MBD2 Ablation Impairs Lymphopoiesis and Impedes Progression and Maintenance Of

Author Manuscript Published OnlineFirst on January 12, 2018; DOI: 10.1158/0008-5472.CAN-17-1434 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

MBD2 ablation impairs lymphopoiesis and impedes progression and maintenance of

T-ALL

Mi 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*, Jianfeng Zhou1,4*

1Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University

of Science and Technology, Wuhan, Hubei, China; 2State Key Laboratory of Experimental

Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of

Medical Sciences and Peking Union Medical College, 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 Medical College, Huazhong University of Science and Technology, Wuhan, Hubei,

China

&Authors contributed equally to this study.

Running title: MBD2 as a potent target in T-ALL

Abbreviations list

MBD2, methyl-CpG-binding domain protein 2; T-ALL, T cell acute lymphoblastic leukemia;

DNMT, DNA methyltransferase; WBC, white blood cell; PB, peripheral blood; DN, double

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 12, 2018; DOI: 10.1158/0008-5472.CAN-17-1434 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

negative; DP, double positive; HSC, hematopoietic stem cells; HPC, hematopoietic progenitor

cells; LT-HSC, long-term HSC; ST-HSC, short-term HSC; MPP, multipotent progenitor cells;

CMP, common myeloid progenitor cells; CLP, common lymphoid progenitor cells; GMP,

granulocyte-monocyte progenitor cells; MEP, megakaryocyte-erythrocyte progenitor cells;

T-ALL, T-cell acute lymphoblastic leukemia; GEP, gene expression profiles; GSEA, Gene set

enrichment analysis; DKK1, Dickkopf-1; SFRP, secreted Frizzled-related protein; ICN1,

intracellular domain of Notch1; CNS, central nervous system; CRISPR/Cas9, clustered

regularly interspaced short palindrome repeats associated nuclease Cas9; ChIP, Chromatin

immunoprecipitation.

Keywords: T-cell acute lymphoblastic leukemia, methyl-CpG-binding domain protein 2,

DNA methylation, epigenetics, leukemia mouse model

Financial support: This work was supported in part by the Key Program of National Natural

Science Funds (NNSF) of China (J Zhou; 81230052, 81630006), Innovative Collaboration

Grant (NNSF) of China (T. Cheng, W. Yuan; 81421002 )，Overseas Collaboration Grand

(NNSF) of China (W. Yuan; 81629001), the General Program of NNSF of China (M Huang;

81270599), the “863” Program of the China Ministry of Science and Technology (L Huang;

2014AA020532), the Youth Science Fund Project of NNSF of China (K Zhou, 81400122; D

Wang, 81300410).

*Corresponding author: Dr. Jianfeng Zhou, Tel and Fax: 86 (27) 83662680, E-mail:

[email protected]; Dr. Weiping Yuan, Tel and Fax: 86 (22) 23909047, E-mail:

[email protected]; Dr. Cong-Yi Wang, Tel and Fax: 86 (27) 83663486, E-mail:

[email protected]

Conflicts of Interest

The authors have no conflicts of interest to declare.

Word count for text: 4960

Figure count: 6

Table count: 1

Abstract

Aberrant DNA methylation patterns in leukemia might be exploited for therapeutic targeting.

In this study, we employed a genetically deficient mouse model to explore the role of the

methylated DNA binding protein MBD2 in normal and malignant hematopoiesis. MBD2

ablation led to diminished lymphocytes. Functional defects of the lymphoid compartment

were also observed after in vivo reconstitution of MBD2-deficient hematopoietic stem cells

(HSC). In an established model of Notch1-driven T cell acute lymphoblastic leukemia

(T-ALL), MBD2 ablation impeded malignant progression and maintenance by attenuating the

Wnt signaling pathway. In clinical specimens of human T-ALL, Wnt signaling pathway

signatures were significantly enhanced and positively correlated with the expression and

function of MBD2. Further, a number of typical Wnt signaling inhibitory genes were

abnormally hypermethylated in primary human T-ALL. Abnormal activation of Wnt signaling

in T-ALL was switched off by MBD2 deletion, partially by reactivating epigenetically

silenced Wnt signaling inhibitors. Taken together, our results define essential roles for MBD2

in lymphopoiesis and T-ALL and suggest MBD2 as a candidate therapeutic target in T-ALL.

Introduction

DNA methylation is introduced by at least three DNA methyltransferases (DNMTs), including

DNMT3a and DNMT3b for de novo methylation, and DNMT1 for methylation

maintenance(1,2). To recognize and “translate” the methylated DNA into signals for

transcriptional repression, DNA methylation must be read by a conserved family of

methyl-CpG-binding domain (MBD) proteins(3). The “reader” proteins of the MBD family in

mammals include five known members named MeCP2, MBD1, MBD2, MBD3, and MBD4,

which recognize and bind methylated CpG sequences, and in turn control gene expression by

interrupting the binding of transcription factors to the corresponding promoter region(4).

Aberrant DNA methylation patterns in tumor cells represent attractive and novel

therapeutic targets(5-7). Therapy with DNMT inhibitors has shown robust clinical activity

and clearly alters the natural progression of several hematopoietic malignancies(8-11). DNMT

inhibitors curtail and even reverse the tumor-associated signature of aberrant DNA

methylation and associated gene silencing in cancer(12,13). Despite rapid clinical progress,

the utility of DNMT inhibitors has been limited by the toxicity and undesired off-target effects

associated with non-specific global demethylation(14). Alternatively, targeting the “reader”

proteins of DNA methylation instead of using DNMT inhibitors might be a highly attractive

strategy for epigenetic therapy(15).

