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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cancer Discov. Author manuscript; available in PMC 2017 May 1.
Published in final edited form as:
PMCID: PMC4861898
NIHMSID: NIHMS775836

Dnmt3a haploinsufficiency transforms Flt3-ITD myeloproliferative disease into a rapid, spontaneous, and fully-penetrant acute myeloid leukemia

Abstract

Cytogenetically normal acute myeloid leukemia (CN-AML) represents nearly 50% of human AML. Co-occurring mutations in the de novo DNA methyltransferase DNMT3A and the FMS related tyrosine kinase 3 FLT3 are common in CN-AML and confer a poorer prognosis. We demonstrate that mice with Flt3-internal-tandem duplication (Flt3ITD) and inducible deletion of Dnmt3a spontaneously develop a rapidly-lethal, completely-penetrant, and transplantable AML of normal karyotype. AML cells retain a single Dnmt3a floxed allele, revealing the oncogenic potential of Dnmt3a haploinsufficiency. FLT3-ITD/DNMT3A-mutant primary human and murine AML exhibit a similar pattern of global DNA methylation associated with changes in the expression of nearby genes. In the murine model, rescuing DNMT3A expression was accompanied by DNA re-methylation and loss of clonogenic potential, suggesting that Dnmt3a-mutant oncogenic effects are reversible. Dissection of the cellular architecture of the AML model using single-cell assays identified clonogenic subpopulations that express genes sensitive to the methylation of nearby genomic loci, and responsive to Dnmt3a levels. Thus, Dnmt3a haploinsufficiency transforms Flt3ITD myeloproliferative disease by modulating methylation-sensitive gene expression within a clonogenic AML subpopulation.

Keywords: Epigenetics, Acute Myeloid Leukemia, Myeloproliferative Neoplasm, Dnmt3a, Flt3

Introduction

FLT3 is one of the most frequently mutated genes in cytogenetically normal acute myeloid leukemia (CN-AML), affecting 37% of cases (13). FLT3 internal tandem duplication (ITD) mutations are associated with a worse overall survival, and constitute an independent prognostic factor for relapse and poor outcome in AML (47). Recently, recurrent mutations were found in the epigenetic regulators DNMT3A and TET2 (2, 3, 811). It is now recognized that DNA methyltransferase DNMT3A is one of the more commonly mutated genes in AML, affecting 23% of cases, and is associated with an unfavorable prognosis (3, 1214). Intriguingly, 36–44% of FLT3 mutant AML patients also carry DNMT3A mutations. Furthermore, the co-occurrence of DNMT3A and FLT3 mutants changes the risk classification to a poorer prognosis (3, 12, 13).

The mammalian DNA methyltransferases DNMT1, DNMT3A and DNMT3B methylate the cytosine residue at 5 position (5mC) in 5′-C-phosphate-G-3′ (CpG) dinucleotides. Methylation of CpG sites at promoter-associated CpG islands is thought to silence expression of neighboring genes. In addition, methylation of CpG dinucleotides can also occur at the gene body or intergenic regions, where it’s role in gene regulation is less clear (15). Different molecular subtypes of CN-AML have highly divergent DNA methylation and transcription profiles (16, 17). Recurring missense mutations in DNMT3A identified in CN-AML act as loss-of-function or dominant negatives, significantly reducing the methyltransferase activity of DNMT3A by approximately 80% (11, 18, 19). While the mechanisms of DNMT3A action in the pathogenesis of AML are still unclear, studies on stem cells demonstrate essential roles for all DNMT proteins. DNMT1 is required to maintain global DNA methylation through successive rounds of cell division, whereas the de novo methyltransferases DNMT3A and DNMT3B are required for mouse embryonic development (20). Serial transplantation of Dnmt3a knockout hematopoietic stem/progenitor cells (HSPC) results in impaired differentiation, enhanced proliferation, and differential methylation of distinct genetic loci resulting in upregulation of stemness genes and proto-oncogenes, and downregulation of differentiation genes (21). Despite this, Dnmt3a knockout HSPC reconstituted mice do not develop AML.

FLT3-ITD mutant receptors undergo ligand-independent receptor dimerization, auto-phosphorylation, and constitutive activation (2224). Mouse knock-in and human cell models of FLT3-ITD demonstrate FLT3 mutant receptors promote aberrant cell proliferation through activation of Stat5, Akt/Erk, and c-Myc pathways, and impair differentiation by altering expression of myeloid transcription factors such as CEBPα (2532). Despite the high frequency of FLT3 mutations in AML, murine models of Flt3ITD develop a mixture of myeloid and lymphoid neoplasms either as a single mutation or in combination with other oncogenic events such as MLL partial tandem duplication (MllPTD) (25, 27, 3335). Importantly, Flt3ITD mice do not progress to leukemia, thus other cooperating mutations must be required for development of AML.

Results

To test whether Flt3 and Dnmt3a mutations cooperate to initiate leukemogenesis we bred mice to combine Flt3ITD knock-in and homozygous Dnmt3a floxed alleles (Dnmt3afl/fl) (Fig. 1A) with Mx1-Cre (MxCre). We find that Flt3+/ITD;Dnmt3afl/fl MxCre mice (with one mutant Flt3 allele) have a long median survival of 227 days and exhibited splenomegaly (Supplementary Fig. S1A). Control Flt3+/ITD;Dnmt3afl/fl mice did not die in this time period. Conversely, doubling the number of Flt3ITD alleles significantly shortened survival with a median of 43 days after birth (Fig. 1B left and lower table). All Flt3ITD/ITD;Dnmt3afl/fl MxCre mice exhibited splenomegaly (Fig. 1B right), and were moribund by 60 days. Secondary and tertiary transplantation of Flt3ITD/ITDDnmt3afl/fl MxCre splenocytes resulted in lethality, albeit with delayed latency (Supplementary Fig. S1B and data not shown). Because the disease occurred spontaneously (pIpC induction of Mx1-Cre expression was not required), we examined the Dnmt3afl/fl alleles (Fig. 1C). As expected, tail DNA from two week-old Flt3ITD/ITD;Dnmt3afl/fl MxCre mice showed amplification of loxP-flanked Dnmt3a exon 18 (Fig. 1C, “floxed” indicates no recombination; “deleted” indicates recombination). However, total or fractionated c-Kit+ (CD117) stem/progenitor enriched splenocytes from Flt3ITD/ITD;Dnmt3afl/fl MxCre de novo or secondary transplant recipients demonstrated both floxed and deleted Dnmt3a alleles (Fig. 1C). In addition, we plated c-Kit+ Flt3ITD/ITD;Dnmt3afl/fl MxCre hematopoietic cells in methylcellulose, isolated DNA from individual colonies, and then analyzed floxed Dnmt3a alleles. Of 77 colonies analyzed, 28 lacked detectable floxed alleles (36%), thus the majority of cells spontaneously deleted only one allele of Dnmt3a (Supplementary Fig. S1C). In agreement with this, the mRNA from c-Kit+ cells from Flt3ITD/ITD;Dnmt3afl/fl MxCre mice showed sporadic low-level expression of Cre, and approximately 50% reduction in Dnmt3a expression (Fig. 1D). These data suggest that sporadic MxCre expression leads to excision of floxed Dnmt3a alleles, and that Flt3ITD/ITD cells with a single Dnmt3a allele may have a selective advantage.

