20–23 12 of NUP98 was fused in-frame to exon 2 of HOXC11 in all three In the present work, we are able to show that the NUP98- cases with t(11;12)(p15;q13). Therefore, this type of fusion may HOXC11 chimeric gene is a recurrent genetic event in acute represent the major form of the NUP98-HOXC11 chimera so far reported. Moreover, two out of three cases had a confirmed myeloid leukemia(AML). More importantly, we identified the deletion of the 30 part of NUP98 gene and more telomeric region fusion between exon 12 of NUP98 and exon 2 of HOXC11 to be of 11p harboring a group of tumor-suppressor genes. Interest- the major form of this gene rearrangement and revealed its ingly, the NUP98-HOXC11 when assayed in a GAL4 aberrant trans-regulatory activity. reporter system, showed an aberrant trans-regulatory activity as compared to the wild-type HOXC11 in both COS-7 and HL-60 cells. Therefore, NUP98-HOXC11 may contribute to the leuke- Materials and methods mogenesis by interfering with the cellular mechanism of transcriptional regulation. Leukemia (2003) 17, 1858–1864. doi:10.1038/sj.leu.2403036 Case history Keywords: NUP98-HOXC11; t(11; 12)(p15; q13); acute myeloid leukemia(AML); trans-regulation Three patients with AML and t(11;12)(p15;q13) chromosomal translocation were selected, and their clinical, hematological and cytogenetic data are summarized in Table 1. In fact, the clinical Introduction data of case 3 were previously reported, but recently reanalyzed according to the WHO classification of leukemia.24,25 Specific chromosomal translocations in hematological malig- nancies, especially those with resultant chimeric genes, are of Cytogenetic analysis great significance for understanding not only pathogenesis of leukemia but also physiological functions of the genes involved. were R-banded on unstimulated bone marrow Nucleoporin 98 (NUP98) is a component of the nuclear pore (BM) cells after 24 h culture. Karyotypes were classified complex (NPC), which conducts RNA and protein traffic according to International System for Cytogenetic between the nucleus and cytoplasm. NUP98 contains a domain Nomenclature. Chromosome painting (CP) was performed by with multiple FG repeats (including FG, FXFG or GLFG), using whole CP probes for (digoxigenin- providing docking sites for nuclear transport, an important labeled, Oncor, USA) and chromosome 11(biotin-labeled, physiological process for cell life.1,2 However, NUP98 calls Cambio, UK). For fluorescence in situ hybridization (FISH) universal attention for its potential in leukemogenesis as a analysis, several BAC/PAC clones (Invitrogen, Carlsbad, CA, promiscuous gene fusing with a number of genomic sites.3 To USA), were selected, labeled with biotin or digoxigenin (Roche, date, at least 14 partner genes have been reported being Indianapolis, USA) and applied to the disrupted region, involved in translocations with NUP98 as the common target. including BAC RP11-496I9 (AC021663), BAC RP11-532E4 They are HOXA9, DDX10, HOXD13, PMX1, RAP1GDS1, (AC061979), PAC PDJ 1075f20 (AC 002536), BAC RP11- TOP1, LEDGF, NSD1, HOXD11, HOXA11, NSD3 , HOXA13, 113A6 (AC 026645), BAC RP11-555F1 (AC16765), BAC HOXC11 and HOXC13.4–17 It has been well established that the RP11-120E20 (AC060812) at 11p15 from telomere to centro- genes, encoding an evolutionarily well-conserved mere, and BAC RP11-972K6 (AC073594) at 12q13. All family of transcription factors, are vital in organogenesis as well procedures were performed according to the manufacturer’s as tumorigenesis.18,19 As a representative type of homeobox recommendations. genes, HOX gene family members share the homeodomain

