Functions of the oncogene EVI1 in primary human CD34+ cells

The EVI1 gene, which codes for a zinc finger transcription factor, plays a well documented role as an oncogene in aggressive forms of human myeloid leukemia. Experiments aimed at better understanding the physiological and pathological functions of this gene have suggested EVI1 to be able to promote the cell cycle, inhibit apoptosis, and block myeloid differentiation. However, the number of these studies is still relatively small, not all have been conducted in cell types relevant to human leukemia, and their results are sometimes contradictory. Open questions therefore remain. These also concern the role of the MDS1/EVI1 protein, which is translated from an alternative EVI1 mRNA variant, and contains an extended amino terminus as compared to EVI1. MDS1/EVI1 acts in a manner different from or even opposite to that of EVI1 in certain contexts, but exhibits properties comparable to those of EVI1 in others. Primary CD34 positive hematopoietic stem/progenitor cells can be isolated from human cord blood and induced to differentiate into various myeloid lineages in vitro in processes that closely recapitulate normal human hematopoiesis. We have used this system to analyze the expression of EVI1 and MDS1/EVI1 during granulocytic, erythroid, monocytic, and dendritic (Langerhans cell) differentiation, and found both EVI1 and MDS1/EVI1 to be expressed at high levels in undifferentiated, multipotent CD34 + cells, and to be downregulated, with lineage specific kinetics, during differentiation. These results not only represent the first analysis of the expression of EVI1 and MDS1/EVI1 during some of the investigated differentiation processes, but also show that the CD34 + cell system is ideally suited for studying the biological functions of EVI1 and MDS1/EVI1 during the proliferation and differentiation of human hematopoietic cells. In the presented proposal, we therefore plan to refine our analyses of the expression of EVI1 and MDS1/EVI1 in primary human CD34 + cells during differentiation into the four lineages mentioned above, and to extend them to the process of megakaryocytic differentiation. The biological functions of EVI1 and MDS1/EVI1 in the CD34 + cell system shall be studied by overexpressing and knocking down their cDNAs/mRNAs using well established retrovirus technology, and analyzing the effects of these manipulations on cellular proliferation and differentiation. The results of these studies are expected to significantly enhance our understanding of the roles of the oncogene EVI1 and its potential antagonist MDS1/EVI1 in normal human hematopoiesis, and provide a basis for the elucidation of their pathological roles in leukemia.

Leukemia results from the malignant transformation of immature precursors of blood cells. One of the earliest developmental stages of healthy hematopoietic cells (a term that refers to both blood cells and their bone marrow resident precursors) are the CD34 positive (CD34+) cells, which are endowed with the ability to mature into any of the cell types present in blood. In a subset of patients with certain types of leukemias, the so-called myeloid leukemias, an increased activity (expression) of the ecotropic viral integration site1 (EVI1) gene is observed. Even though it is well established that such overexpression of EVI1 is associated with an aggressive course of disease and shortened survival of the affected patients, relatively little is known about the mechanism of action of EVI1 in either healthy or malignant cells. Studies on primary (freshly isolated) mouse cells have shown that experimentally increased expression of EVI1 enhanced cellular growth and proliferation, which agrees well with the suspected role of EVI1 in leukemogenesis. In contrast, however, the proliferation of the few investigated human cell lines was inhibited by EVI1. Since cell lines have been cultivated under laboratory conditions for years or decades, they may not always reflect the properties of their cells of origin faithfully. For this reason, we aimed to study the expression and function of EVI1 in primary human CD34+ cells, which are maintained in the laboratory only for a few days, and therefore represent the model that best reflects the conditions within the human organism. Our experiments showed that EVI1 was expressed in CD34+ cells, but downregulated strongly and rapidly during their differentiation into the different myeloid cell types - granulocytes, monocytes, erythrocytes, megakaryocytes, and dendritic Langerhans cells. A similar behaviour was found for MDS1/EVI1, a variant of the EVI1 gene which in certain settings acts in a manner opposite to EVI1. However, the almost identical expression patterns of the two gene variants in the CD34+ cell system suggest that this may not be the case in this context. Furthermore, in contrast to what has been reported for mouse cells, experimental reduction of EVI1 expression in CD34+ cells affected their proliferation barely if at all. Finally, and most interestingly, experimental overexpression of EVI1 in CD34+ cells strongly inhibited their proliferation and/or caused their rapid demise. This indicates that human cells, in contrast to those of the mouse which due to its substantially shorter life span is not naturally susceptible to tumor formation, have developed defense mechanisms that protect them from the action of the aggressive oncogene EVI1. As is the case for other tumor genes, additional genetic alterations are therefore required before EVI1 can reveal its tumor promoting potential. Elucidating these mechanisms will be of fundamental importance in understanding the role of EVI1 in leukemogenesis.