Screening of small molecule inhibitors of the Fanconi Anemia-BRCA pathway

Abstract

4995

Fanconi anemia (FA) is a cancer-susceptibility syndrome characterized by hypersensitivity to DNA crosslinking agents, such as cisplatin and mitomycin C. All 9 known FA proteins cooperate with breast/ovarian cancer susceptibility gene products (BRCA1 and BRCA2) in a common DNA damage-activated signaling pathway called the Fanconi anemia(FA)-BRCA pathway. Seven FA proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG and FANCL) are components of a multi-subunit ubiquitin ligase complex (FA complex) required for monoubiquitination of FANCD2. After DNA damage, FANCD2 is monoubiquitinated and targeted to BRCA1/BRCA2-containing nuclear foci in an ATR kinase-dependent fashion. Importantly, the pathway is inactivated in a wide variety of human cancers by methylation of the FANCF gene. This inactivation causes cisplatin sensitivity in some ovarian cancer cell lines, suggesting an important role of the pathway in cisplatin sensitivity of human tumors. We hypothesized that inhibitors of the FA-BRCA pathway will sensitize cancer cells to cisplatin and, therefore, may be useful for the treatment of cisplatin-resistant cancer. We developed a high-throughput small molecule screen using cells harboring a Green Fluorescent Protein tagged FANCD2 (GFP-FANCD2) and assaying for GFP-FANCD2 nuclear foci formation as a readout, because FANCD2 foci formation is a surrogate marker of the integrity of the pathway. We plated cells with GFP-FANCD2 onto 384-well plates, added small molecules, gamma-irradiated the cells and scored the GFP-FANCD2 nuclear foci. Wells lacking GFP-FANCD2 nuclear foci were scored as positive. We have screened more than 5000 chemicals so far, and found 30 positive chemicals, including kinase inhibitors (wortmannin, H-9, alsterpaullone), geldanamycin, and curcumin. We also found that wortmannin, H-9, and alsterpaullone inhibit ATR-dependent phosphorylation of Chk1. These results together with our previous finding that ATR is required for the activation of the pathway suggest that these drugs inhibit the pathway through inhibition of ATR. Furthermore, we confirmed that alsterpaullone sensitized an ovarian cancer cell line in an FA-BRCA pathway-dependent manner. These findings support the rationale for further screening for FA-BRCA pathway inhibitors.