Review

Oncogene (2007) 26, 6766–6776; doi:10.1038/sj.onc.1210760

Hox genes in hematopoiesis and leukemogenesis

B Argiropoulos1 and R K Humphries1,2

  1. 1Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada
  2. 2Department of Medicine, University of British Columbia, Vancouver, BC, Canada

Correspondence: Dr RK Humphries, Terry Fox Laboratory, British Columbia Cancer Agency, 11th Floor, 675 West 10th Avenue, Vancouver, BC, Canada V5Z 1L3. E-mail: khumphri@bccrc.ca

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Abstract

Gene expression analyses, gene targeting experiments and retroviral overexpression studies in the murine bone marrow transplantation model have provided strong correlative evidence for the involvement of clustered Hox genes in normal hematopoiesis. The data strongly support the hypothesis that the role of Hox genes in normal hematopoiesis is primarily at the level of hematopoietic stem cell function. A large body of evidence now links Hox genes to leukemic transformation including dysregulated HOX expression in leukemic patient samples, their involvement as oncogenic fusion proteins with NUP98 and their requirement for the oncogenicity of Mll fusions. In recent years, much attention has been devoted to the identification and characterization of leukemic stem cells. Given the documented role of Hox genes in hematopoiesis and leukemogenesis, we propose that Hox-dependent pathways are closely linked to the self-renewal program crucial to the origin and function of leukemic stem cells.

Keywords:

self-renewal, NUP98-HOX, Mll, leukemic stem cells

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Introduction

The clustered Hox family of homeobox genes (class I homeobox genes) is an evolutionarily highly conserved set of genes that encode DNA-binding transcription factors that were first identified as key regulators of positional identity along the anterior–posterior body axis of animal embryos (Krumlauf, 1994). In mammals, there are 39 Hox genes organized into four genomic clusters (A–D) located on four different chromosomes and, based on homeobox sequence similarity, consist of 13 paralogous groups, with no cluster containing a full set (Figure 1). Hox genes exhibit a high degree of homology to the clustered homeotic genes (HOM-C) of Drosophila melanogaster, which are located in two gene clusters, the Antennapedia (Ant-C) and bithorax complexes (BX-C). Mammalian paralog groups 1–8 are more closely related to Ant-C genes, while paralog groups 9–13 are more closely related to the Abdominal-B (Abd-B) gene of the BX-C and are thus referred to as Abd-B class Hox genes (Figure 1). Non-clustered or non-Hox homeobox genes (class II homeobox genes) are more divergent, more numerous (approximately 160 in the human genome) (Tupler et al., 2001) and are dispersed throughout the genome. The highly conserved homeobox sequence motif, which encodes the homeodomain, a 60 amino-acid helix-turn-helix DNA-binding domain (Gehring et al., 1994), is the common element defining these two broad classes of genes.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Clustered Hox gene organization. The four Hox clusters each contain 8–11 genes and are located on four different chromosomes. Individual genes in different Hox clusters can be aligned into paralogous groups (identical colors) based on the sequence homology within their homeobox regions, and with the homeotic genes of the Drosophila HOM-C cluster. A box above a gene summarizes the important hematopoietic phenotypes exhibited upon engineered overexpression of that Hox gene. Brackets under a gene, or a group of genes, denote mouse knockout models where the role of the indicated gene(s) was assessed in hematopoiesis. BFU-E, erythroid burst-forming unit; CFU-GM, granulocyte/macrophage colony-forming unit; EPs, erythroid progenitors; LL AML, long latency acute myeloid leukemia; MEP, erythroid/megakaryocytic progenitors; MKs, megakaryocytes; MPs, myeloid progenitors; MPD, myeloproliferative disorder.

Full figure and legend (194K)

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The enigmatic role of Hox genes in normal hematopoiesis

Gene expression analyses of both mouse and human bone marrow (BM) samples revealed that the majority of Hox genes of the A, B and C clusters are expressed in hematopoietic cells and, for the most part, are preferentially expressed in hematopoietic stem cell (HSC)-enriched subpopulations and in immature progenitor compartments and downregulated during differentiation and maturation (Giampaolo et al., 1994, 1995; Moretti et al., 1994; Sauvageau et al., 1994; Kawagoe et al., 1999; Pineault et al., 2002). These observations led to the hypotheses that Hox genes play key functions in early hematopoietic cells, including HSCs, and that dysregulated Hox expression may impact leukemic transformation.

Lessons from Hox overexpression and knockout models

The role of Hox genes in normal hematopoiesis has been extensively investigated through engineered overexpression from retroviral vectors and transgenic knockout and knock-in mouse models. While these studies have provided strong correlative evidence for the involvement of Hox genes in normal hematopoiesis, their precise role(s) and their mechanism(s) of action remain largely unclear.

Overexpression of Hox genes in HSCs from mouse BM, fetal liver and in human cord blood progenitors has provided a rich body of data showing potent effects of Hox on differentiation and proliferation. Details of the specific phenotypes have been extensively reviewed elsewhere and summarized in Figure 1 (Lawrence et al., 1996; Owens and Hawley, 2002; Grier et al., 2005). For example, overexpression of Hoxa10, Hoxb3 and Hoxb6 in mouse BM cells had profound effects such as a block in the differentiation of B- and T cells, impaired erythropoiesis, and induction of myeloproliferative disorders and leukemia. One striking effect of Hox that has emerged from these studies is the potent impact of Hox on HSC self-renewal. This is graphically illustrated by initial findings relating to Hoxb4 and will be discussed in detail below.

The embryonic phenotypes of Hox knockout mouse models have revealed both unique and redundant or overlapping function of Hox genes and their sensitivity to gene dosage as observed by the phenotypic differences between single and compound Hox gene knockouts (Horan et al., 1995a, 1995b; Zakany et al., 1996, 1997; Chen and Capecchi, 1997; Greer et al., 2000; Suemori and Noguchi, 2000). Members of the Hox family also appear to play overlapping and diverse roles in hematopoiesis and challenge efforts to elucidate their specific and combined functions.

We previously documented that Hoxb4 is among the Hox genes expressed in primitive murine and human hematopoietic cells (Sauvageau et al., 1994; Pineault et al., 2002) and demonstrated that enforced overexpression or direct protein delivery of Hoxb4 is sufficient to stimulate marked HSC expansion in vivo and ex vivo without promoting leukemia (Sauvageau et al., 1995; Antonchuk et al., 2001, 2002; Amsellem et al., 2003). These potent effects of Hoxb4 have also been extended to human and nonhuman primate HSCs (Buske et al., 2002; Zhang et al., 2006). As described in more detail in a later section, additional Hox and variant Hox genes have also been shown to be potent stimulators of HSC expansion arguing for their important role in HSC self-renewal. Nevertheless, convincing evidence to ascertain major impacts of Hox on self-renewal or other hematopoietic functions in knockout models has proven somewhat elusive. For example, definitive hematopoiesis was not disrupted in Hoxb4-deficient mice (Brun et al., 2004; Bijl et al., 2006). These mice did, however, exhibit subtle reductions in HSC and progenitor numbers and a modest impairment of competitive repopulating ability (Brun et al., 2004). Compound Hoxb3/b4-/- knockout mice exhibited qualitatively similar but quantitatively more pronounced hematopoietic differences (Bjornsson et al., 2003). Collectively, these studies indicate that Hoxb3 and Hoxb4 are required, but not necessary, for normal HSC function and that their loss may be compensated for by the self-renewal function of other Hox genes.

In a more recent study, a near-complete Hoxb cluster knockout, Hoxb1-hoxb9-/-, revealed that the Hoxb cluster is dispensable for normal hematopoiesis as Hoxb1-hoxb9-/- HSCs as well as wild-type HSCs competed in competitive repopulation assays and retained their full differentiation potential (Bijl et al., 2006). Gene expression profiling of c-kit+ Hoxb1-Hoxb9-/- fetal liver cells revealed a downregulation of most Hoxa genes, and upregulation of three Hoxc genes including the Hoxb4 paralog, Hoxc4. The apparent discrepancy in hematopoietic defects reported by Brun et al. (2004) and Bijl et al. (2006) has been explained in the latter study to be the result of the different gene targeting strategies used, a phenomenon for which a precedent exists. Nevertheless, both studies provide strong support for gene redundancy and compensatory mechanisms, as well as cross-regulatory interactions, among Hox genes. These observations warrant further investigation of the role of other Hox paralog 4 genes and the contribution of other Hoxa and Hoxc genes, by way of complete cluster knockouts, in HSC self-renewal.

