WRN helicase is a synthetic lethal target in microsatellite unstable cancers

Abstract

Synthetic lethality-an interaction between two genetic events through which the co-occurrence of these two genetic events leads to cell death, but each event alone does not-can be exploited for cancer therapeutics¹. DNA repair processes represent attractive synthetic lethal targets, because many cancers exhibit an impairment of a DNA repair pathway, which can lead to dependence on specific repair proteins². The success of poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors in cancers with deficiencies in homologous recombination highlights the potential of this approach³. Hypothesizing that other DNA repair defects would give rise to synthetic lethal relationships, we queried dependencies in cancers with microsatellite instability (MSI), which results from deficient DNA mismatch repair. Here we analysed data from large-scale silencing screens using CRISPR-Cas9-mediated knockout and RNA interference, and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, yet dispensable in models of cancers that are microsatellite stable. Depletion of WRN induced double-stranded DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models required the helicase activity of WRN, but not its exonuclease activity. These findings show that WRN is a synthetic lethal vulnerability and promising drug target for MSI cancers.

Extended Data Fig. 1. Functional genomic screening identifies WRN as a selective dependency with MSI.

a, Screened cell lines plotted by number of deletions and fraction of deletions occurring in microsatellite (MS) regions. Loss of genes involved in MMR are indicated by color. b, Using PCR-based MSI phenotyping, the FDR-adjusted Q values (Benjamini–Hochberg procedure) were plotted against the mean difference of dependency scores between MSI and MSS cell lines for Projects Achilles (n = 19 MSI, 291 MSS) and Project DRIVE (n = 23 MSI, 252 MSS). c, Dependency scores for each RecQ helicase plotted for MSI and MSS cell lines from Projects Achilles and DRIVE (n = as per b). Q values (Wilcoxon rank-sum test) for Achilles/DRIVE = 5.0×10⁻⁸/1.7×10⁻⁸; 0.73/0.52; 0.73/0.85; 0.25/0.73; 0.08/NA for WRN, REQL, BLM, RECQL5, and RECQL4, respectively. Lower, upper hinges: 25th, 75th percentiles, respectively. Lower, upper whiskers: lowest, largest value within 1.5*IQR (inter-quartile range) from the hinge, respectively. d, Sensitivity and positive predictive value of indicated biomarker/genetic dependency relationship. e, Dependency score distributions and associated biomarkers for example biomarker/dependency relationships. Colored regions represent density estimates. Horizontal dashed line: threshold used to separate dependent and non-dependent cell lines. n = 120/546 KRAS hotspot mutants/other; 65/601 BRAF hotspot mutants/other; 86/580 PIK3CA hotspot mutants/other.

Extended Data Fig. 2. MSI cells from MSI-predominant lineages possess greater mutational burden and WRN dependency.

a, WRN dependency scores plotted by lineage, sub-classified by MSI and MSS status. Lower, upper hinges: 25th, 75th percentiles, respectively. Lower, upper whiskers: lowest, largest value within 1.5*IQR (inter-quartile range) from the hinge, respectively. b, MS deletions in cell lines classified as MSS (n = 541), MSI from an infrequent MSI lineage (n = 45), or MSI from an MSI-predominant lineage (n = 54). P value = 1.7×10⁻⁹ (*), Wilcoxon signed-rank test. Lower and upper hinges: 25th and 75th percentiles, respectively. Lower, upper whiskers: lowest and largest value within 1.5*IQR (inter-quartile range) from the hinge, respectively. c, MSI cell lines plotted by their average WRN dependency and number of MS deletions. Lineages are color-labeled. d, MSI cell lines from MSI-predominant lineages are plotted by their average WRN dependency and number of MS deletions. Lineages are color-labeled.

