Sequencing of whole cancer genomes has revealed an abundance of recurrent mutations in gene-regulatory promoter regions, in particular in melanoma where strong mutation hotspots are observed adjacent to ETS-family transcription factor (TF) binding sites. While sometimes interpreted as functional driver events, these mutations are commonly believed to be due to locally inhibited DNA repair. Here, we first show that low-dose UV light induces mutations preferably at a known ETS promoter hotspot in cultured cells even in the absence of global or transcription-coupled nucleotide excision repair (NER). Further, by genome-wide mapping of cyclobutane pyrimidine dimers (CPDs) shortly after UV exposure and thus before DNA repair, we find that ETS-related mutation hotspots exhibit strong increases in CPD formation efficacy in a manner consistent with tumor mutation data at the single-base level. Analysis of a large whole genome cohort illustrates the widespread contribution of this effect to recurrent mutations in melanoma. While inhibited NER underlies a general increase in somatic mutation burden in regulatory elements including ETS sites, our data supports that elevated DNA damage formation at specific genomic bases is at the core of the prominent promoter mutation hotspots seen in skin cancers, thus explaining a key phenomenon in whole-genome cancer analyses.

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. Author summaryCancer is caused by somatic mutations that alter cell behavior. While such mutations typically occur in protein-coding genes, recent studies describe individual positions in gene regulatory regions (promoters) that are recurrently mutated in many independent tumors. This suggests that positive selection could be acting on these non-coding mutations, and that they may contribute to carcinogenesis. However, proper interpretation of recurrent mutations requires a detailed understanding of how such mutations arise in the absence of selection pressures, referred to as mutational heterogeneity. In this paper, we describe a distinct sequence signature that characterizes nearly all highly recurrent promoter mutations in melanoma. Additional analyses support that this sequence mediates an exceptional local vulnerability to UV-induced mutagenesis, explaining why mutations are frequently observed in these positions. Importantly, cultured cells exposed to UV light quickly developed mutations specifically in the expected sites. Our results have important implications for the interpretation of recurrent somatic mutation patterns in non-coding DNA.

High-risk neuroblastoma (NB) often involves MYCN amplification as well as mutations in ALK. Currently, high-risk NB presents significant clinical challenges, and additional therapeutic options are needed. Oncogenes like MYCN and ALK result in increased replication stress in cancer cells, offering therapeutically exploitable options. We have pursued phosphoproteomic analyses highlighting ATR activity in ALK-driven NB cells, identifying the BAY1895344 ATR inhibitor as a potent inhibitor of NB cell growth and proliferation. Using RNA-Seq, proteomics and phosphoproteomics we characterize NB cell and tumour responses to ATR inhibition, identifying key components of the DNA damage response as ATR targets in NB cells. ATR inhibition also produces robust responses in mouse models. Remarkably, a 2-week combined ATR/ALK inhibition protocol leads to complete tumor regression in two independent genetically modified mouse NB models. These results suggest that NB patients, particularly in high-risk groups with oncogene-induced replication stress, may benefit from ATR inhibition as therapeutic intervention.

Somatic mutations can result in the formation of neoantigens, immunogenic peptides that are presented on the tumor cell surface via HLA molecules. These mutations are expected to be under negative selection pressure, but the extent of the resulting neoantigen depletion remains unclear. Based on HLA affinity predictions, we annotated the human genome for its translatability to HLA binding peptides and screened for reduced single nucleotide substitution rates in large genomic datasets from untreated cancers. Apparent neoantigen depletion signals became negligible when considering trinucleotide-based mutational signatures, either due to lack of power or efficient immune evasion mechanisms active early during tumor evolution. Cancer is caused by somatic mutations in driver genes. These genomic alterations result in a selective growth advantage and positive selection of the affected cells 1. With the rise of nextgeneration sequencing technologies, increasing insights into the cancer genome have led to a comprehensive characterization of the frequencies and patterns of somatic mutations across different cancers 2,3. For a tumor to evolve, it also needs to develop ways to avoid immune Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: