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Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver V5Z 1L3,1 Department of Medicine, University of British Columbia, Vancouver V5Z 4E3, British Columbia, Canada2
Received 21 August 2003/ Returned for modification 22 October 2003/ Accepted 20 November 2003
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ABSTRACT |
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INTRODUCTION |
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A central role for Hox genes in hematological malignancies is supported by the frequently observed elevation of Hox gene expression in acute myeloid leukemia (AML) (11, 14, 20) and the common involvement of their upstream regulator, MLL, in both myeloid and lymphoid leukemia (8). A more direct involvement for Hox genes in leukemia is supported by their frequent fusion to the nucleoporin gene NUP98. Hox genes are the most frequent fusion partner in an ever-growing number of NUP98 fusion genes isolated almost exclusively in patients with myeloid leukemia (AML, posttherapy AML, and chronic myeloid leukemia). Reported Hox partners of NUP98 include HOXA9 (3, 21), HOXD13 (27), HOXA13 (9), HOXA11 (9), HOXC11 (35), and HOXC13 (19). All NUP98-Hox fusions reported to date consist of the N terminus of NUP98, containing a region of multiple phenylalanine-glycine (FG) repeats that may act as a transcriptional coactivator through binding to CBP/p300 (13), and the C terminus of the Hox gene product, containing the intact homeodomain and various extents of flanking amino acids. The leukemogenic potential of such chimeric proteins was confirmed for NUP98-HOXA9 (NA9) and NUP98-HOXD13 (ND13) in retrovirally transduced murine bone marrow transplantation models with both genes inducing AML and/or a myeloproliferative syndrome (16, 25). The transforming potentials of NA9 and ND13 were found to be dependent on the ability of their homeodomains to bind DNA (13, 25), and the Pbx interacting motif of HOXA9 in NA9 was found to be dispensable, suggesting that the Hox DNA-binding domain is the minimal and possibly only significant contribution of the Hox fusion partner.
The leukemogenic activity of Hox genes is not restricted to chimeric NUP98-Hox proteins, since HOXB8, HOXA10, HOXB3, and HOXA9 have previously been shown to induce AML upon engineered overexpression (15, 24, 31, 37). This suggests that a key result of NUP98 fusion with Hox is deregulated expression of Hox-responsive genes by the chimeric Hox protein, in which NUP98 replaces the transcriptional activity of the Hox N-terminal region (13). Consistent with this, both HOXA9 and NA9 studied in similar experimental settings are leukemogenic and block myeloid progenitor differentiation in interleukin-3 and granulocyte-macrophage colony-stimulating factor (7, 16). Nevertheless, differences in disease latency, cooperativity with Meis1, and other in vitro growth properties suggest that NUP98-Hox fusions have different properties compared to their normal counterparts. For instance, in vitro, HOXA9 did not replicate the effect induced by NA9, such as proliferation in stem cell factor and granulocytic differentiation block with granulocyte colony-stimulating factor (7).
Intriguingly, so far only Hox genes belonging to the Abd-B-like Hox paralog groups (groups 9 to 13) have been characterized as NUP98 fusion partners, and within those, only members from paralog groups 9, 11, and 13 have been observed. Thus, it may be that only a subset of Hox genes, sharing potential overlap in gene targets and/or pathways, can complement NUP98. Outside of the Abd-B-like Hox genes, it is of interest that HOXB4, while having potent ability to enhance hematopoietic stem cell (HSC) expansion is on its own nonleukemogenic, whereas the near neighbor HOXB3 can cause marked myeloproliferation and ultimately leukemia (1, 31). These findings raise the interesting question as to the intrinsic leukemogenic potential of Hox genes across the clusters and to their leukemogenic potential in the context of fusion genes.
