NUP98–HMGB3: a novel oncogenic fusion

NUP98 is a promiscuous gene involved in chromosomal aberrations with more than 20 different partner genes in a variety of human hematological malignancies.1, 2 These rearrangements all lead to the expression of hybrid proteins that start with the amino-terminal moiety of NUP98. This amino-terminal domain contains multiple copies of a glycine-leucine-phenylalanine-glycine core motif, known to recruit the CBP/p300 acetyltransferases.3 Evidence from overexpressions studies in mouse bone marrow (BM) progenitors indicates that the NUP98-fusion proteins induce leukemic transformation through the upregulation of Hoxa genes and the Hox cofactor Meis1. Expression of Hoxa, most frequently Hoxa7, Hoxa9 and Hoxa10 is postulated to induce a self-renewal stem cell-like program that likely contributes to the leukemic process.4, 5

High mobility group (HMG) proteins are non-histone chromatin-associated proteins that bind to DNA with limited or no sequence specificity. Among them, HMGB proteins are important architectural facilitators of nucleosome remodelling and possibly of transcription factor's interaction with DNA (for a review, see Travers6). The expression of the murine Hmgb3 gene is tightly regulated during hematopoiesis and is required in early steps of hematopoietic stem cell development in which it regulates cell-fate decisions. It is expressed in common myeloid and lymphoid progenitors (CMP and CLP) and except in the erythroid cells, it must be downregulated for the proper differentiation of both lineages to take place.7, 8 Hmgb3 RNA has recently been found to be part of an embryonic stem cell-like transcription signature that was defined in mouse models of mixed-lineage leukemia (MLL)-mediated leukemic transformation and was also reported to be transiently upregulated during myeloid differentiation.9

We earlier reported the involvement of NUP98 in t(X;11)(q28;p15) in a 73-year-old woman with therapy-related acute myeloblastic leukemia (AML) with M4 subtype.1 Fluorescence in situ hybridization experiments performed on metaphasic chromosome of the blast cells permitted the mapping of the translocation breakpoint on Xq28 in which only the HMGB3 gene had the correct transcription orientation (telomere to centromere) that would allow an in-frame fusion to NUP98 (assuming a simple translocation event). Reverse transcriptase-PCR using primers located within NUP98 and HMGB3 exons showed the presence of NUP98–HMGB3 fusion transcript from patient's material and not from control complementary DNA (cDNA) (Figure 1a). Nucleotide sequence analyses of the fragment revealed an in-frame fusion of the exon 11 of NUP98 to the exon 2 of HMGB3 (Figure 1b). No reciprocal HMGB3–NUP98 transcript could be detected (not show). The NUP98–HMGB3 predicted protein is composed of the first 422 amino acids of NUP98 fused to the entire HMGB3 coding sequence, (Figure 1b). Indeed, in the normal HMGB3 transcript, the first coding ATG codon is located in exon 2 after a short untranslated sequence. In the fusion transcript, these nucleotides accommodate a continuous reading frame from exon 11 of NUP98 through HMGB3.

Figure 1
figure1

Molecular breakpoint analysis of the t(X;11)(q28;p15) and bone marrow transplants (BMTs) using NUP98–HMGB3-transduced hematopoietic progenitors. (a) A specific NUP98–HMGB3 product is detected by reverse transcriptase-PCR (RT-PCR) in the patient's sample at diagnosis. PCR with primers located in NUP98 exon 8 (5′-TTGGCCAACAGAATCAGCAGAC-3′) and HMGB3 exon 5 (5′-CCGGGCAACTTTAGCAGGAC-3′) yielded a 983-bp product. Control lane corresponds to the complementary DNA (cDNA) of the human malignant cell line MO7E. (b) Partial nucleotide sequence of the NUP98–HMGB3 chimeric transcript shows that nucleotide 1609 (end of exon 11) of the NUP98 gene and nucleotide 88 of HMGB3 (that is, the start of HMGB3 exon 2) are joined in frame. Schematic representation of the native and chimeric proteins, showing the glycine-leucine-phenylalanine-glycine (GLFG) repeats and RNA binding domain of NUP98 and the two high mobility group (HMG) boxes of HMGB3. (c) Kaplan–Meier survival plot of NUP98–HMGB3 recipients together with control (MSCV) mice. Primary recipients (NUP98–HMGB3 IR, n=6; MSCV, n=3) were obtained by the injection of 5 × 105 transduced Lin– cells into the retroorbital vein of sublethally irradiated mice. Animals were killed because of signs of disease between day 54 and 141 post-transplant. Secondary recipients (NUP98–HMGB3 IIR, n=4) were obtained by the engraftment of 106 bone marrow (BM) cells from primary mice (killed at day 54 post-transplant) into sublethally irradiated animals; they all died between day 68 and 87 post-BMT. (d) Hematological parameters of primary and secondary recipients. *P<0.05 by Mann–Whitney test. (e) Cytological analysis of peripheral blood (PB) and BM cells, evaluated on May–Grünwald–Giemsa staining of smears and cytospin preparations respectively showed an over-representation of mature myeloid cells in the two tissues, associated with the disappearance of the erythroid and megakaryocytic compartments in the BM for primary NUP98–HMGB3 recipient mice compared with MSCV-transduced animals.

