The Role Of MBD2in Hematopoiesis, T Cell Differentiation And T-ALLL

[Objective]T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with poor prognosis and high relapse rate. Although the outcome of T-ALL has moderately improved in the last decade by intensified systemic chemotherapy and other novel avenues, some patients still fail frontline therapies and relapse with a poor prognosis, highlighting the ineffectiveness of current treatments. Thus, the development of more effective and less toxic targets is necessary. Recently, next generation sequencing showed that T-ALL patients were characterized by distinct DNA methylation signatures that exhibited significant correlation with gene expression profiles. It indicated that DNA methylation might exert a key role in T-ALL leukemogenesis. The repressive effects of DNA methylation on gene expression are mediated by methyl-CpG binding domain (MBD) proteins. Compared to traditional DNA methylation inhibitors, targeting MBD2which was the reader of specific DNA methylation would create attractive new approaches for abnormal DNA methylation in tumors. In this thesis, we studied the impact of MBD2on T-ALL initiation and development, decoding the relevant epigenetic mechanism. Additionally, we explored the role of MBD2in hematopoiesis and T cell differentiation, which is essential to identify the clinical significance of MBD2.[Methods]Firstly, the MBD2-deficient mice and wildtype littermates were aquired by crossing. We detected the percentages of HSC, HPC, multilineage cells in bone marrow or peripheral blood by flow cytometry. We then adopted a transplantation strategy to determine the intrinsic role of MBD2in HSCs and progenitors, by noncompetitive and competitive repopulation assays. Secondly, we examined the differentiation of T cells in thymus by flow cytometry under "steady-state". Thymocytes were analyzed6months after transplanting equal numbers of bone marrow cells from WT or MBD2knockout mice mixed with constant fractions of allotypically disparate WT marrow cells and transferred into irradiated WT recipients. We further utilized mouse microarrays to identify the significant changes of signaling pathways and key genes caused by MBD2deletion in thymocyte differentiation. Thirdly, we transduced the bone marrow Lin cells from MBD2-deficient mice or control littermates with the MSCV-ICN1-IRES-GFP vector to establish T-ALL mouse model, and obtained leukemic cells. We observed the mice survival and immunophenotyping, cell proliferation, cell cycle of leukemic cells. Moreover, sorted leukemic cells were subjected to microarrays, to identify the changes of signaling pathways and key genes induced by MBD2knockout, and we used ChIP to verify MBD2was binding to the methylated key genes.[Results]1.Loss of MBD2led to a decreased lymphoid lineages phenotype. In the BMT, the frequencies of donor bone marrow, B-lymphoid cells and HSCs in recipients of MBD2-deficient bone marrow were decreased relative to controls. In the second cBMT, the contribution of MBD2knockout cells to the multilineage was barely detectable and remained low in recipients.2.Loss of MBD2led to increased DN3frequency and decreased DN4frequency through thymocyte differentiation and impaired DN3/DN4transition was further demonstrated in the cBMT, with decreased DN cell proliferation and increased apoptosis. The microarray analysis showed MBD2deletion down-regulated TCF7expression and Wnt signaling pathway.3.Knocking down MBD2in T-ALL mouse model leds to prolong survival, which was more significant in non-irradiated mouse model. The MBD2-null leukemic cell stayed in GO cell cycle and exhibited decreased proliferation ability. The microarray analysis showed MBD2may bind to methylated Wnt inhibitors, and reduction of MBD2inhibited the Wnt signaling pathway.[Conclusions]1.MBD2-null bone marrow is deficient in long-term repopulating ability in vivo and MBD2is required for HSC self-renewal. Loss of MBD2perturbs lymphoid lineages but not the frequencies of HSCs, HPCs.2.Defects of MBD2-null thymocyte development are cell autonomous. Deletion of MBD2down-regulates TCF7to inhibit Wnt signaling pathway.3.MBD2facilitates Notch-induced T-ALL and the activation of Wnt signaling pathway in T-ALL is MBD2dependent, which represents a potential target for promising therapies of T-ALL.