Mutational signature in colorectal cancer caused by genotoxic pks+ E. coli

Abstract

Various species of the intestinal microbiota have been associated with the development of colorectal cancer1,2, but it has not been demonstrated that bacteria have a direct role in the occurrence of oncogenic mutations. Escherichia coli can carry the pathogenicity island pks, which encodes a set of enzymes that synthesize colibactin3. This compound is believed to alkylate DNA on adenine residues4,5 and induces double-strand breaks in cultured cells3. Here we expose human intestinal organoids to genotoxic pks+ E. coli by repeated luminal injection over five months. Whole-genome sequencing of clonal organoids before and after this exposure revealed a distinct mutational signature that was absent from organoids injected with isogenic pks-mutant bacteria. The same mutational signature was detected in a subset of 5,876 human cancer genomes from two independent cohorts, predominantly in colorectal cancer. Our study describes a distinct mutational signature in colorectal cancer and implies that the underlying mutational process results directly from past exposure to bacteria carrying the colibactin-producing pks pathogenicity island.

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Fig. 1: Co-culture of healthy human intestinal organoids with genotoxic E. coli induces DNA damage.
Fig. 2: Long-term co-culture with pks+ E. coli induces SBS-pks and ID-pks mutational signatures.
Fig. 3: Consensus motifs and extended features of SBS-pks and ID-pks mutational signatures.
Fig. 4: SBS-pks and ID-pks mutational signatures are present in a subset of CRC samples from two independent cohorts.

Data availability

Whole-genome sequence data have been deposited in the European Genome–Phenome Archive (https://ega-archive.org); accession number EGAS00001003934. The data used from the Hartwig Medical Foundation and Genomics England databases consist of patient-level somatic variant data (annotated variant call data) and are considered privacy sensitive and available through access-controlled mechanisms. Patient-level somatic variant and clinical data were obtained from the Hartwig Medical Foundation under data request number DR-084. Somatic variant and clinical data are freely available for academic use from the Hartwig Medical Foundation through standardized procedures. Privacy and publication policies, including co-authorship policies, can be retrieved from: https://www.hartwigmedicalfoundation.nl/en/data-policy/. Data request forms can be downloaded from https://www.hartwigmedicalfoundation.nl/en/applying-for-data/. To gain access to the data, this data request form should be emailed to info@hartwigmedicalfoundation.nl, upon which it will be evaluated within six weeks by the HMF Scientific Council and an independent Data Access Board. When access is granted, the requested data become available through a download link provided by HMF. Somatic variant data from the Genomics England data set were analysed within the Genomics England Research Environment secure data portal, under Research Registry project code RR87, and exported from the Research Environment following data transfer request 1000000003652 on 3 December 2019. The Genomics England data set can be accessed by joining the community of academic and clinical scientist via the Genomics England Clinical Interpretation Partnership (GeCIP), https://www.genomicsengland.co.uk/about-gecip/. To join a GeCIP domain, the following steps have to be taken: 1. Your institution has to sign the GeCIP Participation Agreement, which outlines the key principles that members of each institution must adhere to, including our Intellectual Property and Publication Policy. 2. Submit your application using the relevant form found at the bottom of the page (https://www.genomicsengland.co.uk/join-a-gecip-domain/). 3. The domain lead will review your application, and your institution will verify your identity for Genomics England and communicate confirmation directly to Genomics England. 4. Your user account will be created. 5. You will be sent an email containing a link to complete Information Governance training and sign the GeCIP rules (https://www.genomicsengland.co.uk/wp-content/uploads/2019/07/GeCIP-Rules_29-08-2018.pdf). Completing the training and signing the GeCIP Rules are requirements for you to access the data. After you have completed the training and signed the rules, you will need to wait for your access to the Research Environment to be granted. 6. This will generally take up to one working day. You will then receive an email letting you know your account has been given access to the environment, and instructions for logging in (for more detail, see: https://www.genomicsengland.co.uk/join-a-gecip-domain/). Details of the data access agreement can be retrieved from https://figshare.com/articles/GenomicEnglandProtocol_pdf/4530893/5. All requests will be evaluated by the Genomics England Access Review Committee taking into consideration patient data protection, compliance with legal and regulatory requirements, resource availability and facilitation of high-quality research. All analysis of the data must take place within the Genomics England Research Environment secure data portal, https://www.genomicsengland.co.uk/understanding-genomics/data/ and exported following approval of a data transfer request. Regarding co-authorship, all publications using data generated as part of the Genomics England 100,000 Genomes Project must include the Genomics England Research Consortium as co-authors. The full publication policy is available at https://www.genomicsengland.co.uk/about-gecip/publications/. All other data supporting the findings of this study are available from the corresponding author upon request.

