INVOLVEMENT OF THE EVI-2 LOCUS IN MYELOID LEUKEMIA

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Ecotropic viral integration site-2 (Evi-2) was identified as a common site of viral integration in myeloid tumors derived from BXH-2 mice. Characterization of the Evi-2 locus revealed that the viral integrations occurred within 2 novel genes encoding transmembrane proteins (Evi-2a and Evi-2b). Evi-2 was localized to mouse chromosome 11 and to human chromosome 17q11.2. Through the use of Evi-2, the gene for NF1 was cloned and it was revealed that Evi-2a, Evi-2b and a third gene (Omgp) were localized within an intron. Interestingly, it has been observed that patients with NF1 have a higher frequency of developing juvenile myelogenous leukemia, suggesting that whatever gene is involved in the murine myeloid leukemia, could be the same gene involved in human myeloid leukemia. Thus, through the use of a common site of viral integration an important genomic region was identified that is involved in murine and human disease. Our long term goal is to characterize the novel genes found within the Evi-2 locus and to determine their role in normal development and leukemia. Preliminary analysis indicates that it is not the alteration of expression of the NF1 gene, but the alteration of the gene (or genes) in the NF1 intron that contributes to the myeloid leukemia. BXH-2 mice will be aged to isolate additional myeloid tumors and integrations within the Evi-2 locus will be identified. These tumors will be analyzed at both the RNA and protein level to detect alterations in the expression of the genes within the Evi-2 locus. Additionally, we propose to characterize the Evi-2a and Evi-2b genes and their products to determine their role in normal development and myeloid leukemogenesis. The full length mRNA of both genes will be sequenced with the characterization of splicing patterns and delineation of exons. The proteins will be produced in expression vectors and used to generate polyclonal antisera to study the cellular localization of the proteins. Investigation of the function of the proteins will be initiated along with the identification of associated proteins. Additionally, a developmental profile of the expression of both mRNA and protein, through in situ RNA hybridization and immunohistochemical staining, will be determined in order to gain insights into the role these genes play in normal mouse development. The effect of overexpression and loss of expression of these gene products in vitro will be assessed as well as the effect of overexpression in transgenic animals. These studies will elucidate the normal function of the Evi-2 locus and its influence in myeloid neoplasia.