A new translocation t(9;11)(q34;p15) fuses NUP98 to a novel homeobox partner gene, PRRX2, in a therapy-related acute myeloid leukemia

TO THE EDITOR

Acute myelogenous leukemias (AML) are usually associated with recurrent chromosomal rearrangements, which play an important role in diagnosis and prognosis. During the last few years, the nucleoporin gene NUP98 in chromosome band 11p15.5 revealed being implicated in several recurrent chromosomal translocations in AML, myelodysplastic syndrome (MDS), Philadelphia-positive chronic myeloid leukemia (CML) and acute lymphoid leukemia (ALL) in children or adults.1 NUP98 gene encodes a 98 kDa protein component (920 amino acid) of the nuclear pore complex, which appears to function as a docking protein owing to its N-terminal part.2 This protein is involved in mRNA and protein nucleocytoplasmic bidirectional transport, and may play a role in transcription. Since the first t(7;11)(p15;p15) reported in 1996, fusing NUP98 to HOXA9 in myeloid leukemia,3 14 other fusion partners (HOXA11, HOXA13, HOXD11, HOXD13, PMX1, HOXC11, HOXC13, RAP1GDS1, NSD1, NSD3, DDX10, TOP1, LEDGF and ADD3) were described. All these translocations fused the 5′ part of NUP98 to the 3′ part of the partner gene, giving a fusion protein that juxtaposes the N-terminal FG (Phe-Gly) repeats of NUP98, the presumed contact sites for soluble nucleocytoplasmic transport factors, to the C-terminus of the partner gene. Previous reports suggest that patients with NUP98 rearrangement seem to have aggressive course and poor outcome, so it is important to recognize patients with such abnormalities. We identified a novel partner gene for NUP98, which has, to our knowledge, never been involved in malignancies. The patient, a 65-year-old Caucasian female, developed a diffuse large B-cell lymphoma in October 1999, treated with chemotherapy (including alkylants and topoisomerase II inhibitors) and autologous bone marrow transplantation. In February 2004, she developed an acute myelomonocytic leukemia, supposed to be therapy-related. Peripheral blood counts showed hemoglobin 11.4 g/dl, platelets 82.109/l and white blood cells 32.109/l with 2% myelocytes and 4% blasts. Bone marrow was hypercellular, with dysgranulopoiesis and dyserythropoiesis, and contained 24% of blasts positive for myeloperoxidase. In all, 15% of cells were positive for butyrate esterase. The karyotype, obtained using RHG (Reverse Heat Giemsa) banding techniques, showed 46,XX,der(9)(q34) der(11)(p15)[21] on blood cells and 46,XX,der(9)(q34), der(11)(p15)[8]/46,XX[15] on bone marrow cells (Figure 1a), according to the International System for Human Cytogenetic Nomenclature. In front of these 9q and 11p abnormalities, FISH analysis was performed. Whole chromosome 9 and 11 painting probes (Q-Biogen, Illkirch, France) confirmed the t(9;11)(q34;p15). Multi-FISH analysis confirmed the precedent result, without any additional abnormality. The short arm of chromosome 11 carries several tumor suppressor genes (responsible for Beckwith–Wiedmann syndrome, Wilms' tumor, adrenocortical carcinoma, and lung, ovarian and breast cancer), and also contains NUP98 gene at 11p15.5. Therefore, BAC RP11-120E20 and PAC 1173K1 (M Rocchi, Bari, Italy), covering the NUP98 gene, were used and revealed three spots on normal 11, der(11) and der(9) chromosomes, confirming the breakpoint on 11p15.5. BAC RP11-438N5 (11p15.4) (M Rocchi) was used as a control. In association with the centromeric 9 probe (Q-Biogen), they proved that a part of NUP98 signal was on chromosome 9. One ABL signal (9q34.1, LSI BCR/ABL ES dual color translocation probes, Vysis, Downers Grove, IL, USA) was translocated on chromosome der(11), but the BAC RP11-474P12 (M Rocchi) stayed on 9q33.3, indicating that the breakpoint on chromosome 9 was telomeric to 9q33.3 and centromeric to ABL, included in a 6 Mb region (Figure 1b).

Figure 1
figure1

(a) RHG karyotype. 46,XX,t(9;11)(q34;p15). (b) FISH using BAC RP11-474P12 (9q33) and PAC 1173K1 (NUP98). BAC RP11-474P12 (green) localized on normal chromosome 9 and der(9); PAC 1173K1 (red) localized on normal chromosome 11 and splitted on chromosome der(9) and der(11).

