PROJECT SUMMARY Uterine cancer (UC) incidence and death rates are both significantly increasing in the United States. Most UCs are endometrial cancers which arise from the inner lining of the uterus and are defined by several different histologic subtypes, the most common of which is endometrioid. Additionally, survival outcomes are worse for black women. Because of the worsening survival of women with advanced and relapsed endometrial cancer, this P01 grant is focused on targeting replication stress (RS) in order to improve therapeutic outcomes in these patients. RS is defined as the slowing or stalling of the replication fork progression during DNA synthesis and is widely recognized as a significant cause of genomic instability and a critical feature of cancer cells. This P01 grant investigates 3 different strategies for targeting RS in UC with the goal of translating these into effective salvage therapies for women with advanced/relapsed UC. We have assembled a team of internationally recognized researchers from the Dana-Farber Cancer Institute and the Brigham and Women’s Hospital, both in Boston MA, with multidisciplinary expertise in UC, DNA repair and RS, immunotherapy, preclinical models, biostatistics, computational biology, gynecologic pathology, drug development and clinical trials. This P01 grant is being led by Drs. Panagiotis Konstantinopoulos, Joyce Liu, and Ursula Matulonis who are recognized international leaders in the field of gynecologic cancer and endometrial cancer research. This P01 grant consists of 3 Projects and 4 Cores. The leadership team for each Project is comprised of paired investigators with complementary expertise in basic science and clinical/translational research. Project 1 focuses on studying the mechanism of WEE1 inhibition in recurrent USC or p53-mutated UCs and the correlation of WEE1 activity with functional and immunohistochemical (IHC) measures of RS. Based upon the results of a clinical trial of the WEE1 inhibitor adavosertib which previously demonstrated significant clinical activity in USCs, this project leverages a collection of patient-derived xenografts (PDXs), patient-derived organoids (PDOs), and genetically engineered mouse models (GEMMs) of p53-mutated/null endometrial cancer to examine the effects of WEE1 inhibition on functional and IHC measures of RS in the in vitro and in vivo setting. Additionally, through a biopsy-driven investigator-initiated protocol of the WEE1 inhibitor ZN-c3 in USC, Project 1 will examine the effects of WEE1 inhibition on measures of RS in co-clinical PDOs and correlation of these measures with clinical activity. Project 2 focuses on the hypothesis that inhibition of the PI3K pathway can increase RS and therefore create synergy with ATR inhibitors. In previously performed PRISM and CRISPR screens, inhibition of PI3K signaling together with ATR inhibition was identified as potentially synergistic and subsequent experiments have demonstrated synergism between the PI3K ...