Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells characterized by ineffective hematopoiesis and, in the high risk setting, by progression to acute myelogenous leukemia (AML). The DNA hypomethylating agents, 5-azacytidine (5AC) and 5-aza-2’-deoxyazacytidine (DAC), are effective treatments for patients with MDS, increasing time to progression to AML and improving overall response rates and survival. Genome-wide DNA hypermethylation in MDS has been documented. However, promoter methylation signatures in MDS samples have not correlated with the clinical response to epigenetic drug therapies. Recently, attention has been drawn to the potential etiologic role of reduced expression of specific ribosomal proteins in del (5q) MDS, as well as in other bone marrow failure states. We therefore asked whether CD34+ cells from the bone marrow of MDS patients had reduced expression of ribosomal RNA (rRNA) and whether this correlates with hypermethylation of ribosomal DNA (rDNA). Using quantitative SYBR-Green RT-PCR, we found that rRNA expression was significantly reduced in CD34+ hematopoietic progenitor cells from 15/29 MDS patients as compared to normal controls (p value <0.01). We then studied quantitative rDNA methylation using pyrosequencing in DNA from CD34+ cells isolated from 5 normal controls and 6 MDS samples. Two of six MDS samples had reduced rRNA expression and increased DNA methylation, not only within the upstream core element but also in the intergenic rDNA sequences. For each of the CpGs evaluable within these highly CpG rich regions, the extent of DNA methylation in patients with reduced rRNA expression were increased by more than 2-fold compared to that in normal samples. This DNA hypermethylation was not observed in rDNA from 4/6 MDS samples where rRNA expression was not reduced in relation to controls. In addition, cells treated with DAC demonstrated a two-fold induction in rRNA gene expression and an up to four-fold reduction in rDNA promoter methylation as compared to controls, suggesting that promoter methylation may have a direct effect on rRNA expression. We are currently examining MDS patient samples before and after DAC treatment for levels of rDNA methylation and expression. It is possible that the methylation status at the rDNA locus may serve as a better predictor of response to hypomethylating agents than genome-wide methylation status. Regulation of rDNA gene expression is complex, but may be a fundamental underlying mechanism of bone marrow failure in a subgroup of MDS patients and perhaps in other bone marrow failure states.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.