814 Smad4 as a Potential Gatekeeper for Genomic Instability and Mtor-Mediated Tumorigenesis in Esophageal Adenocarcinoma

Abstract

Extensive genomic analysis of patient samples has identified genes whose mutation or loss map malignant progression from Barrett’s metaplasia, through low- (CDKN2A) and high-grade dysplasia (TP53), to invasive adenocarcinoma (SMAD4). Interestingly, loss of SMAD4 has been found to occur exclusively in the invasive disease stage, but the reason for this is unknown. This work aimed to characterise the role of SMAD4 in esophageal adenocarcinoma (EAC) tumorigenesis and identify novel therapeutic targets for SMAD4-deficient EAC. Methods We developed a novel in vivo tumorigenesis model that demonstrates progression of dysplastic Barrett’s esophagus (BE) to invasive EAC upon knockout of SMAD4. We conducted parallel genome-wide CRISPR-Cas9 knockout screens, both in vitro and in vivo, on a background of either wildtype-SMAD4 or SMAD4-knockout dysplastic BE cells to identify co-operative drivers of tumorigenesis in vivo, as well as synthetic lethal interactions to identify potential therapeutic targets in SMAD4-deficient EAC. Functional validation of hits was performed using cell-based assays and drugs targeting candidate molecular targets. Results We identified a synthetic lethal relationship between SMAD4-deficiency and cell cycle checkpoint inhibition, suggesting a role for SMAD4 in maintaining genomic stability and a potential novel therapeutic avenue for SMAD4-deficient EAC. A concurrent in vivo CRISPR-Cas9 tumorigenesis screen produced tumors 4-fold faster than loss of SMAD4 alone and identified regulators of mTOR signalling and SMAD4 as co-operative drivers of tumorigenesis in EAC. Interestingly, these tumorigenic cells exhibited an inherent dependency on specific translation mechanisms downstream of mTOR. Meanwhile, wildtype-SMAD4 BE cells failed to thrive in vivo with mTOR modifications alone, indicating a true co-operative effect at play with SMAD4 loss. Conclusion This study uncovered a potential gatekeeping role of SMAD4 in maintaining genomic stability and inhibiting mTOR-mediated EAC tumorigenesis. In sum, loss of SMAD4 was found to increase genomic instability, thereby rendering EAC cells sensitive to cell cycle checkpoint impediment, whilst simultaneously co-operating with modulated mTOR signalling to promote tumorigenesis in EAC xenograft models.