Figure 1
A rapid, spontaneous, and fully penetrant model of cytogenetically normal AML utilizing Flt3ITD and Dnmt3a haploinsufficiency

Examination of the blood, bone marrow, spleen and liver of moribund Flt3ITD/ITD;Dnmt3afl/fl MxCre mice demonstrated pathologic features of myeloid leukemia. The peripheral blood was notable for anemia and for leukocytosis of neutrophilic and monocytic cells, including immature forms (Supplementary Table S1 and data not shown). Bone marrow contained immature forms/blasts that were greater than 20% of nucleated cells along with numerous intermediate myeloid forms (i.e. non-blast myelomonocytic cells that have not differentiated sufficiently to be distinguished into monocytic or neutrophilic cells by morphology) (36). Similar to Flt3ITD/ITD;Dnmt3afl/fl mice there was a marked decrease in erythroid and lymphoid cells relative to wild-type (WT) marrow (Fig. 1E, top two rows). The massively enlarged spleens of the Flt3ITD/ITD;Dnmt3afl/fl MxCre mice contained areas of predominantly immature cells (similar to those seen in the marrow), but most of the red pulp contained a mix of maturing neutrophilic, monocytic, erythroid and megakaryocytic cells (Fig. 1E, third row). Concordant with the morphology, flow cytometric immunophenotyping of erythroid-depleted bone marrows and spleens of Flt3ITD/ITD;Dnmt3afl/fl MxCre mice showed decreased lymphocytes (data not shown and Supplementary Fig. S1D) and increased monocytic cells (CD11b+, Gr1, B220) in comparison to WT mice, and increased immature cells (Linc-Kit+, abbreviated LK) in comparison to Flt3ITD/ITD;Dnmt3afl/fl mice (Supplementary Fig. S1E–I, Supplementary Table S2). In contrast to the Flt3ITD/ITD;Dnmt3afl/fl mice, myelomonocytic cells aggressively invaded the perivascular and sinusoidal regions of the liver (Fig. 1E, fourth row). These pathologic findings are indicative of myelomonocytic leukemia. The presence of greater than 20% immature forms/blasts in the bone marrows of Flt3ITD/ITD;Dnmt3afl/fl MxCre mice in combination with the rapid lethality observed in the primary animals warranted a diagnosis of acute leukemia (36). In agreement with previous reports, Flt3ITD/ITD;Dnmt3afl/fl control mice (Fig. 1E, second column) showed expansion of both mature and immature myeloid cells, consistent with myeloproliferative neoplasm (MPN) (27). According to these diagnoses, we hereon refer to Flt3ITD/ITD;Dnmt3afl/fl mice as MPN and Flt3ITD/ITD;Dnmt3afl/fl MxCre mice as AML.

To assess the ability of the leukemic cell populations to give rise to colony forming units (CFUs), we seeded purified c-Kit+ and c-Kit cells from moribund mice into methylcellulose. The c-Kit+ fraction from several different donor mice gave rise to significantly more colonies than the c-Kit fraction, compatible with c-Kit+ leukemic blasts (Supplementary Fig. S1J). Moreover, c-Kit+ splenocytes from AML mice had significantly increased colony forming ability compared to c-Kit+ splenocytes from MPN mice (Fig. 1F). No differences in colony morphologies or numbers between c-Kit+ bone marrow and c-Kit+ splenocytes from AML mice were observed (data not shown). We conclude that loss of one Dnmt3a allele is sufficient to increase the clonogenic potential of Flt3ITD myeloid progenitors.

Since FLT3-ITD and DNMT3A mutations are most common in AML patients with normal karyotype, we performed spectral karyotyping analyses on bone marrow from five independent murine AML (Fig. 1G). Four of five AMLs exhibited normal karyotypes and one had a small clone with loss of the Y chromosome (39,X, -Y[4]/40,XY[16]). Note that loss of the Y chromosome is observed in both malignant and non-malignant states, and is not considered to be a driver mutation. Like their human counterparts with similar mutations, the murine model tumors are cytogenetically normal AML.

Since the murine AML express less Dnmt3a mRNA, we next investigated whether our model recapitulated the epigenetic abnormalities seen in human DNMT3A-mutant AML, where activity of the enzyme is compromised due to the dominant-negative nature of the mutation (11, 18, 19). Using Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) (37), we derived the disease-specific DNA-methylation profiles in both human and murine AML with similar mutation profiles. First, we compared primary human AML carrying both FLT3-ITD and DNMT3A-mutations (FLT3-ITD/DNMT3A-mut) versus FLT3-ITD AML lacking mutations in DNMT3A (FLT3-ITD/DNMT3A-wt). To ensure that the DNA methylation differences we observed could only be explained by the DNMT3A status, patients with mutations in other genes related to the DNA methylation pathway (TET2, IDH1 and IDH2) were excluded from both groups. Differentially methylated regions (DMR) with an FDR<10% and a mean methylation difference ≥25% were identified using a beta-binomial model. Patients with FLT3-ITD/DNMT3A-mut presented with a predominantly hypomethylated profile compared to FLT3-ITD/DNMT3A-wt cases (95.9% hypomethylated regions of 25,746 total DMR). These differentially methylated regions in FLT3-ITD/DNMT3A-mut AML were significantly enriched at CpG shores (22% of DMRs at CpG shores versus 15% in background, p<2.2x10−16) and enhancer regions (33% of DMRs at intragenic enhancers versus 19% in background, p<2.2x10−16 and 24% at intergenic enhancers versus 15% in background, p<2.2x10−16) (Fig. 2A–C, Supplementary Table S3). Next, we compared the murine AML versus MPN c-Kit+ splenocytes. Similar to our findings in human AML, Dnmt3a loss was associated with predominance of hypomethylation (80.7% hypomethylated regions of 503 total DMR) with enrichment of DMRs at enhancer regions (9% of DMRs at intragenic enhancers versus 3% background, p=1.519x10−10 and 6% of DMRs at intergenic enhancers versus 2% background, p=6.005x10−7) (Fig. 2D–F, Supplementary Table S3). Importantly, we found that 61.8% of the DMR identified in the murine models (Flt3ITD/ITD;Dnmt3afl/fl MxCre AML versus Flt3ITD/ITD;Dnmt3afl/fl MPN) overlapped with the human FLT3-ITD/DNMT3A-mutant signature.

Figure 2
Genome-wide DNA hypo-methylation associated with DNMT3A mutation in murine and human FLT3-ITD AML

We next determined the DNA methylation status of these DMRs in normal human and murine HSPC. We note that the methylation of a subset of DMR is similar in both DNMA3A-mut leukemic and normal human hematopoietic stem cells (HSC) or murine AML and LinSca-1+c-Kit+ (LSK) cells (Fig. 2B, 2E; “HSPC-like” DMRs) (Supplementary Table S3), suggesting that Dnmt3a-mutant AML cells lack adequate Dnmt3a levels to mediate normal differentiation-associated methylation of these genomic loci. In contrast, other DMR were not found in normal progenitors, and instead displayed methylation patterns unique to the DNMT3A-mutant human and mouse AMLs (Fig. 2B, 2E; “AML-unique” DMRs) (Supplementary Table S3). These data suggest that while Dnmt3a loss results in disease-specific DMRs, it also allows the persistence of HSPC-like methylation profiles in the leukemic blasts despite these cells expressing phenotypic differentiation markers that suggest maturity beyond HSPC.

