Abstract

Mutationally-activated BRAFV600E is detected in ~2% of all human non-small cell lung cancers (NSCLC), and serves as a predictive biomarker for treatment of patients with FDA-approved pathway-targeted therapies that inhibit signaling by the BRAFV600E oncoprotein kinase. In genetically engineered mouse (GEM) models, expression of BRAFV600E in alveolar type 2 (AT2) pneumocytes initiates the development of benign lung tumors that, without additional genetic alterations, rarely progress to malignant lung adenocarcinomas. To identify genes that might cooperate with BRAFV600E for malignant lung cancer progression we employed Sleeping Beauty (SB)-mediated transposon mutagenesis, which dramatically accelerated the onset of lethal lung adenocarcinomas. Amongst the diverse group of genes identified by this in vivo screen was Rbms3 (RNA binding motif single-stranded interacting protein 3), an RNA-binding protein implicated as a possible tumor suppressor. Using CRISPR/CAS9 gene editing we confirmed that RBMS3 silencing cooperated with BRAFV600E to promote progression of malignant lung cancer with a distinct micropapillary architecture. Moreover, RBMS3 silencing also cooperated with BRAFV600E to promote the growth of lung organoids in vitro. BRAFV600E/RBMS3Null lung tumors displayed elevated expression of b-catenin (CTNNB1), suggesting that RBMS3 silencing may result in elevated signaling through the WNT>CTNNB1>c-MYC pathway. Finally, analyses of patient samples in The Cancer Genome Atlas (TCGA) revealed that the region of chromosome 3 encompassing RBMS3 is frequently lost in NSCLC and correlates with poor patient prognosis. Collectively, SB-mediated transposon mutagenesis has revealed the ability of a novel tumor suppressor, RBMS3, to cooperate with BRAFV600E to promote lung carcinogenesis, and suggests that RBMS3 silencing may contribute to malignant progression of numerous human lung cancers. SIGNIFICANCEThe BRAFV600E oncoprotein kinase is a potent initiator of benign lung tumorigenesis, but is insufficient to elicit malignant lung adenocarcinoma without additional cooperating alterations. Sleeping Beauty-mediated transposon mutagenesis has revealed a number of genes that cooperate with BRAFV600E to promote lung cancer progression, in particular Rbms3, which encodes an RNA binding protein. Hence, this genetic screen provides a deeper understanding of the molecular mechanisms underlying BRAFV600E-driven lung carcinogenesis, and is an important step improving our ability to successfully target this disease.