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.
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