Like Hoxb4, Hoxa9 is preferentially expressed in primitive hematopoietic cells and downregulated during differentiation, suggesting a key role in early HSC function (Sauvageau et al., 1994; Pineault et al., 2002). Indeed, Hoxa9-deficient mice exhibited defects in multiple hematopoietic lineages, representing the Hox gene with the most severe hematopoietic phenotypes (Lawrence et al., 1997; Izon et al., 1998). Moreover, competitive repopulation assays revealed that the major defect in Hoxa9-/- animals was a dramatic impairment of their HSCs to repopulate lethally irradiated recipients after bone marrow transplantation (BMT) (Lawrence et al., 2005). One plausible explanation for the severity of the Hoxa9 knockout phenotype relative to the mild Hoxb3/b4-/- or Hoxb4-/- phenotypes is that Hoxa9 is one of the most highly expressed Hox genes in the HSC compartment, and thus, it could potentially be the major determinant of physiologic HSC self-renewal. This is reinforced by evidence that overexpression of Hoxa9 can enhance HSC regeneration in vivo, albeit with additional impacts on differentiation that ultimately lead to leukemia (Thorsteinsdottir et al., 2002), thus implicating it in HSC self-renewal.

Additional Hox knockout models where hematopoiesis was assessed include Hoxa5 (Fuller et al., 1999), Hoxa7 (So et al., 2004), Hoxa10 (Lawrence et al., 2005), Hoxb3 (Ko et al., 2007), Hoxb6 (Shen et al., 1992; Kappen, 2000) and Hoxc8 (Shimamoto et al., 1999) (Figure 1). In summary, their effects range from impaired HSC self-renewal to impaired myelopoiesis or B lymphopoiesis.

Deficiencies of Hox regulators demonstrate a role for Hox genes in HSC expansion

The MLL (mixed-lineage leukemia) gene is the human homolog of the Drosophila Trithorax gene and is the archetypal member of the Trithorax group of genes that encode chromatin modifiers that are required for the proper maintenance of Hox gene expression during development (Schuettengruber et al., 2007). Consistent with a major role for Hox genes in hematopoiesis, it has been shown in the murine system that Mll plays an essential and nonredundant role in definitive hematopoiesis by inducing the proliferation and differentiation of hematopoietic progenitors through the maintenance of Hox gene expression, mainly Hox genes from the Hoxb and Hoxc clusters (Ernst et al., 2004). Indeed, the grossly impaired ability of MLL-/- embryonic bodies to form hematopoietic colonies can be rescued by overexpression of Hoxa9, Hoxa10, Hoxb3 or Hoxb4 (Ernst et al., 2004). Rescue was also observed with the class II homeobox gene cdx4, an upstream regulator of Hox gene expression required for specification of blood progenitors (Davidson et al., 2003; Davidson and Zon, 2006) and when overexpressed is sufficient to induce acute myeloid leukemia (AML) alone and in cooperation with the Hox cofactor Meis1 (discussed below) in a murine BMT model (Davidson et al., 2003; Bansal et al., 2006).

HOX proteins bind target DNA sites in vitro with little obvious selectivity (Hoey and Levine, 1988; Ekker et al., 1994; Mann, 1995). HOX cofactors of the three-amino acid loop extension class have been shown to interact directly with HOX proteins to increase their DNA-binding affinity and specificity and to modify their transregulatory properties (Moens and Selleri, 2006). In general, HOX proteins from paralog groups 1–10 interact physically with PBX1, while those from paralog groups 9–13 interact with MEIS1 (Shen et al., 1997). Thus, loss-of-function mutations of Hox cofactors are predicted to disrupt multiple HOX-dependent pathways. Indeed, Pbx1 and Meis1 mutant animals are embryonic lethal and recapitulate the HSC defects observed in Hox knockout models, indicative of a role for these genes in HSC self-renewal/proliferation (DiMartino et al., 2001; Hisa et al., 2004).

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The unequivocal role of Hox genes in the pathogenesis of acute leukemia

A central role for HOX genes in hematological malignancies is supported by the frequently observed elevation of HOX and MEIS1 gene expression in AML patient samples (Golub et al., 1999; Kawagoe et al., 1999; Lawrence et al., 1999; Afonja et al., 2000; Drabkin et al., 2002). Indeed, HOXA9 is the single most highly correlated gene (out of 6817) for poor prognosis in AML (Golub et al., 1999). Interestingly, gene expression profiling of AML cells with mutations in the nucleophosmin (NPM1) gene, which represent the most frequent acquired molecular abnormalities in AML, are highly associated with a stem cell molecular signature that includes activation of HOX genes and MEIS1 (Alcalay et al., 2005).

HOX gene deregulation has also been documented in lymphoid leukemias (Imamura et al., 2002). Gene expression analysis showed that the whole HOXA gene cluster was dramatically dysregulated in T-cell acute lymphocytic leukemia samples harboring the TCRbeta-HOXA rearrangement (Speleman et al., 2005; Cauwelier et al., 2007). HOXA genes were also found to be upregulated in MLL and CALM-AF10-related T-cell acute lymphoblastic leukemias cases, strongly suggesting that HOXA genes are oncogenic in these leukemias (Soulier et al., 2005).

As described previously, in experimental model systems using retroviral gene transfer into murine BM cells followed by transplantation, we and others have shown that enforced overexpression of select Hox genes, such as Hoxa9, Hoxa10, Hoxb3, Hoxb6 or Hoxb8, confer a growth advantage in vitro and in vivo and leads to long latency leukemia (Sauvageau et al., 1997; Thorsteinsdottir et al., 1997; Kroon et al., 1998; Fischbach et al., 2005). The long latency of disease onset suggests that additional genetic perturbations are required for the progression to overt leukemia. Indeed, co-overexpression of Meis1 collaborates with all Hox genes tested thus far, including the non-leukemogenic Hoxb4, to induce rapid onset AML (Kroon et al., 1998; Thorsteinsdottir et al., 2001; Pineault et al., 2004; Fischbach et al., 2005). These observations strongly indicate that dysregulation of Hox pathways are a dominant mechanism of leukemic transformation.

NUP98-HOX fusion genes

HOX genes have been directly linked to leukemia by virtue of their involvement in leukemia-specific translocations. To date, eight clustered Abd-B HOX genes have been found fused to the nucleoporin gene NUP98 in human leukemia (Figure 1) and have been identified in patients with AML, post-therapy AML and chronic myeloid leukemia (Slape and Aplan, 2004; Nakamura, 2005). The novel fusion oncoproteins contain the N-terminal domain of NUP98, which may confer transcriptional activity on the novel fusion through recruitment of the transcriptional coactivators CREB binding protein and p300 (Kasper et al., 1999) or histone deacetylase 1 (Bai et al., 2006), fused to the C-terminal domain of its HOX partner, which includes the homeodomain. Consistent with a role in modulating transcription, gene expression profiling of NUP98-HOXA9-expressing myeloid cells revealed a significant increase in the number of genes upregulated compared to HoxA9-expressing myeloid cells (Ghannam et al., 2004). It has also been proposed that the transforming potential of NUP98-HOX fusions may reflect their increased protein stability (Calvo et al., 2002; Chung et al., 2006) or their ability to alter nucleocytoplasmic transport of mRNA and/or protein (Lam and Aplan, 2001).

The direct involvement of NUP98-HOX fusion genes in the pathogenesis of AML was demonstrated in the murine BMT model with retroviral overexpression of NUP98-HOXA9 or NUP98-HOXD13, which resulted in a myeloproliferative disease that progressed to AML after a long latency (Kroon et al., 2001; Pineault et al., 2003). Transgenic knock-in models of NUP98-HOXA9 or NUP98-HOXD13 confirmed these observations (Iwasaki et al., 2005; Lin et al., 2005). We have reported that the ability of Hox genes to induce leukemia as NUP98 fusion partners is not restricted to the naturally occurring Abd-B Hox genes and that their potency as single agents to induce leukemia differ among Hox genes, indicating differential and common properties of many Hox genes (Pineault et al., 2004). Strikingly, all tested NUP98-HOX fusions collaborated with Meis1 to accelerate disease onset (Kroon et al., 2001; Calvo et al., 2002; Pineault et al., 2003), arguing that these fusions continue to act in Hox-dependent pathways and point to a potent core property of Hox that we propose may lie in their common ability to intensify or reactivate self-renewal programs.

We have exploited the strong transforming potential of NUP98-HOXD13 and NUP98-HOXA10 to establish transplantable, preleukemic myeloid lines composed of early myeloid progenitors with extensive in vitro self-renewal capacity, short-term myeloid repopulating activity and low propensity for spontaneous leukemic conversion (Pineault et al., 2005). These lines appear to recapitulate the pathologic and clinical attributes of human AML and have provided new opportunities to investigate the molecular mechanisms underlying the progression from a preleukemic to leukemic state upon retroviral transduction with Hox collaborators, such as Meis1 and Flt3 (Pineault et al., 2005; Palmqvist et al., 2006).

MLL fusion genes

Rearrangements involving MLL fusion genes, generated as a consequence of chromosomal translocations fusing N-terminal sequences of MLL to 1 of over 40 functionally diverse group of C-terminal fusion partners (Daser and Rabbitts, 2005), constitute 5% of all AML cases and 22% of those with acute lymphoblastic leukemia (De Braekeleer et al., 2005). Gene expression analyses revealed HOX gene dysregulation in all types of MLL fusion-associated T- and B-cell acute lymphoblastic leukemias (Rozovskaia et al., 2001; Armstrong et al., 2002; Yeoh et al., 2002; Ferrando et al., 2003), implicating HOX genes as integral factors in MLL fusion-associated leukemias. Furthermore, leukemias associated with MLL rearrangements show upregulation of HOXA9 and MEIS1 (Yeoh et al., 2002; Kohlmann et al., 2003; Tsutsumi et al., 2003; Fine et al., 2004), which, as discussed below, may be a common pathway that unifies diverse initiating events in many myeloid leukemias.

