The Methylcytosine Dioxygenase TET3 Is Aberrantly Expressed in Acute Myeloid Leukemia and Promotes AML Growth
Authors: Pulikkottil Jose Alex , Mohr Fabian , Bamezai Shiva , Karlheinz Holzmann , Kohlhofer Ursula , Fend Leticia Quintanilla-Martinez , Iino Tadafumi , Akashi Koichi , Döhner Konstanze , Döhner Hartmut , Feuring-Buske Michaela , Buske Christian , Rawat Vijay P.S. 
Published: October 2019

Abstract
The epigenetic regulator dioxygenase Ten-eleven translocation 3 (TET3) catalyzes the oxidation of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) and plays an important role in histone modification, thereby regulating the gene expression. Epigenetic aberrations play a key role in the pathophysiology of acute myeloid leukemia (AML), however the reasons for the highly distorted epigenome in AML are poorly known. Though in particular TET2 is widely studied in normal and malignant hematopoiesis, the role of TET3 in human myeloid leukemia is still unknown. First we quantified the expression of TET3 in normal murine and human hematopoiesis by quantitative real-time (qRT)-PCR. Among the Tet genes Tet3 showed highest expression in murine hematopoietic stem cells (HSCs) (2-fold and 3-fold compared to Tet1 and Tet2 respectively, p=0.01, p=0.006, n=3). Within the hematopoietic hierarchy highest expression of Tet3 was found in HSCs and lymphoid committed progenitors (CLPs) compared to committed myeloid progenitors (CMPs) (up to ~3.5-fold, p=0.003, n=3). In humans TET3 was higher expressed in CD34+ bone marrow (BM) cells compared to total BM cells (p=0.024, n=3) and within the lineage positive compartment the TET3 was higher expressed in CD33+ myeloid cells compared to lymphoid cells (CD19+ B cells, p=0.008 and CD3+ T cells, p=0.02, n=3). Our qRT-PCR expression data were in agreement with published microarray data demonstrating that TET3 is significantly higher expressed in myeloid cells compared to lymphoid cells (p<0.001, n=5) (Bagger FO et. al., Nucleic Acids Res. 2013). In AML patients TET3 showed a broad range of expression (ΔCT: -1.4 ± 1.4 SD, n=80), but was aberrantly expressed in majority of the AML patients compared to normal total BM (n=3) and CD33+ BM myeloid cells (n=3) (60% and 81% respectively, cut-off for higher expression: 1.5-fold SD of the median). TET3 was higher expressed in 30% of the cases in cytogenetically normal (CN)-AML (n=50) and in 95% of cases in PML-RARa+ AML (n=21) compared to total BM and CD33+ myeloid cells. The AML cell lines OCI-AML3 (NPM1 mutation+), NB4 (PML-RARa+)and KASUMI-1 (AML1-ETO+) showed higher expression of TET3 compared to other AML cell lines (EOL1, Molm 13, MONO-MAC-6 and THP-1) (p<0.001, n=3). shRNA mediated knockdown (KD) of TET3 in human AML cell lines (OCI-AML3, NB4 and KASUMI-1) adversely affected their cell growth (45%-80% reduction compared to scrambled (scr) control, p<0.001, n=3) and clonogenicity (57%-76% reduction, n=3) in vitro. Furthermore, TET3 KD significantly reduced the leukemic engraftment (median 32±17 and 5.6±6 for scr and shRNA respectively) of NB4 and OCI-AML3 in NSG mice (shRNA n=12; scr n=11, p<0.001). Histopathology showed absence or reduced infiltration of leukemic blast cells in different organs of mice transplanted with shRNA expressing AML cells. In contrast, shRNA mediated KD of TET3 in human normal CD34+ cord blood (CB) cells did not impact cell growth in vitro (n=3). TET3 depletion decreased total 5hmC level in the AML cell line NB4 and in parallel changed expression of genes involved in regulation of cell proliferation, MAP kinase activity and cell migration as determined by microarray gene expression analysis (n=3). Constitutive overexpression of wild-type TET3 in human AML cell lines significantly further enhanced their clonogenicity compared to empty vector control (p<0.05, n=3). Interestingly, overexpression of wild-type TET3 or the TET3-CDconstruct containing only the catalytic domain (CD) inhibited cell growth of human CD34+ CB cells and impaired myeloid differentiation in the CFC assays compared to empty vector (EV) (number of colonies EV=68± 5.7SD, TET3=23±1, TET3-CD=36±10, p<0.001, n=3) whereas erythroid colony formation was not affected. These results were also confirmed by flow cytometry with a decrease in the number of CD11b+CD14+ myeloid cells in the colonies (>70% reduction, n=3). In conclusion, our data indicates that ordered expression levels of TET3 are necessary for normal human HSC proliferation and differentiation. The growth and engraftment potential of AML cells are depending on high TET3 expression levels.

Disclosures
Akashi: Shionogi & Co., Ltd: Research Funding; Asahi Kasei Pharma Corporation: Research Funding; Celgene: Research Funding; Astellas Pharma: Research Funding; Kyowa Hakko Kirin: Consultancy, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol Meyers Squibb: Research Funding; Sunitomo Dainippon Pharma: Consultancy. Buske:Celltrion, Inc.: Consultancy, Honoraria.