The Role Of Sirt2-NF-?B/Stat3 In The Occurrence And Development Of MLL-r Acute Myeloid Leukemia And The Regulatory Role Of Sirt2 On C-Myc

Acute myeloid leukemia is a malignant clonal disease of hematopoietic stem/progenitor cells which offen occurs in adults.Leukemia fusion gene can be detected in more than 55%of patients with acute myeloid leukemia.MLL-r is a kind of leukemia fusion gene which caused by rearrangement of the MLL1 gene.Clinically,this kind of patients are tolerant to chemotherapy drugs so that they have a poor prognosis and a high rate of relapse.Therefore,it is urgent to find a new and effective method to treat MLL-r AML.Sirt2 is a member of the NAD⁺-dependent type III histone deacetylase family,which is mainly localized in the cytoplasm and shuttles into the nucleus at the G2/M phase.Sirt2 can involved in regulating the cell cycle,genome stability,cell proliferation,apoptosis,tumorigenesis and other physiological and pathological processes by selectively deacetylating different substrates.The results of the study indicate that Sirt2 is highly expressed in AML patients,and the expression of Sirt2 is higher in relapse patients,and the expression of Sirt2 is negatively correlated with its risk tolerance,and positively correlated with the tolerance of chemotherapeutic drugs.So the high expression of Sirt2 is used as a new biomarker moleculer with poor prognosis to classify the risk level of AML.Nowadays we found that:1.Compared with AML cells,the expression of Sirt2 is higher in MLL-r AML cells;2.Knock out Sirt2 gene in MLL-ENL and MLL-AF9MLL-r AML cells can promote the development of AML,including promoting cell cycle progression,enhancing cell clonality,etc;3.Compared with WT MLL-r AML cells,the acetylation level of NF-?B and Stat3 had risen significantly in sirt2^-/-MLL-r AML cells.NF-?B and Stat3 can enter the nucleus to promote the transcription of target genes in the activated state,so that they can participate in the regulation of cell growth,proliferation and apoptosis.Therefore,we suspect that Sirt2 can inhibits cell cycle progression,cell colony formation and the development of AML by inhibiting the activity of NF-?B and Stat3.In order to verify this conjecture,sirt2^-/-MLL-r AML cells with singular inhibition of NF-?B or Stat3 and co-inhibiting NF-?B and Stat3 were constructed,and we compared their cell cycle and cell clone formation ability.The results showed that in sirt2^-/-MLL-ENL AML cells,whether it inhibited NF-?B or Stat3 alone or co-inhibited NF-?B and Stat3,its cloning formation ability and cell cycle progression were no significant differences with sirt2^-/-MLL-ENL AML cells.Inhibition of NF-?B activity in sirt2^-/-MLL-AF9 AML cells can inhibited cell clonality but did not affect cycle cycle progression;whereas inhibited Stat3 and co-inhibited NF-?B and Stat3,cell coloning formation ability and cell cycle progression were no significant differences with sirt2^-/-MLL-AF9.The results suggest that Sirt2 may regulate the ability of self-renewal and proliferation of MLL-r AML cells and the development of leukemia through other mechanisms,rather than NF-?B and Stat3 signaling pathways.During this experiment,we found that the cloning ability of sirt2^-/-MLL-AF9AML cells was significantly lower than WT MLL-AF9 AML cells,which is contrary to previous findings.After the cells were identified by PCR,we found that the pathogenicity of sirt2^-/-MLL-AF9 AML cells was much weaker than WT MLL-AF9AML cells by mouse tail vein transplantation experiments,which has the same tendency with the change of its clone formation.For these changes in cell clonality and pathogenicity,we hypothesized that Sirt2may have an effect on cell clonality and pathogenicity by regulating the protein level of c-Myc in cells.To verify this hypothesis:1.Detected c-Myc protein levels in WT MLL-r and sirt2^-/-MLL-r AML cells through Western Blot;2.Construct Sirt2^WT OE sirt2^-/-MLL-r and Sirt2^H187Y OE sirt2^-/-MLL-r AML cells to verify whether the level of c-Myc protein in cells is regulated by Sirt2 deacetylase activity through Western Blot;3.In order to investigate whether the degradation of c-Myc is regulated by proteasome and calpain through Western Blot.The results showed that the level of c-Myc decreased significantly in the cells after Sirt2 deletion,and the deacetylase activity of Sirt2 could protect c-Myc from the degradation of proteasome and calpain.May be the lower level of c-Myc in the sirt2^-/-MLL-AF9 cells is the reason of why the clonality and pathogenicity of sirt2^-/-MLL-AF9 AML cells were reduced.