Clinical and molecular effects of azacitidine in the myelodysplastic syndromes

Abstract: The myelodysplastic syndromes (MDS) constitute a heterogeneous group of malignant bone marrow disorders characterized by peripheral cytopenia and increased risk of leukemic progression. In higher-risk MDS, Azacitidine has been shown to prolong survival and modulate the epigenome, although the precise mechanisms by which the drug exerts its effect are unknown. Paper I reports the result from a Nordic study enrolling 30 transfusion-dependent Epo-refractory lower-risk MDS patients. Patients were treated with 6 cycles of Azacitidine and terminated the study if they reached transfusion independence, while non-responding patients received another 3 cycles combined with Epo. Five (21%) and one patient responded after Azacitidine and the combined treatment, respectively, and only 2 patients (10%) responded for more than 6 months. Toxicity was substantial, mainly consisting of infections. We conclude that Azacitidine can be effective in this cohort of patients but that the low response rate and relatively high toxicity precludes its recommendation as standard treatment. Targeted sequencing revealed a high frequency of recurrent MDS mutations without clear relation to response. In paper II we cultured CD34+ progenitors from higher-risk MDS and normal bone marrow (NBM) with or without Azacitidine and studied the effects on DNA methylation and histone acetylation. We showed that the MDS genome at the global level is hypermethylated compared to NBM and that Azacitidine induced profound demethylation. Histone acetylation was decreased by treatment, which theoretically would counteract the transcriptional activation resulting from reduced DNA methylation. To further explore these effects, we repeated the same culture experiment in paper IV to study the effects of Azacitidine on both DNA methylation and gene expression. We confirmed the marked demethylating effect of Azacitidine, and by using RNA seq we could show that Azacitidine significantly increases gene expression but without association with demethylated regions. Interestingly, the repressive histone mark H3K9me3 increased in three demethylated genes without increased expression, providing a potential explanation for the lack of association between demethylation and increased expression. In paper III we searched for factors associated with response to Azacitidine by studying clinical parameters (n=134); mutations (n=90); and DNA methylation (n=42) in patients treated with Azacitidine. Among the clinical variables, only disease duration before treatment predicted for poor response and survival. The group of mutations involved in histone modulation (ASXL1, EZH2, MLL) was associated with prolonged survival, contrasting previous reports on mixed MDS cohorts. Furthermore, DNA methylation profiles differed significantly between responding and non-responding patients. Analysis of 200 differentially methylated regions showed enrichment in pathways involved in differentiation and development. Methylation level of the most significant DMR, the HOXA5/A6-locus, was associated with survival. To summarize, these studies show that epigenetic modifications play a significant role in the pathogenesis and response to treatment in MDS and that further understanding of chromatin modifications will be important in order to develop therapeutic strategies in MDS.