Cooperation of Dnmt3a R878H with Nras G12D promotes leukemogenesis in knock-in mice: a pilot study
DNMT3A R882H, a frequent mutation in acute myeloid leukemia (AML), plays a critical role in malignant hematopoiesis. Recent findings suggest that DNMT3A mutant acts as a founder mutation and requires additional genetic events to induce full-blown AML. Here, we investigated the cooperation of mutant DNMT3A and NRAS in leukemogenesis by generating a double knock-in (DKI) mouse model harboring both Dnmt3a R878H and Nras G12D mutations.
DKI mice with both Dnmt3a R878H and Nras G12D mutations were generated by crossing Dnmt3a R878H knock-in (KI) mice and Nras G12D KI mice. Routine blood test, flow cytometry analysis and morphological analysis were performed to determine disease phenotype. RNA-sequencing (RNA-seq), RT-PCR and Western blot were carried out to reveal the molecular mechanism.
The DKI mice developed a more aggressive AML with a significantly shortened lifespan and higher percentage of blast cells compared with KI mice expressing Dnmt3a or Nras mutation alone. RNA-seq analysis showed that Dnmt3a and Nras mutations collaboratively caused abnormal expression of a series of genes related to differentiation arrest and growth advantage. Myc transcription factor and its target genes related to proliferation and apoptosis were up-regulated, thus contributing to promote the process of leukemogenesis.
This study showed that cooperation of DNMT3A mutation and NRAS mutation could promote the onset of AML by synergistically disturbing the transcriptional profiling with Myc pathway involvement in DKI mice.
KeywordsAcute myeloid leukemia DNMT3A mutation Nras G12D Myc activation
Acute myeloid leukemia
Bone marrow transplantation
Common myeloid progenitors
DNA methytransferase 3A
fetal bovine serum
Hematopoietic stem cells
Lin− Sca-1+ c-Kit+ cells
Red blood cells
White blood cells
DNA methytransferase 3A (DNMT3A), a member of DNA methytransferases family, is responsible for de novo DNA methylation, which is essential for genome regulation and development . DNMT3A mutations have been identified in various hematologic malignancies, with frequencies of 20–25% in AML [2, 3, 4, 5, 6]. The hotspot mutation of DNMT3A in AML occurs at the residue Arginine 882 (R882) [2, 7]. Dnmt3a knock-out mice showed increased self-renewal and impaired differentiation of Hematopoietic stem cells (HSCs) [8, 9, 10]. Mouse models established through retroviral transduction system showed that DNMT3A R882H alone did not develop frank AML, but were susceptive to AML development upon acquisition of additional genetic mutations [11, 12]. Dnmt3a R878H which is homologous with human DNMT3A R882H, only induced moderate AML with an average of 20% immature cells in the bone marrow (BM) and a relatively long latency in the conditional knock-in mice model . DNMT3A mutations were proved to play a key role in clonal hematopoiesis at premalignant stages [14, 15], whereas activated signaling genes including RAS and FLT3 mutations occur in the subsequent process of malignant development . Large scale sequencing of specimens from AML patients has discovered that DNMT3A mutations often coexist with other gene abnormalities, such as FLT3, IDH1/2, NPM1 and RAS [2, 7, 17]. These findings suggest that abnormal DNMT3A acts as a founder mutation and requires additional genetic events to induce an aggressive full-blown AML.
RAS is mutated in ~ 25% of human cancers including AML and other myeloid malignancies [18, 19]. Mutations in NRAS have been identified in AML and coexist with DNMT3A mutations in a portion of AML patients [20, 21]. Mouse models showed that NRAS mutation alone was not sufficient to cause AML [22, 23]. Loss of Dnmt3a and endogenous Kras G12D cooperated to promote myeloid leukemogenesis in mice . Besides, a previous report showed co-expression of DNMT3A R882H and NRAS G12D could induce mouse AML by using a retroviral transduction system, in which the expression of mutant DNMT3A and mutant NRAS were driven by a retroviral promoter instead of the endogenous promoter/enhancer [12, 25].. However, the cooperation of DNMT3A mutation with NRAS mutation under the control of endogenous promoters in inducing AML in mice which mimics human leukemic features and the underlying mechanism remains elusive. In this work, we report that Dnmt3a R878H cooperates with Nras G12D to develop frank AML by establishing a DKI mice model.
