The PARP Inhibitor Olaparib Antagonizes Leukemic Growth Induced By TET1 Overexpression in AML1-ETO Positive Acute Myeloid Leukemia
Authors: Bamezai Shiva , He Jing , Sahin Deniz , Mohr Fabian , Ciccarone Fabio , Vegi Naidu M , Pulikkottil Jose Alex , Mulaw Medhanie A. , Caiafa Paola , Döhner Konstanze , Döhner Hartmut , Feuring-Buske Michaela , Buske Christian , Rawat Vijay P.S. 
Published: October 2019

Abstract
DNA methylation patterns are highly deregulated in human acute myeloid leukemia (AML) cases and stratify AML patient samples into different subgroup. AML1-ETO is the most commonly occurring fusion gene in AML and these AML cases exhibit an aberrant and distinct methylation pattern. So far, the underlying mechanisms for this are only poorly understood. The TET1 dioxygenase has recently emerged as an important epigenetic modifier: by catalyzing the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) TET1 plays an important role in active demethylation, thereby regulating a variety of biological processes. It was linked to tumorigenesis based on the observation that its expression is frequently deregulated in solid cancer. However, the role of TET1 in AML1-ETO+ (AE+)human AML cases is yet unexplored. Using quantitative real time (qRT)- PCR we now show that AE+ AML is characterized by high and aberrant expression of TET1: the gene was significantly higher expressed in the majority of AE+ patients (n=7, p<0.01) compared to other AML subtypes such as inv(16) (n=11), PML-RARα+ (n=31), cytogenetically normal (CN)-AML patients (n=33) and CD34+ normal BM cells (n=4). This observation was consistent with published cDNA microarray data on large patient cohorts (Haferlach et al., JCO 2010, p<0.008 t-test, p<0.01 Anova) and recently published transcriptome data (TCGA) of AML patients. In contrast to TET1, TET2 and TET3 did not show significant higher expression in AE+ patients compared to other AML subtypes. In line with patient data, TET1 was highest expressed in the AE+ AML cell line KASUMI-1 and SKNO-1 compared to other AML cell lines (p<0.05 and n=3). Compared to normal CD34+ and myeloid (CD33+, CD15+ and CD14+) cells (n=3), TET1 was 10-fold and 16-fold higher expressed in AE+ patient samples (n= 7). Aberrant expression of TET1 in AE+ leukemic cells was associated with hypomethylation of its promoter and enrichment for H3K4me3 euchromatic marks at its promoter as determined by LC/MS and ChIP-qPCR respectively. Knockdown (KD) of TET1 mRNA using two short hairpin RNAs (shRNAs) in AE+ AML cell lines impaired their cell growth and clonogenicity by over 50% in vitro (n=3 and p<0.01). shRNA mediated depletion of TET1 did not impact the cell growth and clonogenicity of the TET1 negative cell line RAJI, ruling out off target effects of the shRNAs (n=3). In mice, KD of Tet1 in leukemic bone marrow cells expressing the truncated leukemogenic AML1-ETO9a (AE9a) fusion, dramatically inhibited cell growth (>60% compared to scrambled, n=3, p<0.01), clonogenicity (>50-70% reduction in primary CFCs, p<0.01, n=3) and importantly delayed onset of leukemia in vivo (median survival 35 days for scr vs 80 days for shRNA mice, n=4/arm, p<0.03). Tet1-knock-out c-kit+ hematopoietic stem and progenitor cells (HSPCs) transduced with AE9a showed reduced primary colony formation and impaired serial replanting capacity in vitro compared to AE9a transduced Tet1-wild-type HSPCs (>50% and >70%, respectively; p<0.001, n=3). Global analysis of 5hmC and 5mC levels using hMeDIP/MeDIP-seq performed on TET1 depleted KASUMI-1 cells revealed lower global 5hmC levels and increase in 5mC as compared to cells transduced with scrambled control (n=2). 3324 promoter regions lost 5hmC and gained 5mC upon TET1 depletion (-5kTSS, Fold enrichment cut off <2-fold, q-value<1e5). Recent studies have shown that PARP activity induces TET1 expression by regulating its promoter epigenetically. We could show that aberrant TET1 expression could be antagonized by the PARP inhibitor olaparib in AE+ leukemic cell lines. Furthermore, olaparib treatment decreased 5hmC levels and reduced cell growth and clonogenicity of human AE+ cell lines and of the murine AE9a+ leukemic cell line in vitro (n=3, p<0.01). In conclusion, our data indicates that aberrant TET1 expression contributes to the growth of AE+ AML by maintaining the 5-hydroxymethylome and that the PARP inhibitor olaparib can at least partially antagonize the oncogenic effect of TET in AML.

Disclosures
Mulaw: NuGEN: Honoraria. Buske:Celltrion, Inc.: Consultancy, Honoraria.