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Haploinsufficiency for DNA methyltransferase 3A predisposes hematopoietic cells to myeloid malignancies
Christopher B. Cole, … , Christopher A. Miller, Timothy J. Ley
Christopher B. Cole, … , Christopher A. Miller, Timothy J. Ley
Published September 5, 2017
Citation Information: J Clin Invest. 2017;127(10):3657-3674. https://doi.org/10.1172/JCI93041.
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Research Article Hematology Oncology

Haploinsufficiency for DNA methyltransferase 3A predisposes hematopoietic cells to myeloid malignancies

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Abstract

The gene that encodes de novo DNA methyltransferase 3A (DNMT3A) is frequently mutated in acute myeloid leukemia genomes. Point mutations at position R882 have been shown to cause a dominant negative loss of DNMT3A methylation activity, but 15% of DNMT3A mutations are predicted to produce truncated proteins that could either have dominant negative activities or cause loss of function and haploinsufficiency. Here, we demonstrate that 3 of these mutants produce truncated, inactive proteins that do not dimerize with WT DNMT3A, strongly supporting the haploinsufficiency hypothesis. We therefore evaluated hematopoiesis in mice heterozygous for a constitutive null Dnmt3a mutation. With no other manipulations, Dnmt3a+/– mice developed myeloid skewing over time, and their hematopoietic stem/progenitor cells exhibited a long-term competitive transplantation advantage. Dnmt3a+/– mice also spontaneously developed transplantable myeloid malignancies after a long latent period, and 3 of 12 tumors tested had cooperating mutations in the Ras/MAPK pathway. The residual Dnmt3a allele was neither mutated nor downregulated in these tumors. The bone marrow cells of Dnmt3a+/– mice had a subtle but statistically significant DNA hypomethylation phenotype that was not associated with gene dysregulation. These data demonstrate that haploinsufficiency for Dnmt3a alters hematopoiesis and predisposes mice (and probably humans) to myeloid malignancies by a mechanism that is not yet clear.

Authors

Christopher B. Cole, David A. Russler-Germain, Shamika Ketkar, Angela M. Verdoni, Amanda M. Smith, Celia V. Bangert, Nichole M. Helton, Mindy Guo, Jeffery M. Klco, Shelly O’Laughlin, Catrina Fronick, Robert Fulton, Gue Su Chang, Allegra A. Petti, Christopher A. Miller, Timothy J. Ley

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Figure 2

DNMT3A truncation mutants do not heterodimerize with WT DNMT3A and fail to form mutant DNMT3A homodimers.

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DNMT3A truncation mutants do not heterodimerize with WT DNMT3A and fail ...
(A) AlphaLISA assay schematic of assay for B–E, in which the interaction between DNMT3A-FLAG and DNMT3A-V5 molecules is measured. Anti-FLAG “donor” beads excited by light at 680 nm release singlet oxygen, which can excite anti-V5 “acceptor” beads within 200 nm, leading to emission light at 620 nm. (B) AlphaLISA measurement of dimerization of WT DNMT3A-V5 with DNMT3A-FLAG (WT, Q515*, E616fs, and L723fs), with crosstitration (4:1, 2:1, 1:1, 1:2, 1:4). (C) Quantification of DNMT3A-FLAG:DNMT3A-V5 homodimerization (WT:WT, Q515*:Q515*, E616fs:E616fs, and L723fs:L723fs) when mixed at 1:1 stoichiometric ratio. (D) Oligomerization of WT DNMT3A-V5 and DNMT3A-FLAG (WT, Q515*, E616fs, or L723fs), mixed at a 1:1 stoichiometric ratio, in response to NaCl. (E) WT DNMT3A-V5 and DNMT3A-FLAG (WT, Q515*, E616fs, or L723fs) oligomerization, mixed at a 1:1 stoichiometric ratio, in response to KCl. All data are shown as mean ± SD of 3 independent experiments, each performed in triplicate. *P < 0.05, 2-way ANOVA relative to WT:WT.
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