Friday, May 3, 2024
5/3/2024
Project Summary High-grade serous ovarian cancer (HGSC) is the most common and deadly subtype of ovarian cancer. An oncogenic driver and therapeutic target in HGSC is DNA replication stress (RS). The cellular RS response includes several proteins under investigation as therapeutic targets. Characterization of the oncogenic role of proteins that function in the RS response, and their development as therapeutic targets, will impact HGSC treatment approaches. In this context, RAD9, RAD1, and HUS1 Interacting Nuclear Orphan (RHNO1) plays an important role in the HGSC RS response. RHNO1 interacts with the RAD9-RAD1-HUS1 complex (9-1-1) to promote ATR-CHK1 signaling, homologous recombination (HR) DNA repair, cell survival, and chemotherapy resistance. RHNO1 is commonly overexpressed in HGSC, including in recurrent chemoresistant disease. RHNO1 knockout (KO) in normal fallopian tube epithelial (FTE) cells does not impair cell viability, suggesting that drugs targeting RHNO1 function will have a high therapeutic index. Here, we will investigate the role of RHNO1 in HGSC in vivo and will identify first-in-class inhibitors of the RHNO1 interaction with the 9-1-1 complex. Our overarching goal is to develop a new therapeutic approach for HGSC. Specific Aim 1. Determine the role of RHNO1 in HGSC in vivo using orthotopic xenografting. Orthotopic xenografting is an in vivo approach to determine oncogene function in the context of a human tumor. We will ablate RHNO1 using CRISPR KO in two human HGSC cells lines, OVCAR4 and OVCAR8, that model metastatic, recurrent disease in vivo. We will implant luciferase-tagged cells intraperitoneally (IP) into athymic nude mice, as the peritoneal cavity is the orthotopic site that mimics late stage and recurrent HGSC most relevant for therapeutic development. In Sub-Aim 1A, we will measure tumor growth kinetics using imaging, as well as mouse survival, metastatic growth, and ascites. In Sub-Aim 1B, we will measure biomarkers of proliferation, the RS response, DNA damage, and apoptosis. In Sub-Aim 1C, we will determine the sensitivity of RHNO1 KO and control xenografts to carboplatin or olaparib chemotherapy, agents which have high therapeutic relevance for HGSC. Specific Aim 2. Develop first-in-class compounds that disrupt the RHNO1/9-1-1 interaction. There are no small molecule inhibitors (SMI) of RHNO1 function. Here, we will develop validated RHNO1 mimics that disrupt the RHNO1/9-1-1 interaction and the RS response in HGSC cells, which is expected to inhibit HGSC cell survival. In Sub-Aim 2A, we will conduct a high-throughput screening (HTS) campaign using a 100,000 compound in-house chemical library and a newly developed 9-1-1/RHNO1 fluorescence polarization (FP) assay. In Sub-Aim 2B, we will test the identified hits using an available FP-based counter screen. In Sub-Aim 2C, we will subject hits in complex with 9-1-1 to X-ray crystallography, which will inform future drug design parameters. In Sub-Aim 2D, we will test hit compounds for target engagement in HGSC cells.
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