PARP inhibitors (PARPi) have exhibited clinical success in inherited breast cancers with mutations in homologous recombination (HR) DNA double strand break (DSB) repair genes BRCA 1/2 by synthetic lethality. Novel PARPi have been shown to function not only by catalytic inhibition of PARP 1/2 activity, but also by “trapping” of PARP to sites of DNA damage. Trapping of PARP is a response generated by PARPi that is independent of the inhibition of PARP’s catalytic activity, and leads to lethal unrepaired DSBs during replication. Importantly, PARP trapping ability of PARPi correlates with its cytotoxic effect. In this study, we investigated whether PARP trapping has distinct effects on regulation of DNA DSB repair in breast cancer cells. Major DSB repair pathways in mammalian cells include HR and non-homologous end joining (c-NHEJ). Alternative NHEJ (ALT-NHEJ) is a recently discovered and highly error-prone pathway which requires DNA resection like HR, and utilizes PARP1. Our hypothesis is that PARP1 catalytic inhibition vs ‘trapping’ leads to distinct outcomes on DSB repair, highlighting inhibitor efficacy. To test our hypothesis, we utilized MCF10A (non-tumorigenic), MDA-MB-231 (TNBC/BRCA+), and SUM149PT (TNBC/BRCA-) breast cancer cell lines with stably integrated GFP-reporter plasmids designed to assess interchromosomal activities of the specific DSB repair pathways. For our analysis, we used varying concentrations of Talazoparib (MDV3800), which is a potent PARP trapping agent when used in combination with DNA damaging agents such as methyl methanesulfonate (MMS). First, we determined optimal conditions to induce PARP trapping in cells without leading to significant toxicity. As observed by others, combination treatments with of MMS/MDV3800 led to a significant increase in PARP trapping relative to treatments with individual drugs. Studies were also performed with a potent inhibitor of coenzyme NAD+ (Daporinad/FK866) that blocks poly-ADP ribosylation activity in combination of MMS. We observed significant PARP trapping with FK866/MMS combination treatment, indicating that PARP depletion under the conditions of DNA damage also leads to potent PARP trapping. Next, we investigated the effects of these inhibitors on DSB repair. Surprisingly, combination MDV3800/MMS lead to an overall decrease in HR and increase in ALT NHEJ activity. These results were observed in both our BRCA+/- lines. Similar results are observed between the repair activities when using FK866/MMS treatments. Overall, our results suggest that PARP inhibitors with potent PARP trapping activity increases ALT NHEJ, which may potentially compensate for lack of HR. Studies are ongoing to determine whether targeting ALT NHEJ may increase cytotoxicity in these cells.
Citation Format: Bryan M. Pelkey, Pratik Nagaria, and Feyruz V. Rassool. PARP trapping by PARP inhibitors have distinct effects on HR and ALT NHEJ DSB repair, potentially impacting its therapeutic efficacy in breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2478. doi:10.1158/1538-7445.AM2017-2478