Summary ADP-ribosylation (ADPr) signaling plays a crucial role in the DNA damage response. Inhibitors against the main enzyme catalyzing ADPr after DNA damage – PARP1 – are used as targeted therapies against breast cancers with BRCA1/2 mutations. However, development of resistance to PARP inhibitors (PARPi) is a major obstacle in treating patients. To better understand the role of ADPr in PARPi sensitivity, we used Liquid Chromatography-Mass Spectrometry (LC-MS) for systems level analysis of the ADP-ribosylome in six breast cancer cell lines exhibiting different PARPi sensitivities. We identified 1,632 sites on 777 proteins across all cell lines, primarily on serine residues, with site-specific overlap of targeted residues across DNA damage-related proteins across all cell lines, demonstrating high conservation of serine ADPr signaling networks upon DNA damage. We furthermore observed site-specific differences in ADPr intensities in PARPi-sensitive BRCA mutants, and unique ADPr sites in PARPi-resistant BRCA mutant cells, which we notably show to have low PARG levels and longer ADPr chains on PARP1.

A site-specific analysis of the ADP-Ribosylome unveils Homogeneous DNA Damage-Induced Serine ADP-Ribosylation across wild-type and BRCA-mutant Breast Cancer cell lines