Abstract 2128: Single-cell replication dynamics in genomically unstable cancers

Abstract

Abstract Background: Single-cell whole genome sequencing (scWGS) methods such as direct library preparation (DLP) provide amplification-free capture of cells in all cycle phases and have enabled rich interrogation into the cell to cell genomic diversity of cancer genomes. Previous DLP-driven clonal evolution studies removed S-phase cells as replicated loci confounded phylogenetic inference from somatic copy number aberrations (CNAs), leaving single-cell replication timing (scRT) as an unstudied property. We thus developed a method that assigned S-phase cells to phylogenetic clones and used such assignments to infer scRT profiles in aneuploid cell lines and tumors. We applied this method to determine the relative proliferation rate between clones and obtain a single-cell picture of DNA replication fork progression for genomically unstable cancers. Methods: S-phase cells were assigned to phylogenetic clones based on copy number profile similarity and subsequently scRT profiles were inferred by normalizing out the effects of clonal, subclonal, and cell-specific CNAs before binarizing each genomic bin as replicated or unreplicated. scRT heterogeneity was quantified via a summary statistic representing the aggregate difference between expected and observed replication times (T-width). The scRT profiles were grouped into clone- and sample-level pseudobulks (aggregations of single cells) to determine genomic regions with differential replication timing. We then assessed the ability of S-phase enrichment to predict clonal expansions on time-series patient-derived xenografts of HER2+ and triple negative breast cancers that were either treatment-naive or exposed to cisplatin. In addition, we studied genetically engineered cell lines with inactivated DNA damage response genes (TP53, BRCA1, BRCA2) and primary high-grade serous ovarian cancer samples to quantify scRT heterogeneity at different states of genomic instability. Results: We analyzed 15 datasets containing over 25,000 cells with ~1/3 of cells in S-phase. Time-series datasets revealed that S-phase enriched clones were highly proliferative in the treatment-naive setting and were most sensitive to cisplatin. T-width values calculated from inferred scRT profiles increased as a function of genomic instability, with the degree of heterogeneity varied between genomic regions. Clone-level pseudobulk replication timing profiles revealed that whole-chromosome aneuploidies were more likely to have unperturbed replication timing compared to complex structural variants such as translocations, inversions, and micronuclei. Conclusions: Our time-series results show that S-phase cells enable the prediction of clone-specific proliferation and chemosensitivity using scWGS data from a single time point. The quantification of scRT heterogeneity across our collection of data implicates dysfunctional DNA replication as both driver and consequence of genomic instability. Citation Format: Adam C. Weiner, Andrew McPherson, Sohrab P. Shah. Single-cell replication dynamics in genomically unstable cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2128.