Abstract 1247: Identifying novel regulators of the Unfolded Protein Response (UPR) by genome-scale CRISPR-Cas9 knockout screens

Abstract

Abstract Development and growth of a tumor as well as its ability to metastasize involves a complex relationship with its tissue microenvironment. A proliferating tumor encounters several microenvironmental stress conditions such as hypoxia, lack of nutrients and acidosis. To cope with these conditions, cancer cells have developed elaborate cytoprotective mechanisms which provide them with distinct advantages to thrive. Thus, deciphering the signaling pathways which get activated in the tumor microenvironment has been paramount to develop new therapeutic strategies for treatment. The Unfolded Protein Response (UPR) is an adaptive prosurvival pathway elicited by stresses in the tumor microenvironment (e.g., hypoxia, low glucose) and involves translational and transcriptional activation of effector genes which act to relieve cellular stress and block cancer cell death. We developed a strategy to comprehensively analyze critical mediators of cell fate in response to UPR activation. We have delivered a lentiviral genome-scale CRISPR Cas9 knockout (GeCKOv2) library to Sq20B cells (human squamous head and neck carcinoma) and A375 (human melanoma) cells. The library is targeting 19,050 genes with 123,411 unique guide sequences and enables both negative and positive selection screening. We used the GeCKO v2 library to identify genes essential for triggering the UPR in response to thapsigargin and tunicamycin, known specific activators of ER stress. Our highest-ranking candidates include BIRC5/Survivin, a well-studied molecule that acts as an inhibitor of apoptosis and is highly expressed in cancer cells and eukaryotic translation initiation factor eIF6, whose overexpression increases motility and invasiveness of cancer cells. Our preliminary results indicate that loss of Survivin and eIF6 dramatically enhance sensitization of cells to various ER stress conditions. Moreover, this synergistic outcome is observed when cells are treated with YM155, a small molecule that selectively suppresses Survivin and is used in phase I/II clinical trials. Lastly, morphological changes like endoreduplication are observed after long term absence of Survivin indicating its endogenous ER stress inducing role. Taken together, Survivin and eIF6 are important mediators of survival following ER stress and characterizing the pathways involved can lead to the development of novel targeted agents and therapeutic approaches. Citation Format: Nektaria Maria Leli, Souvik Dey, Lauren Brady, Constantinos Koumenis. Identifying novel regulators of the Unfolded Protein Response (UPR) by genome-scale CRISPR-Cas9 knockout screens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1247. doi:10.1158/1538-7445.AM2017-1247