A novel cell‐free screen identifies a potent inhibitor of the Fanconi anemia pathway

Abstract

The Fanconi Anemia (FA) DNA damage response pathway is involved in the processing of DNA interstrand crosslinks (ICLs). As such, inhibition of the FA pathway could chemosensitize FAcompetent tumor cells to commonly used ICL agents like cisplatin. Moreover, suppression of the FA pathway is synthetic lethal with deficiencies in several other DNA repair pathways, suggesting that FA pathway inhibitors could be used in targeted therapies against specific tumors. To identify such inhibitors, we designed a novel in vitro screening assay utilizing Xenopus egg extracts. Using the DNA-stimulated monoubiquitylation of Xenopus FANCD2 (xFANCD2-L) as readout, a chemical library screen identified DDN (2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone) as a novel and potent FA pathway inhibitor. DDN inhibited xFANCD2-L formation in a dose-dependent manner in both extracts and human cells without disruption of the upstream FA core complex. DDN also inhibited the characteristic subnuclear FANCD2 foci formation following DNA damage. Moreover, DDN displayed a greater synergistic effect with cisplatin in a FA-proficient cancer cell line compared to its FA-deficient isogenic counterpart, suggesting that DDN might be a good lead candidate as cisplatin chemosensitizer in both FA-deficient and FA-competent tumors. This system constitutes the first cell-free screening assay for identifying compounds that inhibit the FA pathway and provides a new biochemical platform for mapping the functions of its various components with specific chemical inhibitors. ' 2008 Wiley-Liss, Inc.Key words: chemosensitization; DNA repair; interstrand crosslinks; cisplatin DNA interstrand crosslink (ICL)-inducing agents such as cisplatin, melphalan and mitomycin C (MMC) remain a mainstay in the treatment of cancers such as multiple myeloma, ovarian, testis, breast, bladder and head and neck cancers. 1-5 However, many cancers become refractory to these drugs by multiple mechanisms during a course of chemotherapy, leading to treatment failure. Resensitizing tumor cells to ICL-inducing agents has great potential for treating a wide variety of cancers. One strategy relies on the discovery of small-molecule inhibitors of proteins that control the cellular response to DNA ICLs. Current evidence suggests that the Fanconi Anemia (FA) DNA damage response pathway (which includes breast cancer susceptibility gene products BRCA2, BRIP1/BACH1 and PALB2/FANCN) is involved in the repair of DNA ICLs. As a consequence, acquired ICL-resistance of cancer cells may be associated with re-activation of the FA pathway in tumors initially deficient in this pathway. 3,6,7 Supporting this idea, epigenetic silencing of FANCF has been shown to occur sporadically in AML 7 and in 17, 21 and 30% of breast, ovarian and cervical cancers, respectively. 8 Loss of FANCD2 expression has also been linked with sporadic breast cancer. 5 Interestingly, a recent report by Kennedy et al. identified a set of DNA repair genes [including ataxia telangectasia-mutated (ATM) and NBS1] that are synthetic lethal w...

Simple vertebrate models for chemical genetics and drug discovery screens: Lessons from zebrafish and Xenopus

“…Cell-free Xenopus extracts can be obtained in large quantities, are easily activated in vitro, and can recapitulate important cellular processes such as cell cycle progression, DNA replication, and centrosome duplication in vitro. Given these properties, it is not surprising that cell-free extracts from Xenopus have been used successfully in chemical screens to identify small-molecule modulators of actin assembly, microtubule stability, ubiquitin-dependent protein degradation, cell cycle machinery, and DNA damage response (Verma et al, 2004;Wignall et al, 2004;Dupre et al, 2008;Landais et al, 2009). Chemical screening in cellfree Xenopus egg extracts takes place in a fully soluble in vitro context.…”

FANCD2 re-expression is associated with glioma grade and chemical inhibition of the Fanconi Anaemia pathway sensitises gliomas to chemotherapeutic agents

“…As such, cells defective in the FA pathway are rendered sensitive to a variety of DNA crosslinking agents, making the FA pathway an attractive target for inhibition in cancer cells. In an attempt to identify novel inhibitors of the FA pathway (FAPi), several groups have carried out large-scale small-molecule library screens [13-16]. These studies have revealed four FAPi that are currently commercially available; curcumin and its monoketone analogues EF-24 & 4H-TTD, and the menadione analogue DDN [13-16].…”

“…In an attempt to identify novel inhibitors of the FA pathway (FAPi), several groups have carried out large-scale small-molecule library screens [13-16]. These studies have revealed four FAPi that are currently commercially available; curcumin and its monoketone analogues EF-24 & 4H-TTD, and the menadione analogue DDN [13-16]. However, the mode of action of these compounds has not been elucidated to date, and curcumin may impact on multiple DNA repair pathways [17].…”

Fanconi anaemia: from a monogenic disease to sporadic cancer

“…This is the case of a natural anti-cancer compound, curcumin, to inhibit FANCD2 monoubiquitination, or the synthetic agent wortmannin, which inhibits kinases, such as ATR and ATM, upstream in the FA/BRCA pathway [53]. Aiming to improve the potency and specifi city of these inhibitors, new compounds, such as monoketone, an analogue of curcumin [54], or the naphthoquinone (DDN) [55], have been more recently developed. Similarly, other approaches aim to sensitise head and neck human cancer cell lines to cisplatin by reducing BRCA1 expression with the histone deacetylase inhibitor phenylbutyrate [56].…”