Molecular Cell Biology

MBD2 Ablation Impairs Lymphopoiesis andImpedes Progression and Maintenance of T-ALLMi Zhou1, Kuangguo Zhou1, Ling Cheng1, Xing Chen1, Jue Wang1,Xiao-Min Wang2, Yingchi Zhang2, Qilin Yu3, Shu Zhang3, Di Wang1,Liang Huang1, Mei Huang1, Ding Ma4, Tao Cheng2, Cong-Yi Wang3,Weiping Yuan2, and Jianfeng Zhou1,4

Abstract

Aberrant DNA methylation patterns in leukemia might beexploited for therapeutic targeting. In this study, we employed agenetically deficient mouse model to explore the role of themethylated DNA binding protein MBD2 in normal and malig-nant hematopoiesis. MBD2 ablation led to diminished lym-phocytes. Functional defects of the lymphoid compartmentwere also observed after in vivo reconstitution of MBD2-defi-cient hematopoietic stem cells (HSC). In an established modelof Notch1-driven T-cell acute lymphoblastic leukemia (T-ALL),MBD2 ablation impeded malignant progression and mainte-nance by attenuating the Wnt signaling pathway. In clinicalspecimens of human T-ALL, Wnt signaling pathway signatureswere significantly enhanced and positively correlated with the

expression and function of MBD2. Furthermore, a number oftypical Wnt signaling inhibitory genes were abnormally hyper-methylated in primary human T-ALL. Abnormal activation ofWnt signaling in T-ALL was switched off by MBD2 deletion,partially by reactivating epigenetically silenced Wnt signalinginhibitors. Taken together, our results define essential roles forMBD2 in lymphopoiesis and T-ALL and suggest MBD2 as acandidate therapeutic target in T-ALL.

Significance: This study highlights a methylated DNA bindingprotein as a candidate therapeutic target to improve the treatmentof T-cell acute lymphoblastic leukemias, as anew startingpoint fordeveloping epigenetic therapy in this and other lymphoid malig-nancies. Cancer Res; 78(7); 1632–42. �2018 AACR.

IntroductionDNA methylation is introduced by at least three DNA methyl-

transferases (DNMT), including DNMT3a and DNMT3b forde novo methylation, and DNMT1 for methylation maintenance(1, 2). To recognize and "translate" the methylated DNA intosignals for transcriptional repression, DNA methylation must beread by a conserved family of methyl-CpG-binding domain(MBD) proteins (3). The "reader" proteins of the MBD familyin mammals include five known members named MeCP2,MBD1, MBD2, MBD3, and MBD4, which recognize and bindmethylated CpG sequences, and in turn control gene expression

by interrupting the binding of transcription factors to the corre-sponding promoter region (4).

Aberrant DNA methylation patterns in tumor cells representattractive and novel therapeutic targets (5–7). Therapy withDNMT inhibitors has shown robust clinical activity and clearlyalters the natural progression of several hematopoietic malignan-cies (8–11). DNMT inhibitors curtail and even reverse the tumor-associated signature of aberrant DNAmethylation and associatedgene silencing in cancer (12, 13). Despite rapid clinical progress,the utility ofDNMT inhibitors has been limited by the toxicity andundesired off-target effects associated with nonspecific globaldemethylation (14). Alternatively, targeting the "reader" proteinsofDNAmethylation instead of usingDNMT inhibitorsmight be ahighly attractive strategy for epigenetic therapy (15).

Of all theMBD proteins, MBD2 has been proposed as themostpromising target (15). Intriguingly, unlike other MBD members,the deletion of MBD2 in mice does not generate any majordeleterious effects (3, 16–19), indicating that MBD2 could bedispensable under normal physiologic conditions and wouldtherefore enable theminimization of off-target toxicity to normaltissues. MBD2 had been shown to mediate the inhibition ofaberrantly methylated tumor suppressor genes by binding tomethylated regulatory promoter regions (20–22), and knock-down of MBD2 could suppress neoplastic cell growth by reacti-vating the transcription of tumor suppressor genes (21, 23).WhenMBD2-deficient mice were crossed with ApcMin/þ (or Min) back-ground mice, the development of intestinal tumors was attenu-ated (22, 24), indicating that MBD2 is crucial for this tumor-promoting effect.

In this study, we sought to investigate the role of MBD2 innormal andmalignant hematopoiesis. Our study showed that the

1Department of Hematology, Tongji Hospital, Tongji Medical College, HuazhongUniversity of Science and Technology, Wuhan, Hubei, China. 2State Key Lab-oratory of Experimental Hematology, Institute of Hematology and Blood Dis-eases Hospital, Chinese Academy of Medical Sciences and Peking Union MedicalCollege, Tianjin, China. 3The Center for Biomedical Research, Tongji Hospital,Tongji Medical College, Huazhong University of Science and Technology,Wuhan, China. 4Cancer Biology Research Center, Tongji Hospital, Tongji MedicalCollege, Huazhong University of Science and Technology, Wuhan, Hubei, China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

M. Zhou and K. Zhou contributed equally to this article.

CorrespondingAuthors: Jianfeng Zhou, Tongji Hospital, Tongji Medical College,Huazhong University of Science and Technology, Wuhan, Hubei 430030, China.Phone: 8627-8366-2437; Fax: 8627-8366-2680; E-mail: jfzhou@tjh.tjmu.edu.cn;Weiping Yuan, wpyuan@ihcams.ac.cn; and Cong-Yi Wang,wangcy@tjh.tjmu.edu.cn

doi: 10.1158/0008-5472.CAN-17-1434

�2018 American Association for Cancer Research.

