Ling Bei

HOXA9 activates transcription of the gene encoding gp91Phox during myeloid differentiation.

The CYBB gene encodes gp91Phox; a component of the phagocyte respiratory burst oxidase. CYBB transcription is restricted to myeloid cells differentiated beyond the promyelocyte stage. In undifferentiated myeloid cells, the homeodomain (HD) transcription factor HoxA10 represses CYBB transcription via a cis element in the proximal promoter. During myelopoiesis, phosphorylation of conserved tyrosine residues in the HD decreases HoxA10 binding to this CYBB cis element. In the current studies, we found HoxA9 activates CYBB transcription in differentiated myeloid cells via the same cis element. We find HoxA9-mediated CYBB-transcription requires Pbx1 but is inhibited by Meis1. Additionally, phosphorylation of the conserved HD tyrosines increases HoxA9 binding to the CYBB promoter. The HOXA9 gene is involved in leukemia-associated translocations with the gene encoding Nup98, a nucleopore protein. We find expression of a Nup98-hoxA9 fusion protein blocks HoxA9-induced CYBB transcription in differentiating myeloid cells. In comparison to HoxA9, Nup98-hoxA9 has greater binding affinity for the CYBB cis element, but binding is not altered by HD tyrosine phosphorylation. Therefore, these studies identify CYBB as a common target gene repressed by HoxA10 and activated by HoxA9. These studies also suggest overexpression of Meis1 or Nup98-hoxA9 represses myeloid-specific gene transcription, thereby contributing to differentiation block in leukemogenesis.

Constitutive activation of SHP2 in mice cooperates with ICSBP deficiency to accelerate progression to acute myeloid leukemia

Myeloproliferative disorders (MPDs) are characterized by cytokine hypersensitivity and apoptosis resistance. Development of a block in myeloid differentiation is associated with progression of MPD to acute myeloid leukemia (AML) and portends poor prognosis. Identifying molecular markers of this transition may suggest targets for therapeutic intervention. Interferon consensus sequence binding protein (ICSBP, also known as IRF8) is an interferon-regulatory transcription factor that functions as a leukemia tumor suppressor. In mice, ICSBP deficiency induces an MPD that progresses to AML over time, suggesting that ICSBP deficiency is sufficient for myeloproliferation, but additional genetic lesions are necessary for AML. Since activity of ICSBP is influenced by tyrosine phosphorylation state, we hypothesized that mutations in molecular pathways that regulate this process might synergize with ICSBP deficiency for progression to AML. Consistent with this, we found that constitutive activation of SHP2 protein tyrosine phosphatase synergized with ICSBP haploinsufficiency to facilitate cytokine-induced myeloproliferation, apoptosis resistance, and rapid progression to AML in a murine bone marrow transplantation model. Constitutive SHP2 activation cooperated with ICSBP deficiency to increase the number of progenitors in the bone marrow and myeloid blasts in circulation, indicating a block in differentiation. Since SHP2 activation and ICSBP deficiency may coexist in human myeloid malignancies, our studies have identified a molecular mechanism potentially involved in disease progression in such diseases.

Identification of a HoxA10 Activation Domain Necessary for Transcription of the Gene Encoding β3 Integrin during Myeloid Differentiation

Transcription of the ITGB3 gene, which encodes β3 integrin, increases during myeloid differentiation. αvβ3 integrin mediates adhesion to fibronectin or vitronectin and regulates various aspects of the inflammatory response in mature phagocytes. In these studies, we found that the homeodomain transcription factor HoxA10 interacted with a specific ITGB3 cis element and activated transcription of this gene during myeloid differentiation. We also found that increased fibronectin adhesion in differentiating myeloid cells was dependent upon this HoxA10-induced increase in β3 integrin expression. We determined that activation of ITGB3 transcription required a HoxA10 domain that was not identical to the “hexapeptide” that mediates interaction of Hox and Pbx proteins. This activation domain was also not identical to a previously identified HoxA10 repression domain that mediates interaction with transcriptional co-repressors. Instead, this HoxA10 activation domain had homology to “PQ” protein-protein interaction domains that have been described previously in other transcription factors. Consistent with this, we found that the HoxA10 PQ-like domain recruited the CREB-binding protein (CBP) to the ITGB3 promoter. This was associated with an increase in local histone acetylation in vivo. In immature myeloid cells, we previously determined that HoxA10 repressed transcription of the CYBB and NCF2 genes, which encode the phagocyte oxidase proteins gp91PHOX and p67PHOX, respectively. Therefore, our studies indicated that HoxA10 either activates or represses gene transcription at various points during myelopoiesis. Our studies also suggested that HoxA10 is a bifunctional protein that is involved in dynamic regulation of multiple aspects of phagocyte phenotype and function.

