PML-RARA requires DNA methyltransferase 3A to initiate acute promyelocytic leukemia
Christopher B. Cole, … , Vincent Magrini, Timothy J. Ley
Christopher B. Cole, … , Vincent Magrini, Timothy J. Ley
Published November 23, 2015
Citation Information: J Clin Invest. 2016;126(1):85-98. https://doi.org/10.1172/JCI82897.
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Research Article Oncology

PML-RARA requires DNA methyltransferase 3A to initiate acute promyelocytic leukemia

Abstract

The DNA methyltransferases DNMT3A and DNMT3B are primarily responsible for de novo methylation of specific cytosine residues in CpG dinucleotides during mammalian development. While loss-of-function mutations in DNMT3A are highly recurrent in acute myeloid leukemia (AML), DNMT3A mutations are almost never found in AML patients with translocations that create oncogenic fusion genes such as PML-RARA, RUNX1-RUNX1T1, and MLL-AF9. Here, we explored how DNMT3A is involved in the function of these fusion genes. We used retroviral vectors to express PML-RARA, RUNX1-RUNX1T1, or MLL-AF9 in bone marrow cells derived from WT or DNMT3A-deficient mice. Additionally, we examined the phenotypes of hematopoietic cells from Ctsg-PML-RARA mice, which express PML-RARA in early hematopoietic progenitors and myeloid precursors, with or without DNMT3A. We determined that the methyltransferase activity of DNMT3A, but not DNMT3B, is required for aberrant PML-RARA–driven self-renewal ex vivo and that DNMT3A is dispensable for RUNX1-RUNX1T1– and MLL-AF9–driven self-renewal. Furthermore, both the PML-RARA–driven competitive transplantation advantage and development of acute promyelocytic leukemia (APL) required DNMT3A. Together, these findings suggest that PML-RARA requires DNMT3A to initiate APL in mice.

Authors

Christopher B. Cole, Angela M. Verdoni, Shamika Ketkar, Elizabeth R. Leight, David A. Russler-Germain, Tamara L. Lamprecht, Ryan T. Demeter, Vincent Magrini, Timothy J. Ley

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Figure 7

DNMT3A is required for the competitive repopulation advantage conferred by PML-RARA and its ability to induce APL in vivo.

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DNMT3A is required for the competitive repopulation advantage conferred ...
(A) Schematic of the experimental design for the results shown in B–E, in which marrow from 2- to 2.5-week-old mice of the indicated genotypes was mixed with WT competitor marrow and transplanted into lethally irradiated recipients (week 0) for monitoring of the relative contribution to PB cells, BM, and spleen (n = 4–21 per genotype). (B) Flow cytometry at the indicated weeks after transplantation demonstrated that the competitive advantage for PR+/– BM in contributing to PB cells was completely abrogated in PR+/– Dnmt3a–/– BM and that Dnmt3a–/– and PR+/– Dnmt3a–/– BM cells had a competitive disadvantage compared with that of WT BM cells in this assay. (C) Examination of chimerism in BM and spleen at 6 months after transplantation indicated a decreased contribution of PR+/– Dnmt3a–/– marrow compared with that observed for PR+/– marrow in both compartments. (D) Characterization of the stem/progenitor compartments in chimeric mice 10 weeks after competitive transplantation. The composition of all compartments was the same for both genotypes, except that PR+/– Dnmt3a–/– donors displayed a significantly increased contribution to the long-term hematopoietic stem cell compartment. LSKs, LinloSca+c-Kit+ cells. (E) Quantification of myeloid progenitor compartments demonstrated no significant differentiation biases in either genotype. (F) Long-term tumor watch of WT animals transplanted with PR+/– or PR+/– Dnmt3a–/– BM demonstrated that 6 of the 16 recipients of PR+/– and 0 of the 13 recipients of PR+/– Dnmt3a–/– BM had succumbed to APL by 1 year after transplantation (P = 0.0336 by Mantel-Cox test). NS indicates that no differences between any 2 genotypes were statistically significant by 2-way ANOVA (B) or 1-way ANOVA (C–E). *P < 0.05 and ***P < 0.001, by 1- or 2-way ANOVA.