Abstract 152: Discovery and characterization of GSK3 allosteric activators

Abstract

Abstract The intestinal epithelium undergoes constant regeneration and is tightly regulated. Disruption of its homeostasis can result in the development of colorectal cancer (CRC). Glycogen synthase kinase 3 (GSK3) is a kinase that plays a central role in multiple signaling pathways, including cellular proliferation, where it inhibits the activity of its substrates. GSK3 dynamics can be described as a multiple input multiple output system. Numerous upstream growth factors can regulate and thus inhibit GSK3 from acting on its many substrates. Mutations in these growth signaling pathways (e.g., Wnt and KRAS) have been well established to have a role in CRC progression. Therefore, aberrantly activated signaling due to these proteins are presumed to result in the constitutive inhibition of GSK3 activity in CRC, thus making it an ideal therapeutic target. The aim of this study is to discover an allosteric GSK3 small molecule activator that can restore GSK3 kinase activity within CRC. Method: We screened 50,000 small molecule compounds using a luciferase-based in-vitro kinase assay. We are further confirming our top hits using both chemical and cell-based assays. For the cell-based assays, we test the compounds in human colonic epithelial cells (HCEC) and its three isogenic cell lines, which mimic CRC progression, and monitor changes in GSK3 kinase activity indirectly, via changes in its substrate protein levels, and directly via a live-cell GSK3 specific reporter. In addition, the top hits are also tested in both normal colon and CRC organoids. Results: Prior to running our large screen, we performed various optimization steps to determine the ideal condition for our screening assay. Our screening assay scored a Z-Score of 0.5 and higher, which indicated that it was a high-quality screening assay. Additionally, we have developed two cell-based orthogonal HTS assays. One using immunofluorescence and single-cell quantification of 24 GSK3 substrates from various pathways in our cell lines. Second, we engineered a GSK3 live-cell reporter and expressed it in the HCEC cells and three isogenic cell lines and organoid models. Impact: There is a need for advancements in preventative or therapeutic targets against CRC. Research indicates that suppression of GSK3 enzymatic activity decreases sensitivity to certain chemotherapeutic and targeted therapies. Therefore, restoring GSK3 enzymatic activity in CRC may re-sensitize tumors to other targeted therapies and establish GSK3 as a potential therapeutic target. Citation Format: Reeba P. Varghese, Curtis A. Thorne. Discovery and characterization of GSK3 allosteric activators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 152.