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.

Figure 7. DNMT3A is required for the competitive repopulation advantage conferred by PML-RARA and its ability to induce APL in vivo.

(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, Lin^loSca⁺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.

Figure 6. DNMT3A is dispensable for leukemia induced by MLL-AF9 overexpression.

(A) Schematic of the experimental design for the results shown in B–E. BM from 2- to 3-week-old mice of the indicated genotypes was harvested and transduced with an MLL-AF9–expressing retrovirus before transplantation into lethally irradiated WT mice. (B) wbc counts 28 days after transplantation demonstrated an equal degree of leukocytosis in recipients of MLL-AF9–transduced WT and Dnmt3a^–/– BM. (C) Spleen weights of moribund animals were not significantly different, regardless of DNMT3A status. (D) MLL-AF9 was able to initiate lethal leukemia with 100% penetrance and equal latency using BM cells with or without Dnmt3a (n = 11 for WT + MLL-AF9; n = 9 for Dnmt3a^–/– + MLL-AF9). NS, by 2-tailed, unpaired t test.

Figure 5. DNMT3B is not required for the aberrant self-renewal ability conferred by PML-RARA in murine BM progenitor cells.

(A) Schematic of the experimental design for the results shown in B–D. BM from 2- to 2.5-week-old mice of the indicated genotypes was plated in MethoCult media containing IL-3, IL-6, and SCF and replated each week. (B) Quantification of colony numbers demonstrated no difference in colony formation in PR^+/– Dnmt3b^fl/fl cells with or without Vav-Cre. (C) Representative flow cytometric plot for the myeloid markers Gr-1 and CD11b after 6 weeks of MethoCult replating demonstrated that self-renewal of myeloid cells from PR^+/– mice was not dependent on Dnmt3b. (D) Graph of CD11b positivity over time. (E) Schematic of the experimental design for the results shown in F. Marrow from 2- to 2.5-week-old mice of the indicated genotypes was retrovirally transduced with MSCV vectors containing WT DNMT3B-IRES-YFP or YFP only and then sorted for YFP⁺ cells and plated in MethoCult media as in A. (F) Quantification of colony numbers at week 4 illustrates that overexpression of DNMT3B was not able to rescue aberrant self-renewal in PR^+/– Dnmt3a^–/– BM cells. n = 3–6 for all experiments. *P < 0.05, by 2-tailed, unpaired t test.

Figure 4. DNA methyltransferase activity of DNMT3A is required for aberrant self-renewal by PML-RARA ex vivo.

(A) Schematic of human DNMT3A cDNAs used in B–E. Mutations in the methyltransferase domain (MTase) from AML patients induced loss of catalytic function (R882H, red arrow) or lacked the methyltransferase domain altogether (Q615*, black arrow). (B) Western blot from PR^+/– or PR^+/– Dnmt3a^–/– BM cells transduced with retroviruses containing human DNMT3A cDNAs and IRES-YFP and then sorted for YFP⁺ cells. Cells were harvested after 1 week in MethoCult medium. The positions of full-length and truncated DNMT3A are indicated on the right. (C) BM from 2- to 3-week-old mice was transduced with the indicated viruses, and GFP⁺ cells were sorted and serially replated in MethoCult medium. Week-4 colony numbers demonstrated that only WT DNMT3A with intact DNA methyltransferase activity was able to restore the colony-forming ability to PR^+/– Dnmt3a^–/– myeloid cells. (D) Flow cytometric analysis of week-4 MethoCult cells demonstrated a persistence of CD11b expression in PR^+/– Dnmt3a^–/– cells, conferred only by WT DNMT3A. (E) Graph of CD11b expression percentages at 4 weeks of serial replating in MethoCult medium. (F) Assay of methylation at a specific HpaII site in the Runx1 locus that is dependent on DNMT3A for methylation in WT BM cells (Site 1 in Supplemental Figure 1E). Reexpression of WT, full-length DNMT3A by retroviral transduction restored a normal level of methylation to these sites after 7 days in MethoCult media. Retroviruses containing mutant DNMT3A (R882H or Q615*) did not restore methylation at this site. n = 3–4 for all experiments. *P < 0.05, **P < 0.005, and ***P < 0.001, by 2-way ANOVA.

Figure 3. Expression analysis of previously identified PML-RARA–dysregulated genes in GMP cells derived from PR^+/–, PR^+/– Dnmt3a^–/–, and Dnmt3a^–/– mice.