Of all the MBD proteins, MBD2 has been proposed as the most promising target(15).

Intriguingly, unlike other MBD members, the deletion of MBD2 in mice does not generate

any major deleterious effects(3,16-19), indicating that MBD2 could be dispensable under

normal physiological conditions and would therefore enable the minimization of off-target

toxicity to normal tissues. MBD2 had been shown to mediate the inhibition of aberrantly

methylated tumor suppressor genes by binding to methylated regulatory promoter

regions(20-22), and knockdown of MBD2 could suppress neoplastic cell growth by

reactivating the transcription of tumor suppressor genes(21,23). When MBD2-deficient mice

were crossed with ApcMin/+ (or Min) background mice, the development of intestinal tumors

was attenuated(22,24), indicating that MBD2 is crucial for this tumor-promoting effect.

In the present study, we sought to investigate the role of MBD2 in normal and malignant

hematopoiesis. Our study showed that the loss of MBD2 significantly impaired lymphoid

hematopoiesis. In Notch1-driven T cell acute lymphoblastic leukemia (T-ALL), MBD2 was

required for the progression and maintenance of leukemia. The present study highlights the

great potential of using MBD2 as a therapeutic target in T cell-related pathologies.

Materials and Methods

Mice. MBD2 deficient (Mbd2-/-) mice on a C57BL/6-CD45.2 genetic background were a gift

kindly provided by Dr. Adrian Bird (Edinburgh University, Edinburgh, UK)(19). All mice

were maintained in a pathogen-free animal facility at Tongji Hospital of Tongji Medical

College, Huazhong University of Science and Technology, Wuhan, China. All animal studies

were approved by the Institutional Committee of Animal Care and Treatment in Tongji

Hospital. Six to eight-week-old male Mbd2-/- mice and WT littermates were used in this study.

Clinical samples. For T-ALL samples, leukocytes were isolated from BM specimens by

Ficoll gradient and stored frozen in aliquots. All specimens were collected before

chemotherapy. Written informed consent was obtained from each patient and healthy

volunteer donor in accordance with the principles expressed in the Declaration of Helsinki,

and the study was approved by the Institutional Review Committee for the use of human

materials at Tongji Hospital. The global gene expression profiles of primary T-ALL samples

were obtained from Gene Expression Omnibus (GEO).

Cell lines. Jurkat (T-ALL) and Molt4 (T-ALL) cell lines were originally purchased from

ATCC (American type culture collection, Manassas, VA) in 2011, authenticated by STR, and

tested to ensure that they were mycoplasma-free by direct culture within 3 months of use. The

cell lines were cultured in RPMI 1640 medium supplemented with 10% FCS (Gibco,

Invitrogen, New York, NY), and used for experimentation within 1 month of being thawed

from frozen stocks.

Non-competitive repopulation assays. 1 × 106 BM cells from Mbd2-/- or littermate WT mice

(both CD45.2+) were injected intravenously into lethally irradiated CD45.1+ recipient animals

(9.5 Gy in two doses, 4 h apart). Peripheral blood (PB) cells were collected 1, 2, 3 and 4

months after transplantation. Five months after transplantation, recipient mice were sacrificed,

and BM cells were collected. The contribution of CD45.2+ donor-derived cells in the PB and

BM of recipient mice was analyzed by flow cytometry. CD45.2 antibody (FITC) was used to

detect the donor cells.

Murine T-ALL model. The retrovirus vector encoding the ICN1 gene was a gift from Dr.

David Scadden (Harvard University, Boston, MA). The MSCV-ICN1-IRES-GFP plasmid was

co-transfected into the package 293T cells with pKat and pCMV-VSV-G via Lipofectamine

2000 (Invitrogen, New York, NY). The supernatant of 293T cells was harvested. The

transduction of Lin- cells from the BM of WT or Mbd2-/- mice with viral supernatant was

performed as previously described(25). Leukemic mice were sacrificed 2~3 months after

transplantation, and the BM cells were harvested as P0 cells. Then, we transplanted P0

leukemic cells into sublethally irradiated (6.5 Gy in one dose, six to eight-week-old) or

non-irradiated recipients (six to eight-week-old) to establish a leukemic mouse model as P1.

Flow cytometry. An LSR II cytometer and FlowJo7.6 software (BD Biosciences, San Jose,

CA) were used for data acquisition and analysis. All antibodies were from BD Biosciences or

e-Bioscience unless otherwise indicated. Cell sorting was performed using a fluorescence

activated cell sorter (FACS, Aria Ⅲ cell sorter, BD Biosciences).

BrdU detection. When the proportion of GFP+ leukemic cells was > 50% in mononuclear

cells of the BM of the WT recipient mice, the mice were given a single pulse administration

of 5-bromo-2’-deoxyuridine (BrdU, Sigma-Aldrich, St. Louis, MO). An intraperitoneal

injection of BrdU (1 mg/6 g) was given 2 hours before harvesting the BM cells. A BrdU-APC

staining kit (BD Biosciences) was used according to the manufacturer’s instructions. BrdU

staining was analyzed using flow cytometry by surface markers and intracellular staining.

Western blotting and antibodies. Protein sample preparation and Western blot (WB) were

performed as previously described(26). Commercial antibodies against the following proteins

were purchased: MBD2 (Santa Cruz, Dallas, Texas); β-catenin, histone H3 and GAPDH (Cell

Signaling Technologies, Danvers, Massachusetts); and GATA2 (Abcam, Cambridge, MA).

