Abstract
WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1,2,3,4,5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.
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Data availability
The structural data have been deposited in wwPDB under IDs 7GQS, 7GQT and 7GQU. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository38 with the dataset identifier PXD046214. These data can be accessed with the following username (reviewer_pxd046214@ebi.ac.uk) and password (5VPbwpt0). Proteomics analysis was performed using the Homo sapiens (2016) and Mus musculus (2017) UniProt Fasta databases. Source data are provided with this paper.
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Acknowledgements
We thank J. Bemis, R. Nishimura, S. Larsen and I. Stiller for programme administration; I. Mochalkin, E. Aitchison, H. Binch, D. Heer, E. Jochnowitz, A. L. Lambert, A. Rufer, R. Thoma, F. Schuler and M. Wittwer for valuable technical and scientific inputs; and B. Cravatt for helpful discussions and review of the manuscript.
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G.M.S., M.P.P. and T.M.K. conceptualized the project. Investigation was carried out by K.A.B., K.N.L., K.T.S., C.-C.W., M.A.H., A.N.S., X.S., T.G., S.K., J.C.G., D.C.R., B.L., M.E.R.-A., D.R.W., C.L.E., S.R., S.M.N., S.M.B., E.T., V.C., H.N.W., M.K.P., J.J.S., M.G.R., M.C., D.B. and I.C. Resources were the responsibility of P.P., C.C. and J.-M.P. K.A.B. wrote the original draft. Writing, review and editing were undertaken by J.P., P.P., J.-M.P., G.M.S., M.P.P. and T.M.K. K.A.B., S.K., J.P., A.T., J.S., L.E.B., R.T.A., D.S.W., G.M.S., M.P.P. and T.M.K. supervised the project.
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K.N.L., K.T.S., M.A.H., A.N.S., X.S., T.G., S.K., J.C.G., D.C.R., B.L., M.E.R.-A., D.R.W., C.L.E., S.R., S.M.N., S.M.B., J.P., V.C., H.N.W., M.K.P., J.J.S., D.S.W., G.M.S., M.P.P. and T.M.K. are current employees of Vividion Therapeutics. K.A.B., A.T., L.E.B., R.T.A., E.T., S.R. and C.-C.W. are former employees of Vividion Therapeutics. P.P., M.G.R., M.C., D.B., C.C. and J.-M.P. are current employees of F. Hoffmann-La Roche, Ltd.
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Extended data figures and tables
Extended Data Fig. 1 Additional TE and helicase activity data.
(a) Live cell and lysate TE50s for an off-target cysteine, TNFAIP3 C54 across all profiled compounds in WRN Type I (blue) and Type II (red) inhibitor series. (b) Ratios of hWRN519–1227 helicase activity without/with 0.2 mM ATP during compound preincubation period versus ratios of TE50s in lysates/live cells show that the ATP-cooperative compounds are the same ones that show enhanced potency in live cells. (c) Global TMT cysteine profiling of VVD-127101. (d) Dose-response curves of target engagement (WRN C727) by VVD-133214 in 4 cell lines: HCT-116 (gray circles), DLD-1 (white squares), SW480 (black triangles), and OCI-AML2 (circles). The comparison between both lysate (black circles) and live cells (white circles) is demonstrated in the screening line, OCI-AML2. (e) Evaluation of VVD-133214 for inhibition of mouse WRN helicase (fragment 486–1232) (mWRN486–1232) and (f) hBLM activity with- and without ATP during compound preincubation period. (g) Lack of WRN helicase inhibition activity by a non-covalent analog of VVD-133214, VVD-129448. (h) Reaction kinetics for VVD-133214 were determined using intact protein mass spectrometry with recombinant WRN. The rate of covalent modification of WRN was observed over time with varying concentrations of inhibitor. Kobs/[I] was determined to be 4848 M−1s−1 (i) Forked DNA binding of hWRN519–1227 in presence of VVD-133214 with- and without ADP preincubation via HTRF. (j) Lack of time-dependent inhibition of WRN helicase activity by VVD-133214 after helicase reaction is initiated. (i) Evaluation of VVD-133214 on helicase activity of several WRN constructs (ATPase domain hWRN532–948, C946S, Helicase Core hWRN519–1093, Helicase Core with HRDC domain hWRN519–1227, and full length WRN hWRN1–1432). Data are presented as means ± SEM.
Extended Data Fig. 2 VVD-133214 inhibits growth in MSI-high cells.
(a) Short-term treatment of Lovo cells with 2 μM VVD-133214 followed by a washout of the compound for a 6-day assay assessing nuclear number (b) and nuclear area (c) (n = 7-8 biologically independent samples). (d) No effect on nuclear area was observed following 4-day treatment of mutant HCT-116 WRN C727A cells at any concentration tested (n = 4 biologically independent samples). Data are means +/− SEM and were analyzed by one-way ANOVA. **** p<0.0001.
