Repeat expansions confer WRN dependence in microsatellite-unstable cancers

The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair 1 – 4 . Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest a...

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Published in	Nature (London) Vol. 586; no. 7828; pp. 292 - 298
Main Authors	van Wietmarschen, Niek, Sridharan, Sriram, Nathan, William J., Tubbs, Anthony, Chan, Edmond M., Callen, Elsa, Wu, Wei, Belinky, Frida, Tripathi, Veenu, Wong, Nancy, Foster, Kyla, Noorbakhsh, Javad, Garimella, Kiran, Cruz-Migoni, Abimael, Sommers, Joshua A., Huang, Yongqing, Borah, Ashir A., Smith, Jonathan T., Kalfon, Jeremie, Kesten, Nikolas, Fugger, Kasper, Walker, Robert L., Dolzhenko, Egor, Eberle, Michael A., Hayward, Bruce E., Usdin, Karen, Freudenreich, Catherine H., Brosh, Robert M., West, Stephen C., McHugh, Peter J., Meltzer, Paul S., Bass, Adam J., Nussenzweig, André
Format	Journal Article
Language	English
Published	London Nature Publishing Group UK 08.10.2020 Nature Publishing Group
Subjects	13 13/1 13/106 13/109 13/31 45 45/15 45/22 45/23 45/29 45/70 45/77 631/337/1427 631/67/1504 Apoptosis Ataxia Telangiectasia Mutated Proteins - metabolism Biomarkers Cancer Cell cycle Cell Line, Tumor Chromosomes Chromosomes, Human - genetics Chromosomes, Human - metabolism Chromothripsis Deoxyribonucleic acid Dependence Depletion Dinucleotide Repeats - genetics DNA DNA Breaks, Double-Stranded DNA Cleavage DNA damage DNA helicase DNA Repeat Expansion - genetics DNA Replication DNA-Binding Proteins - metabolism Endodeoxyribonucleases - metabolism Endonucleases - metabolism Gene deletion Genomes Genomic Instability Humanities and Social Sciences Humans Insertion Kinases Microsatellite instability multidisciplinary Mutation Neoplasms - genetics Nuclease Recombinases - metabolism RecQ protein Replication forks Science Science (multidisciplinary) Unwinding Werner Syndrome Helicase - metabolism
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Summary:	The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair 1 – 4 . Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear. Here we show that TA-dinucleotide repeats are highly unstable in MSI cells and undergo large-scale expansions, distinct from previously described insertion or deletion mutations of a few nucleotides 5 . Expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and require unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to cleavage by the MUS81 nuclease, leading to massive chromosome shattering. These findings identify a distinct biomarker that underlies the synthetic lethal dependence on WRN, and support the development of therapeutic agents that target WRN for MSI-associated cancers. In cells with microsatellite instability, expanded TA-dinucleotide repeats form cruciform structures that stall replication forks and cause chromosome shattering in the absence of the WRN helicase.
Bibliography:	Author contributions N.v.W. set up the project, performed END-seq and flow cytometry experiments upon WRN, MUS81 and SLX4 depletion, and performed preliminary analysis of END-seq data; W.J.N. performed MUS81–EME1 in situ END-seq and PCR; A.T. performed END-seq, Southern blotting and designed ATR-mutant WRN cDNA; E.M.C. generated the inducible WRN shRNA in KM12 and HCT116 cells, performed and analysed the HSEC western blot and viability experiments, long-read sequencing, and analysed the CCLE and WRN dependency data; E.C. performed ATRi END-seq experiments, western blotting, and metaphase analysis. V.T. performed RPA ChIP–seq; K. Foster performed the HSEC and long-read sequencing experiments; N.W. performed western blotting and helped to generate WRN(3A) and WRN(6A) cells; J.N. and J.K. analysed the CCLE and WRN dependency data; S.S. analysed END-seq, RPA ChIP–seq experiments; W.W. analysed WGS, PacBio coverage across repeats, deletion breakpoints in MSI cancers, and performed quantitative modeling; F.B. analysed nucleotide composition of broken versus non-broken repeats and replication timing; E.D. performed ExpansionHunter and exSTRa bioinformatic analysis; M.A.E. supervised computational work; K.G., Y.H., A.A.B., J.T.S. and N.K. analysed the data and designed bioinformatic pipelines; R.L.W. prepared WGS libraries; A.C.-M. and K. Fugger provided recombinant MUS81–EME1; J.A.S. provided recombinant WRN; B.E.H. provided advice about PCR across repeats; K.U. provided advice about repeat expansion biology; C.H.F. provided advice about secondary structure biology; R.M.B. provided advice about WRN helicase; S.C.W. provided advice about structure specific nucleases and recombination intermediates; P.J.M. helped design in situ experiments with recombinant proteins; P.S.M. provided advice on WGS experiments and analyses; A.J.B. and A.N. supervised the project; N.v.W., W.J.N., A.T., E.M.C., A.J.B. and A.N. wrote the manuscript with comments from the other authors. N.v.W., S.S., W.J.N., A.T. and E.M.C. contributed equally; E.C. and W.W. contributed equally as second authors.
ISSN:	0028-0836 1476-4687
DOI:	10.1038/s41586-020-2769-8