DNA polymerase ε and δ exonuclease domain mutations in endometrial cancer

Abstract

Accurate duplication of DNA prior to cell division is essential to suppress mutagenesis and tumour development. The high fidelity of eukaryotic DNA replication is due to a combination of accurate incorporation of nucleotides into the nascent DNA strand by DNA polymerases, the recognition and removal of mispaired nucleotides (proofreading) by the exonuclease activity of DNA polymerases δ and ε, and post-replication surveillance and repair of newly synthesized DNA by the mismatch repair (MMR) apparatus. While the contribution of defective MMR to neoplasia is well recognized, evidence that faulty DNA polymerase activity is important in cancer development has been limited. We have recently shown that germline POLE and POLD1 exonuclease domain mutations (EDMs) predispose to colorectal cancer (CRC) and, in the latter case, to endometrial cancer (EC). Somatic POLE mutations also occur in 5-10% of sporadic CRCs and underlie a hypermutator, microsatellite-stable molecular phenotype. We hypothesized that sporadic ECs might also acquire somatic POLE and/or POLD1 mutations. Here, we have found that missense POLE EDMs with good evidence of pathogenic effects are present in 7% of a set of 173 endometrial cancers, although POLD1 EDMs are uncommon. The POLE mutations localized to highly conserved residues and were strongly predicted to affect proofreading. Consistent with this, POLE-mutant tumours were hypermutated, with a high frequency of base substitutions, and an especially large relative excess of G:C>T:A transversions. All POLE EDM tumours were microsatellite stable, suggesting that defects in either DNA proofreading or MMR provide alternative mechanisms to achieve genomic instability and tumourigenesis.

Figure 1.

POLE exonuclease mutations in endometrial cancer affect conserved residues required for proofreading. (A) Localization of POLE mutations within the exonuclease domain (residues 268–471). Conserved exo motifs I–V are highlighted in pink, and the exo I motif active sites at codons 275 and 277 are shown in red. Mutations in endometrioid and serous tumours are indicated by open and closed circles, respectively. (B) Sequence alignment demonstrates that Asp275 and Pro286 are invariant in POLE orthologues. (C) Structural assessment of POLE EDMs using a composite of the conserved yeast Pol δ structure (PDB 3IAY) and the ssDNA component of the T4 polymerase complex (PDB 1NOY). Mutated residues are highlighted. With the exception of Arg446, which is solvent exposed and located away from the active site and polymerase domain, all changes are likely to distort the active site architecture. Pro286 flanks the exo I motif with its side chain 2.6 Å from the nascent DNA strand, while the side chain of active site residue Asp275 lies 3.7 Å from the DNA backbone (C, inset). Substitution of either residue will result in steric and electrostatic perturbation of exonuclease function.

Figure 2.

Hypermutation of POLE EDM endometrial cancers is associated with a relative excess of G:C>T:A transversions associated with bias of flanking A:T base pairs. The frequency of each type of base change is shown as a proportion of the total number of substitutions detected by Ion Torrent sequencing. Tumours without DNA polymerase EDM (A–C) display a preponderance of C:G>T:A transitions, while POLE EDM tumours with Pro286Arg changes (D–F) are characterized by an relative increase in the proportion of G:C>T:A transversions. (G) Examination of the sequence context of G:C>T:A transversions in POLE p.Pro286Arg EDM cancers revealed an apparent over-representation of A:T base pairs 5′ and 3′ to the mutated base. The low frequency of G:C>T:A changes precluded analysis of flanking sequence in the POLE EDM non-mutants. For clarity in (G), all nucleotides are shown relative to the mutated guanine.

Figure 3.

Whole exome data from the TCGA EC study confirm disproportionate increase in G:C>T:A transversions in hypermutant POLE EDM cancers with variation by the EDM mutation type. The frequency of each type of base change is shown as a proportion of all substitutions for (A) cancers without POLE/POLD1 mutations (n = 215), (B) POLE EDM tumours with p.Pro286Arg (n = 8) and (C) p.Val411Leu (n = 5) changes. Examination of sequence context of G:C>T:A transversions by POLE EDM status (D–F) confirmed enrichment of A:T base pairs 5′ and 3′ to mutated G:C base pairs, greatest in the adjacent residue. For clarity in (D–F) all nucleotides are shown relative to the mutated guanine. Error bars in (A–C) indicate 95% confidence intervals.