Use of DNA demethylating agent 5-azacytidine (AZA) in high risk Myelodysplastic syndromes (MDS) represents an important strategy yet not fully understood at molecular levels. Previous work demonstrated that normal progenitors treated by DNA demethylation agent Decitabine upregulated a key hematopoietic transcription factor PU.1. This was potentiated in presence of granulocyte colony stimulating factor (G-CSF) prior the DNA demethylation (Hu et al. 2010). Similarly, Decitabine administered to multiple myeloma cells resulted in DNA demethylation and upregulation of PU.1 gene (Tatetsu et al. 2007). To advance our understanding of molecular events mediated by AZA in MDS we measured effect of AZA on transformed cell lines (OCI-M2, SKM-1, MOLM-13, HNT-34, F-36P) and monitored the hematopoietic transcriptional program. We herein present evidence that AZA, while inducing efficiently cell cycle arrest and apoptosis of MDS tumor cells, stimulates also the expression of PU.1 and its target C/EBPa that is followed by acquisition of the myeloid differentiation program. Gene derepression of PU.1 is marked by epigenetic modifications at the upstream regulatory element (URE) of PU.1. G-CSF potentiated effects of AZA in MOLM-13 and SKM-1 cells. The effects of G-CSF and other cytokines (such as M-CSF or GM-CSF) on monocytic versus granulocytic lineage commitment of MDS cells are currently tested. To advance our study on the patient level we used primary high risk MDS CD34+ cells (N=36). Tumor cells with the lowest PU.1 expression displayed partial methylation at URE and incomplete sensitivity to AZA/G-CSF-mediated in vitro differentiation. We observed that patients with permissive chromatin at URE and higher PU.1 (and C/EBPa) levels responded significantly better to AZA/G-CSF in vitro. Currently we enrolled more patients in order to ask whether permissive PU.1 locus predicts responsiveness to AZA and whether it associates with different clinical patient outcome. (Grants: IGA NT10310-3/2009, MPO FR-TI2/509, GAUK251135 82210 & 251070 45410, SVV-2011-262507).
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.