Titration of oncogenic RAS alters senescent state and influences tumour initiation

Abstract Oncogenic RAS-induced senescence (OIS) is an autonomous tumour suppressor mechanism associated with pre-malignancy 1-3 . Achieving this phenotype typically requires a high level of oncogenic stress, yet the phenotype provoked by lower oncogenic dosage remains unclear. Here we develop oncogenic RAS-dose escalation models in vitro and in vivo, revealing a RAS-dose-driven non-linear continuum of downstream phenotypes. In a hepatocyte OIS model in vivo, where ectopic expression of NRAS G12V fails to induce tumours in part due to OIS-driven immune clearance 4 . scRNA-seq analyses reveal distinct hepatocyte clusters with typical OIS or progenitor-like features, corresponding to high- and intermediate-NRAS G12V levels, respectively. Remarkably, when titered down, NRAS G12V -expressing hepatocytes become immune-resistant, and develop tumours. Time-series monitoring at single-cell resolution identifies two distinct tumour types: early-onset aggressive undifferentiated and late-onset differentiated hepatocellular carcinoma (HCC). The molecular signature of each murine tumour type is associated with different progenitor features and enriched in distinct human HCC subclasses. Our results define the oncogenic dosage-driven OIS spectrum, reconciling the senescence and tumour initiation phenotypes in early tumorigenesis.