(A and B) Using Affymetrix Mouse Exon 1.0ST arrays, we interrogated gene expression in GMP cells purified from BM cells of mice transplanted with WT (n = 4), PR^+/– (n = 2), PR^+/– Dnmt3a^–/– (n = 4), or Dnmt3a^–/– (n = 3) marrow cells that were harvested 6–8 weeks later. (A) Mean fold changes for 239 probe sets previously found to be significantly dysregulated in GMP cells derived from the BM of 6- to 8-week-old Ctsg-PML-RARA mice (labeled as PR^+/–a) versus WT GMP cells (n = 4 for each; ref. and Supplemental Table 3). The mean fold changes (compared with those for WT GMP cells) for the 112 probe sets that were upregulated in the PR^+/–a mice are shown in the left panel, and the mean fold changes for the 127 probe sets that were downregulated are shown on the right. Fold changes for the PR^+/–b, PR^+/– Dnmt3a^–/–, and Dnmt3a^–/– probe sets were calculated by comparison with the WT data from the current set of experimental data. (B) Heatmap of Z-scored data using the same 239 PR^+/–-dysregulated probe sets defined in Wartman et al. (31) and displaying the average values obtained from the arrays generated from GMP-purified cells with the designated genotype used in this study. The list of genes with significant variation in expression levels was generated using a fold change of 2 and an FDR criterion of 0.05 or less. Bar plot P values (***P < 0.001) are based on Student’s paired t test, with a 2-tailed distribution.

Figure 2. DNMT3A is required for the aberrant self-renewal ability of PML-RARA–expressing mouse BM cells ex vivo.

(A) RT-PCR for PML-RARA expression in BM cells derived from Ctsg-PML-RARA mice (PR^+/–) or Ctsg-PML-RARA mice that were also deficient for DNMT3A (PR^+/– Dnmt3a^–/–). (B and C) BM from 2- to 2.5-week-old mice of the indicated genotypes was transplanted into lethally irradiated WT recipients in a noncompetitive transplantation. (B) Quantification of cell numbers in the mature myeloid compartment (Gr-1⁺, left panel) versus the mature B cell compartment (B220⁺, right panel) at 10 weeks after transplantation demonstrated the ability of Dnmt3a^–/– donor stem cells to engraft and contribute normally to both myeloid and lymphoid lineages (see also Supplemental Figure 3A). (C) Quantification of the indicated progenitor and stem cell compartments showed no significant differences for any genotype 10 weeks after transplantation. MEPs, megakaryocyte-erythroid progenitors. (D–F) BM from 2- to 2.5-week-old mice of the indicated genotypes was plated in MethoCult media containing IL-3, IL-6, and SCF and replated weekly. (D) Quantification of colony numbers demonstrated a loss of colony formation by PR^+/– Dnmt3a^–/– cells. (E) Representative flow cytometric plot for the myeloid markers Gr-1 and CD11b demonstrated a loss of myeloid cells from PR^+/– Dnmt3a^–/– mice after 2 weeks of replating in MethoCult media. (F) Graph of CD11b positivity over time. n = 3–6 for all experiments. *P < 0.05 and ***P < 0.001 for PR^+/– versus all other genotypes, by 2-way ANOVA.

Figure 1. DNMT3A is required for aberrant self-renewal ability conferred by PML-RARA on hematopoietic progenitor cells.

(A) Schematic of the experimental design for results shown in B–E. BM from 2- to 2.5-week-old mice of the indicated genotypes was transduced with the indicated retroviruses, plated in MethoCult media containing IL-3, IL-6, and SCF, and then replated each week. (B) A YFP-expressing control vector did not induce replating in WT or Dnmt3a^–/– cells. (C and D) Dnmt3a was unnecessary for aberrant self-renewal driven by retroviruses expressing MLL-AF9 (C) or RUNX1-RUNX1T1 (AML-ETO) (D). (E) Loss of Dnmt3a eliminated replating driven by a PML-RARA–containing retrovirus. (F) Representative flow cytometric plots for the myeloid markers Gr-1 and CD11b for week 1 versus week 4 of replating. (G) Flow data for CD11b positivity at week 4 of replating, demonstrating loss of myeloid cells in Dnmt3a^–/– marrow transduced with PML-RARA. n = 3–6 for all experiments. *P < 0.05 and ***P < 0.001 by 2-tailed, unpaired t test.