Rawat VP et al. (2010), The vent-like homeobox gene VENTX promotes huma...

XB-ART-42032

Proc Natl Acad Sci U S A 2010 Sep 28;10739:16946-51. doi: 10.1073/pnas.1001878107.

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The vent-like homeobox gene VENTX promotes human myeloid differentiation and is highly expressed in acute myeloid leukemia.

Rawat VP , Arseni N , Ahmed F , Mulaw MA , Thoene S , Heilmeier B , Sadlon T , D'Andrea RJ , Hiddemann W , Bohlander SK , Buske C , Feuring-Buske M .

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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.

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Species referenced: Xenopus
Genes referenced: cd34 cd38 flt3 itgam runx1 ventx2.2

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	Fig. 1. (A) Expression of VENTX in total BM vs. CD34+ BM progenitors and CD34−/38+ BM cells and in (B) GlyA+ erythroid, CD33+ myeloid, and CD19+ lymphoid cells (C). VENTX expression in hematopoietic subpopulations derived from the peripheral blood. Expression analyses were performed by TaqMan qRT–PCR, and δCT values were obtained by normalization to β-actin. Bars represent average expression ±SD; the number of the tested samples is indicated. Note that all δCT values are inversely correlated to the expression level.
	Fig. 2. VENTX expression in AML cell lines and primary AML samples. (A) Box plots of mRNA expression levels (microarray signal intensity values) of VENTX in normal bone marrow (nBM) (n = 6), chronic myelocytic leukemia (CML) and AML (10 cases each) with normal karyotype (AML_nk), AML with complex karyotype (AML_comp), AML with mixed lineage leukemia (MLL) rearrangement (AML_MLL), AML with CBFB/MYH11 fusion (AML_M4), AML-M3 with PML/RARA fusion (AML_M3), AML with AML1/ETO fusion (AML_M2), and AML with normal karyotype and FLT3 length mutation (AML_FLT3). The normalized and variance stabilized expression values are shown on a logarithmic scale (log 2). Solid bar represents median, boxes represent the 25–75% quantile range (inter-quantile range, IQR), whiskers represent the 1.5-fold IQR, small circles represent outliers. Ten samples are included in each group. (B) Quantitative expression level of VENTX in AML patients with either AML1-ETO translocation or normal karyotype and NPMc+/FLT3-LM− or NPMc−/FLT3-LM+. Diamonds indicate single patients; bars indicate median expression level. (C) Quantitative VENTX expression in human normal CD34+ enriched BM and sorted BM subpopulations of AML1-ETO positive AML patients. Expression analyses were performed by TaqMan qRT–PCR, and δCT values were obtained by normalization to β-actin. Note that all δCT values are inversely correlated to the expression level.
	Fig. 3. (A) Morphology of colonies and colony numbers derived from CB cells transduced with VENTX compared with the GFP control. Bars represent the average colony number of 11 independent experiments ±SEM. A macroscopic picture of primary colonies and magnification of one representative GFP+ colony are shown. (B) Immunophenotypic analysis of primary colonies by glycophorin A and CD11b staining. Bars represent the average percentage of positively stained cells ±SD.
	Fig. 4. Overexpression of VENTX in human CB cells increases myeloid engraftment in mice. (A) Immunophenotypic analysis of human CB cells 9–12 wk after transplantation in NOD/SCID mice transduced with either GFP or VENTX. Bars represent the average percentage of cells positive for the respective marker ±SEM. (B) Lymphoid/ myeloid (CD19/CD15) ratio of the same mice. Whiskers indicate SD.
	Fig. 5. Proliferation of AML cell lines after shRNA-mediated silencing of VENTX expression. Down-regulation of VENTX expression by shRNA V73 and V77-inhibited proliferation of the VENTX-expressing AML cell lines MV4-11, NB4, and HL60. In contrast, treatment with scrambled shRNA had no inhibitory effect (control). Values are represented as ±SD.

References [+] :

Antonchuk, HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. 2002, Pubmed