PROJECT SUMMARY DNA replication stress is one of the mechanistic underpinnings of many genotoxic chemotherapies which cause replication fork stalling. Stalled forks frequently reverse to form four-way junction structures, as a natural coping mechanism, to allow repair of the DNA lesions ahead of the forks and avoid catastrophic fork collapse. However, fork reversal is a double-edged sword because the nascent DNA in the reversed arm is susceptible to excessive nuclease resection if not securely protected which can lead to severe genome instability. Many factors play tug- of-war at stalled forks to strengthen or weaken their stability, and their balance in the cell determines the fate of replication forks during stress and cellular outcome upon chemotherapy treatments. In this study, we investigate the molecular mechanisms of a novel replication fork regulator named profilin-1 (Pfn1). As a well-known actin- binding protein, Pfn1 plays an essential role in actin polymerization and dynamics. Paradoxically, it also has well- documented but poorly understood anticancer activities including the ability to sensitize cancer cells to chemotherapy treatments. In recently published work, we demonstrated for the first time that the anticancer effects of Pfn1 stem, at least partially, from its nuclear functions that are spatially and mechanistically distinct from its cytoplasmic function in actin regulation. We showed that nuclear Pfn1 directly interacts with ENL in the Super Elongation Complex (SEC) and inhibits the ability of SEC to drive transcriptional elongation of various cancer genes including MYC. We also presented clinical evidence that nuclear Pfn1 level is frequently decreased in cancer due to the upregulation of its nuclear exporter exportin-6 (XPO6), whose deletion increases nuclear Pfn1 level and decreases tumor growth. These findings establish the notion that Pfn1 has fundamentally important and cancer-relevant functions in the nucleus, and set the stage for further discovery of its involvement in additional nuclear processes. In this grant, we present novel evidence that nuclear Pfn1 promotes normal DNA replication but causes fork destabilization during stress and increases cellular sensitivity to replication stress- inducing chemotherapies including PARP inhibitors. We hypothesize that nuclear Pfn1 has context-dependent effects on DNA replication forks. Under normal conditions, it promotes fork progression by increasing chromatin relaxation through SNF2H. Under stressed conditions, it promotes fork reversal by stimulating SNF2H and increases fork resection by suppressing BOD1L, leading to fork destabilization. Aim 1: Determine the effect of nuclear Pfn1 on BOD1L-dependent replication fork protection. Aim 2: Define the role of Pfn1/SNF2H axis in replication fork remodeling and stability. Aim 3: Understand the chemotherapy-sensitizing ability of nuclear Pfn1. Work proposed in this grant has the potential to generate important mechanistic insight...