DNMT3A mutation leads to leukemic extramedullary infiltration mediated by TWIST1
DNMT3A mutations are frequently discovered in acute myeloid leukemia (AML), associated with poor outcome. Recently, a relapse case report of AML extramedullary disease has showed that AML cells harboring DNMT3A variation were detected in the cerebral spinal fluid. However, whether a causal relationship exists between DNMT3A mutation (D3Amut) and extramedullary infiltration (EMI) is unclear.
We took advantage of DNMT3A (R882C) mutation-carrying AML cell strain, that is, OCI-AML3, assessing its migration ability in vitro and in vivo. By RNA interfering technology and a xenograft mouse model, we evaluated the effect of DNMT3A mutation on cell mobility and explored the possible mechanism.
OCI-AML3 displayed extraordinary migration ability in vitro and infiltrated into meninges of NOD/SCID mice after intravenous transfusion. We found that this leukemic migration or infiltration capacity was significantly compromised by the knockdown of DNMT3A mutant. Notably, TWIST1, a critical inducer of epithelial–mesenchymal transition, which underlies the metastasis of carcinomas, was highly expressed in association with R882 mutations. Abrogation of TWIST1 in DNMT3A mutated cells considerably weakened their mobility or infiltration.
Our results demonstrate that D3Amut in OCI-AML3 strain enhances leukemic aggressiveness by promoting EMI process, which is partially through upregulating TWIST1.
KeywordsDNMT3A mutation Acute myeloid leukemia Extramedullary infiltration TWIST1
Acute myeloid leukemia
Central nervous system
Positron emission tomography-computed tomography
Acute myeloid leukemia (AML) is a group of subtypes that share common features with various manifestations. Extramedullary infiltration (EMI) is a specific symptom of bone marrow diseases, such as myeloid sarcoma, leukemia cutis, and central nervous system (CNS) leukemia. The prognosis of extramedullary event is controversial but generally considered an advanced malignancy and indicator of poor outcome [1, 2]. The mortality rate caused by EMI, to some extent, is reduced by the means of standard systemic chemotherapy combined with local treatment, such as intrathecal injection and skin radiation . However, extramedullary relapse after chemotherapy, even hematopoietic stem cell transplantation, is still common [4, 5].
Several lines of clinical analyses demonstrated that the patients with abnormal karyotypes, such as t (8; 21), inv (16), and 11q23 translocations, tend to have extramedullary diseases . With regard to immunophenotype, CD56-positive leukemic cells are prone to infiltrate . Additionally, a family of matrix metalloproteinases (MMPs) is considered to facilitate cell invasion into soft tissues and CNS [7, 8, 9]. This evidence confirms that molecular markers are useful to predict leukemic progressive invasiveness.
Recently, a case report on an AML-M2 patient relapsed with CNS leukemia after achieving complete remission (CR) has attracted attention. Although no DNMT3A mutation (D3Amut) is detected in the bone marrow and her buccal mucosal cells at diagnosis, deletion of exon 18 in DNMT3A is observed in the cerebral spinal fluid (CSF) on relapse stage . However, the mechanism on how the chemo-resistant subclone with D3Amut could emerge in CNS remains unknown.
Mutated DNMT3A is highly relevant to higher WBC counts, older age, and shorter survival in AML with mutations compared with those with wild-type (WT) DNMT3A [11, 12]. Mutated DNMT3A occurs in hematopoietic stem cells and is considered a driver mutation in initiating leukemia . D3Amut is relatively obstinate. It can persist in cases with morphologically CR  and be closely associated with disease relapse or progression [15, 16]. Interestingly, this mutation has been frequently identified in myelomonocytic and monoblastic phenotypes of AML (AML-M4/M5) . With these two subtypes, patients are more likely to have EMI presentation [2, 17]. Nevertheless, whether D3Amut takes part in EMI process is unclear.
In the present study, D3Amut could promote cell migration. OCI-AML3, a leukemia cell line harboring the hotspot DNMT3A R882C mutation , could proliferate in NOD/SCID mice and induce paralysis and finally death. Paralysis symptom was mentioned in a previous study . Our investigation demonstrated that this particular symptom is caused by murine CNS leukemia, which could be attributed to the cells bearing D3Amut. Intriguingly, an epithelial–mesenchymal transition (EMT) inducer, TWIST1, is activated upon D3Amut and could facilitate aberrant leukemic cell migration.
