Loss of chromosome 7 (monosomy 7) and deletion of a segment of the long arm [del(7q)] are recurring cytogenetic abnormalities in de novo and therapy-related myeloid malignancies that are associated with a poor prognosis. In previous studies supported by this award, cytogenetic analysis delineated a commonly-deleted segment (CDS) in patients with myeloid disorders characterized by a del(7q) within band q22 that accounts for most cases, and a second CDS in bands q32-34. Using an ordered set of yeast artificial chromosome clones as probes, fluorescence in situ hybridization experiments were then performed on leukemias with deletion breakpoints within 7q22. These studies implicated a ~2.5 Mb CDS as harboring a tumor suppressor gene (TSG) that is inactivated in myeloid malignancies. During this period of support, we have extensively characterized this CDS, identified and cloned 19 known and novel genes from the interval, analyzed leukemia samples for mutations in these candidate TSGs, and performed Taqman real time quantitative polymerase chain reaction experiments to measure expression levels in normal and leukemic human bone marrows. These studies did not uncover either pathogenic mutations or a consistent reduction in expression levels in any candidate TSG. We also harnessed chromosome engineering technology to introduce loxP sites flanking a ~2 Mb syntenic interval on mouse chromosome 5, and have generated conditional and germline A5 mice. We are currently pursuing a number of strategies to identify candidate TSGs and cooperating genes in these strains. Our underlying hypotheses are: (1) that a myeloid TSG resides either within this 7q22 CDS or in a DNA segment proximal to this CDS;and, (2) that this gene is either inactivated or demonstrates reduced expression in myeloid malignancies with monosomy 7 or by a del(7q). We will exploit reagents that we have generated and a diverse collection of leukemia samples to pursue this hypothesis through two aims: (1) to use heterozygous and homozygous A5 mice to identify candidate human 7q22 TSGs and other genes that cooperate with the A5 deletion in leukemogenesis;and (2) to integrate new data developed by Projects 2 and 3 and by other researchers to prioritize candidate myeloid TSGs in the interval immediately proximal to the 7q22 CDS that might cooperate with loss of the 7q22 CDS. We will interrogate human leukemia specimens for mutations in genes located in a 2.1 Mb interval immediately proximal to the current CDS, and develop reagents for modeling loss of this more proximal segment in the mouse. Project 4 is highly interactive with Projects 2 and 3;together, these projects seek to identify the spectrum of genetic mutations, and genetic pathways leading to alkylating-agent induced t-AML.
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