Abstract
Histone Deacetylase inhibitors (HDACi) affect gene expression through acetylation of histone and non-histone proteins which could alter their cellular activity. We have previously shown that HDACi trigger DNA damage responses in leukemia cells, and actual DNA Double Strand Breaks (DSBs) which are significantly increased and persist longer compared with normal cells. The mechanism by which this DNA damage occurs is still unknown. Non Homologous End Joining (NHEJ) is one of the main pathways for the repair of DSBs in mammalian cells. We have recently shown that NHEJ activity is abnormal in myeloid leukemias in that classical NHEJ that is Ku and DNA-PKcs dependent, is down regulated, and compensatory repair of DSBs occurs via alternative (Alt) NHEJ pathways involving Poly-ADP-Ribose Polymerase-1 (PARP-1) and DNA LigaseIIIα. Alt NHEJ is known to generate large deletions and translocations in cancers and leukemias. To determine whether HDACi may decrease the capacity of leukemic cells to repair DSBs by direct acetylation of NHEJ proteins, K562 and HL60 leukemia cell lines were treated with Trichostatin A (TSA, 300nM for 1 and 6 hours) followed by immunoprecipitation and western blot analysis for acetylated NHEJ proteins. TSA treatment of K562 and HL60 show differential acetylation of classical NHEJ protein Ku70 and Alt NHEJ protein PARP-1, compared with controls. To investigate whether TSA treatment alters the binding of Ku70 and PARP-1 to DSBs, we used a chromatin immunoprecipitation (CHIP) assay in K562 cell line stably transfected with DRNeo that can be induced to express a single DSB. Strikingly, CHIP analysis shows that PARP-1 is increased at the DSB after TSA treatment while Ku70 is not. To determine whether DSB repair activity is altered after HDACi treatment, we performed a plasmid based NHEJ repair assay in our leukemia cell lines after treatment with TSA. Both leukemia cell lines demonstrate a significant decrease in the capacity to repair DSBs following HDACi treatment. The same result can be obtained by the use of another HDACi entinostat. These data suggest that HDACi treatment leads to an increased presence of PARP-1 at DSBs, preventing subsequent critical repair steps in leukemia cells. Importantly, AML cells from patients treated with entinostat in vivo show significantly increased co-localization of PARP-1 and γH2A.x, a marker for DSBs, compared with pretreatment controls, confirming our in vitro data in leukemia cell lines. Overall, our results suggest that HDACi result in both a physical and functional alteration of proteins participating in both classical and Alt NHEJ, leading to a loss of binding of Ku70 and an abnormal persistence of PARP-1 at DSBs. Our data supports the idea that acetylated PARP-1 prevents repair of DSBs by Alt NHEJ, leading to a persistence of DSBs in leukemia cells treated with HDACi.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 688.
- ©2010 American Association for Cancer Research
Volume 70, Issue 8 Supplement