As translocations involving NUP98 always end up at fusion transcript and no partner gene on 9q34 was described previously, we used 3′RACE (Rapid Amplification of cDNA Ends) to identify a potential fusion transcript. Total RNA was reverse transcribed using the adaptator oligo-dT primer 5′-IndexTermGAC CAC GCG TAT CGA TGT CGA CTT TTT TTT TTT TTT TTV-3′ (5′/3′ RACE kit, Roche, Basel, Switzerland), and cDNA was then amplified using the adaptator primer 5′-IndexTermGAC CAC GCG TAT CGA TGT CGA C-3′ (5′/3′ RACE kit, Roche) and an NUP98 gene-specific forward primer 5′-IndexTermCTG CTC CTA CTG GGA CTA CTA TT-3′ (nt 640–662, NM_016320). Electrophoresis of the 3′RACE PCR product showed a band of approximately 1600 bp, which was subcloned. Colonies with recombinant plasmid containing the PCR products were selected for sequencing, using primer T3 (5′-IndexTermAAT TAA CCC TCA CTA AAG GG-3′) and T7 (5′-IndexTermGTA ATA CGA CTC ACT ATA GGG C-3′). The nucleotide (nt) sequence of the 3′RACE clone diverged from the germline NUP98 sequence at nucleotide 1444. A BLAST search (National Center for Biotechnology Information) allowed us to discover a new partner gene for NUP98, PRRX2, a class II homeobox gene localized in 9q34.1, as it was suggested by FISH analysis. The NUP98 exon 11 (nt 1443, NM_016320) was fused in frame to the PRRX2 exon 2 (nt 487, NM_016307) (Figure 2a). The predicted chimeric protein would contain the N-terminus of NUP98, including the FG repeats, fused to the part of PRRX2 containing the homeodomain (Figure 2b). To confirm this result, reverse transcriptase-PCR was performed. For NUP98-PRRX2 transcript, NUP98 sense primer 5′-IndexTermGGA GTA ACC CAA GCC TCA CAG C-3′ (nt 1134–1155) and PRRX2 antisense primer 5′-IndexTermCTT TCA TTC CTG CGG AAC TT-3′ (nt 718–699, NM_016307) were used. For PRRX2-NUP98 fusion transcript, PRRX2 sense primer 5′-IndexTermCTC CTG GAC CTG GAA GAG GT-3′ (nt 354–373) and NUP98 antisense primer 5′-IndexTermGAC ATC GGA TTC CGG AAG AG-3′ (nt 1696–1677) were used. RT-PCR allowed to detect the 542 bp NUP98-PRRX2 fusion transcript at the expected size, but not the 386 bp PRRX2-NUP98 reciprocal fusion transcript (Figure 2c), as it was frequently observed in translocation involving NUP98. The absence of the reciprocal PRRX2-NUP98 fusion transcript indicates that NUP98-PRRX2 is the leukemia-associated transcript.

Figure 2
figure2

(a) NUP98-PRRX2 fusion cDNA sequence. NUP98 breakpoint localized in exon 11 (nt 1443, NM_016320). PRRX2 breakpoint localized at exon 2 (nt 487, NM_016307). The fusion sequence has been deposited in the Genbank database with the Genbank Accession number AY662674. (b) Structure of the wild-type proteins and predicted chimeric protein. The predicted chimeric protein contains the N-terminus of NUP98, including the FG repeats (docking site) fused to the part of PRRX2 containing the homeodomain. FG, Phe-Gly repeats; GLEBS, RAE1 binding domain; HD, homeodomain. (c) RT-PCR NUP98-PRRX2 and PRRX2-NUP98. Detection of NUP98-PRRX2 fusion transcript at the expected size (542 bp), but not PRRX2-NUP98. SF, Small fragment molecular weight marker.

This is the first time, to our knowledge, NUP98 is fused to PRRX2 by a t(9;11)(q34;p15), and even PRRX2 is involved in malignancy. NUP98 gene has been found at breakpoints of several distinct recurrent translocations in patients with both de novo and therapy-related MDS/AML and T-ALL. The report from the international workshop of Chicago in 2002 considered 11p15 rearrangements as rare (3.3%) recurring balanced chromosome abnormality in therapy-related leukemia, suggesting that genotoxic chemotherapeutic agents may play an important role in generating chromosomal rearrangements involving NUP98.

