User:Igor Landais/Development of an in vitro screen for compounds that modulate the FA/BRCA DNA damage response pathway

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Re-sensitizing tumor cells that have become resistant to common chemotherapeutic DNA interstrand crosslinking agents like cisplatin and melphalan has great potential for treating a wide variety of cancers. One approach is to inhibit DNA damage response pathways involved in DNA interstrand crosslink (ICL) repair. In particular, the breast cancer associated (BRCA)/ Fanconi Anemia (FA) DNA damage response pathway is required for cell survival after exposure to DNA ICLs. We designed a novel in vitro assay using Xenopus egg extracts to identify small molecules that suppress activation of the BRCA/FA pathway. The assay is based on the monitoring of the DNA-substrate stimulated monoubiquitylation of a protein central to the pathway, FANCD2. To assess the validity of the system, we screened analogs of curcumin, an inhibitor of the FA pathway recently identified in cell-based assays (Chirnomas, et al., 2006). Among 38 analogs tested, 4 displayed higher activity than curcumin in the Xenopus assay. These results were confirmed in human cells since 3 of the compounds sensitized Fanconi anemia-competent cells to crosslinking agents at concentrations 10-50 times lower than curcumin (the fourth is still under investigation). In contrast, these compounds did not sensitize the isogenic Fanconi-deficient cell line, suggesting that they target the BRCA/FA pathway. We also screened 372 compounds from targeted small molecule libraries and identified several previously unknown inhibitors of the BRCA/FA pathway. Follow-up analysis in human cells demonstrated that at least one compound inhibited the DNA damage-induced monoubiquitylation and foci formation of FANCD2 and chemosensitized cells to DNA crosslinking agents. This approach provides the first example of a rapid and inexpensive in vitro assay for identifying compounds that modulate the BRCA/FA pathway, bridging the gap between biochemical and cell-based assays. Furthermore, because Xenopus egg extracts are naturally synchronized in S-phase, this assay is uniquely suited for identification and targeting of modifications occurring during S-phase. Indeed, in addition to FANCD2 monoubiquitylation, we found that at last 7 other DNA repair proteins are post-translationally modified in our assay. Hence, this system has a strong potential for development as a high-thoughput and multiple target screening tool.


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