Recurrent promoter mutations in melanoma are defined by an extended context-specific mutational signature

Abstract

Sequencing of whole tumor genomes holds the promise of revealing functional somatic regulatory mutations, such as those described in the TERT promoter. Recurrent promoter mutations have been identified in many additional genes and appear to be particularly common in melanoma, but convincing functional data such as influence on gene expression has been more elusive. Here, we show that frequently recurring promoter mutations in melanoma occur almost exclusively at cytosines flanked by a distinct sequence signature, TTCCG, with TERT as a notable exception. In active, but not inactive, promoters, mutation frequencies for cytosines at the 5' end of this ETS-like motif were considerably higher than expected based on a UV trinucleotide mutational signature. Additional analyses solidify this pattern as an extended context-specific mutational signature that mediates an exceptional position-specific vulnerability to UV mutagenesis, arguing against positive selection. We further use ultra-sensitive amplicon sequencing to demonstrate that cell cultures exposed to UV light quickly develop subclonal mutations specifically in affected positions. Our findings have implications for the interpretation of somatic mutations in regulatory regions, and underscore the importance of genomic context and extended sequence patterns to accurately describe mutational signatures in cancer.

Fig 1. A distinct sequence signature characterizes nearly all highly recurrent somatic promoter mutations in melanoma.

Whole genome sequencing data from 38 melanomas were analyzed for individual recurrently mutated bases in promoter regions. (a) All highly recurrent mutations within +/- 500 bp from TSSs ordered by recurrence (number of mutated tumors). ^aRecurrence of each mutation. ^bChromosome. ^cReference base. ^dVariant base. ^eSequence context, showing pyrimidine-containing strand with respect to the central mutated base (gray). The motif CTTCCG is highlighted in yellow. ^fDistance to the nearest TSS in GENCODE 17. ^gClosest gene. Cancer gene census genes [42] in blue. ^hGenes were sorted by mean expression (all samples) and assigned to tiers 1 to 3 with 3 being the highest. ⁱP-value for differential expression of the gene comparing tumors with and without the mutation (two-sided Wilcoxon test). ^{j, k, l, m}Second closest TSS, if within 500 bp. ⁿA previous analysis in a larger cohort failed to show significant differential expression [10]. (b) All mutations occurring within +/- 500 bp of a TSS while overlapping with the motif NCTTCCGN. The distance to the nearest TSS and the degree of recurrence (number of mutated tumors) is indicated. (c) Similar to panel b, but instead showing mutations not overlapping NCTTCCGN. (d) Positional distribution across the sequence NCTTCCGN for mutations listed in panel a.

Fig 2. Positive correlation between promoter hotspot mutations and total mutational load across melanomas.

(a) Bars, left axis: Number of mutations occurring in the established recurrent CTTCCG-related promoter positions (> = 3 tumors) in each of the 38 samples. Line, right axis: Total mutational load per tumor (number of mutations across the whole genome). (b) Presence of TERT promoter mutations and mutations in known driver genes are indicated for all samples.

Fig 3. Recurrent mutations at CTTCCG sites are observed only near active promoters.

(a-c) Genes were assigned to three expression tiers by increasing mean expression across the 38 melanomas. The graphs show, on the x-axis, the distance to the nearest annotated TSS for all mutations overlapping with or being adjacent to the motif CTTCCG across the whole genome, separately for each expression tier. The level of recurrence is indicated on the y-axis.

Fig 4. Mutation probabilities for CTTCCG-related sequence contexts compared to trinucleotides in melanoma.

The mutated position in each sequence context is shaded in gray. Bar colors indicate the substituting bases (mainly C>T). Mutation probabilities were calculated genome-wide (a), or only considering mutations less than 500 bases from TSS of genes with a low (b), middle (c) or high (d) mean expression level.

Fig 5. Mutation probabilities for CTTCCG-related sequence contexts compared to trinucleotides in cSCC tumors with NER deficiency.

5 cSCC tumors with defective global NER[18] were screened for mutations within 500 bp upstream of the TSSs, considering only genes in the upper expression tier as defined earlier based on TCGA data. Template (a) and non-template strands (b), with respect to the transcription direction of the downstream gene, were considered separately. The mutated position in each sequence context is shaded in gray. Bar colors indicate the substituting bases.

Fig 6. UV exposure of cultured cells induces mutations specifically at CTTCCG-related promoter hotspot sites.

(a) Human cells (A375 melanoma cells or HaCat keratinocytes) were subjected to daily UV doses (254 nm, 36 J/m² once a day, 5 days a week). An ultrasensitive amplicon sequencing protocol, SiMSen-Seq[28], was used to assay for subclonal mutations in two of the established promoter hotspot sites after 5 or 10 weeks. (b) 16 different conditions (+/- UV, two regions, two time points, and two cell lines) were sequenced at 2.5M to 4.8M reads per library. Minimum 20 times oversampling was required, resulting in 36k-82k error-corrected reads per library. (c) Example of raw and corrected mutation frequencies upstream of RPL13A (HaCat cells, 10 weeks UV exposure). (d-e) Subclonal mutations at or near CTTCCG hotspots upstream of RPL13A or DPH3, after 5 or 10 weeks of UV exposure. The CTTCCG elements are indicated, and other possible UV-susceptible sites (cytosines flanking pyrimidines) are underscored. The amplicon sizes were 49 and 36 bp, respectively.