Code availability

All analysis scripts are available at https://github.com/ToolsVanBox/GenotoxicEcoli.

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Acknowledgements

We thank J. H. J. Hoeijmakers, P. Knipscheer and J. I. Garaycoechea for discussions on DNA damage, and P. Robinson, K. Vervier, T. Lawley, and M. Stratton for explorative analysis and discussions. This publication and the underlying study have been made possible partly on the basis of the data that Hartwig Medical Foundation and the Center of Personalised Cancer Treatment (CPCT) have made available to the study. This research was made possible through access to the data and findings generated by the 100,000 Genomes Project. The 100,000 Genomes Project is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The 100,000 Genomes Project is funded by the National Institute for Health Research and NHS England. The Wellcome Trust, Cancer Research UK and the Medical Research Council have also funded research infrastructure. The 100,000 Genomes Project uses data provided by patients and collected by the National Health Service as part of their care and support. This work was supported by CRUK grant OPTIMISTICC (C10674/A27140), the Gravitation projects CancerGenomiCs.nl and the Netherlands Organ-on-Chip Initiative (024.003.001) from the Netherlands Organisation for Scientific Research (NWO) funded by the Ministry of Education, Culture and Science of the government of the Netherlands (C.P.-M., J.P.), the Oncode Institute (partly financed by the Dutch Cancer Society), the European Research Council under ERC Advanced Grant Agreement no. 67013 (J.P., T.M., H.C.), a VIDI grant from the NWO (no. 016.Vidi.171.023) to R.v.B. that supports A.R.H. and NWO building blocks of life project: Cell dynamics within lung and intestinal organoids (737.016.009) (M.H.G.). With financial support from ITMO Cancer AVIESAN (Alliance Nationale pour les Sciences de la Vie et de la Santé, National Alliance for Life Sciences & Health) within the framework of the Cancer Plan (HTE201601) (G.D., R.B.) as well as Howard Hughes Medical Institute, Mathers Foundation, and NIH-1R01DK115728-01A1 (Y.M., K.C.G.).

Author information

C.P.-M., J.P., A.R.H. and H.C. conceived the study; C.P.-M., J.P., A.R.H., R.v.B. and H.C. wrote the manuscript; A.R.H., H.M.W., F.M. and R.v.B. performed signature analysis; A.R.H., A.v.H., H.M.W., J.N., C.G., P.Q., M.G., M.M. and E.C. provided access to and analysed patient WGS data; G.D. and R.B. isolated bacterial strains and generated knockouts; C.P.-M., J.P., T.M., R.v.d.L., M.H.G. and S.v.E. established and performed organoid cloning experiments; C.P.-M., J.P. and J.B. performed organoid co-culture experiments; P.B.S., F.L.P., J.T. and R.J.L.W. performed bacteria validation and assays. Y.M. and K.C.G. provided and advised on the use of the Wnt surrogate-Fc fusion reagent.

Correspondence to Ruben van Boxtel or Hans Clevers.

Ethics declarations

Competing interests

H.C. is inventor on several patents related to organoid technology; his full disclosure is given at https://www.uu.nl/staff/JCClevers/. M.M. is scientific advisory board chair and a consultant for OrigiMed, receives research support from Bayer, Janssen, and Ono, and receives royalty payments from Labcorp. H.C and K.C.G are co-founders of Surrozen.

Additional information

Peer review information Nature thanks Bogdan Fedeles, Christian Jobin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Co-culture with genotoxic pks+ E. coli induces DNA interstrand crosslinks in healthy human intestinal organoids.

a, Representative images (out of n = 5 organoids per group) of DNA interstrand crosslink formation after 1 day of co-culture, measured by FANCD2 immunofluorescence (green). Nuclei were stained with DAPI (blue). Yellow boxes represent inset area. Scale bars, 50 μm (main image); 10 μm (inset). Experiment was repeated independently twice with similar results. b, Gating strategy to select epithelial cells (left) and to quantify viable cells (right). c, Mean ± s.d. viability of intestinal organoid cells after 1, 3 or 5 days of co-culture (n = 3 technical replicates) (bacteria eliminated after 3 days of co-culture). Points are independent replicates.

Extended Data Fig. 2 Genotoxic pks+ E. coli induce SBS-pks and ID-pks mutational signatures after long-term co-culture with wild-type intestinal organoids.

a, Ninety-six-trinucleotide mutational spectra of SBSs in each of the three individual clones sequenced per condition. Top three, dye; middle three, pksΔclbQ E. coli; bottom three, pks+ E. coli. b, Total 96-trinucleotide mutational spectra of organoids injected with pks+ E. coli or pksΔclbQ E. coli from which SBSs in dye-injected organoids have been subtracted. c, Heatmap depicting cosine similarity between 96-trinucleotide mutational profiles of organoids injected with dye, pks+ E. coli or pksΔclbQ E. coli. d, Indel mutational spectra plots from each of the three individual clones sequenced per condition. Top three, dye; middle three, pksΔclbQ E. coli; bottom three, pks+ E. coli. e, Total indel mutational spectra of organoids injected with pks+ E. coli and pksΔclbQ E. coli from which indels in dye-injected organoids have been subtracted. f, Heatmap depicting cosine similarity between indel mutational profiles of organoids injected with dye, pks+ E. coli or pksΔclbQ E. coli.