Generation of DKI mice
All mouse experiments were performed according to the guide of laboratory animal care and use standards, and were approved by the animal use committee of Shanghai Jiao Tong University. And all animals were maintained with sterilized water and food in the specific pathogen free circumstance in Research Center for Experimental Medicine at Rui Jin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Mx1-Cre; Dnmt3a R878H KI C57 mouse model was established as described in our previous work . Mx1-Cre; Nras G12D KI C57 mice were generously provided by Ren Lab from Shanghai Institute of Hematology. The Mx1-Cre; Dnmt3aR878H/+ KI mice were crossed with the Mx1-Cre; Nras G12D KI mice to obtain DKI mice harboring both Dnmt3a R878H and Nras G12D mutations. Cre expression was induced through intraperitoneal injection of 250 μg Polyinosinic-polycytidylic acid (pIpC) every other day for two times at 4 weeks old. The mice were monitored for leukemia development and sacrificed for phenotypic analysis 4 months after pIpC injection. The mice were sacrificed after the study. The method of euthanasia used to sacrifice the mice was cervical dislocation.
Flow cytometric analysis
Peripheral blood (PB) was obtained from the tail vein of mice, red blood cells (RBCs) were lysed by RBC Lysis Buffer prior to staining. BM cells were flushed out from the tibias and femurs, and suspended in PBS buffer with 2% fetal bovine serum (FBS). The spleen cells were suspended as a single cell suspension in PBS buffer with 2% FBS. Cells were washed and resuspended in PBS buffer containing 1% FBS and subsequently stained with fluorochrome-conjugated antibodies (Biolegend) as following: PE anti-mouse Gr-1, APC anti-mouse Mac-1, BV421 anti-mouse B220, FITC anti-mouse CD3, FITC anti-mouse Lineage, PE anti-mouse Sca-1, APC anti-mouse c-Kit, APC-Cy7 anti-mouse CD48, BV421 anti-mouse CD150, PE-Cy7 anti-mouse Sca-1, BV786 anti-mouse c-Kit, APC anti-mouse CD16/32, PE anti-mouse CD34. Flow cytometry was performed on LSRFortessa (BD), and data were analyzed by using FlowJo software (Tree Star, Ashland, OR).
Bone marrow transplantation (BMT)
BM cells were isolated from the tibias and femurs of diseased DKI mice and mice at the same age of the other groups including Dnmt3aR878H/+, NrasG12D/+ and WT mice (CD45.2+), respectively. BMT was then performed by injecting 4 × 105 BM cells suspended in PBS into the tail vein of sublethally irradiated (350 cGy) recipient mice (CD45.1+) at 2–3 months old.
All the BM cells isolated from the various genotype mice were suspended in PBS buffer containing 1% FBS and subsequently stained with PE-conjugated Gr-1 antibody for half an hour. The cells were then washed and resuspended in 1–2 ml PBS buffer with 2% FBS. Gr-1+ cells were sorted by FACS ArialII (BD).
RNA was extracted from Gr-1+ cells using TRIzol-isopropanol precipitation. The quality of the RNA was checked using Nano drop, Qubit and Agilent 2100 Bioanalyzer. The mRNA of the qualified sample was enriched with mRNA Capture Beads, and then the fragmentation of mRNA was realized by the action of high temperature and metal ions. Using mRNA as template, a single chain cDNA was synthesized by six base random primers, followed by two strand cDNA synthesis reaction, then VAHTSTM DNA Clean Beads was used to purify double chain cDNA. The purified double strand cDNA was first repaired (poly A was added and sequenced), and VAHTSTM DNA Clean Beads was used to resize the fragment size. Finally, PCR amplification was carried out and the PCR products were purified by VAHTSTM DNA Clean Beads. The obtained library was then checked using Agilent High Sensitivity DNA Reagent with a 2100 Bioanalyzer, and 200-bp paired-end sequencing was carried out on an Illumina HiSeq.
Quantitative real-time RT-PCR
The cDNA was synthesized used M-MLV reverse transcriptase (Invitrogen). RT-PCR was performed as described using reagents according to instructions of the manufacturer (Hieff TM qPCR SYBR® Green Master Mix; Yeasen). Reactions were performed on ABI PRISM 7500 Fast Real-Time PCR System or Applied Biosystems ViiA™ 7 Real-Time PCR System. Data were analyzed using formula 2−ΔΔCt.
Western blot analysis
Total BM cells were lysed by RIPA Lysis Buffer (Beyotime). Protein assay was performed on Tecan infinite 200 Microplate Reader by BCA Protein Assay Kit (Beyotime). Antibodies were purchased from Cell Signaling Technology (anti-β-actin) and Abcam (anti-cMyc, anti-p62-cMyc).
Kaplan-Meier survival analysis was performed and survival differences between groups were assessed with the Log-rank test, assuming significance at P < 0.05. Unpaired 2-tailed Student’s t-test was used to determine the significance between two data sets, assuming significance at P < 0.05.