Human AML is known to exhibit altered DNA methylation relative to normal HSPC (38, 39). As expected, we find that human FLT3-ITD AML exhibits extensive global DNA methylation changes compared to normal human HSC, irrespective of DNMT3A status (Supplementary Fig. 2A–B). However, comparison of FLT3-ITD DNMT3A-wild-type versus FLT3-ITD DNMT3A-mutant AML reveals a more restricted pattern of predominantly hypomethylated DNA associated with DNMT3A mutation (Supplementary Fig. S2C). Similarly, comparison of murine MPN or AML versus wild-type LSK marrow cells reveals dramatic differences in global DNA methylation patterns (Supplementary Fig. S2D–E); however, comparing murine MPN versus murine AML reveals a more subtle global consequence of Dnmt3a haploinsufficiency, predominantly resulting in DNA hypomethylation (Supplementary Fig. S2F).

To understand whether these changes in global DNA methylation correspond to changes in gene expression, we performed RNA sequencing on c-Kit+ splenocytes from murine AML and MPN (Fig. 2G, Supplementary Table S4). Similar to our findings in the methylome, the expression of some genes in murine AML blasts is similar to LSK marrow cells (“HSPC-like” Fig. 2G, Supplementary Table S4), while a group of mostly upregulated genes are uniquely expressed in AML compared to LSK or MPN (“AML-unique” Fig. 2G, Supplementary Table S4). In agreement with prior studies indicating that Dnmt3a regulates HSPC function (21, 40), Gene set enrichment analyses (GSEA) demonstrated that genes upregulated in murine AML are enriched for HSPC gene expression as well as genes downregulated during myeloid development (Supplementary Fig. S3A, Supplementary Table S4) (41, 42). To evaluate the relevance of murine Flt3ITD/ITD;Dnmt3afl/fl MxCre AML gene expression in human AML, we identified genes that are significantly upregulated in murine AML versus MPN, then curated a gene-set of their human orthologs (Supplementary Table S4). When we compare gene expression data from human FLT3-ITD AML with either wild-type or mutant DNMT3A, GSEA demonstrates that gene expression in DNMT3A-mutant AML is significantly enriched for this gene set (Fig. 2H). We then performed correlation (Fig. 2I) and GSEA analyses (Fig. 2J, Supplementary Table S4) between murine AML DMRs and gene expression, finding that the majority of overexpressed genes (60%) overlap with sites of DNA hypomethylation events in the murine AML (Fig. 2I, upper left quadrant red dots). Finally, we validated candidate genes with Flt3ITD/ITD;Dnmt3afl/fl MxCre AML-unique (Emilin2 and Pim1) and HSPC-like (Gata3) DMRs and associated gene upregulation (Fig. 2K). We conclude that deletion of a single Dnmt3a allele results in functional Dnmt3a haploinsufficiency, and in the context of Flt3ITD/ITD recapitulates a pattern of methylation and gene expression seen in human AML with FLT3-ITD and DNMT3A mutations.

GSEA also revealed that murine AML-upregulated genes are enriched for c-Myc-target-gene sets (Supplementary Fig. S3B and Supplementary Table S4). Therefore, we genetically incorporated a knock-in allele that generates an eGFP-c-Myc fusion protein from the endogenous c-Myc locus (c-MyceGFP;Flt3ITD/ITD;Dnmt3afl/fl MxCre AML vs. c-MyceGFP;Flt3ITD/ITD;Dnmt3afl/fl MPN) (43). We found significantly increased levels of c-MyceGFP expression in c-Kit+ splenocytes from AML versus MPN mice (Supplementary Fig. S4A); however, only a subpopulation of c-Kit+ splenocytes from either genotype expressed c-MyceGFP. In contrast, when AML splenocytes are grown in cytokine rich media in vitro the majority express c-MyceGFP, but treatment with a Flt3 inhibitor (Quizartinib/AC220) downregulated c-MyceGFP (Supplementary Fig. S4B). Flow cytometric analysis of phospho-Stat5, a downstream effector of Flt3ITD (28), was uniformly activated in c-Kit+ AML splenocytes (Supplementary Fig. S4C). These data suggest that c-MyceGFP is a potential biomarker of Flt3ITD signaling, but that there is significant heterogeneity in AML cellularity and/or integration of Flt3ITD and loss of Dnmt3a signaling. To assess this possibility, we dissected the cellular architecture of the murine AML model using single-cell RNA-Sequencing (scRNA-Seq) coupled to complementary single-cell assays. Using the Iterative Clustering and Guide-gene Selection (ICGS) algorithm in AltAnalyze (44), unsupervised analysis of 96 individual c-Kit+ AML splenocyte libraries (generated from two independent mice) identified 7 distinct cell populations. Based on gene expression and ontology analysis in GO-Elite (45) the cell clusters were assigned identities similar to their normal counterparts (macrophage-dendritic precursors “MDP” (46), neutrophil precursors, monocyte progenitors, and four distinct HSPC-like populations) (Fig. 3A, Supplementary Table S5). We note that more mature cell types express c-Myc with low incidence and amplitude (e.g. MDP and neutrophil precursors), while the more immature populations show high c-Myc incidence and amplitude (Fig. 3B). However, the bioinformatics assignment of a mature cellular identity does not necessarily obviate the leukemogenic potential of an AML subpopulation. Therefore, we performed flow cytometric sorting and CFU assays to assess the clonogenic potential of ICGS-defined murine AML subpopulations. Using discovered candidate markers from the ICGS clusters (c-Kit, Cxcr4, c-Myc, and Il18r1) (Fig. 3B), ten AML subpopulations were sorted using combinations of these markers (Fig. 3C; representative flow plots). While c-Kit+ AML cells contain the clonogenic fraction (Fig. 3D compare 1 vs. 3), c-Kit+c-MyceGFP cells were significantly enriched for clonogenicity (Fig. 3D compare 2 and 3 vs. 4). Thus, although c-Kit+ splenocytes uniformly show activated Stat5 downstream of Flt3ITD signaling, the heterogeneity in integrating that signal to provide c-Myc protein accumulation appears to be context dependent and most probably relates to the progenitor versus precursor-like state of the Flt3ITD/ITD;Dnmt3afl/fl MxCre AML cell.

Figure 3
Single-cell RNA-Seq to determine the cellular structure of the Flt3ITD/ITD;Dnmt3afl/fl MxCre AML model

We find, in contrast to c-Myc, Cxcr4+ expressing AML cells had reduced clonogenicity in all populations as compared to populations without surface Cxcr4 expression (Fig. 3D compare 3 vs. 6, 4 vs. 9, 5 vs. 7, and 8 vs. 10). Notably, while both c-Myc and Cxcr4 are expressed at high amplitude and incidence in several ICGS clusters, only c-Myc is expressed at high incidence in the HSPC-like-1 cluster (Fig. 3B). Searching for another HSPC-like-1-enriched marker with amenable reagents for flow cytometry, we focused upon Interleukin 18 receptor 1 (Il18r1, Il18rα, or CD218a) (Fig. 3B). While Il18rα is expressed at very high levels in Flt3ITD/ITD;Dnmt3afl/fl MxCre AML, role for Il18rα in normal HSPC or AML has yet to be clearly defined (47). Similar to c-Kit+c-MyceGFP cells, c-Kit+Il18rα+ cells were enriched for clonogenicity (Fig. 3D population 3 vs. 5); however, AML cells that were triple-positive (c-Kit+c-MycGFPIl18rα+Cxcr4) were the most significantly enriched for leukemia clonogenicity (Fig. 3D population 8). In summary, the Flt3ITD/ITD;Dnmt3afl/fl MxCre AML model consists of functionally heterogeneous c-Kit+ stem/progenitor/precursor populations with enriched clonogenicity corresponding to HSPC-like cluster gene expression.

