Dangerous liaisons: cooperation between Pbx3, Meis1 and Hoxa9 in leukemia

Homeobox (HOX) genes have a longstanding association with human acute leukemias. In particular, high expression of the HOXA9 gene is a highly significant marker of poor prognosis in acute myeloid leukemia,1 and dysregulation of HOXA9 appears to play a central role in several distinct leukemias. These include acute myeloid leukemia and acute lymphoblastic leukemias caused by translocations of the Mixed Lineage Leukemia (MLL) gene,42 fusions of the HOXA9 gene that produce a novel HOXA9-NUP98 fusion protein in acute myeloid leukemia,5 and T-cell acute lymphoblastic leukemias that have translocations between the TCRβ and HOXA9/A10 loci.6 Interestingly however, despite this seemingly central role in a subset of acute leukemias, Hoxa9 expression alone is only weakly oncogenic in mouse leukemia models and usually requires a second “hit” via overexpression of Meis1,87 or in some cases Pbx3.9 Much work has been done trying to understand the molecular function of the HOXA9 protein. MEIS1 and PBX3 are both members of the TALE (three amino acid loop extension) homeodomain-containing family of proteins and are able to modulate HOXA9 binding to DNA.10 In a rigorous ChIP-seq experiment in mice, Hoxa9 was shown to co-bind with Meis1 at a large number of enhancer regions that control the activity of several key oncogenes,11 but it was unknown if Pbx3 could contribute to this gene regulatory activity. In this issue of Haematologica, Garcia-Cuellar et al. extend our knowledge of this very important protein complex by showing that the TALE protein PBX3 is able to stabilize the TALE protein MEIS1 and contribute to HOXA9-mediated activation of gene targets and subsequent leukemogenesis.12 Understanding the details of how this trimeric protein complex forms and interacts has the potential to aid the development of novel therapeutic inhibitors.13

Normal function of Hox genes

The most famous HOX genes are the clustered Hox genes originally discovered in Drosophila melanogaster, in which they function as developmental regulators of body segment identity specification, along the anterio-posterior axis.14 They similarly control body patterning in mammals, and also have a key role in controlling cell identity and differentiation of hematopoietic stem cells and progenitors.15 Murine developmental studies have shown that Hoxa gene cluster expression is generally high in primitive hematopoietic populations and is subsequently down-regulated in more differentiated bone marrow cells.16

HOX proteins all contain a homeodomain, a protein domain with known DNA binding activity. Because of this, HOX proteins are widely regarded to be transcription factors. Direct evidence for their role in transcriptional regulation comes from the fact that both Hoxa9 and Meis1 have been shown to bind directly to enhancer regions, and inactivation of these proteins can cause both up-regulation and down-regulation of their target genes.11 However, it is also worth noting that evidence exists for HOX function that does not require DNA binding. They have been shown to interfere with CBP-mediated transcriptional activation (via interactions with the HOX homeodomain) by blocking histone acetyltransferase activity.17 Additionally, Hoxa9 can regulate hematopoietic stem cell and progenitor activity through direct down-regulation of the cell-cycle regulator Geminin, via association with a ubiquitin ligase complex.18 Although these alternate mechanisms of HOX molecular function are potentially very interesting, they have not been fully elucidated and most research has focused on the role of HOX proteins in transcriptional regulation.

If HOX proteins function primarily as transcription factors, this raises a potential problem. Individual HOX proteins all have highly conserved homeodomains with very similar DNA binding activities, and yet they often display significantly different phenotypes. This raises an interesting question: if HOX proteins do function primarily as transcription factors, what controls their phenotypic specificity¿

HOXA9 DNA binding and TALE family proteins

Early in vitro DNA binding experiments with HOX proteins identified the cooperation of a PBX family cofactor (extradenticle in fly) that contributes to both the specificity and selectivity of these DNA interactions. Further experiments suggested an interesting model whereby HOX protein-mediated activation/repression of target gene transcription could be switched, by co-binding or absence (respectively) of extradenticle.19 Human HOXA9, MEIS1 and PBX2 were shown to form a trimeric DNA-binding complex, and further studies confirmed an important role of HOXA9/MEIS1 in leukemia.2120 However, exactly which factors control HOXA9 activity in mammalian systems and how much this activity affects normal hematopoiesis versus leukemogenesis is not completely understood.