Extended Data Fig. 3 Time course of p53 pathway activation, cell cycle arrest, and apoptosis by VVD-133214.
Time course of dose-response of Navitoclax (a,c) or VVD-133214 (b,d) for apoptosis as measured by Real-time Glo Annexin V assay in MSI-high HCT-116 cells (a,b) or MSS SW480 cells (c,d). DNA damage pathway activation time course assessed by Simple Western™ following 2 μM VVD-133214 treatment in MSI-High HCT-116 (e), MSI-high LoVo (f), or MSS SW480 (g). Tubulin was used as a protein loading control. Columns represent individual samples. Dose-response of VVD-133214 or etoposide at 2 μM after 48 h of treatment for flow cytometry based cell cycle assessment of 2 N and 4 N DNA content in SW48 cells (h). (i) Percentage of cells in S-phase by EdU labeling. (j) Percentage of cells in mitosis by phospho-histone H3 (Ser10) staining. (k) Gating strategies for determining DNA content, EdU incorporation and phospho-Histone H3 (Ser10). (l) Time course of cycloheximide chase in HCT-116 cells upon treatment with 1 μM VVD214. WRN degradation occurs similarly in the presence or absence of cycloheximide suggesting that it is not translationally mediated. Cyclin D1 was utilized as a marker for cycloheximide activity (half-life of approximately 30 min). (m-o) HCT-116 cells were synchronized for 12 h with 0.5 mM mimosine, washed out, treated with 2 μM VVD-133214, 10 μM etoposide or DMSO and then analyzed over time for cell cycle progression (flow cytometry) (m) and WRN protein expression (Simple Western) (n). (o) Quantitation of the Simple Western data depicted in (n). Data are presented as means ± SEM.
Extended Data Fig. 4 DNA damage and repair response in MSI-high cells.
(a) Representative images of phospho-H2AX (Ser139) in MSI-high RKO cells with VVD-133214 or etoposide for 96 h. (b-e) Quantitation of Simple Western data from Fig. 4d showing effects of VVD-133214 treatment on phospho-p53 (Ser15) (b), total p53 (c), p21 (d), and WRN (e) in HCT-116 (MSI), RKO (MSI), and SW480 (MSS) cells. (f) Time course of compound-induced loss of WRN protein in HCT-116 cells treated with VVD-133214 or Etoposide (western blot quantitation normalized to tubulin expression; n = 1). (g) Rescue of VVD-133214-induced loss of WRN protein in the presence of 10 μM Bortezomib. Time points beyond 8 h could not be accurately assessed due to induction of apoptosis at later time points when using Bortezomib in HCT-116 cells. (h) Kinetic analysis of WRN protein and mRNA levels in Nocodazole synchronized HCT-116 cells demonstrating that WRN protein and mRNA are not cell cycle regulated (n = 4, technical replicates). Micrographs are representative of two independent experiments with similar results.
Extended Data Fig. 5 Tumor growth inhibition and PD in colorectal cancer xenograft models in mice.
VVD-133214 was dosed daily at 2.5, 5, 10, or 20 mg/kg via oral administration for 4 days. HCT-116 tumor tissue was collected at 2 or 24 h after the last dose (n = 4 group) and p-p53 (Ser15) (a) and p21 (b) normalized to tubulin were quantified by Simple Western. Tumor growth was assessed in female homozygous Foxn1 <nu> mice dosed orally with 2.5, 5, 10, or 20 mg/kg VVD-133214 every day for 3 weeks and p-p53 (Ser15), p21, and WRN protein levels were measured by Simple Western with tubulin as a loading control (c). Treatment did not impact body-mass (d). Additional models (n = 4–8/group) were tested for tumor growth inhibition in mice including MSI-H LoVo (e) and SW48 (f) and MSS SW480 (g). Markers of WRN inhibition were unchanged in MSS SW480 xenografts (h). Data are means +/− SEM. Data were analyzed by one-way ANOVA.
Extended Data Fig. 6 Characterization of PDX models.
(a) Description of PDX models. TMB = tumor mutational burden. (b) History of treatment for immunotherapy-refractory PDX model. (c-j) Spider plots of individual mice bearing the indicated PDX models treated with once-daily oral dosing of VVD-133214 at 20 mg/kg. CR = complete response.
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Baltgalvis, K.A., Lamb, K.N., Symons, K.T. et al. Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase. Nature (2024). https://doi.org/10.1038/s41586-024-07318-y
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DOI: https://doi.org/10.1038/s41586-024-07318-y
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