绘制Li-Fraumeni综合征中的杂合性缺失以揭示肿瘤发生的早期分子驱动因素

Li-Fraumeni syndrome (LFS) is a cancer predisposition syndrome caused by germline mutations in TP53. Mutant p53 impairs DNA damage repair, causing dysregulation in cell growth and division. LFS patients have a 40% chance of developing cancer during childhood/early adolescence, and an almost 100% lifetime risk of developing a variety of cancers. A recent study found that 86% of tumors in LFS patients exhibit loss of the wild-type (WT) allele (loss of heterozygosity (LOH)), which was absent in healthy tissue, indicating specificity to pre-malignant and malignant cells. LOH appeared to occur many years before tumor diagnosis, likely in utero or early life, suggesting it plays an early role in LFS precancer development and later tumorigenesis. In cancers with somatic TP53 mutations, LOH is an early event in tumor evolution, leading to detrimental genomic events and accelerated tumor development. While the aftermath of TP53 LOH has been explored in sporadic cancers, the extent and contribution of LOH to cancer evolution and development in LFS remains poorly understood. We leveraged LFS patient-derived skin fibroblasts, collected either pre- or post-cancer diagnosis, to map LOH in cell culture over time. Droplet digital PCR was used to determine the allelic ratio between the WT and mutated copy of TP53. Interestingly, a significant increase in the mutated TP53 allele was seen in fibroblasts collected from patients years after diagnosis, suggesting loss of the WT allele, which was not observed in fibroblasts collected prior to cancer diagnosis. Single-cell RNA sequencing (scRNA-seq) will be used to identify gene expression at an individual cell level to distinguish between cells which have undergone allelic change and those which have not. This method will help identify transcriptomic changes, with subsequent GO analysis revealing biological pathway changes before, during, and after loss of WT TP53, generating specific LOH signatures in vitro. This signature identified will be mapped back to an in vivo LFS mouse model (Trp53 R172H/WT ), probing for these in vitro transcriptomic signatures in scRNA-seq data across embryonic, post-natal and post-cancer development. LOH of WT TP53 precedes tumorigenesis, many years before tumor diagnosis in LFS. Knowing this, it is important to better understand mechanisms influencing, contributing, and responding to LOH in LFS. This is critical for cell evolution and precancer development in LFS, offering insight into opportunities for tumor prevention or interception. This project is generating the first map of LOH in LFS patient-derived fibroblasts, integrating allelic analyses and transcriptomic data, providing critical insights into the earliest stages of precancer evolution. By uncovering pathways that are differently regulated across distinct LOH states, the mechanisms of precancer development and potential therapeutic targets will be revealed.