Correspondence: Dr S-J Chen, Shanghai Institute of Hematology, RNA isolation Ruijin Hospital, Shanghai Second Medical University (SSMU), 197 Rui Jin Road II, Shanghai 200025, People’s Republic of China; Fax: +86 21 6474 3206 Mononuclear cells (MNC) were obtained from BM samples of The first four authors contributed equally to this work patients by Ficoll density centrifugation, and were frozen and Received 2 December 2002; accepted 24 April 2003 stored in liquid nitrogen. Total RNA was extracted from the NUP98/HOXC11 fusion in AML B-W Gu et al 1859 stored MNC samples using TRIzol Reagent (Invitrogen, Carls- gene) coding sequences were obtained by RT-PCR on RNA bad, CA, USA) RNA was quantitated, aliquoted and stored at samples extracted from leukemia cells of patients and Hela cell À701C for further use. line, respectively. The wild-type NUP98 cDNA was kindly provided by Dr van Deursen JM (Department of Pediatrics and 0 0 Analysis of genomic DNA fragments spanning Adolescent Medicine, Rochester, USA). The 5 and 3 primers NUP98-HOXC11 breakpoints used for PCR contained unique BamH1 and Not1 restriction sites to facilitate cloning into the pBIND plasmid (Promega) High molecular weight genomic DNA was extracted from inframe with sequences encoding the GAL4 DNA-binding frozen leukemia cells. Genomic DNA (10 mg) was digested, domain. respectively, with BamHI and BglII restriction enzymes, separated by 0.7% agarose gel electrophoresis, transferred onto a Hybond-XL membrane (Amersham-Pharmacia, Uppsala, Cell culture and transient transfection assay Sweden). Hybridization with a-32P-dCTP-labeled probe was performed at 681C in ExpressHyb Hybridization Solution COS-7 cells were grown in Dulbecco’s modified Eagle’s (Clontech). Autoradiography was performed using Molecular medium supplemented with 10% fetal bovine serum (FBS), Imager FX System (Bio-Rad). A cDNA fragment covering exons while HL-60 cells were cultured in RPMI-1640 medium with 11–14 of NUP98 gene was used as a probe to detect NUP98 10% FBS. For transient transfection, the cells were placed in six- rearrangement. well plates for 24 h and were then transfected with 1 mg of each pBIND fusion plasmid, 1 mg5Â GAL4-pG5-luc (Promega) plasmid, and 0.1 mg pRL-SV40 plasmid (Promega) using Super- cDNA cloning of the NUP98 fusion partner gene Fect transfection reagent (QIAGEN, German) according to the manufacturer’s recommendations. Cells were harvested 36 h A30-rapid amplification of cDNA end (RACE) was performed after transfection. Luciferase activity was measured in a using the 30-RACE system (Invitrogen) with NUP98 gene specific luminometer with Dual-Luciferase Reporter Assay System primers N-1 (50-GTGCACCTTCATTTGGTACAACC-30) and N-2 (Promega) according to the manufacturer’s protocol. Each (50-CTCTTGGTGCTGGACAGGCATC-30) and adaptor primers transfection was performed in duplicate and repeated three AP (50-GGCCACGCGTCGACTAGTAC(T) -30) and UAP (50- 17 times. Reporter gene-derived firefly luciferase activity was then (CUA) GGCCACGCGTCGACTAGTAC-30). The 30-RACE pro- 4 normalized to Renilla luciferase derived from pRL-SV40. ducts were separated by electrophoresis and subcloned into the pGEMT-easy vector (Promega, Madison, WI, USA). The plasmid clones were