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loss of MBD2 significantly impaired lymphoid hematopoiesis. InNotch1-driven T-cell acute lymphoblastic leukemia (T-ALL),MBD2 was required for the progression and maintenance ofleukemia. This study highlights the great potential of usingMBD2as a therapeutic target in T-cell–related pathologies.

Materials and MethodsMice

MBD2deficient (Mbd2�/�)mice on aC57BL/6-CD45.2 geneticbackground were a gift kindly provided by Dr. Adrian Bird(Edinburgh University, Edinburgh, United Kingdom; ref. 19). Allmice were maintained in a pathogen-free animal facility at TongjiHospital of Tongji Medical College, Huazhong University ofScience and Technology, Wuhan, China. All animal studies wereapproved by the Institutional Committee of Animal Care andTreatment in Tongji Hospital. Six- to eight-week-oldmaleMbd2�/�

mice and WT littermates were used in this study.

Clinical samplesFor T-ALL samples, leukocytes were isolated frombonemarrow

specimens by Ficoll gradient and stored frozen in aliquots. Allspecimenswere collectedbefore chemotherapy.Written informedconsent was obtained from each patient and healthy volunteerdonor in accordance with the principles expressed in the Decla-rationofHelsinki, and the studywas approvedby the InstitutionalReview Committee for the use of human materials at TongjiHospital. The global gene expression profiles of primary T-ALLsamples were obtained from Gene Expression Omnibus (GEO).

Cell linesJurkat (T-ALL) and Molt4 (T-ALL) cell lines were originally

purchased from ATCC in 2011, authenticated by short tandemrepeat, and tested to ensure that they were mycoplasma-free bydirect culture within 3 months of use. The cell lines were culturedin RPMI1640 medium supplemented with 10% FCS (Gibco,Invitrogen), and used for experimentation within 1 month ofbeing thawed from frozen stocks.

Noncompetitive repopulation assaysA total of 1 � 106 bone marrow cells from Mbd2�/� or

littermate WT mice (both CD45.2þ) were injected intravenouslyinto lethally irradiated CD45.1þ recipient animals (9.5 Gy in twodoses, 4 hours apart). Peripheral blood cells were collected 1, 2, 3,and 4 months after transplantation. Five months after transplan-tation, recipient mice were sacrificed, and bonemarrow cells werecollected. The contribution of CD45.2þ donor-derived cells in theperipheral blood and bone marrow of recipient mice was ana-lyzed by flow cytometry. CD45.2 antibody (FITC) was used todetect the donor cells.

Murine T-ALL modelThe retrovirus vector encoding the ICN1 gene was a gift from

Dr. David Scadden (Harvard University, Boston, MA). TheMSCV-ICN1-IRES-GFP plasmid was cotransfected into the pack-age 293T cells with pKat and pCMV-VSV-G via Lipofectamine2000 (Invitrogen). The supernatant of 293T cells was harvested.The transduction of Lin� cells from the bone marrow of WT orMbd2�/�mice with viral supernatant was performed as describedpreviously (25). Leukemic mice were sacrificed 2–3 months aftertransplantation, and the bone marrow cells were harvested as

P0 cells. Then, we transplanted P0 leukemic cells into sublethallyirradiated (6.5 Gy in one dose, 6- to 8-week-old) or nonirradiatedrecipients (6- to 8-week-old) to establish a leukemicmousemodelas P1.

Flow cytometryAn LSR II cytometer and FlowJo7.6 software (BD Biosciences)

were used for data acquisition and analysis. All antibodies werefrom BD Biosciences or eBioscience unless otherwise indicated.Cell sorting was performed using a fluorescence activated cellsorter (FACS, Aria Cell Sorter, BD Biosciences).

BrdUrd detectionWhen the proportion of GFPþ leukemic cells was >50% in

mononuclear cells of the bone marrow of the WT recipient mice,the mice were given a single pulse administration of 5-bromo-20-deoxyuridine (BrdUrd, Sigma-Aldrich). An intraperitoneal injec-tion of BrdUrd (1mg/6 g) was given 2 hours before harvesting thebone marrow cells. A BrdUrd-APC staining kit (BD Biosciences)was used according to the manufacturer's instructions. BrdUrdstaining was analyzed using flow cytometry by surface markersand intracellular staining.

Western blotting and antibodiesProtein sample preparation and Western blot analysis were

performed as described previously (26). Commercial antibodiesagainst the following proteinswere purchased:MBD2 (SantaCruzBiotechnology); b-catenin, histone H3, and GAPDH (Cell Sig-naling Technology).

qPCRTotal RNA was isolated from cells using TRIzol and reverse

transcription-PCR (RT–PCR) was then performed. qPCR wasconducted using an ABI Prism 7900 Sequence Detection System(Applied Biosystems) with SYBR Green Supermix (Applied Bio-systems) following the manufacturer's instructions. All data werenormalized using the endogenous GAPDH control, and therelative gene expression was calculated using the DDCt method.All primer sequences are summarized in Supplementary Table S1.

IHCTissue biopsy samples were subjected to IHC to detect the

expression of GFP using a GFP antibody (MBL) diluted 1:1,000.Themicroscope used for imagingwas anOLYMPUS BX51with anOLYMPUS DP72 camera (Olympus).

MicroarrayTotal RNAwas extractedwith TRIzol fromGFPþ T-ALL cells and

double positive (DP, CD4þCD8þ) thymocytes in TRIzol (Invitro-gen). The cDNAwas amplified before array analysis, and 1.5 mg ofcDNA from each sample was hybridized to GeneChip MouseGenome 430 2.0 microarrays (Affymetrix) according to the man-ufacturer's instructions. Genes with a P value below 0.01 anda fold change greater than 2.0 were considered significant.Hierarchical clustering was generated using cluster 3.02 (Stan-ford University, Stanford, CA). Gene-set enrichment analysis(GSEA) was used to identify classes of genes significantlyenriched within genes being regulated upon MBD2 knockout.Microarray raw data are available for download at Gene Expres-sion Omnibus (http://ncbi.nlm.nih.gov/geo) under accessionnumber GSE105763.