HoxA10 represses gene transcription in undifferentiated myeloid cells by interaction with histone deacetylase 2

The homeodomain proteins, HoxA10 and Pbx1a, interact with negative cis elements to repress gene transcription in undifferentiated myeloid cells. The CYBB and NCF2 genes, which encode the gp91PHOX and p67PHOX proteins, are two such HoxA10-Pbx1a target genes. In previous studies, we found that HoxA10-Pbx1a represses transcription of these genes by two mechanisms: competition for DNA binding with transcriptional activators and endogenous repression activity. In these studies, we identify a novel molecular mechanism of endogenous transcriptional repression by HoxA10-Pbx1a. Endogenous repression activity of other Hox-Pbx1a complexes requires recruitment of transcriptional co-repressor proteins by Pbx1a. In contrast, our investigations have determined that HoxA10 has Pbx1a-independent endogenous repression activity. We find that this transcriptional repression activity is abrogated by histone deacetylase inhibitors, suggesting involvement of co-repressor proteins. Consistent with this, we identify HoxA10 amino acids 224–249 as a Pbx1-independent repression domain, which interacts with histone deacetylase 2. We have determined that this HoxA10 domain is not conserved with other Abd Hox proteins, although homology exists with other transcription factors and co-repressors. Understanding the roles different Hox proteins play in myeloid differentiation is a challenging problem. Our results suggest that insight into this problem can be obtained from biochemical characterization of the various molecular mechanisms of Hox protein function.

Constitutively Active SHP2 Cooperates with HoxA10 Overexpression to Induce Acute Myeloid Leukemia

The homeodomain transcription factor HoxA10 is maximally expressed in myeloid progenitor cells. Sustained HoxA10 expression during differentiation has been described in poor prognosis human acute myeloid leukemia (AML). Consistent with this, engineered overexpression of HoxA10 in murine bone marrow induces a myeloproliferative disorder that progresses to AML over time. This murine model suggests that HoxA10 overexpression is sufficient for myeloproliferation but that differentiation block, and therefore AML, requires acquisition of additional mutations. In myeloid progenitor cells, HoxA10 represses transcription of genes that encode phagocyte effector proteins such as gp91PHOX and p67PHOX. Tyrosine phosphorylation of HoxA10 during myelopoiesis decreases binding to these target genes. In immature myeloid cells, HoxA10 also activates transcription of the DUSP4 gene that encodes Mkp2, an anti-apoptotic protein. HoxA10 binding to the DUSP4 promoter decreases during myelopoiesis. Therefore, both myeloid-specific gene repression and DUSP4 activation by HoxA10 decrease during myelopoiesis. This results in phenotypic differentiation and facilitates apoptosis as differentiation proceeds. HoxA10 is de-phosphorylated by SHP2 protein-tyrosine phosphatase in myeloid progenitors. This mechanism maintains HoxA10 in a nonphosphorylated state in immature, but not differentiating, myeloid cells. Constitutively active SHP2 mutants have been described in human AML, which dephosphorylate HoxA10 throughout myelopoiesis. In this study, we hypothesize that constitutive SHP2 activation synergizes with HoxA10 overexpression to accelerate progression to AML. Because both HoxA10 overexpression and constitutive SHP2 activation are found in poor prognosis human AML, these studies contribute to understanding biochemical aspects of disease progression in myeloid malignancy.

