DNA methylation and 5-azacytidine in myelodysplastic syndromes : Pharmacodynamic, mechanistic and clinical studies

Abstract: Promoter DNA hypermethylation and hence silencing of e.g. tumour suppressor genes is considered to be an important step in carcinogenesis and has been associated with poor outcome in patients with myelodysplastic syndromes (MDS). In contrast to many other chemotherapeutic agents, the DNA hypomethylating compound 5-azacytidine has a positive therapeutic effect in patients with high-risk myelodysplastic syndromes (MDS), however, the lack of knowledge about its mechanism of action hampers clinical development of the drug. The first aim of the thesis was to assess the pharmocodynamic properties of 5-azacytidine, and the mechanism of 5-azacytidine-induced apoptosis as well as its link with DNA methylation. Secondly, we investigated the role of gene-specific methylation status for the outcome of induction chemotherapy in a cohort of patients high-risk MDS and MDS-AML, and assessed global and gene-specific (p15INK4B) methylation patterns in another cohort of patients with low and intermediate-risk MDS. 5-azacytidine induced dose-dependent hypomethylation and growth inhibition, as well as a complex pattern of apoptotic mechanisms regulated by multiple and separate pathways. Furthermore, we demonstrated a possible link between apoptosis and hypomethylation. We showed that optimal cellular effects of 5-azacytidine could be achieved by shorter exposure times than expected. Translated to shorter treatment duration in patients, this may eventually lead to superior and more cost-effective and convenient clinical treatment schedules. P15INK4B methylation plus methylation of CDH or HIC gene was more frequent in patients with conventional high-risk features, such as adverse karyoptype and high percentage of marrow blasts. DNA hypermethylation of multiple surrogate genes correlated with the percentage of blasts and marrow CD34 expression, but not with the degree of inefficient haematopoiesis. We demonstrated a significant negative impact of promoter methylation status on the outcome of conventional induction chemotherapy in high-risk MDS and MDS-AML, a finding that may have significant impact on the clinical management of patients with high-risk myeloid disease. Global and gene-specific DNA methylation pattern in MDS correlated with each other in the sense that more advanced MDS, characterized by p15INK4B methylation showed higher global DNA methylation than those with unmethylated p15INK4B. Global methylation status did not correlate with conventional risk factors, but was more pronounced in patients with severe erythroid failure. Patients with stable disease maintained their global DNA methylation level over time, and disease progression was associated with an increase in global DNA methylation in only half of the cases. To conclude, these studies provide important knowledge about the cellular mechanisms of 5-azacytidine and role of DNA methylation in patients with MDS and MDS-AML.