DNMT3A R882H Overexpression Leads To Hematopoietic and Skin Alterations In Transgenic Mice

Somatic mutations in the DNA methyltransferase gene, DNMT3A, have been identified in >30% of de novo AML cases with a normal karyotype, and in >10% of patients with MDS. To understand whether mutations in DNMT3A alter hematopoiesis, we generated a transgenic mouse model capable of overexpressing either wild type human DNMT3A or the most common DNMT3A mutation found in AML cases (R882H, a hypomorphic variant that acts as a potent dominant negative inhibitor of WT DNMT3A, D. Germain and T. Ley, unpublished). Full-length human cDNAs encoding WT or R882H DNMT3A were cloned into a mammalian expression vector directly downstream from a tetracycline responsive element. This facilitates the inducible expression of DNMT3A upon the expression of the rtTA coactivator and the presence of Doxycycline (Dox). A single founder line for the WT DNMT3A allele and two founder lines for the R882H DNMT3A allele were established in the C57Bl6/J background and subsequently shown to express the transgene in the bone marrow when crossed to mice that carry the Rosa26-rtTA allele, which is expressed ubiquitously (including in the skin, see below). In the unfractionated bone marrow of Dox treated mice carrying the WT DNMT3A transgene, western blots revealed that human DNMT3A was expressed 3.5 fold higher than endogenous murine DNMT3A; bone marrow from Dox treated transgenic lines with R882H expressed a 4.5 fold excess of human DNMT3A in line 1, and a 16 fold excess in line 2.

No phenotypes have been observed for mice in any of the 8 experimental and control genotypes without Dox chow (since the transgenes are not expressed). No consistent abnormalities in CBCs or in marrow morphology or progenitor numbers have been observed in mice treated for 4-8 weeks with 1% Dox chow. A tumor watch has therefore been established with all mice on Dox chow (WT Tg x rtTA, n=24; R882H Tg line 1 x rtTA, n=20; R882H Tg line 2 x rtTA, n=36). One of 36 R882H line 2 x rtTA mice became moribund at 8 weeks on Dox chow. After 6 months of Dox chow, 13/36 (36.1%) DNMT3A R882H mice from line 2 were sacrificed after becoming moribund, or died before analysis could be performed. Four of seven analyzed prior to death demonstrated a leukocytosis (WBC range 30.8-40.32), the presence of blast-like and immature myeloid cells in the peripheral blood (2/7 mice), mild splenomegaly (3/7 mice, range 0.2g to 0.25g), shifts toward the myeloid lineage in the bone marrow as assessed by CD11b+ (4/7 mice, range 69.0-84.3% versus 53.24% +/- 8.69% SD in control mice), and splenic infiltration by CD11b+ cells (2/7 mice, >10% composition). Four of the 7 moribund mice presented with anemia, with hemoglobin (Hgb) levels ranging from 3.2-10.4 g/dl. Six of 7 mice had erythroid maturation defects in the marrow, where 3/7 specifically displayed an accumulation of proerythroblasts (CD71 high/ Ter119 low). A single mouse from the lower expressing DNMT3A R882H line (line 1) became moribund at 9 months and was found to have severe anemia (Hgb=3.6), splenomegaly (0.7g), splenic infiltration of mature myeloid cells, and altered erythropoiesis.

Some doubly transgenic mice expressing the high level of DNMT3A R882H (line 2) developed extensive alopecia as early as 3 weeks on Dox chow. Examination of skin sections from these mice showed epidermal hyperplasia and acanthosis, a mild mixed inflammatory infiltrate, and follicular plugging; the penetrance of the skin phenotype is ∼60% in this line at 3 months (21/36 mice affected). The alopecia was completely reversible upon withdrawal of Dox. Mice from R882H Line 1 developed a milder form of alopecia, with a latency of 3 months and a penetrance of 60% (12/20 affected). No skin phenotype was apparent in mice overexpressing WT DNMT3A (or in any mice from any genotype not on Dox chow). Taken together, these data suggest that the overexpression of DNMT3A R882H in the skin may cause dominant negative effects on Dnmt3a in skin stem/progenitor cells, affecting their ability to differentiate into hair follicles. Further experiments are in progress to confirm this hypothesis.

Our data suggest that DNMT3A R882H overexpression may affect both hematopoietic and skin stem/progenitor cells in a dose dependent manner, perhaps by exerting a dominant negative effect on the function of endogenous Dnmt3a. Experiments currently being performed will determine whether these phenotypes are cell-autonomous in both compartments in vivo.