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
Authors: Bamezai Shiva , He Jing , Rajput Pradeep , Sahin Deniz , Pulikkottil Alex Jose , Mohr Fabian , Vegi Mahalakshmi Naidu , Singh Rana Pratap , Thakral Deepshi , Gupta Ritu , Buske Christian , Rawat Vijay Pal Singh 
Published: December 2024

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 (TET1FL) dioxygenase catalyzes the conversion of 5mC to 5hmC and plays an important role in active demethylation, thereby regulating a variety of biological processes. TET1FL was linked to tumorigenesis based on the observation that its expression was frequently reported as deregulated in AML. Recently, a novel TET1 isoform (TET1ALT) 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 TET1ALT in human AML cases is yet unexplored. In this study by using the quantitative real time (qRT)-PCR we now show that the TET1ALT isoform is severalfold overexpressed in the majority of AML patients (n=80) compared to TET1FL and normal BM. Surprisingly, the expression of TET1FL 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, TET1ALT is more than 50-fold higher expressed in relapsed AML patients compared to TET1FL (n=7). In line with qRT-PCR data, TET1ALT is aberrantly overexpressed at the protein level in AML patients and AML cell lines. The TET1ALT promoter was enriched for H3K4me3 euchromatic marks in AML patients (n=4). Knockdown (KD) of TET1ALT 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&lt;0.01). KD of TET1FL using shRNA specifically targets the Exon 2 of TET1FL (absent in TET1ALT isoform) did not impact the AML cell growth in proliferation assays. TET1ALT-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 TET1ALT promoted the growth of human cell lines and murine ex vivo AML cell lines harbouring MLL-AF9 and AML-1ETOA9a translocations while TET1FL inhibited growth. Similarly, in normal murine hematopoietic stem progenitor cells (HSPCs), overexpression of Tet1ALT increased and Tet1FL suppressed growth in vitro and in vivo. suggesting that TET1FL exhibited tumour suppressor activity and TET1ALT played an important growth-promoting role in AML. RNA-Seq (n=2) of TET1ALT mRNA-depleted AML cells showed the differential expression of 2941 genes (p=0.05, FDR=0.05), with 1344 genes being downregulated. TET1ALT 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 TET1ALT 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 TET1ALT 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, TET1ALT expression, target genes expression, and reduced cell growth and clonogenicity of human and murine AML cell lines in vitro (n=3, p&lt;0.01). In conclusion, our data indicates that aberrant TET1ALT expression not the TET1FL 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 TETALT in AML.