The leukemic potential of intact Hox genes, such as HOXB3 and HOXA9, and NUP98 fusions with HOXA9 or HOXD13 has been shown to be increased by co-overexpression of Meis1 (15, 22, 25, 36) as evidenced by decreased disease latency. The basis for this cooperativity is unclear because, although in vitro studies have suggested that Meis1 expression can synergize with Hox genes in promoting a stronger differentiation block of Hox-transformed myeloid progenitors (5), it does not show leukemogenic activity on its own in vivo (16, 25). Understanding the role of Meis1 is further complicated by the fact that several of the known collaborating Hox proteins involved lack the ability to physically interact with Meis1, as in the case of HOXB3 and ND13. Finally, it is not clear whether all NUP98-Hox fusions could be strongly complemented by Meis1 since the leukemogenic potential of NA9 was only weakly augmented by Meis1, whereas Meis1 strongly complemented ND13 (16, 25).
The principal objectives of the work described here were to test whether the preferential identification of Abd-B-like Hox genes as NUP98 fusion partners in human leukemias is a consequence of their special intrinsic properties or, on the contrary, whether other Hox genes might share the functional capacity to form oncogenic NUP98 fusion genes. The leukemogenic potential of various NUP98-Hox fusion genes representing Hox genes of different paralog groups and different leukemogenic potentials was tested in vivo, alone and in concert with Meis1, to enlarge the knowledge of the role of Meis1 in Hox-related leukemia. Furthermore, in order to gain further insight into the mechanism(s) of Hox-mediated leukemia, we analyzed the impact of the native and NUP98-fused Hox genes on bone marrow differentiation and proliferation.
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MATERIALS AND METHODS |
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A 1:1 mixture of producers of the NUP98-Hox viruses (with the green fluorescent protein [GFP] marker) and Meis1 virus (with the YFP marker) was used to coinfect bone marrow cells. Loosely adherent and nonadherent cells were harvested from the cocultures after 2 days and cultured for 48 h in the same medium without protamine sulfate. Where indicated, transduced cells were highly purified based on expression of GFP, YFP, or both fluorescent proteins with a FACS-Vantage (Becton Dickinson, Mississauga, Canada), as previously described (25).
Bone marrow transplantation and monitoring of recipients.
Purified GFP+ and/or nonpurified (unsorted cells) transduced bone marrow cells were injected into the tail vein of irradiated (900 cGy of 137Cs 
-radiation) recipient F1 B6C3 mice. Mice transplanted with singly transduced bone marrow cells received from 1.4 x 105 to 3.5 x 105 GFP+ cells/transplant and mice injected with doubly transduced cells received from 0.2 x 104 to 8.0 x 104 GFP+/YFP+ bone marrow cells. For peripheral blood, bone marrow, and spleen cell analysis, single-cell suspensions were stained with the following monoclonal antibodies: phycoerythrin-labeled Gr-1, Mac-1, B220, Ter-119, and c-Kit (all obtained from Pharmingen, San Diego, Calif.). The immunoglobin E (IgE) receptor was detected with a polyclonal biotinylated anti-IgE receptor antibody (StemCell Technologies Inc.) followed by streptavidin-phycoerythrin (Pharmingen, San Diego, Calif.). Morphological analysis of peripheral blood, bone marrow, and spleen cells and histological analysis were performed as previously described (25).
In vitro assays. Cell proliferation was assessed in the same media described above. Differentiation of clonogenic progenitors was analyzed by plating cells in 1 ml of methylcellulose culture medium per petri dish in standard conditions (Methocult M3434, StemCell Technologies Inc.), containing 10 ng of murine interleukin-3 per ml, 10 ng of human interleukin-6 per ml, 50 ng of murine stem cell factor per ml, and 3 U of human erythropoietin per ml. Colonies were scored microscopically with standard criteria after 8 to 10 days.
CFU-S assay. GFP+ purified bone marrow cells were first cultured for 7 days before injection. The total cell progeny from the starting equivalent (day 0) of 6 cells up to 1 x 106 cells were then injected into irradiated recipient mice. The recovery of CFU-spleen (CFU-S) cells was measured by determining the number of macroscopic colonies on the spleen at day 12 postinjection after fixation in Telleyesnickzky's solution.