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Global methylation profilingTen T-ALL patient bonemarrow samples and ten normal CD3þ

bone marrow cells from healthy donors were collected, and all T-ALL bone marrow samples had more than 80% leukemic cells.Genomic DNA was extracted as described previously. All caseswere analyzed by an Illumina Human Methylation 450K (Illu-mina Inc.) according to the manufacturer's instructions. Signalintensities were obtained from GenomeStudio (Illumina), con-verted to b values, filtered, and normalized to remove biasesbetween Infinium I and II probes. A DNA methylation b valuewas reported as a DNA methylation index ranging from 0 (non-methylated) to 1 (completely methylated). CpG probes withaberrant methylation in T-ALL cells compared with those innormal T cells were identified as having a P < 0.01 and a meanb value difference > 0.2. Hierarchical clustering was generatedusing cluster 3.02 (Stanford University, Stanford, CA).

DNA bisulfite sequencing analysisGenomic DNA was extracted using a QIAamp DNA mini kit

(Qiagen) and then subjected to bisulfite conversion using an EZDNA Methylation Kit (Zymo) followed by PCR of the targetedsequence. The PCR amplifications were analyzed by agarose gelelectrophoresis and then subcloned into the pEASY-T1 SimpleCloning Vector (TransGen Biotech). Ten positive clones derivedfrom each PCRproduct were randomly selected forDNA sequenc-ing analysis.

Chromatin immunoprecipitation assayThe experiment was performed with a chromatin immunopre-

cipitation (ChIP) assay kit (Millipore). The primers used in ChIPassays are listed in Supplementary Table S2. The anti-MBD2 anti-body used in ChIP assays was purchased from Bethyl Laboratory.

ResultsMBD2 deficiency leads to diminished lymphocytes

To assess MBD2 expression in hematopoietic cells, we searchedpublic databases (27) of the MBD protein family and found thatMBD2was expressed at significantly higher levels in morematureblood cell lineages than in HSCs/HPCs in normal mouse andhuman hematopoiesis (Fig. 1A and B; Supplementary Fig. S1A–S1H). The role of MBD2 in hematopoiesis was explored byutilizingMbd2�/�mice andWT littermates. The deletion ofMBD2alleles and abolished transcriptional expression were examined(Supplementary Fig. S2A and S2B). No significant difference inplatelets or red blood cells was observed between Mbd2�/� andWTmice (Supplementary Fig. S3A and S3B). The cellularity of thebone marrow, spleen, and thymus was also unaffected by MBD2deletion (Supplementary Fig. S3C). However, compared withWTmice,Mbd2�/� mice showed a dramatically reduced white bloodcell count in peripheral blood compared with their WT litter-mates,whichwas attributable to strikingly decreased lymphocytescounts (Fig. 1C). Flow cytometry analysis corroborated thesefindings (Fig. 1D). Nevertheless, the frequency of lymphocytesor myeloid cells in the bone marrow was equivalent betweenMbd2�/� and WT mice (Fig. 1E). Further examination of DN(double negative, CD4�CD8�) and DP (CD4þCD8þ) thymo-cytes showed increased DN and decreased DP thymocytes inMbd2�/� mice relative to WT mice, although the differences werenot statistically significant (Fig. 1F). The absolute number ofsingle positive CD4þ or CD8þ thymocytes was equivalent

between the two groups (Fig. 1G). In HSC/HPC pools, no signif-icant differences in the frequencies of HSCs andHPCs in the bonemarrow were observed between Mbd2�/� and WT mice (Fig. 1Hand I). Therefore, MBD2 deletion led to diminished numbers oflymphocytes but did not alter the size of the HSC and HPCcompartments.

Loss of MBD2 significantly impairs the reconstitution capacityof HSCs, which results in a diminished lymphoid compartmentin vivo

Because the Mbd2�/� mouse is deficient for MBD2 in bothblood cells and the hematopoietic microenvironment (19), weadopted a transplantation strategy to address the direct effectsof MBD2 deletion on HSCs/HPCs. There was no significantdifference in the homing efficiency of donor cells to the recip-ient bone marrow between Mbd2�/� and WT mice (Fig. 2A). Innoncompetitive transplantation, MBD2 ablation dramaticallydiminished CD45.2þ cell reconstitution compared with WTlittermates in the peripheral blood of recipient mice (Fig. 2B).Five months after transplantation, the engraftment of B andT lymphocytes but not myeloid cells remained dramaticallyimpaired by MBD2 deletion in the peripheral blood (Fig. 2C);in the bone marrow, the engraftment of total CD45.2þ and Blymphocytes was impaired (Fig. 2D). In various lymphoidorgans of recipient mice, MBD2 deletion dramatically impededthe reconstitution of CD45.2þ lymphocytes (Fig. 2E). More-over, the engraftment of HSCs/HPCs, with the exception ofCLPs in the bone marrow, was also impeded by the loss ofMBD2 (Fig. 2F and G). Therefore, MBD2 loss significantlyimpaired the reconstitution capacity of HSCs/HPCs, whichresulted in a diminished lymphoid compartment.