HoxA10 Regulates Transcription of the Gene Encoding Transforming Growth Factor β2 (TGFβ2) in Myeloid Cells

HoxA10 is a homeodomain transcription factor that is maximally expressed in myeloid progenitor cells. HoxA10 is overexpressed in a poor prognosis subset of human acute myeloid leukemia (AML) and in vivo overexpression of HoxA10 in murine bone marrow induces myeloid leukemia. HoxA10 contributes to myeloid progenitor expansion and differentiation block, but few target genes have been identified that explain the influence of HoxA10 on these processes. The current study identifies the gene encoding transforming growth factor β2 (TGFβ2) as a HoxA10 target gene. We found that HoxA10 activated TGFβ2 transcription by interacting with tandem cis elements in the promoter. We also determined that HoxA10 overexpression in myeloid progenitor cells increased Tgfβ2 production by the cells. Tgfβ2 stimulates proliferation of hematopoietic stem and progenitor cells. Therefore, these studies identified autocrine stimulation of myeloid progenitors by Tgfβ2 as one mechanism by which HoxA10 expands this population. Because HoxA proteins had not been previously known to influence expression of pro-proliferative cytokines, this has implications for understanding molecular mechanisms involved in progenitor expansion and the pathobiology of AML.

HoxA10 Activates CDX4 Transcription and Cdx4 Activates HOXA10 Transcription in Myeloid Cells

HoxA10 is a homeodomain transcription factor that influences a number of developmental processes, including hematopoiesis. During definitive hematopoiesis, expression of HoxA10 is maximal in committed myeloid progenitor cells and decreases as differentiation proceeds. Aberrantly increased expression of HoxA10 was found in bone marrow cells in a poor prognosis subset of human acute myeloid leukemia (AML). Consistent with this, AML developed in mice transplanted with HoxA10-overexpressing bone marrow. However, relatively few target genes have been identified that explain the role of HoxA10 in leukemogenesis. In the current study, we identified CDX4 as a HoxA10 target gene. Cdx4 is a homeodomain transcription factor that was also implicated in myeloid leukemogenesis. Although relatively few Cdx4 target genes have been identified, Cdx4 was known to influence HOX gene transcription. We identified a HoxA10-binding cis element in the CDX4 promoter that activated transcription. We also identified a Cdx4-binding cis element that activated the HOXA10 promoter. Therefore, increased Cdx4 expression in HoxA10-overexpressing cells augmented transcription of the endogenous HOXA10 gene. Increased endogenous HoxA10 in these cells induced additional CDX4 transcription. We found that Cdx4 influenced transcription of HoxA10 target genes in a HoxA10-dependent manner. Similarly, HoxA10 influenced transcription of HOX genes in a Cdx4-dependent manner. We previously found that HoxA10-overexpressing myeloid progenitors were hypersensitive to a variety of cytokines. In the current studies, we found that Cdx4 knockdown decreased cytokine hypersensitivity of HoxA10-overexpressing cells. Therefore, these studies identified a positive feedback relationship between HoxA10 and Cdx4, which potentially amplified the contribution of either transcription factor to the pathogenesis of AML.

HoxA10 Protein Regulates Transcription of Gene Encoding Fibroblast Growth Factor 2 (FGF2) in Myeloid Cells

Background: HoxA10 is a homeodomain transcription factor that regulates myeloid progenitor cell expansion and contributes to myeloid leukemogenesis. Results: HoxA10 activates the FGF2 (fibroblast growth factor 2) gene in myeloid progenitor cells. Conclusion: Increased production of Fgf2 by HoxA10-overexpressing myeloid progenitor cells stimulates proliferation through an autocrine mechanism. Significance: Signaling pathways that are activated by Fgf2 may be rational therapeutic targets for leukemia. HoxA10 is a member of a highly conserved family of homeodomain transcription factors that are involved in definitive hematopoiesis and implicated in the pathogenesis of acute myeloid leukemia (AML). During normal hematopoiesis, HoxA10 facilitates myeloid progenitor expansion and impedes myeloid differentiation. To better understand the molecular mechanisms that control these events, we have been identifying and characterizing HoxA10 target genes. In this study, we identified the gene encoding fibroblast growth factor 2 (Fgf2 or basic fibroblast growth factor) as a target gene that is relevant to the biological effects of HoxA10. We identified two cis elements in the proximal FGF2 promoter that are activated by HoxA10 in myeloid progenitor cells and differentiating phagocytes. We determined that Fgf2 expression and secretion are regulated in a HoxA10-dependent manner in these cells. We found that increased Fgf2 production by HoxA10-overexpressing myeloid progenitor cells induced a phosphoinositol 3-kinase-dependent increase in β-catenin protein. This resulted in autocrine stimulation of proliferation in HoxA10-overexpressing cells and hypersensitivity to other cytokines that share this pathway. Therefore, these studies identified expression of Fgf2 as a mechanism by which HoxA10 controls the size of the myeloid progenitor population. These studies also suggested that aberrant production of Fgf2 may contribute to leukemogenesis in the subset of AML with dysregulated Hox expression. Therapeutic targeting of Fgf2-stimulated signaling pathways might be a rational approach to this poor prognosis subset of AML.

