Abstract 1974: Nuclear BCL2 inhibits PARP1 enzymatic activity, DNA repair, and necrotic cell death

BCL-2 is overexpressed in many types of cancer through a variety of genetic, epigenetic and posttranscriptional mechanisms. The physiologic role of BCL2 is to suppress apoptosis by sequestering BH3 domain-containing pro-apoptotic factors. We demonstrate that some tumor cells with BCL2 overexpression have constitutive localization of BCL2 within the nucleoplasm and that DNA damage induces mobilization of BCL2 to the chromatin. To identify factors that mediate this response, we irradiated lymphoid tumor cells and then performed immunoprecipitation of the chromatin protein fraction using an anti-BCL2 antibody. Mass spectrometry of a unique protein isolated only after DNA damage identified PARP1 (poly(ADP) ribose polymerase 1), an enzyme involved in various cellular processes, including base excision repair and alternative nonhomologous end-joining. In response to DNA damage, PARP1 utilizes NAD to add poly(ADP) ribose (PAR) onto acceptor proteins including itself, which provides a scaffold for additional damage response factors. We confirmed the BCL2-PARP1 interaction in additional tumor cell lines and transgenic mouse embryonic fibroblasts that overexpress BCL2 but lack the apoptosis effectors BAX and BAK. Purified BCL2 and PARP1 interacted directly in vitro independently of both DNA and NAD. Small molecule PARP inhibitors did not interfere with the BCL2-PARP1 complex, suggesting that the interaction is not dependent on PAR. In contrast, the BH3 mimetic ABT-737 blocked the BCL2-PARP1 interaction in a dose-dependent manner, indicating that PARP1 binding by BCL2 involves the BCL2 BH3-binding pocket. In fact, PARP1 contains 9 consensus BH3 motifs (LXXXXD). Interaction with PARP1 was specific to BCL2 and was not observed with the anti-apoptotic factors BCL-xL or MCL1. Using an anti-PAR enzyme immunoassay, the addition of BCL2 to PARP1 inhibited PARP1 activity in vitro. This effect was reversed by adding ABT-737. In addition, we used the alkaline comet assay to determine the effect of BCL2-PARP1 interaction on base excision repair. PARP1-mediated repair of single-strand DNA breaks induced by hydrogen peroxide was attenuated in BCL2 overexpressing cells. ABT-737 increased PARP1-mediated repair, both in tumor cells rendered resistant to apoptosis through the addition of a caspase inhibitor and in embryonic fibroblasts that overexpress BCL2 but lack BAX and BAK. In the latter cells, restoration of PARP1-dependent repair by ABT-737 promoted necrotic cell death through the exhaustion of cellular NAD. This suggests that nuclear BCL2 blocks clastogen-mediated killing by inhibiting PARP1 activity, even in cells that are resistant to BCL2 inhibitors through loss of apoptosis effectors or overexpression of antiapoptotic factors like MCL1. As such, inhibition of BCL2 interactions could synergize with DNA damaging agents independently of effects on apoptosis. 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 1974.