Loss of MBD2 significantly delays Notch1-inducedleukemogenesis, and MBD2 is critical for the maintenanceof T-ALL

Because MBD2 loss led to a pronounced decrease in T lym-phocytes, we asked whether MBD2 would be essential for T-ALLleukemogenesis in vivo. A Notch1-induced mouse T-ALL modelwas generated (Fig. 3A). Consistent with previous studies, micetransplantedwith ICN1-infectedWTcells showed an expansion inthe percentage of CD4þCD8þ cells in the bone marrow andperipheral blood two weeks after transplantation. Recipient micetransplanted with ICN1-infected Mbd2�/� cells developed leuke-mia with amuch longer latency (Fig. 3B and C). All recipientmiceeventually developed signs of overt T-ALL anddiedwithin 70 daysof transplantation (Fig. 3C). The longer latency for leukemia inMbd2�/� group was apparently not due to a weakened homingefficiency (Fig. 3D).

To determine the functional impact of MBD2 deletion on themaintenance of Notch1- driven T-ALL leukemia, a P1 leukemicmousemodelwas generated (Fig. 3A). In the subirradiatedmodel,mice in the Mbd2�/� group had a significantly longer overallsurvival (Fig. 3E). Strikingly, in the nonirradiated model, mice inthe Mbd2�/� group regenerated T-ALL with an extremely longerlatency and had a significantly longer overall survival (Fig. 3F).Moreover, while WT leukemic cells continued to retain theirpotent capacity to develop leukemia, 40% of Mbd2�/� leukemiccells did not ultimately generate T-ALL (Fig. 3F). No leukemic cellinfiltration in the bone marrow or spleen was detected in theMbd2�/� group 120 days after transplantation (SupplementaryFig. S4).

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To assess the malignant features ofMbd2�/� T-ALL cells, in vivoproliferation, apoptosis, and the frequencies of central nervoussystem (CNS) infiltration were compared between Mbd2�/� andWT T-ALL cells in nonirradiated recipients. In vivo BrdUrd incor-poration studies showed that Mbd2�/� T-ALL cells grew muchmore slowly (Fig. 3G), but no significant difference in apoptosiswas observed (Supplementary Fig. S5A and S5B). Amazingly, theT-ALL mice transplanted with WT cells presented with CNSleukemia infiltration much more frequently thanMbd2�/� T-ALLmice (Fig. 3H and I).

MBD2 ablation impedes the progression of Notch1-drivenT-ALL by inhibiting the Wnt signaling pathway

To explore potential mechanisms underlying the anti-ALLeffects of MBD2 ablation, leukemic cells were harvested frommice transplanted with WT or Mbd2�/� T-ALL cells, and theirglobal gene expression profiles (GEP) were compared. Normal

DP thymocytes were isolated and used as a normal control. Thedifferent GEPs of WT T-ALL and DP cells generated leukemia-associated gene signatures. Intriguingly, MBD2 ablationreversed 26% of the leukemia-associated gene signatures inT-ALL cells (Fig. 4A), leading to the upregulation of 1,987 genesand the downregulation of 938 genes. However, most genes inMbd2�/� DP cells remained unchanged compared with WT DPcells (Supplementary Fig. S6A), and MBD2 ablation did notaffect the expression of other MBD family members in DP andT-ALL cells (Supplementary Fig. S6B). GSEA revealed thatMBD2 loss in T-ALL cells resulted in a significantly attenuatedWnt signaling pathway signature and the inhibition of cellproliferation (Fig. 4B). Moreover, MBD2 deletion in leukemiccells led to the substantially altered expression of a number ofcritical Wnt signaling regulators (Fig. 4C; Table 1; refs. 28, 29).Interestingly, MBD2 loss in T-ALL cells led to increased Notchpathway (Supplementary Fig. S6C and S6D), indicating a

Figure 1.

Loss of MBD2 leads to a decreased lymphoid lineages phenotype. A and B, Analysis of MBD2 expression levels in murine or human purified hematopoietic cells(from the Bloodspot database). LT-HSC, long-term hematopoietic stem cell; ST-HSC, short-term HSC; LMPP, lymphoid-primed multipotent progenitorcell; CLP, common lymphoid progenitor cell; GMP, granulocyte-monocyte progenitor cell; MEP, megakaryocyte-erythrocyte progenitor cell; GRANU, granulocyte;MONO, monocyte. C,WT andMbd2�/�mice were examined for hematopoietic cell counts of peripheral blood (n¼ 10 mice per group). WBC, white blood cell; LYM,lymphocyte; NEUT, neutrophil; MONO,monocyte.D andE, The percentages of B and T lymphocytes, andmyeloid cells among themononuclear cells of the peripheralblood (PB; D) and bone marrow (BM; E) of WT and Mbd2�/� mice (n ¼ 5 mice per group). F and G, The counts or percentages of thymocyte subsets of DN/DP (F)and CD4þCD8�/CD4�CD8þ (G) in Mbd2�/� and WT mice (n ¼ 5 per genotype) were analyzed by flow cytometry. H and I, The counts of HSCs (H) or HPCs(I) in the bone marrow of WT and Mbd2�/� mice (n ¼ 5 per group). Error bars, SEM; � , P < 0.05; �� , P < 0.005; ��� , P < 0.001, two independent experiments.

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potential negative correlation between Notch and Wnt signal-ing activity in T-ALL (30, 31).

Previously, the abnormal epigenetic modification of variouscritical Wnt signaling regulators was shown to be responsible forthe aberrant activation of Wnt signaling in cancer cells (28, 32).Therefore, we further validated the microarray data by examiningthe expression of 9 typical Wnt signaling regulators using qPCR(Fig. 4D). Notably, MBD2 deficiency inhibited Wnt signaling atmultiple levels by affecting the transcriptional expression of bothnegative and positive Wnt signaling regulators (Fig. 4E). Accord-ingly, the expression of CD44, a downstream target gene of theWnt signaling pathway (29), was downregulated more than 14-fold inMbd2�/� T-ALL cells comparedwith that inWT T-ALL cells.These data indicated that MBD2 deletion impeded the progres-sion of T-ALL by inhibiting the Wnt signaling pathway.

