DNMT3A Arg882 Mutation Drives Chronic Myelomonocytic Leukemia Through Disturbing Gene Expression/DNA Methylation in Hematopoietic Cells

DNMT3A Arg882 mutation drives chronic myelomonocytic leukemia through disturbing gene expression/DNA methylation in hematopoietic cells

Jie Xua,1, Yue-Ying Wanga,1,2, Yu-Jun Daia,1, Wu Zhanga, Wei-Na Zhanga, Shu-Min Xionga, Zhao-Hui Gub, Kan-Kan Wanga, Rong Zengc, Zhu Chena,b,2, and Sai-Juan Chena,b,2

aState Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; bKey Laboratory of Ministry of Education of Systems Biomedicine, Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China; and cKey Laboratory of Systems Biology, Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai 200031, China

Contributed by Zhu Chen, January 16, 2014 (sent for review December 2, 2013)

The gene encoding DNA methyltransferase 3A (DNMT3A) is mu- the transforming potentials of hematopoietic stem/progenitor tated in ∼20% of acute myeloid leukemia cases, with Arg882 (R882) cells (HSPCs). We also examined the possible mechanisms of as the hotspot. Here, we addressed the transformation ability of the these abnormal potentials at transcriptome, epigenetics, and DNMT3A-Arg882His (R882H) mutant by using a retroviral transduc- protein–protein interaction levels in transformed HSPCs with tion and bone marrow transplantation (BMT) approach and found the DNMT3A-R882H mutation. that the mutant gene can induce aberrant proliferation of hemato- Results poietic stem/progenitor cells. At 12 mo post-BMT, all mice developed chronic myelomonocytic leukemia with thrombocytosis. RNA DNMT3A-R882H Mutation Enhances Proliferation Potential of Hema- microarray analysis revealed abnormal expressions of some he- topoietic Cells. To explore the in vivo transformation effect of matopoiesis-related genes, and the DNA methylation assay DNMT3A mutation, we carried out BMT experiments using identified corresponding changes in methylation patterns in murine bone marrow (BM) cells retrovirally transduced with gene body regions. Moreover, DNMT3A-R882H increased the mutated or WT DNMT3A or empty (vehicle) retroviral constructs. GFP was used to trace transduced cells. We found that CDK1 protein level and enhanced cell-cycle activity, thereby cells in peripheral blood (PB) with DNMT3A-R882H could contributing to leukemogenesis. compete effectively with the cells without GFP and presented an increasing proliferative potential, whereas proliferative capacities genomic variation | epigenetic abnormality | leukemogenic effect were gradually reduced in cells overexpressing WT DNMT3A (Fig. 1A). However, at 6 mo post-BMT, no obvious changes were ob- NA methylation represents one of the major epigenetic served in hemoglobin level, white blood cell (WBC) count, or platelet count (Fig. S1A). To evaluate the possible effect of R882H Dmodifications and plays a key role in a number of regulatory + mechanisms of life processes (1–3). In mammals, the executors of on the stemness of HSPCs, GFP BM cells from each group were isolated for cfu assay. The DNMT3A protein was overex- genome methylation are members of the DNA methyltransferase + (DNMT) family, including DNMT1, DNMT3A and DNMT3B. It pressedinsortedGFP BM cells from both the WT and mutant has been well established that DNMT3A forms complex with DNMT3A groups (Fig. S1B). Of note, cells with DNMT3A- R882H formed statistically more CFU-G (granulocyte), CFU-M DNMT3L to catalyze the de novo DNA methylation (4, 5). Both DNMT3A and DNMT3B show high expression levels at the early embryogenesis, and their expressions are down-regulated along Significance with the embryonic development and cell differentiation (6, 7). It is well known that all blood-cell lineages originate from the Epigenetic modifications are required for the regulation of multipotent hematopoietic stem cells (HSCs). A number of hematopoiesis. DNA methyltransferase 3A (DNMT3A), a critical regulations are involved in directing HSCs activities, and the epigenetic modifier responsible for de novo DNA methylation, epigenetic modifications are of great importance (8, 9). It has been was reported recently to be a frequently mutated gene in shown that loss of Dnmt3a in a Dnmt3a-conditional knockout hematopoietic malignancies. However, the role of mutated mouse results in progressive impairment of HSCs differentiation DNMT3A in hematopoiesis remains largely unknown. Here we and expansion (10). Notably, DNMT3A recently has been reported show that the Arg882 (R882) mutation of DNMT3A disrupts the to be mutated in up to 20% of cases of acute myeloid leukemia normal function of this enzyme and results in chronic myelo- (AML), mostly in cases with monocytic lineage (AML-M5 or -M4) monocytic leukemia (CMML) in mice. Meanwhile, the gene and clinical features including old age, normal karyotype, leuko- expression, DNA methylation, and protein–protein interaction cytosis and thrombocytosis, and poor prognosis (11, 12). Although assays suggest that DNMT3A R882 mutation drives CMML by a variety of DNMT3A mutations have been identified, the majority disturbing the transcriptional expression/DNA methylation (∼50%) affect Arg882 (R882) located at the catalytic domain, and program and cell-cycle regulation of hematopoietic cells. This the most common substitution is Arg882His (R882H) (11, 13, 14). study may shed light on the function of DNMT3A mutant in It also has been suggested that R882 mutation may interfere with myeloid leukemogenesis. oligomerization of DNMT3A and thereby exert an aberrant effect on its enzymatic function (15). Evidence has been obtained sup- Author contributions: Y.-Y.W., Z.C., and S.-J.C. designed research; J.X., Y.-Y.W., Y.-J.D., W.Z., porting DNMT3A mutations as the fundamental genetic event at W.-N.Z., S.-M.X., Z.-H.G., K.-K.W., and R.Z. performed research; J.X., Y.-Y.W., Z.C., and S.-J.C. the initiation stage of AML pathogenesis (16, 17). However, the in analyzed data; and J.X., Y.-Y.W., Z.C., and S.-J.C. wrote the paper. vivo transformation power of DNMT3A mutations needs to be The authors declare no conflict of interest. addressed, and the relevant molecular and cellular mechanisms of 1J.X., Y.-Y.W., and Y.-J.D. contributed equally to this work. these mutations in AML pathogenesis remain obscure. 2To whom correspondence may be addressed. E-mail: [email protected], zchen@ In the present work, using retroviral transduction and bone stn.sh.cn, or [email protected]. marrow transplantation (BMT) technology, we were able to in- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. vestigate the in vivo effect of the DNMT3A-R882H mutation on 1073/pnas.1400150111/-/DCSupplemental.

