Inferring active mutational processes in cancer using single cell sequencing and evolutionary constraints.

Ongoing mutagenesis in cancer drives genetic diversity throughout the natural history of cancers. As the activities of mutational processes are dynamic throughout evolution, distinguishing the mutational signatures of 'active' and 'historical' processes has important implications for studying how tumors evolve. This can aid in understanding mutagenic states at the time of presentation, and in associating active mutational process with therapeutic resistance. As bulk sequencing primarily captures historical mutational processes, we studied whether ultra-low-coverage single-cell whole-genome sequencing (scWGS), which measures the distribution of mutations across hundreds or thousands of individual cells, could enable the distinction between historical and active mutational processes. While technical challenges and data sparsity have limited mutation analysis in scWGS, we show that these data contain valuable information about dynamic mutational processes. To robustly interpret single nucleotide variants (SNVs) in scWGS, we introduce ArtiCull, a method to identify and remove SNV artifacts by leveraging evolutionary constraints, enabling reliable detection of mutations for signature analysis. Applying this approach to scWGS data from pancreatic ductal adenocarcinoma (PDAC), triple-negative breast cancer (TNBC), and high-grade serous ovarian cancer (HGSOC), we uncover temporal and spatial patterns in mutational processes. In PDAC, we observe a temporal increase in mismatch repair deficiency (MMRd). In cisplatin-treated TNBC patient-derived xenografts, we identify therapy-induced mutagenesis and inactivation of APOBEC3 activity. In HGSOC, we show distinct patterns of APOBEC3 mutagenesis, including late tumor-wide activation in one case and clade-specific enrichment in another. Additionally, we detect a clone-specific increase in SBS17 activity, in a clone previously linked to recurrence. Our findings establish ultra-low-coverage scWGS as a powerful approach for studying active mutational processes that may influence ongoing clonal evolution and therapeutic resistance.