Abstract A33: Therapeutic potential of sapacitabine in ovarian cancers defective in homologous recombination

Abstract

Abstract Background: Sapacitabine is an oral nucleoside analogue that is metabolised in-vivo to CNDAC (2′-C-cyano-2′-deoxy-1-β-D-arabino-pentofuranosylcytosine). It has a novel mechanism of action by causing single-strand breaks and replication-associated double-strand breaks (DSBs) after its incorporation into DNA. DSB, if unrepaired, are cytotoxic and are predominantly repaired by homologous recombination (HR). Over 50% of high-grade serous ovarian cancers are estimated to be defective in HR, only a small proportion of which are accounted for by germline BRCA mutations. HR-defective ovarian cancers have been shown to be sensitive to PARP inhibitors ex-vivo and platinum in-vivo and we therefore hypothesised that HR defective ovarian cancers would be sensitive to CNDAC and aimed to explore the potential use of CNDAC in ovarian cancer stratified for HR function. Methods: Primary cultures (PCO) were generated from ascitic fluid collected from patients undergoing ovarian cancer surgery, characterised and confirmed to be ovarian and epithelial in origin based upon morphology, expression of immunofluorescent-labelled markers and cross-referencing with formal examination of FFPE tissue and cytology of ascites. The functional status of HR was determined for each cell line and PCO culture by quantification of nuclear Rad51 focus formation following induction of DNA DSB. SRB assays were used to calculate the GI50 for cisplatin and CNDAC, (Cyclacel, UK), in cell lines nad PCO cultures. Results: As hypothesised, HR defective cell lines were highly sensitive to CNDAC (VC8 BRCA2 mutant: GI50=366 nM) in comparison to their matched HR competent cell lines (VC8-BRCA2 reconstructed: GI50=18 μM; VC8-PARPi-resistant BRCA revertant: GI50=3 μM). Sensitivity to CNDAC in patient PCO cultures (n=39) was heterogeneous with a mean GI50 of 297 nM (95% CI 136-646 nM). Stratifying PCO cultures according to their HR status demonstrated greater sensitivity in HR defective cultures with a mean GI50 of 135 nM (46-394; n=12) in defective cultures, compared to 477 nM in HR competent cultures (164-1386; n=27). Of the 34 cisplatin resistant PCO cultures (GI50 >5 μM), 20 (59%) remained sensitive to CNDAC with a mean GI50 of 426 nM (148-1223; n=13) in HR competent cultures and a GI50 of 230 nM (70-759; n=7) in HR defective cultures. CNDAC sensitivity was evaluated in base excision repair (BER) defective EM9 cell line (GI50=374 nM) compared with parental wild type AA8 cells (GI50=6.9 μM), p<0.0001. Using detection of 8-hydroxy-2′-deoxyguanosine (8-OHdG) as an indicator of BER functional status in PCO cultures, a trend between BER dysfunction and sensitivity to CNDAC was seen with a mean GI50 of 42 nM (18-97; n=2) in BER defective PCO cultures in comparison to 187 nM (74-473; n=15) in BER competent cultures. Conclusions: Cell lines defective in HR or BER are highly sensitive to CNDAC suggesting a possible use of HR or BER status as biomarkers for the stratified use of CNDAC in the treatment of ovarian cancer. When applied to ex-vivo studies HR status as a single marker cannot reliably predict sensitivity and a panel of pathways may therefore be needed to enable reliable treatment stratification. Nevertheless, stratification based on these markers is likely to permit enrichment of patients more likely to respond to CNDAC. Additionally CNDAC may have a role in the treatment of platinum resistant disease as approximately 60% of patients with resistant disease may benefit. Further work is required to explore the possibility of CNDAC as a chemosensitiser in platinum sensitive disease and as a second line agent in platinum resistant disease. Citation Format: Rachel L. O'Donnell, James CC Murray, Adriana GB Buskin, Sheelagh Frame, David G. Blake, Michelle Dixon, Aiste McCormick, Angelika Kaufmann, E Ruth Plummer, Richard J. Edmondson, Nicola J. Curtin. Therapeutic potential of sapacitabine in ovarian cancers defective in homologous recombination. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A33.