2620–2625 | PNAS | February 18, 2014 | vol. 111 | no. 7 www.pnas.org/cgi/doi/10.1073/pnas.1400150111 Downloaded by guest on September 27, 2021 in the vehicle group (P < 0.001 and P = 0.003, respectively). More- + + + + over, the proportion and number of GFP (Mac-1 Gr-1 F4/80 ) monocytes were much higher in DNMT3A-R882H mice than in vehicle group (P = 0.007 and P = 0.018, respectively) (Fig. 2E and Fig. S1E). These results suggested that DNMT3A mutation positively regulates cell growth in monocytic/megakaryocytic lineages. When the BM of mice was examined 1 y after BMT, significantly increased numbers of blasts were noted in all nine DNMT3A-R882H mice (11.5% on average in DNMT3A-R882H mice, versus 4.5% on average in the vehicle group; P < 0.001). The blasts in the DNMT3A-R882H mice were characterized by large size, high nuclear/cytoplasmic ratio, and fine chromatin. The nuclei of some blasts were indented, reminiscent of the morphology of monoblasts. Among mature myeloid cells, the percentage of monocytes (3.4%) was significantly increased in the DNMT3A-R882H group as compared with the vehicle group (1.0%) (P < 0.001) (Fig. 2 F and G). As observed in PB at 1 y + post-BMT, the percentage of GFP cells was much higher in BM cells with the R882H mutation than in the vehicle group. + Immunophenotype analysis of these GFP BM cells showed that + + + the Mac-1 Gr-1 F4/80 population was increased significantly in DNMT3A-R882H mice as compared with the vehicle group (P < 0.001) (Fig. 2H), providing strong evidence that the DNMT3A mutation promotes the proliferation of a myeloid compartment, monocytic lineage in particular. We performed bubble PCR to identify the retroviral integrating sites. A single integration site Fig. 1. Phenotypic analysis in mice at 6 mo post-BMT. (A) Average percent + of GFP cells in PB after BMT (n = 8–10 recipients per group). (B) Increased + myeloid colony formation by GFP DNMT3A-R882H BM cells in methylcel- lulose compared with that by WT or vector groups. (C) Representative flow cytometry plots of BM cells from mice at 6 mo post-BMT. (D) Average ratios of PB GFP+ cells after secondary transplantation (vector: n = 7; DNMT3A WT: n = 3; DNMT3A-R882H: n = 10). ***P < 0.001.

