Outcompeting oncogenesis: oesophageal cancer prevention by manipulating mutant fitness.

Background: Squamous cancer of the oesophagus is a major cause of global cancer death due to late presentation and ineffective treatment. During normal ageing the oesophagus becomes colonised by mutant clones which compete for space so that only the fittest cells persist. Selected clones include those carrying mutants that promote transformation, such as TP53 and others with mutants that appear to be anti-oncogenic, such as NOTCH1. Aims: Reducing the proportion of oncogenic mutants in the tissue will cut the population at risk of transformation. In the competitive environment of the oesophagus this may be achieved by making oncogenic mutants less fit or other cells fitter, so that the potentially oncogenic cells are outcompeted and lost from the tissue. Methods: A new 3D cell culture method, epithelioids, allows long term culture of human and mouse oesophageal epithelium without passaging. This will be used to identify the genes that determine the competitive fitness of wild type, p53 mutant and Notch1 mutant cells using genome scale CRISPR screens in transgenic mouse cells. Candidate genes will be validated by testing whether they cause clonal expansions in mouse and human oesophagus using sequencing. Functional studies of novel fitness regulators will be performed and a ‘fitness map’ for wild type oesophagus constructed. Druggable genes and pathways which selectively regulate the fitness of p53 and Notch1 mutant cells will be identified, and agents tested in mouse and human epithelioid cultures and in transgenic mice for their ability to alter mutant fitness. Genome instability develops early in squamous carcinogenesis following biallelic p53 disruption. This process will be tracked in long term epithelioid cultures, using CRISPR/Cas9 gene editing to delete p53 in oesophageal cells and then analysing the resulting genome changes with combined clonal and single cell genomic sequencing. The effects of competition on the selection of genome unstable clones will be studied in mixed epithelioid cultures containing p53 cells and other competitive mutants. The agents regulating mutant fitness will be evaluated for their ability to deplete p53 genome unstable clones. Finally, lesions that resemble microscopic intra-epithelial neoplasms form in epithelioids cultured from mutated cells. The mechanism of lesion formation will be studied with genetic screens, requirements for further transformation determined and agents that may prevent this identified. Outputs Agents that alter the competitive fitness of mutants will be identified for future evaluation, with the goal of depleting oncogenic mutants from the oesophagus and reducing cancer risk.