sequenced using a Sequence dye terminator Results cycle sequencing kit (Perkin-Elmer, Foster City, CA, USA). Region containing NUP98 gene was disrupted by Reverse transcription-polymerase chain reaction t(11;12)(p15;q13) (RT-PCR) The results of the cytogenetic analysis of R-banding on BM Reverse transcription was performed on 1 mg of total RNA. After cells from three cases revealed a t(11;12)(p15;q13) (Table 1, a 10 min at 251C, RNA was incubated for 50 min at 451C with representative karyotype of case 1 being shown in Figure 1a). In 50 U of Superscript II Reverse transcriptase (Invitrogen), 20 U of case 1, CP analysis further confirmed the translocation between RNase inhibitor (Invitrogen), 2.5 mM random hexamers and chromosomes 11 and 12 (Figure 1b). Although FISH analysis dNTP at 0.5 mM in a final volume of 20 ml. Then, the reverse with BAC RP11-120E20 containing the NUP98 gene showed transcriptase was denatured by heating for 5 min at 851C. A two pairs of signals, the intensity of the signals on normal volume of 2 ml of the reverse transcription product was used for chromosome 11 being much stronger than those of the der(11) PCR. PCR amplification was carried out for 35 cycles (Figure 1c). On the other hand, FISH analysis with clones at (denaturation at 941C for 25 s, annealing at 571C for 45 s and distal region of chromosome 11, such as BAC RP11-496I9, BAC elongation at 721C for 60 s), followed by predenaturation at RP11-532E4, BAC RP11-113A6, BAC RP11-555F1 and PAC PDJ 941C for 5 min and elongation at 721C for 10 min) using a 1075f20, showed only one pair of signals (Figure 1d), in contrast GeneAmp PCR system 9600 (Perkin-Elmer). For NUP98- to two pairs in normal control (Figure 1e). We speculated two 0 HOXC11 transcript, the primers were N-1 and H-1 (5 - interrelated events in this case: a translocation t(11;12)(p15;q13) 0 TTACAGCAGAGGATTTCCCG-3 ). For the reciprocal HOXC11- disrupting the NUP98 gene and a deletion of distal region 0 NUP98 transcript, the primers were N-3 (5 -CTAGGGATGGTT downstream of the chromosomal break on der(11). The 0 0 CATCGTC-3 ) and H-2 (5 -ATGGATCCATGTTTAACTCGGTC molecular cytogenetic analysis of case 2 was consistent with 0 AACCT-3 ). The PCR products were separated by electrophor- that of case 1 (data not shown). esis and sequenced. To confirm the accuracy of the RT-PCR results, Southern analysis of the PCR products was performed using the oligonucleotide probes P1 (corresponding to the Genomic structure of the t(11;12)(p15;q13) joining of NUP98 exon 12 to HOXC11 exon 2) and P2 translocation breakpoints (corresponding to the theoretical joining of HOXC11 exon 1 32 to NUP98 exon 13) labeled with g- P-ATP using T4 poly- To confirm that NUP98 was directly involved in the nucleotide kinase. t(11;12)(p15;q13), Southern analysis was conducted on NUP98 gene. As shown on Figure 2, a cDNA probe encom- Construction of expression plasmids passing exons 11–14 revealed a rearranged band in BglII- digested DNA from case 1, whereas no abnormal bands were cDNAs containing full-length NUP98-HOXC11, wild-type detected in DNA analyzed by BamHI. Restriction mapping thus HOXC11 and HOXC11-HD (only containing HD of HOXC11 located the breakpoint to intron 12 of the NUP98 (Figure 2a).