(monocyte/macrophage), and CFU-GM colonies than those from − + + the WT and vehicle groups (Fig. 1B). Lin Sca-1 C-kit (LSK) − + + + − cells and long-term HSCs (Lin Sca-1 C-kit CD150 CD48 )were significantly increased in the DNMT3A-R882H group as compared with the WT group. In addition, myeloid progenitor (Mac- + 1lowC-kit ) cells also showed an apparent increase, and mature myeloid cells were comparatively decreased in DNMT3A-R882H mice (Fig. 1C). To assess further the ability of the DNMT3A

mutation to provide HSPCs with an advantage in growth and GENETICS + survival, we transplanted 1 × 106 GFP BM cells from the first generation of mice in each group into the secondary recipient mice treated with sublethal irradiation. In the second-generation + mice, the percentage of GFP cells was maintained at a higher level in the DNMT3A-R882H group but dropped rapidly in the WT group (Fig.1D). + At 1 y after BMT, remarkable differences of GFP cells appeared among the three groups: PB cells carrying DNMT3A- + R882H increased continuously, GFP PB cells with WT DNMT3A almost died out, and those from vehicle group remained at the same level as 6-mo post-BMT (Fig. S1C). Morphological analysis of PB smears with Wright’s staining revealed striking features in the DNMT3A-R882H group, notably significantly increased granu- locytes and monocytes, and a few early myeloid elements could be found (Fig. 2A). The number of monocytes was greater than 109/L (Fig. 2B), and the number of platelets increased significantly (Fig. 2C), but no statistically significant changes in hemoglobin were observed (Fig. 2D). In agreement with the morphology data, + the sorted GFP WBCs showed increased monocytes in all nine Fig. 2. Morphological and immunophenotypical analyses at 1 y post-BMT. (A) Morphological comparison of PB in DNMT3A-R882H and vector groups. DNMT3A-R882H mice investigated (Fig. S1D), and the number of (B) Statistical analysis of cell counts in PB. (C and D) Absolute values of pla- lymphocytes was reduced significantly; thus the ratio between gran- telets (C) and hemoglobin (D). (E) Representative flow cytometry plots of ulocytes/monocytes and lymphocytes was reversed in DNMT3A- murine PB. (F) Morphological analysis of BM in the DNMT3A-R882H and R882H mice as compared with the vector group (Fig. 2B).An vector groups. Wright’s staining was used in BM cytospin preparations. (G) analysis of the development of myeloid lineages showed that, within + + + Statistical analysis of the numbers of blasts and monocytes in BM. (H) Rep- GFP clusters, the percentage and number of Mac-1 Gr-1 cells resentative flow cytometry plots of BM from mice at around 1 y post-BMT was statistically significantly higher in DNMT3A-R882H mice than (vector group: n = 8 mice; DNMT3A-R882H group: n = 9 mice).

Xu et al. PNAS | February 18, 2014 | vol. 111 | no. 7 | 2621 Downloaded by guest on September 27, 2021 was identified in each sample from three mice at 12 mo post- BMT. These sites were located at different chromosomes (Table S1). Taken as a whole, these features in DNMT3A-R882H mice at 1 y post-BMT represented a disease phenotype mimicking chronic myelomonocytic leukemia (CMML) in humans. The advantage in cellular proliferation provided by DNMT3A- R882H also was demonstrated in a distinct set of experiments using in vitro NIH3T3 cell lines stably expressing either DNMT3A-R882H or WT DNMT3A. In a colony-formation assay, NIH3T3 cells expressing DNMT3A-R882H formed many more colonies than those expressing WT DNMT3A (Fig. S2).

