Abstract 1146: Development of CRISPR human Skp2 knock-in in the prostate of mouse and the associated prostate organoids for testing Skp2 targeting agents

Abstract

Abstract S-phase kinase associated protein 2 (Skp2) is a promising drug target as studies have demonstrated that Skp2 is required for spontaneous tumor development that occurs in the retinoblastoma protein (pRb), Pten or p53 deficient mice. We therefore have aimed to establish CRISPR human Skp2 (hSkp2) knock-in in the prostate of mouse to examine its role in prostate carcinogenesis. In addition, Skp2 overexpressing organoids are being developed for convenient and efficient screening of specific Skp2 inhibitors. cDNA of hSkp2 and porcine teschovirus-1 (P2A) was introduced into immediate upstream of mouse probasin coding sequencing using CRISPR knock-in targeting approach to establish transgenic mice specifically overexpressing hSkp2 in the prostate glands. Quantitative PCR, western blot and immunohistochemistry (IHC) were used to identify the expression levels of Skp2 and histopathological analyses were performed after H&E staining. Prostate glands were dissected and digested for organoid culture, then morphological features and viability of organoids were evaluated following treatments of potential Skp2 inhibitors. Prostate glands and other tissues from mice of three transgenic lines which were genotyped positive were dissected and hSkp2 was detected in ventral, dorsal and lateral prostate lobes, but not in testis, kidney, spleen, and liver. Line 10092 was found to have the highest expression of hSkp2 among these three lines. Prostate weight slightly increased in transgenic mice compared to that of wild-type control mice. Hyperplasia, prostatic intraepithelial neoplasia (PIN), and carcinoma were noted, which suggested that overexpression of hSkp2 in the prostate of mice promoted proliferation and prostate tumorigenesis. Organoids that were derived from the prostate of hSkp2 transgenic mice recapitulated prostatic phenotypic features and maintained a high level of hSkp2. A novel Flavokawain A derivative was found to selectively alter the morphology of hSkp2 organoids and decrease the viability. Prostatic lesions found in the hSkp2 knock-in mice demonstrated a potential role of hSkp2 in early prostate carcinogenesis and organoids overexpressing hSkp2 have the utility for screening out compounds targeting Skp2 in prostate cancer. Citation Format: Liankun Song, Shan Xu, Kia Arabzadehkaffash, Ali Fazelpour, Dongjun Fu, Matthew Tippin, Xiaolin Zi. Development of CRISPR human Skp2 knock-in in the prostate of mouse and the associated prostate organoids for testing Skp2 targeting agents [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1146.