The t(3;5)(q25.1;q34) chromosomal translocation produces a fusion protein consisting of the nucleolar phosphoprotein nucleophosmin (NPM) and myelodysplasia/myeloid leukemia factor 1 (MLF1), which is associated with the formation of myelodysplastic syndrome and acute myeloid leukemia. Fusion of MLF1 to nucleophosmin alters MLF1 localization and presumably interferes with endogenous MLF1 function. MLF1 levels are elevated in a significant portion of patients with acute myeloid leukemia, suggesting aberrant MLF1 expression may result in oncogenic transformation. Previous studies have determined that MLF proteins are involved in the growth and differentiation of hematopoietic cells and in transcriptional regulation. However, the endogenous functional roles of MLF in normal cellular development and leukemic hematopoietic development have not been well characterized. Therefore, this proposal describes experiments that will characterize the endogenous functions of MLF using the model organism Drosophila melanogaster and determine how leukemia-associated mlf mutations alter its normal functions. To address these goals, the following specific aims will be examined: identification and characterization of novel proteins that physically interact with MLF and the NPM-MLF fusion protein;characterization of the role of MLF in gene expression;and determination of the in vivo consequences of MLF overexpression and expression of the mutant NPM-MLF fusion protein. Novel proteins that interact differentially with dMLF and the NPM-dMLF leukemic fusion protein will be identified using mass spectrometry analysis. The identified dMLF-interacting proteins will be tested for their abilities to genetically suppress or enhance the cellular proliferation defects observed in dmlf mutants. The role of dMLF in transcriptional regulation will be characterized through the use of ChIP-Seq to determine the genetic loci where dMLF binds chromatin and by identifying any changes in gene expression in dmlf mutants as compared to wild type animals using RNA-Seq. Additionally, the novel interaction of dMLF with the SAGA histone acetyltransferase coactivator complex that was identified in the Workman laboratory will be characterized to determine the involvement of dMLF in SAGA-mediated gene regulation. As overexpression of hMLF1 and the expression of NPM-MLF1 are associated with the formation of leukemia, transgenic animals overexpressing dMLF and NPM-dMLF will be examined to determine the effects of these mutations on transcriptional regulation and cellular proliferation in hematopoietic cells. In sum, these studies will characterize the functional roles of dMLF and leukemia-associated mlf mutations in transcriptional regulation and cellular proliferation in vivo. Through the identification of protein interactions and gene expression levels that differ in mlf mutants, these studies could provide new targets for the treatment or cure of acute myeloid leukemia. The long-term goal of these studies is to determine how hMLF1 is involved in the formation of leukemia and to apply this knowledge to inhibit the expansion of immature blood cells characteristic of acute myeloid leukemia.
Mutations in the gene called human myelodyplasia/myeloid leukemia factor 1, abbreviated MLF1, are associated with the development of acute myeloid leukemia, a cancer of the blood. As little is known about the role of MLF proteins in the formation of cancer, the studies described in this proposal aim to determine how MLF functions in normal and cancerous cells. Through developing a better understanding of how MLF proteins are involved in cancer formation, these studies could determine new targets for the treatment and/or cure for acute myeloid leukemia.
|Dyer, Jamie O; Dutta, Arnob; Gogol, Madelaine et al. (2017) Myeloid Leukemia Factor Acts in a Chaperone Complex to Regulate Transcription Factor Stability and Gene Expression. J Mol Biol 429:2093-2107|