Myeloproliferative Disease and Myeloid Leukemia in Dnmt3a Haploinsufficient Mice

Recent whole genome sequencing efforts by our group and others have identified recurrent mutations in the DNA methyltransferase DNMT3A in various hematologic malignancies, including acute myeloid leukemia (AML), MPN, MDS, CMML, and T-ALL. The most common point mutation in AML, R882H, is nearly always heterozygous in AML, and has been demonstrated to have a dominant negative effect on the methylation activity of the remaining wild-type allele (Russler-Germain et al. Cancer Cell 2014). However, other heterozygous mutations produce frameshifts, premature stop codons, or deletions of the entire coding sequence of the gene, strongly suggesting that these mutations lead to simple haploinsufficiency for DNMT3A. In order to test the hypothesis that Dnmt3a haploinsuffiency may initiate AML, we performed a long-term tumor watch comparing wild-type mice (Dnmt3a+/+) to mice carrying one wild-type Dnmt3a allele and one knockout allele that contains a neomycin-resistance cassette inserted into the sequence coding for the catalytic domain of the protein, producing a true null allele (Dnmt3a+/- mice). At 6 weeks of age, Dnmt3a+/- mice have normal hematopoiesis, with no detectable differences from wild-type littermates in myeloid, lymphoid, erythroid, or stem/progenitor populations in the bone marrow or spleen. However, after 1.5 years of age, 15/43 Dnmt3a+/- mice (35%) became moribund and were euthanized for pathologic evaluation (Figure 1). Affected mice generally exhibited massive splenomegaly with myeloid infiltrates in spleen, liver, and other extramedullary organs. At conclusion of the tumor watch at two years, all remaining mice were euthanized, and similar pathologic findings were observed in an additional 9 Dnmt3a+/- mice, for an overall disease penetrance of 24/43 (56%). Flow cytometric evaluation of splenic tumors demonstrated positivity for the myeloid markers Gr-1 and CD11b, and the presence of a subpopulation of cells double-positive for the mature myeloid marker Gr-1 and the progenitor cell marker CD34. Based on flow cytometric and morphologic findings, 16 splenic tumors were definitively classified according to the Bethesda criteria and were found to exhibit myeloid proliferation/MPD (11/16), myeloid leukemia with maturation (2/16), MPD-like myeloid leukemia (2/16), or myeloid sarcoma (1/16). 6 of 16 tumors tested were able to successfully engraft and lead to lethal disease in sublethally irradiated wild-type recipients, providing further evidence that these tumors represent transplantable, cell-autonomous myeloid malignancies. Analysis of exome sequencing data from 5 primary tumor samples is ongoing. One MPD-like myeloid leukemia was engrafted into four separate wild-type recipients, and all 5 tumors were sequenced. Collectively, this set of samples was found to contain mutations in the Ras/MAPK pathway, including the canonical gain-of-function mutation Kras G12C, a Ptpn11 E76K mutation, and a missense mutation in the tumor suppressor Neurofibromatosis 1 (Nf1). A second, independent primary tumor was also found to contain an activating Kras G13D mutation. Examination of the Dnmt3a locus in all 5 sequenced samples revealed no evidence of mutations or loss of heterozygosity of the residual wild-type Dnmt3a allele. Importantly, 9/51 AML samples with DNMT3A mutations in the TCGA AML cohort also contained activating NRAS or KRAS mutations. These data strongly suggest that Ras/MAPK pathway mutations can cooperate with Dnmt3a haploinsufficiency to induce AML in C57Bl/6 mice and in humans.

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Figure 1

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