Tet1 promotes leukemic growth of AML1-ETO+ AML via 5-hydroxymethylcytosine marks and its oncogenic role and high expression can be antagonized using Olaparib, an inhibitor of its binding partner PARP1

AML1-ETO (AE) is the most commonly occurring fusion gene in AML and shows a distinct methylation pattern, the underlying mechanisms for which is poorly understood. TET1 dioxygenase regulates methylation patterns via conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), thereby regulating a variety of biological processes. However, the role of TET1 in AE+ AML cases is yet unexplored. Using qRT- PCR we observed that TET1 was significantly higher expressed in the majority of AE+ patients compared to other AML subtypes and CD34+ normal BM cells. This observation was consistent with published cDNA microarray and transcriptome data. Knockdown (KD) of TET1 using two shRNAs in AE+ AML cell lines impaired their cell growth and clonogenicity in vitro. KD of Tet1 in AE9a+ murine leukemic cell line inhibited its clonogenicity in vitro and delayed onset of leukemia in vivo. Tet1-ko mouse derived HSPCs transduced with AE9a showed impaired serial replating capacity compared to AE9a transduced Tet1-wt HSPCs in vitro. hMeDIP and MeDIP-seq performed on TET1 depleted KASUMI1 cells revealed 8,562 5hmC-enriched promoters (-5kbTSS) in the scrambled arm and a 50% decrease in 5hmC-enriched promoters in KD arm. In RNA-seq the genes associated with AML, Pleuripotency and WNT signaling were downregulated upon TET1 KD and over 200 of these genes exhibited loss of 5hmC on their promoters. MeDIPseq and RNA-seq data revealed a global decrease of promoter methylation and increased expression of myeloid differentiation associated genes. IP analysis confirmed that TET1 physically interacted with PARP1 in AE+ cell line. Oncogenic TET1 expression was antagonized by PARP inhibitor Olaparib in AE+ human leukemic cell lines, which induced reduction of H3K4me3 marks on the TET1 promoter and 5hmC levels in AML cell lines. Furthermore, Olaparib treatment decreased cell growth and clonogenicity of human and murine AE+ cell lines. In conclusion, our data indicate that aberrant TET1 expression contributes to the growth of AE+ AML by maintain 5hmC marks and that the PARP inhibitor olaparib can at least partially antagonize the oncogenic effect of TET in AML.