Extended Data Fig. 3. WRN depletion preferentially impairs MSI cell viability.

a, IB. WRN, GAPDH levels 4 days following sgRNA transduction. b, Relative viability following sgRNA transduction in a competitive growth assay. Center values: means ± SEM (n = 6 biological replicates). P values (two-way ANOVA) between sgWRNs and negative controls at day 10: 0.37 (∗), 1.2×10⁻⁷ (†), 0.23 (‡), 2.7×10⁻¹⁹ (§). c, Clonogenic assay following sgRNA transduction. Non-targeting negative control (shRFP), pan-essential control (shPSMD2), and 2 shRNAs targeting WRN (shWRN1, shWRN2). d, Relative staining intensity of the clonogenic assay. Data shown: means ± SEM (n = 3 technical replicates). For Extended Data Fig. 3, representative data from one experiment are shown. All experiments were performed three times.

Extended Data Fig. 4. WRN depletion preferentially induces cell cycle arrest and apoptosis in MSI cells.

a, Gating strategy. For cell cycle analyses (top), debris and dead cells were excluded based on forward scatter-area (FSC-A) and side scatter-area (SSC-A) profiles. Subsequently, singlets were identified based on FSC-A and forward scatter-height (FSC-H) profiles. These singlets were then analyzed for DAPI (DNA content) and EdU-Alexa Fluor 647 (EdU-647) staining intensities. EdU-647-positive cells (cells exhibiting higher staining intensity than unstained cells) were classified as ‘S-phase’. EdU647-negative cells were classified either as ‘G1-phase’ or ‘G2/M-phase’ based on their DNA content. For apoptosis analyses (bottom), debris were excluded based on FSC-A and SSC-A profiles. The remaining samples were analyzed for Annexin V-FITC and propidium iodide (PI) staining intensities. Subsequently, Annexin V-FITC-positive cells and PI-positive cells (cells exhibiting higher staining intensity than unstained cells) were identified. Based on the positivity of these markers, cells were classified into either of the following three categories: Viable (Annexin V-negative, PI-negative), Early Apoptosis (Annexin V-positive, PI-negative), and Late Apoptosis/Nonapoptotic death (PI-positive). b, Cell cycle evaluation 4 days after sgRNA transduction. P values (two-way ANOVA) comparing sgCh2–2 vs sgWRNs for % S-phase cells: 0.16 (∗), 0.67 (†), 6.1×10⁻⁷ (‡), 3.5×10⁻⁴ (§) 0.69 (||), 2.6×10⁻⁶ (¶). c, Annexin V/propidium iodide (PI) staining evaluating early apoptosis and late apoptosis/non-apoptotic cell death 7 days following sgRNA transduction. P values (two-way ANOVA between sgCh2–2 and sgWRNs for % dying/dead cells): 0.10 (∗), 0.41 (†), 3.4×10⁻³ (‡); 3.6×10⁻⁴ (§), 0.57 (||), 3.6×10⁻⁵ (¶). d, Annexin V/PI staining 4 and 8 days following shRNA transduction. P values (two-way ANOVA comparing shRFP and shWRNs): 1.3×10⁻³ (SW837 day 4), 1.6×10⁻² (SW837 day 8), 1.2×10⁻⁶ (KM12 day 4), 4.3×10⁻⁹ (KM12 day 8). Three biological replicates are presented in tandem for b, c, d. For Extended Data Fig. 4, representative data from one experiment are shown. All experiments were performed twice.

Extended Data Fig. 5. WRN depletion activates a p53 response in MSI cells.

