TET1 Promotes Leukemic Growth in T-ALL Via Maintenance of 5-Hydroxymethylation Marks and Can be Antagonized By the PARP Inhibitor Olaparib

Abstract

T-cell acute lymphoid leukemia (T-ALL) is an aggressive hematological cancer predominantly occurring in children and adolescents. T-ALL is characterized by aberrant expression of genes involved in T-cell differentiation and self-renewal accompanied by aberrant DNA cytosine methylation patterns. The mechanisms underlying these aberrations are not well understood. The Ten-eleven translocation 1 (TET1) enzyme is an iron-dependent dioxygenase that catalyzes the oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and plays a key role in active DNA demethylation and regulation of gene expression. Using quantitative real time (qRT)-PCR we now demonstrate that T-ALL is characterized by high and aberrant expression of TET1: TET1 was significantly and >20-fold higher expressed in human T-ALL cell lines compared to other B-lymphoid or myeloid leukemia cell lines (p<0.001, n=3). This was confirmed in primary patient samples, in which TET1 was more than 10-fold higher expressed in 95% of T-ALL cases (n=10) compared to other leukemia subtypes (B-ALL, n=29; AML, n=39; CLL, n=23, p<0.001). Furthermore, expression levels of TET1 in T-ALL patient samples were over 50-fold higher than in normal PB and BM derived healthy CD3+ T cells (n=6, p<0.001) in line with published cDNA microarray data from a large patient cohort of 174 T-ALL cases (Haferlach et al., JCO 2010). Knockdown (KD) of TET1 mRNA using two independent shRNAs in T-ALL cell lines adversely affected their cell growth (>70% reduction compared to scrambled, p<0.001, n=4) clonogenicity (>70% reduction compared to scrambled, p<0.001, n=4) and blocked cells in the G2/M phase of the cell cycle in vitro (n=3). Furthermore, TET1 KD significantly reduced the leukemic engraftment potential of T-ALL cell line Jurkat in xenografts (shRNA, n=16; scrambled, n=8). Global analysis of 5hmC levels using hMeDIP-seq performed on TET1 depleted Jurkat cells revealed lower global 5hmC levels as compared to cells transduced with scrambled control (n=2). Promoters (-5kb TSS) of 5,181 genes showed reduction of 5hmC marks (log2 fold diff.; p-value < 1e-10). Global 5mC analysis (MeDIP-seq) revealed 12,007 genes that gained 5mC marks at their promoters as a result of TET1 KD (log2 fold diff. > 1.5; p-value < 1-10). Of note, genes demonstrating a loss of 5hmC marks and corresponding a gain in 5mC marks on their promoters belonged to gene families involved innucleosome organization (HIST family), DNA damage response (DDR)(RAD, PARP), T-cell development (NOTCH3, RUNX1, LEF1) and homeobox genes (q-value < 1e-5). These changes were paralleled by changes in expression after TET1 depletion as measured by RNA-Seq in Jurkat cells: GO TERM analysis (log2 fold diff. > 1.5; p<0.05) revealed deregulation of genes belonging to nucleosome assembly, DNA damage, nucleosome organization and transcriptional deregulation in cancer. In line with these observations these pathways showed differential expression in T-ALL patients divided into the categories high vs. low TET1 expression based on previously published microarray data (Haferlach et al., JCO 2010) (median as cut-off; n=174)(log2 fold diff. >1.5; p<0.05).The aberrantly high TET1 expression in T-ALL cell lines shares a potential causal relationship with the hypomethylated status of the TET1 promoter (determined by LC/MS), and the enrichment of euchromatic marks such as H3K4me3 and acetylated H3 as determined by ChIP-qPCR. Since it was shown that PARP activity positively influences TET1 expression by regulating its promoter epigenetically, we tested the impact of the PARP inhibitor olaparib on TET1 activity in our model: of note, aberrant expression of TET1 could be reverted by the PARP inhibitor olaparib, which induced reduction of euchromatic marks on the TET1 promoter, in parallel with decreased TET1 expression and protein levels as well as 5hmC levels in Jurkat cells without affecting expression of other TET family members. Olaparib treatment caused a >75% reduction in cell growth and clonogenicity of T-ALL cell lines in vitro (n=3) and delayed leukemic engraftment in NSG mice (42%±8 in scrambled vs 9%±7 for the shRNAs, n=4 per arm; p<0.001). In conclusion, these data demonstrate for the first time that TET1 plays an important oncogenic role in T-ALL. We also identify PARPs as upstream regulators of TET1 in T-ALL, opening the way to antagonize TET1 pharmacologically in this poor prognosis leukemia. DisclosuresMulaw:NuGEN: Honoraria. Buske:Celltrion, Inc.: Consultancy, Honoraria.