Abstract
Objective: The Fanconi anemia (FA) pathway is a DNA damage response network involved in the cellular resistance against DNA interstrand crosslinks (ICLs). A recent study showed that the FA pathway is synthetic lethal with several other DNA repair genes (Kennedy, 2007) such as ATM, NBS1, RAD54B and TP53BP1. Defects in those genes have been linked to a wide range of inherited and sporadic hematological malignancies including B-CLL, ALL, AML, CML, non-Hodgkin lymphoma, mantle cell lymphoma and multiple myeloma. FA pathway inhibitors may therefore selectively kill malignant cells bearing these defects. Curcumin, a natural product, was the first identified FA pathway inhibitor with activity in the micromolar range in cells (Chirnomas, 2006). However, the poor bioavailability of curcumin hinders its clinical efficacy. Identification of a curcumin analog with better activity, bioavailability and low toxicity could overcome this obstacle. We recently developed a cell-free assay for FA pathway function using Xenopus egg extracts to test the activity of curcumin analogs. As a pilot study we evaluated how well the assay identified inhibitors of the FA pathway in human cells.
Methods: Fourteen curcumin analogs previously assayed in the NCI anticancer cell line screen (Adams, 2004) were tested for their activity on the FA pathway. Xenopus egg extracts were used to measure the relative inhibitory activity of the analogs on FANCD2 monoubiquitylation (FANCD2-L) and phosphorylation of other DNA damage response proteins. The underlying mechanism of inhibition was explored by testing the integrity of the core complex, the recruitment of the core complex to DNA and chromatin, and analyzing DNA replication and proteasome activity. Activity of several analogs was confirmed in HeLa cells by evaluation of the inhibition of hydroxyurea (HU)-induced FANCD2-L and FANCD2 foci.
Results: EF24 (Adams, 2005) and three structurally similar analogs were 10 times more active than curcumin for FANCD2-L inhibition in Xenopus extracts. These analogs inhibited Mre11 phosphorylation at similar concentrations but had no effect on RPA32 and H2AX phosphorylation. In contrast to curcumin, EF24 did not display significant proteasome inhibition activity and did not affect integrity of the core complex or its recruitment to DNA and chromatin, ruling out these mechanisms to explain inhibition of the FA pathway. In HU-treated HeLa cells, EF24 strongly inhibited FANCD2-L and FANCD2 foci with an IC50 of 350 nM, confirming the results observed in Xenopus extracts.
Conclusions: EF24 is a more potent FA pathway inhibitor than curcumin both in Xenopus extracts and in human cells, and as such may be effective as a single agent in targeted therapies against hematological malignancies deficient in ATM, NBS1, RAD54B or TP53BP1. In addition, this study demonstrates that Xenopus extracts are a powerful tool to identify and evaluate small molecules that modulate the FA and other DNA damage response pathways.
REV1 is important for the ATR-Chk1 DNA damage response pathway in Xenopus egg extracts