a, Phospho-p53 (S15) IF following sgRNA transduction in ovarian cell lines (50 µm scale bar). b, Nuclear phospho-p53 (S15) staining intensity per cell following WRN knockout compared to control sgRNA. Mean log fold-change: 0.059 (OVK18), −0.037 (ES2). Difference in log fold-change between OVK18 and ES2; P value (contrast test of least-squares means) < 2×10^-16. n = (# cells with sgCh2–2, sgWRN2, sgWRN3) for OVK18 (3982, 1143, 2740), ES2 (4916, 3072, 3690). c, p21 IF following sgRNA transduction in colon cell lines (50 µm scale bar). KM12 is a p53-impaired MSI cell line. d, Nuclear p21 staining per cell. Mean log fold-change following WRN knockout compared to control in SW48 cells compared to either SW620 (P value < 2×10⁻¹⁶; contrast test of least-squares means) or KM12 cells (P value < 2×10⁻¹⁶; contrast test of least-squares means). Mean log fold-change: 0.13 (SW48), −0.016 (SW620), −0.032 (KM12). n = for SW48 (16203, 7617, 13257), SW620 (7278, 13768, 11576), KM12 (16117, 14200, 11301). e, p21 IF following sgRNA transduction in ovarian cell lines (50 µm scale bar). f, Nuclear p21 staining intensity per cell. Mean log fold-change following WRN knockout compared to control in OVK18 compared to ES2; P value (contrast test of least-squares means) < 2×10^-16. Mean log-fold change: 0.157 (OVK18), −0.010 (ES2). n = for OVK18 (3436, 5876, 8275), ES2 (9117, 6834, 11576). g, WRN dependency for cells lines classified as MSS (n = 514), MSI from an infrequent MSI lineage (n = 6, 8 for p53-intact and impaired), or MSI from an MSI-predominant lineage (n = 23, 13 for p53-intact and impaired) and further sub-classified by p53 status. For b, d, f; lower error bar, box lower limit, bar, box upper limit, upper error bar, dots: 1st, 25th percentiles, median, 75th, 99th percentiles, outliers. For g, lower and upper hinges: 25th and 75th percentiles, respectively. Lower, upper whiskers: lowest and largest value within 1.5*IQR (inter-quartile range) from the hinge, respectively. For Extended Data Fig. 5, representative data from one experiment are shown. All experiments (a–f) were performed twice.

Extended Data Fig. 6. WRN depletion preferentially induces double strand breaks in MSI cells.

a, Nuclear ɣH2AX foci per cell following sgRNA transduction in colon cell lines. b, ɣH2AX IF following sgRNA transduction in ovarian cell lines. c, Nuclear ɣH2AX staining intensity per cell following sgRNA transduction. Difference in log fold-change between OVK18 and ES2; P value (contrast test of least-squares mean) < 2×10^-16. Mean log fold-change: 0.147 (OVK18), 0.055 (ES2). n = (# cells with sgCh2–2, sgWRN2, sgWRN3) for OVK18 (2612, 4823, 6164), ES2 (6429, 6469, 6388). Lower error bar, box lower limit, bar, box upper limit, upper error bar, dots: 1st, 25th percentiles, median, 75th, 99th percentiles, outliers. d, Nuclear ɣH2AX foci per cell following sgRNA transduction in ovarian cell lines. e, Fluorescence of Apple-53BP1 foci in colon cell lines exogenously expressing Apple-53BP1(truncated). f, Nuclear Apple-53BP1 foci per cell following sgRNA transduction in colon cell lines. g, Fluorescence of Apple-53BP1 foci following sgRNA transduction in ovarian cell lines exogenously expressing Apple-53BP1(truncated). h, Nuclear Apple-53BP1 foci per cell in ovarian cell lines. For Extended Data Fig. 6, representative data from one experiment are shown. All experiments were performed twice.

Extended Data Fig. 7. WRN depletion preferentially induces double-strand break responses in MSI cells.

a, Phospho-ATM (S1981) IF following sgRNA transduction in colon cell lines. b, Nuclear phospho-ATM (S1981) foci per cell following sgRNA transduction in colon cell lines. c, Phospho-ATM (S1981) IF following sgRNA transduction in ovarian cell lines. d, Nuclear phospho-ATM (S1981) foci per cell following sgRNA transduction in ovarian cell lines. e, ɣH2AX, phospho-Chk2, total-Chk2, WRN, GAPDH levels following shRNA transduction. For Extended Data Fig. 7, representative data from one experiment are shown. All experiments were performed twice.

Extended Data Fig. 8. WRN protein is preferentially recruited to DNA in MSI cells.

a, Telomere PNA-FISH of metaphase spreads ± dox-induction of shWRN1. Hollow triangles: chromosomal breaks. Filled triangles: chromosomal fragments. b, WRN IF following shWRN1 or control shRNA (shWRN1-C911). c, WRN IF (20 µm scale bar). d, Analyses of WRN co-localization to the nucleolar marker, fibrillarin, by Pearson’s co-localization coefficients. Data shown: means ± SEM (n = 5 biological replicates). P values (two-tailed t-test): 1.0×10⁻³ (∗), 4.3×10⁻⁵ (†); 0.014 (‡). For Extended Data Fig. 8, representative data from one experiment are shown. All experiments were conducted twice.

