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IntroductionIn recent years, substantial progress has been made in understanding the biology of acute myeloid leukemia (AML). One of the pathogenetic hallmarks of AML are chromosomal translocations generating leukemogenic fusion genes that often act as aberrant transcription factors. 1 The second key genetic characteristics in AML are mutations, particularly those found in patients with normal karyotype and affecting the receptor tyrosine kinase FLT3 or the nucleophosmin protein (NPM1). Beside these structural genetic changes, large-scale gene expression analyses of cDNA samples from patients with AML have demonstrated that deregulated expression of nonaltered genes characterizes many AML cases. The most prominent example for this is the deregulated expression of homeobox genes in AML. [2][3][4] Homeobox genes form a highly conserved family of transcription factors known to be key regulators of normal hematopoietic stem cell and progenitor development. 5 Several studies have demonstrated that aberrant HOX gene expression profoundly perturbs normal murine and human hematopoietic development and causes leukemia in mice. [5][6][7][8][9] The aberrant expression of homeobox genes such as HOXA9 and HOXA10 is strongly associated with certain AML subtypes characterized by MLL fusion genes, NPM1 mutations (NPMc ϩ ), and by more rare translocations such as the translocation t(10;11)(p13q14) generating the CALM-AF10 fusion gene. 4,[10][11][12][13] All together, deregulated homeobox gene expression characterizes more than every third case of AML. So far, it is largely unknown how the aberrant expression of homeobox genes is initiated in the malignant clone. In cases with 11q23 chromosomal translocations, it is thought that aberrant function of the MLL gene, a known positive upstream regulator of HOX gene expression, is responsible for the perturbed expression of these key regulatory genes of early hematopoietic development. 14 In contrast, the aberrant HOX gene expression in patients with AML with normal karyotype and NPM1 mutation is not well understood. 15 In particular, the patients with NPMc ϩ AML demonstrate that aberrant HOX gene expression cannot be just explained by the stage of differentiation at which the leukemic clone is arrested: NPMc ϩ patients are CD34 Ϫ in more than 95% of patients, and represent therefore a cell stage in which HOX genes are normally silenced. 8,16 Another gene family critically involved in Hox gene regulation is the family of the so-called ParaHox genes, comprising the different "caudal-related homeobox genes" such as CDX1, CDX2, and CDX4, and the GSH2 homeobox gene. 17 Several experimental systems have demonstrated that loss of Cdx2 causes homeotic alterations and posterior shifts in Hox expression domains, 18 and that consensus-binding sites for the 3 Cdx homologs are present in the promoters of multiple Hox genes. [19][20][21][22] Expression of Cdx2 is tightly restricted to intestinal development in the adult. 23 Aberrant expression of CDX2 is associated with intestinal metaplasia, 24,25...

The t(10;11)(p13-14;q14-21) translocation, giving rise to the CALM-AF10 fusion gene, is a recurrent chromosomal rearrangement observed in patients with poor prognosis acute myeloid leukemia (AML). Although splicing of the CALM-AF10 fusion transcripts has been described in AML patients, the contribution of different CALM and AF10 domains to in vivo leukemogenesis remains to be defined. We therefore performed detailed structure-function studies of the CALM-AF10 fusion protein. We demonstrate that fusion of the C-terminal 248 amino acids of CALM, which include the clathrin-binding domain, to the octapeptide motif-leucine-zipper (OM-LZ) domain of AF10 generated a fusion protein (termed CALM-AF10 minimal fusion (MF)), with strikingly enhanced transformation capabilities in colony assays, providing an efficient system for the expeditious assessment of CALM-AF10-mediated transformation. Leukemias induced by the CALM-AF10 (MF) mutant recapitulated multiple aspects of full-length CALM-AF10-induced leukemia, including aberrant Hoxa cluster upregulation, a characteristic molecular lesion of CALM-AF10 leukemias. In summary, this study indicates that collaboration of the clathrin-binding and the OM-LZ domains of CALM-AF10 is sufficient to induce AML. These findings further suggest that future approaches to antagonize CALM-AF10-induced transformation should incorporate strategies, which aim at blocking these key domains.

Recent data indicate that a variety of regulatory molecules active in embryonic development may also play a role in the regulation of early hematopoiesis. Here we report that the human Vent-like homeobox gene VENTX, a putative homolog of the Xenopus xvent2 gene, is a unique regulatory hematopoietic gene that is aberrantly expressed in CD34 + leukemic stem-cell candidates in human acute myeloid leukemia (AML). Quantitative RT-PCR documented expression of the gene in lineage positive hematopoietic subpopulations, with the highest expression in CD33 + myeloid cells. Notably, expression levels of VENTX were negligible in normal CD34 + /CD38 − or CD34 + human progenitor cells. In contrast to this, leukemic CD34 + / CD38 − cells from AML patients with translocation t(8,21) and normal karyotype displayed aberrantly high expression of VENTX. Gene expression and pathway analysis demonstrated that in normal CD34 + cells enforced expression of VENTX initiates genes associated with myeloid development and down-regulates genes involved in early lymphoid development. Functional analyses confirmed that aberrant expression of VENTX in normal CD34 + human progenitor cells perturbs normal hematopoietic development, promoting generation of myeloid cells and impairing generation of lymphoid cells in vitro and in vivo. Stable knockdown of VENTX expression inhibited the proliferation of human AML cell lines. Taken together, these data extend our insights into the function of embryonic mesodermal factors in human postnatal hematopoiesis and indicate a role for VENTX in normal and malignant myelopoiesis.human | myelopoiesis | Xvent | VENTX2