The Ink4a/Arf locus operates as a regulator of the circadian clock modulating RAS activity

The mammalian circadian clock and the cell cycle are two major biological oscillators whose coupling influences cell fate decisions. In the present study, we use a model-driven experimental approach to investigate the interplay between clock and cell cycle components and the dysregulatory effects of RAS on this coupled system. In particular, we focus on the Ink4a/Arf locus as one of the bridging clock-cell cycle elements. Upon perturbations by the rat sarcoma viral oncogene (RAS), differential effects on the circadian phenotype were observed in wild-type and Ink4a/Arf knock-out mouse embryonic fibroblasts (MEFs), which could be reproduced by our modelling simulations and correlated with opposing cell cycle fate decisions. Interestingly, the observed changes can be attributed to in silico phase shifts in the expression of core-clock elements. A genome-wide analysis revealed a set of differentially expressed genes that form an intricate network with the circadian system with enriched pathways involved in opposing cell cycle phenotypes. In addition, a machine learning approach complemented by cell cycle analysis classified the observed cell cycle fate decisions as dependent on Ink4a/Arf and the oncogene RAS and highlighted a putative fine-tuning role of Bmal1 as an elicitor of such processes, ultimately resulting in increased cell proliferation in the Ink4a/Arf knock-out scenario. This indicates that the dysregulation of the core-clock might work as an enhancer of RAS-mediated regulation of the cell cycle. Our combined in silico and in vitro approach highlights the important role of the circadian clock as an Ink4a/Arf-dependent modulator of oncogene-induced cell fate decisions, reinforcing its function as a tumour-suppressor and the close interplay between the clock and the cell cycle network.Author summary: In mammals, the circadian clock controls the punctual regulation of biological processes, which, in turn, affect physiology and behaviour, allowing for the synchronisation of internal time to environmental light-dark cycles. Malfunctions of the circadian clock are associated with pathological phenotypes including cancer. Given the range of molecular time-dependent processes, including metabolism, DNA repair, and the cell cycle, the clock is hypothesised to act as a tumour suppressor. With the help of mathematical modelling and whole-genome analysis combined with machine learning, we investigated the RAS-dependent dysregulation of the circadian clock. We find that the tumour-suppressor Ink4a/Arf acts as a key mediator of RAS oncogene-induced changes in the circadian system, thereby mediating the interplay between the clock and the cell cycle.