Q‒PCR. Total RNA was isolated from cells using TRIzol and reverse

transcription-polymerase chain reaction (RT‒PCR) was then performed. Q‒PCR was

conducted using an ABI Prism 7900 Sequence Detection System (Applied Biosystems, Grand

Island, NY) with SYBR Green Supermix (Applied Biosystems) following the manufacturer’s

instructions. All data were normalized using the endogenous GAPDH control, and the relative

gene expression was calculated using the ΔΔCt method. All primer sequences are summarized

in Supplemental Table S1.

Immunohistochemistry (IHC). Tissue biopsy samples were subjected to IHC to detect the

expression of GFP using a GFP antibody (MBL, Tokyo, Japan) diluted 1:1000. The

microscope used for imaging was an OLYMPUS BX51 with an OLYMPUS DP72 camera

(Olympus, Tokyo, Japan).

Microarray. Total RNA was extracted with TRIzol from GFP+ T-ALL cells and double

positive (DP, CD4+CD8+) thymocytes in TRIzol (Invitrogen, New York, NY). The cDNA was

amplified before array analysis, and 1.5 μg of cDNA from each sample was hybridized to

GeneChip® Mouse Genome 430 2.0 microarrays (Affymetrix, Santa Clara, CA) according to

the manufacturer’s instructions. Genes with a P value below 0.01 and a fold change greater

than 2.0 were considered significant. Hierarchical clustering was generated using cluster 3.02

(Stanford University, CA). Gene set enrichment analysis (GSEA) was used to identify classes

of genes significantly enriched within genes being regulated upon MBD2 knockout.

Microarray raw data are available for download at Gene Expression Omnibus

(http://ncbi.nlm.nih.gov/geo) under accession number GSE105763.

Global methylation profiling. Ten T-ALL patient BM samples and ten normal CD3+ BM

cells from healthy donors were collected, and all T-ALL BM samples had more than 80%

leukemic cells. Genomic DNA was extracted as previously described. All cases were analyzed

by an Illumina Human Methylation 450K (Illumina Inc., San Diego, CA) according to the

manufacturer’s instructions. Signal intensities were obtained from GenomeStudio (Illumina),

converted to β values, filtered, and normalized to remove biases between Infinium I and II

probes. A DNA methylation β value was reported as a DNA methylation index ranging from 0

(non-methylated) to 1 (completely methylated). CpG probes with aberrant methylation in

T-ALL cells compared with those in normal T cells were identified as having a P < 0.01 and a

mean β value difference > 0.2. Hierarchical clustering was generated using cluster 3.02

(Stanford University, CA).

DNA bisulfite sequencing analysis. Genomic DNA was extracted using a QIAamp DNA

mini kit (QIAGEN, Valencia, CA) and then subjected to bisulfite conversion using an EZ

DNA Methylation Kit (Zymo, Orange, CA) followed by PCR of the targeted sequence. The

PCR amplifications were analyzed by agarose gel electrophoresis and then subcloned into the

pEASY-T1 Simple Cloning Vector (TransGen Biotech, Beijing, China). Ten positive clones

derived from each PCR product were randomly selected for DNA sequencing analysis.

Chromatin immunoprecipitation (ChIP) assay. The experiment was performed with a ChIP

assay kit (Millipore, Bedford, MA). The primers used in ChIP assays are listed in

Supplemental Table S2. The anti-MBD2 antibody used in ChIP assays was purchased from

Bethyl Laboratory (Bethyl Laboratories, Montgomery, TX).

Results

MBD2 deficiency led to diminished lymphocytes

To assess MBD2 expression in hematopoietic cells, we searched public databases(27) of the

MBD protein family and found that MBD2 was expressed at significantly higher levels in

more mature blood cell lineages than in HSCs/HPCs in normal mouse and human

hematopoiesis (Figure 1A‒B, Figure S1A‒H). The role of MBD2 in hematopoiesis was

explored by utilizing Mbd2-/- mice and WT littermates. The deletion of MBD2 alleles and

abolished transcriptional expression were examined (Figure S2A‒B). No significant

difference in platelets or red blood cells was observed between Mbd2-/- and WT mice (Figure

S3A‒B). The cellularity of the BM, spleen and thymus was also unaffected by MBD2

deletion (Figure S3C). However, compared with WT mice, Mbd2-/- mice showed a

dramatically reduced white blood cell count in PB compared to their WT littermates, which

was attributable to strikingly decreased lymphocytes counts (Figure 1C). Flow cytometry

analysis corroborated these findings (Figure 1D). Nevertheless, the frequency of lymphocytes

or myeloid cells in the BM was equivalent between Mbd2-/- and WT mice (Figure 1E). Further

examination of DN (double negative, CD4-CD8-) and DP (CD4+CD8+) thymocytes showed

increased DN and decreased DP thymocytes in Mbd2-/- mice relative to WT mice, although the

differences were not statistically significant (Figure 1F). The absolute number of single

positive CD4+ or CD8+ thymocytes was equivalent between the two groups (Figure 1G). In

HSC/HPC pools, no significant differences in the frequencies of HSCs and HPCs in the BM

were observed between Mbd2-/- and WT mice (Figure 1H‒I). Therefore, MBD2 deletion led to

diminished numbers of lymphocytes but did not alter the size of the HSC and HPC

compartments.

