MDS1-EVI1 and EVI1 Overexpression Results in Changes in the Behavior of Murine Hematopoietic Cells.

We have found non-random patterns of retroviral integration in long-term hematopoietic repopulating cells in the rhesus macaque, with frequent integration events of MLV vectors into the MDS1-EVI1 gene complex. These findings, along with reports regarding frequent integration events in the same gene complex in patients with chronic granulomatous disease receiving MLV-transduced hematopoietic cells in a clinical trial and the ability of MLV vectors activating expression of this gene via integration to immortalize primary murine bone marrow cells, suggests these gene products could have important roles in normal and leukemic hematopoiesis. Expression from this gene complex can result in translation of at least three distinct proteins: MDS1, EVI1, and MDS1-EVI1. EVI1 has been the most studied protein of this locus. Its overexpression, as a consequence of chromosomal rearrangement or viral integration, is associated with leukemia. MDS1-EVI1 contains a PR domain that is lacking in EVI1 and is thought to possibly be antagonistic to EVI1, however the location of the integrations in our prior rhesus studies would indicate that overexpression of either gene product could be immortalizing. Both proteins share the same expression profile in normal tissues as well as most reports of myeloid leukemias. To investigate the impact of the three gene products on hematopoietic cells, we cloned murine mds1, evi1, and mds1-evi1 into the pMIEV-GFP retroviral vector and produced ecotropic vector particles. These were used to transduce the murine BaF3 hematopoietic cell line as a model to study the impact of expression of these various gene products. Gene expression analysis using Afflymetrix arrays demonstrated that both EVI1 and MDS1-EVI1 expression produced dramatic changes in gene expression profiles of these cells, compared to MDS1 and control vector. For instance, EVI1 transduced cells overexpressed oncogenes such as small G proteins belonging to the RAS family. There was modulation of genes implied in hematopoiesis, apoptosis, TGF beta signaling, and cell cycle. To assess changes in cell cycling of transduced BaF3 cells we used a flow cytometric assay, which unraveled an arrest in G1 phase only when EVI1 was overexpressed. These changes were concomitant to an increased metabolic activity as measured by an MTT assay. Further studies of these different pathways have to be performed in order to confirm the results obtained by the DNA chips analysis. Primary murine bone marrow cells could be immortalized after transduction by both EVI1 and MDS1-EVI1 vectors, compared to MDS1 and control vectors. Mice have been transplanted with primary bone marrow cells transduced with all vectors, and are being followed for hematopoietic changes or leukemia. In conclusion, both MDS1-EVI1 and EVI1 overexpression appear to result in marked changes in the behavior of primitive hematopoietic cells.