DNMT3A-R882H Mutation Causes a Transcriptional Alteration. To explore the mechanism underlying DNMT3A-R882H phenotypic defects, we performed gene-expression microarray assay + using the GFP BM cells harvested at 6 mo and 1 y post-BMT. At 6 mo post-BMT, supervised analysis revealed 1,618 and 1,247 genes with differential expression in the DNMT3A-R882H group as compared with the WT DNMT3A and vehicle groups, respectively (Datasets S1 and S2). The expression levels of 738 genes in DNMT3A-R882H mice were different from the levels in both the WT and vehicle groups (Dataset S3). These results indicate that the DNMT3A mutant exerts a dramatic effect on transcriptional regulation. Analysis based on Gene Ontology (GO) clas- sification and the Hematopoietic Fingerprints Database (18) Fig. 3. Transcriptional changes in murine BM at 6 mo and 1 y post-BMT. (A) revealed 14 differentially expressed genes in the “long-term HSC Heatmap representation of mouse long-term HSC regulatory genes identified + subgroup,” and 11 of these genes were well annotated. Genes as being differentially expressed in GFP vector, WT, and DNMT3A-R882H such as Mpl and Hlf were reported to be associated with hema- mouse BM cells at 6 mo post-BMT. Red indicates up-regulated genes; green topoietic malignancies, as well (19, 20). Notably, 10 of the 11 indicates down-regulated genes. (B) Quantitative RT-PCR results for six genes genes were up-regulated in DNMT3A-R882H cells (Fig. 3A), in the BM samples of distinct groups at 6 mo post-BMT. (C) Heatmap for gene = = suggesting that cells transduced with DNMT3A-R882H acquired expression in BM samples with DNMT3A-R882H (n 3) and vector (n 3) at characters of stemness. Interestingly, a group of genes related to 1 y post-BMT. Genes associated with myeloid function are clustered. (D)GO hematopoietic proliferation and differentiation were dysregu- analysis of up-regulated genes in mice at 1 y post-BMT found that a series of functional genes positively regulating myeloid lineage development was acti- lated dramatically, with the functions of myeloid lineage being vated in DNMT3A-R882H mice. (E) Quantitative RT-PCR of representative genes activated and the activities of lymphoid lineage repressed (Table closely associated with myeloid lineage development in murine BM samples at S2). A portion of the differentially expressed genes was validated + 1 y post-BMT showed the same patterns observed in microarray analysis. in murine GFP BM samples (Fig. 3B). We also compared the mRNA levels of some differentially expressed genes in the OCI- AML3 cell line, a human AML strain harboring the DNMT3A- or mutated DNMT3A constructs. Indeed, unique DNA hypo- and Arg882Cys (R882C) mutation, which was infected by lentivi- hypermethylation features were identified in murine CMML BM rus expressing shRNA against mRNAs encoding both WT and cells. On the whole, there was no significant change between vector DNMT3A-R882C, with the levels in control cells expressing and mutant groups (Fig. S4A), but the change in hypo- and scramble shRNA. Quantitative RT-PCR results indicated that the hypermethylation patterns took place regionally throughout the expression of genes related to transcriptional regulation, including κ genome (Fig. 4A). We then looked at methylation changes in four FOS, MYC, NF- B,andATF3, was increased in cells with DNMT3A regions defined by the distance from CpG islands (21), namely the knockdown (Fig. S3A). CpG island, Shore, Shelf, and Open Sea regions, the latter three At 1 y post-BMT, 6,544 genes in the DNMT3A-R882H group being respectively 2 kb, 2–4 kb, and more than 4 kb from the CpG had an expression pattern that differed from that in the vector island. Most hypo- and hypermethylation patterns were detected in group; 3,680 genes were up-regulated, and 2,864 were down- the Open Sea region (Fig. S4B). No differences in the distribution regulated (Dataset S4). We further found that a group of genes of hypo- and hypermethylation status were found in any of these known to be activated in leukemia progression was up-regulated C dramatically in mutant mice (Fig. 3C). For example, Hox family four regions (Fig. S4 ). Next, we scrutinized the DNA hypo- and genes and Idh1, which have been found to be activated in human hypermethylation distribution in the context of gene structure. AML with DNMT3A-R882 mutations, were overexpressed. Mll1 Four relevant regions were defined: promoter, gene body, the and Mllt6 were down-regulated, supporting a previous observa- transcriptional termination region (TTR), and the intergenic re- tion that DNMT3A mutation and MLL abnormalities are mu- gion. The number of methylated cytosines was much higher than tually exclusive (11). On the other hand, a series of genes that the number of demethylated ones in CMML mice as compared with play key roles in T-cell differentiation was down-regulated (Fig. the vehicle group. However, DNA hypo- and hypermethylations S3B). The pathways involved in granulocytic/monocytic lineage were distributed differently in the four regions. As shown in Fig. 4 B differentiation were mostly up-regulated (Fig. 3D), whereas gene and C, hypomethylation was concentrated mainly in gene body clusters related to lymphocytes, mainly to T-cell development, areas, and more hypermethylation was detected in the inter- were largely repressed (Fig. S3C). Some key genes were vali- genic region, suggesting that DNMT3A-R882H could induce dated by quantitative RT-PCR (Fig. 3E and Fig. S3D). abnormal modifications of methylation selectively. Notably, most hematopoiesis-related genes that displayed up-regula- DNMT3A-R882H Mutation Induces Modifications of Genomic Methylation tion in the DNMT3A-R882H group, such as Hoxa3, Hoxa6, Idh1, Patterns. It was predicted previously that the DNMT3A-R882 mu- Flt3,andHlf, were hypomethylated in the gene body (Fig. 4D). In tation could disrupt the tetramerization of the enzyme, and that this contrast, a cluster of suppressed genes that regulate lymphocyte disruption might explain its reduced activity (15). To evaluate development, such as Notch1, Notch3, Gata3,andTcf7, showed thepossibleinjurytogenomicmethylationcausedbyDNMT3A- more hypermethylated status in gene body regions (Fig. S4D). R882H, methylated DNA immunoprecipitation (MeDIP) sequen- These results suggest that aberrant activity of DNMT3A-R882H cing was carried out for murine BM cells transduced with vehicle can target mainly the gene body region, where abnormal