Loss of MBD2 significantly impaired the reconstitution capacity of HSCs, which

resulted in a diminished lymphoid compartment in vivo

Because the Mbd2-/- mouse is deficient for MBD2 in both blood cells and the hematopoietic

microenvironment(19), we adopted a transplantation strategy to address the direct effects of

MBD2 deletion on HSCs/HPCs. There was no significant difference in the homing efficiency

of donor cells to the recipient BM between Mbd2-/- and WT mice (Figure 2A). In

non-competitive transplantation, MBD2 ablation dramatically diminished CD45.2+ cell

reconstitution compared to WT littermates in the PB of recipient mice (Figure 2B). Five

months after transplantation, the engraftment of B and T lymphocytes but not myeloid cells

remained dramatically impaired by MBD2 deletion in the PB (Figure 2C); in the BM, the

engraftment of total CD45.2+ and B lymphocytes was impaired (Figure 2D). In various

lymphoid organs of recipient mice, MBD2 deletion dramatically impeded the reconstitution of

CD45.2+ lymphocytes (Figure 2E). Moreover, the engraftment of HSCs/HPCs, with the

exception of CLPs in the BM, was also impeded by the loss of MBD2 (Figure 2F‒G).

Therefore, MBD2 loss significantly impaired the reconstitution capacity of HSCs/HPCs,

which resulted in a diminished lymphoid compartment.

Loss of MBD2 significantly delayed Notch1-induced leukemogenesis, and MBD2 is

critical for the maintenance of T-ALL

Because MBD2 loss led to a pronounced decrease in T lymphocytes, we asked whether

MBD2 would be essential for T-ALL leukemogenesis in vivo. A Notch1-induced mouse

T-ALL model was generated (Figure 3A). Consistent with previous studies, mice transplanted

with ICN1-infected WT cells showed an expansion in the percentage of CD4+CD8+ cells in

the BM and PB two weeks after transplantation. Recipient mice transplanted with

ICN1-infected Mbd2-/- cells developed leukemia with a much longer latency (Figure 3B‒C).

All recipient mice eventually developed signs of overt T-ALL and died within 70 days of

transplantation (Figure 3C). The longer latency for leukemia in Mbd2-/- group was apparently

not due to a weakened homing efficiency (Figure 3D).

To determine the functional impact of MBD2 deletion on the maintenance of Notch1-

driven T-ALL leukemia, a P1 leukemic mouse model was generated (Figure 3A). In the

subirradiated model, mice in the Mbd2-/- group had a significantly longer overall survival

(Figure 3E). Strikingly, in the non-irradiated model, mice in the Mbd2-/- group regenerated

T-ALL with an extremely longer latency and had a significantly longer overall survival

(Figure 3F). Moreover, while WT leukemic cells continued to retain their potent capacity to

develop leukemia, 40% of Mbd2-/- leukemic cells did not ultimately generate T-ALL (Figure

3F). No leukemic cell infiltration in the BM or spleen was detected in the Mbd2-/- group 120

days after transplantation (Figure S4).

To assess the malignant features of Mbd2-/- T-ALL cells, in vivo proliferation, apoptosis,

and the frequencies of central nervous system (CNS) infiltration were compared between

Mbd2-/- and WT T-ALL cells in non-irradiated recipients. In vivo BrdU incorporation studies

showed that Mbd2-/- T-ALL cells grew much more slowly (Figure 3G), but no significant

difference in apoptosis was observed (Figure S5A‒B). Amazingly, the T-ALL mice

transplanted with WT cells presented with CNS leukemia infiltration much more frequently

than Mbd2-/- T-ALL mice (Figure 3H‒I).

MBD2 ablation impeded the progression of Notch1-driven T-ALL by inhibiting the Wnt

signaling pathway

To explore potential mechanisms underlying the anti-ALL effects of MBD2 ablation,

leukemic cells were harvested from mice transplanted with WT or Mbd2-/- T-ALL cells, and

their global gene expression profiles (GEPs) were compared. Normal DP thymocytes were

isolated and used as a normal control. The different GEPs of WT T-ALL and DP cells

generated leukemia-associated gene signatures. Intriguingly, MBD2 ablation reversed 26% of

the leukemia-associated gene signatures in T-ALL cells (Figure 4A), leading to the

upregulation of 1,987 genes and the downregulation of 938 genes. However, most genes in

Mbd2-/- DP cells remained unchanged compared to WT DP cells (Figure S6A), and MBD2

ablation did not affect the expression of other MBD family members in DP and T-ALL cells

(Figure S6B). GSEA revealed that MBD2 loss in T-ALL cells resulted in a significantly

attenuated Wnt signaling pathway signature and the inhibition of cell proliferation (Figure

4B). Moreover, MBD2 deletion in leukemic cells led to the substantially altered expression of

a number of critical Wnt signaling regulators (Figure 4C, Table 1)(28,29). Interestingly,

MBD2 loss in T-ALL cells led to increased Notch pathway (Figure S6C‒D), indicating a

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

Previously, the abnormal epigenetic modification of various critical Wnt signaling

regulators was shown to be responsible for the aberrant activation of Wnt signaling in cancer

cells(28,32). Therefore, we further validated the microarray data by examining the expression

of 9 typical Wnt signaling regulators using q‒PCR (Figure 4D). Notably, MBD2 deficiency

inhibited Wnt signaling at multiple levels by affecting the transcriptional expression of both

negative and positive Wnt signaling regulators (Figure 4E). Accordingly, the expression of

CD44, a downstream target gene of the Wnt signaling pathway(29), was downregulated more

than 14-fold in Mbd2-/- T-ALL cells compared with that in WT T-ALL cells. These data

indicated that MBD2 deletion impeded the progression of T-ALL by inhibiting the Wnt

signaling pathway.