To establish the transforming properties of the NUP98–HMGB3 fusion, murine primary BM hematopoietic progenitors were transduced with a retroviral vector murine stem cell virus (MSCV) co-expressing the NUP98–HMGB3 fusion cDNA and the green fluorescent protein (GFP), a MSCV–NUP98–HOXA9 used as a reference or the empty MSCV alone as negative control. Viral supernatants were obtained as described.10 Transduced cells were split in two and one-half was seeded in methylcellulose medium for serial replating assays. We observed that NUP98–HMGB3-transduced progenitors formed moderate numbers of colonies (90% GFP+) until but not beyond the third round of replating (not shown). Cells transduced with NUP98–HOXA9 grew beyond five replating, whereas for empty vector-transduced cells, no colony was observed after the second replating. This indicates that NUP98–HMGB3 is a weak oncogene in vitro. The other half of NUP98–HMGB3-transduced cells was engrafted into sublethally irradiated mice. Mice transplanted with progenitors transduced with the empty MSCV (n=3) remained free of hematological disease up to 12 months after transplantation. Mice engrafted with progenitors transduced with the NUP98–HOXA9 fusion developed a myeloproliferative disease (MPD) that progressed to AML with long latency (n=2; median 250 days) consistent with previous reports11 (not shown). Mice that received NUP98–HMGB3-transduced cells (n=6) rapidly died with a median survival of 112 days (range, 60–140 days) (Figure 1c). They developed hyperleucocytosis, anemia with mucous paleness and dyspnea, thrombopenia and splenomegaly (Figure 1d). Femurs of NUP98–HMGB3 mice were conspicuously discolored reflecting a block in terminal erythropoiesis, as confirmed by fluorescence-activated cell sorting (FACS) quantitative analyses performed on BM erythroid progenitors (not shown). BM smears from femurs showed cytological abnormalities that affected immature and mature myeloid cells, suggesting a MPD-like leukemia according to the Bethesda classification12 (Figure 1e). In agreement, when compared with control mice, FACS analysis of NUP98–HMGB3 hematopoietic organs showed an increased number of GFP+Gr1+CD11b+ mature myeloid cells into the peripheral blood (PB), spleen and BM of sick mice (Figure 2a).

Figure 2
figure2

Cytometric analyses of the degree of hematopoietic proliferation induced by NUP98–HMGB3-fusion and quantification of Hoxa genes expression. (a) Fluorescence-activated cell sorting (FACS) analysis of the bone marrow (BM), spleen and peripheral blood (PB) showed an enrichment for mature Gr-1+CD11b+ myeloid cells in primary NUP98–HMGB3-transduced mice, compared with mice engrafted with MSCV-transduced progenitors. (b) FACS analysis of the LinSca-1+c-Kit+ (LSK) and myeloid progenitor (MP) populations in the BM. MPs from primary NUP98–HMGB3 recipients are enriched in the granulocyte-macrophage progenitor (GMP) subset whereas common myeloid progenitors (CMPs) and megakaryocyte-erythroid progenitors (MEPs) are virtually absent. (c) Real-time reverse transcriptase-PCR (RT-PCR) analysis of endogenous Hoxa (Hoxa5, Hoxa7, Hoxa9, Hoxa10), Meis1, Pbx1 and Pbx3 genes expression in engrafted mice. Accumulation of transcript was quantified in primary (NUP98–HMGB3 IR n=3) and secondary (NUP98–HMGB3 IIR n=3) recipients, compared with NUP98–HOXA9 recipients. Levels of expression are standardized to Abl and expressed relative to the expression levels measured in MSCV-engrafted mice. Values shown are mean±s.d. from two independent experiments.

