Colibactin DNA damage signature indicates causative role in colorectal cancer

Background:

Colibactin is a toxin produced by species of E. coli expressing a biosynthetic gene cluster (pks), known as pks+ E. coli. Colibactin causes double-stranded breaks in human genomic DNA, and has been associated with colorectal cancers. Recent advances, including the elucidation of the chemical structure of colibactin, have clarified the mechanism of colibactin action – it alkylates DNA using a cyclopropane “warhead”, and identification of DNA adducts revealed linkages to adenine bases (Xue and Kim, Science 2019). However, it has been unclear whether there is a sequence preference of colibactin, and there is limited evidence for colibactin-induced DNA damage in human cancers. In this preprint, Dziubańska-Kusibab and Berger et al identify the target site preferences of colibactin and identify enrichment of mutations at colibactin sites in colorectal and other cancers.

 

Key Findings:

• Colibactin has a specific target motif for DNA damage.

The authors used BLISS, a sequencing-based method to identify double-stranded break (DSB) sites in colibactin-expressing pks+ E coli infected cells. Analysis of the DSB sites showed enrichment of an AT-rich motif, which the authors narrowed to a hexanucleotide AAWWTT motif. This motif is predicted to harbor particularly narrow minor groove widths, and highly negative electrostatic potential, along with other DNA structural parameters that are highly unusual. These shape characteristics enable the fit of colibactin, as determined by molecular modeling, in a position that allows the reactive “warhead” to attack the DNA at the characterized N3-targeting position of adenine.

• Colibactin target motifs are enriched in colorectal cancers

Using the newly defined AAWWTT colibactin motif, the authors examined whole-exome sequences (across tumor types, from TCGA) and whole-genome sequences (from colorectal cancers) to identify whether mutations were enriched at colibactin targeting sites. In both datasets, colorectal cancer showed enrichment for mutations at colibactin motifs. This mutational enrichment was detected at higher rates in hypermutator POLE mutant tumors, but not in microsatellite-instabile (MSI) tumors. Additionally, previously characterized mutational signatures of colorectal cancer matched the colibactin targeting motif.

 

Importance:

This preprint further clarifies the mechanism and DNA binding preferences of colibactin, a prototypical bacterial genotoxin with strong links to human disease. Additionally, it expands from association studies linking colibactin prevalence among colorectal cancer patients to identify evidence of colibactin-induced mutations in human patients. The past decade has seen thorough investigation into colibactin, from DNA breaks in vitro, mouse models of colorectal cancer, epidemiological studies, detailed chemical analysis, and now evidence of colibactin mutation signatures in large cohorts. This may be the strongest evidence to date of the breadth of colibactin impact on human health and disease.

 

Moving Forward / Questions for Authors:

• The mutational signature associated with colibactin was enriched in colorectal cancer, but also in stomach and endometrial cancers by both mutational signature and SNV analysis. Is there evidence for colibactin among stomach and vaginal/uterine microbes (either coli such as UPEC, or other species such as Klebsiella), or an association of these tumor types with microbial composition?
• With a defined sequence preference for colibactin now available, it will be interesting to see the effect of DNA modifications on colibactin activity. Does modification of adenine bases impact their targetability, or alter the shape of the DNA groove to make it less suited for colibactin binding? Do coli protect themselves from “self-infection” by modifying colibactin motifs in their DNA?

 

References:

• Xue M*, Kim CS*, et al. “Structure elucidation of colibactin and its DNA cross-links” Science 2019 doi:10.1126/science.aax2685

 

doi: https://doi.org/10.1242/prelights.14960

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