There are two categories of NUP98 fusion partners: the homeobox genes in about half cases (Table 1) and the nonhomeobox genes. The known nonhomeobox genes such as DDX10, RAP1GDS1, LEDGF, NSD1 and ADD3 all seem to code for proteins that share regions with a significant probability of adopting a coiled-coil conformation that may have a role in the formation of multimeric complexes that facilitate interaction with other transcription factors or cofactors. Homeobox genes are a class of transcription factors characterized by the presence of a conserved DNA-binding homeodomain, and distributed in two main groups: the class I, organized in four clusters: A (7p14–15), B (17q21.3), C (12q13.3), D (2q31.1); and the class II, scattering throughout the genome, including PRRX1 and PRRX2. NUP98-HOX fusion deregulates expression of Hox-responsive genes by the chimeric Hox protein, in which NUP98 replaces the transcriptional activity of the Hox N-terminal region. The Hox partner gene has an intrinsic leukemogenic potential responsible for the ability of Hox genes to induce leukemia as NUP98 fusion partners. The fusion protein may inhibit HOX-mediated terminal differentiation and/or cause aberrant nucleocytoplasmic transport. The mechanism by which the NUP98-HOXA9 fusion protein causes AML has been studied by expressing NUP98-HOXA9 in myeloid cells, and by identifying their target genes using microarray analysis.4 Expression of 102 genes were modified, suggesting an aberrant transcriptional function. Those genes are frequently implicated in increased cell proliferation and survival, suggesting a possible leukemogenic pathway. They are also implicated in drug metabolism, explaining association with etoposide treatment and its incidence in Asian population.

Table 1 NUP98 Homeobox partner genes

PRRX2 (PMX2) and PRRX1 (PMX1) code for DNA-associated proteins, which are members of the paired family of homeobox proteins and are expressed during embryogenesis. In 1999, PRRX1 appeared to be involved in translocation t(1;11)(q23;p15) in AML,5 which fused in frame PRRX1 and NUP98, conserving the N-terminal docking region of NUP98 and the homeodomain of PRRX1. The chimeric protein was under the control of NUP98 promoter, upregulating the homeodomain expression and altering the transcriptional activity of PRRX1. The authors suggested that the result may be a deregulation of target genes in bone marrow cells. The fusion protein may act as an oncogenic transcriptor factor, and the same hypothesis may be carried out for NUP98-PRRX2. PRRX2 was firstly described in human in 1998, while Stelnicki et al searched for transcriptional regulatory genes. They identified and cloned a partial cDNA designed PRRX2 because of its overall identity of 74% to murine prrx2. Norris et al6 cloned the entire gene and characterized the expression of human PRRX2 in embryonic and adult tissues by RT-PCR. In mice, prrx2 and prrx1 are expressed during embryogenesis, predominantly in mesenchyme-specific patterns. The prrx2 null mice are morphologically normal, in contrast with prrx1 null mice, but prrx1−/− prrx2−/− double mutant mice showed many novel abnormalities in addition to an aggravation of the prrx1 single mutant phenotype. prrx1 and prrx2 control cell proliferation during mandibular arch morphogenesis by indirect upstream regulation on sonic hedgehog (Shh).7 In prrx1−/− prrx2−/− mutant mice, reduction of Shh expression leads to reduction of mesenchymal proliferation in the medial–oral region and causes malformation of the mandibular arch. Shh belongs to the Shh–patched–gli pathway, which plays an important role in the development of many tissues and organs during embryogenesis. Dysregulation of this pathway leads to several human diseases, including birth defects and solid cancers.8 In muscle, skin or nervous system tumors, mutations in Shh signaling pathway appear to result in the activation of this pathway and thus, in increased expression of the transcription factor Gli1. Shh has never been described to be involved in hematological malignancies, but is known to act as negative regulators of thymocyte development, and to induce the proliferation of primitive human hematopoietic cells. A possible interaction between NUP98-PRRX2 fusion protein and Shh deserves to be studied.

We found a new translocation t(9;11)(q34;p15) in a t-AML, fusing NUP98 with the class II homeobox gene PRRX2. As its homologue PRRX1, PRRX2 is a DNA-binding transcription factor essential for fetal development. Further studies are needed to confirm the recurrence of the t(9;11)(q34;p15) in t-AML/MDS or even in de novo AML, and to define its prognosis. Transformation assays in cell lines and transgenic mice studies would be interesting to understand the leukemogenicity of the NUP98-PRRX2 fusion transcript. More generally, there is probably some more NUP98 partner genes that have not been already described, involved in small or cryptic translocations occurring in the 11p15 band.

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Acknowledgements

We thank Seiamak BAHRAM for reading the manuscript, and Mariano ROCCHI for providing BACs and PACs.

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Correspondence to M Lessard.

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Gervais, C., Mauvieux, L., Perrusson, N. et al. A new translocation t(9;11)(q34;p15) fuses NUP98 to a novel homeobox partner gene, PRRX2, in a therapy-related acute myeloid leukemia. Leukemia 19, 145–148 (2005). https://doi.org/10.1038/sj.leu.2403565

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