Rearrangements of the ALL-1/MLL1 gene, resulting in production of ALL-1 fusion proteins, underlie the vast majority of infant acute leukemias and of a substantial part of a therapy-related leukemia. The way by which the fusion proteins trigger leukemia is not known. Here we attack this issue in several directions: 1) we have recently found that expression of ALL-1 fusion proteins in leukemic and transfected cells results in upregulation of a specific micro RNA. miR-191. We found that the upregulation is due to recruitment of ALL-1 fusion proteins, together with the general processor of microRNAs-Drosha, to the miR-191 locus. Since Drosha is not recruited with normal ALL-1 to the same locus (nor was it shown to be bound to other rniR loci), we consider this recruitment as very significant in miR-191 upregulation. Included in the proposal are experiments concerning potential association between ALL-1 fusion proteins and Drosha, involvement of Drosha's recruitment in miR-191 overexpression in solid tumors, direct role of miR-191 in onset of acute leukemia, effect of miR-191 overexpression on differentiation of hematopoietic precursor cells, and identification of miR-191 target gene(s). 2) We have used a combination of conventional columnes, immunearflnlty purification, and mass spectrometry analysis to identify a series of proteins associated with ALL-1/AF4 within a multiprotein complex. The identified proteins will be examined by multiple approaches to determine whether they constitute integral components of the ALL-1/AF4 complex. A follow-up will focus on interesting components. 3) Following our success in bringing off a genome-wide location analysis of normal ALL-1, we will extend it to a global analysis of the gene targets of ALL-1 fusion proteins ALL-1/AF4 and ALLVAF9 in leukemic lymphoid and myeloid cell lines, respectively.
