Sustained NF-kB activation allows mutant alveolar stem cells to co-opt a regeneration program for tumor initiation [Expt1]
Tissue homeostasis relies on meticulously coordinated fate determination by resident stem cells through a tissue-wide feedback loop.  Dysregulation of this process is closely associated with tumorigenesis, yet the mechanisms distinguishing these phenomena remain in question. Here, we integrate comparative analyses of in vivo large-scale quantitative clonal dynamics and single-cell transcriptomics to unveil a key evolutionary trajectory within a single alveolar type II (AT2) stem cell, driving tissue-wide neoplastic transformation in the lung. Tracking thousands of AT2 clones throughout murine adulthood uncovers two independent homeostatic AT2 subpopulation scharacterised by unique cellular kinetics. Notably, a small AT2 subset undergoes substantial clonal expansion upon KrasG12D activation. The mutant AT2 cells follow a differentiation trajectory via emergent regenerative cell states, yet exhibit remarkable cell state equivalency with stochastic fate transitions, driving abnormal expansion with halted differentiation into mature AT1 cells. The genetic deletion of Il1r1 signalling impedes oncogenic expansion by disrupting this reprogramming trajectory, underscoring its significance in tumour initiation. Significantly, our analysis of oncogene-associated multi-colour reporter mouse models reveals a tissue- wide systemic injury response elicited by oncogenic clones in neighbouring wildtype AT2 cells, modulating their fitness programme. Moreover, we identify sustained NF-kB activation in reprogrammed cell states, setting neoplastic transformation apart from normal regeneration by preventing oncogenic exit into the differentiated AT1 state. Collectively, this work delineates the common mechanisms governing lineage infidelity across diverse tissues and proposes a compelling target amenable to therapeutic manipulation. Overall design: Lineage-labelled RFP+ or YFP+ cells were isolated from the distal lungs of either Sftpc-CreERT2; Confetti or Sftpc-CreERT2; R2Kras mice administered with 3 x 0.2mg/gbw tamoxifen and isolated at various timepoints post-induction as indicated. Individual libraries were prepared for each sample and sequenced via 10x Genomics.