Abstract C51: High sensitivity of testicular germ cell tumors to PARP inhibitor olaparib alone and in combination with cisplatin

Abstract

Abstract Cisplatin is among the most widely used antitumor agents, but its use, is limited by inherent or acquired resistance at tolerable doses for most tumors. Thus, the improved understanding of the mechanisms underlying cisplatin sensitivity and resistance holds enormous therapeutic potential. Testicular germ cell tumors (TGCTs) represent an ideal platform for gaining such insight; since they are a model of cisplatin-sensitivity with favorable responses observed in most patients with advanced disease. However, up to 30% of TGCTs show inherent or acquired cisplatin resistance during treatment. Cisplatin, is a DNA-damaging agent, which binds to form both intra- and inter-strand cross-links (ICLs). Eukaryotic cells repair ICL-induced double strand breaks (DSBs) by multiple pathways. The key factors for such repair are XPF/ERCC1, as well as homologous recombination (HR) proteins. Studies have shown that the inhibitor of the enzyme Poly ADP ribose polymerase PARP [PARPi] AZD2281 (olaparib) has the capacity to kill tumors carrying mutations in the HR repair genes. Using TGCT embryonal carcinoma (EC) as a model, we investigated the molecular mechanisms involved in the cisplatin-resistant phenotype of TGCTs, and the role of Olaparib in their therapy outcome. We compared five EC cell lines which display up to 6 fold difference in their IC50 response to cisplatin. Using FACS analyses of γH2Ax distribution, we found that by 72h after damage, the more resistant TGCT cell lines do repair ICLs, but in the TGCTs, with lower IC50 values, γH2Ax signal persisted in S/G2 phases of the cell cycle and cells were eliminated by apoptosis. What makes TGCT cell lines different from each other? TGCTs have been reported to express low levels of ERCC1 and XPF proteins. We analyzed XPF and ERCC1 expression in our cell lines. We did not observed any clear correlation between cell line sensitivity to treatment and protein expression. Since HR is also involved in the resolution of ICL damage, we tested the abilities of different TGCTs to assemble Rad51 foci. The analyses revealed that the most cisplatin-sensitive TGCT cell lines were unable to efficiently assemble Rad51 foci, indicating a defect in initiating HR repair. To further test HR proficiency, we analyzed the responses to olaparib. We found that some of TGCT cell lines were also sensitive to olaparib. Importantly, the TGCT cell lines that were more resistant to cisplatin were also more resistant to olaparib. We further tested if the sensitivity of TGCTs to PARPi might be also linked to a reduced expression of ERCC1/XPF. We found that Ercc1-/- ES cells are also sensitive to the drug. This suggests that TGCT sensitivity to PARPi is linked to both HR deficiency and low ERCC1/XPF expression. An analysis of PARP expression and activity in TGCTs, revealed that cell lines that were more sensitive to PARPi, expressed higher basal levels of the protein; such cells had higher levels of PARP activity after damage. This indicates that differential responses of TGCTs to olaparib treatment are also physiologically linked to PARP protein expression levels. We asked if the combination of olaparib and cisplatin may cause an increased sensitivity of TGCT cells to treatment. We found that TGCT cell lines that were more resistant to olaparib or cisplatin monotherapy, became much more sensitive to combination therapy. In conclusion our results demonstrate that 1) TGCTs can be defective in multiple steps of ICL repair, 2) TGCTs are sensitive to olaparib monotherapy, 3) cisplatin/olaparib combination therapy decrease the survival of TGCT cell lines. Supported by AIRC grant 4765 to MB and Memorial Sloan Kettering grant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C51.