Our hope of curing leukemia rests on our understanding of the molecular mechanisms involved in leukemogenesis. Insight into this can be garnered from the study of nonrandom chromosomal translocations that occur in different subsets of leukemia. The t(3;21) occurs in the setting of therapy-related AML, the blast crisis phase of CML, and de novo AML, and is associated with a particularly poor prognosis. This translocation results in the production of the 190 kDa AML1/MDS1/EVII (AME) fusion protein. This protein has been shown to induce leukemia in mice following transplantation of transduced bone marrow cells into irradiated recipients, and to immortalize murine bone marrow cells as assessed by a serial replating assay. The mechanism by which this protein transforms cells is not known. We propose three specific aims to address this mechanism. The first goal is to use in vitro replating assay to assess the transforming capability of AME mutants that lack key functional domains. AME is known to contain a number of domains within the AML1, MDS1 and EVIl portions of the protein. We will test the ability of these mutants to transform mouse bone marrow cells in vitro, using the serial replating assay, to determine which of these functions is important for transformation. The second goal is to determine the components of protein complexes that contain AME from transformed hematopoietic cells. We have created a C-terminally tagged version of AME that is capable of transforming as assessed by the in vitro replating assay. This tag is tandem affinity purification (TAP) tag allows for rapid purification of protein complexes from crude cell extracts. In we will purify complexes from cells expressing transforming mutant forms of AME. This will allow us to identify interactions that are essential for transformation. Using mass spectrophotometric techniques, we will determine the components of these complexes. The third goal is to use short hairpin inhibitory RNAs to downregulate AME in AME-transformed hematopoietic cells, and determine the changes in gene expression by microarray analysis. We will also develop an inducible RNA system in AME-transformed cells, to allow rapid suppression of AME. This, in conjunction with time course microarray analysis, will allow us to determine, in a temporal manner, the changes in gene expression that ensue from loss of AME.
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| Zhang, Yi; Stehling-Sun, Sandra; Lezon-Geyda, Kimberly et al. (2011) PR-domain-containing Mds1-Evi1 is critical for long-term hematopoietic stem cell function. Blood 118:3853-61 |