Positive correlation between MBD2 and the Wnt signalingpathway in human T-ALL

On the basis of our results in murine T-ALL models, we furtherexplored the global gene expression profiles of 174 primary T-ALLsamples to determine the potential clinical relevance of MBD2 inhuman T-ALL. GSEA revealed that compared with normal bonemarrow, human T-ALLs showed significantly increased Wnt sig-naling pathway signature (GEO GSE13204; Fig. 5A and B). Tofurther confirmenhancedWnt signaling in T-ALL,we analyzed theb-catenin protein level and found that b-catenin was higher inprimary T-ALL samples than in normal T cells samples (Supple-mentary Fig. S7A). To determine the possible correlation betweenMBD2 and the Wnt signaling pathway in human T-ALL, MBD2and TCF7 expression levels were extracted from 174 primaryT-ALL samples, which are available in a publicly accessible data-

base (GEO GSE13204). Pearson correlation analysis (P � 0.05)showed that MBD2 expression was positively correlated withTCF7 expression, a known important Wnt transcriptional factor(33), among these primary T-ALL samples (Fig. 5C). The positivecorrelation of TCF7 expression with MBD2 expression in T-ALLwas again observed in our primary T-ALL patient cohort (n ¼ 20;Supplementary Fig. S7B). On the other hand, our data showedthat the TCF7 expression level was significantly higher in T-ALL,while the MBD2 expression level was not significantly differentbetween T-ALL and normal T cells, illustrating that MBD2 wasprobably heterogeneously expressed in leukemia (SupplementaryFig. S7C). Furthermore, using the Wnt signaling gene set fromKEGG (Kyoto Encyclopedia of Genes and Genomes, Supplemen-tary Table S3), unsupervised hierarchical clustering analysis ofprimary T-ALL samples (GEO GSE62156, GSE33469) was per-formed. The differential signatures of Wnt signaling stratifiedT-ALLs into two distinct clusters, cluster 1 T-ALLs display distinctMBD2 expression levels relative to T-ALLs within cluster 2 (Sup-plementary Fig. S8A and S8B).

Next, we sought to address howMBD2 correlated with theWntsignaling. As attempts with siRNA failed to yield greater than 50%knockdown of MBD2 protein in T-ALL cell lines, a clusteredregularly interspaced short palindrome repeat associated nucleaseCas9 (CRISPR/Cas9) strategy (34) was used to generate a stablecell line with haploid deletion of theMBD2 gene (SupplementaryFig. S9A and S9B). Interestingly, a haploid deletion of theMBD2gene in Jurkat cells caused a complete loss of endogenous MBD2protein (Fig. 5D). Notably, the transcription levels of a positiveWnt regulator (TCF7; ref. 33) and downstream Wnt target genes(CD44, c-MYC; refs. 29, 35) were significantly decreased uponMBD2 depletion in Jurkat cells (Fig. 5E). To further address the

Figure 2.

MBD2 deficiency impairs HSC reconstitution capacity. A, Homing assay. Bars indicate the percentage of homing CFSEþ mononuclear cells found in the bonemarrow of recipient mice. B, Each lethally irradiated CD45.1þ mouse recipient was transplanted with 1 � 106 bone marrow cells harvested fromCD45.2þ WT or Mbd2�/� donor mice. Transplanted mice were monitored by flow cytometry for the engraftment of CD45.2þ cells in peripheral blood (PB) atthe indicated time points. C and D, Transplanted mice were sacrificed five months after transplantations and monitored for the engraftment of CD45.2þ

cells in the peripheral blood (C) or bone marrow (BM; D). E–G, Five months after transplantations, the engraftment of CD45.2þ thymocytes in the thymus,CD4þCD8�/CD4�CD8þ T lymphocytes in lymph nodes and the spleen (E), and CD45.2þ HSCs/HPCs (F and G) in the bone marrow was quantified.Data represents the mean � SEM of five replicates determined from 5 mice over two independent experiments. � , P < 0.05; �� , P < 0.005.

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consequence of MBD2 deletion on Wnt signaling pathway activ-ity, we determined the protein levels of b-catenin, the pivotalmediator of Wnt signaling (36, 37). MBD2-deficient Jurkat cellshad significantly decreased expression of both total and nuclearb-catenin, and decreased cell proliferation with G0–G1 cell-cyclearrest, indicating inactive Wnt signaling in the absence of MBD2(Fig. 5F and G). Similar results were observed in mutant Molt4clones andWT clones (Fig. 5H and I). Furthermore, the decreasedexpression of Wnt target genes as well as the decreased cellproliferation could be significantly rescued by transfection withlentivirus encoding human b-catenin into MBD2-deficient T-ALLcells (Fig. 5G and J). Overexpression of b-catenin in MBD2-deficient Jurkat cells did not affect MBD2 ablation (Fig. 5K).Taken together, these results indicated that human T-ALLs showsignificantly increased Wnt signaling pathway signatures, whichwere positively correlated with MBD2 expression.

