Heterozygous Dnmt3a Mutation Induces Expansion of Hematopoietic Stem Cell Pool in a Murine Model

Somatic mutations in DNMT3A, a member of the DNA methyltransferase family, have been identified in various kinds of hematologic malignancies including acute myeloid leukemia (AML), acute lymphoblastic leukemia, myeloproliferative neoplasms, and aplastic anemia. Especially, in cytogenetically normal AML, DNMT3A mutations are detected in 15-20% of cases and associated with poor prognosis. Approximately 40 to 60% of DNMT3A mutations reside at Arg882 (R882), forming hot spot mutation site, implying gain-of-functional property of the mutation in development of leukemia. So far, a functional role of DNMT3A mutation in leukemogenesis has been investigated by overexpressing DNMT3A R882 mutant or Dnmt3a knockout in murine hematopoietic cells. However, the consequenses of DNMT3A R882 mutant with endogenous expression have been largely unknown. To elucidate this, we generated a novel mouse model for Cre-mediated conditional expression of Dnmt3a R878C mutant (homologous to human R882C) from the endogenous locus of Dnmt3a. For hematopoietic cell-specific mutation in vivo, we crossed these mice with Vav-Cre mice. By analyzing genotypes of offspring derived from the pair of Dnmt3a^R878C/wt; Vav-Cre^wt/wt mice and Dnmt3a^R882C/wt; Vav-Cre^tg/wt mice, it is revealed that mice harboring homozygous mutation of Dnmt3a in hematopoietic cells (DNMT3A^R882C/R882C; Vav-Cre^tg/wt) were not born for now. In contrast, Dnmt3a^R882C/wt; Vav-Cre^tg/wt (hereafter Dnmt3a R878C) mice were normally born and they did not show any hematological or other disorders at least until 40 weeks after birth. Additionally, frequencies of peripheral blood mature cells in each lineage are not altered in Dnmt3a R878C mice compared to Dnmt3a^wt/wt Vav-Cre^tg/wt (hereafter control mice). To further investigate the effect of DNMT3A R882 mutation, we sacrificed Dnmt3a R878C and control mice at 8-12 weeks after birth and compared the distribution of hematopoietic cells by using flow cytometry. Although there was no obvious difference in the number of mononuclear cells in the whole bone marrow, we found a significant increase of the frequency of long-term hematopoietic stem cells (defined by CD150⁺ CD48^- Lineage^- c-Kit⁺ Sca-1⁺) in Dnmt3a R878C mice compared to control mice (0.040% and 0.019%, p = 0.022). The frequencies of other progenitors including short-term hematopoietic stem cells, multipotent progenitors, or Lineage^- c-Kit⁺ Sca-1⁺ (LSK) cells were not changed between Dnmt3a R878C mice and control mice. Moreover, in order to determine whether DNMT3A mutation leads to a qualitative difference in HSCs, we performed colony forming assay. While control LSK cells could not form colonies at fifth round of replating, Dnmt3a R878C LSK cells could be serially replated up to five passages. Additionally, the numbers of colonies in each round were much higher in Dnmt3a R878C cells compared to control cells, suggesting HSCs with heterozygous Dnmt3a R878C mutation have aberrantly enhanced self-renewal capacity. Currently, we are performing competitive bone marrow transplantation assay to evaluate the functional role of DNMT3A mutation in vivo. We transplanted 500,000 bone marrow cells from Dnmt3a R878C or control mice, which are Ly5.2 background, together with the same number of Ly5.1-horboring competitor cells into Ly5.1-harboring lethally-irradiated recipient mice. Thus far, Peripheral blood chimerisms of donor cells have tended to be higher in recipients of Dnmt3a R878C mutant cells compared to control mice, suggesting that heterozygous Dnmt3a R878C mutation contribute to expansion of hematopoietic stem cell pool. Collectively, heterozygous Dnmt3a R878C mutant enhanced self-renewal capacity of HSCs and resulted in accumulation of HSCs in vivo, similar to mice models with overexpression of DNMT3A R882 mutants. Our results revealed that endogenous expression of DNMT3A mutant is sufficient for expansion of HSCs, revealing functional contribution of DNMT3A mutation in forming the backbone of premalignant status of HSCs.