Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal disorders characterized by progressive peripheral blood cytopenias, hypercellular or normocellular bone marrow, morphologic abnormalities of the erythroid and megkaryocytic lineages, and propensity to progress to acute leukemia. Despite the high morbidity and mortality associated with these disorders, the molecular mechanisms underlying their pathogenesis and progression remain incompletely understood. Mutations involving the transcription factor Runx-1 have recently been identified in a large number of MDS cases, especially those that progress to leukemia. This proposal uses Runx-1's recently discovered role in normal megakaryopoiesis as a model to better understand the mechanisms by which it may be perturbed by MDS associated mutations. Many transcription factors, in addition to binding DMA, make important protein-protein interactions that modulate their activity. Preliminary studies using gel filtration chromatography indicate that Runx-1 participates in at least two stable multiprotein complexes of approximately 150-300 kDa and >669 kDa in a murine megakaryoblastic cell line. This proposal utilizes new powerful proteomic technology to identify components of these complexes and probe their functional significance. It takes advantage of a technique we developed for metabolic biotin tagging of recombinant proteins in mammalian cells. This high affinity tag is combined with a FLAG epitope for tandem affinity purification of complexes under native conditions. Whole lane LC/MS/MS mass spectrometry is then used to comprehensively identify associated proteins in a non-biased manner. After validation of results, the functional significance of identified proteins is probed, and the effect of MDS-associated Runx-1 mutations on their interactions examined. Our preliminary results support recent reports of a physical interaction between Runx-1 and GATA-1, a zinc finger transcription factor central to erythroid and megakaryocyte terminal maturation. The interacting domains of these molecules will be further defined, and mutations that disrupt binding identified in a yeast altered specificity mutant screen. These mutants will then be used to test the hypothesis that Runx-1 :GATA-1 interactions are functionally important in normal megakaryopoiesis, and may be disrupted by MDS-associated mutations. These studies should provide important insights that will facilitate the design of novel therapies for MDS.