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

Based on our results in murine T-ALL models, we further explored the global gene expression

profiles of 174 primary T-ALL samples to determine the potential clinical relevance of MBD2

in human T-ALL. GSEA revealed that compared with normal BM, human T-ALLs showed

significantly increased Wnt signaling pathway signature (GEO GSE13204, Figure 5A‒B). To

further confirm enhanced Wnt signaling in T-ALL, we analyzed the β-catenin protein level

and found that β-catenin was higher in primary T-ALL samples than in normal T cells samples

(Figure S7A). To determine the possible correlation between MBD2 and the Wnt signaling

pathway in human T-ALL, MBD2 and TCF7 expression levels were extracted from 174

primary T-ALL samples, which are available in a publicly accessible database (GEO

GSE13204). Pearson’s correlation analysis (P ≤ 0.05) showed that MBD2 expression was

positively correlated with TCF7 expression, a known important Wnt transcriptional factor(33),

among these primary T-ALL samples (Figure 5C). The positive correlation of TCF7

expression with MBD2 expression in T-ALL was again observed in our primary T-ALL

patient cohort (n = 20, Figure S7B). On the other hand, our data showed that the TCF7

expression level was significantly higher in T-ALL, while the MBD2 expression level was not

significantly different between T-ALL and normal T cells, illustrating that MBD2 was

probably heterogeneously expressed in leukemia (Figure S7C). Furthermore, using the Wnt

signaling gene set from KEGG (Kyoto Encyclopedia of Genes and Genomes, Supplemental

Table S3), unsupervised hierarchical clustering analysis of primary T-ALL samples (GEO

GSE62156, GSE33469) was performed. The differential signatures of Wnt signaling stratified

T-ALLs into two distinct clusters, cluster 1 T-ALLs display distinct MBD2 expression levels

relative to T-ALLs within cluster 2 (Figure S8 A‒B).

Next, we sought to address how MBD2 correlated with the Wnt signaling. Since attempts

with siRNA failed to yield greater than 50% knockdown of MBD2 protein in T-ALL cell lines,

a clustered regularly interspaced short palindrome repeat associated nuclease Cas9

(CRISPR/Cas9) strategy(34) was used to generate a stable cell line with haploid deletion of

the MBD2 gene (Figure S9A‒B). Interestingly, a haploid deletion of the MBD2 gene in Jurkat

cells caused a complete loss of endogenous MBD2 protein (Figure 5D). Notably, the

transcription levels of a positive Wnt regulator (TCF7)(33) and downstream Wnt target genes

(CD44, c-MYC)(29,35) were significantly decreased upon MBD2 depletion in Jurkat cells

(Figure 5E). To further address the consequence of MBD2 deletion on Wnt signaling pathway

activity, we determined the protein levels of β-catenin, the pivotal mediator of Wnt

signaling(36,37). MBD2-deficient Jurkat cells had significantly decreased expression of both

total and nuclear β-catenin, and decreased cell proliferation with G0/G1 cell cycle arrest,

indicating inactive Wnt signaling in the absence of MBD2 (Figure 5F‒G). Similar results

were observed in mutant Molt4 clones and WT clones (Figure 5H‒I). Furthermore, the

decreased expression of Wnt target genes as well as the decreased cell proliferation could be

significantly rescued by transfection with lentivirus encoding human β-catenin into

MBD2-deficient T-ALL cells (Figure 5G, 5J). Overexpression of β-catenin in MBD2-deficient

Jurkat cells did not affect MBD2 ablation (Figure 5K). Taken together, these results indicated

that human T-ALLs show significantly increased Wnt signaling pathway signatures, which

were positively correlated with MBD2 expression.

Abnormally hypermethylated negative Wnt regulators require MBD2 to activate Wnt

signaling in human T-ALL cells

MBD2 binds to methylated CpG islands and translates the methylated DNA into signals for

transcriptional repression(21). Therefore, we hypothesized that the promoter regions of some

genes were methylated in T-ALL and were read by MBD2. To investigate this, we subjected

primary human T-ALL samples and CD3+ BM T-lymphocytes to genome-wide DNA

methylation profiling analysis. Compared with normal CD3+ T-lymphocytes, primary T-ALL

cells showed hypermethylation of the promoter regions of approximately 2,800 genes and

hypomethylation of 596 gene promoters (Figure 6A). Consistent with previous reports,

PTPN6 and GALNT6 were hypermethylated and hypomethylated(38,39), respectively, in

T-ALL (Figure 6B). Strikingly, a number of typical Wnt signaling inhibitors, were

hypermethylated to a greater extent than that of the positive control (PTPN6) in human

T-ALL (Figure 6B). In line with these findings, the transcription levels of these Wnt signaling

inhibitors were consistently lower in T-ALL samples relative to normal CD3+ T-lymphocytes

(Figure 6C). Next, we sought to address whether MBD2 plays a role in regulating the

transcriptional expression of typical Wnt signaling inhibitors. The transcription levels of these

negative Wnt regulators were consequently reactivated upon MBD2 depletion, and the

upregulation of SFRP5 was shown to be the same in both mutant clones (Figure 6D),

suggesting that the knockdown of MBD2 could effectively relieve the transcriptional

suppression of several abnormally methylated genes.

