Abstract 2751: Functional assessment of STK11 C-terminal domain variants

Abstract STK11 mutations in KRAS-driven lung adenocarcinomas (LUADs) are associated with aggressive tumor phenotypes characterized by increased risk of metastasis and lower overall and progression free survival. STK11 is a serine-threonine kinase that is comprised of an N-terminal domain containing a nuclear localization signal, a kinase domain, and a C-terminal domain (CTD). In LUAD, STK11 loss of function impacts many aspects of coordinated cell motility and promotes alterations characteristic of metastasis including cell polarity and adhesion. Both kinase activity and subcellular localization are important for STK11 tumor suppressor function. Our objective was to develop a reliable method to identify pathogenic STK11 mutations. Using archival data from the University of Vermont Medical Center, we identified 28 STK11 missense variants that had not yet been functionally characterized. We evaluated the functional impact of these variants on STK11 kinase activity through detection of autophosphorylation using an in-vitro kinase assay, as well as their ability to induce p53 activity through a luciferase reporter. Of the variants studied, 5 were CTD mutations. These variants retained kinase activity and the ability to bind STK11’s partners STRAD-alpha and MO25 in co-immunoprecipitation assays. Although the CTD of STK11 has no catalytic activity, it is thought to be integral for translocation to the cytoplasm where it can colocalize with actin and the cell membrane. STK11 dependent regulation of directional migration requires both proper localization to the actin cytoskeleton and kinase activity. Moreover, it has been reported that the C-terminal polybasic motif of STK11 (aa403-426), consisting of 3 Lysine and 5 Arginine residues is key for its localization and activation of AMPK at the plasma membrane. Therefore, when ascertaining functional status of STK11 in vivo, it is important to consider kinase independent STK11 functions. In LUAD cell lines, we show through immunofluorescent microscopy that expression of a truncated construct encoding STK11 with a CTD deletion results in nuclear sequestration. Additionally, point mutants in 3 of the polybasic motif residues (R409W, K416E and K423E) retain kinase activity and STRAD-alpha binding, but also lead to nuclear sequestration - highlighting the importance of a multipronged approach when assessing STK11 function. Future studies will assess the ability of these C-terminal domain mutants to phosphorylate cytoplasmic substrates, and their impact on cell migration and invasion. Additionally, we will utilize the TurboID proximity ligation system to assess the effect of C-terminal domain point mutations on binding partner accessibility. Citation Format: Gopika Nandagopal, Sean Lenahan, Hannah Ross, Hailey Sarausky, David Seward, Paula Deming. Functional assessment of STK11 C-terminal domain variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2751.