Abstract B33: PARP-dependent co-modulation of DNA repair and microglial activity as a dual-pronged anti-glioblastoma treatment strategy
Authors: Sinha Asha , Katyal Sachin , Kauppinen Tiina 
Published: April 2017

Abstract
Introduction: Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with poor treatment outcomes and prognoses. Treatment usually consists of surgery, radiation and chemotherapy, which often result in severe systemic side effects frequently accompanied by tumor recurrence of highly radioresistant tumor cell populations. PARP-1 is a nuclear enzyme crucial for DNA damage repair (DDR) response. PARP-1 inhibition is a highly studied adjuvant to many anti-cancer treatment strategies as this arrests DNA damage repair thus enhancing the efficacy of conventional chemoradiotherapy at lower doses. However, PARP inhibition also promotes anti-inflammatory responses. Cancer cells suppress the immune cells ability to recognize and eliminate the tumor and promote their release of cancer-supporting growth factors and promote angiogenesis. Microglia the immune cells of the brain, normally protect the brain from pathogens and help in tissue recovery, but when associated with GBM cells, microglia obtain anti-inflammatory state that can promote tumor growth.
We are developing a novel bimodal immunotherapeutic/DNA repair suppressive approach to treating GBM through modulation of microglial PARP-1 activity while concurrently sensitizing GBM to DNA damaging therapeutics. We hypothesize that while PARP-1 inhibition can enhance efficacy of chemoradiotherapy it might further suppress microglial ability to promote tumor elimination. Furthermore PARP-1 inhibitors' efficacy in preventing DNA repair is partially linked to their ability to trap PARP-1 at DNA break-site, which might be detrimental for the viability of non-cancerous brain cells.
Objectives:
1) to establish the effects of PARP-1 inhibition alone, and in combination with targeted microglial PARP-1 activation, on microglial ability to eliminate glioblastoma cells in vitro (tumor cells vs. healthy glial cells).
2) to identify how microglial PARP-1 modulation (on/off) impacts glioblastoma elimination in vivo (tumor vs. brain).
Methods: We will use GBM cells co-cultured with primary microglial cells in the presence of PARP inhibitors and observe their effect on the proliferation and viability of GBM cells. Efficacy of PARP-DNA trapping and corresponding DNA damage repair activity utilizing a variety of PARP inhibitors will be tested by specific biochemical and genotoxicity assays which directly quantify the degree of PARP-trapping in the glial cells and their impact on tumor cell genotoxcity. These studies take advantage of newly-developed and unique high-throughput DNA damage repair assays that we have been optimized to determine potential synergy of PARP-inhibitors with existing chemotherapeutic agents.
Results: DNA damage assays (gamma H2AX foci and alkaline comet analysis) show that co-treatment of GBM cells (U87 and U251) with the PARP-inhibitor Olaparib and the chemotherapeutic agent Camptothecin (CPT) significantly increased the level of DNA damage when compared to CPT treatment alone. In the co-culture experiments, microglia enhanced CPT-induced GBM cell death when treated with Olaparib at 50-100 fold lower doses. Real time co-culture experiments also showed that Olaparib does not jeopardize microglial ability to actively phagocytize GBM cells.
Conclusion: Preliminary results show that Olaparib is effective in sensitizing GBM cells to chemotherapy while microglia appear to enhance this effect. Further studies linking this combination therapy with microglia-mediated GBM elimination will help to elucidate the mechanism of this bimodal effect. This study emphasizes the importance of: (1) identifying novel effects of PARP inhibition on microglia in association with GBM and (2) the importance of developing microglia-mediated immunotherapeutic intervention to overcome the limitations of current therapies to significantly improve patient outcome.
Citation Format: Asha Sinha, Sachin Katyal, Tiina Kauppinen. PARP-dependent co-modulation of DNA repair and microglial activity as a dual-pronged anti-glioblastoma treatment strategy [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 B33.