Abstract 4159: Characterization of three recurring STK11/LKB1 mutants in lung adenocarcinoma
Authors: Engel Brienne E. , Schabath Matthew B. , Thompson Zachary J. , Eschrich Steven A. , Brantley Stephen G. , Belock Anastasia R. , Berglund Anders , Gray Jhanelle E. , Beg Amer A. , Haura Eric B. , Cress W. Douglas 
Published: May 2015

Abstract
The STK11 gene encodes the tumor suppressor serine/threonine protein kinase 11 also known as liver kinase β1 (LKB1). The STK11 gene product regulates cellular energy metabolism and cell polarity by activating AMP-activated protein kinase (AMPK) and other members of the AMPK family. Although rare in most human cancers, mutations in the STK11 gene are third most frequent in lung adenocarcinoma after KRAS and TP53.
Mutational status for STK11, EGFR, and KRAS was determined in tumor DNA from 468 lung adenocarcinoma patients and STK11 gene expression measured on an Affymetrix GeneChip microarray. Immunohistochemistry (IHC) was performed on a subset of the patients (N = 140). Survival analyses were performed using Kaplan-Meier survival curves and Cox proportional hazard regression. Three recurring mutations (D194Y, P281fs*6, F354L) were evaluated via Western Blot for AMPK phosphorylation. These mutants were also evaluated via immunoprecipitation and Western Blot for binding to two accessory subunits (MO25 and STRAD) that, along with STK11, make up the active trimeric complex.
STK11 was mutated in 17.5% of all tumors evaluated with 66 different STK11 mutations noted, only 8 of which occurred more than once. STK11 mutant samples had lower levels of STK11 protein as measured by IHC (&lt; 2+: 50.0% mutant vs. 17.9% WT, P &lt; 0.001). Among three STK11 mutants that were characterized, D194Y and F354L were both able to bind MO25 and STRAD, but these interactions were lost in P281fs*6. The P281fs*6 mutant lacks kinase activity while the D194Y mutant has dominant negative activity, suppressing AMPK activation by WT protein. P281fs*6 mutant tumors had lower gene expression compared to other mutants while WT tumors had the highest expression. D194Y mutant tumors had expression levels similar to WT tumors, higher than the average mutant expression. STK11 gene expression for F354L tumors was higher than the mutant tumor average, but lower than WT. The F354L variant is able to phosphorylate AMPK indicating a potential single nucleotide polymorphism (SNP) supported by its appearance in the 1000 Genomes Project. The F354L variant is more prevalent in lung adenocarcinoma patients compared to the general population which may suggest that it is a genetic susceptibility factor. Interestingly, when all STK11 mutant samples were combined and compared to WT tumors, STK11 mutant tumors were significantly associated with a reduced risk of death (HR = 0.28; 95% CI 0.10 - 0.78) among stage I and II patients adjusted for prognostic factors.
This study revealed that STK11 mutant tumors as a whole had lower levels of STK11 protein as measured by IHC, and were associated with a significantly reduced risk of death among early stage patients with lung adenocarcinoma. Mutation status and protein levels alone cannot be used to make treatment decisions, however, as the three recurring mutants characterized caused three disparate effects on the STK11 pathway.
Citation Format: Brienne E. Engel, Matthew B. Schabath, Zachary J. Thompson, Steven A. Eschrich, Stephen G. Brantley, Anastasia R. Belock, Anders Berglund, Jhanelle E. Gray, Amer A. Beg, Eric B. Haura, W. Douglas Cress. Characterization of three recurring STK11/LKB1 mutants in lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4159. doi:10.1158/1538-7445.AM2014-4159