Overexpression of MN1 is a negative prognostic factor in patients with acute myeloid leukemia (AML) with normal cytogenetics. We have previously demonstrated that overexpression of MN1 is sufficient as a single genetic event to induce AML in mice with very short latency (

Blood 110:1639, 2007
). To investigate the effects of MN1 on the functional activity of primitive human hematopoietic cells, we transduced CD34+ human cord blood cells with an oncoretrovirus (3 experiments) or lentivirus (3 experiments) encoding GFP ± MN1. Immediately post-transduction primary colony-forming cell (CFC) assays showed no significant differences in the number or type of colonies generated from the MN1 and control-transduced cells. However, the number of CFCs detectable in secondary assays of the MN1-transduced cells was 17-fold higher than from the GFP-transduced cells and this difference was found to increase a further 234-fold as detected by tertiary CFC assays. To assess the effect of MN1 on cells that can be propagated for more prolonged periods on mouse fibroblast feeders engineered to produce human Steel factor, IL-3 and G-CSF, 104 unsorted cord blood cells were plated into such cultures and the number and types of cells present was then determined 8 weeks later. The remarkable potency of MN1 in stimulating the output of primitive cells under these long-term culture (LTC) conditions was evident from the 1700 greater increase in total cell output and 277-fold higher output of CFCs as compared to control cultures and an accompanying large expansion of CD34+ cells (representing up to 18 % of the final population) whereas CD34+ cells were no longer detectable in the control cultures. Further analysis by limit dilution assay (2 experiments) showed that the frequency of cells able to produce CFCs for at least 6 weeks in these stromal based cultures was increased 10-fold (1/7656 control cells vs 1/718 MN1-transduced cells). In addition the CFC output of each of these was enhanced, on average, 6-fold. Remarkably, these effects of MN1 were sustained for a further 16 weeks as shown by the analysis of the cells they produced in secondary and tertiary LTCs. Overall this resulted in a cumulative 6250-fold increase in total cells over 24 weeks, whereas control cells declined to undetectable levels within 16 weeks. Cells harvested from the secondary and tertiary LTCs initiated with MN1-transduced cells also showed a continuing output of 878,967 ± 332,300 and 264,458 ± 120,600 CFCs after 16 and 24 weeks, respectively. This study sets the stage for further investigation of the effect of MN1 on human NOD/SCID/γcnull mouse repopulating cells and the molecular mechanisms by which MN1 blocks the differentiation fate of primitive human hematopoietic cells and how this may be related to its contribution to the genesis of poor prognosis AML.

Topics:
cd34 antigens, chlorofluorocarbons, dilution technique, granulocyte colony-stimulating factor, interleukin-3, leukemia, myelocytic, acute, mn1 gene, prognostic factors, protein overexpression, recombinant granulocyte colony stimulating factor

Disclosures: No relevant conflicts of interest to declare.

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2008, The American Society of Hematology
2008
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