Abnormally hypermethylated negative Wnt regulators requireMBD2 to activate Wnt signaling in human T-ALL cells

MBD2 binds to methylated CpG islands and translates themethylated DNA into signals for transcriptional repression (21).Therefore, we hypothesized that the promoter regions of somegenes were methylated in T-ALL and were read by MBD2. Toinvestigate this, we subjected primary human T-ALL samples and

CD3þ bone marrow T lymphocytes to genome-wide DNA meth-ylation profiling analysis. Compared with normal CD3þ T lym-phocytes, primary T-ALL cells showed hypermethylation of thepromoter regions of approximately 2,800 genes and hypomethy-lation of 596 gene promoters (Fig. 6A). Consistent with previousreports, PTPN6 and GALNT6 were hypermethylated and hypo-methylated (38, 39), respectively, in T-ALL (Fig. 6B). Strikingly, anumber of typicalWnt signaling inhibitors were hypermethylatedto a greater extent than that of the positive control (PTPN6) inhuman T-ALL (Fig. 6B). In line with these findings, the transcrip-tion levels of these Wnt signaling inhibitors were consistentlylower in T-ALL samples relative to normal CD3þ T lymphocytes(Fig. 6C). Next, we sought to address whether MBD2 plays a rolein regulating the transcriptional expression of typical Wnt signal-ing inhibitors. The transcription levels of these negative Wntregulators were consequently reactivated upon MBD2 depletion,and the upregulation of SFRP5 was shown to be the same in bothmutant clones (Fig. 6D), suggesting that the knockdown ofMBD2could effectively relieve the transcriptional suppression of severalabnormally methylated genes.

To validate the methylation microarray and transcriptiondata, we analyzed the methylation of the 342-bp promoter ofSFRP5 by bisulfite sequencing (Fig. 6E). While a low methyl-ation rate was detected in normal CD3þ T cells, significantly

Figure 3.

MBD2 is critical for the progression and maintenance of Notch1-induced T-ALL in vivo. A, Experimental design of an MBD2-deficient murine Notch1-inducedT-ALL model. B, The dynamics of GFPþ leukemic cells appearing in the peripheral blood in WT orMbd2�/� group (n¼ 15 per group) are shown. C, The Kaplan–Meiersurvival curves for recipient mice after transplantation are shown (n � 8 per group, two independent experiments). D, Leukemic cell homing analysis (n ¼ 5 pergenotype, two independent experiments). E and F, The Kaplan–Meier survival curves for sublethally irradiated (E) or nonirradiated (F) recipient mice aftertransplantation are shown (n � 8 per group, three independent experiments). G, Shown are the mean percentages � SEM of BrdUrdþ cells within the total GFPþ

leukemia cells (left) and representative histograms (right) for the cohort (n ¼ 5 per group, two independent experiments) of Mbd2�/� versus WT leukemic mice. H,Representative hematoxylin andeosin staining (H&E, left) andGFP IHC(right) images (�100and�600)of infiltrationofT-ALLcells in thebrainmeningeal spacesofWTleukemic mice and Mbd2�/� leukemic mice. B, brain; M, meninges. Scale bar, 40 mm. I, The numbers of mice with meningeal leukemia detected in WT or Mbd2�/�

leukemic mice (n ¼ 18 per group, two independent experiments). Error bars, SEM; � , P < 0.05; �� , P < 0.005; ��� , P < 0.001.

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increased methylation in the same region was detected in T-ALL(Fig. 6F and G; Supplementary Fig. S10A and S10B). To confirmwhether MBD2 deletion induced a change in DNA methylationlevels of SFRP5, which might regulate SFRP5 through demeth-ylation way, we performed bisulfite sequencing on parent, WT,and mutant Jurkat clones, as well as murine T-ALL cells. Wefound that MBD2 deletion did not affect the methylation rateof the SFRP5 promoter region (Fig. 6H). Next, we used ChIPassays to determine whether MBD2 regulates the transcription-al expression of SFRP5 by directly binding to its promoterregion. FGF19 was selected as a positive control, and GAPDHwas selected as a negative control (40). A definite binding ofMBD2 protein to the SFRP5 promoter region was detected inJurkat T-ALL cells (Fig. 6I), supporting the hypothesis thatMBD2 suppressed the transcriptional expression of SFRP5 bydirectly binding to its promoter in leukemic cells.

DiscussionAlthoughMBD2, one of the "readers" of DNAmethylation, has

been regarded as themost promising target for the next generationof DNA demethylation therapy (15, 24), its role in hematopoiesishas not been comprehensively investigated. In this study, weutilized MBD2 knockout mice to investigate the role of MBD2in normal and malignant hematopoiesis, and provided strongevidence to support the potential of MBD2 as a therapeutic targetfor T-ALL.

The sequential steps of hematopoiesis are precisely con-trolled by several regulatory mechanisms, in which epigeneticmechanisms play a vital role (41, 42). Despite substantialadvances in understanding the functional role of DNA meth-ylation regulators in hematopoiesis (43–45), little is knownabout the role of the MBD family in hematopoiesis. In publiclyavailable data, MBD2 was expressed at significantly higherlevels in more mature blood cell lineages than in HSCs/HPCs,indicating that MBD2 might play an essential role in thesequential steps of hematopoiesis. In our study, while MBD2knockout mice were viable and did not develop any obviousillness, MBD2 deficiency in a "steady-state" gave rise to pro-foundly diminished lymphocytes. Importantly, MBD2 deletiondid not alter the frequencies of HSCs/HPCs. In transplantationstudies, the reconstitution capacities of various HSC/HPCsubsets were impaired to various degrees by MBD2 ablation.

Figure 4.

MBD2 ablation partially reverses T-ALL–associated gene signatures and attenuated Wnt signaling. A, Hierarchical clustering of normal CD4þCD8þ DP thymocytesof WT normal mice and T-ALL cells sorted from P1 leukemic mice. B, GSEA plot showing the decreased expression of signaling by Wnt, Benporathproliferation, and KEGG DNA replication signatures in Mbd2�/� cells relative to WT T-ALL cells. NES, normalized enrichment score. C, Scatter plots of expressionprofiling for WT or Mbd2�/� T-ALL groups in microarrays. D, Validation of the microarray data of typical Wnt signaling regulators by qPCR. Green bars,negative regulators; red bars, positive regulators. The level of transcripts in WT T-ALL cells was set at 1.0. E, MBD2 deletion increased the expression of negative(green elliptic) Wnt regulators and decreased the expression of positive (red elliptic) Wnt regulators in T-ALL cells. Red arrows, increased expression; blackarrows, decreased expression. Error bars, SEM; � , P < 0.05; �� , P < 0.005; ��� , P < 0.001.

