The bone marrow microenvironment plays an important role in the pathogenesis and perpetuation of stem cell defects in Myelodysplastic Syndrome (MDS). However, while distinct cytogenetic alterations have been described in the stem cell compartment in MDS, the bone marrow (BM) stroma has never been shown to be part of the clone. Thus, aberrant epigenetic alterations may be responsible for altered function of BM stroma in MDS. DNA methyl transferase (DNMT) inhibitors, which are therapeutically effective in MDS, can potentially affect both hematopoietic cells and the stroma, providing further rationale for studying DNA methylation profiles of BM stroma in MDS. To accomplish this aim, adherent non-hematopoietic cells were isolated from MDS patients and controls by immunomagnetic CD45 negative selection and then used for whole genome methylation studies using the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR) . Global epigenetic profiling revealed that MDS stroma (n=7) was epigenetically distinct from normal BM stroma (n=3) (ANOVA, P<0.0001). Many novel genes were differentially methylated in MDS stroma, though majority were found to be hypermethylated compared to normal controls. Growth regulators and transcription factors such as BMP-9, PAX-4, EIF2B1, BATF-1 TNF, HGF, CXCL12, SOCS2, ITGA10, were most significantly hypermethylated.
In subsequent studies, we profiled stroma from another set of MDS patients treated with the DNMT inhibitor, 5-Azacytidine (5-Aza) (n=4). In contrast to untreated MDS patients, the 5-Aza treated MDS patients and healthy controls (p = NS) were epigenetically similar. These 5-Aza exposed stroma cells did not demonstrate increased cytosine methylation thus suggesting that these cells were also targeted by DNMT depletion by the drug.
Finally, the ability of MDS and control stromal cells to support the growth of healthy CD34+ cells was tested by in-vitro colony formation assay. MDS stromal cell co-culture led to significant reduction in erythroid and myeloid colony formation, demonstrating the MDS stroma contributes to ineffective hematopoiesis seen in this disease. Strikingly, pretreatment of MDS stromal cells with 5-aza increased the colony formation seen in co-cultures, demonstrating again that DNMT inhibitors exert effects on the microenvironment.
Thus our results reveal for the first time that MDS is characterized by widespread aberrant epigenetic changes in the BM microenvironment. Our results also demonstrate that DNMT inhibitors can alter the epigenomic profiles of stromal cells, contributing in part to their clinical efficacy. Overall, these studies underscore the importance of studying the entire BM, including the microenvironment to improve our understanding of the pathophysiology of MDS and therapy.
Citation Format: Tushar D. Bhagat, Matthias Bartenstein, Yiting Yu, A. Mario Marcondes, Davendra Sohal, John Greally, H Joachim Deeg, Amit Verma. Myelodysplasia bone marrow stromal cells have widespread aberrant hypermethylation that is targeted by treatment with DNMT inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1503. doi:10.1158/1538-7445.AM2013-1503