Abstract 5231: Small-molecule inhibitors of SCF-Skp2-Cks1 ubiquitin E3 ligase stabilize nuclear p27kip1 as a novel therapeutic approach to endometrial cancer
Authors: Egbuta Chinaza , Dubrulle Julien , Tellechea Ana , Manzanares Miguel A. , Li Yunfeng , Duan Shanshan , Dickson John K. , Meniconi Mirco , Huang Kuo-sen , Huang Xinyan , Shohdy Nadim , Pagano Michelle , Hao Bing , Mancini Michael A. , Gold Leslie I. 
Published: August 2018

Abstract
We have previously shown that ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor, p27kip1 (p27), critical for G1 arrest, is involved in the pathogenesis of Type I endometrial cancer (ECA; 80% of ECAs), which is estrogen (E2) induced and usually preceded by hyperplasia. In vitro, E2 induces MAPK phosphorylation of p27 at Thr187, which is required for ubiquitylation of p27 by the SCF-Skp1-Skp2-Cks1E3 ligase to signal its degradation by proteasomes. We have shown that the absence of nuclear p27 in human endometrial hyperplasia and ECA is due to high SCF-Skp2-Cks1 E3 ligase activity, exemplified in vitro by a decrease in nuclear p27 and proliferation of ECA primary cells and cell lines. The 3D structure of Skp2 and Cks1 harboring p27 was previously solved. Two approaches were used to identify small-molecule inhibitors of the SCF-Skp2-Cks1 E3 ligase (Skp2E3LIs) that block p27 ubiquitylation: (i) structure-based virtual screen targeting the protein interaction surfaces formed by Skp2-Cks1 (the p27 binding pocket) and separately, the Skp1-Skp2 interface; and (ii) ligand-based virtual screen of previously published compounds, which target the p27 pocket within Skp2-Cks1 (blocked E2-induced endometrial hyperplasia in mice with an increase in nuclear p27) or the Skp1-Skp2 interface (blocked tumor growth in mice). The virtual screening hits (207 compounds) from both approaches were subjected to time-resolved-FRET (Skp2-Cks1 pocket interface) and ELISA (Skp1-Skp2 interface) for evaluation of their physical binding to Skp1-Skp2-Cks1 (SSC) complex. 35 hits that elicited IC50s ≤120 µM in both assays were further evaluated by high-content image analysis (HCA) for toxicity, uridine (EdU) uptake, and number of p27 fluorescent nuclei. We confirmed 3 Skp2E3LIs that show IC50s less than 60 µM (E12, E17, E27) are nontoxic by trypan blue, have EC50s for growth inhibition ≤15 µM, increase nuclear p27, and block retinoblastoma protein (pRb) phosphorylation, preventing S phase progression. Importantly, Skp2E3LIs block both E2-induced degradation of nuclear p27 and proliferation and induce an increase in nuclear p27 in 3D spheroids composed of ECA cells and ECA cancer-associated fibroblasts (CAFs). The lead compounds will be co-crystallized with the SSC complex and together with SAR studies will be optimized (i.e., biologic activities and druggability parameters) for therapeutic development. SKp2E3LIs represent a new class of specific E3 ligase inhibitors that prevent degradation of cell cycle proteins involved in growth control. Therefore, lead compounds have the potential to be a major therapeutic advancement over current general proteasome inhibitors that indiscriminately block protein degradation including oncogenes. Skp2E3LIs are promising new therapeutics for ECA and other cancers characterized by high Skp2 levels and loss of nuclear p27.
Citation Format: Chinaza Egbuta, Julien Dubrulle, Ana Tellechea, Miguel A. Manzanares, Yunfeng Li, Shanshan Duan, John K. Dickson, Mirco Meniconi, Kuo-sen Huang, Xinyan Huang, Nadim Shohdy, Michelle Pagano, Bing Hao, Michael A. Mancini, Leslie I. Gold. Small-molecule inhibitors of SCF-Skp2-Cks1 ubiquitin E3 ligase stabilize nuclear p27kip1 as a novel therapeutic approach to endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5231.