Abstract 3785: Olaparib increases the effectiveness of radiation in hypoxic tumor cells in xenograft models of human non-small-cell lung cancer

Abstract

Abstract Purpose: Poly (ADP-ribose) polymerase (PARP) detects DNA single strand breaks and facilitates their repair. Recently, PARP inhibitors have been shown to potentiate radiotherapy in mouse models of human non-small-cell lung cancer (NSCLC) by compromising DNA repair and increasing tumor vascular perfusion. This study examined radiosensitization by PARP inhibition in xenograft models of NSCLC with differing levels of hypoxia. Methods: For in vitro studies, human NSCLC cell lines Calu-6 and Calu-3 were treated with olaparib (5 µM) or vehicle prior to ionizing radiation (IR). Cytotoxicity was evaluated by clonogenic survival assays and DNA damage repair was assessed by γH2AX foci immunofluorescence staining. For in vivo efficacy studies, Calu-6 or Calu-3 subcutaneous xenografts (100 mm3) were established in 6-8 week old female BALB/c nude mice. Animals (n=48) were then randomized into four groups (i) control, (ii) olaparib (50 mg/kg, d1-3), (iii) IR (10 Gy, d1) and (iv) combination treatment. Subsequent tumor growth was monitored for up to 42 days. In parallel, for pharmacodynamic studies, tumor bearing animals were sacrificed 24h and 72h after treatment (n=3 for each treatment and time point). Tumor sections were evaluated by immunohistochemistry (IHC). Results: In vitro, both NSCLC cell lines were significantly radiosensitized by olaparib. Sensitization enhancement ratios were 1.9 (Calu-6) and 2.2 (Calu-3). Furthermore, olaparib markedly inhibited DNA damage repair in both Calu-6 and Calu-3 cells 24h post-IR (p<0.01). Growth of Calu-6 and Calu-3 xenografts was reduced by IR alone, but unaffected by olaparib treatment alone. Surprisingly, although olaparib significantly increased radiation-induced growth delay in Calu-6 xenografts (p<0.01), there was no significant effect in Calu-3 xenografts, in contrast to the in vitro data. IHC staining for vascularity (CD31) and hypoxia (CA9) showed that Calu-3 xenografts were well-oxygenated without evidence of necrotic tissue, whereas Calu-6 xenografts had large areas of hypoxia surrounding regions of necrosis. RAD51 expression (a protein involved in homologous recombination, HR) was selectively down-regulated in hypoxic regions of Calu-6 tumors. 24h and 72h after IR, olaparib treated Calu-6 xenografts displayed significantly higher levels of γH2AX (DNA double strand breaks) and cleaved caspase 3 (apoptosis) staining in CA9 positive (hypoxic) cells compared with the hypoxic regions in xenografts treated with IR alone. Conclusion: Our data identify a novel mechanism that contributes to radiosensitization by PARP inhibition in NSCLC. Olaparib selectively increased DNA damage and apoptosis in radioresistant hypoxic tumor cells with decreased HR potential. Our data also suggest that tumor hypoxia may be a potential biomarker of differential benefit from the combination of olaparib with radiotherapy. Citation Format: Yanyan Jiang, Tom Verbiest, Aoife M. Devery, Sivan M. Bokobza, Anika M. Weber, Anderson J. Ryan. Olaparib increases the effectiveness of radiation in hypoxic tumor cells in xenograft models of human non-small-cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3785. doi:10.1158/1538-7445.AM2014-3785