Table 1. List of relevant genes with respect to the Wnt signaling pathway inmicroarrays of WT and Mbd2�/� murine T-ALL groups

Downregulated in T-ALL Mbd2�/� Upregulated in T-ALL Mbd2�/�

Gene symbol Fold change Gene symbol Fold change

Tcf7 0.32 Cdkn1a 2.10Wnt8b 0.36 Sox13 12.48Wnt5b 0.45 Dkkl1 7.29Cd44 0.07 Cdh1 3.30

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Nevertheless, as far as myeloid hematopoiesis is concerned,MBD2-deficient mice display a rather weak phenotype, asMBD2-deficient HSCs/HPCs ultimately had normal productionof myeloid progeny despite the impaired function of HSCs/HPCs. However, MBD2 loss dramatically impaired the capacityof HSCs to form lymphoid progeny. Previously, redundancywas reported among various MBD proteins in mediating theirrepression of methylated genes (21). For example, MBD3 hasbeen shown to partially compensate for the phenotypes causedby MBD2 deficiency (3, 19). It is therefore possible that theweak phenotype of myeloid hematopoiesis in MBD2-deficient

mice is due to the presence of a redundant mechanism. Incontrast, MBD2 appears to play an indispensable role inlymphopoiesis.

Beyond the impact on normal T lymphopoiesis, MBD2 abla-tion substantially impeded the progression of Notch1-driven T-ALL. Although MBD2 deletion is not sufficient to block the onsetof T-ALL in mice, it significantly delayed Notch1-induced leuke-mogenesis. Strikingly,Mbd2�/� T-ALL cells weremuch less aggres-sive than Mbd2þ/þ T-ALL cells in vivo. More importantly, MBD2was critical for the maintenance of T-ALL in nonirradiated reci-pients. To explore underlying mechanisms of MBD2 in T-ALL, we

Figure 5.

MBD2 is positively correlated with Wnt signaling in human T-ALLs. A and B, GSEA plot showing the increased expression of Wnt signaling pathway signaturesin T-ALL relative to normal bone marrow. C, Positive correlation of TCF7 with MBD2 in 174 T-ALL samples. Statistics are from Pearson correlation analysis.D, MBD2 expression in isogenic clones and parental Jurkat was determined by Western blot analysis. E, The transcriptional expression levels of TCF7, CD44, andc-MYC were determined in isogenic Jurkat clones. F, Expression levels of total and nuclear b-catenin protein in isogenic clones and parental Jurkat cells weredetermined by Western blot analysis. GAPDH and Histone H3 were used as internal controls. G, Lentiviral particles containing the GV358 expression vectorencoding human b-catenin and the control lentiviruswere transfected intoMBD2-deficient Jurkat cells (Jurkatmut1). The cell cycle was analyzed in Jurkat wt1, Jurkatmut1, Jurkat mut1 b-catenin, and Jurkat mut1 Ctrl cells using propidium iodide staining. Typical flow cytometry profiles show representative data from threeindependent experiments. H and I, Expression levels of MBD2, total and nuclear b-catenin protein in isogenic clones and parental Molt4 cells were determined byWestern blot analysis. J, qPCR confirmed the restoration of b-catenin at the mRNA level. The transcription levels of TCF7 and downstream Wnt target geneswere analyzed by qPCR. K, Western blot analysis of b-catenin and MBD2 expression in Jurkat wt1, Jurkat mut1, Jurkat mut1 b-catenin, and Jurkat mut1 Ctrl cells.GAPDH served as a control. Error bars, SEM; � , P < 0.05; �� , P < 0.005; ��� , P < 0.001.

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exploited an integrated strategy consisting of microarrays, path-way enrichment analysis, expression validation of target genes inT-ALL mice or cell lines, and global DNA methylation analysis inclinical samples to investigate targeted genes and related signalingpathways. In the T-ALL mouse model, we found that MBD2ablation inhibited Wnt signaling at multiple layers and reversedthe leukemia-associated gene signatures. The Wnt signaling path-way regulates the stability of coactivator b-catenin and thusactivates the Lef/Tcf family of transcription factors and the expres-sion of a set of target genes, which in turn regulates cell prolif-

eration, behavior, and survival (46). Our in vitro study showedthat MBD2 ablation led to decreased accumulation of nuclearb-catenin, a hallmark of activeWnt signaling (37), resulting in thesuppression of transcription factor TCF7 and Wnt target genes.The functional restoration of Wnt signaling by overexpressingb-catenin led to the restoration of the Wnt signaling target genesand cell proliferation in MBD2-deficient T-ALL cells, clearly dem-onstrating the positive correlation between MBD2 and Wntsignaling. Interestingly, in this study, MBD2 loss in T-ALL miceled to increased Notch pathway and decreased Wnt signaling

Figure 6.