Mice transplanted with BM cells engineered to overexpress various MLL fusion genes develop leukemias, and in the case of the MLL-ENL fusion, expression of Hox genes and Hox cofactors were required for the initiation and maintenance of transformation of myeloid progenitor cells (Ayton and Cleary, 2003; Zeisig et al., 2004; Horton et al., 2005). Specifically, retroviral transduction of MLL-ENL into wild type or Hoxa9-/- primary myeloid progenitors revealed the necessity of Hoxa9 in MLL-ENL-mediated leukemogenesis (Ayton and Cleary, 2003). Moreover, MLL-ENL immortalization of myeloid progenitor cells could be substituted for by overexpression of HoxA9 and Meis1 (Zeisig et al., 2004), and was dependent on the activity of the oncoprotein c-Myc (Schreiner et al., 2001), a downstream effector of Hoxb4 that is sufficient to induce HSC self-renewal (Satoh et al., 2004). By contrast, analogous studies in a germline mouse model of Mll-AF9-mediated leukemogenesis suggest that Hoxa9 is not necessary for leukemia but does influence the phenotype of the leukemia (Kumar et al., 2004). Gene expression profiling of Mll-AF9-expressing BM cells revealed overexpression of the Hoxa cluster genes, Hoxa5 to Hoxa9, which the authors propose represents the Mll-AF9- triggered 'Hox code' that defines this oncogene. Redundancy and/or overlapping function(s) among HOX were proposed to account for the loss of Hoxa9. Collectively, these data indicate that MLL fusion genes contribute to pathogenesis of leukemia through dysregulated Hox gene expression.

Meis1 and Flt3 in Hox-mediated leukemia

The proto-oncogene Meis1 was first identified, together with Hoxa7 and Hoxa9, as a common viral integration site in myeloid leukemic cells of BXH-2 mice (Moskow et al., 1995). We and others have subsequently demonstrated that overexpression of Meis1 collaborates with multiple native and NUP98-HOX fusion genes to accelerate the onset of AML in the murine BMT models (Kroon et al., 1998, 2001; Thorsteinsdottir et al., 2001; Pineault et al., 2003, 2004; Fischbach et al., 2005). Curiously, wild-type Meis1 has no transforming activity alone but can be engineered to do so when fused to the potent transactivating domain of VP16 (Mamo et al., 2006; Wang et al., 2006), thus linking its oncogenicity to transcriptional activation of its downstream target genes. However, collaboration with Hox may be central as suggested by frequent insertional activation of Hoxa genes in the VP16-Meis1 model (Mamo et al., 2006).

The molecular mechanisms responsible for the collaboration of Hox or NUP98-HOX fusions with Meis1 in leukemic transformation are not fully understood. A plausible hypothesis posits that deregulated expression of Hox or NUP98-HOX fusion genes program an increase in self-renewal potential and/or a block in hematopoietic differentiation thus establishing a pre-leukemic population of BM cells, and deregulated Meis1 expression programs a proliferative and/or survival advantage to these preleukemic cells leading to the emergence of leukemic stem cells (LSCs). This hypothesis is corroborated by the finding that MEIS1 upregulates the pro-proliferative and pro-survival receptor tyrosine kinase Flt3 in Hoxa9 and NUP98-HOX models of leukemia, indicating that Flt3 is a major downstream effector of the leukemic collaboration between Hox and Meis1 (Wang et al., 2005, 2006; Palmqvist et al., 2006). Consistent with this, elevated wild-type FLT3 levels are frequently observed in AML samples and correlate with elevated expression of HOX and MEIS1 (Ozeki et al., 2004; Quentmeier et al., 2004).

We have recently demonstrated that overexpression of wild-type Flt3 is sufficient to collaborate with NUP98-HOX fusions to induce AML (Palmqvist et al., 2006). However, disease induced with NUP98-HOX+Flt3 develops with a longer latency than NUP98-HOX+Meis1, suggesting that Meis1 triggers additional leukemogenic pathways. This is supported by (1) the identification of other Meis1-regulated genes implicated in stem cell biology, including CD34 (Wang et al., 2005) and c-Myb (Hess et al., 2006), and (2) by the dispensability of Flt3 in HoxA9+Meis1 (Morgado et al., 2007) and NUP98-HOXD13+Meis1-mediated leukemogenesis (Argiropoulos and Humphries, submitted). Further identification and characterization of Meis1-regulated genes and the regulatory pathways they control is essential to resolve the molecular mechanisms underlying the potent effect of Meis1 on Hox and NUP98-HOX-mediated leukemogenesis.

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HOX as potent stimulators of HSC expansion

The ability of HSCs to provide for the sustained production of all blood lineages is accomplished by a balance between extensive HSC expansion characterized by purely symmetrical self-renewal divisions that occurs during embryogenesis and at times of hematopoietic stress, and the homeostatic maintenance of HSC numbers that likely reflect asymmetrical self-renewal divisions that give rise to one HSC and one committed progenitor cell.

As mentioned previously, it now appears that Hoxb4 is among the most potent stimulators, so far known, of HSC expansion both in vitro and in vivo, likely reflecting its ability to promote symmetrical self-renewal divisions. Engineered retroviral overexpression of Hoxb4 in the mouse BMT model induced a 1000-fold net increase in transduced HSCs within 3–5 months after transplantation versus 20-fold for the control (Sauvageau et al., 1995; Thorsteinsdottir et al., 1999; Antonchuk et al., 2001). In addition, the rate and magnitude of HSC regeneration was dramatically enhanced in the Hoxb4-transduced animals compared to the control. We subsequently demonstrated that Hoxb4 induced a rapid (12 days) 40-fold ex vivo expansion of HSCs, which reconstituted both myeloid and lymphoid compartments in vivo without inducing leukemia (Antonchuk et al., 2002). Investigation into the molecular mechanisms underlying Hoxb4-mediated HSC expansion revealed that DNA binding was essential and that physical interaction with the HOX cofactor PBX1 was dispensable (Beslu et al., 2004). Interestingly, suppression of Pbx1 expression enhanced in vivo Hoxb4-mediated HSC expansion 20- to 50-fold greater than HOXB4 alone (Krosl et al., 2003). Curiously, Pbx-/- BM cells show no proliferative advantage. The Hoxb4 (high)–Pbx1 (low) collaboration has recently been extended to ex vivo cultures, achieving a remarkable 100 000-fold expansion in a 2-week time period while preserving their stem cell properties (Cellot et al., 2007). These data suggest that PBX1 activity negatively modulates the growth-enhancing effects of HOXB4.

Our finding that a subset of NUP98-HOX fusion genes blocked hematopoietic differentiation when retrovirally overexpressed in murine BM (Pineault et al., 2004, 2005) prompted us to test whether these fusions may be used to stimulate HSC expansion. Intriguingly, both engineered NUP98-HOXB4 and NUP98-HOXA10HD (NUP98 fused only to the homeobox of HOXA10) were extremely potent stimulators of ex vivo HSC expansion with net levels of 300-fold for NUP98-HOXB4 and >2000-fold for NUP98-HOXA10HD demonstrated in 7 days in culture (Ohta et al., 2007). Like Hoxb4, both fusions reconstituted normally both myeloid and lymphoid compartments in vivo without inducing leukemia. The level of ex vivo HSC expansion obtained for NUP98-HOXA10HD and Hoxb4 (high)–Pbx1 (low) are unprecedented and, according to the reported doubling time of HSCs (Uchida et al., 2003), are near-maximal symmetrical expansions.

Together, the ability to exploit Hox and NUP98-HOX fusion genes to expand HSCs at near-maximal levels, thus producing a large number of pluripotent and differentiation-competent HSCs, will provide tools to elucidate the molecular mechanisms of HSC self-renewal and will have major clinical implications with respect to BMT therapy. Moreover, as a key function of Hox is their ability to impact on self-renewal, this logically implies that this might underpin their role in leukemia.

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HOX and the leukemic stem cell

The field of oncology is undergoing a revolution in considering in which stem cells are seen as the key determinant of the origin and nature of cancer. This concept gained enormous impetus with the early findings in the field of leukemia by Bonnet and Dick (1997) who demonstrated that the cell capable of initiating human AML in the NOD/SCID mouse model is extremely rare, is immunophenotypically similar to normal NOD/SCID-repopulating HSCs and is capable of differentiating (asymmetric division) in vivo into leukemic blast cells. These and more recent findings (Hope et al., 2004) strongly suggest that leukemic cells exist in a hierarchical state in which only a small number of cells are able to self-renew extensively, and thus capable of initiating and maintaining the leukemic clone. This hierarchical model of leukemia in which a rare stem cell initiates and maintains disease has since been extended to a range of other tumors including multiple myeloma (Matsui et al., 2004), mammary carcinomas (Ponti et al., 2005), colon cancer (O'Brien et al., 2007), prostate cancer (Xin et al., 2005) and brain tumors (Singh et al., 2004). Such findings raise great interest in both the target cells that can give origin to LSCs and the mechanisms underlying their key properties such as self-renewal.