2622 | www.pnas.org/cgi/doi/10.1073/pnas.1400150111 Xu et al. Downloaded by guest on September 27, 2021 the issue in NIH3T3 cells by transfection assay. Compared with the controls, CDK1 increased in NIH3T3 cells overexpressing DNMT3A-R882H but not in cells overexpressing DNMT3A WT. Interestingly, when we applied RNAi to silence WT or mutant DNMT3A, the CDK1 protein level decreased substantially when DNMT3A-R882H was inhibited but not when WT DNMT3A was inhibited (Fig. 5 D and E). We also examined the possible effect of DNMT3A mutation on cell-cycle status in OCI-AML3 cells. In OCI- AML3 cells with knockdown of DNMT3A mutant, the compartment at the G0/G1 phase was much higher, and that at S phase was much smaller, than in control cells treated with scramble shRNA (Fig. 5 F and G). We found the expression levels of both total and phosphorylated CDK1 proteins were reduced after cells were treated with 5-Aza, an inhibitor of DNA methyltransferases (Fig. 5H). Moreover, 5-Aza–treated OCI-AML3 cells were largely blocked at the G1 phase (Fig. 5I). Together, these data suggest that the aberrant increase of CDK1 in the presence of the DNMT3A mutant can promote cell- cycle activity.

Fig. 4. Genomic methylation patterns at 1 y post-BMT. (A) Hypo- and hypermethylation peak counts obtained from three mice for each group are shown in the histogram. DNMT3A-R882H versus control (vector) peaks were considered as hypermethylation peaks; control (vector) versus DNMT3A- R882H peaks were considered as hypomethylation peaks. (B and C) The average proportions of peaks in each region defined by genomic structure are shown in pie charts (B) and a histogram (C). Peak ratios of hypo- and hypermethylation are shown, respectively. Hypomethylation was concentrated mainly in gene body areas, and hypermethylation was detected more frequently in intergenic regions. (D) Hoxa3, Hoxa6, Idh1, Flt3, and Hlf genes showed a local hypomethylation pattern in the gene body whereas Mpl exhibited a hypomethylation status in promoter region in DNMT3A-R882H mice compared with vector group.

DNA methylation patterns—either hypomethylation or hypermethylation—can be produced.