Leukemia NUP98/HOXC11 fusion in AML B-W Gu et al 1860 It was reasoned that if this rearrangement resulted from a gene fusion, then exon 12 of NUP98 should be joined to the partner gene. CR

Cloning of fusion transcripts in t(11;12)(p15;q13) leukemia patients

Next, a 30-RACE was performed to determine the partner gene cytarabine fused to NUP98 gene, most likely on chromosome 12q13. A RACE product of about 400 bp was identified. Sequence analysis revealed that the 50 portion of RACE product was a part of the cDNA sequence of NUP98 gene, while the 30 portion was a part of the cDNA sequence of HOXC11 gene on chromosome 12q13 (Figure 3a). Importantly, the fusion was an in-frame one between exon 12 of the NUP98 gene and exon 2 of HOXC11 NegativePositive NA Daunorubicin, NA Positive THP, Ara-C Died gene (Figure 3a). Next, a BAC clone RP11-972K6 corresponding to the HOXC11 displayed fused signal to that of NUP98 in FISH analysis (Figure 1f). Subsequently, using appropriate primers, we amplified the whole NUP98-HOXC11 fusion transcript, which includes the N-terminal FG domain of the NUP98 and the C-terminal HD of HOXC11 gene (Figure 3b).

Examination of NUP98-HOXC11 fusion gene in patients with t(11;12) Hypercellular marrow with increased blast cells (67%) Hypercellular marrow with increased blast cells (32%), megakaryocytes and megaloblastic erythropoiesis Hypercellular marrow with increased blast cells (73%) To examine if NUP98-HOXC11 transcripts existed in other two

/L, patients (cases 2 and 3) with t(11;12)(p15;q13), RT-PCR was /L, 9 /L, 9 9 carried out to amplify the fusion region, which was then subject 10 10 10 Â

 to Southern analysis with specific oligonucleotide probes. The  result showed that these two patients, like case 1, also had NUP98-HOXC11 transcripts and the fusion occurred exactly /L, B 16%, /L, B 31%,

/L, N 16%, between NUP98 exon 12 and HOXC11 exon 2 (Figure 3c). Of 9 9 9 10 10 note, no reciprocal HOXC11-NUP98 transcript could be 10 Â Â

 obtained among all three patients in this series (Figure 3c). N 34%, L 42%, M 8% N 35%, L 27%, M 3% L 70.5%, M 13.5% Plt 277 Plt 5.0 Plt 262 N-terminal of NUP98 modulates the trans-regulatory activity of HOXC11

Since HOXC11 is an important , its fusion to NUP98 may change the biological functions in terms of the regulation of the downstream genes. Since the target genes physiologically regulated by HOXC11 have not yet been reported, we were not able to construct a reporter system with HOXC11 binding sites. In addition, a reporter construct with PBX1-HOXA9 binding motifs showed no response to HOXC11 in transient transfection assay (data not shown). Hence, an artificially constructed system used by several groups in previous studies on NUP98-related fusion genes was also used in the present work to address the trans-regulatory activities of NUP98-HOXC11. We therefore prepared a series of expression vectors in which the GAL4 DNA-binding domain was in-frame fused to NUP98-HOXC11, NUP98, HOXC11 and HOXC11- HD, and then tested the trans-regulatory effect of these constructs on luciferase-based GAL4 reporter system in COS-7 cells and the myeloid cell line HL-60, respectively. Similar to the previous report of the HOXA9 and PMX1, wild-type HOXC11 construct exhibited strong trans-repressing activity in 7,37 Clinical data of three cases with t(11;12)(p15;q13) GAL4 reporter system. Apparently, this trans-repressive activity mainly resulted from the C-terminal homeodomain of HOXC11, according to the data of HOXC11-HD. However, NUP98-HOXC11 behaved more likely as a trans-activator, since Table 1 Case no.1 Age/sex Diagnosis Karotype 22 years/F M2a 46,XX,t(11;12)(p15;q13)[10]/46,XX[3] Hb 86 g/L, WBC 3.9 Peripheral blood examinations Bone marrow examinations Myeloperoxidase Treatment Follow-up 3 41 years/FNA: not available; Hb: hemoglobin; Plt: M2 platelet; B: blast cells; N: neutrophils; L: lymphocytes; M: monocytes. 46,XX,t(11;12)(p15;q13)[20] Hb 102 g/L, WBC 5.7 2 40 years/M M5b 46,XY,t(11;12)(p15;q13)[9]/46,XY[11] Hb 96 g/L, WBC 211 it drove higher reporter than the control (Figure

Leukemia NUP98/HOXC11 fusion in AML B-W Gu et al 1861

Figure 1 Conventional and molecular cytogenetic analysis of case 1. (a) Representative karyotype showing t(11;12)(p15;q13). (b) CP analysis of the metaphase. The red arrow indicates der(11) and the green arrow indicates der(12). The chromosomes displaying only red color and green color (yellow arrows) are normal chromosomes 11 and 12, respectively. (c) FISH signals of BAC RP11-120E20 containing NUP98 gene were observed on normal chromosome 11 and der(11). Note the intensity of the signals on normal chromosome 11(opened arrow) was much stronger than that on der(11) (filled arrow). (d) FISH analysis with PAC1075f20 located downstream of chromosome 11 breakpoint showed signals only on normal chromosome 11 but not on der(11). (e) FISH analysis with the same probe as in (d) showed signals on both normal chromosome 11 in control sample. (f) FISH analysis using probes containing both NUP98 (green) and HOXC11 (red) showed fusion signals (arrows) in interphases in case 1.