In recent years, investigations into the origin of human LSCs have revealed that these cells are strikingly similar to normal HSCs, with respect to their ability for self-renewal, cell-surface markers and differentiation capacities (Blair et al., 1998; Miyamoto et al., 2000; Hope et al., 2004; Taussig et al., 2005). Thus, LSCs may be derived from normal HSCs that have acquired an enhanced self-renewal program followed by additional transforming mutations. Alternatively, LSCs may be derived from committed progenitors or lineage-positive cells that have reacquired the ability to self-renew and, subsequently, to proliferate or inhibit apoptosis. In either scenario, there is great interest in elucidating the mechanisms that are responsible for intensifying or reactivating the self-renewal program.

Taking into account the role of Hox genes in HSC self-renewal, we propose that dysregulation of HOX activity may be a central mechanism underlying the self-renewal capacity of LSCs, regardless of origin. Support for this hypothesis comes from multiple murine studies demonstrating that purified committed hematopoietic progenitors, with limited self-renewal capacities, engineered to overexpress the Hox gene regulators MLL-AF9 (Krivtsov et al., 2006), MLL-ENL (Cozzio et al., 2003) or MLL-GAS7 (So et al., 2004) can be transformed into LSCs. In the latter study, leukemic transformation of committed progenitors by MLL-GAS7 was Hoxa7 or Hoxa9-independent. However, considering the high degree of redundancy in HOX function this most likely remains a Hox-dependent transformation. Furthermore, overexpression of the oncogene MOZ-TIF2, but not BCR-ABL, in committed myeloid progenitors such as the common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs), which have no self-renewing capacity, can induce leukemia in mice by imparting self-renewal potential to these progenitors (Huntly et al., 2004). We predict that MOZ-TIF2, like the fusion oncoprotein MOZ-CBP (MYST3-CREBBP), establishes a 'Hox code' conducive to extensive self-renewal (Camos et al., 2006). Indeed, MOZ is a regulator of Hox genes during development (Miller et al., 2004; Crump et al., 2006). The inability of BCR-ABL to turn on the self-renewal program in any progenitor cell population suggests that the cellular target of BCR-ABL-mediated leukemogenesis is the normally self-renewing HSC. The HSC as a cellular target for leukemic transformation has also been described for JunB (Passegue et al., 2004) and STAT5-mediated leukemogenesis (Kato et al., 2005).

More recently, it has been demonstrated that MLL-AF9 leukemia can be initiated in myeloid cells distal to the committed progenitors (Somervaille and Cleary, 2006). These leukemia-initiating cells were relatively abundant and most likely accumulated additional genetic events through the serial BMTs to become fully competent LSCs. Consistent with the hypothesis that Hox genes drive the self-renewal program in LSCs of any origin, Hoxa gene expression was elevated in these lineage positive LSCs. Similarly, it has been shown that the LSC in murine CALM/AF10-positive AML was positive for the lymphoid associated surface antigen B220, raising the possibility that the LSC in a subset of AMLs is a lymphoid progenitor or a myeloid progenitor with lymphoid characteristics (Deshpande et al., 2006). Although the level of Hox gene expression was not reported in these lineage-positive LSCs, it would be of interest to determine if their novel ability to self-renew is a result of dysregulation of Hox gene expression, more precisely, genes of the Hoxa cluster. Indeed, CALM/AF10 is a known upstream regulator of Hoxa gene expression (Dik et al., 2005; Bergeron et al., 2006; Okada et al., 2006).

It is becoming increasingly obvious that the mechanisms underlying HSC self-renewal control extend beyond the function of Hox. For example, the Polycomb group gene Bmi1 has emerged as a crucial regulator of HSC and LSC self-renewal (Lessard and Sauvageau, 2003; Park et al., 2003; Iwama et al., 2004). Bmi1 negatively regulates Hox gene expression during development (van der Lugt et al., 1996), thus it is surprising that its role in self-renewal has not been linked to Hox gene regulation. Rather, Bmi1 controls cellular proliferation by silencing the CDKN2A locus that encodes the cell-cycle inhibitor genes p16Ink4a and p19ARF (Jacobs et al., 1999). Other pathways implicated in HSC self-renewal control include the Wnt (beta-catenin) (Reya et al., 2003), Notch (Varnum-Finney et al., 2000) and Shh (Bhardwaj et al., 2001) signaling pathways. Dissection of these pathways and elucidation of their interplay will be of great interest.

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Toward a mechanistic understanding of Hox genes

On the basis of our current understanding of the role of Hox genes in normal hematopoiesis and leukemogenesis, we hypothesize that a core function of Hox and NUP98-HOX fusions is the ability to initiate and maintain a state of self-renewal that is necessary but not sufficient for the development of overt leukemia.

A major focus of future research will be to explore the mechanism(s) by which Hox genes initiate and/or intensify self-renewal in normal HSCs or in later progenitors and how this may impact on the origin and function of LSCs. Many avenues of investigation remain open. Chief among these are to identify the cell targets within the hematopoietic hierarchy that can respond to Hox pathways and thus give origin to LSCs. The collaborating genes necessary for frank leukemic transformation in concert with Hox remain to be elucidated fully although much progress in this area is being made (Iwasaki et al., 2005; Jin et al., 2007). Crucial for further insight will be the delineation of the target genes regulated by Hox of which there is still little known, and identification of the likely protein interactions, extending beyond the known HOX cofactors, that mediate the transcriptional activity of HOX. Elucidation of these issues will undoubtedly shed light into the mechanism of Hox-induced self-renewal and into the mechanisms underlying the emergence of the LSC in leukemia.

