SMARCA4, STK11, and KEAP1 co-loss associates with poor prognosis and upregulation of the TGF-β pathway in lung adenocarcinoma (LUAD) - RNA-seq

Background Lung adenocarcinoma (LUAD) is clinically and molecularly defined by oncogenic driver mutations, whose identification has led to the development of driver-targeted therapies and substantial improvements in prognosis for subsets of LUAD patients. However, recent studies synthesizing LUAD tumor sequencing with clinical outcomes have identified intradriver genetic and phenotypic heterogeneity that underlie differential responses to targeted therapy. Genetic inactivation or loss of SMARCA4, which has been observed to co-occur with loss-of-function mutations in STK11 and KEAP1, is especially predictive of poor prognosis and shorter overall survival in LUAD patients, regardless of driver status. Thus, we sought to examine the clinical features and functional associations of SMARCA4 deficiency in LUAD, both on its own and co-incident with STK11/KEAP1 loss-of-function. Methods We examined the apparent correlation between SMARCA4 loss and poor prognosis through MSK-IMPACT clinicogenomic analysis and transcriptomic profiling of curated clinical samples. We additionally generated isogenic cell line models with genetic knockouts of SMARCA4, STK11, and KEAP1 to capture phenotypic and transcriptional features of our mutationally-defined genotypes of interest in vitro and in vivo. Lastly, we interrogated the functional dependency of SMARCA4/STK11/KEAP1 triple mutant models on TGF-β signaling to assess its potential as a therapeutic target. Results In our clinicogenomic analysis, we observed that remarkably short overall survival and higher frequency of metastasis to multiple sites distinguish SMARCA4/STK11/KEAP1 triple mutant LUAD. Uniquely in the SMARCA4/STK11/KEAP1 triple knockout models, we observed in vitro metastatic behaviors, including enhanced migration and invasion, and lowered organotropism in an in vivo intracardiac xenograft metastasis assay. RNA-Seq and DNase-Seq of these in vitro models and clinical samples identified upregulation of TGF-β signaling and EMT-associated gene expression as a signature unique to SMARCA4/STK11/KEAP1 triple loss, which correlated with concomitant changes in chromatin accessibility. In vitro small molecule inhibition of TGF-β signaling revealed enhanced dependence on this pathway for survival and migration in triple knockout models, nominating TGF-β as a novel therapeutic target in SMARCA4/STK11/KEAP1 co-mutant LUAD. Conclusions We identify SMARCA4/STK11/KEAP1 triple mutant LUAD as a prognostically significant disease subset and nominate TGF-β signaling as a potential therapeutic target.