The Vent-Like Homeobox Gene Ventx2 Is a Novel Candidate for a Human Hematopoietic Regulatory Protein.

Abstract Identification of the genes which are critically involved in normal and leukemic hematopoiesis is a major goal for experimental and clinical hematology. Recent data indicate that a variety of regulatory molecules active in early development may also play a role in the maintenance of hematopoietic stem cells with repopulating activity. Since it was shown, that the Xvent-2 homeobox gene is part of the BMP-4 signalling pathway in Xenopus it is of particular interest to examine the expression profile and function of the human homologue Ventx2 in hematopoietic development. We first analysed Ventx2 expression by RT-PCR in CD3, CD19 and CD33 cells highly purified by FACS sort from peripheral blood of healthy donors. Expression of Ventx2 was detected in T- and B- as well as differentiated myeloid cells indicating that Ventx2 is expressed in multiple hematopoietic lineages. Furthermore, VENTX2 expression was recurrently detected in bone marrow samples from AML patients at diagnosis as determined by RT-PCR (n=6). In an attempt to characterize the functional relevance of Ventx2 expression for hematopoietic development we retrovirally engineered murine hematopoietic progenitor cells to constitutively express the gene using a MSCV-based retroviral construct with an IRES-EGFP cassette. Successfully transduced cells were injected into lethally irradiated mice or used for in vitro experiments. At the level of the clonogenic progenitor VENTX2 induced a 3fold increase in the number of CFU-G (n=5; p<0.001) compared to the GFP control (62 versus 25 CFU-G, respectively, per 1000 initially plated cells) without increasing the total number of colonies, indicating that VENTX2 promoted granulocytic differentiation in vitro. Re-plating assays confirmed the effect of the homeobox gene with an over 9fold increase in the number of secondary CFU-G (511 vs. 54, respectively, per 1000 initially plated cells). When the effect of VENTX-2 on the frequency of LTC-IC was determined by limiting dilution assay (n=2), no major differences were detected between the homeobox gene and the control arm (453 LTC-IC vs. 801 LTC-IC per 1x106 cells, respectively, p = n.s.). Furthermore, the number of colonies generated per LTC-IC did not significantly differ between the two arms (17 colonies for VENTX2 and 26 colonies for the control). In NOD/SCID mice VENTX2 induced a 2.9fold increase in the proportion of CD15+ mature myeloid cells within the GFP-positive compartment (n=7) compared to the control (n=9)(6.4 % vs. 2.2 %, respectively; p<0.02), translating into 4 x 106 (± 1 x 106) human CD15+ /GFP+ cells per mouse in the VENTX2 group compared to 2x106 cells (± 6 x 105) in the control. These findings characterize VENTX2 as a novel regulatory protein in human hematopoiesis and add information about the role of non-clustered homeobox genes in early blood development.