4a and b). That NUP98-HOXC11 could deviate the trans- some 12 was located within the 30 end of the exon 1 of repressing activity of HOXC11 was further confirmed by the HOXC11.16 In that particular case, to salvage the otherwise cotransfection assays of both plasmids (Figure 4a). Of note, the disrupted ORF, a complex mechanism was involved where a wild-type NUP98, when fused with GAL4 DNA-binding stretch of DNA sequence from intron 12 of NUP98, immediately domain, showed a strong trans-activating property. It is possible upstream of the truncated exon 1 of HOXC11, was spliced into that the altered trans-regulatory activity of fusion protein be the fusion transcript. caused by the addition of N-terminal portion of NUP98. It is well known that in leukemia, reciprocal or balanced translocations account for most of the chromosomal transloca- tions, while nonreciprocal or unbalanced ones are rare. Of Discussion particular note is that the distal portion of the breakpoints on chromosome 11p in two cases (cases 1 and 2) here studied was In this work, we identified, through positional candidate deleted at molecular cytogenetic level. The absence of the strategy, the NUP98-HOXC11 fusion as a result of the HOXC11-NUP98 fusion transcript in all three patients further t(11;12)(p15;q13) in three adult AML patients, confirming that supports this observation. Therefore, the t(11;12)(p15;q13) in this molecular abnormality is a recurrent genetic event in human most patients should be an unbalanced translocation. It is leukemogenesis. However, the phenotypes of these three cases interesting to point out that the lost part in our cases is an belonged to two quite distinct subtypes of AML, two with AML- important region of tumor-suppressor gene (TSG). At least six M2 and one with AML-M5, implying that NUP98-HOXC11 TSGs have been found at 11p15.5, namely TSSC1–6. FISH fusion could be a relatively late event in the leukemogenic analysis with BAC RP11-555F1, containing TSSC3, revealed process. Of note, the chimeric protein in our three cases all only one pair of signals in case 1, on the contrary to two pairs of resulted from a fusion between exon 12 of NUP98 and exon 2 of signals in normal control, confirming a loss of this TSG. The fact HOXC11, contrarily to a very recently reported pediatric AML that a recently reported NUP98-HOXC13 fusion gene cloned patient with NUP98-HOXC11 in whom the break on chromo- from a patient with t(11;12)(p15;q13) was also accompanied by

