Deficiency of nucleotide excision repair is associated with mutational signature observed in cancer

Abstract

Nucleotide excision repair (NER) is one of the main DNA repair pathways that protect cells against genomic damage. Disruption of this pathway can contribute to the development of cancer and accelerate aging. Mutational characteristics of NER-deficiency may reveal important diagnostic opportunities, as tumors deficient in NER are more sensitive to certain treatments. Here, we analyzed the genome-wide somatic mutational profiles of adult stem cells (ASCs) from NER-deficient Ercc1 ^-/Δ mice. Our results indicate that NER-deficiency increases the base substitution load twofold in liver but not in small intestinal ASCs, which coincides with the tissue-specific aging pathology observed in these mice. Moreover, NER-deficient ASCs of both tissues show an increased contribution of Signature 8 mutations, which is a mutational pattern with unknown etiology that is recurrently observed in various cancer types. The scattered genomic distribution of the base substitutions indicates that deficiency of global-genome NER (GG-NER) underlies the observed mutational consequences. In line with this, we observe increased Signature 8 mutations in a GG-NER-deficient human organoid culture, in which XPC was deleted using CRISPR-Cas9 gene-editing. Furthermore, genomes of NER-deficient breast tumors show an increased contribution of Signature 8 mutations compared with NER-proficient tumors. Elevated levels of Signature 8 mutations could therefore contribute to a predictor of NER-deficiency based on a patient's mutational profile.

Figure 1.

Experimental setup and tissue-specific expression of Ercc1 in mouse ASCs. (A) Schematic overview of the experimental setup used to determine the mutational patterns in single ASCs from the liver and small intestine of mice. Biopsies from the liver and small intestine of six 15-wk-old female mice (three Ercc1^−/Δ mice and three WT littermates) were cultured in bulk for ∼1.5 wk to enrich for ASCs. Subsequently, clonal organoids were derived from these bulk organoid cultures and expanded for ∼1 mo, until there were enough cells to perform both WGS and RNA sequencing. As a control sample a biopsy of the tail of each mouse was also subjected to WGS. (B) Box plots depicting normalized Ercc1 expression in ASC organoid cultures derived from liver and small intestine of Ercc1^−/Δ mice (n = 3 and n = 3, respectively) and WT littermates (n = 3 and n = 4, respectively). Asterisks represent significant differences (P < 0.05, negative binomial test). (C) Western blot analysis of ERCC1 in Ercc1^−/Δ and WT small intestinal and liver mouse organoids.

Figure 2.

Somatic mutation rates in the genomes of ASCs from liver and small intestine of WT and Ercc1^−/Δ mice. (A) Mean number of base substitutions, (B) double base substitutions, (C) indels, and (D) SVs acquired per autosomal genome per week in ASCs of WT liver (n = 3), Ercc1^−/Δ liver (n = 3), WT small intestine (n = 2), and Ercc1^−/Δ small intestine (n = 3). Error bars represent standard deviations. Asterisks represent significant differences (q < 0.05, two-sided t-test, FDR correction). (n.s.) Nonsignificant (q ≥ 0.05, two-sided t-test, FDR correction).

Figure 3.

Mutational patterns of base substitutions acquired in the genomes of ASCs from liver and small intestine of WT and Ercc1^−/Δ mice. (A) Mean relative contribution of the indicated mutation types to the mutation spectrum for each mouse ASC group. Error bars represent standard deviations. The total number of mutations and total number of ASCs (n) per group is indicated. Asterisks indicate significant differences in mutation spectra (q < 0.05, χ² test, FDR correction). (B) Relative contribution of the indicated COSMIC mutational signatures to the average 96-channel mutational profiles of each mouse ASC group. Asterisks indicate significantly different signature contributions; P-values were obtained using a bootstrap resampling approach (Methods; Supplemental Fig. S6E,F). (C) Absolute contribution of the indicated COSMIC mutational signatures to the average 96-channel mutational profiles of each mouse ASC group. (D) Absolute contribution of two mutational signatures that were identified by nonnegative matrix factorization (NMF) analysis of the average 96-channel mutational profiles of each mouse ASC group. (E) Relative contribution of each indicated context-dependent base substitution type to mutational Signature 8 and Signature 8*.

Figure 4.

Mutational consequences of XPC^KO in human intestinal organoid cultures in vitro. (A) Targeting strategy for the generation of XPC^KO organoid cultures using CRISPR-Cas9 gene-editing. (B) Western blot analysis of XPC in human XPC^WT and XPC^KO organoids. (C) Number of base substitutions, (D) double base substitutions, and (E) Signature 8 mutations acquired per autosomal genome per week in human XPC^WT ASCs (n = 3) and an XPC^KO ASC (n = 1) in vitro.

Figure 5.

Mutation accumulation in predicted NER-deficient and NER-proficient breast cancer whole-genomes. (A) Relative contribution of each indicated context-dependent base substitution type to the average 96-channel mutational profiles of NER-deficient and NER-proficient breast cancer samples. (B) Number of Signature 8 mutations in NER-deficient and NER-proficient breast cancer whole-genomes (n = 27 and n = 43, respectively). Asterisk indicates significant difference (P < 0.05, Wilcoxon rank-sum test).