Reversible Opto-Chemical activation of KRASG12V signaling with near single-cell precision

Abstract

KRAS mutations drive some of the most lethal carcinomas, and genomic and inducible systems have established many of the cellular and tissue-level consequences. However, these approaches operate at the level of oncogene expression, allowing for cellular adaptation that masks the individual role of KRAS oncoprotein signaling. Here, we developed a reversible Opto-Chemical system to activate KRAS signaling by chemically translocating a cytosolic mutant KRASG12V G-domain to the plasma membrane upon light or small-molecule input. In MDCK cells, the G-domain plasma membrane recruitment activated downstream signaling and reduced collective migration. In mouse small Intestinal Organoids, G-domain recruitment promoted increased crypt size and number under Epidermal Growth Factor-deprived conditions. We further showed that the increased number of crypts depended on continuous KRASG12V signaling. Finally, under the same deprived conditions, localized activation in just one budding crypt promoted crypt formation compared to controls. This system decouples oncoprotein activity from oncogene expression, allowing to investigate the KRAS signaling contribution to early epithelial transformation.