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References

  1. Afonja O, Smith Jr JE, Cheng DM, Goldenberg AS, Amorosi E, Shimamoto T et al. (2000). MEIS1 and HOXA7 genes in human acute myeloid leukemia. Leuk Res 24: 849–855. | Article | PubMed | ISI | ChemPort |
  2. Alcalay M, Tiacci E, Bergomas R, Bigerna B, Venturini E, Minardi SP et al. (2005). Acute myeloid leukemia bearing cytoplasmic nucleophosmin (NPMc+AML) shows a distinct gene expression profile characterized by up-regulation of genes involved in stem-cell maintenance. Blood 106: 899–902. | Article | PubMed | ISI | ChemPort |
  3. Amsellem S, Pflumio F, Bardinet D, Izac B, Charneau P, Romeo PH et al. (2003). Ex vivo expansion of human hematopoietic stem cells by direct delivery of the HOXB4 homeoprotein. Nat Med 9: 1423–1427. | Article | PubMed | ISI | ChemPort |
  4. Antonchuk J, Sauvageau G, Humphries RK. (2001). HOXB4 overexpression mediates very rapid stem cell regeneration and competitive hematopoietic repopulation. Exp Hematol 29: 1125–1134. | Article | PubMed | ISI | ChemPort |
  5. Antonchuk J, Sauvageau G, Humphries RK. (2002). HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell 109: 39–45. | Article | PubMed | ISI | ChemPort |
  6. Armstrong SA, Staunton JE, Silverman LB, Pieters R, den Boer ML, Minden MD et al. (2002). MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia. Nat Genet 30: 41–47. | Article | PubMed | ISI | ChemPort |
  7. Ayton PM, Cleary ML. (2003). Transformation of myeloid progenitors by MLL oncoproteins is dependent on Hoxa7 and Hoxa9. Genes Dev 17: 2298–2307. | Article | PubMed | ISI | ChemPort |
  8. Bai XT, Gu BW, Yin T, Niu C, Xi XD, Zhang J et al. (2006). Trans-repressive effect of NUP98-PMX1 on PMX1-regulated c-FOS gene through recruitment of histone deacetylase 1 by FG repeats. Cancer Res 66: 4584–4590. | Article | PubMed | ISI | ChemPort |
  9. Bansal D, Scholl C, Frohling S, McDowell E, Lee BH, Dohner K et al. (2006). Cdx4 dysregulates Hox gene expression and generates acute myeloid leukemia alone and in cooperation with Meis1a in a murine model. Proc Natl Acad Sci USA 103: 16924–16929. | Article | PubMed | ChemPort |
  10. Bergeron J, Clappier E, Cauwelier B, Dastugue N, Millien C, Delabesse E et al. (2006). HOXA cluster deregulation in T-ALL associated with both a TCRD-HOXA and a CALM-AF10 chromosomal translocation. Leukemia 20: 1184–1187. | Article | PubMed | ISI | ChemPort |
  11. Beslu N, Krosl J, Laurin M, Mayotte N, Humphries KR, Sauvageau G. (2004). Molecular interactions involved in HOXB4-induced activation of HSC self-renewal. Blood 104: 2307–2314. | Article | PubMed | ISI | ChemPort |
  12. Bhardwaj G, Murdoch B, Wu D, Baker DP, Williams KP, Chadwick K et al. (2001). Sonic hedgehog induces the proliferation of primitive human hematopoietic cells via BMP regulation. Nat Immunol 2: 172–180. | Article | PubMed | ISI | ChemPort |
  13. Bijl J, Thompson A, Ramirez-Solis R, Krosl J, Grier DG, Lawrence HJ et al. (2006). Analysis of HSC activity and compensatory Hox gene expression profile in Hoxb cluster mutant fetal liver cells. Blood 108: 116–122. | Article | PubMed | ISI | ChemPort |
  14. Bjornsson JM, Larsson N, Brun AC, Magnusson M, Andersson E, Lundstrom P et al. (2003). Reduced proliferative capacity of hematopoietic stem cells deficient in Hoxb3 and Hoxb4. Mol Cell Biol 23: 3872–3883. | Article | PubMed | ISI | ChemPort |
  15. Blair A, Hogge DE, Sutherland HJ. (1998). Most acute myeloid leukemia progenitor cells with long-term proliferative ability in vitro and in vivo have the phenotype CD34(+)/CD71(-)/HLA-DR. Blood 92: 4325–4335. | PubMed | ISI | ChemPort |
  16. Bonnet D, Dick JE. (1997). Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 3: 730–737. | Article | PubMed | ISI | ChemPort |
  17. Brun AC, Bjornsson JM, Magnusson M, Larsson N, Leveen P, Ehinger M et al. (2004). Hoxb4-deficient mice undergo normal hematopoietic development but exhibit a mild proliferation defect in hematopoietic stem cells. Blood 103: 4126–4133. | Article | PubMed | ISI | ChemPort |
  18. Buske C, Feuring-Buske M, Abramovich C, Spiekermann K, Eaves CJ, Coulombel L et al. (2002). Deregulated expression of HOXB4 enhances the primitive growth activity of human hematopoietic cells. Blood 100: 862–868. | Article | PubMed | ISI | ChemPort |
  19. Calvo KR, Sykes DB, Pasillas MP, Kamps MP. (2002). Nup98-HoxA9 immortalizes myeloid progenitors, enforces expression of Hoxa9, Hoxa7 and Meis1, and alters cytokine-specific responses in a manner similar to that induced by retroviral co-expression of Hoxa9 and Meis1. Oncogene 21: 4247–4256. | Article | PubMed | ISI | ChemPort |
  20. Camos M, Esteve J, Jares P, Colomer D, Rozman M, Villamor N et al. (2006). Gene expression profiling of acute myeloid leukemia with translocation t(8;16)(p11;p13) and MYST3-CREBBP rearrangement reveals a distinctive signature with a specific pattern of HOX gene expression. Cancer Res 66: 6947–6954. | Article | PubMed | ISI | ChemPort |
  21. Cauwelier B, Cave H, Gervais C, Lessard M, Barin C, Perot C et al. (2007). Clinical, cytogenetic and molecular characteristics of 14 T-ALL patients carrying the TCRbeta-HOXA rearrangement: a study of the Groupe Francophone de Cytogenetique Hematologique. Leukemia 21: 121–128. | Article | PubMed | ISI | ChemPort |
  22. Cellot S, Krosl J, Chagraoui J, Meloche S, Humphries RK, Sauvageau G. (2007). Sustained in vitro trigger of self-renewal divisions in Hoxb4hiPbxl(10) hematopoietic stem cells. Exp Hematol 35: 802–816. | Article | PubMed | ISI | ChemPort |
  23. Chen F, Capecchi MR. (1997). Targeted mutations in hoxa-9 and hoxb-9 reveal synergistic interactions. Dev Biol 181: 186–196. | Article | PubMed | ISI | ChemPort |
  24. Chung KY, Morrone G, Schuringa JJ, Plasilova M, Shieh JH, Zhang Y et al. (2006). Enforced expression of NUP98-HOXA9 in human CD34(+) cells enhances stem cell proliferation. Cancer Res 66: 11781–11791. | Article | PubMed | ISI | ChemPort |
  25. Cozzio A, Passegue E, Ayton PM, Karsunky H, Cleary ML, Weissman IL. (2003). Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors. Genes Dev 17: 3029–3035. | Article | PubMed | ISI | ChemPort |
  26. Crump JG, Swartz ME, Eberhart JK, Kimmel CB. (2006). Moz-dependent Hox expression controls segment-specific fate maps of skeletal precursors in the face. Development 133: 2661–2669. | Article | PubMed | ISI | ChemPort |
  27. Daser A, Rabbitts TH. (2005). The versatile mixed lineage leukaemia gene MLL and its many associations in leukaemogenesis. Semin Cancer Biol 15: 175–188. | Article | PubMed | ISI | ChemPort |
  28. Davidson AJ, Ernst P, Wang Y, Dekens MP, Kingsley PD, Palis J et al. (2003). cdx4 mutants fail to specify blood progenitors and can be rescued by multiple hox genes. Nature 425: 300–306. | Article | PubMed | ISI | ChemPort |
  29. Davidson AJ, Zon LI. (2006). The caudal-related homeobox genes cdx1a and cdx4 act redundantly to regulate hox gene expression and the formation of putative hematopoietic stem cells during zebrafish embryogenesis. Dev Biol 292: 506–518. | Article | PubMed | ISI | ChemPort |
  30. De Braekeleer M, Morel F, Le Bris MJ, Herry A, Douet-Guilbert N. (2005). The MLL gene and translocations involving chromosomal band 11q23 in acute leukemia. Anticancer Res 25: 1931–1944. | PubMed | ISI | ChemPort |
  31. Deshpande AJ, Cusan M, Rawat VP, Reuter H, Krause A, Pott C et al. (2006). Acute myeloid leukemia is propagated by a leukemic stem cell with lymphoid characteristics in a mouse model of CALM/AF10-positive leukemia. Cancer Cell 10: 363–374. | Article | PubMed | ISI | ChemPort |
  32. Dik WA, Brahim W, Braun C, Asnafi V, Dastugue N, Bernard OA et al. (2005). CALM-AF10+ T-ALL expression profiles are characterized by overexpression of HOXA and BMI1 oncogenes. Leukemia 19: 1948–1957. | Article | PubMed | ISI | ChemPort |
  33. DiMartino JF, Selleri L, Traver D, Firpo MT, Rhee J, Warnke R et al. (2001). The Hox cofactor and proto-oncogene Pbx1 is required for maintenance of definitive hematopoiesis in the fetal liver. Blood 98: 618–626. | Article | PubMed | ISI | ChemPort |
