DNA methyltransferases (DNMT) play an important role in regulation of DNA methylation and mutations of DNMT3A are frequently found in AML. We analyzed the effects of DNMT3B overexpression on leukemogenesis using a tetracycline-inducible DNMT3B mouse model. To analyze the impact of DNMT3B overexpression on leukemogenesis, we retrovirally co- transduced lineage-negative bone marrow cells of wildtype and DNMT3B transgenic mice with an MSCV- cMyc-bcl2 or MSCV-MLL-AF9-hCD4 and an MSCV- tTA- GFP expressing vector. Using these conditions, doxycycline suppressed DNMT3B expression whereas absence of doxycycline led to overexpression of DNMT3B on the mRNA and protein level. To verify that expression of DNMT3b in this mouse model was able to induce DNA methylation, we performed Reduced Representation Bisulfite Sequencing (RRBS) on cMyc-Bcl2 leukemic bone marrow cells. Analysis of the global methylation levels revealed a strong hypermethylation in DNMT3b overexpressing bone marrow cells. Differentially methylated regions (DMR) were defined as those regions with at least 30% difference in methylation levels. DMRs were mainly located in promoter regions and first introns and the heatmap of these DMRs confirms distinct hypermethylation in DNMT3b transgenic cells whereas very few regions become hypomethylated in transgenic cells when compared to wildtype cells. This specific methylation pattern could be responsible for altered gene expression and consecutively altered (stem) cell characteristics.
To analyze the influence of changed DNA methylation levels induced by expression of DNMT3b on leukemogenesis, sorted GFP+cells were transplanted into sublethally irradiated wildtype recipients. Mice with and without DNMT3B expression developed leukemia with splenomegaly and a tendency of delayed leukemogenesis in DNMT3B overexpressing mice. Mice with cMyc-Bcl2- and MLL-AF9-induced leukemia showed a clear myeloid phenotype with enhanced expression of the stem cell marker c-kit on MLL-AF9 leukemic cells. To consider the leukemogenic capacity, we transplanted the leukemic blasts into secondary recipients. cMyc-Bcl2 induced leukemia of both, wildtype and DNMT3b recipients was transplantable and lethal. DNMT3B tg leukemic cells were severely impaired in leukemia development in secondary recipients. Secondary recipients of cMyc-Bcl2 induced leukemic DNMT3B cells died significantly later (p= 0.04).
As differential methylation can also have an effect on the responsiveness to chemotherapy, spleen cells from MLL-AF9 leukemic mice were treated with different cytotoxic and demethylating drugs to see if DNMT3b induced hypermethylation confers a resistance or increased sensitivity to chemotherapeutic treatment. DNMT3b overexpressing cells were more sensitive to treatment with Azacytidine and Decitabine.
Taken together, these findings demonstrate that expression of DNMT3b in a doxycycline-regulatable mouse model induces widespread DNA hypermethylation, whereas a few small regions become hypomethylated when compared to wildtype cells. This specific methylation pattern is associated with delayed leukemogenesis and DNMT3b expression significantly impaired leukemia maintenance. In addition, response to chemotherapeutic treatment is altered by DNMT3b induced hypermethylation which underlines the importance of epigenetic regulatory mechanisms as promising targets for new therapeutic strategies in AML.
Müller-Tidow:Celgene: Lecture fees Other; Deutsche Forschungsgemeinschaft: Research Funding.
Asterisk with author names denotes non-ASH members.