c-myb and Mmll continue to be the focus of the laboratory since we have found that these two loci are important to the development of promonocytic leukemia. Both are targets of insertional mutagenesis by retroviruses in our animal model. Our laboratory has shown that the c-myb oncogene can be activated in promonocytic leukemias when the retrovirus integrates into the locus. Most of our initial studies were carried out in leukemic cells that had virus inserted into the 5' end of the gene. This caused constitutive mRNA expression by a mechanism referred to as promoter insertion. In vitro data suggest that the consequence of uncontrolled expression of this gene, which encodes a transcription factor, is the inhibition of growth arrest that occurs at end stages of myeloid cell differentiation. More recently, our laboratory has focused on alternative forms of activation which occur in other leukemias when the virus integrates into exons 9 or 11. Results show that leukemic cells with such alterations are oncogenic due to overexpression of c-myb as in the previously studied leukemias. However, in these cells overexpression occurs at both the mRNA and protein levels and the increases in mRNA are due to changes in mRNA stability instead of increases in rate of transcription. The normal half-life for c-myb mRNA is approximately 40 min as we have shown in M1 cells following actinomycin D treatment. This is extended to about 5 fold in these leukemias due to the loss of 3' sequences. Previously we reported that c-Myb protein, truncated at its carboxy-terminus (CT) because of integration of virus, is more resistant to proteolysis, having a half-life which is significantly longer than that of wild-type c-Myb. In addition, it was reported that c-Myb is degraded by the ubiquitin-26S proteasome pathway. We have extended these studies now by localizing determinants in the CT important to proteolysis by the proteasome, one overlapping the putative leucine zipper and one at the extreme terminal CT end. This year we have also provided evidence that phosphorylation is important in the regulation of c-Myb proteolysis since the protein can be induced to more rapidly degrade when cells are treated with okadaic acid, a phosphatase inhibitor. We are currently looking at whether induction of differentiation or progression of the cell cycle influences the regulation of c-Myb proteolysis. The Mmll locus was identified as a target of insertional mutagenesis in many leukemia's that lack integration within the c-myb locus. It maps 20kb upsteam of the c-myb promoter and we are currently attempting to determine if changes in this region can effect expression of c-myb through the promoter or elongation block. Furthermore, we are mapping DNase hypersensitivity sites in this region to look for hot spots of regulatory activity.