Southern blot analysis. Genomic DNA was isolated with DNAzol reagent as recommended by the manufacturer (Invitrogen), and Southern blot analysis was performed as previously described (29). The stable integration of the provirus was confirmed by digesting the DNA with NheI (cutting in each long terminal repeat) and then the DNA-containing membrane was probed with 32P-labeled GFP cDNA. For clonality analysis DNA from NUP98-Hox plus Meis1 primary or secondary transplant recipients was digested with EcoRI and probed with a 32P-labeled NUP98 cDNA. DNA from HOXB4-plus-Meis1- and HOXA10-plus- Meis1-transplanted mice was digested with XhoI and EcoRI and hybridized to HOXB4- or HOXA10-specific probes, respectively.
Western blot analysis. Transduced BaF3 cells (infected with the same procedure as that for bone marrow) were lysed (400 mM NaCl, 0.1% Triton, 0.1 M HEPES, pH 7.5). Cell lysate was loaded onto a 10% Bis-Tris polyacrylamide gel (NuPAGE [NP0303], Invitrogen) and blotted to a Biotrace polyvinylidene difluoride membrane (Pall Corporation, Ann Arbor, Mich.). A monoclonal anti-Flag (M2, Sigma) and a donkey horseradish peroxidase-conjugated anti-mouse immunoglobulin antibody were then used (Jackson ImmunoResearch Lab. Inc., West Grove, Pa.). Protein expression was detected with an enhanced Luminol reagent (Renaissance, Boston, Mass.).
Statistical analysis. Data were statistically tested with the t test for dependent or independent samples (Microsoft Excel). Differences with P values <0.05 were considered statistically significant.
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RESULTS |
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Expression of NA10 provided freshly transduced bone marrow cells with a strong growth advantage, as evidenced by the increasing proportion of transduced GFP+ cells over time in liquid culture (Fig. 1D). This effect was similar to that induced by ND13 and provided initial support for HOXA10 as an oncogenic NUP98 fusion partner. To determine whether the growth advantage could translate into leukemogenic activity in vivo, lethally irradiated mice were reconstituted with NA10-transduced bone marrow cells.
NA10-transduced bone marrow cells reconstituted the hematopoietic system of irradiated mice efficiently, with the proportion (Table 1) and expression levels of GFP+ mononuclear cells (Fig. 1E) similar to that observed for ND13 control-transplanted mice. No significant differences were observed in the number of white blood cells and red blood cells in the NA10 and control HOXA10 and GFP recipients 8 weeks posttransplant (Table 1). However, as first detected at 24 weeks, three out of eight healthy-looking NA10 mice displayed elevated white blood cell numbers (14 x 106 to 35 x 106/ml) due to increased myeloid cells (Gr1+ and/or Mac1+), consistent with a mild myeloproliferative condition, as previously observed in some ND13 and NUP98-HOXA9 mice (16, 25).
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25%) of blast cells in the marrow of the sick NA10 recipients (Fig. 3A; Table 2). The myeloid nature of the leukemic cells was confirmed by flow cytometry (Fig. 2B), which revealed that the majority of the leukemic bone marrow cells stained positive for Mac1, Gr1, and c-Kit, although a small degree of phenotypic heterogeneity was observable among different mice. This phenotypic variability likely reflects small differences in the predominant stage of differentiation impairment influenced by the level of NA10 expression or the nature of an extra mutation(s) acquired during leukemia progression (see below).
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Fusion of the nonleukemogenic HOXB4 gene to NUP98 does not cause AML. The results presented above supported the hypothesis that Abd-B-like Hox genes have common transforming properties that can be complemented by fusion to NUP98. However, it remained possible that any Hox gene, regardless of its intrinsic leukemogenic potential, could form an oncogenic fusion gene with NUP98. To investigate this, we engineered a fusion between NUP98 and HOXB4, a Hox gene previously found to be nonleukemogenic in transplant models and not yet implicated in human leukemia.
