Dissecting cross‐lineage tumourigenesis under p53 inactivation through single‐cell multi‐omics and spatial transcriptomics

Abstract Background Tumour suppressor genes, exemplified by TP53 (encoding the human p53), function as critical guardians against tumourigenesis. Germline TP53 ‐inactivating mutations underlie Li‐Fraumeni syndrome, a hereditary cancer predisposition disorder characterised by early‐onset pan‐tissue malignancies. However, the context‐dependent tumour‐suppressive mechanisms of p53 remain incompletely elucidated. This study aims to investigate the disruption of cellular homeostasis and tumourigenic mechanisms following p53 inactivation across distinct cell lineages. Methods Trp53 (encoding mouse p53) knockout mouse model was employed to study molecular alterations under p53‐deficient conditions. Multi‐omics analyses – including single‐cell transcriptomics, single‐cell ATAC‐seq, spatial transcriptomics, whole genome sequencing, and CUT&Tag – were integrated to construct a Trp53 functional cell landscape. Deep learning‐based gene network models were employed to reconstruct p53 regulatory networks and simulate in silico perturbations caused by p53 loss. Results Our analyses revealed transitional dynamics in immune, stromal, and epithelial cells from normal physiology to p53‐deficient states and subsequent tumourigenesis. These transitions implicated critical pathways such as cell cycle regulation, stress response, metabolic reprogramming, and immune modulation, displaying both lineage‐conserved and lineage‐specific features. Tumour‐prone cell populations exhibiting elevated differentiation plasticity were identified across lineages within tumourigenic trajectories. Spatial transcriptomic profiling confirmed the emergence of thymic tumour‐initiating T‐cell clusters characterised by deterministic chromatin architectural disruptions under p53‐loss pressure. Notably, we uncovered a recurrent upregulation signature of ribosomal protein genes as an early pivotal molecular event preceding malignant transformation in p53‐deficient oncogenesis. Finally, we decoded the p53 downstream regulatory network and computationally evaluated the perturbation effects of genetic inactivation at single‐cell resolution. Conclusions Our results elucidate the multiscale consequences of p53 inactivation while providing valuable resources for understanding tumour predisposition associated with p53‐inactivating mutations and developing clinical interception strategies. Key points Construction of a Trp53 functional cell landscape utilising single‐cell multi‐omics and spatial omics technologies. Reconstruction of p53 downstream regulatory relationships with lineage heterogeneity via machine learning‐based gene network modelling. Dissection of shared and lineage‐specific features during cross‐lineage tumourigenesis under p53 deficiency.