ADP-ribosylome analysis reveals homogeneous DNA-damage-induced serine ADP-ribosylation across wild-type and BRCA-mutant breast cancer cell lines.

ADP-ribosylation (ADPr) signaling plays a crucial role in DNA damage response. Inhibitors against the main enzyme catalyzing ADPr after DNA damage, poly(ADP-ribose) polymerase 1 (PARP1), are used to treat patients with breast cancer harboring BRCA1/2 mutations. However, resistance to PARP inhibitors (PARPi) is a major obstacle in treating patients. To understand the role of ADPr in PARPi sensitivity, we use liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze ADPr in six breast cancer cell lines exhibiting different PARPi sensitivities. We identify 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. Furthermore, we observe site-specific differences in ADPr intensities in PARPi-sensitive BRCA mutants and unique ADPr sites in PARPi-resistant BRCA-mutant HCC1937 cells, which have low poly(ADP-ribose) glycohydrolase (PARG) levels and longer ADPr chains on PARP1.

Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose).

Intrinsically disordered proteins can phase separate from the soluble intracellular space, and tend to aggregate under pathological conditions. The physiological functions and molecular triggers of liquid demixing by phase separation are not well understood. Here we show in vitro and in vivo that the nucleic acid-mimicking biopolymer poly(ADP-ribose) (PAR) nucleates intracellular liquid demixing. PAR levels are markedly induced at sites of DNA damage, and we provide evidence that PAR-seeded liquid demixing results in rapid, yet transient and fully reversible assembly of various intrinsically disordered proteins at DNA break sites. Demixing, which relies on electrostatic interactions between positively charged RGG repeats and negatively charged PAR, is amplified by aggregation-prone prion-like domains, and orchestrates the earliest cellular responses to DNA breakage. We propose that PAR-seeded liquid demixing is a general mechanism to dynamically reorganize the soluble nuclear space with implications for pathological protein aggregation caused by derailed phase separation.

CDK-mediated activation of the SCF(FBXO) (28) ubiquitin ligase promotes MYC-driven transcription and tumourigenesis and predicts poor survival in breast cancer.

SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCF(FBXO28) activity and stability are regulated during the cell cycle by CDK1/2-mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F-box mutant unable to support MYC ubiquitylation results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCF(FBXO28) plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer.