Characterizing mutational signatures at hotspots of genetic instability in human cancer genomes

Passenger mutations serve as a valuable record of the underlying mutational forces that shape the cancer genome and their characteristic imprints can be studied through a process known as mutational signatures, which reflect the activity of endogenous mechanisms (e.g., DNA repair function) and exogenous exposures (e.g., carcinogens). Non-B DNA structures are alternative DNA conformations that arise at specific repetitive sequence contexts and represent hotspots of genome instability. These include G-quadruplexes, Z-DNA, hairpins, cruciforms, slipped DNA, and H-DNA, which are widespread in the human genome and have been implicated in transcriptional regulation, splicing modulation, and replication dynamics. Previous work by our group and others has demonstrated that non-B DNA motifs are enriched at sites of both germline and somatic variation and are drivers of localized mutability across cancer genomes. However, the precise mechanisms driving mutagenesis at non-B DNA structures are poorly understood, representing a critical gap in our knowledge. The innovation of this project lies in integrating large-scale cancer genome analyses, gene perturbation experiments, and environmental mutagen exposure data to uncover the mutational processes that cause genomic instability at non-B DNA structures. We hypothesize that distinct non-B DNA structures have different susceptibilities to mutational processes and are preferentially associated with specific mutational signatures in cancer. The significance of this project lies in providing the first comprehensive mechanistic analysis of how non-B DNA structures drive cancer mutagenesis, promoting a more holistic understanding of genome instability in oncogenesis. In Aim 1, we will characterize non-B DNA motifs in silico across 17,517 cancer genomes and extract and compare mutational signatures at non-B DNA versus matched control regions. We will quantify the extent to which specific non-B DNA motifs are associated with known mutational signatures in a cancer-type- specific and pan-cancer manner. In Aim 2, we will determine how deficiencies in key DNA repair pathways impact non-B DNA-associated mutagenesis by integrating WGS data from CRISPR-Cas9 knockout cell lines. We will also assess DNA mismatch repair- and homologous recombination-deficiency signatures in tumor data to identify clinically relevant mutational patterns at non-B DNA loci. In Aim 3, we will evaluate how non-B DNA sequences modulate the mutational impact of 79 environmental mutagens and determine whether certain mutagens preferentially induce mutations at specific non-B DNA motifs and whether these align with patterns observed in human tumors. Finally, for Aims 2 and 3, we will conduct a focused set of targeted experiments to validate and reinforce our key findings. The objective of this project is to identify and characterize the critical pathways through which non-B DNA structures promote genome instability in cancer. Our findings will directly contribute to our long-term goal of informing precision approaches to cancer risk assessment and targeted therapy development. Project Number: 1R21CA314232-01 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Ilias Georgakopoulos-Soares (+1 co-PI) | Institution: UNIVERSITY OF TEXAS AT AUSTIN, AUSTIN, TX | Award Amount: $396,377 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZRG1 BTC-V (02)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11390635