PROJECT SUMMARY Oncogenic stimuli in early cancerous lesions, such as driven by RAS mutations, triggers oncogene-induced senescence (OIS), which acts as a barrier to prevent malignant transformation. Establishment and maintenance of OIS is dependent on the sustained DNA damage response (DDR) that stems from aberrant DNA replication and accumulation of DNA replication stress. Hence, progression to malignancy requires DDR inactivation and escape from senescence, which, when accompanied by genome instability, accelerates carcinogenesis. For instance, gains in RAS are prevalent in metastatic prostate cancer, suggesting that this aberration may be a driver of the prostate cancer progression and aggressiveness. However, the precise nature of oncogene-induced replication stress and how it affects replication fork integrity remains unclear. While recent studies have highlighted replication fork reversal as a means to stabilize stressed forks and promote fork restart, whether oncogenic signaling disrupts fork protection mechanisms, thereby culminating into fork destabilization remains unclear. Furthermore, whether there exists a distinct program that transmits upstream oncogenic signaling to a replication fork to cause fork instability is an important outstanding question. These knowledges are essential for understanding how premalignant lesions restrain from developing cancer, which would help design therapeutics that sensitizes cells to oncogene-induced replication stress. The goal of this project is to explicate the mechanism that links oncogene-induced replication stress to DNA replication fork integrity. We propose that TIMELESS (TIM) in the fork protection complex, an integral constituent of the replisome, is a key determinant of modulating OIS and transformation. Our preliminary studies reveal that OIS induced by HRASG12V is accompanied by TIM downregulation in non-transformed cells, which is mediated by PARP1-dependent poly(ADP-ribosyl)ation onto TIM, a novel TIM post-translational modification that primes TIM for proteasomal degradation. Conversely, TIM is upregulated in OIS-bypassed clones and in the KrasG12D prostate cancer model, indicating that TIM activity at stalled forks may determine the responses to oncogene- induced replication stress and resistance to OIS. We thus hypothesize that deprotection of stalled forks via TIM downregulation is an unappreciated oncogene-induced mechanism that contributes to DNA replication fork instability to cause OIS. To test this idea in prostate cancer, we will 1) determine how deprotection of stalled forks is linked to senescence induced by HRASG12V and other oncogenes in cellular models; 2) explicate the mechanism of TIM downregulation mediated by RAS-PI3K-PARP1 proteolytic signaling; 3) determine the role of TIM in bypassing senescence and promoting tumorigenesis using KrasG12D-driven prostate cancer organoid and mouse models. We expect to reveal how modulation of TIM, or stalled fork integrity in genera...