To validate the methylation microarray and transcription data, we analyzed the methylation

of the 342-bp promoter of SFRP5 by bisulfite sequencing (Figure 6E). While a low

methylation rate was detected in normal CD3+ T cells, significantly increased methylation in

the same region was detected in T-ALL (Figure 6F‒G and Figure S10A‒B). To confirm

whether MBD2 deletion induced a change in DNA methylation levels of SFRP5, which might

regulate SFRP5 through demethylation way, we performed bisulfite sequencing on parent,

WT and mutant Jurkat clones, as well as murine T-ALL cells. We found that MBD2 deletion

did not affect the methylation rate of the SFRP5 promoter region (Figure 6H). Next, we used

ChIP assays to determine whether MBD2 regulates the transcriptional expression of SFRP5

by directly binding to its promoter region. FGF19 was selected as a positive control, and

GAPDH was selected as a negative control(40). A definite binding of MBD2 protein to the

SFRP5 promoter region was detected in Jurkat T-ALL cells (Figure 6I), supporting the

hypothesis that MBD2 suppressed the transcriptional expression of SFRP5 by directly binding

to its promoter in leukemic cells.

DISCUSSION

Although MBD2, one of the “readers” of DNA methylation, has been regarded as the most

promising target for the next generation of DNA demethylation therapy(15,24), its role in

hematopoiesis has not been comprehensively investigated. In the present study, we utilized

MBD2 knockout mice to investigate the role of MBD2 in normal and malignant

hematopoiesis, and provided strong evidence to support the potential of MBD2 as a

therapeutic target for T-ALL.

The sequential steps of hematopoiesis are precisely controlled by several regulatory

mechanisms, in which epigenetic mechanisms play a vital role(41,42). Despite substantial

advances in understanding the functional role of DNA methylation regulators in

hematopoiesis(43-45), little is known about the role of the MBD family in hematopoiesis. In

publicly available data, MBD2 was expressed at significantly higher levels in more mature

blood cell lineages than in HSCs/HPCs, indicating that MBD2 might play an essential role in

the sequential steps of hematopoiesis. In our study, while MBD2 knockout mice were viable

and did not develop any obvious illness, MBD2 deficiency in a “steady-state” gave rise to

profoundly diminished lymphocytes. Importantly, MBD2 deletion did not alter the

frequencies of HSCs/HPCs. In transplantation studies, the reconstitution capacities of various

HSC/HPC subsets were impaired to various degrees by MBD2 ablation. Nevertheless, as far

as myeloid hematopoiesis is concerned, MBD2-deficient mice display a rather weak

phenotype, since MBD2-deficient HSCs/HPCs ultimately had normal production of myeloid

progeny despite the impaired function of HSCs/HPCs. However, MBD2 loss dramatically

impaired the capacity of HSCs to form lymphoid progeny. Previously, redundancy was

reported among various MBD proteins in mediating their repression of methylated genes(21).

For example, MBD3 has been shown to partially compensate for the phenotypes caused by

MBD2 deficiency(3,19). It is therefore possible that the weak phenotype of myeloid

hematopoiesis in MBD2-deficient mice is due to the presence of a redundant mechanism. By

contrast, MBD2 appears to play an indispensable role in lymphopoiesis.

Beyond the impact on normal T lymphopoiesis, MBD2 ablation substantially impeded the

progression of Notch1-driven T-ALL. Although MBD2 deletion is not sufficient to block the

onset of T-ALL in mice, it significantly delayed Notch1-induced leukemogenesis. Strikingly,

Mbd2-/- T-ALL cells were much less aggressive than Mbd2+/+ T-ALL cells in vivo. More

importantly, MBD2 was critical for the maintenance of T-ALL in non-irradiated recipients. To

explore underlying mechanisms of MBD2 in T-ALL, we exploited an integrated strategy

consisting of microarrays, pathway enrichment analysis, expression validation of target genes

in T-ALL mice or cell lines, and global DNA methylation analysis in clinical samples to

investigate targeted genes and related signaling pathways. In the T-ALL mouse model, we

found that MBD2 ablation inhibited Wnt signaling at multiple layers and reversed the

leukemia-associated gene signatures. The Wnt signaling pathway regulates the stability of

co-activator β-catenin and thus activates the Lef/Tcf family of transcription factors and the

expression of a set of target genes, which in turn regulates cell proliferation, behavior and

survival(46). Our in vitro study showed that MBD2 ablation led to decreased accumulation of

nuclear β-catenin, a hallmark of active Wnt signaling(37), resulting in the suppression of

transcription factor TCF7 and Wnt target genes. The functional restoration of Wnt signaling

by overexpressing β-catenin led to the restoration of the Wnt signaling target genes and cell

proliferation in MBD2-deficient T-ALL cells, clearly demonstrating the positive correlation

between MBD2 and Wnt signaling. Interestingly, in this study, MBD2 loss in T-ALL mice led

to increased Notch pathway and decreased Wnt signaling signatures. It has been reported that

Wnt and Notch fulfill opposing functions in normal development: Wnt in proliferation and

Notch in differentiation in the thymus. In cancer studies, Notch signaling induces

differentiation and acts as a tumor suppressor by inhibiting β-catenin-mediated signaling in

the skin, and Notch1 signaling retains the capability to suppress the expression of Wnt target

genes in colorectal cancers(30,31). These findings further imply that Notch and Wnt signaling

activity might have a negative correlation in the development of many kinds of diseases.

