5-Azacytidine and G-CSF Derepressed Chromatin Structure of PU.1 and Its Targets Cebpa and Cbfb In Myelodysplastic Syndrome (MDS)

Abstract 124

The myelodysplastic syndrome (MDS) represents a heterogeneous disorder characterized by ineffective hematopoiesis and evolution to acute myelogenous leukemia that is strikingly refractory to current therapeutic approaches. Novel epigenetic drugs including DNA-methyltransferase inhibitor 5-Azacitidine (5-AZA, Vidaza) are currently considered to improve clinical response in patients with MDS. MDS is characterized by abnormal differentiation and blocked maturation responsive to 5-AZA, therefore we studied major regulator of hematopoietic differentiation, transcription factor PU.1 as a candidate target of the epigenetic therapy. Transcription factor PU.1 represents very important myelo-lymphoid regulator of differentiation. PU.1 expression is regulated by Upstream Regulatory Element (URE) and its deletion in mouse caused downregulation of PU.1 leading to acute leukemia (Rosenbauer 2004). Our laboratory recently demonstrated that PU.1 in murine acute leukemic cells binds and promotes derepression of CCAAT/enhancer binding protein (C/EBP) alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) (Burda 2009) that encode two key hematopoietic transcription factors involved in myeloid differentiation. Furthermore, transcriptional regulation through PU.1 binding sites of Cebpa and Cbfb loci involves quantitative increases in a transcriptionally active chromatin mark: acetylation of histone H3 lysine K9. Others reported that Cebpa expression is augmented by G-CSF (Dahl 2003). To determine if 5-AZA regulates PU.1 and its targets we determined their expression and chromatin structure following the 5-AZA treatment in MDS patient-derived blasts and in cell lines derived from MDS (MOLM-13, OCI-M2, SKM-1) and AML (K562). Our data provide evidence that in the chosen cell lines and in so far limited number of patients-derived cells (N=4) the gene expression of PU.1 and its direct targets Cebpa and Cbfb is stimulated by 5-AZA and this effect is further enhanced by G-CSF. Furthermore, marks of activated chromatin structure including histone H3K9 hyperacetylation and H3K4 hypermethylation are increased at the URE of the PU.1 gene again documenting its transcriptional activation. Conversely, levels of H3K9 methylation at URE are significantly reduced upon 5-AZA treatment documenting 5-AZA stimulates loss of repressive chromatin structure near PU.1 gene. These observations are currently compared with responsiveness of the patients to 5-AZA in vivo and expanded to larger set of patients. Our data collectively supports importance of the chromatin structure upstream of PU.1 gene and of its direct targets Cebpa and Cbfb in patients with MDS that may add to better understanding of effectiveness of epigenetic therapy in MDS. (Grants # IGA 10310-3, MSMT 2B06077, SVV-2010-254260507, MPO FR-TI2/509, GAUK 251135 82210).