Abstract B26: Development of a proximity ligation assay for the detection of PARP-1 trapped to chromatin

Background : PARP inhibitors (PARPi) have been under clinical investigation as anti-cancer therapies both as monotherapy and in combination. The two basic mechanisms underlying PARP inhibitor activity are catalytic inhibition and PARP-1 trapping. PARPi trap PARP-1 onto base excision repair intermediates, contributing to potentiation of DNA damage by alkylating agents. The role of PARP-1 trapping with regard to efficacy and tolerability in the clinic is still under investigation. All PARPis are capable of PARP-1 trapping; however, different clinical-stage PARPi trap with dramatically different efficiencies. High efficiency trapping agents trap at doses similar to those required for catalytic inhibition while low efficiency trapping agents trap only at doses much higher than those required for inhibition. Being able to image trapped PARP-1 complexes in the cellular context would be beneficial for identifying other proteins associated with these complexes and may have clinical application in the detection of trapped PARP in tumor biopsies. To date, detection of PARP-1 complexes has been limited to low through-put, techniques that disrupt cellular architecture and lack the sensitivity to detect PARP-1 trapping in the absence of supraphysiologic doses of alkylating agents. More sensitive methods for quantifying trapped PARP1 complexes with spatial resolution are needed to enable further understanding of PARP-1 trapping in cancer and normal cells. The objective of this study was to develop an assay capable of identifying cellular PARP-1 trapped to chromatin with enhanced sensitivity and higher assay throughput. Methods : Duolink is a 96-well plate adaptable system that uses a proprietary antibody/oligonucleotide conjugate that, when localized within 40 angstroms, will anneal, amplify and hybridize to generate a quantitative proximity ligation assay (PLA) signal. To develop a Duolink trapping assay, antibodies to PARP-1, histone H2A.X and phosphorylated (Ser139) H2A.X were identified and optimized for labeling concentrations, endogenous target protein recognition and suitability for high content imaging. Duolink PLA was performed per the manufacturer9s protocol. Automated image acquisition by Cell Insight and data analysis were performed using Cellomics View Software. PARP1 trapping was also evaluated by cellular fractionation and Western blot. Results : Binding to chromatin places PARP-1 in close proximity to histone H2A.X, presumably within the distance required for successful Duolink oligonucleotide annealing and amplification reactions. Co-treating cells with alkylating agents and PARPi induces a significant increase in phosphorylation of H2A.X (Ser139) as well as trapped PARP1 complexes. Comparative analysis of Western blot and PLA signals in HeyA8 cells treated with different PARPi/alkylating agent combinations revealed positive rank-order correlation in trapping activity. Resolution of in vitro single agent PARP-1 trapping by Western blot has been elusive but PLA detection of single agent PARP1 trapping was observed. Conclusion : The PLA trapping assay provides an improved methodology for evaluation of endogenous PARP-1 complexes bound to chromatin and may have utility for clinical tissue specimens where detection of PARP-1 trapping may lead to insights regarding efficacy and tolerability of PARP inhibitors. Disclosures : All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: Todd Hopkins, Barrett Ainsworth, Vivek Abraham, David Maag, Eric Johnson, Julie Wilsbacher. Development of a proximity ligation assay for the detection of PARP-1 trapped to chromatin [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B26.