HoxA10 influences protein ubiquitination by activating transcription of ARIH2, the gene encoding Triad1.

HoxA10 is a homeodomain transcription factor that is maximally expressed in myeloid progenitor cells. An increase in HoxA10 expression correlates with poor prognosis in human acute myeloid leukemia (AML). Consistent with this scenario, HoxA10 overexpression in murine bone marrow induces a myeloproliferative neoplasm that advances AML over time. Despite the importance of HoxA10 for leukemogenesis, few genuine HoxA10 target genes have been identified. The current study identified ARIH2, the gene encoding Triad1, as a HoxA10 target gene. We identified two distinct HoxA10-binding cis elements in the ARIH2 promoter and determined that HoxA10 activates these cis elements in myeloid cells. Triad1 has E3 ubiquitin ligase activity, and we found that HoxA10-overexpressing myeloid cells exhibited a Triad1-dependent increase in protein ubiquitination. Therefore, these studies have identified the regulation of protein ubiquitination as a novel function of Hox transcription factors. Forced overexpression of Triad1 has been show previously to inhibit colony formation by myeloid progenitor cells. In contrast, HoxA10-overexpressing myeloid progenitor cells exhibited increased proliferation in response to low doses of various cytokines. We found that Triad1 knockdown further increased cytokine-induced proliferation in HoxA10-overexpressing cells. Therefore, these studies have identified a HoxA10 target gene that antagonizes the overall influence of overexpressed HoxA10 on myeloproliferation. This result suggests that the consequences of HoxA10 overexpression reflect a balance between the target genes that facilitate and antagonize proliferation. These results have implications for understanding the mechanisms of leukemogenesis in AML with Hox overexpression.

Fas associated phosphatase 1 (Fap1) influences βcatenin activity in myeloid progenitor cells expressing the Bcr-abl oncogene.

Background: Increased Fap1 expression in chronic myeloid leukemia (CML) is associated with Fas resistance. Results: Fap1 also increases βcatenin activity in CML by inactivating Gsk3β, a serine/threonine kinase that effects inhibitory phosphorylation of βcatenin. Conclusion: Fap1 influences cell proliferation and survival by regulating βcatenin protein stability. Significance: Fap1 contributes to the pathogenesis of CML by inhibiting apoptosis and increasing βcatenin activity. Increased βcatenin activity correlates with leukemia stem cell expansion and disease progression in chronic myeloid leukemia (CML). We found previously that expression of the CML-related Bcr-abl oncoprotein in myeloid progenitor cells increases expression of Fas-associated phosphatase 1 (Fap1). This resulted in Fap1-dependent resistance to Fas-induced apoptosis in these cells. Fap1 also interacts with the adenomatous polyposis coli (Apc) protein, but the functional significance of this interaction is unknown. Apc participates in a complex that includes glycogen synthase kinase β (Gsk3β) and βcatenin. Assembly of this complex results in phosphorylation of βcatenin by Gsk3β, which facilitates βcatenin ubiquitination and degradation by the proteasome. In this study, we found increased association of Fap1 with the Apc complex in Bcr-abl+ myeloid progenitor cells. We also found Fap1-dependent inactivation of Gsk3β and consequent stabilization of βcatenin in these cells. Consistent with this, Bcr-abl+ cells exhibited a Fap1-dependent increase in βcatenin activity. Our studies identified Fap1-dependent Gsk3β inactivation as a molecular mechanism for increased βcatenin activity in CML.