Werner syndrome helicase has a critical role in DNA damage responses in the absence of a functional fanconi anemia pathway

Abstract

Werner syndrome is genetically linked to mutations in WRN that encodes a DNA helicase-nuclease believed to operate at stalled replication forks. Using a newly identified small-molecule inhibitor of WRN helicase (NSC 617145), we investigated the role of WRN in the interstrand cross-link (ICL) response in cells derived from patients with Fanconi anemia, a hereditary disorder characterized by bone marrow failure and cancer. In FA-D2(-/-) cells, NSC 617145 acted synergistically with very low concentrations of mitomycin C to inhibit proliferation in a WRN-dependent manner and induce double-strand breaks (DSB) and chromosomal abnormalities. Under these conditions, ataxia-telangiectasia mutated activation and accumulation of DNA-dependent protein kinase, catalytic subunit pS2056 foci suggested an increased number of DSBs processed by nonhomologous end-joining (NHEJ). Rad51 foci were also elevated in FA-D2(-/-) cells exposed to NSC 617145 and mitomycin C, suggesting that WRN helicase inhibition interferes with later steps of homologous recombination at ICL-induced DSBs. Thus, when the Fanconi anemia pathway is defective, WRN helicase inhibition perturbs the normal ICL response, leading to NHEJ activation. Potential implication for treatment of Fanconi anemia-deficient tumors by their sensitization to DNA cross-linking agents is discussed.

Figure 1. NSC 617145 inhibits cell proliferation in a WRN-specific manner

Panel A, HeLa cells were treated with DMSO or indicated NSC 617145 concentration for 0–3 days. Panel B, HeLa cells were exposed to 1.5 μM NSC 617145 for 3 days, replenished with media lacking NSC 617145, and allowed to recover for 0–3 days. Panel C, HeLa cells either untransfected (HeLa), transfected with non-specific siRNA (NS siRNA) or WRN specific siRNA (WRN siRNA) were treated with DMSO or 1.5 μM NSC 617145 for indicated number of days. Cell proliferation was then determined with WST-1 reagent. Percent proliferation was calculated. Experiments were repeated three times, and error bars indicate standard deviation. This applies to all figures.

Figure 2. NSC 617145 induces WRN to bind chromatin and WRN degradation

Panel A, HeLa cells were exposed to increasing concentrations (0.75, 1.0, 1.5, and 2.0 μM) of NSC 617145 for 4 hours. Chromatin-bound and nuclear soluble fractions were prepared and 10 μg of protein was resolved by SDS-PAGE, followed by analysis for WRN protein by immunoblotting. A representative Western blot from one of three independent experiments is shown. Histone H3 and TopoI served as markers for the chromatin and soluble nuclear fractions, respectively. Panel B, Whole cell extracts were prepared from HeLa cells exposed to NSC 617145 (0.75 μM) or DMSO for 6 hours and proteins resolved by SDS-PAGE followed by Western blot analysis for WRN. As indicated, the proteasome inhibitor MG132 (10 μM) was added to the cell culture at the same time as NSC 617145. Beta actin and RECQL1 served as a loading control. Panel C, Immunoprecipitated WRN obtained from an equivalent amount of protein derived from extracts of HeLa cells that had been exposed to 0.75 μM NSC 617145 or DMSO for 4 hours was tested for helicase activity on a forked duplex DNA substrate as described in Experimental Procedures. A representative helicase gel and quantitative data from at least three independent experiments are shown.

Figure 3. NSC 617145 exposure enhances sensitivity of FA mutant cells to MMC in a WRN-dependent manner

Panel A, HeLa cells were treated with NSC 617145 (0.5 μM), MMC (9.4 nM), or both compounds for 3 days. Panel B, FA-D2^−/− and FA-D2^+/+ cells were treated with NSC 617145 (0.125 μM), MMC (9.4 nM), or both compounds for 2 days. Panel C, FA-A^−/− and FA-A^+/+ cells were treated with NSC 617145 (0.75 μM), MMC (9.4 nM), or both compounds for 2 days. Panel D, FA-D2^−/− and FA-D2^+/+ cells lines transfected with WRN siRNA were treated with NSC 617145 (0.125 μM), MMC (9.4 nM), or both compounds for 2 days. Panel E, FA-D2^−/− and FA-D2^+/+ cells lines transfected with NS siRNA were treated with NSC 617145 (0.125 μM), MMC (9.4 nM), or both compounds for 2 days. Cell proliferation was determined. Percent proliferation was calculated.

Figure 4. Increased accumulation of γ–H2AX foci in FA-D2 mutant cells co-treated with NSC 617145 and MMC

Panel A, FA-D2^−/− and FA-D2^+/+ cells were treated with NSC 617145 (0.125 μM), MMC (9.4 nM), or both compounds for 2 days. Cells were stained with anti-γ–H2AX antibody or DAPI, and imaged. Panel B, Percentage of cells with γ–H2AX foci (≤15 or >15, as indicated).

Figure 5. FA-D2 mutant cells co-treated with NSC 617145 and MMC display ATM activation and increased chromosomal instability

Panel A, FA-D2^−/− and FA-D2^+/+cells were co-treated with 0.125 μM NSC 617145 and 9.4 nM MMC for 3 hours. Cell lysates were prepared and analyzed by immunoblotting by using anti-pATM Ser1981 antibody. As a control, blot was reprobed with anti-ATM antibody. Arrow indicates phosphorylated ATM. Panel B, FA-D2^−/− and FA-D2^+/+ cells were treated with NSC 617145 (0.125 μM), MMC (9.4 nM), or both compounds for 2 days. Chromosome spreads were analyzed. Arrow indicates chromatid break, chromatid loss, or radial. Each radial is counted as equivalent to two chromatid breaks. Panel C, Chromatid breaks per metaphase.

Figure 6. NSC 617145 exposure elicits NHEJ pathway in FA deficient cells upon co-treatment with MMC

Panel A, FA-D2^−/− and FA-D2^+/+ cells were treated with NSC 617145 (0.125 μM), MMC (9.4 nM), or both compounds for 2 days. Cells were stained with anti-DNA-PKcs-pS2056 antibody, or DAPI (Panel A). Percentage of cells with DNA-PKcs-pS2056 foci (≤15 or >15, as indicated) is shown (Panel B).

Figure 7. Increased accumulation of Rad51 foci in FA-D2 mutant cells co-treated with NSC 617145 and MMC

FA-D2^−/− and FA-D2^+/+ cells were treated with NSC 617145 (0.125 μM), MMC (9.4 nM), or both compounds for 2 days. Cells were stained with anti-Rad51 antibody or DAPI (Panel A). Percentage of cells with Rad51 foci (≤15 or >15, as indicated) is shown (Panel B).