The overall goal of this application is to determine the role of topoisomerase (topo) IIbeta in all trans retinoic acid (ATRA)-induced differentiation of acute myeloid leukemia (AML) cells, within the broader context of identifying signaling mechanisms involved in myeloid differentiation. In studies employing pharmacologic inhibition or down- regulation with targeted si-RNA of topo IIbeta in a panel of AML cell lines we established that topo IIbeta is required for survival of ATRA-differentiated cells. Preliminary studies to identify the mechanisms by which deficiency in topo IIbeta promotes ATRA-induced apoptosis revealed down regulation of the redox regulator, peroxiredoxin 2 (PRDX2), and ATRA-induced accumulation of reactive oxygen species (ROS) and up regulation of regulator of G-protein signaling (RGS2), which is involved in myeloid differentiation and stress response. Thus, our working hypothesis is that ATRA- induced differentiation of APL cells requires topo IIbeta and/or PRDX2 as survival signals, in the absence of which the cell death pathway is activated via a mechanism involving accumulation of ROS and up-regulation of RGS2. To test this hypothesis we will use models of AML cells that differentially express topo IIbeta, PRDX2 or RGS2 to determine the functional significance of down regulation of PRDX2 and ATRA-induced up-regulation of RGS2. Specifically we will determine ATRA-induced differentiation, ROS accumulation and apoptosis following a) down regulation of PRDX2 or over expression of RGS2 in topo IIbeta expressing AML cells, or b) over expression of PRDX2 or down regulation of RGS2 in topo IIbeta deficient AML cells. We will next determine whether deficiency in topo IIbeta and PRDX2 leads to activation of the extrinsic and/or intrinsic caspase pathway following ATRA-induced differentiation and whether cross- talk between these pathways is involved. For these studies we will examine activation of TRAIL, caspases (9, 8 and 3) and PARP, cleavage of BID and release of cytochrome c in ATRA treated topo IIbeta -deficient AML cells. Further we will examine the effect of inactivating caspase 9 or 8 on ATRA-induced apoptosis in topo IIbeta -deficient cells. The effect of ATRA treatment on differentiation, growth arrest and apoptosis in the absence or presence of the topo IIbeta catalytic inhibitor will be determined in patient derived myeloid leukemias. These results will be corroborated with those in AML cell lines. The proposed studies should provide important information on novel targets regulating ATRA-induced differentiation, growth arrest and apoptosis of AML cells. In the long term, identification of these targets would assist in the development of novel strategies for treatment of AML secondary to myelodysplastic syndrome. ? ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA117928-02
Application #
7254799
Study Section
Basic Mechanisms of Cancer Therapeutics Study Section (BMCT)
Program Officer
Mufson, R Allan
Project Start
2006-07-01
Project End
2011-05-31
Budget Start
2007-06-01
Budget End
2008-05-31
Support Year
2
Fiscal Year
2007
Total Cost
$266,285
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
135781701
City
Cleveland
State
OH
Country
United States
Zip Code
44195
Grozav, Adrian G; Willard, Belinda B; Kozuki, Toshiyuki et al. (2011) Tyrosine 656 in topoisomerase II? is important for the catalytic activity of the enzyme: Identification based on artifactual +80-Da modification at this site. Proteomics 11:829-42
Chikamori, K; Grozav, A G; Kozuki, T et al. (2010) DNA topoisomerase II enzymes as molecular targets for cancer chemotherapy. Curr Cancer Drug Targets 10:758-71
Grozav, Adrian G; Chikamori, Kenichi; Kozuki, Toshiyuki et al. (2009) Casein kinase I delta/epsilon phosphorylates topoisomerase IIalpha at serine-1106 and modulates DNA cleavage activity. Nucleic Acids Res 37:382-92
Chikamori, K; Hill, J E; Grabowski, D R et al. (2006) Downregulation of topoisomerase IIbeta in myeloid leukemia cell lines leads to activation of apoptosis following all-trans retinoic acid-induced differentiation/growth arrest. Leukemia 20:1809-18