Leukemic cell lines
Human AML cell lines (OCI-AML3, Kasumi-1, NB4, THP-1, and U937) were all suspended and cultured in RPMI-1640 medium (Invitrogen, Grand Island, USA) with 10 % FBS (Invitrogen, Grand Island, USA). OCI-AML3 strain was kindly provided by Dr. Lan Wang (Shanghai Institutes for Biological Sciences, China). The four other cell lines were obtained from Shanghai Institute of Hematology. Logarithmically growing cells were used for the experiments.
Primary AML blasts
Total bone marrow cells were collected from diagnosed AML patients. These fresh cells were immediately purified via density gradient centrifugation using Ficoll. Leukemia blasts were harvested in the mononuclear layer for experiments or storage. All patients provided written informed consent for the use of their AML samples under a protocol approved by the ethics committee of Shanghai Institute of Hematology. Human primary AML samples were obtained in accordance with the ethical guidelines established by Shanghai Institute of Hematology.
AML mouse model
Human AML cell strains OCI-AML3, U937, and THP-1 with or without exogenous plasmids transduction were prepared in about (1–10) × 106 number. Cells were injected into lethally irradiated 8-week-old NOD/SCID mice through tail veins. Around 1 month post xenografting or at the time of paralysis, leukemic cells in murine peripheral blood, bone marrow, spleen, or brain were examined. All animal experiments were carried out in accordance with the approved guidelines provided by the Laboratory Animal Resource Center of Shanghai Jiao Tong University School of Medicine.
The procedures of siRNA transfection and lentivirus-mediated shRNA infection were described previously . The sequences of DNMT3A siRNA oligomers and shRNA primers were according to previous study . The sequences of human TWIST1 siRNA oligomers were as follows:
The primers for human TWIST1 shRNA were as follows:
Real-time quantitative PCR
cDNA templates were prepared after RNA extraction and reverse transcription. Amplification was performed on a real-time PCR system (Applied Biosystems 7500, USA). The whole procedure was according to the manual of SYBR® Premix Ex Taq™ kit (Takara RR420A, Japan). Relative expression was calculated using the formula of 1/2△△Ct.
Transwell® Permeable Supports (Costar 3421, Corning, USA) was prepared to test cell migration. The assay was carried out following the protocol provided by the manufacturer.
About 6-cm diameter dish (Falcon, Bedford, USA) was fully grown with adherent cells, and DMEM (Invitrogen, Grand Island, USA) medium with 10 % FBS was replaced with pure DMEM medium. Cells were starved for 16 h in an incubator at 37 °C and 5 % CO2. Subsequently, the monolayer cells in the middle of the dish were scratched using a sterile tip, and the dish was continuously incubated for 12 h. Finally, cell number in the wound was observed using a microscope (Nikon, TS100, NY, USA).
Flow cytometry and cell sorting
Immunophenotyping assays were all analyzed on LSRII Flow Cytometer (Franklin Lakes, NJ, USA). Flow data were further analyzed by FlowJo software (TreeStar, Ashland, OR, USA). Antibodies were as follows: human CD44-APC and human CD45-PE (BD Pharmingen™, NJ, USA). Leukemic cells carrying green fluorescence proteins (GFPs) or red fluorescence proteins were detected and selected by MoFlo flow-sorter (Beckman coulter, Fullerton, CA, USA).
SDS-PAGE gels were prepared depending on protein size. Electrophoresis and transmembrane were carried out on a protein electrophoresis and blotting system (Bio-Rad, Hercules, CA, USA). The signals were visualized using a chemiluminescence detector (LAS-4000, FUJIFILM). The antibodies used in this study were DNMT3A, SNAIL, Flag, GAPDH (Cell Signaling, Danvers, USA), TWIST1, VIMENTIN (Santa Cruz, Santa Cruz, USA), and β-actin (Sigma, St. Louis, MO, USA).
Bone marrow cytospins and sections were subject to Wright–Giemsa staining for morphological analyses. Leukemic cells with Wright–Giemsa staining are large and with round shape, pale blue cytoplasm, and pink nucleus.
Immunohistochemical staining and immunofluorescence
Murine brain sections and spinal cord slices were prepared for HE staining and human CD44 (Santa Cruz) immunohistochemical staining. Exogenous cells labeled by hCD44 in CNS were surrounded with brownish red color. Murine brain or BM slices were observed under Leica TCS SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) after incubation with GFP (Cell Signaling) or human CD44 antibody.
