Monoketone analogs of curcumin, a new class of Fanconi anemia pathway inhibitors

Abstract

Background: The Fanconi anemia (FA) pathway is a multigene DNA damage response network implicated in the repair of DNA lesions that arise during replication or after exogenous DNA damage. The FA pathway displays synthetic lethal relationship with certain DNA repair genes such as ATM (Ataxia Telangectasia Mutated) that are frequently mutated in tumors. Thus, inhibition of FANCD2 monoubiquitylation (FANCD2-Ub), a key step in the FA pathway, might target tumor cells defective in ATM through synthetic lethal interaction. Curcumin was previously identified as a weak inhibitor of FANCD2-Ub. The aim of this study is to identify derivatives of curcumin with better activity and specificity.

Results: Using a replication-free assay in Xenopus extracts, we screened monoketone analogs of curcumin for inhibition of FANCD2-Ub and identified analog EF24 as a strong inhibitor. Mechanistic studies suggest that EF24 targets the FA pathway through inhibition of the NF-kB pathway kinase IKK. In HeLa cells, nanomolar concentrations of EF24 inhibited hydroxyurea (HU)-induced FANCD2-Ub and foci in a cell-cycle independent manner. Survival assays revealed that EF24 specifically sensitizes FA-competent cells to the DNA crosslinking agent mitomycin C (MMC). In addition, in contrast with curcumin, ATM-deficient cells are twofold more sensitive to EF24 than matched wild-type cells, consistent with a synthetic lethal effect between FA pathway inhibition and ATM deficiency. An independent screen identified 4H-TTD, a compound structurally related to EF24 that displays similar activity in egg extracts and in cells.

Conclusions: These results suggest that monoketone analogs of curcumin are potent inhibitors of the FA pathway and constitute a promising new class of targeted anticancer compounds.

Figure 1

Curcumin analogs efficiently inhibit xFANCD2 monoubiquitylation in Xenopus extracts. A) Inhibition of xFANCD2-Ub and xMRE11 phosphorylation by curcumin anlogs. IC₅₀ values were determined using immunoblots of plasmid-activated extracts treated with a range of concentrations for each compound (0, 10, 25, 60, 150, 400, 1000, 3000 μM). A representative experiment is shown (out of 3 repeats). B) Structure of curcumin and monoketone analogs. C) Curcumin analogs do not affect RPA₃₂ and H2AX phosphorylation. Activated extracts were treated with 1 mM each compound and phosphorylation of RPA₃₂ and H2AX monitored by immunoblot.

Figure 2

Proteasome inhibition activity of EF24 and related curcumin analogs in Xenopus extracts. A) Inhibition of caspase- and chymotrypsin-like proteasome activities and xFANCD2-Ub by MG132, curcumin and curcumin analogs. Proteasome activities and xFANCD2-Ub were monitored using fluorogenic probes and immunoblotting, respectively. IC₅₀ values were determined for each compound using a range of concentrations and represented in histogram graphs. For MG132, numbers above caspase-like and chymotrypsin-like histograms indicate the actual IC₅₀ values. A representative experiment is shown (out of 3 repeats). B) Inhibition of proteasome activities by EF31 and AS153-4 compared to curcumin.

Figure 3

EF24 does not inhibit xFANCD2-Ub through modulation of a phosphorylation event, but IKK inhibition might play a role. A) EF24 inhibits the IKK kinase in Xenopus extracts. Extracts treated as indicated were analyzed by immunoblot using xFANCD2, IκB-α and tubulin-α antibodies. IκB-α protein level was used as readout for monitoring IKK inhibition. B) The specific IKK inhibitor compound BMS-345541 (IKK inhibitor III, Calbiochem) inhibits xFANCD2-Ub in extracts. Experiment was performed as in (A). The structure of BMS-345541 is shown. The star denotes a non-specific band used as loading control. C) EF24-dependent inhibition of xFANCD2-Ub is not affected by co-treatment with tautomycin (phosphatase inhibitor), caffeine (kinase inhibitor) and SAP (shrimp alkaline phosphatase). Extracts treated as indicated were analyzed by immunoblot using xFANCD2 and xMRE11 antibodies. xMRE11 phosphorylation status was used to monitor the efficiency of tautomycin, caffeine and SAP treatments.

Figure 4

EF24 inhibits hFANCD2-Ub and foci formation in HeLa cells and is more active than curcumin. A) Hydroxyurea-induced FANCD2-Ub in HeLa cells is inhibited by EF24 at lower concentrations than curcumin. hFANCD2-Ub was monitored by immunoblot and densitometry analysis to determine IC₅₀. B) Hydroxyurea-induced hFANCD2 foci formation is inhibited by EF24. hFANCD2 foci were detected by immunofluorescence and the percentage of cells with more than 5 foci was determined for each treatment. Histograms represent the average of 3 experiments. Error bars represent standard deviation.

Figure 5

EF24 sensitizes the HSC 72OT+A cell line to MMC but not its FA-deficient counterpart, HSC 72OT. A) Effect of the combination of MMC and EF24 treatment on the viability of HSC 72OT and HSC 72OT+A cells. HSC 72OT (patient-derived FANCA-deficient cell line) and HSC 72OT+A (FANCA-complemented isogenic cell line) were treated with various concentrations of MMC only (solid lines) or MMC + 100 nM EF24 (dashed lines). Cell viability was measured after 3 days by MTS assay. B) Similar experiment was performed using EF24 only.

Figure 6

EF24 but not curcumin or DRB is more toxic to ATM-deficient mouse kidney cells than wild-type cells. A) 309_{ATM KO} and 334_{ATM WT} cells were treated with various concentrations of EF24. Cell viability was measured after 3 days by MTS assay. Each point represents the mean of 3 repeats. Error bars represent standard deviation. B) Similar experiment was performed using curcumin. C) Similar experiment was performed using DRB, a caseine kinase II inhibitor that does not inhibit the FA pathway in Xenopus extracts.

Figure 7

Monoketone analogs of curcumin form a new class of FA pathway inhibitors. A) Identification of 4H-TTD in an independent screen for FA pathway inhibitors. Each compound of row E of the NCI Challenge Set library plate was tested at 0.5 mM in Xenopus extracts for inhibition of xFANCD2-ub and xMRE11-P. Arrow indicates the active compound. The chemical structure of 4H-TTD is shown. B) Comparison of xFANCD2-Ub inhibition activity of 4H-TTD, EF24 and curcumin in Xenopus extracts. xFANCD2-Ub IC₅₀ were determined as in Fig. 1A. C) 4H-TTD inhibits the FA pathway in HeLa cells. IC₅₀ was determined as in Fig. 4A. D) 4H-TTD is more toxic to 309_{ATM KO} than 334_{ATM WT} cells.

Figure 8

Summary of the mechanistic findings presented in this paper. The ATM and FA pathways (white boxes/arrows) are two DNA damage response pathways that display synthetic lethal relationship. Inhibition of the FA pathway may therefore be used as a targeted therapy to selectively kill ATM-deficient tumors. Several enzymatic complexes have been proposed to modulate the FA pathway (light grey boxes). Monitoring the effect of the compounds used in this study (dark grey boxes) toward those enzymatic activities suggests that (i) in contrast with cell-based assays, the proteasome is not required for FA pathway activation in Xenopus extracts, (ii) in contrast with curcumin, EF24 is a weak proteasome inhibitor, (iii) EF24 does not inhibit the FA pathway through disruption of the core complex, and (iv) curcumin, EF24 and BMS-345541 might inhibit the FA pathway through inhibition of IKK.