Extended Data Fig. 9. Paralog dependencies and hypermutation alone cannot explain the WRN/MSI relationship.

a, Estimated association between WRN dependency and MSI status after controlling for loss of indicated genes (linear model effect size estimates plotted against significance). If loss of a gene can fully account for the MSI/WRN relationship, the difference in dependency and significance would be 0. Genes whose loss are typically associated with insertion/deletion (indel) mutations (> half of loss events) are highlighted in red. n = 51 MSI, 541 MSS. b, Average WRN dependency score for MSS and MSI lines stratified by POLE status (n = 4, 5, 35, 497, 2, 12, 5, 10, 22 cell lines per category in order of left to right). Lower and upper hinges: 25th and 75th percentiles, respectively. Lower and upper whiskers: lowest and largest value within 1.5*IQR (inter-quartile range) from the hinge, respectively.

Extended Data Fig. 10. MMR deficiency contributes to WRN dependency.

a, Flow-cytometric host cell reactivation assay measuring the ability of the indicated cell lines to repair a G:G mismatch on plasmid reporter, thereby activating the fluorescence reporter and measuring MMR activity. P values (two-tailed t-test): 5.5×10⁻² (∗), 2.3×10⁻³ (†). P values (two-way ANOVA): 3.6×10⁻⁸ (‡). Data shown: means ± SEM from three independent experiments. b, IB. ɣH2AX, WRN, MLH1, MSH3, GAPDH levels following shRNA transduction in HCT116 ± MMR restoration. c, Relative viability of HCT116 derivatives 7 days following sgRNA transduction. P values (two-way ANOVA): 5.7×10⁻²⁰ (∗ vs †), 3.3×10⁻¹² († vs ‡), 1.6×10⁻¹⁶ († vs §). Data shown: means ± SEM (n = 6 biological replicates). c, IB. ɣH2AX, WRN, MLH1, MSH3, GAPDH levels following shRNA transduction in HCT116 derivatives. d, Fifteen-day clonogenic assay following shRNA transduction. e, Relative staining intensity of the clonogenic assay. P values (two-way ANOVA) comparing 3.6×10⁻⁶ (∗ vs †), 8.5×10⁻⁸ († vs ‡), 2.8×10⁻⁸ († vs §). Data shown: means ± SEM (n = 3 biological replicates). For b–f, representative data from one experiment are shown. All experiments were conducted twice except a, which was conducted thrice.

Fig. 1. Genome-scale functional genomic screening identifies genes synthetic lethal with MSI.

a, Analyses schematic. Cell lines were grouped by feature. Dependency scores were analyzed to identify feature-specific genetic dependencies. b, Cell lines plotted by number of deletions and fraction of deletions in microsatellite (MS) regions. MSI classification by next generation sequencing (NGS) and multiplex polymerase chain reaction (PCR) are indicated. c, False discovery rate adjusted (FDR) Q values (Benjamini–Hochberg method) plotted against the mean difference of dependency scores between MSI and MSS cell lines for Projects Achilles (n = 32 MSI, 412 MSS) and DRIVE (n = 34 MSI, 327 MSS).

Fig. 2. WRN is a synthetic lethal partner with MSI.

