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Molecular Cell Biology
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, and Jianfeng Zhou1,4
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 malig- nant hematopoiesis. MBD2 ablation led to diminished lym- phocytes. Functional defects of the lymphoid compartment were also observed after in vivo reconstitution of MBD2-defi- cient hematopoietic stem cells (HSC). In an established model of Notch1-driven T-cell acute lymphoblastic leukemia (T-ALL), MBD2 ablation impeded malignant progression and mainte- nance 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. Furthermore, a number of typical Wnt signaling inhibitory genes were abnormally hyper- methylated 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.
Significance: This study highlights a methylated DNA binding protein as a candidate therapeutic target to improve the treatment of T-cell acute lymphoblastic leukemias, as anew startingpoint for developing epigenetic therapy in this and other lymphoid malig- nancies. Cancer Res; 78(7); 1632–42. �2018 AACR.
Introduction DNA methylation is introduced by at least three DNA methyl-
transferases (DNMT), 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 corre- sponding 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 malignan- cies (8–11). DNMT inhibitors curtail and even reverse the tumor- associated signature of aberrant DNAmethylation and associated gene silencing in cancer (12, 13). Despite rapid clinical progress, the utility ofDNMT inhibitors has been limited by the toxicity and undesired off-target effects associated with nonspecific global demethylation (14). Alternatively, targeting the "reader" proteins ofDNAmethylation instead of usingDNMT inhibitorsmight be a highly attractive strategy for epigenetic therapy (15).
Of all theMBD proteins, MBD2 has been proposed as themost 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 physiologic conditions and would therefore enable theminimization 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 knock- down of MBD2 could suppress neoplastic cell growth by reacti- vating the transcription of tumor suppressor genes (21, 23).When MBD2-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 in normal andmalignant hematopoiesis. Our study showed that the
1Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University 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 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.
Note: Supplementary data for this article are available at Cancer Research Online (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: email@example.com; Weiping Yuan, firstname.lastname@example.org; and Cong-Yi Wang, email@example.com
�2018 American Association for Cancer Research.
Cancer Res; 78(7) April 1, 20181632
on March 24, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from
Published OnlineFirst January 12, 2018; DOI: 10.1158/0008-5472.CAN-17-1434
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. This study highlights the great potential of usingMBD2 as a therapeutic target in T-cell–related pathologies.
Materials and Methods Mice
MBD2deficient (Mbd2�/�)mice on aC57BL/6-CD45.2 genetic background were a gift kindly provided by Dr. Adrian Bird (Edinburgh University, Edinburgh, United Kingdom; ref. 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-oldmaleMbd2�/�
mice and WT littermates were used in this study.
Clinical samples For T-ALL samples, leukocytes were isolated frombonemarrow
specimens by Ficoll gradient and stored frozen in aliquots. All specimenswere collectedbefore chemotherapy.Written informed consent was obtained from each patient and healthy volunteer donor in accordance with the principles expressed in the Decla- rationofHelsinki, and the studywas approvedby 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 in 2011, authenticated by short tandem repeat, and tested to ensure that they were mycoplasma-free by direct culture within 3 months of use. The cell lines were cultured in RPMI1640 medium supplemented with 10% FCS (Gibco, Invitrogen), and used for experimentation within 1 month of being thawed from frozen stocks.
Noncompetitive repopulation assays A total of 1 � 106 bone marrow cells from Mbd2�/� or
littermate WT mice (both CD45.2þ) were injected intravenously into lethally irradiated CD45.1þ recipient animals (9.5 Gy in two doses, 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 were collected. The contribution of CD45.2þ donor-derived cells in the peripheral blood and bone marrow of recipient mice was ana- lyzed 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 cotransfected into the pack- age 293T cells with pKat and pCMV-VSV-G via Lipofectamine 2000 (Invitrogen). The supernatant of 293T cells was harvested. The transduction of Lin� cells from the bone marrow of WT or Mbd2�/�mice with viral supernatant was performed as described previously (25). Leukemic mice were sacrificed 2–3 months after transplantation, and the bone marrow cells were harvested as
P0 cells. Then, we transplanted P0 leukemic cells into sublethally irradiated (6.5 Gy in one dose, 6- to 8-week-old) or nonirradiated recipients (6- to 8-week-old) to establish a leukemicmousemodel as P1.
Flow cytometry An LSR II cytometer and FlowJo7.6 software (BD Biosciences)
were used for data acquisition and analysis. All antibodies were from BD Biosciences or eBioscience unless otherwise indicated. Cell sorting was performed using a fluorescence activated cell sorter (FACS, Aria Cell Sorter, BD Biosciences).
BrdUrd detection When 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 ad