Leukemia NUP98/HOXC11 fusion in AML B-W Gu et al 1862

Figure 3 (a) Nucleotide and protein sequences surrounding the NUP98-HOXC11 fusion region. Note the fusion was in-frame, so that the open reading frames of both genes in the fusion transcript were retained. The arrows indicate the breakpoint and fusion sites of the NUP98-HOXC11 gene. The underlined sequences indicate the location of the oligonucleotide probe P1. (b) Schematic representation Figure 2 (a) Upper part: restriction map of the breakpoint region of of the NUP98-HOXC11 fusion proteins and its wild-type counterparts. NUP98 gene in t(11;12)(p15;q13). The vertical arrows indicate the The locations of the primers used for RT-PCR are indicated by genomic breakpoints in t(11;12). Bg and Ba indicate the BglII and horizontal arrows. HD denotes the homeodomain, RD the RNA- BamHI restriction sites, respectively. Lower part: organization of binding domain, and arrows the joining site. (c) Examination of HOXC11 gene and location of the chromosomal breakpoint. (b) NUP98-HOXC11(left panel) and HOXC11-NUP98 (right panel) fusion Southern blot analysis of the leukemia cells from case 1. A rearranged transcripts by RT-PCR in three patients with t(11;12)(p15;q13). The band of about 4.5 kb was detected by BglII digestion using a NUP98 PCR products were transferred to nylon membranes by Southern cDNA probe (see part A). C1 and N denote case 1 patient and a blotting and hybridized with a g-32P -ATP-labeled probe P1 (open normal control sample, respectively. arrow indicating the specific signal of hybridization). 1, 2 and 3: results of cases 1, 2 and 3, respectively; 4: a negative control; M: size marker. a deletion of the reciprocal fusion gene,17 suggests that this concomitant event may be the basis for future work in translocation involving NUP98 gene. The situation is also reminiscent of another nonreciprocal translocation found in differentiation, suggesting a lineage determination function of alveolar soft part sarcoma, a rare solid tumor, with these genes. The interesting point for HOX genes to be involved der(17)t(X;17)(p11.2;q25) and the resultant ASPL-CTFE3 fusion in oncogenesis is that the molecular mechanisms could be either gene.26,27 Of note, a TSG was lost at 17q25.3-qter. Interest- deregulation of structurally normal protein or formation of ingly, a balanced t(X;17)(p11.2;q25) with ASPL-CTFE3 fusion chimeric genes.33 For example, altered expression of these gene was found in papillary renal adenocarcinomas.28,29 It is genes was demonstrated in the involvement of leukemogenesis postulated that the selective pressure for an unbalanced and some HOXC genes are involved in lymphomagenesis.34 translocation might be a result of loss of those tumor Moreover, overexpression of HOXC11 was reported previously suppressors. We therefore hypothesize that the deletion of the in aplasia/hypoplasia of the fibula and severe malformations of telomeric region on chromosome 11p accompanying the appendicular skeleton in mice.35 On the other hand, the t(11;12)(p15;q13) may also contribute to the pathogenesis in fusion of HOX gene to the NUP98 represents a distinct those leukemia patients. mechanism in disturbing the cellular program of transcription. The discovery of HOXC11 as a novel biological factor to be In NUP98-HOXA9 fusion, the prototype fusion protein invol- reckoned with in leukemogenesis further strengthens the ving a component of NPC and a HOX family member, the trans- concept that abnormal transcriptional regulation plays a key repression activity of HOXA9 was modified and the fusion role in malignant transformation. It has been shown that most protein showed some trans-activating effect over target genes in HOX gene family members including HOXC11 are transcription some experimental settings.36 Here we show that, as in the case repressors.30–32 Expression of several HOX genes (located on of the NUP98-HOXA9 fusion, linking NUP98 to HOXC11 HOXA and HOXB loci) varies with hematopoietic stem cell changes HOXC11 into an activator. It was suggested that

Leukemia NUP98/HOXC11 fusion in AML B-W Gu et al 1863 SIH. We thank all members of the SIH and SKLMG for their support.

References

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(1) Control of pG5- 1264–1267. luc plasmid; (2) pBIND; (3) pBIND-NUP98; (4) pBIND-HOXC11; (5) 12 Taketani T, Taki T, Shibuya N, Ito E, Kitazawa J, Terui K et al. The pBIND-HOXC11-HD and (6) pBIND-NUP98-HOXC11. These data HOXD11 gene is fused to the NUP98 gene in acute myeloid represent the mean7SD from three independent experiments. leukemia with t(2;11)(q31;p15). Cancer Res 2002; 62: 33–37. 13 Suzuki A, Ito Y, Sashida G, Honda S, Katagiri T, Fujino T et al. t(7;11)(p15;p15) Chronic myeloid leukaemia developed into blastic transformation showing a novel NUP98/HOXA11 fusion. NUP98 is able to bind some components of nuclear Br J Haematol 2002; 116: 170–172. 37 coactivator (CoA) complex, such as CBP. The fusion of NUP98 14 Rosati R, La Starza R, Veronese A, Aventin A, Schwienbacher C, to HOX family member therefore provides the access to the CoA Vallespi T et al. NUP98 is fused to the NSD3 gene in acute activity. 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