PARP1 protein complexes and post-translational modifications as a predictor of response to PARP inhibitors in BRCA-linked ovarian carcinoma Ou Deng, Sweta Dash, Thales Nepomuceno, Bin Fang, Douglas Marchion, John Koomen, Alvaro N. Monteiro*, Uwe Rix* 1Moffitt Cancer Center, Tampa, FL BACKGROUND: Several PARP inhibitors (PARPis) are approved for treatment of BRCA1/2-mutant epithelial ovarian cancers (EOC) and produce significant responses (40-60%), but resistance remains a challenge. Several studies have reported mechanisms of acquired resistance to PARPis, but mechanisms of primary resistance are still poorly understood. 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. METHODS: Cell viability was determined by CellTiterGlo and crystal violet assays. Chemical, ADP-ribosylation and phosphoproteomics were performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein complexes and signaling effects were validated by immunoblotting. RESULTS: Cell viability of primary patient-derived EOC cell lines and an isogenic EOC cell line pair for BRCA1 deletion confirmed the expected correlation between PARPi response and BRCA1/2 status. Phosphoproteomics and MS-based quantification of ADP-ribosylation identified multiple post-translational modifications (PTMs) in BRCA-linked ovarian cancer cells that are potentially relevant for PARPi response, such as DNAPK, which is critical for non-homologous end joining (NHEJ), and AKT/mTOR signaling. In addition, chemical proteomics with rucaparib and olaparib using tumor samples and isogenic cell lines detected Ku70 and Ku80, important elements of NHEJ DNA damage repair, as components of PARP complexes, which are differentially represented in BRCA1-null compared to BRCA1-wild-type (wt) cells. Co-immunoprecipitation indicated inverse participation of Ku70 in complexes with PARP1 vs. PARP2 between BRCA1-null and wt cells and that targeting DNAPK or AKT enhances PARPi efficacy. CONCLUSION: BRCA-linked ovarian cancers that do not respond to PARPi displayed significant changes in PARPi-engaged protein complexes as well as PTMs relevant for DNAPK and AKT signaling. Drug combinations that target these pathways enhanced PARPi efficacy.