  34. Drabkin HA, Parsy C, Ferguson K, Guilhot F, Lacotte L, Roy L et al. (2002). Quantitative HOX expression in chromosomally defined subsets of acute myelogenous leukemia. Leukemia 16: 186–195. | Article | PubMed | ISI | ChemPort |
  35. Ekker SC, Jackson DG, von Kessler DP, Sun BI, Young KE, Beachy PA. (1994). The degree of variation in DNA sequence recognition among four Drosophila homeotic proteins. EMBO J 13: 3551–3560. | PubMed | ISI | ChemPort |
  36. Ernst P, Mabon M, Davidson AJ, Zon LI, Korsmeyer SJ. (2004). An Mll-dependent Hox program drives hematopoietic progenitor expansion. Curr Biol 14: 2063–2069. | Article | PubMed | ISI | ChemPort |
  37. Ferrando AA, Armstrong SA, Neuberg DS, Sallan SE, Silverman LB, Korsmeyer SJ et al. (2003). Gene expression signatures in MLL-rearranged T-lineage and B-precursor acute leukemias: dominance of HOX dysregulation. Blood 102: 262–268. | Article | PubMed | ISI | ChemPort |
  38. Fine BM, Stanulla M, Schrappe M, Ho M, Viehmann S, Harbott J et al. (2004). Gene expression patterns associated with recurrent chromosomal translocations in acute lymphoblastic leukemia. Blood 103: 1043–1049. | Article | PubMed | ISI | ChemPort |
  39. Fischbach NA, Rozenfeld S, Shen W, Fong S, Chrobak D, Ginzinger D et al. (2005). HOXB6 overexpression in murine bone marrow immortalizes a myelomonocytic precursor in vitro and causes hematopoietic stem cell expansion and acute myeloid leukemia in vivo. Blood 105: 1456–1466. | Article | PubMed | ISI | ChemPort |
  40. Fuller JF, McAdara J, Yaron Y, Sakaguchi M, Fraser JK, Gasson JC. (1999). Characterization of HOX gene expression during myelopoiesis: role of HOX A5 in lineage commitment and maturation. Blood 93: 3391–3400. | PubMed | ISI | ChemPort |
  41. Gehring WJ, Affolter M, Burglin T. (1994). Homeodomain proteins. Annu Rev Biochem 63: 487–526. | Article | PubMed | ISI | ChemPort |
  42. Ghannam G, Takeda A, Camarata T, Moore MA, Viale A, Yaseen NR. (2004). The oncogene Nup98-HOXA9 induces gene transcription in myeloid cells. J Biol Chem 279: 866–875. | Article | PubMed | ISI | ChemPort |
  43. Giampaolo A, Pelosi E, Valtieri M, Montesoro E, Sterpetti P, Samoggia P et al. (1995). HOXB gene expression and function in differentiating purified hematopoietic progenitors. Stem Cells 13(Suppl 1): 90–105. | PubMed | ISI | ChemPort |
  44. Giampaolo A, Sterpetti P, Bulgarini D, Samoggia P, Pelosi E, Valtieri M et al. (1994). Key functional role and lineage-specific expression of selected HOXB genes in purified hematopoietic progenitor differentiation. Blood 84: 3637–3647. | PubMed | ISI | ChemPort |
  45. Golub TR, Slonim DK, Tamayo P, Huard C, Gaasenbeek M, Mesirov JP et al. (1999). Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science 286: 531–537. | Article | PubMed | ISI | ChemPort |
  46. Greer JM, Puetz J, Thomas KR, Capecchi MR. (2000). Maintenance of functional equivalence during paralogous Hox gene evolution. Nature 403: 661–665. | Article | PubMed | ISI | ChemPort |
  47. Grier DG, Thompson A, Kwasniewska A, McGonigle GJ, Halliday HL, Lappin TR. (2005). The pathophysiology of HOX genes and their role in cancer. J Pathol 205: 154–171. | Article | PubMed | ISI | ChemPort |
  48. Hess JL, Bittner CB, Zeisig DT, Bach C, Fuchs U, Borkhardt A et al. (2006). c-Myb is an essential downstream target for homeobox-mediated transformation of hematopoietic cells. Blood 108: 297–304. | Article | PubMed | ISI | ChemPort |
  49. Hisa T, Spence SE, Rachel RA, Fujita M, Nakamura T, Ward JM et al. (2004). Hematopoietic, angiogenic and eye defects in Meis1 mutant animals. EMBO J 23: 450–459. | Article | PubMed | ISI | ChemPort |
  50. Hoey T, Levine M. (1988). Divergent homeo box proteins recognize similar DNA sequences in Drosophila. Nature 332: 858–861. | Article | PubMed | ISI | ChemPort |
  51. Hope KJ, Jin L, Dick JE. (2004). Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity. Nat Immunol 5: 738–743. | Article | PubMed | ISI | ChemPort |
  52. Horan GS, Kovacs EN, Behringer RR, Featherstone MS. (1995a). Mutations in paralogous Hox genes result in overlapping homeotic transformations of the axial skeleton: evidence for unique and redundant function. Dev Biol 169: 359–372. | Article | ISI | ChemPort |
  53. Horan GS, Ramirez-Solis R, Featherstone MS, Wolgemuth DJ, Bradley A, Behringer RR. (1995b). Compound mutants for the paralogous hoxa-4, hoxb-4, and hoxd-4 genes show more complete homeotic transformations and a dose-dependent increase in the number of vertebrae transformed. Genes Dev 9: 1667–1677. | Article | PubMed | ISI | ChemPort |
  54. Horton SJ, Grier DG, McGonigle GJ, Thompson A, Morrow M, De Silva I et al. (2005). Continuous MLL-ENL expression is necessary to establish a 'Hox Code' and maintain immortalization of hematopoietic progenitor cells. Cancer Res 65: 9245–9252. | Article | PubMed | ISI | ChemPort |
  55. Huntly BJ, Shigematsu H, Deguchi K, Lee BH, Mizuno S, Duclos N et al. (2004). MOZ-TIF2, but not BCR-ABL, confers properties of leukemic stem cells to committed murine hematopoietic progenitors. Cancer Cell 6: 587–596. | Article | PubMed | ISI | ChemPort |
  56. Imamura T, Morimoto A, Takanashi M, Hibi S, Sugimoto T, Ishii E et al. (2002). Frequent co-expression of HoxA9 and Meis1 genes in infant acute lymphoblastic leukaemia with MLL rearrangement. Br J Haematol 119: 119–121. | Article | PubMed | ISI | ChemPort |
  57. Iwama A, Oguro H, Negishi M, Kato Y, Morita Y, Tsukui H et al. (2004). Enhanced self-renewal of hematopoietic stem cells mediated by the polycomb gene product Bmi-1. Immunity 21: 843–851. | Article | PubMed | ISI | ChemPort |
  58. Iwasaki M, Kuwata T, Yamazaki Y, Jenkins NA, Copeland NG, Osato M et al. (2005). Identification of cooperative genes for NUP98-HOXA9 in myeloid leukemogenesis using a mouse model. Blood 105: 784–793. | Article | PubMed | ISI | ChemPort |
  59. Izon DJ, Rozenfeld S, Fong ST, Komuves L, Largman C, Lawrence HJ. (1998). Loss of function of the homeobox gene Hoxa-9 perturbs early T-cell development and induces apoptosis in primitive thymocytes. Blood 92: 383–393. | PubMed | ISI | ChemPort |
  60. Jacobs JJ, Kieboom K, Marino S, DePinho RA, van Lohuizen M. (1999). The oncogene and polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus. Nature 397: 164–168. | Article | PubMed | ISI | ChemPort |
  61. Jin G, Yamazaki Y, Takuwa M, Takahara T, Kaneko K, Kuwata T et al. (2007). Trib1 and Evi1 cooperate with Hoxa and Meis1 in myeloid leukemogenesis. Blood 109: 3998–4005. | Article | PubMed | ISI | ChemPort |
  62. Kappen C. (2000). Disruption of the homeobox gene Hoxb-6 in mice results in increased numbers of early erythrocyte progenitors. Am J Hematol 65: 111–118. | Article | PubMed | ISI | ChemPort |
  63. Kasper LH, Brindle PK, Schnabel CA, Pritchard CE, Cleary ML, van Deursen JM. (1999). CREB binding protein interacts with nucleoporin-specific FG repeats that activate transcription and mediate NUP98-HOXA9 oncogenicity. Mol Cell Biol 19: 764–776. | PubMed | ISI | ChemPort |
  64. Kato Y, Iwama A, Tadokoro Y, Shimoda K, Minoguchi M, Akira S et al. (2005). Selective activation of STAT5 unveils its role in stem cell self-renewal in normal and leukemic hematopoiesis. J Exp Med 202: 169–179. | Article | PubMed | ISI | ChemPort |
  65. Kawagoe H, Humphries RK, Blair A, Sutherland HJ, Hogge DE. (1999). Expression of HOX genes, HOX cofactors, and MLL in phenotypically and functionally defined subpopulations of leukemic and normal human hematopoietic cells. Leukemia 13: 687–698. | Article | PubMed | ISI | ChemPort |
  66. Ko KH, Lam QL, Zhang M, Wong CK, Lo CK, Kahmeyer-Gabbe M et al. (2007). Hoxb3 deficiency impairs B lymphopoiesis in mouse bone marrow. Exp Hematol 35: 465–475. | Article | PubMed | ISI | ChemPort |
  67. Kohlmann A, Schoch C, Schnittger S, Dugas M, Hiddemann W, Kern W et al. (2003). Molecular characterization of acute leukemias by use of microarray technology. Genes Chromosomes Cancer 37: 396–405. | Article | PubMed | ISI | ChemPort |
  68. Krivtsov AV, Twomey D, Feng Z, Stubbs MC, Wang Y, Faber J et al. (2006). Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature 442: 818–822. | Article | PubMed | ISI | ChemPort |