a, Relatively viability 8 days following sgRNA transduction. Negative controls targeting chromosome 2 intergenic sites: sgCh2–2, sgCh2–4. Pan-essential controls: sgPolR2D, sgMyc. WRN sgRNAs: sgWRN1, sgWRN2, sgWRN3. b, (left) Immunoblot (IB). WRN, GAPDH levels in KM12 expressing indicated WRN cDNA. (right) IB. WRN levels following sgRNA transduction. c, Relative viability 9 days following sgRNA transduction in KM12 expressing Cas9 and GFP or indicated WRN cDNA. d, KM12 xenograft growth ± dox-induction of shWRN1 or seed control (shWRN1-C911). e, IB. WRN, ɣH2AX, GAPDH levels in KM12 xenografts. f, Representative images (200 µm scale bar) and g, viability of CCLF_CORE_0001_T 9 days following shRNA induction relative to Dox(−). Data shown: means ± SEM (a, c, d, g). n = 3 (a), 6 (c) biological replicates, 5 (4 from d15–18), 5, 4, 4 tumors for shWRN1/Dox(−), Dox(+), shWRN1-C911/Dox(−), Dox(+), respectively (d), 2 biological replicates with 3 technical replicates each (g). P values: two-way ANOVA between sgWRNs and negative controls (a), two-tailed t-test for sgWRN-EIJ values between mock and indicated WRN cDNA (c), likelihood ratio test comparing Dox(+) vs (−) growth rates (d), two-tailed t-test between shWRN and its seed control (g). For Fig. 2, representative data from one experiment are shown. All experiments were performed three times except for e-g (twice) and d (once).

Fig. 3. WRN depletion in MSI cells induces cell cycle arrest, apoptosis, and a p53 response.

a, GSEA enrichment/depletion scores in WRN-depleted OVK18 cells plotted against WRN-depleted SW48 cells. Signature enrichment plots for Hallmark gene sets shown for WRN-depleted OVK18 and SW48 cells. n = 2 biological replicates. b, phospho-p53 (S15) immunofluorescence (IF) following sgRNA transduction (50 µm scale bar). c, Nuclear phospho-p53 (S15) staining intensity per cell. Lower error bar, box lower limit, bar, box upper limit, upper error bar, dots: 1st, 25th percentiles, median, 75th, 99th percentiles, outliers, respectively. Mean log intensity change following WRN knockout compared to control sgRNA in MSI versus MSS cells; P < 2×10⁻¹⁶, contrast test of least-squares means. Mean log-fold change: 0.21 (KM12), 0.10 (SW48), 0.034 (SW620). n = (# cells with sgCh2–2, sgWRN2, sgWRN3) for KM12 (7080, 14410, 15921), SW48 (15329, 9491,13196), SW620 (27374, 23898, 28808). For Fig. 3, representative data from one experiment are shown. All experiments were performed twice except for a (once).

Fig. 4. WRN depletion in MSI cells leads to accumulation of double strand DNA breaks.

a, IB. ɣH2AX, phospho(T86)- and total- Chk2, WRN, GAPDH levels following WRN knockout. Etoposide and hydroxyurea were used to generate DSB and replication stress, respectively. b, ɣH2AX IF following sgRNA transduction (50 µm scale bar). c, Nuclear ɣH2AX staining intensity per cell. Lower error bar, box lower limit, bar, box upper limit, upper error bar, dots: 1st, 25th percentiles, median, 75th, 99th percentiles, outliers, respectively. Mean log intensity change following WRN knockout compared to control in MSI compared with MSS cells; P < 2×10⁻¹⁶, contrast test of least-squares means. Mean log fold-change: 0.39 (KM12), 0.33 (SW48), −0.10 (SW620). n = (cells with sgCh2–2, sgWRN2, sgWRN3) for KM12 (3029, 8880, 6887), SW48 (13246, 4553, 7216), SW620 (9071, 5174, 3853). d, Telomere PNA-FISH of metaphase spreads ± dox-induction of shWRN1. Hollow triangles: chromosomal breaks. Filled triangles: chromosomal fragments. e, Metaphase spread DNA damage pattern per cell. n = 2 independent experiments presented in tandem. f, Relative viability of HCT116 ± MMR restoration 7 days following shRNA transduction. Negative control: shRFP. Pan-essential controls: shPSMD2, shRPS6. WRN shRNA: shWRN1, shWRN2. Data shown: means ± SEM (n = 6 biological replicates). P values: two-tailed t-test for % cells with DNA damage (e), two-way ANOVA between shWRNs and shRFP (f). For a–d, f, representative data from one experiment are shown. For e, data from two independent experiments are shown. All experiments were performed twice.