Given the association between DMR and gene expression (Fig. 2), and the considerable cellular heterogeneity in gene expression (Fig. 3), we next determined whether manipulating Dnmt3a levels would reverse methylation, gene expression and/or clonogenicity. To this end, we rescued Dnmt3a haploinsufficiency in the murine AML by forced expression of DNMT3A (Fig. 4A). Compared to empty vector (EV) control transduced AML, DNMT3A rescue did not alter differentiation or colony numbers in the initial plating (Fig. 4B, left bar graphs). Importantly, DNMT3A rescue ablated clonogenicity upon replating (Fig. 4B, right line graphs), and was associated with DNA hypermethylation (Fig. 4C, Supplementary Table S6). In particular, 74/260 of the differentially methylated regions in AML were reverted to methylation levels similar to MPN and 40 new hypermethylated regions were identified (Fig. 4D). As in the de novo murine AML cells, there was a significant enrichment of DMRs overlapping with both intra- and intergenic enhancers (12% of DMRs at intragenic enhancers versus 3% background, p<2.2x10−16 and 8% of DMRs at intergenic enhancers versus 2% background, p<2.2x10−16) (Fig. 4E). In agreement with the association between HSPC-like gene expression and clonogenicity, prior to rescue the HSPC-like AML populations expressed genes proximal to re-methylated regions at a significantly higher level compared to more mature precursors (Fig. 4F, Supplementary Fig. S4D compare clusters c3–c7 versus c1 and c2). For example, we consistently observed DNA hypomethylation of the Gata3, Cxxc5, and Hdac7 gene loci in both human and murine Dnmt3a-mutant AML (Supplementary Table S3). In addition, GATA3 and CXXC5 were overexpressed in human FLT3-ITD/DNMT3A-mut AML (Supplementary Table S4 GSEA leading edge from Fig. 2H), while Gata3, Cxxc5, and Hdac7 were all significantly overexpressed murine AML (Fig. 2I, Supplementary Table S4). Moreover, Gata3, Cxxc5, and Hdac7 were expressed almost exclusively in the HSPC-like murine AML subpopulations (Fig. 3B, Supplementary Fig. S4E) and are hypermethylated upon DNMT3A rescue (Fig. 4D). These data suggest that genes that respond in this manner may have central roles in CN-AML downstream of DNMT3A and FLT3. We conclude that DNA hypomethylation downstream of Dnmt3a loss-of-function is reversible.

Figure 4
Rescuing DNMT3A expression is associated with reversion of DNA hypo-methylation and loss of clonogenicity

Since DNMT3A rescue diminished the colony forming ability of Flt3ITD/ITD;Dnmt3afl/fl MxCre AML we next sought to delineate genes downstream of Dnmt3a loss-of-function that might be important for leukemic potential. To this end, we examined the clonogenicity of murine c-Kit+ AML cells upon shRNA-mediated knockdown of genes that are overexpressed and hypomethylated in murine AML, and/or DMR reverted upon DNMT3A rescue (e.g. Gata3, Emilin2, Hdac7, Cxxc5, Alpk3, and Ttyh2). We also examined knockdown of Il18r1 (Il18rα) on colony formation since we find this cell surface receptor is expressed on HSPC-like populations with increased clonogenicity (Fig. 3D). Repression of Emilin2 and Cxxc5 by multiple independent hairpins caused significant reduction in colony formation, while Il18r1 knockdown had no effect (Fig. 4G and Supplementary Fig. S4F). Even though we did observe some reduction in colony formation with Gata3 and Alpk3 shRNA, these effects were less consistent across multiple hairpins in multiple mouse AMLs (data not shown); however, we cannot discount the possibility that these genes may also play a role in Flt3ITD/ITD;Dnmt3afl/fl MxCre AML. In sum, these data suggest Il18rα simply serves as a surface marker on AML cells with increased clonogenic capacity, while Cxxc5 and Emilin2, which are specifically expressed in HSPC-like AML populations, have an important functional role in self-renewal and proliferation in vitro. In conclusion, Dnmt3a haploinsufficiency collaborates with Flt3ITD to form a rapidly lethal, fully penetrant, AML. Dnmt3a loss is associated with reversible changes in DNA methylation leading to signaling programs distinct from Flt3ITD alone that promote leukemogenesis.

Discussion

Flt3ITD mutation combined with other oncogenic lesions such as Tet2−/− (16), MllPTD (35), and Npmc+ (48) progress to AML or mixed lineage leukemias in mice. The uniquely rapid lethal nature of the Flt3ITD/ITD;Dnmt3afl/fl MxCre AML model may be due to Dnmt3a control of HSC self-renewal. Deletion of Dnmt3a (Dnmt3a−/−) specifically expands HSC and myeloproliferation in vivo, but does not produce acute leukemia (21, 49). The addition of KRasG12D mutation with Dnmt3a−/− in mice significantly accelerates MDS/MPN development compared to KRasG12D alone (50). Yet only ~30% of Dnmt3a−/−;KRasG12D MxCre mice progress to AML and require pIpC to induce complete Dnmt3a deletion. The DNMT3A-R882H mutation found in human AML acts as a dominant negative that reduces, but does not completely block, the activity of DNMT3A (18). Moreover, the prognosis for human FLT3-ITD AML is worsened with the addition of a DNMT3A mutation (3, 12, 13). In parallel to human FLT3-ITD/DNMT3A-mutant CN-AML, Flt3ITD/ITD;Dnmt3afl/fl MxCre mice spontaneously select for Dnmt3a haploinsufficiency –rather than a complete loss– that transforms Flt3ITD MPN into a rapidly lethal AML with 100% penetrance. Moreover, a high FLT3-ITD allelic ratio predicts poor survival in human AML (51). Thus, the requirement for two Flt3ITD alleles to form AML (in cooperation with Dnmt3a loss) in this mouse model is consistent with human disease. The pattern of methylation was similar to human AML with FLT3-ITD and mutations in DNMT3A. In addition, there was significant correlation between the expression of some genes with nearby changes in DNA methylation in Flt3ITD/ITD;Dnmt3afl/fl MxCre AML compared to Flt3ITD/ITD;Dnmt3afl/fl MPN, indicating that these changes in DNA methylation do not reflect non-specific methylation losses but rather play a functional role in the development of leukemia.

We used single cell RNA-Seq to dissect the cellular architecture of the leukemia. While cancer stem cell markers are usually not conserved across different cancers (46), c-Kit expression is a known marker of leukemia stem cells (LSC) in other AML models. In agreement with this, our functional assays revealed tremendous enrichment for clonogenicity in the c-Kit+ population. However, the scRNA-Seq analysis revealed significant heterogeneity within the c-Kit+ leukemic splenocytes, with many subpopulations expressing transcriptomes similar to myeloid precursors and possessing little clonogenicity. In contrast, expression of Il18rα (CD218a) significantly enriched clonogenic potential in our murine AML model. Manipulation of Il18r1 (Il18rα) by shRNA in Flt3ITD/ITD;Dnmt3afl/fl MxCre AML cells had no significant effect on colony forming ability. Therefore, Il18rα is a candidate surface marker of a rare leukemia stem cell population, but is likely not driving the functional clonogenic output. In addition to Il18rα and c-Kit, the most highly clonogenic subpopulation also accumulated c-Myc protein and expressed significantly higher levels of HSPC-like genes, whose associated DMR were reverted upon DNMT3A rescue. Therefore, this rare putative LSC population contained active oncogenic signaling downstream of mutant Dnmt3a and Flt3ITD, demonstrating the power of single cell analyses to resolve both biological and molecular complexity.