  69. Kroon E, Krosl J, Thorsteinsdottir U, Baban S, Buchberg AM, Sauvageau G. (1998). Hoxa9 transforms primary bone marrow cells through specific collaboration with Meis1a but not Pbx1b. EMBO J 17: 3714–3725. | Article | PubMed | ISI | ChemPort |
  70. Kroon E, Thorsteinsdottir U, Mayotte N, Nakamura T, Sauvageau G. (2001). NUP98-HOXA9 expression in hemopoietic stem cells induces chronic and acute myeloid leukemias in mice. EMBO J 20: 350–361. | Article | PubMed | ISI | ChemPort |
  71. Krosl J, Beslu N, Mayotte N, Humphries RK, Sauvageau G. (2003). The competitive nature of HOXB4-transduced HSC is limited by PBX1: the generation of ultra-competitive stem cells retaining full differentiation potential. Immunity 18: 561–571. | Article | PubMed | ISI | ChemPort |
  72. Krumlauf R. (1994). Hox genes in vertebrate development. Cell 78: 191–201. | Article | PubMed | ISI | ChemPort |
  73. Kumar AR, Hudson WA, Chen W, Nishiuchi R, Yao Q, Kersey JH. (2004). Hoxa9 influences the phenotype but not the incidence of Mll-AF9 fusion gene leukemia. Blood 103: 1823–1828. | Article | PubMed | ISI | ChemPort |
  74. Lam DH, Aplan PD. (2001). NUP98 gene fusions in hematologic malignancies. Leukemia 15: 1689–1695. | Article | PubMed | ISI | ChemPort |
  75. Lawrence HJ, Christensen J, Fong S, Hu YL, Weissman I, Sauvageau G et al. (2005). Loss of expression of the Hoxa-9 homeobox gene impairs the proliferation and repopulating ability of hematopoietic stem cells. Blood 106: 3988–3994. | Article | PubMed | ISI | ChemPort |
  76. Lawrence HJ, Helgason CD, Sauvageau G, Fong S, Izon DJ, Humphries RK et al. (1997). Mice bearing a targeted interruption of the homeobox gene HOXA9 have defects in myeloid, erythroid, and lymphoid hematopoiesis. Blood 89: 1922–1930. | PubMed | ISI | ChemPort |
  77. Lawrence HJ, Rozenfeld S, Cruz C, Matsukuma K, Kwong A, Komuves L et al. (1999). Frequent co-expression of the HOXA9 and MEIS1 homeobox genes in human myeloid leukemias. Leukemia 13: 1993–1999. | Article | PubMed | ISI | ChemPort |
  78. Lawrence HJ, Sauvageau G, Humphries RK, Largman C. (1996). The role of HOX homeobox genes in normal and leukemic hematopoiesis. Stem Cells 14: 281–291. | PubMed | ISI | ChemPort |
  79. Lessard J, Sauvageau G. (2003). Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature 423: 255–260. | Article | PubMed | ISI | ChemPort |
  80. Lin YW, Slape C, Zhang Z, Aplan PD. (2005). NUP98-HOXD13 transgenic mice develop a highly penetrant, severe myelodysplastic syndrome that progresses to acute leukemia. Blood 106: 287–295. | Article | PubMed | ISI | ChemPort |
  81. Mamo A, Krosl J, Kroon E, Bijl J, Thompson A, Mayotte N et al. (2006). Molecular dissection of Meis1 reveals 2 domains required for leukemia induction and a key role for Hoxa gene activation. Blood 108: 622–629. | Article | PubMed | ISI | ChemPort |
  82. Mann RS. (1995). The specificity of homeotic gene function. BioEssays 17: 855–863. | Article | PubMed | ISI | ChemPort |
  83. Matsui W, Huff CA, Wang Q, Malehorn MT, Barber J, Tanhehco Y et al. (2004). Characterization of clonogenic multiple myeloma cells. Blood 103: 2332–2336. | Article | PubMed | ISI | ChemPort |
  84. Miller CT, Maves L, Kimmel CB. (2004). moz regulates Hox expression and pharyngeal segmental identity in zebrafish. Development 131: 2443–2461. | Article | PubMed | ISI | ChemPort |
  85. Miyamoto T, Weissman IL, Akashi K. (2000). AML1/ETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 8;21 chromosomal translocation. Proc Natl Acad Sci USA 97: 7521–7526. | Article | PubMed | ChemPort |
  86. Moens CB, Selleri L. (2006). Hox cofactors in vertebrate development. Dev Biol 291: 193–206. | Article | PubMed | ISI | ChemPort |
  87. Moretti P, Simmons P, Thomas P, Haylock D, Rathjen P, Vadas M et al. (1994). Identification of homeobox genes expressed in human haemopoietic progenitor cells. Gene 144: 213–219. | Article | PubMed | ISI | ChemPort |
  88. Morgado E, Albouhair S, Lavau C. (2007). Flt3 is dispensable to the Hoxa9/Meis1 leukemogenic cooperation. Blood 109: 4020–4022. | Article | PubMed | ISI | ChemPort |
  89. Moskow JJ, Bullrich F, Huebner K, Daar IO, Buchberg AM. (1995). Meis1, a PBX1-related homeobox gene involved in myeloid leukemia in BXH-2 mice. Mol Cell Biol 15: 5434–5443. | PubMed | ISI | ChemPort |
  90. Nakamura T. (2005). NUP98 fusion in human leukemia: dysregulation of the nuclear pore and homeodomain proteins. Int J Hematol 82: 21–27. | Article | PubMed | ISI | ChemPort |
  91. O'Brien CA, Pollett A, Gallinger S, Dick JE. (2007). A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445: 106–110. | Article | PubMed | ISI | ChemPort |
  92. Ohta H, Sekulovic S, Bakovic S, Eaves CJ, Pineault N, Gasparetto M et al. (2007). Near maximal-expansions of hematopoietic stem cells in culture using NUP98-HOX fusions. Exp Hematol 35: 817–830. | Article | PubMed | ISI | ChemPort |
  93. Okada Y, Jiang Q, Lemieux M, Jeannotte L, Su L, Zhang Y. (2006). Leukaemic transformation by CALM-AF10 involves upregulation of Hoxa5 by hDOT1L. Nat Cell Biol 8: 1017–1024. | Article | PubMed | ISI | ChemPort |
  94. Owens BM, Hawley RG. (2002). HOX and non-HOX homeobox genes in leukemic hematopoiesis. Stem Cells 20: 364–379. | Article | PubMed | ISI | ChemPort |
  95. Ozeki K, Kiyoi H, Hirose Y, Iwai M, Ninomiya M, Kodera Y et al. (2004). Biologic and clinical significance of the FLT3 transcript level in acute myeloid leukemia. Blood 103: 1901–1908. | Article | PubMed | ISI | ChemPort |
  96. Palmqvist L, Argiropoulos B, Pineault N, Abramovich C, Sly LM, Krystal G et al. (2006). The Flt3 receptor tyrosine kinase collaborates with NUP98-HOX fusions in acute myeloid leukemia. Blood 108: 1030–1036. | Article | PubMed | ISI | ChemPort |
  97. Park IK, Qian D, Kiel M, Becker MW, Pihalja M, Weissman IL et al. (2003). Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature 423: 302–305. | Article | PubMed | ISI | ChemPort |
  98. Passegue E, Wagner EF, Weissman IL. (2004). JunB deficiency leads to a myeloproliferative disorder arising from hematopoietic stem cells. Cell 119: 431–443. | Article | PubMed | ISI | ChemPort |
  99. Pineault N, Abramovich C, Humphries RK. (2005). Transplantable cell lines generated with NUP98-Hox fusion genes undergo leukemic progression by Meis1 independent of its binding to DNA. Leukemia 19: 636–643. | Article | PubMed | ISI | ChemPort |
  100. Pineault N, Abramovich C, Ohta H, Humphries RK. (2004). Differential and common leukemogenic potentials of multiple NUP98-Hox fusion proteins alone or with Meis1. Mol Cell Biol 24: 1907–1917. | Article | PubMed | ISI | ChemPort |
  101. Pineault N, Buske C, Feuring-Buske M, Abramovich C, Rosten P, Hogge DE et al. (2003). Induction of acute myeloid leukemia in mice by the human leukemia-specific fusion gene NUP98-HOXD13 in concert with Meis1. Blood 101: 4529–4538. | Article | PubMed | ISI | ChemPort |
  102. Pineault N, Helgason CD, Lawrence HJ, Humphries RK. (2002). Differential expression of Hox, Meis1, and Pbx1 genes in primitive cells throughout murine hematopoietic ontogeny. Exp Hematol 30: 49–57. | Article | PubMed | ISI | ChemPort |
  103. Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G, Coradini D et al. (2005). Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res 65: 5506–5511. | Article | PubMed | ISI | ChemPort |
  104. Quentmeier H, Dirks WG, Macleod RA, Reinhardt J, Zaborski M, Drexler HG. (2004). Expression of HOX genes in acute leukemia cell lines with and without MLL translocations. Leuk Lymphoma 45: 567–574. | Article | PubMed | ISI | ChemPort |
  105. Reya T, Duncan AW, Ailles L, Domen J, Scherer DC, Willert K et al. (2003). A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 423: 409–414. | Article | PubMed | ISI | ChemPort |
  106. Rozovskaia T, Feinstein E, Mor O, Foa R, Blechman J, Nakamura T et al. (2001). Upregulation of Meis1 and HoxA9 in acute lymphocytic leukemias with the t(4:11) abnormality. Oncogene 20: 874–878. | Article | PubMed | ISI | ChemPort |
  107. Satoh Y, Matsumura I, Tanaka H, Ezoe S, Sugahara H, Mizuki M et al. (2004). Roles for c-Myc in self-renewal of hematopoietic stem cells. J Biol Chem 279: 24986–24993. | Article | PubMed | ISI | ChemPort |
