Abstract A119: A proteomics-based approach to identify biomarker profiles of homologous recombination deficiency (HRD) in breast cancer cell lines

Abstract

Abstract Background: The oral PARP inhibitor olaparib (AZD2281, KU-0059436) has been shown to have single agent clinical activity in tumors from patients with hereditary BRCA mutations (Fong et al., 2009). Pre-clinical studies have also shown sensitivity to PARP inhibition in cells deficient in non-BRCA components of the homologous recombination (HR) DNA repair and signalling pathways. These include proteins such as NBS1, ATM and CHK2 (McCabe et al., 2006), suggesting the broader clinical potential of PARP inhibitors in tumors that are HR deficient (HRD). Thus the ability to identify tumors with HRD will be critical to realising the full potential of this targeted therapy. A proteomics study analysing isogenic breast cancer cell lines in which key HR gene expression had been stably knocked down was performed to identify a broad protein biomarker profile which may contribute to this patient selection strategy. This study also offered the opportunity to identify novel HR associated proteins and a greater understanding of DNA damage responses. Methods: Nuclear or phospho-protein enriched samples from stable RNAi knockdown breast cancer cell lines (CAL51 and/or MCF7) for 6 key HR genes (BRCA1, BRCA2, ATM, CHEK2, MRE11, NBS1) were analysed using 2 dimensional-difference gel electrophoresis (2D-DIGE) in comparison with parental wild type and/or non specific control cells. The identities of proteins of interest were determined by mass spectrometry. Further analysis of these results was undertaken through bioinformatics approaches utilising the Ingenuity Pathway Analysis (IPA) and GeneGo software tools. Results: Significant differences in protein abundance were observed in 308 proteins through these proteomic analyses. In depth bioinformatics analysis of these results determined their functional roles, their relationship to the double strand break repair (DSBR) pathways such as HR and non homologous end joining (NHEJ) and DNA damage response (DDR) signalling through network modelling. Where no direct relationship was apparent for these putative HRD associated proteins, their linkage was explored back to the specific HR gene knocked down in the cell line that gave the protein abundance change result. The most consistent cellular functions and pathways associated with the protein changes across the different gene knock downs included cell death, post-translational modification and protein folding. Conclusions: The abrogation of key genes in the HR pathway leads to wide ranging effects on downstream cellular functions including cell death, post-translational modification and protein folding. These candidate biomarkers of HRD are being developed into a biomarker profile to predict response to olaparib in the clinic. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A119.