The correlation between reverted DMR and significantly diminished clonogenicity in our murine AML suggests that the methylome changes downstream of mutant DNMT3A in human AML are probably disease-driving and reversible. Cxxc5 is an exemplary candidate because it conforms to many criteria of a functionally important putative Dnmt3a target gene in AML; hypomethylated and overexpressed (HSPC-like) in murine and human AML, DMR reverted upon DNMT3A rescue, and knockdown of Cxxc5 (similar to DNMT3A rescue) significantly diminished AML colony forming ability. The CXXC-type zinc finger protein Cxxc5 is a pleiotropic factor that can interact with Dishevelled to inhibit Wnt/β-catenin signaling (52, 53), binds to CpG-rich DNA sequences to regulate gene expression (54), and is a biomarker and potential therapeutic target in leukemia (5557). The extracellular matrix protein Emilin2, also described as an inhibitor of Wnt signaling (58), is hypomethylated and overexpressed in AML, and is important for AML clonogenicity. Since both Cxxc5 and Emilin2 play a role in Wnt signaling and c-Myc is a target of the Wnt pathway, we monitored the level c-MycGFP and do not find that shRNA-knockdown of either Cxxc5 or Emilin2 disrupt c-Myc accumulation (data not shown). Though this does not preclude a possible role for either Cxxc5 or Emilin2 in Wnt signaling, more work needs to be done to fully understand the roles and therapeutic potential of Cxxc5 and Emilin2 in FLT3-ITD DNMT3A mutant AML.

Our data suggest that the functional implications of some cancer-specific epigenetic alternations (e.g. DNMT3A mutations) may extend beyond that of persisting clones from the pre-malignant state (59, 60) to a continuing role in AML disease pathogenesis and maintenance. We note recent progress in small molecule inhibitors therapeutically targeting proteins that create and/or maintain epigenetic modifications in cancer. Given the acutely rapid and spontaneous development of AML in the Flt3ITD/ITD;Dnmt3afl/fl MxCre mice, and the plasticity of DNA methylation within the resulting AML, we conclude that this new murine model is suitable for testing future therapeutic methods of manipulating Dnmt3a, which may provide new opportunities for clinical intervention in CN-AML.

Methods

Animals

Mice were bred and housed by Cincinnati Children’s Hospital Medical Center (CCHMC) Veterinary Services, and mouse manipulations were performed according to protocol reviewed and approved by the Children’s Hospital Research Foundation Institutional Animal Care and Use Committee (Protocol Number IACUC 2013-0090). c-MyceGFP (43), Flt3ITD (27), Dnmt3aflox (61), Mx1Cre (62), mice were maintained on the C57BL/6-Ly5.2 (CD45.2) background. CD45.1+ C57BL/6-Ly5.1 (JAX and CCHMC Comprehensive Mouse Core) transplant recipient mice were between 1–3 months of age and given 700 Rads 5 hours prior to tail vein injection of single cell suspensions.

Bone Marrow and Leukemic Splenocyte Isolation

Moribund mice were euthanized with carbon dioxide and cervical dislocation. Cytospin Buffer containing 10% FBS (HyClone), 2% BSA Fraction V (ThermoFisher Scientific), and Cell Dissociation Buffer (Gibco) in PBS was used for all cell isolations, washes, and cytospins under sterile conditions. Bone marrow cells were isolated from freshly sacrificed mice by crushing bones using a mortar and pestle. Splenocytes were isolated from freshly sacrificed mice and crushed between two glass slides. Red blood cells were lysed using Ammonium-Chloride-Potassium (ACK) buffer (Gibco), washed, and filtered to obtain single cells suspensions for downstream applications.

Histology and cytospins

Bone marrow, spleen, liver, and sternum here harvested from moribund Flt3ITD/ITD;Dnmt3afl/fl MxCre and age-matched wild-type or Flt3ITD/ITD;Dnmt3afl/fl controls. Sternum was decalcified then spleen, liver, and sternum were paraffin embedded and sections were stained with hematoxylin & eosin (H&E) (CCHMC pathology core) and cytospins were stained with Wright Giemsa using the Camco Stain Pak (Cambridge Diagnostic Products). Cytospins of bone marrow and spleen were performed both before and after red blood cell lysis. Photographs were taken on a Nikon Eclipse 80i microscope with a Nikon Digital Sight camera using NIS-Elements F4.30 software at a resolution of 2560 × 1920. Using Adobe Photoshop CS2, auto contrast was applied to H&E images, unsharp mask was used to improve image clarity, images were re-sized and set at a resolution of 300 pixels/inch, and variations was used to adjust color balance and brightness. Scale bar represents 30μm in each image. At least 3–5 mice per group were examined for histologic, morphologic, peripheral blood, and flow cytometric findings for acute myelomonocytic leukemia diagnosis.

Spectral Karyotyping Analyses

To characterize the cytogenetic pattern of AMLs from Flt3ITD/ITD;Dnmt3afl/fl MxCre mice, G-banding and SKY analysis was performed using the ASI SkyPaint assay for mouse chromosomes as described previously (63) on fresh or viably frozen bone marrow and spleen cells moribund mice with leukemia. Five independently derived AMLs (20 metaphases per case) from the Flt3ITD/ITD;Dnmt3afl/fl MxCre model were analyzed. The analysis of 20 cells excludes an abnormal clone of ≥14% with 95% confidence.

Flow cytometry and cell sorting

All flow cytometric staining, sorting, and analyses were performed by resuspending cells in 1X FACS Buffer (1% FBS, 0.01% NaN3 in DPBS). To isolated c-Kit+ leukemic stem/progenitor populations, freshly isolated single cells suspensions were incubated with CD117 MicroBeads and separated on an AutoMACS Pro separator (Miltenyi, San Diego, CA) according to manufacturer specifications.

To prepare for flow cytometric analysis of stem/progenitor populations, single cell suspensions were first stained with a cocktail of biotin-conjugated antibodies against markers of lineage positive cell types: anti-CD3 (clone 145-2C11, BioLegend, San Diego, CA), anti-CD4 (clone RM4-5, eBioscience, San Diego, CA), anti-CD8 (clone 53-6.7, Becton, Dickinson and Company, Franklin Lakes, NJ), anti-CD11b (clone M1/70, Becton, Dickinson and Company), anti-CD19 (clone 6D5, BioLegend), anti-Gr1 (clone RB6-8C5, BioLegend), anti-Ter119 (clone Ter-119, BioLegend) and anti-CD45R (clone RA3-6B2, BioLegend). Following two washes in 1X FACS Buffer, lineage stained cells were stained with: Streptavidin-conjugated APC-Cy7 (Becton, Dickinson and Company), PerCP-eFluor710-conjugated anti-CD16/32 (clone 93, eBioscience), APC-conjugated anti-CD117 (clone 2B8, Becton, Dickinson and Company), PE-Cy7-conjugated anti-Sca1 (clone D7, Becton, Dickinson and Company) and BV421-conjugated anti-CD34 (clone RAM34, Becton, Dickinson and Company). Alternatively, for analysis of L-GMP populations, a modified lineage cocktail was used, which excluded anti-CD11b and anti-Gr1 biotin-conjugated antibodies. Furthermore, lineage stained cells were incubated with the same antibodies described above but with BV605-conjugated anti-CD11b (clone M1/70, Becton, Dickinson and Company) and PE-Cy5-conjugated anti-Gr1 (clone RB6-8C5, eBioscience) added and BV650-conjugated anti-CD117 (clone RAM34, Becton, Dickinson and Company) substituted for the APC-conjugate. Antibodies were used at 1:100 dilutions, except for anti-CD34, which was used at 1:50 in a staining volume of 50μl/5x106 cells. All results reported for LK, GMP, and LSK populations exclude CD11b and Gr1 along with other linage markers indicated above either as biotin or fluorochrome conjugates.

