Abstract 3859: Histone deacetylase inhibitors promote persistent binding of PARP-1 to DNA double strand breaks in chromatin, thus decreasing repair via non-homologous end joining

Abstract

Abstract Histone deacetylase inhibitors (HDACi) induce acetylation of histone and non-histone proteins, and trigger an increase in DNA double-strand breaks (DSBs) in addition to widespread acetylation. Additionally, research has also shown that post-translational modifications, such as acetylation/deacetylation of DSB repair proteins, influence their overall function. The focus of our study is to determine how HDACi influence the repair of DSBs by non-homologous end joining (NHEJ) and thus the potential genomic instability of cancer cells. We have previously demonstrated that HDAC inhibition in leukemia cells (K562 and HL60) with trichostatin A (TSA) results in differential acetylation of Ku70 and PARP1, and this is associated with decreased repair via classical NHEJ (c-NHEJ). Our new studies, using a chromosomally integrated reporter for measuring c-NHEJ, confirms that treatments with TSA significantly downregulate c-NHEJ activity. Recent reports have indicated that Ku70 and PARP compete for binding to DSBs following DNA damage. Since the structure of chromatin can impede the accessibility of these repair proteins, we questioned whether HDAC inhibitors directly (via acetylation) or indirectly (via DSB induction), influence the recruitment of repair factors to sites of DNA damage in chromatin. To test this, we performed subcellular fractionation of TSA-treated K562 cells into cytoplasmic, nuclear and chromatin fractions and by Western blotting analysis observed a significantly increased presence of several c-NHEJ proteins (Ku70, Ku80 and 53BP1) and ALT NHEJ protein PARP1. Interestingly, homologous recombination (HR) DSB repair factor RAD51 is also present in chromatin following TSA treatment. Also, chromatin localization of DSB repair proteins increases with higher doses of TSA. To investigate whether TSA treatment alters the binding of these DNA repair proteins to DSBs, we used a chromatin immunoprecipitation (ChIP) assay in K562 cell line with stable chromosomal integration of a c-NHEJ reporter. Here, a DSB can be induced in a site-specific manner and recruitment of DSB repair components can be studied at the site of a DSB in the c-NHEJ reporter. Our ChIP analysis shows that of all DSB repair proteins tested, PARP-1, is most significantly increased and persists at the DSB after TSA treatment. These results suggest that increased binding of PARP1 to DSBs block subsequent repair steps, a phenomenon similar to PARP1 trapping observed with the potent PARP inhibitors. The results presented here provide a mechanism by which HDACi treatment increases cytotoxic DSBs in leukemia cells. Citation Format: Carine Robert, Pratik K. Nagaria, Ivana Gojo, Feyruz Rassool. Histone deacetylase inhibitors promote persistent binding of PARP-1 to DNA double strand breaks in chromatin, thus decreasing repair via non-homologous end joining. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3859. doi:10.1158/1538-7445.AM2015-3859