In contrast to NA10, NB4 did not induce leukemia even after follow-up of 1 year (Fig. 2A). This lack of leukemogenic activity was not attributable to low-level engraftment or expression levels, as evident from GFP expression in peripheral blood mononuclear cells (Table 1 and Fig. 1E). During that observation period, three NB4 and two GFP recipients died of unknown causes with no evidence of myeloproliferative disorder or hematological abnormalities. Clonogenic progenitor assays and fluorescence-activated cell sorter (FACS) analysis of the marrow of one of the NB4 mice revealed no apparent perturbation, and all secondary bone marrow transplants remained healthy (>47 weeks). These results indicate that the fusion of NUP98 to HOXB4 was without detectable leukemogenic activity and thus suggest that not all Hox genes can form leukemogenic NUP98 fusions.
To discriminate between the possibilities that HOXB4 could not complement NUP98 due to lack of intrinsic leukemogenic activity or that only Abd-B genes are able to collaborate with NUP98, we engineered a fusion between NUP98 and another Antennapedia-like Hox gene, HOXB3, that on its own has been shown to induce AML. Low viral titers resulted in sufficient numbers of GFP+ NB3 cells to transplant only two mice; nevertheless, one of two NB3 recipients developed leukemia, as evidenced by a high white blood cell count (110 x 106/ml), low red blood cell count (5.3 x 109 red blood cells/ml), splenomegaly (0.58 g), and a proportion of blasts of 21% in its marrow at 
235 days posttransplant, a latency similar to that reported for the intact HOXB3 gene (31). Together, these data suggest that the ability of Hox genes to form leukemogenic fusions with NUP98 is not restricted to Abd-B-like Hox genes, but may rather be determined by the intrinsic leukemogenic potential of the Hox gene fused.
Redundant ability of Hox and NUP98-Hox genes to cause AML in collaboration with Meis1 is independent of their intrinsic leukemogenic potential. The Hox cofactor Meis1 has been shown to reduce the time of disease onset for several Hox-induced leukemias, suggesting redundancy in the ability of Hox genes to collaborate with Meis1. A second important observation arising from these studies is that the strength of this cooperation seems to correlate with the intensity of the leukemogenic potential of the Hox gene involved. In order to clarify these issues, we investigated the potential synergy between Meis1 and NA10 (a strong oncogene), NB3, and NB4 (nononcogenic). In addition, to determine whether the synergy between Meis1 and the fusion genes reflects an intrinsic property inherited from their normal Hox counterparts, both HOXA10 and HOXB4 were coexpressed with Meis1 in vivo.
Bone marrow cells were cotransduced with the various NUP98-Hox-IRES-GFP and Meis1-IRES-YFP retroviruses and injected into irradiated mice. Strikingly, all fusion and Hox genes tested collaborated with Meis1 to induce leukemia, as evidenced by reduced disease latency or, in the case of NB4 and HOXB4, conversion to leukemogenic activity (Fig. 4A and B). Expression of Meis1 alone did not cause disease, as previously reported (15). Moreover, the median survival seen with Meis1 in combination with NA10 or HOXA10 was much shorter than in combination with NB3, HOXB4, or NB4 (Fig. 4B), reflecting the previously documented different leukemogenic potencies of the Hox genes by themselves. Moreover, although as single factors NB4 and HOXB4 were unable to induce leukemia, both genes were equally capable of inducing the disease with indistinguishable latency in combination with Meis1 (Fig. 4A and B; Table 3). This ability of nonleukemogenic Hox genes to cause AML in collaboration with Meis1 further supports the redundancy of such collaboration.
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Strong leukemogenic potential of Abd-B-like Hox genes correlates with their robust ability to promote cell growth and block differentiation. Based on the results described above, we speculated that the high leukemogenic potential of Abd-B-like Hox genes might be explained by their strong ability to promote cell proliferation and/or block differentiation.