Around 1 month post xenografting, paralyzed mice transplanted with OCI-AML3 strains were used for PET-CT scanning (Siemense, Inveon PET-CT, USA) after injecting fluorodeoxyglucose into mice bodies via tail veins. Except for the brain, heart, and bladder, the sites presented in highlight indicated a concentration of leukemic cells caused by their active metabolism.
Human AML cells carrying luciferase reporter were transplanted into NOD/SCID mice. Luciferase substrate was injected into living animals before imaging. In vivo imaging system (Xenogen IVIS Spectrum, PerkinElmer) was used for catching the fluorescence from the whole body.
DNMT3A R882 mutation enhances cell migration in vitro
To further assess the effect of D3Amut on cell migration, we overexpressed the WT and mutant DNMT3A in U937, which exhibited low migration capacity (Fig. 1a, c). Repeated independent transwell assays showed that the numbers of migrated U937 cells stably expressing DNMT3A R882C were higher than those without D3Amut (p < 0.001) (Fig. 1c). In addition to leukemia cells in suspension culture, the effect of D3Amut on migration behavior in adherent cells was also investigated. To eliminate the possible effect of endogenous DNMT3A protein, we tested a series of solid tumor cell lines and found little DNMT3A expression in MDA-MB-231, a breast cancer cell line (Additional file 1: Figure S1d). Scratch-wound experiment was conducted, and cell motility was efficiently increased in MDA-MB-231 cells expressing D3Amut but not in those expressing WT DNMT3A (Fig. 1d). Taken together, these data suggest that DNMT3A R882 mutation contributes to enhance the migration of malignant cells.
EMI of OCI-AML3 cells in mice
To explore the cause of paralysis presentation, we proposed the possibility of CNS abnormality caused by extramedullary leukemia. Positron emission tomography-computed tomography (PET-CT) was performed to investigate the hypermetabolic regions that are suggestive of tumor infiltration. Compared with those of normal controls, the spinal cords of sick mice contained more radioactive substances (Fig. 2b), supporting the infiltration of OCI-AML3 cells into CNS. To detect the disease status at the entire organism level, we transfected luciferase reporter lentivirus vector into OCI-AML3 cells and subsequently transplanted the cells into the mice. All transplanted animals developed a paralysis syndrome at 1 month post xenografting. Xenografts were observed to infiltrate the brains, spinal cords, and BMs by using bioluminescent imaging (Fig. 2c). Flow cytometry showed quite a lot of xenografted cells in the brain when hCD44 antigens were labeled (Fig. 2d). To examine whether other leukemia cell lines could cause CNS infiltration, we used 5 × 106 cells of U937 and THP-1 lines, both from acute monocytic leukemia patients, for transplantation. Around 20 days post xenografting, U937 cell-transplanted mice developed severe leukemia, but few exogenous cells were detected in the brain (Fig. 2d). In THP-1 cell-transplanted mice, no leukemia infiltration was observed in cerebral tissues at 40 days post xenografting when the mice became moribund (Fig. 2d). The above phenomenon demonstrates that OCI-AML3 cells could infiltrate into murine CNS and induce characteristic clinical symptoms.
CNS-infiltrating leukemic cells concentrate in murine meninges
Abrogation of D3Amut exerts an anti-infiltration effect
TWIST1 represents a downstream target of D3Amut
TWIST1 mediates enhanced cell migration ability in vivo
Genetic alterations are now regarded as important biomarkers for disease evaluation and prognosis assessment. In addition to chromosomal disorders, growing mutation information is recognized in EMI procedure. Some case reports found FLT3-ITD and NPM1 variations in myeloid sarcomas with high frequencies of 15 and 14.4 %, respectively. Extramedullary tumors that carry these two abnormalities are mostly accompanied with cytogenetically normal AML and represent short lifespan, although NPM1 mutation is considered a good prognostic indicator [22, 23]. This observation demonstrates that genetic mutation may independently affect disease progression in systemic leukemia with EMI.
In the present study, genetic lesion located in exon 18 of DNMT3A can promote leukemic cell migration. Meningeal leukemia, where EMI is displayed, could be determined in our NOD/SCID mice transplanted with human leukemic cells carrying D3Amut. Hence, we provide a strong evidence to support the clinical discovery of D3Amut in CSF from CNS relapse patient . Importantly, about 20 % of our AML cases with D3Amuts and whose genetic profiles have been reported before  showed CNS leukemia when CSF was detected during disease courses.