DNMT3A-R882H Mutation Increases CDK1 Protein Level and Enhances GENETICS Cell-Cycle Activity. To explore mechanisms for enhanced cell proliferation other than aberrant DNA methylation and gene transcription, we investigated partner proteins interacting with WT DNMT3A and DNMT3A-R882H. We performed immunoprecipitation and mass spectrometry analysis in transfected 293T cells. In total, 1,565 proteins were identified, and 1,562 proteins were quantified by label-free quantification (LFQ) with a false-discovery rate of 0.01 (Dataset S5). Seven candidate proteins including DNMT3A with P < 0.05 were identified (Table S3). Among them, Fig. 5. Protein–protein and functional interaction between the DNMT3A CDK1, one of the key proteins that promote the cell cycle, caught mutant and CDK1. (A) Interaction of DNMT3A with CDK1. Lysates from 293T our attention. In the validation test, we found that both WT cells transfected with plasmids encoding Flag-DNMT3A were immunopreci- DNMT3A and DNMT3A-R882H could form a complex with pitated using anti-Flag and analyzed by Western blot using the indicated CDK1, but the binding capacity of DNMT3A-R882H to CDK1 antibodies. (B and C) Lysates from OCI-AML3 cells transfected with two was higher than that of WT DNMT3A (Fig. 5A). To test the siRNAs (siRNA1 and 2) against different DNMT3A regions were Western functional significance of such binding, we knocked down the ex- blotted with the indicated antibodies. (D and E) Analysis of lysates from pression potential of both WT and mutant DNMT3A in the OCI- NIH3T3 cells stably expressing DNMT3A WT or the R882H mutant. These cells AML3 cell line by RNAi technology. We found that both total were transfected with scramble siRNA or DNMT3A siRNA1, and their lysates CDK1 and its phosphorylated form were decreased markedly when were Western blotted with antibodies against the indicated proteins. Vec- the DNMT3A mutant was silenced by two different siRNAs (Fig. 5 tor-transfected cells were used to estimate the endogenous CDK1 level compared with levels in cells transfected with WT or mutant DNMT3A.(F) B and C). Because OCI-AML3 cells can express both WT and OCI-AML3 cells were infected with lentiviruses expressing scramble shRNA mutant DNMT3A, we further investigated whether WT or mutated (control) or two shRNAs (shRNA 1 and 2) targeting different DNMT3A DNMT3A might exert an effect on CDK1 protein levels. However, regions. (G) OCI-AML3 cells infected with lentiviruses expressing control or specific siRNA targeting the R882 point mutation and the corre- two different DNMT3A shRNAs were labeled with BrdU and 7-AAD for flow sponding WT sequence failed to reduce protein-expression levels, cytometry analysis. (H and I) Western blot analysis of indicated proteins (H) and the effort to make an antibody to distinguish WT from mutant and propidium iodide (PI) staining flow cytometry analysis (I) in OCI-AML3 DNMT3A protein was not successful. Therefore we tried to address cells treated by DMSO or 5-Aza (0.8 μM) for 48 h.