All three novel fusion genes enhanced the proliferation of transduced bone marrow cells in short-term liquid culture, as evidenced by the increased proportion of GFP+ cells over time (Fig. 1D and data not shown). The total expansion of GFP+ cells (i.e., total number of GFP+ cells after 14 days/input GFP+ cells) induced by NA10 was, however, consistently greater (10- to 50-fold higher) than that induced by NB3 and NB4 and similar to that obtained for ND13 (Fig. 6A). Moreover, this differential growth-promoting effect induced by the various fusion genes was also seen in more primitive cells, as shown by day 12 CFU-S. As previously reported, GFP cultures yielded a limited number of day 12 CFU-S cells following 1 week of culture. In contrast, all fusion genes tested produced a prominent increase, an average of 4,700-fold, in the yield of CFU-S-like cells (Fig. 6B). Again, the magnitude of the impact was greater for NA10, which produced the highest day 12 CFU-S recovery, matching that observed with ND13 (Fig. 6B), whereas NB4 consistently produced the smallest yield in all experiments (n = 3), followed by NB3.
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DISCUSSION |
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Decisively, the Abd-B-like Hox genes formed NUP98 chimeric proteins with the greatest impact on proliferation and differentiation in vitro and leukemogenic potency in vivo, which may account for the fact that to date, only Hox genes belonging to this group have been found rearranged with NUP98 in human leukemia. Nevertheless, the leukemogenic potentials observed for NUP98-HOXB4 and HOXB4 with Meis1 support a model in which many if not all Hox genes may have the not previously appreciated capacity to contribute to leukemic transformation and point to striking overlap in properties.
Intrinsic leukemogenic potential of intact Hox genes correlates with their ability to induce AML as NUP98 fusion partners. The novel NA10 fusion gene induced AML with kinetics similar to that of ND13, indicating that the reported leukemogenic potential of HOXA10 (37) is retained in the NA10 fusion. Our data further imply that the ability to form leukemogenic NUP98-Hox fusion genes is not restricted to the already identified Hox genes, but rather seems to be a redundant property of Abd-B-like Hox genes. These results are consistent with reported functional identity between paralogous as well as nonparalogous Abd-B-related Hox genes (10, 38, 39). Abd-B-like Hox genes are derived from a common ancestor, and therefore their homeodomains are closely related, which may indicate that their redundant function in leukemogenesis is mediated through the regulation of a similar or overlapping set of genes and/or pathways that control hematopoietic cell growth and differentiation.
Our results showing strong leukemogenic potential for NA10 but not for NB4 suggest that the intrinsic leukemogenicity of NUP98 fusions is determined primarily by the Hox fusion partner. Moreover, such intrinsic leukemogenicity appears not to be restricted to Abd-B-like Hox genes, as suggested by leukemia arising in an NB3 recipient. This differential ability of NB3 and NB4 to cause leukemia is consistent with the specific effects of their native counterparts (30, 31) and further demonstrates that closely related Hox genes are not completely functionally equivalent (26, 40).
AML is characterized by the uncontrolled proliferation of myeloid cells that accumulate at different stages where their further differentiation is blocked. The high correlation observed between the leukemogenic potential of the native and NUP98-Hox genes and their ability to increase self-renewal and block differentiation argues that the latter characteristics determine the Hox leukemogenic potential. Thus, the intrinsic ability of ND13 and NA10 and that reported for NA9 (7) to sustain increased self-renewal and strongly block bone marrow differentiation appear to be a unifying property that determines their potent leukemogenicity.
Redundant ability of Hox genes to collaborate with Meis1 to induce AML. Despite considerable differences in the function and structure of the native and Hox-fused genes studied in this work, they were all able, though with different latencies, to cause AML in collaboration with Meis1. Importantly, our data demonstrating the capacity of NB4 and B4 to cause AML with Meis1 indicate that even Hox genes that do not cause leukemia on their own possess the redundant ability of becoming leukemogenic in the presence of this, and possibly other, cofactors.
