Department of Pathology

Chromosomal rearrangements lead to the formation of abnormal fusion proteins.Fifteen different chromosomal rearrangements involving the Nup98 gene have been described, of which 13 are associated with AML. Leukemias associated with Nup98 gene rearrangements tend to be aggressive, with a poor response to therapy. Nup98 gene rearrangements result in the formation of fusion proteins that consist of Nup98 fused to one of 15 partner proteins. The mechanisms by which these proteins transform cells are not clear. Our research aims to elucidate the mechanisms by which Nup98 fusion proteins induce leukemia in order to pave the way for the development of more effective therapies. The best characterized Nup98 fusion is Nup98-HOXA9, which contains the DNA-binding homeodomain (HD) of the homeobox transcription factor HOXA9 and the N-terminal portion of Nup98. Our current studies are focused on this protein.

We have recently shown that Nup98 acts as an aberrant transcription factor in a myeloid cell line, with a stronger and wider transcriptional activity than wild-type HOXA9. Thus Nup98-HOXA9 is a gain-of-function mutant, in contrast to other aberrant transcription factors in AML that appear to act as dominant negative suppressors of their wild-type counterparts. In order to understand the mechanisms by which Nup98-HOXA9 transforms myeloid cells we are pursuing the following approaches:

1. We will identify genes that are directly regulated by Nup98-HOXA9 using inducible systems of Nup98-HOXA9 expression in primary human cells and in cell lines. The Nup98-HOXA9 DNA-binding sites in their promoter regions will be determined using chromatin immunoprecipitation, luciferase, and gel-shift assays.

2. We have shown that Nup98-HOXA9 acts predominantly as a transcriptional activator, with a wider and stronger transcriptional effect than wild-type HOXA9. This could be due to differences in DNA binding and/or recruitment of co-activators/co-repressors by the two proteins. We will assess differences in DNA binding using gel-shift assays; and differences in protein recruitment will be determined by proteomic analysis of HOXA9 and Nup98-HOXA9 immunoprecipitates from myeloid cells.

3. Nup98-HOXA9 will be expressed in primary human hematopoietic cells in order to assess its effects on proliferation, cell cycle, differentiation, and apoptosis. Simultaneously, RNA will be isolated and subjected to microarray analysis to identify gene expression patterns associated with the biological effects.

These studies should clarify the mechanisms and pathways involved in Nup98-HOXA9 leukemogenesis and suggest possible targets for therapeutic intervention.