Xu et al. PNAS | February 18, 2014 | vol. 111 | no. 7 | 2623 Downloaded by guest on September 27, 2021 Discussion between proliferation and differentiation of hematopoietic cells Leukemia represents a group of heterogeneous clonal malig- may be broken, leading to disease progression. nancies of HSPCs. Abnormalities of transcriptional factors and Notably, the pattern of DNA methylation also was found to be of protein tyrosine kinases have been considered two categories aberrant in DNMT3A-R882H–transformed cells. Previous work of major molecular events in leukemogenesis. However, over the suggested that R882 is important for the oligomerization of – past few years, a large body of evidence has been generated sup- DNMT3A because it is located at the surface of protein protein porting the involvement of epigenetic abnormalities in both DNA interaction of the enzyme, and the oligomer form of the enzyme and histone modifications in leukemia and lymphoma (22, 23). is essential for its function (4, 15). It is reasonable to hypothesize High-throughput parallel sequencing has revealed mutations of a that DNMT3A-R882H could affect DNA methylation, probably third group of genes, such as EZH2, TET2, IDH1/2,andDNMT3A, via a dominant-negative effect (32). In fact, an altered genomic involved in epigenetic regulations (11, 13, 24–26). Some of these DNA methylation pattern characterized by regionally modified gene mutations have been shown to cause genome-wide aberration patterns of hypo- and hypermethylation existed in mouse HSPCs. of epigenetic modifications (27) and may play a role as disease Notably, there was no major change in the methylation pattern at drivers (17, 28). CpG islands, and the abnormal hypo- and hypermethylation Among the leukemia-associated epigenetic regulators, muta- status existed mostly in regions where CpG islands were sparse. tions of DNMT3A have drawn particular attention because of This observation was consistent with a recent study on genomics their preferential correlation with the important physiological and epigenomics analysis in a large series of AML (33). One functions of the gene. Several lines of evidence suggest that interesting finding in our work is that hypomethylation is present in the gene body regions of a number of genes that are key in mutated DNMT3A might be present in the initial clone of leu- hematopoietic regulation and whose expression is up-regulated kemia cells (29). In the present work, to address the transforming significantly in transformed HSPCs. It was hypothesized pre- potential of DNMT3A mutants in a definitive way, we used viously that DNMT3A might exert a selective effect on DNA a murine BMT model with HSPCs transduced by DNMT3A- methylation (7), and our data support this notion. Moreover, the R882H. Interestingly, by 6 mo post-BMT, murine hematopoietic methylation pattern in gene body regions seems to correlate well cells with the DNMT3A-R882H mutation showed an obvious with a gene-expression profile in which myeloid genes are up- growth advantage, and the unique features of the HSPCs with regulated and lymphoid genes are down-regulated. Therefore we mutant DNMT3A could be transmitted to other mice via sec- speculate that DNMT3A may be responsible for the balance ondary BMT. At 1 y post-BMT, the hematopoietic cells with this between myeloid and lymphoid commitment in HSPCs. hotspot mutant persisted and gradually acquired a growth ad- It has been proposed that the DNMT3A mutant also may vantage over the residual normal blood cells. Importantly, a function by forming a protein complex with other components of CMML-like disease phenotype appeared at this stage, as defined the cell transcription machinery to change the expression of by the 2008 World Health Organization nomenclature for hu- certain genes (34). Indeed, CDK1 is among proteins interacting > 9 < man setting ( 10 monocytes/L in PB, with 20% blasts in both with DNMT3A-R882H. It is well known that, as one of the key PB and BM and dysplasia in more myeloid lineages, without regulators of the cell cycle, CDK1 binds to cycline A and cycline BCR-ABL1 fusion gene or PDGFRA/PDGFRB rearrangement). B and regulates the mitotic division of the cell (35, 36). Inter- Interestingly, this murine CMML phenotype was accompanied estingly, our experiments showed that DNMT3A-R882H forms by thrombocytosis, which was reminiscent of the clinical features a complex with CDK1 more readily than does WT DNMT3A. of AML with DNMT3A mutation (12). That the CMML-like Moreover, the stability of both total CDK1 and its active form is phenotype developed in all recipient mice of DNMT3A mutant increased in OCI-AML3 cells that have the potential of expressing within a similar latent time and that the retroviral integration both WT and mutant DNMT3A. In the NIH3T3 cells, over- sites for this mutant were on different chromosomes in the mice expression of DNMT3A-R882H, but not WT DNMT3A, induces investigated provide strong evidence that this mutation alone is a higher level of CDK1, whereas knockdown of the DNMT3A capable of initiating leukemia. DNMT3A-R882H therefore should mutant, but not the WT protein, leads to a significant reduction of be considered not only as driving leukemogenesis but also as di- CDK1. A possible explanation for this observation might be that recting the survival/growth advantage of the myelomonocytic the long-time existence of DNMT3A-R882H changes the protein lineage, with specific clinical manifestations. However, this phe- expression/stability program in the cells to some extent, so that the notype corresponds to a chronic hematopoietic disorder, and level of CDK1 becomes dependent on to the level of the DNMT3A further genetic events, in addition to DNMT3A mutations, may mutant. Of note, knocking down the potential of expressing both be required for full-blown AML to occur. WT and the DNMT3A mutant in OCI-AML3 strains reduces the Now that the transformational ability of DNMT3A mutation cell-cycle activity. Finally, our study provides evidence that 5-Aza, has been established, how might the DNMT3A mutant impact as DNMTs inhibitor, might be used in the clinical treatment of genome-wide transcriptional expression and epigenetic regula- AML with DNMT3A mutations. tion? We systematically investigated gene-expression profiles of hematopoietic cells at 6 or 12 mo post-BMT and found that the Materials and Methods expression of a series of genes associated with long-term hema- Plasmid Construction. The full-length coding sequence of human DNMT3A- topoiesis, such as Mpl and Hlf, was up-regulated significantly at WT cDNA was cloned into the BglII and EcoRI multiple cloning sites of ret- 6 mo post-BMT. Of note, Mpl codes for thrombopoietin receptor roviral vector MSCV-IRES-GFP (MigR1) or CMV4-Flag. The constructs con- and regulates growth not only of the megakaryocytic lineage- taining the DNMT3A R882H mutant were generated by site mutagenesis generating platelets but also other myeloid progenitors (30, 31). (Quick Change). Interestingly, at 1 y post-BMT, in addition to continuous over- expression of Mpl, pathways involved in granulocytic/monocytic Retroviral Preparations and Protein Expression Analysis. Retrovirus was pro- lineage proliferation, including Hoxb2, Hoxa9, and Meis1, were duced by transfection of the retroviral construct into the packaging cell line activated, and those involved in platelet activation, including Plat-E by calcium phosphate precipitation. Retroviral supernatants were harvested at 48 h after transfection, and the viral titer was determined by Pdgfb, and Itgb3, were up-regulated. In contrast, gene clusters flow cytometry using infected NIH3T3 cells. The cell lysates were processed related to lymphocytes, and mainly to T-cell development, were for Western blot analysis. The following primary antibodies were used: anti- largely repressed. Thus, a dynamic change in the gene-expression DNMT3A (Cell Signaling), anti-CDK1 (Cell Signaling), phospho-CDK1 (Cell program seems to emerge over the disease process. At a relatively Signaling), and β-actin (Santa Cruz Biotechnology). early stage, DNMT3A-R882H may exert an effect on gene expression at whole-genome level and initiate abnormal proliferation Murine Bone Marrow Transduction and Transplantation. BM cells were isolated of HSPCs. With the clonal expansion of HSPCs harboring the from the 6- to 8-wk-old BALB/c donor mice which were pretreated with DNMT3A mutant, the relative balance of transcriptional controls 5-fluorouracil (250 mg/kg) 5 d before operation. The primary murine BM cells