BM progenitor populations were analyzed by FACS (Figure 2b). The LinSca1c-Kit+CD34+FcγRII/IIIhigh subset, corresponding to the granulocyte-macrophage progenitors,13 was markedly expanded in the BM of NUP98–HMGB3 mice compared with control mice. In contrast, the common myeloid progenitors (CMP) and megakaryocyte-erythroid progenitors (MEPs) populations were virtually absent (Figure 2b) indicating that NUP98–HMGB3 expression is associated with preferential expansion of the myelo-monocytic lineages, as observed in several mouse models of MLL-induced leukemias.14 The malignant nature of NUP98–HMGB3-induced hemopathy was further established by engraftment of primary MPD cells into secondary recipients. All engrafted mice developed an MPD-like leukemia of similar immunophenotype (not shown) with shorter latency than the primary disease (median survival time of 80 days) (Figure 1c). Taken together, these results show that NUP98–HMGB3 acts as an oncogene responsible for a rapid and transplantable MPD-like leukemia in recipient mice, which is associated with defects in the differentiation of myelo-monocytic cells.

Mouse models developed to analyze the oncogenic activity of MLL and NUP98 fusions showed the overexpression of Hoxa9 and Meis1 in blast cells;4, 5, 15, 16, 17, 18 in addition, Hoxa9 and Meis1 overexpression was associated with the emergence but not the maintenance of MLL leukemic stem cells.9 We thus measured the expression levels of genes of the Hoxa cluster as well as the genes encoding for the HOX cofactors Meis1, Pbx1 and Pbx3 in BM cells from primary and secondary NUP98–HMGB3 recipients. We compared these values to NUP98–HOXA9 leukemic mice. Relative to NUP98–HOXA9, NUP98–HMGB3 BM cells showed much weaker expression (approximately 10- to 90-fold) of Hoxa5, Hoxa7 and Hoxa9 (Figure 2c). In contrast, roughly similar transcription of Hoxa10 was observed in all samples, whereas Meis1 expression was detected only in NUP98–HOXA9 leukemic cells and Pbx3 transcripts levels were similarly upregulated in both leukemia models compared with control. As NUP98–HMGB3-expressing blast cells upregulate Hoxa9 only weakly and retain wild-type levels of Meis1, we infer that transformation mediated by the fusion does not involve deregulated activity of the canonical Hoxa–Meis1 pathway. These results suggest that several transformation pathways might be involved in the leukemogenic properties of NUP98 fusions. This would be consistent with recent work in mice. Indeed, co-expression of the Hmgb3 Myb, and Cbx5 genes is sufficient to induce HoxA/Meis1-independent immortalization of mouse myeloid progenitors, pointing to a critical role for these genes in the leukemic process.9 Thus, we suggest that the ectopic expression of HMGB3, as a result of the fusion with NUP98, can bypass the requirement of a concomitant Hoxa9 and Meis1 misregulation in human AML. To our knowledge, this is the first report of a genetic alteration of an HMGB gene in human hematological malignancies. It is also the first example of a NUP98 leukemogenic fusion whose expression is not associated with a strong Hoxa and concomitant Meis1 expression. Together, the data indicate that further investigations of HMGB3-related leukemia may facilitate the analysis of conserved ‘terminal’ mechanisms of AML uncoupled from upstream events.

Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

We thank Julie Bergeron for help in analyses of PB and BM smears. AP acknowledges the support from the Fondation pour la Recherche Medicale (FRM; Paris, France) and Association pour la recherche sur le Cancer (ARC; Villejuif, France). This work was funded by grants from ARC and Institut National du Cancer (INCa).

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Correspondence to V Penard-Lacronique or S P Romana.

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Petit, A., Ragu, C., Della-Valle, V. et al. NUP98–HMGB3: a novel oncogenic fusion. Leukemia 24, 654–658 (2010). https://doi.org/10.1038/leu.2009.241

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