DNMT3B Expression Delays Leukemogenesis,

Abstract 3446

Acute myeloid leukaemia (AML) is a malignant disease with poor prognosis, which is, among other biological features, characterized by epigenetic changes including alterations in DNA methylation. 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 DNMT overexpression on leukemogenesis using an inducible DNMT3B mouse model (Linhart et al., 2007). To analyse the impact of DNMT3B overexpression on leukemia we retrovirally co-transduced lineage-negative bone marrow cells of wildtype and DNMT3B^tg mice with a MSCV-cMyc-bcl2 and a MSCV-tTA-GFP containing vector. Under these conditions, doxycycline suppressed DNMT3B expression whereas absence of doxycycline led to overexpression of DNMT3B on the mRNA and protein level. DNMT3B overexpression was not toxic since colony formation in vitro did not differ between DNMT3B expressing and physiologically expressing cells. To analyze leukemogenesis, 5 × 10⁴ sorted GFP-positive cells were transplanted into sublethally irradiated wildtype recipients. Both recipients of transduced wildtype cells and recipients of transduced DNMT3B^tg cells developed leukemia with a tendency of delayed leukemogenesis in DNMT3B overexpressing mice. GFP positive leukemic cells were sorted and doxycycline regulated DNMT3B expression was verified by Western blot analysis in vitro. To determine the repopulation capacity of the leukemic cells we performed transplantation of GFP-positive primary leukemia cells into secondary wildtype recipients. Leukemia of both, wildtype and DNMT3B-overexpressing donors was transplantable and lethal. However, DNMT3B^tg leukemic cells were severely impaired in leukemia development in secondary recipients. Secondary recipients of leukemic DNMT3B^tg cells died significantly later (p= 0.02). Taken together, these findings demonstrate that DNMT3B expression impairs leukemia maintenance. Loss of DNMT activity might contribute to the pool size of leukemia initiating cells.