An Aberrantly Overexpressed Short Isoform of TET1 Lacking a DNA Binding Domain Drives Aberrant 5-Hydroxymethylation Marks, Oncogenic Pathways, and Growth in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a malignancy of the hematopoietic system and still characterized by a high mortality today. Aberrant epigenetic marks in the form of skewed DNA methylation marks (5-methylcytosine or 5mC) and demethylation marks (5-hydroxymethylcytosine or 5hmC) have been implicated in the prognosis, origin, and propagation of AML, therapy resistance, and as a potential therapeutic target. So far, the underlying mechanisms for this are poorly understood. The full length TET1 (TET1^FL) dioxygenase catalyzes the conversion of 5mC to 5hmC and plays an important role in active demethylation, thereby regulating a variety of biological processes. TET1^FL was linked to tumorigenesis based on the observation that its expression was frequently reported as deregulated in AML. Recently, a novel TET1 isoform (TET1^ALT) with a unique transcription start site resulted in a protein with a distinct translation start site, lacking the CXXC domain but retaining the catalytic domain was reported overexpressed in solid cancers (Good CR et al NAR, 2017). However, the role of TET1^ALT in human AML cases is yet unexplored. In this study by using the quantitative real time (qRT)-PCR we now show that the TET1^ALT isoform is severalfold overexpressed in the majority of AML patients (n=80) compared to TET1^FL and normal BM. Surprisingly, the expression of TET1^FL is either low or not detectable in the majority of AML patients. This is confirmed by exon array and RNA-Seq data of AML patients and in functionally validated AML-LSC subpopulation (n=5). Moreover, TET1^ALT is more than 50-fold higher expressed in relapsed AML patients compared to TET1^FL (n=7). In line with qRT-PCR data, TET1^ALT is aberrantly overexpressed at the protein level in AML patients and AML cell lines. The TET1^ALT promoter was enriched for H3K4me3 euchromatic marks in AML patients (n=4). Knockdown (KD) of TET1^ALT mRNA using two short hairpin RNAs (shRNAs) in human AML cell lines impaired cell growth and clonogenicity by over 50% in vitro (n=3 and p<0.01). KD of TET1^FL using shRNA specifically targets the Exon 2 of TET1^FL (absent in TET1^ALT isoform) did not impact the AML cell growth in proliferation assays. TET1^ALT-depleted AML cells exhibited a significant increase in the G1 phase and a decrease in the S phase of the cell cycle compared to cells transduced with scrambled shRNA.The overexpression of TET1^ALT promoted the growth of human cell lines and murine ex vivo AML cell lines harbouring MLL-AF9 and AML-1ETOA9a translocations while TET1^FL inhibited growth. Similarly, in normal murine hematopoietic stem progenitor cells (HSPCs), overexpression of Tet1^ALT increased and Tet1^FL suppressed growth in vitro and in vivo. suggesting that TET1^FL exhibited tumour suppressor activity and TET1^ALT played an important growth-promoting role in AML. RNA-Seq (n=2) of TET1^ALT mRNA-depleted AML cells showed the differential expression of 2941 genes (p=0.05, FDR=0.05), with 1344 genes being downregulated. TET1^ALT depletion reduced the expression of AML-related genes like ERG, MSI2, YES1, CDK6 and STAT5B and pathways associated with spliceosome, cancer, transcriptional misregulation, and AML. CellRadar gene signature analysis of differentially expressed genes showed that downregulated genes are enriched for HSC, CMP, and GMP signatures, whereas upregulated genes are primarily enriched for monocyte signatures Co-expression analyses showed that TET1^ALT expression significantly correlated with the expression of the above mentioned genes in AML patients. These genes also lost 5hmC marks on their promoters in hMeDIP-seq analysis of TET1^ALT KD AML cell line, specifically genes involved in the pathobiology of AML such as ERG, YES1, and CCND2. Our bioinformatics analysis shows that PARP gene expression significantly positively correlated with TET1 expression and not with other TET members in AML patients. PARP inhibitor Olaparib treatment significantly, decreased 5hmC levels, TET1^ALT expression, target genes expression, and reduced cell growth and clonogenicity of human and murine AML cell lines in vitro (n=3, p<0.01). In conclusion, our data indicates that aberrant TET1^ALT expression not the TET1^FL contributes to the growth of AML by maintaining the 5-hydroxymethylome and that the PARP inhibitor Olaparib can at least partially antagonize the oncogenic effect of TET^ALT in AML.