Nautral products for targeting Skp2 in cancer interception

Project Summary Prostate cancer (PCa) is the second most common cancer in American men and is responsible for 3% of total deaths and 10% of deaths by cancer. Most patients experience a slow-progressing disease and are assigned to active surveillance. These patients are spared immediate radical prostatectomy (RP) or radiation that is used for patients that have more advanced disease. Because assignment to active surveillance is imperfect, some patients are unnecessarily overtreated, with the burdens of unnecessary side effects, cost, and lower quality of life, whereas a few patients that need prompt radical treatment may experience delays. The limited treatment options available for aggressive disease usual fail within a few years. Thus, there is an unmet clinical need for low toxicity preventive/interceptive agents or natural products that prevent PCa progression either during active surveillance or after RP or radiation treatment. S phase kinase-associated protein 2 (Skp2) is a putative oncogene that targets several tumor suppressors for degradation. The Skp2 gene is amplified in several human cancers. In PCa, Skp2 is overexpressed in pre- malignant high-grade prostatic intraepithelial neoplasia (HG-PIN) and in primary tumors, suggesting a role in the early stages of PCa. Skp2 deletion blocks tumorigenesis in Rb1, p53, or Pten deficient mouse models, suggesting that Skp2 may serve as an “Achilles' heel” drug target against pRb, p53 and/or Pten-deficient cancer. However, inhibitors that disrupt Skp2 protein-protein interactions or cause degradation through the proteolysis targeting chimera (PROTAC) have had limited success. Thus, we propose to instead screen directly for natural products that target Skp2 for degradation. In a collaboration with the National Center for Advancing Translational Sciences (NCATS), we will use 500,000 natural product samples that are known to include highly diversified chemotypes with cancer-preventive properties. To implement the screen, we have developed a cell-based luciferase assay for degradation of Skp2 and several other resources including our large PCa patient cohort, our prostate-specific human Skp2 knock-in mouse model of early prostate carcinogenesis, our PCa patient-derived organoid cultures, and patient-derived xenograft (PDX) models. In Specific Aim 1 (UG3) we will optimize our cell-base luciferase assay, develop a prototype HTS assay for screening skp2 degraders, and perform mechanistic studies of products that have potential utility. In Specific Aim 2 (UG3) we will quantitate Skp2 protein in PCa progression. This is a step towards identifying patients that would benefit from interception via a Skp2 degrader, which will be important for developing a novel clinical trial. In Specific Aim 3 (UH3) we will complete a full-scale HTS of up to 500,000 natural products with NCATS. We will perform activity-based characterization and bioassay-directed isolation, structural elucidation, and perform mechanistic studies of leads generated during screening. In Specific Aim 4 (UH3) we will determine the in vivo chemopreventive efficacy, toxicity, and mechanisms of two confirmed compounds through dietary administration in a novel prostate-specific human Skp2 knock-in mouse carcinogenesis model and in PDX models. Impact: Positive results from this study will identify novel natural products for interception of progression in early stage PCa. Measuring Skp2 in a large PCa patient population will allow identification of patients most likely to respond, allowing development of a novel clinical trial.