To prepare for flow cytometric analysis of mature populations, single cell suspensions were incubated with FcγRII/III Block (clone 2.4G2, Becton, Dickinson and Company) and a combination of: Pacific Blue-conjugated anti-CD11b (cloneM1/70, BioLegend), PE-Cy7-conjugated anti-Gr1 (clone RB6-8C5, BioLegend), APC-conjugated anti-CD3 (clone 145-2C11, Becton, Dickinson and Company) or FITC-conjugated anti-CD3 (clone 145-2C11, Becton, Dickinson and Company), PE-conjugated anti-CD45R (clone RA3-6B2) or AlexaFluor700-conjugated anti-CD45R (clone RA3-6B2, BioLegend), PerCP-Cy5.5-conjugated anti-CXCR4 (clone L276F12, BioLegend) PE-conjugated anti-IL18R (clone P3TUNYA, eBioscience), and APC-conjugated anti-CD117 (clone RAM34, Becton, Dickinson and Company) or BV650-conjugated anti-CD117 (clone RAM34, Becton, Dickinson and Company). Antibodies were used at 1:100 dilutions in a staining volume of 50 μl for up to 5x106 cells.

Cell sorting was performed on a MoFloXDP (Beckman Coulter, Brea, CA) or BD FACSAria II with a 70μm or 100μm nozzle. Sorted populations of interest were collected in DPBS buffer containing 50% FBS (Hyclone). Flow cytometric analyses were performed on a FACS LSRII or LSR Fortessa (Becton, Dickinson and Company). Data were analyzed with FlowJo Software (TreeStar, Ashland, OR).

In preparation for flow cytometric analysis of phospho-STAT5 quantitation, one million freshly isolated c-Kit+ splenocytes were added to each well of a 96-well round bottom plate and centrifuged at 300xg at 4°C for 5 minutes. Cells were next fixed in 100μl of 4% methanol-free formaldehyde (Pierce) for 20 minutes at room temperature and then centrifuged as before. Cells were washed in 200 μl 1X FACS Buffer. Next, cells were permeabilized for 10 minutes at 4°C in 200μl of 90% methanol (Fisher) then washed twice. Cells were stained with PE-conjugated anti-phosphoY694-Stat5 antibody (clone 47/Stat5-pY694, Becton, Dickinson and Company) at a 1:100 dilution in 100μl of 1X FACS buffer for 1 hour on ice. Cells were washed twice prior to flow analysis.

AC220 treatment

Prior to in vitro treatment with AC220 (Ambit Biosciences), cryopreserved splenocytes were thawed and cultured overnight in StemSpan (Stem Cell Tech) supplemented with recombinant mouse IL3 (10 ng/ml), IL6 (20 ng/ml), and SCF (25 ng/ml) (Miltenyi). Cells were then replated at 1 million cells per ml with either 0.1% DMSO or 1nM AC220 and incubated for 5 hours at 37°C. Cells were then washed once in 1X FACS and stained with APC-conjugated anti-CD117 (clone RAM34, Becton, Dickinson and Company) at a 1:100 dilution in 100μl of 1X FACS buffer for 1 hour on ice. Cells were washed twice prior to flow analysis.

RNA Isolation, cDNA synthesis, and TaqMan Assay

RNA was extracted using TriZol or TriZol LS (Ambion) as previously described (64). Total RNA was converted into cDNA using the High Capacity cDNA Archive Kit (Life Technologies) for standard gene expression analyses. All gene expressions were quantified using TaqMan assays (Life Technologies) with the 2x Universal TaqMan Master Mix (Life Technologies) according to the manufacturers instructions on the StepOne Plus Instrument (Applied Biosystems, Inc.) using equal amounts of starting cDNA. Standard inventoried gene expression TaqMan assays recommended by the manufacturer (Life Technologies) for best coverage were purchased when available. For human gene expression assays: DNMT3A (Hs01027166_m1). For mouse gene expression assays: Dnmt3a (Mm00432881_m1), Emilin2 (Mm00467425_m1), Pim1 (Mm00435712_m1), Gata3 (Mm00484683_m1), Cxxc5 (Mm00505000_m1), Il18r1 (Mm00515178_m1). Gene expression data was analyzed using the comparative Ct method (2−ΔΔCt). Human SDHA (Hs00417200_m1) and mouse Sdha (Mm01352366_m1) served as loading controls.

Bulk RNA Sequencing Analyses

c-Kit+ splenocytes were harvested from three independent Flt3ITD/ITD;Dnmt3afl/fl MxCre or age-matched Flt3ITD/ITD;Dnmt3afl/fl mice. RBCs were lysed with ACK, and 5x106 leukemia cells were lysed with TriZol (Invitrogen). RNA quality and quantity were measured by Agilant Bioanalyzer at CCHMC Microarray Core. Then samples with a RIN>8.0 were processed by poly-A selection, stranded True-seq protocol and RNA library sequencing using paired-end 75bp reads at a depth of 40 million paired-reads per sample (Illumina HiSeq 2500) by the CCHMC Sequencing Core. Sorted LSK, CMP and GMP bulk RNA sequenced samples, obtained using the same library preparation and sequencing protocol were obtained from the gene expression omnibus dataset GSE70235. The fastq files were aligned to mouse genome Mm10 using BowTie2 and TopHat 2 (65, 66). Gene level RPKM (Reads Per Kilobase of transcript per Million mapped reads) were obtained using AltAnalyze 2.0.9 (44) to identify differentially expressed genes between Flt3ITD/ITD;Dnmt3afl/fl MxCre AML and Flt3ITD/ITD;Dnmt3afl/fl MPN using default parameters, including a moderated t-test with Benjamini-Hochberg multiple testing correction, raw p-value cut-off of 0.05, RPKM>1 and a differential expression cut-off of 2-fold. WT LSK samples were also displayed as normal baseline comparators. HOPACH (Hierarchical Ordered Partitioning And Collapsing Hybrid) clustering in AltAnalyze was used to identify clusters of gene expression in AML, MPN, and WT LSK.

