PROJECT SUMMARY/ABSTRACT Oncogene-induced senescence (OIS) is triggered by aberrant activation of oncogenes such as the mutant form of RAS and acts as a key barrier to inhibit the progression of preneoplastic lesions. OIS is accompanied by elevated DNA replication stress and activation of the DNA damage response (DDR); seminal studies have shown that replication-associated DNA damage is a prerequisite for establishing OIS, where cells must be actively replicating leading to a rise in replication stress that triggers DDR in response to oncogene-induced DNA hyper- replication. Conversely, bypass of OIS allows the proliferation and oncogenic transformation of damaged cells with genome instability. While these ideas underscore OIS as an anti-cancer barrier imposed by DNA damage checkpoints, the precise nature of oncogenic stimuli that culminate in aberrant DNA replication and activation of the DDR at stalled replication forks remains elusive. In addition, whether there exists distinct cellular signaling cascade that relays upstream oncogenic signaling to a replication fork to cause DNA replication fork instability is yet to be discovered. In this application, we propose to explicate the mechanisms that link oncogene-induced replication stress to DNA replication fork integrity. Specifically, we reason that TIMELESS (TIM) in the fork protection complex, an essential scaffold of the replisome necessary for replication fork progression and checkpoint activation, is a key determinant of OIS. Our preliminary data indicate that OIS induced by RAS activation is associated with downregulation of TIM that is linked to a novel posttranslational modification, i.e. PARP1-dependent polyADP-ribosylation, of TIM and its proteolytic degradation. We hypothesize that failure to protect stalled forks via suppression of TIM activity is an oncogene-induced mechanism that contributes to the accumulation of replication stress and DNA fork instability. We will use HRASG12V as a model oncogene to induce OIS in non-transformed human fibroblasts. In the first aim, we will identify the mechanism of HRASG12V-induced OIS established by TIM downregulation and determine the role of TIM knockdown in triggering DNA replication fork instability and senescence. I will also investigate whether TIM overexpression overcomes DNA replication fork instability, and thus the onset of OIS. A variety of molecular and cellular techniques will be employed, including DNA combing analysis of DNA replication fork resection, to evaluate the capacity of TIM-depleted cells to prevent fork degradation in response to oncogene activation. In the second aim, we will identify the mechanism of TIM downregulation during OIS, focusing on characterizing the polyADP-ribosylation and proteolytic degradation of TIM triggered by the HRASG12V-driven signaling cascade. In the third aim, we will elucidate the role of TIM in promoting tumorigenesis in the Kras-driven prostate cancer organoid model to define TIM as a majo...