Loss of DNA replication fork protection by TIMELESS degradation supports oncogene-induced senescence

Oncogene-induced senescence (OIS) is a potent barrier that limits tumorigenesis. While persistent DNA replication stress accompanied by cellular hyperproliferation is considered an underlying basis of OIS, the molecular mechanisms through which oncogenes elicit genome instability remain unclear. Here, we identify a distinct cellular process in which DNA replication fork stalling is linked to senescence in response to oncogenic signaling. We demonstrate that TIMELESS (TIM), an essential regulatory component of the fork protection complex that supports the integrity of the replisome, is downregulated in non-transformed human cell lines that undergo OIS by HRAS^G12V expression. Loss of TIM is sufficient to induce senescence, while ectopic expression of TIM delays the induction of OIS via enhancing protection of stalled forks. Furthermore, TIM levels are restored in cells capable of bypassing OIS, indicating that TIM regulation modulates the onset of OIS. Mechanistically, HRAS^G12V expression induces cellular poly(ADP-ribosyl)ation mediated by PARP1, which prompts poly(ADP-ribose)/PAR-dependent TIM degradation. Together, our study uncovers RAS-mediated proteolytic signaling as a key determinant of OIS centered at stalled DNA replication forks. We propose the existence of an oncogenic signaling cascade that actively suppresses replisome activity, contributing to the DNA replication stress that drives OIS.