Single Cell RNA Sequencing Analyses

Single CD117+ (c-Kit+) cells were prepared using the C1 Single-Cell Auto Prep System (Fluidigm, San Francisco, CA), according to the manufacturer’s instructions. In short, sorted cells were counted and resuspended at a concentration of 333,333 cells per 1 ml DPBS then loaded onto a primed C1 Single-Cell Auto Prep Integrated Fluidic Chip for mRNA-Seq (5–10 μm). After the fluidic step, cell separation was visually scored to identify wells containing single live cells. In two independent rounds, 52 and 56 cells were captured. Cells were lysed on the chip and reverse transcription was performed using the Clontech SMARTer® Kit with the mRNA Seq: RT + Amp (1771x) according to the manual. After the reverse transcriptase step, cDNAs were transferred to a 96-well plate and diluted with 6μl C1 DNA Dilution Reagent. cDNAs were quantified using the Quant-iT PicoGreen® dsDNA Assay Kit (Life Technologies, Grand Island, NY) and Agilent High Sensitivity DNA Kit (Agilent Technologies (Santa Clara, CA). Libraries for the first 48 visually validated cells from each of the two independent captures were prepared using the Nextera XT DNA Library Preparation Kit (Illumina Inc., Santa Clara, CA). In each single-cell library preparation, a total of 125pg cDNA was tagmented at 55°C for 20 minutes. Next, libraries were pooled and purified on the AMPure® bead-based magnetic separation Kit prior to a final quality control check using both the Qubit® dsDNA HS Assay Kit (Life Technologies, Grand Island, NY) and Agilent High Sensitivity DNA Kit. The majority of cDNA fragments were confirmed to be between 200–602 base pairs (bp), qualifying the libraries for sequencing. All 96 single-cell RNA-Seq libraries were sequenced on one HiSeq 2500 gel. Gene expression was quantified as transcripts per million (TPM) using RSEM (RNA-Seq by Expectation-Maximization) alignment and quantification (67). The predominant gene expression signatures were obtained from the ICGS (Iterative Clustering and Guide-gene Selection) module of AltAnalyze 2.0.9 (http://altanalyze.org/ICGS.html). For ICGS analysis, genes with a minimum TPM>1 and fold>4 in at least 3 cells were retained for analysis, with a minimum Pearson correlation cutoff of 0.4 and conservative exclusion of cell-cycle effects selected as the parameters. Associated cell population predictions were obtained the GO-Elite (45) module in AltAnalyze and verified based on prior literature. All associated expression plots display gene expression values as the median subtracted or un-subtracted log2 TPM values. The genes associated with DNMT3A rescued AML reverted DMRs are displayed in Violin plots comparing the expression of these genes in the original single cell RNA-Seq AML dataset across all clusters using the vioplot library in R.

Human AML specimens

Genomic DNA was isolated from bone marrow mononuclear cells from AML leukemia patients seen either at Erasmus Medical Center, Rotterdam or enrolled in the E1900 clinical trial from the Eastern Cooperative Oncology Group. Informed consent was obtained prior to sample banking. IRB approval for this study was obtained at the University of Michigan and Weill Cornell Medical Center. This study was performed in accordance to the declaration of Helsinki. All specimens analyzed age-matched consisted of >90% blasts. A total of 32 patients were included (Supplementary Table S7). Sixteen patients were FLT3-ITD/DNMT3A-wt while 13 patients were FLT3- ITD/DNMT3A-mut. All patients were wild-type for TET2, IDH1 and IDH2. Patients with FLT3-TKD were also excluded. Bone marrow from normal donors was obtained after IRB approval from the Johns Hopkins Sidney Kimmel Cancer Center Specimen Accessioning Core. Mononuclear cells were isolated by density centrifugation using Ficoll-Paque PLUS (GE Healthcare) and then labeled for CD34 positive selection using the CD34 MicroBead kit (Miltenyi Biotec) and frozen viable in FBS + 10% DMSO. Upon thawing, CD34+ cells were labeled using the Aldefluor kit (Stem Cell Technologies), CD34-BV421 or CD34-PE-Cy7, CD45-PE, CD38-APC (BD Biosciences) and 7-Aminoactinomycin D (7-AAD; Life Technologies). Hematopoietic stem cells (HSC) were identified as ALDHhighCD34+CD38CD45+, and sorted on a Beckman Coulter MoFlo in the Cell Sorting Core Facility at the Johns Hopkins Bloomberg School of Public Health. Gene expression data for these AML patients was previously published (68) (GEO accession number: GSE6891).

DNA Isolation and Dnmt3a Genotyping

DNA was isolated from c-Kit+ splenocytes (ERRBS) or single cell suspensions from individual colonies grown for 7 days in methylcellulose (genotyping) using the Qiagen Puregene kit according to manufacturer’s instructions. For small cell numbers glycogen was added to help precipitate DNA. ERRBS data for wild-type murine LSK cells were previously published (69) (GEO accession number: GSE57114). For genotyping the Dnmt3afl/fl-F 5′-TGT GGC ATC TCA GGG TGA T -3′, Dnmt3afl/fl-R 5′-CTC AGG CCC TCT AGG CAA G -3′, and Dnmt3aKO-R 5′-GAA GCA GCA GTC CCA GGT AG -3′ were used.

DNA methylation by ERRBS

Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) libraries were generated from 25ng of genomic DNA as previously described (37) for the human specimens, and sequenced on a HiSeq-2000 sequencer. Murine libraries were prepared in the same way but using the indexed adaptors and sequenced with two libraries per lane on a HiSeq-2500 sequencer (70). Reads were aligned against a bisulfite-converted version of the hg19 or mm9 builds of the genome using Bowtie followed by methylation percentage calling using Bismark (71). Conversion rates were >99.8 for all samples. Only genomic regions with coverage between 10X and 500X were included for downstream data analysis.

DNA methylation data analysis

ERRBS data analysis was performed using R statistical software version 3.0.3 and the MethylSig 0.1.3 package (72) for differential methylation calling using the beta binomial method. The MethylKit 0.9.2.5 package (73) was used for downstream gene-, CpG-, and enhancer region annotation analysis and, chromosome ideogram representations. Differentially methylated regions (DMR) were identified by first summarizing the methylation status of genomic regions into 25-bp tiles and then identifying regions with absolute methylation difference ≥25% and false discovery rate (FDR) <10%. DMRs were annotated to GENCODE genes using a custom script with the following criteria: (i) DMRs overlapping with a gene were annotated to that gene, (ii) intergenic DMRs were annotated to all neighboring genes within a 50-kb window, and (iii) if no gene was detected within a 50-kb window, then the DMR was annotated to the nearest transcription start site (TSS) (74). Statistical significance for comparisons between all measured CpGs (all tiles) and DMRs at genomic, CpG, and enhancers regions was calculated using upper and lower tail binomial tests, percent of total DMR and p-values are reported.

Virus Production and Transduction

For virus production, Lenti-X 293T cells (Clonetech) were seeded at 4x106 cells/10cm dish in standard growth medium DMEM (ThermoFisher Scientific) with 10% heat-inactivated FBS (Atlanta Biologics), Penicillin-Streptomycin (Pen-Strep) (ThermoFisher Scientific), and L-Glutamine (ThermoFisher Scientific). The following day, cells were replenished with fresh Opti-MEM GlutaMAX media (ThermoFisher Scientific) supplemented with 10% heat-inactivated FBS (Atlanta Biologics) at least two hours prior to transfection. MSCV-DNMT3A-GFP and empty vector MSCV-GFP retroviral plasmids were a kind gift from Dr. Grant Challen (Washington University, St. Louis). For retroviruses, 10μg of DNMT3A and empty vector (EV) retroviral vectors were co-transfected with 10μg retroviral packaging and envelope plasmid pCL-Eco (75) into Lenti-X 293T cells using Transit-LT1 (Mirus) according to manufacturer instructions. Virus-containing supernatant was collected 48 and 72 hours post-transfection, and either used immediately or frozen at −80°C in aliquots. For lentiviruses, gene targeting shRNA or non-targeting control lentiviral vectors were co-transfected with lentiviral packaging plasmid Δ8.9 (76) and envelope plasmid VsVg (77) using TransIT-LT1 (Mirus Bio) into Lenti-X 293T cells (Clonetech). Virus-containing supernatant was collected 48 and 72 hours post-transfection, and either used immediately or frozen at −80°C in aliquots. Two independent shRNA against Cxxc5 (TRCN0000143369, TRCN0000219528), Emilin2 (TRCN0000257587, TRCN0000246910), Il18r1 (TRCN0000068103, TRCN0000068105) were purchased from the CCHMC Lenti shRNA viral Core or Sigma.

