Project Abstract. High-grade serous ovarian carcinoma (HGS-OvCa) is the most malignant form of ovarian cancer. Among the most aggressive HGS-OvCa tumors are those that harbor genomic amplification and overexpression of CCNE1, the gene that encodes for cyclin E1, a key cell cycle regulator. This challenging HGS-OvCa subset carries poor outcomes after standard cytotoxic chemotherapy, and is associated with high proliferative rate, rapid development of platinum resistance, and de novo resistance to poly ADP (ribose) polymerase inhibitors. Despite intense efforts, targeted therapies for the treatment of CCNE1-amplified HGS-OvCa remain elusive, in part, due to the paucity of druggable molecular targets. As such there remains an urgent unmet medical need for the development of new therapies for CCNE1-amplified HGS-OvCa and other cancers marked by CCNE1 overexpression. Promising preclinical evidence demonstrates that knockdown or inhibition of cyclin-dependent kinase (CDK2), the catalytic kinase partner of cyclin E1, selectively kills CCNE1-amplified ovarian cancer cell lines, highlighting a potential dependency associated with CCNE1 amplification. However, efforts to directly target CDK2 with pharmacological agents have been plagued by difficulties in achieving specificity for CDK2. We recently employed an alternative strategy of selectively inhibiting CDK7, a key upstream activator of CDK2, to achieve selective killing of CCNE1-amplified ovarian cancer cells. In proof-of-principle studies, YKL-5-124, a new CDK7 inhibitor with superior selectivity over existing inhibitors of its kind, led to pronounced tumor shrinkage in a human xenograft mouse models of CCNE1-amplified HGS-OvCa. The primary goal of the proposed research is to expand on our preliminary findings by elucidating the underlying principles governing CCNE1-amplified HGS-OvCa sensitivity to CDK7 inhibition. This knowledge will then be leveraged to guide further preclinical inquiry into targeting CDK7 in CCNE1-amplified HGS-OvCa. Herein we propose to identify (1) HGS-OvCa cancer cells and genetic backgrounds that are sensitive to YKL-5-124; (2) biomarkers that correlate with drug response; and (3) combination strategies that augment or expand drug response (Aim 1). While YKL-5-124 displays potent in vivo activity in mice, we will continue to optimize these CDK7 inhibitors for improved pharmacokinetics to further the preclinical development of this chemical series (Aim 2). Lastly, we will evaluate YKL-5-124 (or a further in vivo optimized analog) in mouse models of CCNE1-amplified and non-amplified HGS- OvCa (Aim 3). To accomplish these goals we have assembled a multi-disciplinary team with expertise in medicinal chemistry (Nathanael Gray, Stanford); cell and systems biology (Caitlin Mills and Peter Sorger, Harvard Medical School); mouse models in ovarian cancer (Panagiotis Konstantinopoulos, DFCI); and translational and clinical ovarian research (Ursula Matulonis, DFCI). This research describe...