Extended Data Fig. 3 Genotoxic pks+ E. coli and isogenic strain reconstituted with pksΔ clbQ:clbQ induce SBS-pks and ID-pks mutational signatures after co-culture.

a, Ninety-six-trinucleotide mutational spectra of SBSs in three individual clones from the independent human healthy intestinal organoid line ASC-6a co-cultured for three rounds with pks+ or pksΔclbQ E. coli. b, Top, total 96-trinucleotide mutational spectra from the three clones co-cultured with from pks+ or pksΔclbQ E. coli shown in a. Bottom, resulting 96-trinucleotide mutational spectrum from ASC-6a organoids co-cultured with pks+ E. coli after the subtraction of background mutations from three parallel pksΔclbQ E. coli co-cultures (cosine similarity to SBS-pks = 0.77). c, Indel mutational spectra from the three independent ASC-6a clones co-cultured for three rounds with pks+ or pksΔclbQ E. coli. d, Top, total indel mutational spectra from the three clones co-cultured with pks+ or pksΔclbQ E. coli shown in c. Bottom, resulting indel mutational spectrum from the independent ASC-6a organoids co-cultured with pks+ E. coli after the subtraction of background mutations from three parallel pksΔclbQ E. coli co-cultures (cosine similarity to ID-pks = 0.93). e, Ninety-six-trinucleotide mutational spectra from three individual clones of the ASC-5a line co-cultured for three rounds with the isogenic recomplemented E. coli strain pksΔclbQ:clbQ. f, Top, total 96-trinucleotide mutational spectrum from the three clones co-cultured with pksΔclbQ:clbQ E. coli shown in e. Bottom, resulting mutational spectrum after subtracting pksΔclbQ background (cosine similarity to SBS-pks = 0.95). g, Indel mutational spectra from three individual clones of the ASC-5a line co-cultured for three rounds with the isogenic recomplemented E. coli strain pksΔclbQ:clbQ. h, Top, total indel mutational spectrum from the three clones co-cultured with pksΔclbQ:clbQ E. coli shown in g. Bottom, resulting mutational spectrum after subtracting pksΔclbQ background (cosine similarity to ID-pks = 0.95).

Extended Data Fig. 4 Detailed sequence context for ID-pks and longer deletions by length.

a, Ten-base up- and downstream profile shows an upstream homopolymer of adenosines that favours induction of T deletions. The length of the adenosine stretch decreases with increasing T homopolymer length (1–8, top left to bottom right).

Extended Data Fig. 5 Signature extraction and clonal contribution of SBS-pks in CRC metastases.

a, De novo NMF-SBS-pks signature extracted by NMF on all 496 CRC metastases in the HMF data set. b, Cosine similarity scores between the de novo extracted SBS signature in a and COSMIC SigProfiler signatures, including our experimentally defined SBS-pks signature (left). c, Relative contribution of SBS-pks to clonal (corrected variant allele frequency >0.4, blue) and subclonal fractions (corrected variant allele frequency <0.2, red) of mutations in the 31 SBS/ID-pks high CRC metastases from the HMF cohort. Box, upper and lower quartiles; centre line, mean; whiskers, largest value no more than 1.5 times the interquartile range extending from the box; points, individual CRC metastases.

Extended Data Table 1 SBS-pks and ID-pks levels across tissue types

Supplementary information

Reporting Summary

41586_2020_2080_MOESM2_ESM.xlsx

Supplementary Table 1 Mutations matching pks motifs in driver genes in colorectal cancer. List of the number of mutations matching the SBS-pks or ID-pks motifs and total number of mutations within the top 50 driver genes present in colorectal cancer. Dataset obtained from the IntOGen cancer mutation database25.

Supplementary Table 2 Protein coding sequence mutations matching the SBS/ID-pks motif. List of all mutations from all SBS/ID-pks high CRC samples matching the SBS/ID-pks extended motif and leading to changes in protein coding regions of the genome.

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Pleguezuelos-Manzano, C., Puschhof, J., Rosendahl Huber, A. et al. Mutational signature in colorectal cancer caused by genotoxic pks+ E. coli. Nature (2020). https://doi.org/10.1038/s41586-020-2080-8

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