Abstract B033: Single-nuclei profiling of LFS development reveals tumour susceptibility

Abstract Li-Fraumeni Syndrome (LFS), caused by pathogenic germline Tp53 mutations, is a pediatric cancer predisposition disorder associated with markedly elevated risk of early-onset tumors, particularly rhabdomyosarcoma (RMS). Our study examined how developmental disruption may prime the tissues of LFS individuals for tumorigenesis. Single-nuclei RNA sequencing (snRNA-seq) was performed on RMS tumors, distal muscle from mice matched to RMS tumors, and cancer-free WT and LFS mouse muscle to define lineage-specific transcriptomic signatures of LFS-RMS. Embryonic limb buds were collected at embryonic days 10, 12, 14, and 16, subjected to snRNA-seq, and used to reconstruct developmental trajectories. These lineage-specific signatures were mapped back onto both WT and LFS embryonic lineages to determine whether oncogenic programs were detectable in utero and to identify the timing of their emergence. A stress-primed transcriptional program was identified as a defining feature of LFS tissues. This program distinguished precancer from postcancer states, becoming amplified in both RMS tumors and distal muscle matched to tumors. In LFS embryos, stage-specific alterations were observed in cell type proportions and maturation, including the emergence of unique myogenic and mesenchymal subpopulations absent in both WT embryos and postnatal muscle, reflecting developmental plasticity restricted to the LFS embryonic context. A lag in the transition to mature myogenic cells was detected at E14, preceded by a spike in the LFS stress-primed signature at E12 during windows of active lineage specification and proliferation. Pathway analyses of lineage-specific signatures indicated widespread dysregulation, with particularly strong changes in RNA-associated and developmental programs, further defining this stress-sensitive transcriptional state. These findings provide the first single-nuclei resolution map of embryogenesis in LFS and provide evidence that LFS-associated oncogenic programs are prenatally encoded. By linking altered lineage dynamics, developmental plasticity, and stress-primed transcriptional states to the emergence of RMS, this work establishes a developmental framework for understanding pediatric cancer predisposition. The results highlight potential windows for early detection and suggest opportunities to target embryonically primed programs for intervention in LFS. Citation Format: Ashby Kissoondoyal, Paula R. Quaglietta, Brianne Laverty, David Malkin. Single-nuclei profiling of LFS development reveals tumour susceptibility [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Cancer Evolution: The Dynamics of Progression and Persistence; 2025 Dec 4-6; Albuquerque, NM. Philadelphia (PA): AACR; Cancer Res 2025;85(23_Suppl):Abstract nr B033.