TET2 Downregulation Leads to AML Cells Sensitive to Decitabine

Background : Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and have very lethal rate. Chemotherapy is the main method to treat AML, but the complete remission rate is still not very optimal. With the development of genetic and molecular biology technologies, more and more molecular biomarkers are found, some of them are useful for us to evaluate the prognosis and can help us to tailor the treatment plan for different patients. TET2, a member of the ten-eleven-translocation(TET) family genes which can modify DNA by catalyzing the conversion of 5mehtyl-cytosine(5-mC) to 5-hydroxymethyl-cytosine(5-hmC), is often inactivated through loss-of-function mutation and deletion in myeloid malignancies. Recent clinical research reported that the lower the expression of TET2 in MDS and AML patients, the better the response to decitabine( DAC ,a demethylation agent) will be. However, the mechanism of the phenomenon is still unknown. Our investigation is trying to uncover the mechanism how TET2 protein levels are negatively related with AML sensitivity to decitabine.

Methods: We detected TET2 mRNA expression level in acute leukemia cell lines, bone marrow AML specimens and peripheral blood mononuclear cells from healthy donors by semiquantitative real time polymerase chain reaction (qRT-PCR). Western blot is also applied to detect TET2 protein expression. In order to access TET2 methylation status ,we used the methylation-specific PCR . And we also checked the mutant status of TET2 in U937 and KG-1 cell line. CCK8 and flow cytometry are used to detect cell proliferation rate，cell apoptosis, and cell cycle profile. Also, we developed TET2 knock-down and overexpression lentivirus to transfect AML cell lines to explore the mechanism why TET2 expression level is related to the response of DAC. Last, gene array is used to compare gene expression level changes between TET2 knock-down cell lines(or TET2 overexpression cell lines) and the control cell lines.

Results:The AML cell lines(KG-1,U937,Kasumi,HL-60,THP-1) and AML patients specimens express lower TET2 than that of PBMC from the healthy donor(P<0.05). Among AML cell lines ,U937 barely expresses TET2, while KG-1 expresses TET2 relatively higher than other AML cell lines. The methylation-specific PCR showed that TET2 in U937 was partially methylated while KG-1 was not. After using decitabine to treat U937 cell line, the TET2 methylation status was attenuated . And all the exons of TET2 were not detected any mutation in KG-1 AND U937.  Then, we used CCK8 to compare the response difference to DAC between U937 and KG-1 and found that U937 is much more sensitive to DAC rather than KG-1(P<0.05). Next, we constructed a TET2 shRNA to transfect KG-1, both qRT-PCR and WB were used to verification the knock-down efficiency.Again,CCK8 told us that KG-1 TET2 knock-down cells was more sensitive to DAC than KG-1 NC cells. Flow cytometry identified that cell cycle profile were altered between KG-1 TET2 knock-down cells and KG-1 NC cells. Gene array(KG-1 TET2 KD and KG-1 NC) showed that the expression levels of cell cycle related genes(e.g. CCNB2,RBL1), DNA replication related genes(e.g. PRIM1,RCF3,FEN1) and many other function genes were changed between the knock-down and control cell line. 

Conclusion: Our study showed that the sensitivity to decitabine of AML cell lines is related to TET2 expression level, knock-down TET2 in KG-1 can increase its vulnerability to decitbine. And the mechanism may be related to the changing expression levels of the genes which regulating cell cycles and DNA replication.