Project Summary DNA hypomethylation is a critical epigenetic alteration in cancer, and is associated with altered gene expression, genomic instability, tumor progression, and poor survival. Despite extensive data documenting its occurrence, the underlying mechanism(s) driving DNA hypomethylation remains unclear. We have spent several years studying DNA hypomethylation in epithelial ovarian cancer (EOC), with an emphasis on the most prevalent and deadly subtype of EOC, high-grade serous ovarian cancer (HGSC). Recently, we have focused on identifying underlying mechanisms driving DNA hypomethylation in HGSC. Our preliminary data point towards a novel and intriguing possibility: cyclin E1 (CCNE1) and DNA replication stress (DRS) play a role. CCNE1 expression is widely increased in cancer and promotes DRS and genomic instability (GI). DRS is an important cancer phenotype characterized by fork stalling and/or collapse, and GI is a molecular hallmark of HGSC. While DRS is known to promote GI, whether it promotes altered epigenetic states, including DNA hypomethylation, is unknown. Based on preliminary data that includes studies of primary HGSC, HGSC cell lines, and immortalized fallopian tube epithelial (FTE) cells, an HGSC precursor cell model, we hypothesize that CCNE1 promotes DNA hypomethylation via the induction of DRS. We will test this hypothesis in two complementary and novel Specific Aims. Specific Aim 1: Determine the genomic relationship between the sites of CCNE1-induced DRS, DNA damage, and DNA hypomethylation, using FTE cells. We will express CCNE1 in FT282 cells, a validated HGSC precursor cell model, and will map the genomic sites of DRS using RAD9 ChIP-seq, DNA damage using -H2AX-ChIP-seq, and DNA hypomethylation using hybrid-capture DNA methyl-seq. We will use bioinformatics and biostatistics to test the genomic overlap and relative enrichments between mapped sites. This strategy will provide the first genome-wide assessment of the link between DRS, DNA damage, and altered DNA methylation. Specific Aim 2: Mechanistically link CCNE1-induced DRS and DNA hypomethylation using FTE cells. We will use the same experimental model system as in Aim 1, FT282-CCNE1 cells. We will inhibit the DRS using several approaches: 1) treatment with the Cdc7/Cdki PHA-767491, which reduces origin firing, 2) media supplementation with exogenous nucleosides, which restores replication fork rate, 3) treatment with cordeycepin, which inhibits transcriptional elongation and replication-transcription collisions, and 4) expression of RNAseH1, which resolves RNA-DNA hybrids (R-loops) and also inhibits such collisions. Following confirmation of DRS inhibition, we will determine the resulting impact on DNA methylation. This strategy will mechanistically link discrete aspects of CCNE1-induced DRS to the induction of DNA hypomethylation.