Abstract 5143: Using a multipronged approach to characterize C-terminal domain polybasic motif STK11 variants

Abstract KRAS-driven lung adenocarcinomas (LUADs) with concurrent STK11 loss of function (LoF) mutations are associated with aggressive tumors with increased risk of metastasis and lower overall and progression free survival. In LUAD, STK11 LoF impacts many aspects of coordinated cell motility and promotes alterations characteristic of metastasis. STK11 is a serine-threonine kinase that is comprised of an N-terminal, kinase, and C-terminal domain (CTD). With the goal of developing a method to identify pathogenic STK11 mutations, we previously evaluated functional impact of variants identified from the University of Vermont Medical Center on STK11 kinase activity using in-vitro kinase assays, and luciferase reporter assays. Here, we saw that CTD variants retained kinase activity and the ability to form the STK11 heterotrimeric complex. Although the CTD has no catalytic activity, it is thought to be integral for STK11 localization to the plasma membrane and cytoskeleton, where it negatively regulates the activity of focal adhesion kinase (FAK). Recent work by others has shown that a region of the CTD, the polybasic motif (PBM), (aa403-426) is key for localization of STK11 to the plasma membrane. Therefore, we reasoned that STK11 variants in the CTD would demonstrate altered subcellular localization and cytoplasmic functions. Indeed, we show in cell lines through immunofluorescent microscopy that expression of a truncated construct encoding STK11 with a CTD deletion results in full nuclear sequestration. Additionally, point mutants in 4 of the polybasic motif residues (R409W, K416E, A417S and K423E) retain kinase activity, but lead to partial nuclear sequestration despite their ability to bind heterotrimeric complex partners STRAD-α and MO25. This suggests a novel mode of dysregulated localization, independent of the known mechanism of STK11 export into the cytoplasm through binding of STRAD-α. This phenotype is exacerbated in a double mutant of STK11 PBM sites K416E and K423E. As FAK activity is known to be negatively regulated by STK11 in the cytoplasm, we used a KRAS-driven LUAD cell model system with (STK11-WT) and without STK11 (∆STK11) to monitor pY397 levels via SDS-Page and western blot analysis. This is a known FAK autophosphorylation site and was used as a proxy for active FAK. Under glutamine starved conditions known to activate STK11, the STK11 WT cells demonstrate a decrease in pY397-FAK levels. However, the levels of pY397-FAK in ∆STK11 cells were significantly elevated. To determine if this elevated pY397-FAK phenotype is recapitulated in PBM mutants, we are generating a stable K416E_K423E double mutant cell line which will allow us to ascertain the ability of this mutant to regulate FAK activity. Ongoing experiments examine the migratory and invasive ability of the WT-expressing vs. PBM mutants. Additionally, we are utilizing the TurboID system to identify PBM-dependent STK11 interacting proteins. Citation Format: Gopika Nandagopal, Peyton Jenkins, Olivia Morton, Shannon Prior, David Seward, Paula Deming. Using a multipronged approach to characterize C-terminal domain polybasic motif STK11 variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5143.