  108. Sauvageau G, Lansdorp PM, Eaves CJ, Hogge DE, Dragowska WH, Reid DS et al. (1994). Differential expression of homeobox genes in functionally distinct CD34+ subpopulations of human bone marrow cells. Proc Natl Acad Sci USA 91: 12223–12227. | Article | PubMed | ChemPort |
  109. Sauvageau G, Thorsteinsdottir U, Eaves CJ, Lawrence HJ, Largman C, Lansdorp PM et al. (1995). Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo. Genes Dev 9: 1753–1765. | Article | PubMed | ISI | ChemPort |
  110. Sauvageau G, Thorsteinsdottir U, Hough MR, Hugo P, Lawrence HJ, Largman C et al. (1997). Overexpression of HOXB3 in hematopoietic cells causes defective lymphoid development and progressive myeloproliferation. Immunity 6: 13–22. | Article | PubMed | ISI | ChemPort |
  111. Schreiner S, Birke M, Garcia-Cuellar MP, Zilles O, Greil J, Slany RK. (2001). MLL-ENL causes a reversible and myc-dependent block of myelomonocytic cell differentiation. Cancer Res 61: 6480–6486. | PubMed | ISI | ChemPort |
  112. Schuettengruber B, Chourrout D, Vervoort M, Leblanc B, Cavalli G. (2007). Genome regulation by polycomb and trithorax proteins. Cell 128: 735–745. | Article | PubMed | ISI | ChemPort |
  113. Shen WF, Detmer K, Mathews CH, Hack FM, Morgan DA, Largman C et al. (1992). Modulation of homeobox gene expression alters the phenotype of human hematopoietic cell lines. EMBO J 11: 983–989. | PubMed | ISI | ChemPort |
  114. Shen WF, Montgomery JC, Rozenfeld S, Moskow JJ, Lawrence HJ, Buchberg AM et al. (1997). AbdB-like Hox proteins stabilize DNA binding by the Meis1 homeodomain proteins. Mol Cell Biol 17: 6448–6458. | PubMed | ISI | ChemPort |
  115. Shimamoto T, Tang Y, Naot Y, Nardi M, Brulet P, Bieberich CJ et al. (1999). Hematopoietic progenitor cell abnormalities in Hoxc-8 null mutant mice. J Exp Zool 283: 186–193. | Article | PubMed | ISI | ChemPort |
  116. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T et al. (2004). Identification of human brain tumour initiating cells. Nature 432: 396–401. | Article | PubMed | ISI | ChemPort |
  117. Slape C, Aplan PD. (2004). The role of NUP98 gene fusions in hematologic malignancy. Leuk Lymphoma 45: 1341–1350. | Article | PubMed | ISI | ChemPort |
  118. So CW, Karsunky H, Wong P, Weissman IL, Cleary ML. (2004). Leukemic transformation of hematopoietic progenitors by MLL-GAS7 in the absence of Hoxa7 or Hoxa9. Blood 103: 3192–3199. | Article | PubMed | ISI | ChemPort |
  119. Somervaille TC, Cleary ML. (2006). Identification and characterization of leukemia stem cells in murine MLL-AF9 acute myeloid leukemia. Cancer Cell 10: 257–268. | Article | PubMed | ISI | ChemPort |
  120. Soulier J, Clappier E, Cayuela JM, Regnault A, Garcia-Peydro M, Dombret H et al. (2005). HOXA genes are included in genetic and biologic networks defining human acute T-cell leukemia (T-ALL). Blood 106: 274–286. | Article | PubMed | ISI | ChemPort |
  121. Speleman F, Cauwelier B, Dastugue N, Cools J, Verhasselt B, Poppe B et al. (2005). A new recurrent inversion, inv(7)(p15q34), leads to transcriptional activation of HOXA10 and HOXA11 in a subset of T-cell acute lymphoblastic leukemias. Leukemia 19: 358–366. | Article | PubMed | ISI | ChemPort |
  122. Suemori H, Noguchi S. (2000). Hox C cluster genes are dispensable for overall body plan of mouse embryonic development. Dev Biol 220: 333–342. | Article | PubMed | ISI | ChemPort |
  123. Taussig DC, Pearce DJ, Simpson C, Rohatiner AZ, Lister TA, Kelly G et al. (2005). Hematopoietic stem cells express multiple myeloid markers: implications for the origin and targeted therapy of acute myeloid leukemia. Blood 106: 4086–4092. | Article | PubMed | ISI | ChemPort |
  124. Thorsteinsdottir U, Kroon E, Jerome L, Blasi F, Sauvageau G. (2001). Defining roles for HOX and MEIS1 genes in induction of acute myeloid leukemia. Mol Cell Biol 21: 224–234. | Article | PubMed | ISI | ChemPort |
  125. Thorsteinsdottir U, Mamo A, Kroon E, Jerome L, Bijl J, Lawrence HJ et al. (2002). Overexpression of the myeloid leukemia-associated Hoxa9 gene in bone marrow cells induces stem cell expansion. Blood 99: 121–129. | Article | PubMed | ISI | ChemPort |
  126. Thorsteinsdottir U, Sauvageau G, Hough MR, Dragowska W, Lansdorp PM, Lawrence HJ et al. (1997). Overexpression of HOXA10 in murine hematopoietic cells perturbs both myeloid and lymphoid differentiation and leads to acute myeloid leukemia. Mol Cell Biol 17: 495–505. | PubMed | ISI | ChemPort |
  127. Thorsteinsdottir U, Sauvageau G, Humphries RK. (1999). Enhanced in vivo regenerative potential of HOXB4-transduced hematopoietic stem cells with regulation of their pool size. Blood 94: 2605–2612. | PubMed | ISI | ChemPort |
  128. Tsutsumi S, Taketani T, Nishimura K, Ge X, Taki T, Sugita K et al. (2003). Two distinct gene expression signatures in pediatric acute lymphoblastic leukemia with MLL rearrangements. Cancer Res 63: 4882–4887. | PubMed | ISI | ChemPort |
  129. Tupler R, Perini G, Green MR. (2001). Expressing the human genome. Nature 409: 832–833. | Article | PubMed | ISI | ChemPort |
  130. Uchida N, Dykstra B, Lyons KJ, Leung FY, Eaves CJ. (2003). Different in vivo repopulating activities of purified hematopoietic stem cells before and after being stimulated to divide in vitro with the same kinetics. Exp Hematol 31: 1338–1347. | Article | PubMed | ISI | ChemPort |
  131. van der Lugt NM, Alkema M, Berns A, Deschamps J. (1996). The polycomb-group homolog Bmi-1 is a regulator of murine Hox gene expression. Mech Dev 58: 153–164. | Article | PubMed | ChemPort |
  132. Varnum-Finney B, Xu L, Brashem-Stein C, Nourigat C, Flowers D, Bakkour S et al. (2000). Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling. Nat Med 6: 1278–1281. | Article | PubMed | ISI | ChemPort |
  133. Wang GG, Pasillas MP, Kamps MP. (2005). Meis1 programs transcription of FLT3 and cancer stem cell character, using a mechanism that requires interaction with Pbx and a novel function of the Meis1 C-terminus. Blood 106: 254–264. | Article | PubMed | ISI | ChemPort |
  134. Wang GG, Pasillas MP, Kamps MP. (2006). Persistent transactivation by meis1 replaces hox function in myeloid leukemogenesis models: evidence for co-occupancy of meis1-pbx and hox-pbx complexes on promoters of leukemia-associated genes. Mol Cell Biol 26: 3902–3916. | Article | PubMed | ISI | ChemPort |
  135. Xin L, Lawson DA, Witte ON. (2005). The Sca-1 cell surface marker enriches for a prostate-regenerating cell subpopulation that can initiate prostate tumorigenesis. Proc Natl Acad Sci USA 102: 6942–6947. | Article | PubMed | ChemPort |
  136. Yeoh EJ, Ross ME, Shurtleff SA, Williams WK, Patel D, Mahfouz R et al. (2002). Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling. Cancer Cell 1: 133–143. | Article | PubMed | ISI | ChemPort |
  137. Zakany J, Fromental-Ramain C, Warot X, Duboule D. (1997). Regulation of number and size of digits by posterior Hox genes: a dose-dependent mechanism with potential evolutionary implications. Proc Natl Acad Sci USA 94: 13695–13700. | Article | PubMed | ChemPort |
  138. Zakany J, Gerard M, Favier B, Potter SS, Duboule D. (1996). Functional equivalence and rescue among group 11 Hox gene products in vertebral patterning. Dev Biol 176: 325–328. | Article | PubMed | ISI | ChemPort |
  139. Zeisig BB, Milne T, Garcia-Cuellar MP, Schreiner S, Martin ME, Fuchs U et al. (2004). Hoxa9 and Meis1 are key targets for MLL-ENL-mediated cellular immortalization. Mol Cell Biol 24: 617–628. | Article | PubMed | ISI | ChemPort |
  140. Zhang XB, Beard BC, Beebe K, Storer B, Humphries RK, Kiem HP. (2006). Differential effects of HOXB4 on nonhuman primate short- and long-term repopulating cells. PLoS Med 3: e173. | Article | PubMed | ChemPort |
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Acknowledgements

We apologize to authors whose work could not be cited due to space constraints. We acknowledge all members of our laboratory for stimulating discussions. We also thank Kim Gall for critical reading of the manuscript. Our work cited is supported by grants from the National Cancer Institute of Canada with funds from the Terry Fox Foundation, Genome Canada/BC, the Canadian NCE Stem Cell Network and the National Institutes of Health. BA is a recipient of a postdoctoral fellowship from the Leukemia Research Fund of Canada.