2624 | www.pnas.org/cgi/doi/10.1073/pnas.1400150111 Xu et al. Downloaded by guest on September 27, 2021 + then were infected with retroviruses and transplanted into lethally irradiated Microarray Expression Profiling. Total RNAs from murine GFP BM samples recipient mice (details are given in SI Materials and Methods). Recipient mice were prepared and, after quality control, subjected to the Agilent whole in secondary transplantation assay were treated by sublethally irradiation. mouse genome 4 × 44K Array GeneChip microarray test according to the All animal experiments were approved by the Department of Animal Ex- manufacturer’s instructions (Agilent). The scanning procedure was per- perimentation at Shanghai Jiao Tong University School of Medicine. formed using an Agilent Microarray Scanner (Agilent G2565BA) at the KangCheng Bio-Tech Corporation in Shanghai, China. Agilent Feature Ex- Mouse Bone Marrow Colony-Forming Cell Assays. Assays were performed traction Software was used for data analysis. according to the manufacturer’s instructions (MethoCult GF M3434; STEMCELL + Technologies). Briefly, 2 × 104 GFP BM cells were mixed with 1.1 mL MethoCult Methylation Analysis by MeDIP Sequencing. A total of 3 μg DNA prepared + medium and plated in a 35-mm culture dish. Triplicate cultures for each sample from sorted murine GFP BM for each mouse was used for MeDIP se- were performed. Colonies were counted at day 8 after plating. quencing. Detailed procedures are available in SI Materials and Methods.

Morphological and Immunophenotypic Analysis of Hematopoietic Cells. Murine Mass Spectrometry Analysis. Lysate of 293T cells transfected with pFLAG- PB cells and BM cells were prepared for analysis. Smears and cytospin CMV4 or DNMT3A WT-pFLAG-CMV4 constructs were immunoprecipitated by preparations were subject to Wright’s staining for routine cell morphology. anti-Flag M2 Affinity Gel (Sigma-Aldrich) and analyzed by mass spectrometry. Single-cell suspensions were incubated with antibodies and harvested for Details are available in SI Materials and Methods. flow cytometry, as described in SI Materials and Methods. ACKNOWLEDGMENTS. We thank colleagues from Shanghai Institute of Cell-Cycle Detection. Cells were stained with propidium iodide or BrdU/7-AAD, Hematology, particularly Dr. R.-B. Ren for constructive discussions, T. Zhen and the assays were performed using flow cytometry according to the and J.-Y. Shi for assistance in biological experiments, and J.-Q. Mi and manufacturers’ instructions (BD Sciences and BD Pharmingen). B. Chen for hematological morphological analysis, and. C. Li from Shanghai Institutes for Biological Science for technical support. This work was supported by the Chinese National Key Basic Research Project 973 Grants Lentivirus-Mediated shRNA Infection and siRNA Transfection Assay. Lentiviruses 2010CB529200 and 2013CB966800; Ministry of Health Grant 201202003; the expressing scramble shRNA or DNMT3A shRNA were generated, and cells were Mega-projects of Scientific Research for the 12th Five-Year Plan Grant transfected with siRNA oligomer using Lipofectamine 2000 Transfection Re- 2013ZX09303302; the State Key Laboratories Project of Excellence Grant agent (Invitrogen). Details are given in SI Materials and Methods. 81123005; the National Natural Science Foundation of China for Excellent Young Scholars Grant 81222004; the Shanghai Municipal Commission for the Rising-Star Program (follow-up support) Grant 12QH1401500; the Immunoprecipitation and Immunoblotting. 293T cells (1 × 107) overexpressing Special Foundation for Doctor Discipline of Ministry of Education of China DNMT3A-flag were washed twice with cold PBS and suspended in 1 mL lysis Grant 20120073110074; the Innovation Foundation for Doctoral Students buffer (Thermo). Lysates were harvested and incubated overnight with anti- of Shanghai Jiao Tong University School of Medicine Grant BXJ201208; FLAG M2 Affinity Gel (Sigma-Aldrich). Gels with immunoprecipitated prod- and the Samuel Waxman Cancer Research Foundation Co-Principal Inves- ucts were washed for immunoblotting. tigator Program.

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