The Role Of Methyl-CpG-binding Domain 2(MBD2) In MLL-rearranged Acute Myeloid Leukemia And Studying On The Underlying Mechanisms

[Objective]Many previous studies have been demonstrated that epigenetics plays an important role in the process of tumorigenesis.Characterized by the formation of MLL fusion proteins at the 11q23 chromosomal site,acute myeloid leukemia with MLL fusion proteins(MLL myeloid leukemia),is a malignant myeloid leukemia that is rapidly advancing in adults and has very poor prognosis.With the deepening progress of relevant mechanisms,the correlation between histone methylation(H3K79)abnormalities and MLL leukemia was confirmed,and then targeted therapies such as DOT1 L inhibitors were developed.As the drug metabolism or kinetics in vivo and other reasons,as of now no DOT1 L inhibitors formally entered clinical trials.The new experimental study found abnormal DNA methylation in the genome of patients with MLL myeloid leukemia,suggesting that DNA methylation may be another target of MLL treatment.Methyl-Cp G-binding domain 2(MBD2)is a specific DNA methylation reader,that affects the regulation of DNA methylation in gene expression by interfering with the recognition and readout function of MBD2.Unlike conventional DNMTs inhibitors may cause the hypomethylation of the entire genome,MBD2 has become a potential therapeutic target for abnormal DNA methylation.This article studied the role of MBD2 in the pathogenesis of MLL myeloid leukemia and its mechanism,and explored the feasibility of MBD2 as a target for treatment of MLL myeloid leukemia.[Methods]MBD2 knockout mice were crossed with C57 mice,which with the same background,to obtain hybrid mice,thus obtaining the same batch of MBD2 knockout mice and the corresponding wild type mice.The MLL myeloid leukemia mouse model was established,the development and changes of MLL myeloid leukemia in MBD2 knockout and control groups were observed,the survival period was observed;the immunophenotype,cell cycle and proliferation of leukemia cell population was detected by flow cytometry.The leukemic cells were sorted out and analyzed by using expression microarray to analyze the key pathways involved in MLL affected by MBD2 and the differences of related gene expression.Further,we used CRISPR / CAS9,a third-generation gene editing technology,to construct a cell model of MBD2 knockout in acute myeloid leukemia cell line THP1 with MLL rearrangement to verify the effect of MBD2 knockout on related pathways and genes in vitro.[Results]1.MLL-AF9 leukemia animal model was successfully established in MBD2 knockout mice and corresponding wild type mice by infecting mice with retrovirus containing MLL-AF9 fusion gene.2.In mouse MLL myeloid leukemia model,MBD2 knockout mice were found to have significantly longer survival and lighter infiltration of leukemia than WT mice;3.Loss of MBD2 caused leukemic cells in mice decreased proliferation,cell cycle arrest;leukemia cell sorting were sent for microarray analysis,and a total of 1878 differential genes were detected,the results showed MBD2 knockout promoted differentiation of tumor cells;4.MBD2 knockout in human MLL myeloid leukemia cell line THP1 was found to block the cell cycle,inhibit cell proliferation and restore the expression of cycle related CDKI molecules,and chromatin immunoprecipitation(Ch IP)experiments showed that MBD2 was closely binding to P57.[Conclusion]1.In mouse MLL-AF9 leukemia model,MBD2 knockout can be partially reversed MLL myeloid leukemia fingerprint;2.MBD2 knockout in mouse MLL-AF9 leukemia model was found to promote myeloid differentiation,slow down the proliferation of leukemia cells and reduce the expression of leukemia stem cell related genes,promote the expression of gene which suggesting a good prognosis of AML;3.MBD2 knockout in THP1,the human MLL-AF9 myeloid leukemia cell line,blocked cell cycle and inhibited cell proliferation,and it is showed that MBD2 may be a potential therapeutic target for the treatment of MLL myeloid leukemia.