On-target mutations confer resistance to WRN helicase inhibitors in Microsatellite Unstable Cancer Cells
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Abstract
Werner helicase inhibitors (WRNi) are in clinical development for microsatellite-unstable (MSI) tumors with defective DNA mismatch repair. Here, we investigate how cancer cell evolution shapes response to WRN inhibition and informs potential resistance mechanisms. Genome-wide CRISPR screens combined with WRN knockout did not identify bypass mechanisms, underscoring WRN’s essential, non-redundant function in MSI cells. Pharmacogenomic screens identified modulators of WRNi sensitivity, including SMARCAL1 , which links it to WRN-MSI synthetic lethality. Semi-saturation mutagenesis of WRN and prolonged drug treatment identified on-target WRN mutations driving acquired resistance to multiple WRNi in vitro and in vivo, which was mitigated by combination with standard chemotherapies. Some resistance mutations conferred broad cross-resistance, whereas others preserved sensitivity to alternative clinical-grade WRNi with distinct mechanism of action. Our findings could inform clinical trial design by suggesting the feasibility of real-time tracking of emerging resistance and enabling early therapeutic adaptations.
Significance
We present the first exploration of how MSI cancer cells evolve under the selective pressure of WRN helicase inhibition, providing a framework for understanding adaptive responses to this newly identified synthetic-lethal dependency. This study identifies on-target WRN mutations as key drivers of resistance in MSI cancers, supporting the use of combination strategies with other standard-of-care treatments to prevent resistance. It highlights how mutation tracking can guide therapeutic switching to clinically available WRN inhibitors with distinct mechanisms of action, thereby refining clinical development and potentially improving biomarker-informed patient outcomes.
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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/20506943.
Picco et al. present a framework for monitoring and designing treatment strategies to respond to WRN inhibitor resistance as it emerges, using screening and co-treatment approaches to dissect the distinct mechanisms of action underlying resistance. The comparison of resistance arising from genetic knockdown versus pharmacological inhibition provides an essential resource for distinguishing mechanisms underlying global WRN function from those specific to the inhibited WRN. Additionally, the base editing approach across multiple cancer cell lines and patient-derived xenograft models focuses the work on biologically accessible mutations, providing a valuable tool for understanding mechanisms …
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/20506943.
Picco et al. present a framework for monitoring and designing treatment strategies to respond to WRN inhibitor resistance as it emerges, using screening and co-treatment approaches to dissect the distinct mechanisms of action underlying resistance. The comparison of resistance arising from genetic knockdown versus pharmacological inhibition provides an essential resource for distinguishing mechanisms underlying global WRN function from those specific to the inhibited WRN. Additionally, the base editing approach across multiple cancer cell lines and patient-derived xenograft models focuses the work on biologically accessible mutations, providing a valuable tool for understanding mechanisms of action. This work is a substantial resource applicable across treatment paradigms and will inform clinical decision-making as WRN inhibitors advance.
Minor points:
The base editing screen showed broad coverage for WRN. Among the identified hits, were any mutations tested in a homozygous background? Whether the rescue effect is sufficient to replace wild-type activity would be relevant to assess. Similarly, do all in vivo and in vitro resistant samples harbor a single mutant allele while maintaining the wild-type allele, or do dual mutations emerge in the same clone?
In Figure 4B, the effects of C727R in the top panel do not appear to confer resistance to the covalent inhibitor VVD-133214, with no increase in relative IC₅₀ across different cell lines. However, the bottom panel shows some effect. This discrepancy makes the figure difficult to interpret. Clarification in the figure legend of what the top versus bottom panels represent, along with additional detail in the text or methods on how the over 150 assay conditions are summarized in this heatmap, would improve interpretability.
Editorial points:
The visualization of the binding pocket in Figure 4A does not clearly illustrate the proposed changes in the hydrophobic cage. The supplementary figure images are clearer. An improved comparison of the pocket changes between wild-type and mutant structures would strengthen the interpretability of the structural predictions.
Another preprint (https://www.biorxiv.org/content/10.64898/2026.01.22.700152v1 ) has some similar analyses and both strongly implicated SMARCAL1. But they differ in the specifics of DNA damage response/NEHJ pathway genes implicated in resistance with implications for companion therapies. They also differ in the discussion of the importance of P53. The authors may wish to comment about these differences and whether they result from different cell backgrounds, analyses, or other factors.
Competing interests
The authors declare that they have no competing interests.
Use of Artificial Intelligence (AI)
The authors declare that they did not use generative AI to come up with new ideas for their review.
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