Abstract 2747: PARP inhibitors act synergistically with cisplatin and doxorubicin in BRCA competent cells by compromising chemotherapy-induced replication arrest

Abstract

Abstract PARP inhibitors as monotherapy have emerged as promising antitumor drugs in particular for malignancies with nonfunctional BRCA1/2 proteins. Furthermore, preclinical data as well as first results from clinical studies suggest that PARP inhibition can potentiate cytotoxic effects of conventional chemotherapy independently of the BRCA status. Mechanisms underlying these combinations have predominantly been attributed to an enhancement of DNA damage through interference with DNA repair. Apart from its central function in DNA damage repair processes the enzymatic function of PARP1/2, namely Poly(ADPribosy)lation (PARylation), PARP has also been implicated in stabilizing replication fork arrest upon Topoisomerase I (TopoI) poisoning. We aimed to determine if the function of PARP1/2 at the stalled replication fork might be important for synergistic effects of PARP inhibitors with widely used chemotherapeutics other than TopoI inhibitors. For this, the BRCA competent ovarian and colon cancer cell lines OVCAR8, OVCAR3, HCT116, and LOVO as well as primary tissue samples from ovarian carcinoma patients were treated with cisplatin, doxorubicin, etoposide, 5-FU, and paclitaxel for one hour in presence or absence of the three commonly used PARP1/2 inhibitors Rucaparib, PJ34, and A966492. DNA fiber spreading analyses, quantification of PARylation and 53BP1/γH2AX spot formation, as well as analysis of DNA strand break repair capacity were performed within the first hours after treatment. Furthermore, cell lines were analyzed for long term survival using colony forming assays. Importantly, PARylation was only observed at early time points after treatment with cisplatin and doxorubicin, but not upon 5-FU, etoposide and paclitaxel. Consequently, PARP inhibition had only synergistic effects on long term survival together with cisplatin and doxorubicin but not with the other tested chemotherapeutics. Inhibition of PARylation caused a significant delay of cisplatin adduct removal and strand break repair upon doxorubicin. More importantly, combination of PARP inhibitors with both cisplatin and doxorubicin led to double strand break formation selectively in S/G2 phase cells as detected by 53BP1/γH2AX spots in Geminin positive cells. This was caused by a complete circumvention of chemotherapy-induced fork slowing in cell lines and primary tumor cells. The inability of initiating replication fork arrest upon chemotherapy in cells with blocked PARP1/2 activity was confirmed by the presence of new origin firing and significantly reduced replication stops. In conclusion our work implicates that PARP inhibitors have significant effects not only in BRCA deficient cells but also in proliferating BRCA competent cells when combined with adduct-forming, replication stress-inducing chemotherapeutics. Citation Format: Lea Schaaf, Matthias Schwab, Simon Heine, Walter E. Aulitzky, Heiko van der Kuip. PARP inhibitors act synergistically with cisplatin and doxorubicin in BRCA competent cells by compromising chemotherapy-induced replication arrest. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2747.