The findings in T-ALL mice and T-ALL cell lines are clinically relevant. In hematological

malignancies, it has been reported that the aberrant activation of Wnt signaling is frequent and

is associated with a poor prognosis in ALL due to abnormal DNA methylation(47). SFRP4

was found to be frequently methylated in CLL samples(48), while WIF1 methylation was a

poor prognostic 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 pathway signatures, which were positively correlated with the

expression and function of MBD2. Our global DNA methylation analysis on human primary

T-ALL revealed an abnormal methylation signature that facilitated an aberrant activation of

Wnt signaling. Strikingly, this abnormal activation of Wnt signaling in T-ALL could be

effectively switched off by the deletion of MBD2, partially by reactivating epigenetically

silenced Wnt signaling inhibitors, which subsequently impeded the progression of

Notch1-driven T-ALL. The SFRP family members possess a domain similar to one in the

Wnt-receptor Frizzled protein and can inhibit Wnt receptor binding to downregulate pathway

signaling. Epigenetic loss of SFRP may provide constitutive activation of Wnt signaling in the

evolution of cancers(50). In our study, bisulfite sequencing analysis of the promoter regions

demonstrated direct evidence that SFRP5 was remarkably hypermethylated in primary T-ALL

samples, and MBD2 deletion did not induce a change in its DNA methylation level. The

results of ChIP assay further illustrated that MBD2 likely functions as a reader of DNA

methylome by binding to the methylated CpG elements of the targeted gene SFRP5.

Our findings have important clinical implications. First, compared with DNMT knockout

mice and knockout mice for other MBD family members, MBD2 knockout mice were

reported to have a surprisingly weak phenotype(43,44). The surprisingly weak phenotype of

MBD2 deficiency and the requirement of MBD2 for the maintenance of Notch1-driven

T-ALL should allow the maximization of anti-T-ALL activity and the minimization of

off-target toxicity in normal tissues. The present study has highlighted MBD2 as an attractive

therapeutic target for T-ALL. Second, although aberrant DNA methylation is increasingly

being recognized as a common feature in ALL, there is little evidence available to support a

potential role of demethylating therapy in treating lymphoid malignancies. The targeting of

MBD2 in T-ALL provides a new starting point for the potential application of demethylating

therapy in T-ALL and other lymphoid malignancies. Finally, the interesting finding that

MBD2 impact T lymphopoiesis strongly argues for the clinical relevance of MBD2 as a

potentially valuable epigenetic target for the treatment of T cell-related disease states, ranging

from autoimmunity to transplantation.

Acknowledgements

We thank all of the members of Department of Hematology and Cancer Biology Research

Center at Huazhong University of Science and Technology for their helpful discussions, and

the members of State Key Laboratory of Experimental Hematology at Chinese Academy of

Medical Sciences and Peking Union Medical College for their technical assistance. This work

was supported in part by the Key Program of National Natural Science Funds (NNSF) of

China (J Zhou; 81230052, 81630006), Innovative Collaboration Grant (NNSF) of China (T.

Cheng, W. Yuan; 81421002 ), Overseas Collaboration Grand (NNSF) of China (W. Yuan;

81629001), CAMS Innovation Fund for Medical Sciences, CIFMS (W. Yuan, X. Wang;

2017-I2M-3-015, T. Cheng; 2016-I2M-1-017), the General Program of NNSF of China (M

Huang; 81270599), the “863” Program of the China Ministry of Science and Technology (L

Huang; 2014AA020532), the Youth Science Fund Project of NNSF of China (K Zhou,

81400122; D Wang, 81300410).

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Table 1 List of relevant genes with respect to the Wnt signaling pathway in microarrays

of WT and Mbd2-/- murine T-ALL groups.

Down-regulated in T-ALL Mbd2-/- Up-regulated in T-ALL Mbd2-/-

Gene symbol Fold change Gene symbol Fold change

TCF7 0.32 Cdkn1a 2.10

Wnt8b 0.36 Sox13 12.48

Wnt5b 0.45 Dkkl1 7.29

Cd44 0.07 Cdh1 3.30

Figure legends

Figure 1. Loss of MBD2 led to a decreased lymphoid lineages phenotype. (A‒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 progenitor cell; CLP, common lymphoid progenitor cell;

GMP, granulocyte-monocyte progenitor cell; MEP, megakaryocyte-erythrocyte progenitor cell;

GRANU, granulocyte; MONO, monocyte. (C) WT and Mbd2-/- mice were examined for

hematopoietic cell counts of PB (n = 10 mice per group). WBC, white blood cell; LYM,

lymphocyte; NEUT, neutrophil; MONO, monocyte. (D‒E) The percentages of B and T

lymphocytes, and myeloid cells among the mononuclear cells of the PB (D) and BM (E) of

WT and Mbd2-/- mice (n = 5 mice per group). (F‒G) The counts or percentages of thymocyte

subsets of DN/DP (F), and CD4+CD8-/CD4-CD8+ (G) in Mbd2-/- and WT mice (n = 5 per

genotype) were analyzed by flow cytometry. (H‒I) The counts of HSCs (H) or HPCs (I) in the

BM of WT and Mbd2-/- mice (n = 5 per group). Error bars, SEM; *, P < 0.05; **, P < 0.005;

***, P < 0.001, two independent experiments.

Figure 2. MBD2 deficiency impaired HSC reconstitution capacity. (A) Homing assay.

Bars indicate the percentage of homing CFSE+ mononuclear cells found in the BM of

recipient mice. (B) Each lethally irradiated CD45.1+ mouse recipient was transplanted with

1×106 BM cells harvested from CD45.2+ WT or Mbd2-/- donor mice. Transplanted mice were

monitored by flow cytometry for the engraftment of CD45.2+ cells in PB at the indicated time

points. (C‒D) Transplanted mice were sacrificed five months after transplantations and

monitored for the engraftment of CD45.2+ cells in the PB (C) or BM (D). (E‒G) Five months

after transplantations, the engraftment of CD45.2+ thymocytes in the thymus,

CD4+CD8-/CD4-CD8+ T lymphocytes in lymph nodes and the spleen (E) and CD45.2+

HSCs/HPCs (F‒G) in the BM was quantified. Data represents the mean ± SEM of five

replicates determined from 5 mice over 2 independent experiments. *, P < 0.05; **, P <

0.005.

