A ‘Synthetic-Sickness’ Screen for Senescence Re-engagement Targets in Mutant Cancer Backgrounds. Homo sapiens

Senescence is a universal barrier to immortalisation and tumorigenesis. As such, interest in the use of senescence-induction in a therapeutic context has been gaining momentum in the past few years; however, senescence and immortalisation remain underserved areas for drug discovery owing to a lack of robust senescence inducing agents and an incomplete understanding of the signalling events underlying this complex process. In order to address this issue we undertook a large-scale morphological siRNA screen for inducers of senescence phenotypes in the human melanoma cell line A375P. Following rescreen and validation in a second cancer cell line, HCT116 colorectal carcinoma, a panel of 16 of the most robust hits were selected for further validation based on significance and the potential to be targeted by drug-like molecules. Using secondary assays for detection of senescence biomarkers p21, 53BP1 and senescence associated beta-galactosidase (SA?Gal) in a panel of HCT116 cell lines carrying cancer-relevant mutations, we show that partial senescence phenotypes can be induced to varying degrees in a context dependent manner, even in the absence of p21 or p53 expression. However, proliferation arrest varied among genetic backgrounds with predominantly toxic effects in p21 null cells, while cells lacking PI3K mutation failed to arrest. Furthermore, we show that the oncogene ECT2 is a robust inducer of senescence in all mutant backgrounds tested and highlight a dependence on activating KRASG13D for growth suppression and a complete senescence response, suggesting a potential mechanism to target mutant KRAS signalling through ECT2 in cancers that are reliant on activating KRAS mutations and remain refractory to current treatments. Overall design: HCT116 colorectal cancer cells with heterozygous KRAS mutation and KRAS+/- cells in which the mutation is knocked out were reverse transfected with 50nM siRNA against ECT2 or scrambled control. 5 days post transfection, cells were harvested for RNA extraction.