CONICET | Buscador de Institutos y Recursos Humanos

The leukemias are malignant diseases of hematopoietic cells in which the proper balance between proliferation, differentiation and apoptosis is no longer operative. Synthetic glucocorticoids, like dexametasone (dex), are frequently used in the treatment of hematopoietic diseases due to its proapoptotic properties, and although their mechanism of action on leukemic cells is unclear, these steroid hormones could represent an alternative and promising therapy. The trithorax protein ASH2L is considered to be one of the core subunits of all known H3K4-‐ specific MLL/SET methyltransferase complexes and functions as an oncoprotein that is overexpressed in promyelocytic leukemia. The Nuclear Coactivator 6, NCoA6, is essential for nuclear receptor function in vivo and is overexpressed in various human cancers. The main goal of this project is to study the role of ASH2L and NCoA6 in GR-‐mediated apoptosis in human leukemic cells. Our previous results demonstrated that U937 cell line undergoes a significant cell death after dex treatment, and this correlated with the downregulation of the antiapoptotic isoform Bcl-‐XL, that is overexpressed in myeloid leukemia. Interestingly, both ASH2L and NCOA6 showed to be involved as putative epigenetic modulators in this response to glucocorticoids, contributing to transcriptional output regulation. Importantly, we demonstrated a novel endogenous interaction between GR, ASH2L and NCoA6 that is even stronger in the presence of hormone. Chromatin immunoprecipitation experiments reveal GR recruitment to bcl-‐X HREs (+10, +18 and +58 kbp from TSS) and also to the promoter region in the absence of hormone. Upon dex treatment, GR is displaced from chromatin when ASH2L and NCoA6 are expressed. We discovered that knockdown of ASH2L increases the binding of GR to HREs, but abolishes receptor recruitment to promoter region. In addition, we found that NCoA6 expression is essential for GR stability at chromatin. By chromosome conformation capture (3C) experiments we demonstrated interaction of the +58 region with bcl-‐X promoter, implying direct regulation of bcl-‐X expression by this distal GR enhancer. Overall, our data reveals the existence of a transcriptional looping model dedicated to inducing apoptosis of leukemic cells through bcl-‐XL downregulation. We predict that NCoA6 is a central component of the complex, and is required for GR and ASH2L recruitment to glucocorticoid-‐target genes. Further characterization of this complex could thus be instrumental in defining a general epigenetic mechanism that identifies attractive targets for therapeutic strategies in myeloid leukemia.