Work from Calvo et al. showing that Meis1 may further block differentiation of myeloid progenitors overexpressing HOXA9 (5), coupled to the ability of HOXB4 to transform fibroblasts in vitro (17), could in part explain the leukemogenic activity of HOXB4 and NB4 in collaboration with Meis1. Meis1 has been shown in myeloid cells to be associated with ternary complexes containing Pbx and Hox proteins (34), in which Meis1 directly interacts with Hox proteins from paralog groups 9 to 13, or indirectly associates with paralogs 1 to 8 through Pbx (33). NA10 and NA9 both retain the Pbx interacting motifs and therefore, Meis1 may indirectly interact with these proteins through the formation of trimer complexes containing Pbx, as suggested for A9-mediated transformation of myeloid progenitors (32). Arguing against the likelihood that such direct interactions are the key to leukemic transformation, several of the fusions tested (NB3, NB4, ND13, ND13hd, and NA10hd) lacked both known Pbx1- and Meis1-interacting motifs. Nevertheless, the longer latency observed for NA10hd/Meis1 (lacking a Pbx-interacting motif) compared to NA10/Meis1 (containing a Pbx-interacting motif) suggests that interaction with Pbx proteins, though dispensable, may accelerate disease development.
How Meis1 accelerates the onset of Hox-mediated AML is still unclear. Whether Meis1 functions to increase the strength of the transcriptional activity of Hox genes on Hox-regulated promoters or works through different target genes needs to be resolved. Several non-mutually exclusive explanations could be proposed. First, in view of work from Saleh et al. showing that under some circumstances Pbx1 can repress Hox gene-mediated transcription by interacting with multiple histone deacetylases (28) and a recent report showing increased ability of HOXB4 to enhance self-renewal of HSC when Pbx1 was downregulated (18), one could propose that Meis1, known to form strong heterodimers with Pbx1, might bind and titrate off Pbx proteins. Another possibility is that the exogenous Meis1 physically interacts with endogenously expressed Hox proteins also implicated in leukemia, such as Hoxa7 and Hoxa9 (7, 25). Meis1 might also directly bind endogenous Hox-responsive genes and/or different genes, through Hox-independent activity (23). This possibility is supported by data indicating that Homothorax, a Drosophila Meis1 orthologue, possesses transcriptional activation activity of its own (12).
The NUP98-Hox fusion gene transformation models exploited here should now prove useful to dissect the molecular and cellular pathways involved in the redundant and differential leukemogenic potential of Hox genes and to provide new strategies to elucidate the role of collaborating genes.
Supplementary materials.Primer sequences to amplify indicated Hox regions for NUP98-HOX fusion constructs were as follows: NUP98 portion, forward, 5-AGTCGGATCCTTTAACAAATCATTTGGAACACCCTT-3, and reverse, 5-TTTAAGAATTCTACTGGGGCCTGGGG-3; HOXB4 Exon2, forward, 5-CCCAGGAATTCGTAAACCCCAATTACGCC-3, and reverse, 5-ACATAAAAATGCGGCCGCTAGAGCGCGCGGGGGCC-3; HOXB3 Exon4, forward, 5-ATACCCGAATTCGCAGAGGGCTGTGGT-3, and reverse, 5-CCCATCCCCTAATCCTCGAGCGC-3; HOXA10 Exon2, forward, 5-TTATCCGAATTCGGCAATTCCAAAGGTGAA-3, and reverse, 5-TTCAATAATGCGGCCGCTCATCAGGAAAAATT-3; HOXA10 homeodomain, homeobox (hd) A10hd; forward, 5-ACTGATGAATTCAAGAGTGGTCGGAAGAAGCGC-3; reverse, 5-ATAAAAATGCGGCCGCTCACATTTTCTTCAGTTT-3; HOXD13 homeodomain, forward, 5-ACTGATGAATTCCGAAGAGGGAGGAAGAAG-3, and reverse, 5-ACTGATCTCGAGTCATTTCTTGTCCTTCACTCTTCGGT-3.
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ACKNOWLEDGMENTS |
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We thank Peter Aplan for initial NUP98-HOXD13 cDNAs; Gayle Thornbury, Rick Zapf, and Cam Smith for cell sorting; Patty Rosten and Jason Wong for technical assistance; and Colleen Mackinnon for secretarial support.
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FOOTNOTES |
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