PROJECT 2: PROJECT SUMMARY Treatment of high-grade serous ovarian cancer (HGSC) has been advanced by targeting specific genomic alterations with DNA damage response (DDR) inhibitors. We found that combination PARP and ATR inhibition (PARPi-ATRi) overcomes acquired PARPi resistance with a 50% overall response rate in BRCA-mutant HGSC in the CAPRI investigator-initiated clinical trial (SPORE, Project 2, 2018-2023). We and others have now discovered that CCNE1 amplification (CCNE1Amp) emerges with PARPi resistance using deep molecular profiling of BRCA-mutant patient tumor samples. In addition, CCNE1Amp is a known subset of HGSC that are homologous recombination proficient and resistant to both chemotherapy and PARPi, indicated that CCNE1AMP may participate in acquired PARPi resistance. Overall, CCNE1Amp HGSC patients have poor overall survival and need new therapeutic options. The WEE1 kinase is a regulator of cell cycle progression and inhibition of this kinase causes aberrant DNA synthesis, particularly in CCNE1Amp cells. Indeed, WEE1 inhibitors (WEE1i) have shown monotherapy activity in CCNE1 overexpressing HGSC in the clinic. ZN-c3 is a next-generation WEE1i demonstrating significantly less hematologic toxicity in Phase I trials than prior WEE1i, such as AZD1775. ZN-c3 is moving forward into a Phase II clinical trial in CCNE1-overexpressing HGSC. We proposed to develop CCNE1 as a predictive biomarker of WEE1i benefit as well as identify additional biomarkers that increase durable responses to treatment (Aims 1 and 3). Because emergence of resistance to WEE1i monotherapy is possible, we propose to identify new strategies to overcome or prevent resistance using a new drug combination and exploring the potential mechanisms of WEE1i resistance (Aim 2). Because the mechanism of action of WEE1i treatment is related to its ability to cause defects in DNA replication fork, factors that respond to WEE1i treatment at DNA replication forks are candidate regulators of drug responsiveness. We will use the identification of such factors to prioritize candidate biomarkers of responsiveness as indicated by tumor genomic and gene expression studies (Aim 3). In summary these studies will develop CCNE1 as a biomarker for WEE1i monotherapy for HGSC and test new treatment options for CCNE1Amp HGSCs after progression on WEE1i. In addition, these studies will identify novel candidate biomarkers of responsiveness beyond CCNE1 overexpression, while simultaneously investigating the mechanisms that regulate the effectiveness of these drugs.