Aberrant hypermethylation of Wnt inhibitors in human T-ALL is read by MBD2. A, Global methylation profiling. B, Mean b values of Wnt signaling inhibitors.C, Validation of the microarray data by qPCR analysis. D, The transcriptional expression of Wnt signaling inhibitors was determined in isogenic Jurkat clones.E, The CpG islands (�425–695) and selected regions for bisulfite sequencing (39–381) in the SFRP5 promoter region. TSC, translation start codon. F, Shown are thedifferent methylation profiles of the SFRP5 promoter detected in normal CD3þ T cells and T-ALL samples (n ¼ 10 per group). G, The average methylationlevels for normal CD3þ T cell, T-ALL, and Jurkat cells are shown.H, Left, the average SFRP5methylation levels of Jurkat cells, WT Jurkat cells (wt1, wt2), andmutatedJurkat cells (mut1, mut2). Right, the average SFRP5 methylation levels of WT and Mbd2�/� T-ALL cells from leukemic recipients of P1 generation. I, Bindingof MBD2 to the SFRP5 promoter was examined by ChIP assay. DNA levels were normalized to 100% of input. FGF19 was selected as a positive control andGAPDH was selected as a negative control. Error bars, SEM; � , P < 0.05; �� , P < 0.005; ��� , P < 0.001.

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signatures. It has been reported that Wnt and Notch fulfill oppos-ing functions in normal development: Wnt in proliferation andNotch in differentiation in the thymus. In cancer studies, Notchsignaling induces differentiation and acts as a tumor suppressorby inhibiting b-catenin-mediated signaling in the skin, andNotch1 signaling retains the capability to suppress the expressionof Wnt target genes in colorectal cancers (30, 31). These findingsfurther imply that Notch and Wnt signaling activity might have anegative correlation in the development of many kinds ofdiseases.

The findings in T-ALL mice and T-ALL cell lines are clinicallyrelevant. In hematologic malignancies, it has been reported thatthe aberrant activation of Wnt signaling is frequent and is asso-ciated with a poor prognosis in ALL due to abnormal DNAmethylation (47). SFRP4 was found to be frequently methylatedin CLL samples (48), while WIF1 methylation was a poor prog-nostic factor for acute promyelocytic leukemia (49). In this study,analysis of the global gene expression profiles in 174 primary T-ALL samples revealed significantly increased Wnt signaling path-way signatures, which were positively correlated with the expres-sion and function of MBD2. Our global DNA methylation anal-ysis on human primary T-ALL revealed an abnormal methylationsignature that facilitated an aberrant activation of Wnt signaling.Strikingly, this abnormal activation of Wnt signaling in T-ALLcould be effectively switched off by the deletion of MBD2,partially by reactivating epigenetically silenced Wnt signalinginhibitors, which subsequently impeded the progression ofNotch1-driven T-ALL. The SFRP family members possess adomain similar to one in the Wnt-receptor Frizzled protein andcan inhibit Wnt receptor binding to downregulate pathway sig-naling. Epigenetic loss of SFRP may provide constitutive activa-tion of Wnt signaling in the evolution of cancers (50). In ourstudy, bisulfite sequencing analysis of the promoter regionsdemonstrated direct evidence that SFRP5 was remarkably hyper-methylated in primary T-ALL samples, and MBD2 deletion didnot induce a change in its DNA methylation level. The results ofChIP assay further illustrated that MBD2 likely functions as areader of DNA methylome by binding to the methylated CpGelements of the targeted gene SFRP5.

Our findings have important clinical implications. First, com-pared with DNMT knockout mice and knockout mice for otherMBD family members, MBD2 knockout mice were reported tohave a surprisingly weak phenotype (43, 44). The surprisinglyweak phenotype of MBD2 deficiency and the requirement ofMBD2 for the maintenance of Notch1-driven T-ALL should allowthe maximization of anti-T-ALL activity and the minimization ofoff-target toxicity in normal tissues. This study has highlighted

MBD2 as an attractive therapeutic target for T-ALL. Second,although aberrant DNA methylation is increasingly being recog-nized as a common feature in ALL, there is little evidence availableto support a potential role of demethylating therapy in treatinglymphoidmalignancies. The targeting ofMBD2 in T-ALL providesa new starting point for the potential application of demethylat-ing therapy in T-ALL and other lymphoid malignancies. Finally,the interestingfinding thatMBD2 impact T lymphopoiesis strong-ly argues for the clinical relevance of MBD2 as a potentiallyvaluable epigenetic target for the treatment of T-cell–relateddisease states, ranging from autoimmunity to transplantation.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Zhou, W. Yuan, J. ZhouDevelopment of methodology:M. Zhou, K. Zhou, J. Wang, Y. Zhang, S. ZhangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Zhou, K. Zhou, L. Cheng, X. Chen, Q. YuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Zhou, K. Zhou, W. YuanWriting, review, and/or revision of the manuscript: M. Zhou, K. Zhou,W. Yuan, J. ZhouAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X.-M. Wang, S. Zhang, D. Wang, L. Huang,M. Huang, D. Ma, T. Cheng, C.-Y. WangStudy supervision: W. Yuan, J. Zhou

AcknowledgmentsThis work was supported in part by the Key Program of National Natural

Science Funds (NNSF) of China (81230052 and 81630006 to J. Zhou),Innovative Collaboration Grant (NNSF) of China (81421002 to T. Cheng andW. Yuan), Overseas Collaboration Grand (NNSF) of China (81629001 to W.Yuan), the General Program of NNSF of China (81270599 to M. Huang), the"863" Program of the China Ministry of Science and Technology(2014AA020532 to L. Huang), the Youth Science Fund Project of NNSF ofChina (81400122 to K. Zhou; 81300410 to D. Wang). We thank all of themembers of Department of Hematology and Cancer Biology Research Center atHuazhong University of Science and Technology for their helpful discussions,and the members of State Key Laboratory of Experimental Hematology atChinese Academy of Medical Sciences and Peking Union Medical College fortheir technical assistance.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 26, 2017; revised November 23, 2017; accepted January 9,2018; published OnlineFirst January 12, 2018.

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