Transduction of c-Kit+ leukemic splenocytes with retroviruses was performed using the spinfection method. Tissue culture plates were pre-coated with 5μg/cm2 recombinant Retronectin (Takara) overnight at 4°C, then blocked with 2% BSA (ThermoFisher Scientific) in PBS (ThermoFisher Scientific) for 30 minutes at RT. Unconcentrated fresh or freeze/thawed retroviral supernatant was spun onto the retronectin at 1,000g for 2 hours at 4°C. Supernatant was removed and a second spin was performed with additional retrovirus supernatant. Viral supernatant was removed and 1x106 c-Kit+ AML cells in 0.5ml standard growth medium were spun onto the retrovirus/retronectin coated wells at 1,000g for 10 minutes at RT. Then plates were removed overnight to 37°C 5% CO2. The spinfection protocol was repeated again the next day. 48 hours post-transduction cells were sorted for GFP+ expression, and 2,000 cells were plated in methylcellulose M3434 supplemented in triplicate. After 7 days colonies were enumerated, single cell suspensions were made, and 2,000 cells were serially replated in triplicate. Extra cells not replated were mixed with cell lysis solution (Qiagen) (for DNA isolation) or lysed in TriZol (for RNA isolation).

For lentiviral transduction, c-Kit+ leukemic splenocytes freshly harvested from moribund AML mice were cultured overnight in standard growth medium RPMI 1640 (Thermo Fisher Scientific) containing 20% FBS (HyClone) supplemented with mouse recombinant SCF (60ng/ml), IL6 (20ng/ml), and IL3 (20ng/ml) (Miltenyi Biotech). 1x106 c-Kit+ leukemia cells in 400μl standard growth medium containing FBS were mixed with 100μl lentiviral supernatant. Cytokines (60ng/ml SCF, 20ng/ml IL6 and IL3) and polybrene (10μg/ml, Sigma) were added to the final volume. Cells were incubated with virus for 6 hours at 37°C with 5% CO2, then virus supernatant was replaced with standard growth medium containing FBS and cytokines. Cells were incubated for 48 hours, then for an additional 48 hours with 1.5μg/ml puromycin in fresh growth medium. 4,000 puromycin-selected cells were plated in triplicate in methylcellulose M3434 (Stem Cell Technologies) for colony forming assay. Colonies were enumerated after 7 days. Extra cells (not plated for colony assays) were lysed in TriZol (for RNA isolation).

GSEA Analyses

Log2 RNA-Seq expression derived from c-Kit+ leukemic splenocytes from three independent Flt3ITD/ITD;Dnmt3afl/fl MxCre AML mice and three Flt3ITD/ITD;Dnmt3afl/fl MPN mice was imported into GSEA v.2.2.0 (41, 42). A fold-change ranked gene list was created comparing the AML vs. MPN using the Diff_of_Classes ranking metric and genes were sorted by descending order real mode. GSEA analyses were run using 1,000 gene_set permutations of gene sets in the website gene matrix database with the weighted enrichment statistic. Results are reported for the most highly positively correlated and negatively correlated hematopoietic-related gene sets with leading edge analyses.

Genes upregulated in Flt3ITD/ITD;Dnmt3afl/fl MxCre AML versus murine MPN were tested for enrichment in human FLT3-ITD/DNMT3A-mut AML in GSEA as described above using a pre-ranked fold change gene expression list in descending order comparing FLT3-ITD/DNMT3A-mut AML versus FLT3-ITD/DNMT3A-wt AML. Genes upregulated in Flt3ITD/ITD;Dnmt3afl/fl MxCre AML both legacy and unique groups were used as the testing gene set. A gene set of nearest gene neighbors to hypo-methylated regions in Flt3ITD/ITD;Dnmt3afl/fl MxCre AML versus MPN was tested for enrichment in against a fold-changed ranked gene expression list from RNA-Seq. in Flt3ITD/ITD;Dnmt3afl/fl MxCre AML versus Flt3ITD/ITD;Dnmt3afl/fl MPN as described above.

In Vitro Colony Forming Assays

Equal numbers of cells were mixed with methylcellulose M3434 complete with cytokines SCF, IL6, IL3, and EPO (Stem Cell Technologies) according to manufacturers instructions in triplicate. 2,000 c-Kit+ or c-Kit cells where indicated were seeded per plate. On day 7 immortalized/transformed colonies >500 cells were enumerated. Colonies were dissociated with PBS, single cell suspensions were counted and either equal numbers of cells were replated, or used in other indicated downstream applications. Each group was performed in triplicate and results were repeated in independent experiments at least twice.

Statistical Analyses

The absolute cell numbers of mouse normal, MPN, and AML total bone marrow and spleen counts were converted to natural log prior to statistical evaluation to normalize the variance between groups. Significant differences between normal, MPN, and AML groups for each population were examined by two-way ANOVA with Tukey’s multiple comparisons correction. The average cell counts are shown for at least 3 mice/group. The percentage of total bone marrow and spleen LK, GMP, and LSK and spleen CD11b+Gr1+CD3, CD11b+Gr1CD3, and CD11b+Gr1B220 populations are shown for at least 3 mice/group. Significant differences between normal, MPN, and AML groups for each population were examined by two-way ANOVA with Tukey’s multiple comparisons correction. Descriptions of statistical analyses used can also be found in the figure legends.

Significance

Dnmt3a haploinsufficiency results in reversible epigenetic alterations that transform Flt3-ITD mutant myeloproliferative neoplasm into acute myeloid leukemia.

Supplementary Material

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6

7

Acknowledgments

The authors would like to acknowledge the Cornell University Epigenetics Core, the University of Michigan Epigenomics Core facility, the Cincinnati Children’s Hospital Pathology, Flow Cytometry, Gene Expression, and Comprehensive Mouse Cores. We also thank Dr. Peter Valk (Erasmus University Medical Center, Netherlands) for human AML samples and gene expression data. c-MyceGFP knock-in mice were a kind gift from Barry Sleckman (Washington University, St. Louis). Dnmt3afl/fl mice were a kind gift from David Hildeman (CCHMC). DNMT3A retroviral constructs were a kind gift from Grant Challen (Washington University, St. Louis).

Grant Support

The research was supported by Ladies Auxiliary to the Veterans of Foreign Wars (LAVFW) Postdoctoral Fellowship (to S.E.M.), CancerFree KIDS (Loveland, Ohio), University of Cincinnati training grant NIEHS T32ES007250 (to S.E.M.), CCTST Just-InTime Core Grant Program (University of Cincinnati and CCHMC), Leukemia and Lymphoma Society Translational Research Program Award, NHLBI Progenitor Cell Biology Consortium U01HL099997 (to N.S.) and NIH R01CA159845 and R21CA186945 (to H.L.G.).

Footnotes

Conflict of Interest Statement

The authors have no conflicts to disclose.

Accession Numbers

RNA sequencing (GEO accession GSE77849) and ERRBS data (GEO accession GSE77026) can be accessed in the Gene Expression Omnibus.

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