Dnmt3a R878H cooperates with Nras G12D to shorten the lifespan of DKI mice
Dnmt3a R878H cooperates with Nras G12D to induce a full-blown AML
DKI mice develop more aggressive AML than Dnmt3aR878H/+ mice
Leukemic cells from DKI mice show significant advantage of proliferation
Cooperation of Dnmt3a R878H and Nras G12D causes transcriptional alteration
Cooperation of Dnmt3a R878H and Nras G12D activates Myc pathway
As one of common epigenetic alterations in AML, DNMT3A mutation has been demonstrated to play an important role in the pathogenesis of leukemia. Many studies have suggested that mutant DNMT3A requires additional gene mutations to cause full-blown AML. In this regard, a previous report showed the cooperation of DNMT3A mutation and RAS mutation in leukemogenesis by using retroviral transduced mouse model . However, the conditional KI approach is better than retroviral transduction system to recapitulate human leukemic features in mice. In this study, we created the first conditional DKI mouse model expressing Dnmt3a R878H and Nras G12D under the control of endogenous promoters by crossing Dnmt3aR878H/+ KI mice and NrasG12D/+ KI mice. By generating the conditional DKI mouse model, we discovered that cooperation of Dnmt3a R878H and Nras G12D could lead to a much earlier onset and more severe AML characterized by significantly increased WBCs, elevated immature cells, splenomegaly and shortened survival time, compared with Dnmt3aR878H/+ or NrasG12D/+ KI mice. In this pilot study, preliminary data derived from 10 mice per group were statistically analyzed.
Dnmt3aR878H/+ mice have been reported to develop moderate AML with considerably increased hematopoietic stem and progenitor cells especially LSK cells which were demonstrated to harbor leukemia-initiating cells . Here, we showed that the LSK compartment was significantly enlarged in DKI AML mice compared with the diseased Dnmt3aR878H/+ mice, suggesting that DNMT3A mutation with acquisition of additional genetic abnormality such as NRAS mutation could significantly promote the leukemogenic transformation and proliferation of hematopoietic cells.
We investigated the mechanism underlying the leukemic phenotype induced by the cooperation of Dnmt3a R878H and Nras G12D. DKI mice showed much more up-regulated genes contributing to positive regulation of growth, negative regulation of differentiation and negative regulation of apoptosis process than Dnmt3aR878H/+ KI mice and NrasG12D/+ KI mice. Interestingly, the genes associated with phosphorylation and protein activation cascade were also up-regulated in DKI mice, indicating abnormal phosphorylation and protein activation caused by the cooperation of Dnmt3a and Nras mutation. We found that Dnmt3a mutation together with Nras mutation could not only increase the expression of Myc oncogene and Myc target genes, but also could induce the s62 phosphorylation of cMyc which has been reported to play a key role in increasing the protein stability and activating the Myc oncogenic signature [44, 45]. Therefore, the activation of Myc pathway may contribute to the pathogenesis of full-blown AML caused by the cooperation of Dnmt3a mutation and Nras mutation.
In this work, we generated a novel DKI mouse model to investigate the function and mechanism of Dnmt3a mutation under the presence of Nras mutation in inducing a full-blown AML. We discovered that Dnmt3a mutation and Nras mutation could cooperate to induce a much more severe AML than Dnmt3a or Nras mutation alone. In the mechanism study, we revealed that DKI mice showed significantly altered gene expression patterns with involvement of Myc pathway activation, indicating a potential therapeutic target in DNMT3A mutation-related leukemia which needs to be further investigated.
In conclusion, we discovered that cooperation of Dnmt3a mutation with Nras mutation could synergistically induce AML under the control of endogenous promoter/enhancer in mice, and activation of Myc pathway is one of the key players in the disease mechanism. Our study thus provides a unique mouse model that recapitulates many aspects of human AML and a potential therapeutic target for DNMT3A mutation-related leukemia.
We thank Dr. Zhu Chen and Saijuan Chen for constructive discussions, and Hui Sun for technical assistance of biological information.
XS, JM and YW designed the research. XS, YY, SS, SW, WZ, LP, TH and RZ performed the research. XS, YY, RR, and YW analyzed the data. XS and YW wrote the paper. YW and JM conceived and supervised the project. All authors read and approved the final manuscript.
This work was supported by the National Natural Science Foundation of China (81570151, 81770182), Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant (20152507), Shanghai Jiao Tong University Tang Scholar Program, and SMC-Morningstar Young Scholars Program. The funders had no role in the design of the study, collection, analysis, interpretation of data or in writing the manuscript.
Ethics approval and consent to participate
All mouse experiments were performed according to the guide of laboratory animal care and use standards, and were approved by the animal use committee of Shanghai Jiao Tong University.
Consent for publication
The authors declare that they have no competing interests.
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