Abstract P018: Integrative proteomics of PARP1 protein complexes and post-translational modifications implicates DDR and AKT-mTOR signaling in mediating response or primary resistance of ovarian carcinoma cells to PARP1 inhibitors

Abstract

Abstract Background: About 40-50% of epithelial ovarian cancers (EOC) show defects in DNA repair by homologous recombination (HR), which are mostly associated with BRCA1/2 loss-of-function mutations. The PARP inhibitors (PARPis) olaparib, niraparib and rucaparib were recently approved for treatment of ovarian cancer patients with platinum sensitivity and recurrent ovarian cancer who carry inactivating BRCA1/2 mutations. These targeted drugs produce significant response rates ranging from 40-60% in patients with BRCA-linked advanced EOCs, but resistance is a continuing challenge. Whereas several studies have reported various mechanisms of acquired resistance to PARPis, the mechanisms of primary resistance are still poorly understood. Our goal is to develop predictors of PARPi response and to identify new targets for combination therapy to overcome primary resistance. We apply a novel integrated proteomics approach to develop mechanism-based biomarkers of response or primary resistance and to identify new therapeutic targets for rational combination approaches that can overcome resistance to single agent PARPi therapy. METHODS: The isogenic EOC cell line pair UWB1.289 with BRCA1 deletion (parental; UWB) and reconstituted with ectopic BRCA1 (UWB+B) was used. The effects of FDA approved PARPis on BRCA1-null and BRCA1-reconstituted UWB1.289 cells regarding short- and long-term cell viability were determined by CellTiterGlo and crystal violet assays. Chemical proteomics, global phosphoproteomics and ADP-ribosylation proteomics were used to identify the components of PARP1-based multiprotein complexes as well as protein post-translational modifications in the DNA damage signaling network in BRCA1/2-linked EOC cells. Specific PARP1-engaged protein complexes were further determined by immunoblotting. Frozen BRCA1-proficient and deficient ovarian cancer patient tumor samples collected at the time of debulking were also characterized by chemical proteomics. RESULTS: Cell viability assays confirmed the expected correlation between PARPi response and BRCA1/2 status. Chemical proteomics followed by validation with co-immunoprecipitation revealed differential composition of the PARP1/2-Ku70/Ku80 protein complexes in PARPi-sensitive UWB compared to UWB+B cells. Global phosphoproteomics and ADP-ribosylation proteomics further indicated that rucaparib induced the cell cycle and c-NHEJ pathways in UWB cells, but down-regulated the MAPK pathway in UWB+B cells. In addition, our results showed that inhibition of AKT PARylation and AKT-mTOR signaling may help to preserve cell viability in UWB+B cells after rucaparib treatment. Consistently, synergy with DNAPKi and AKTi was more pronounced in UWB+B cells. CONCLUSION: Ovarian cancers that do not respond to PARPi displayed significant changes in PARPi-engaged protein complexes as well as post-translational protein modifications. The combination of chemical, phospho- and ADP-ribosylation proteomics can generate a systems view of PARP1 complexes and diverse drug compensatory signaling in EOC. Citation Format: Ou Deng, Sweta Dash, Thales Nepomuceno, Bin Fang, Douglas Marchion, John Koomen, Alvaro N. Monteiro, Uwe Rix. Integrative proteomics of PARP1 protein complexes and post-translational modifications implicates DDR and AKT-mTOR signaling in mediating response or primary resistance of ovarian carcinoma cells to PARP1 inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P018.