TET2 Downregulation Promotes AML Cell Proliferation Via Pim-1 Expression
Authors: Chen Qingxiao , He Jingsong , Guo Xing , Chen Jing , Lin Xuanru , Huang Xi , Yan Haimeng , Yang Li , Li Yi , Zhang Enfan , Yang Yang , Huang He , Cai Zhen 
Published: October 2019

Abstract
Background: Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults which is still incurable although novel drugs and new combination of chemotherapies are used . With the development of genetic and molecular biology technologies, more and more genes are found to be related to leukemogenesis and drug resistance of AML. TET2, a member of the ten-eleven-translocation gene family which can modify DNA by catalyzing the conversion of 5-mehtyl-cytosine to 5-hydroxymethyl-cytosine , is often inactivated through mutation or deletion in myeloid malignancies. Recent research reported that TET2 knock-down can promote proliferation of hematopoietic stem cells and leukemic cells. Also, several clinical studies showed that patients with TET2 mutation or low levels of TET2 expression have more aggressive disease courses than those with normal levels of TET2. However, the mechanism of the phenomenon is unknown. Our aim is to uncover how TET2 protein level is negatively correlated with AML cell proliferation and to provide a better view of target therapy in AML.
Methods: We determined the expression levels of TET2 and other target genes in acute leukemia cell lines, bone marrow AML specimens, and peripheral blood mononuclear cells from healthy donors by qRT-PCR and Western blot. We also determined the mutation status of TET2 in AML cell lines. CCK8 and flow cytometry were used to determine cell proliferation, cell apoptosis, and cell cycle profile. Methylation-specific PCR were used to examine the methylation status in gene promoter regions. Also, we developed TET2 knock-down lentivirus to transfect AML cell lines to examine the effect of TET2 depletion. Last, RNA-seq was used to compare gene expression level changes between TET2 knock-down cell lines and the control cell lines.
Results: AML cells from AML cell lines (KG-1,U937, Kasumi, HL-60, THP-1, and MV4-11) and AML patients' specimens expressed lower levels of TET2 than those of PBMC from the healthy donor (P<0.05). Among AML cell lines, U937 barely expressed TET2, while KG-1 expressed TET2 at a relatively higher level than those of other AML cell lines. We constructed a TET2 shRNA to transfect KG-1,THP-1,MV-4-11,Kasumi,and HL-60, and used qRT-PCR and western blot to verify the knock-down efficiency. CCK8 confirmed that knocking down TET2 could increase leukemia cell proliferation (P<0.05). Flow cytometry showed that cell cycle profile was altered in TET2 knock-down cells compared to the negative control cells. In order to identify target genes, we performed RNA-seq on wildtype and TET2 knockdown KG-1 cells and found that the expression of cell cycle related genes, DNA replication related genes, and some oncogenes were changed. We focused on Pim-1, an oncogene related to leukemogenesis, which was significantly up-regulated in the RNA-seq profile. Western blot and qPCR verified the RNA-seq results of Pim-1 expression in the transfected cells . Also, AML patients' bone marrow samples (n=35) were tested by qPCR and 28 of them were found to express low TET2 but high Pim-1 with the other 7 being opposite. For detailed exploration in expression regulation of Pim-1 via TET2, we screened genes affecting Pim-1 expression and found SHP-1, a tumor suppress gene which is often silenced by promoter methylation in AML. Western blot band of SHP-1 was attenuated in TET2 knockdown KG-1 cells. Moreover, methylation-specific PCR showed that after knocking down TET2 in KG-1 cell line, the promoter regions were methylated much more than the control cells. These results indicated that the function of TET2 in epigenetic modulation plays an important role in regulating Pim-1 expression. Finally, using flow cytometry and CCK8 we surprisingly found that knocking down TET2 expression could lead leukemic cells (KG-1, THP-1 and MV-4-11) more sensitive to Pim-1 inhibitor (SGI-1776 free base) and decitabine (a demethylation agent treating MDS and AML) (P<0.05).
Conclusion: Our study showed that knocking down TET2 promoted leukemic cell proliferation. This phenomenon may correlate to Pim-1 up-regulation. Our clinical data also showed that the expression of TET2 and Pim-1 have an inverse relationship. The mechanism of TET2 regulating Pim-1 expression may be related to the epigenetic modulation function of TET2. Finally, we found TET2 downregulation could increase leukemia vulnerability to Pim-1 inhibitor and decitbine, and provide a novel view of target therapy in AML.

Disclosures
No relevant conflicts of interest to declare.