D3Amuts are frequently detected in cases diagnosed with M4 or M5 subtypes of AML . D3Amut alone could induce aggressive proliferation of differentiated monocytes , thereby suggesting that this mutation underlies the development of monocytic blasts . MLL abnormalities, which are mutually exclusive to D3Amuts in M4/M5 variants , are related to extramedullary disease . Our results demonstrate that D3Amut, which represents another group of AML patients with monocytic involvement, might also be associated with EMI.
The role of DNMT3A in cell invasion has been observed in lung cancer. Deletion of DNMT3A promotes tumor progression and enables cells to invade into bronchiole. Remarkably, a pool of genes in charge of cell adhesion and motion is highly expressed in DNMT3A-knockout mice . Therefore, DNMT3A variation may enhance tumor cell invasiveness through altering migrating mechanisms . In our leukemic EMI model, an EMT inducer TWIST1 is highly expressed in OCI-AML3 strains and AML patients’ bone marrow samples because of D3Amuts. EMT occurs in the initiation of metastasis for cancer progression. It enables carcinoma cells to escape cell-cell adhesion and gain migratory phenotype. EMT involvement has been experimentally proven in solid tumors . Recently, a group from Italy reported that EMT-like processes are relevant to acute promyelocytic leukemia development or progression . This result implicates that EMT regulator TWIST1 causes leukemia invasive behavior. Our data further suggest that the aggressive migratory behavior reminiscent of TWIST1 also exists in extramedullary leukemia and could be induced by DNMT3A R882 mutation.
DNMT3A is an epigenetic modifier, and mutation on its catalytic domain can decrease enzymatic activities and affect epigenetic modifications. We analyzed the methylation level of TWIST1 genes in a set of primary AML samples with normal karyotype from the TCGA AML cohort . This set includes 27 and 49 samples with DNMT3A R882 mutations and WT DNMT3A, respectively. Notably, in a region within 500-bp up- and downstream of gene transcriptional start site, R882 mutation group showed hypomethylation (Additional file 1: Figure S7). We suppose that D3Amut may lead to the demethylation of TWIST1 gene, thereby increasing its expression in leukemic cells. Interestingly, SHI-1, a cell line harboring MLL-AF6 translocation derived from an AML-M5 patient, can also invade murine brain . MLL is a histone modifying gene, and MLL rearrangement interferes the normal function of MLL. Therefore, EMI, as one of the features of AML-M4/M5 subtypes, may be partly attributed to epigenetic deregulation.
EMI is one of the reasons for the relapsed and refractory AML. Clinical studies have demonstrated that cells bearing DNMT3A mutant are resistant to conventional chemotherapy but sensitive to high-dose of daunorubicin-based regimen [30, 31]. We suppose that dose-escalated therapy might be useful for clearance of DNMT3A mutated cells, thus disrupting cell mobility. Importantly, in our assays, abrogation of DNMT3A mutant or TWIST1 in leukemic cells reveals an anti-infiltration effect, thereby providing a possible theoretical basis for clinical transformation.
In summary, our work first links D3Amut to leukemic cell migration and demonstrate that D3Amut in OCI-AML3 strain enhances leukemic aggressiveness by promoting EMI process, which is partially through upregulating TWIST1. Therefore, AML patient with this variation should be given further attention to the possibility of EMI, and D3Amut in extramedullary tumor is worth to detecting in further study. In addition, the inhibition of EMT inducer TWIST1 may be potential therapeutic target of EMI in AML.
We thank Dr. Lan Wang (Shanghai Institutes for Biological Sciences, China) for providing the OCI-AML3 cell line.
This work was supported by the Chinese National Key Basic Research Project (973:2013CB966803), National Natural Science Foundation of China (81400106, 81170519), Doctoral Innovation Fund Projects from Shanghai Jiao Tong University School of Medicine (BXJ201407), and Shanghai Young Doctor Training Program (2015).
Availability of data and material
All data generated and analyzed during our study are included in this published article and its supplementary information file—Additional file 1.
JX and WZ designed the study. JX and WZ performed the experiments and interpreted the data. X-QL and WL performed the DNA methylation analysis. J-QM and J-ML provided the clinical samples. JZ, ZC, and S-JC provided the conceptual advice, designed the experiments, and wrote the manuscript. JX, WZ, and X-JY supervised the entire project and wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
All patients provided written informed consent for the use of their AML samples under a protocol approved by the ethics committee of Shanghai Institute of Hematology. Human primary AML samples were obtained in accordance with the ethical guidelines established by Shanghai Institute of Hematology. All animal experiments were carried out in accordance with the approved guidelines provided by the Laboratory Animal Resource Center of Shanghai Jiao Tong University School of Medicine.
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