MEIS1 first became implicated in leukemia as a result of experiments using the BXH2 murine myeloid leukemia model to identify disease genes by proviral tagging. Hoxa9, Hoxa7 and Meis1 were almost always targeted for activation in these leukemias,22 and synergy between Hoxa9 and Meis1 expression is required to produce aggressive leukemia in mice.7 Pbx1 fails to copy this requirement, despite it having a role in maintaining definitive hematopoiesis.23

The discovery that PBX3 (not PBX1 or PBX2) is an important player in HOX-dysregulated leukemias reconciled the apparent primacy of Hoxa9 and Meis1-mediated transformation, with earlier studies that demonstrated Pbx involvement in the Hoxa9 trimeric complex.249 While these studies showed that PBX3 was needed for HOXA9-mediated induction of leukemia, the molecular mechanism for this contribution was not completely understood.

Pbx3 contributes to Hoxa9 leukemogenesis through stabilization of the Meis1 protein

In this issue of Haematologica, Garcia-Cuellar and colleagues make an important step towards elucidating the regulation of Hox function through Hoxa9-Meis1-Pbx3 interactions. They were able to show that a direct interaction with Pbx3 protects Meis1 from ubiquitination and subsequent proteasome-mediated degradation, thus crucially extending the half-life of the Meis1 protein.12 Interestingly, making mutations in the Meis1 protein that disrupt this Pbx3-Meis1 interaction domain (i.e. Meis1Δ for short) abrogated Meis1 ubiquitination and degradation, suggesting that Pbx3 may compete directly with a ubiquitin ligase for binding to the same region of Meis1. However, they went on to show that Meis1 stabilization alone is not sufficient for increased Meis1 activity, as Meis1D mutants were unable to cooperate with Hoxa9 in colony-forming assays or in vivo leukemia assays. They further showed that Pbx3-Meis1 binding is required for an efficient Meis1-Hoxa9 interaction, indicating that the overall effect is that Pbx3 stabilizes Meis1 protein levels, as well as enhancing Meis1-Hoxa9 interactions and subsequent gene regulatory activity12 (see Figure 1). Additionally, when Hoxa9 and Pbx3 are co-expressed, they also cause increased expression of the Meis1 gene, suggesting that there is another layer of cooperation between these factors.

Figure 1.Schematic representation of TALE protein cooperation. Meis1-Pbx3 dimerzation (highlighted in blue) stabilizes Meis1 protein levels and enhances Hox-mediated transformation by increasing transcription of Meis1 target genes such as Flt3 and Trib2. Unbound Meis1 is ubiquitinated and subsequently degraded by the proteasome. Mutating the Pbx interaction domain of Meis1 (Meis1D) also prevents Meis1 ubiquitination. Ub: ubiquitin.

What does all this mean for the function of Hoxa9 and the promotion of leukemogenesis¿ The authors were able to show that while Hoxa9/Pbx3 or Hoxa9 alone are unable to produce leukemia in mice even after 180 days, Hoxa9 expression along with both Pbx3 and Meis1 shows some degree of cooperation. This contrasts with the results of Li et al. who were able to see a cooperative effect between Hoxa9 and Pbx3 without Meis1,9 but this could potentially be explained by differences in expression levels between the two different model systems used. Whatever the final explanation might be, the role of Pbx3 in enhancing Hoxa9/Meis1-mediated leukemogenesis provides novel insights into how these two TALE cofactors mediate the transcriptional function and leukemic role of the Hoxa9 protein.

Conclusions

These findings provide a mechanistic analysis for the long-standing enigma of the trimeric Hoxa9-Meis1-Pbx3 complex in leukemia. They suggest a number of specific molecular mechanisms by which Pbx3 individually supports the function of Meis1, and the complex as a whole, and explore the mechanisms by which Pbx3 may be contributing to HOXA9 molecular function.

In doing so, the work by Garcia-Cuellar and colleagues provides a better understanding of the complex interactions that control HOXA9 protein activity, an entity that plays an important role in a number of severe hematologic malignancies. This knowledge could provide further possible avenues for targeting the HOX-mediated leukemic transcription program. As new therapeutic technologies mature, such as small peptide inhibitors, detailed knowledge of the molecular interaction between oncogenic transcription factors and their cofactors will be essential in the design of novel therapeutics.

Acknowledgments

TAM and RMWT are supported by the Medical Research Council UK.

Footnotes

• Financial and other disclosures provided by the authors using the ICMJE (www.icmje.org) Uniform Format for Disclosure of Competing Interests are available with the full text of this paper at www.haematologica.org.