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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 1

Truncated DNMT3A proteins are absent in AML cells, but stable in HEK293T cells, and lack de novo DNA methyltransferase activity.

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Truncated DNMT3A proteins are absent in AML cells, but stable in HEK293T...
(A) Western blot of endogenous DNMT3A (top panel) or actin (bottom panel) from primary AML bone marrow samples (DNMT3AWT/WT, DNMT3AWT/Q515*, DNMT3AWT/E616fs, and DNMT3AWT/L723fs). Asterisks indicate predicted positions of DNMT3A based on corresponding cDNAs (B). (B) Western blot of exogenous DNMT3A produced by WT, Q515*, E616fs, and L723fs DNMT3A cDNAs expressed in HEK293T cells. (C) In vitro methylation of a linearized plasmid DNA substrate (pcDNA3.1) by recombinant full-length human WT or mutant DNMT3A (Q515*, E616fs, or L723fs). Time-course assays using 1 μg of total protein per 35 μl reaction (250 nM). (D) In vitro methylation of a linearized plasmid DNA substrate (pcDNA3.1) by recombinant full-length human WT or mutant DNMT3A (Q515*, E616fs, or L723fs). Dose response with fixed 16-hour incubation. All experiments were independently performed 3 times, and data for C and D are shown as mean ± SEM of 3 independent experiments, each performed in triplicate. *P < 0.05, 2-way ANOVA relative to WT DNMT3A.
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