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-induced

T-ALL model. (B) The dynamics of GFP+ leukemic cells appearing in the PB in WT or

Mbd2-/- group (n = 15 per group) are shown. (C) The Kaplan-Meier survival curves for

recipient mice after transplantation are shown (n ≥ 8 per group, 2 independent experiments).

(D) Leukemic cell homing analysis (n = 5 per genotype, 2 independent experiments). (E‒F)

The Kaplan-Meier survival curves for sublethally irradiated (E) or non-irradiated (F) recipient

mice after transplantation are shown (n ≥ 8 per group, 3 independent experiments). (G)

Shown are the mean percentages ± SEM of BrdU+ cells within the total GFP+ leukemia cells

(left panel) and representative histograms (right panel) for the cohort (n=5 per group, 2

independent experiments) of Mbd2-/- versus WT leukemic mice. (H) Representative

hematoxylin and eosin staining (HE, left panel) and GFP immunohistochemistry (IHC, right

panel) images (×100 and ×600) of infiltration of T-ALL cells in the brain meningeal spaces of

WT leukemic mice and Mbd2-/- leukemic mice. B, brain; M, meninges; T, T-ALL cells. Scale

bar, 40μm. (I) The numbers of mice with meningeal leukemia detected in WT or Mbd2-/-

leukemic mice (n = 18 per group，2 independent experiments). Error bars, SEM; *, P < 0.05;

**, P < 0.005; ***, P < 0.001.

Figure 4. MBD2 ablation partially reversed T-ALL-associated gene signatures and

attenuated Wnt signaling. (A) Hierarchical clustering of normal CD4+CD8+ DP thymocytes

of WT normal mice and T-ALL cells sorted from P1 leukemic mice. (B) GSEA plot showing

the decreased expression of signaling by Wnt, Benporath proliferation and KEGG DNA

replication signatures in Mbd2-/- cells relative to WT T-ALL cells. NES, normalized

enrichment score. (C) Scatter plots of expression profiling for WT or Mbd2-/- T-ALL groups in

microarrays. (D) Validation of the microarray data of typical Wnt signaling regulators by

q‒PCR. 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; Black arrows, decreased

expression. Error bars, SEM; *, P < 0.05; **, P < 0.005; ***, P < 0.001.

Figure 5. MBD2 was positively correlated with Wnt signaling in human T-ALLs. (A‒B)

GSEA plot showing the increased expression of Wnt signaling pathway signatures in T-ALL

relative to normal BM. (C) Positive correlation of TCF7 with MBD2 in 174 T-ALL samples.

Statistics are from Pearson’s correlation analysis. (D) MBD2 expression in isogenic clones

and parental Jurkat was determined by WB. (E) The transcriptional expression levels of TCF7,

CD44 and c-MYC were determined in isogenic Jurkat clones. (F) Expression levels of total

and nuclear β-catenin protein in isogenic clones and parental Jurkat cells were determined by

WB. GAPDH and Histone H3 were used as internal controls. (G) Lentiviral particles

containing the GV358 expression vector encoding human β-catenin and the control lentivirus

were transfected into MBD2-deficient Jurkat cells (Jurkat mut1). The cell cycle was analyzed

in Jurkat wt1, Jurkat mut1, Jurkat mut1 β-catenin and Jurkat mut1 Ctrl cells using PI staining.

Typical flow cytometry profiles show representative data from three independent experiments.

(H‒I) Expression levels of MBD2, total and nuclear β-catenin protein in isogenic clones and

parental Molt4 cells were determined by WB. (J) q‒PCR confirmed the restoration of

β-catenin at the mRNA level. The transcription levels of TCF7 and downstream Wnt target

genes were analyzed by q‒PCR. (K) WB analysis of β-catenin and MBD2 expression in

Jurkat wt1, Jurkat mut1, Jurkat mut1 β-catenin and Jurkat mut1 Ctrl cells. GAPDH served as

a control. Error bars, SEM; *, P < 0.05; **, P < 0.005; ***, P < 0.001.

Figure 6. Aberrant hypermethylation of Wnt inhibitors in human T-ALL is read by

MBD2. (A) Global methylation profiling. (B) Mean β values of Wnt signaling inhibitors. (C)

Validation of the microarray data by q‒PCR 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 the different methylation profiles of the

SFRP5 promoter detected in normal CD3+ T cells and T-ALL samples (n = 10 per group). (G)

The average methylation levels for normal CD3+ T cell, T-ALL and Jurkat cells are shown. (H)

Left, the average SFRP5 methylation levels of Jurkat cells, WT Jurkat cells (wt1, wt2) and

mutated Jurkat cells (mut1, mut2). Right, the average SFRP5 methylation levels of WT and

Mbd2-/- T-ALL cells from leukemic recipients of P1 generation. (I) Binding of 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 and GAPDH was selected as a negative control.

Error bars, SEM; *, P < 0.05; **, P < 0.005; ***, P < 0.001.

MBD2 ablation impairs lymphopoiesis and impedes progression and maintenance of T-ALL

Mi Zhou, Kuangguo Zhou, Ling Cheng, et al.

Cancer Res Published OnlineFirst January 12, 2018.

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