Abstract 144: Survivin, a novel mediator of the UPR identified by a functional genome wide CRISPR/Cas9 based knock out screen

Abstract

Abstract Tumor growth and metastasis involve a complex relationship with the tissue microenvironment. A proliferating tumor encounters several stresses from the microenvironment like hypoxia, lack of nutrients and acidosis. To cope with these conditions, cancer cells can co-opt existing cytoprotective mechanisms, such as the Unfolded Protein Response (UPR). The UPR involves transcriptional and translational activation of signaling pathways designed to relieve cellular stress and attenuate cell death; although, in the case of unresolved or chronic ER stress UPR can promote cell death. However, the mechanisms by which the UPR influences cell fate in the presence of ER stress are poorly understood. To address this, we have used a functional CRISPR-based genetic knockout screen, to determine novel regulators of UPR and the mechanisms involved to control cellular fate following chronic ER stress. We delivered a lentiviral genome-wide CRISPR-Cas9 knockout library to two cell lines and identified sgRNAs which are over-represented (pro-apoptotic genes), or underrepresented (pro-survival genes) following ER stress induced by thapsigargin and tunicamycin. One of the highest-ranking gene candidates was Survivin/BIRC5, a protein which is overexpressed in tumor cells compared to normal tissues. Survivin was first identified as an inhibitor of apoptosis but also participates in the regulation of the cell cycle. Survivin held a high position in the negative ranking scale of all screens suggesting a possible prosurvival role in response to ER stress. In agreement, our results show that genetic knock down or chemical inhibition of Survivin led to sensitization of cells to ER stress. Moreover, both in vitro an in vivo data support that PERK (a key sensor of the third arm of the UPR cascade) is responsible for this sensitization. Interestingly, we observed that ER stress in turn leads to downregulation of Survivin in a dose and time dependent fashion, both in the level of protein but also RNA, employing a variety of mechanisms like protein translation, degradation and RNA synthesis. Foreseeably, PERK knock down reverses the aforementioned effect supporting its role as a key participant in the interplay between Survivin and ER stress. Though the balance between those two is still to be understood our results demonstrate that Survivin holds a novel function as an ER stress regulator but also targeting of PERK and Survivin in a clinical setting could reveal new windows in therapeutic intervention in malignancy. Citation Format: Nektaria Maria Leli, Souvik Dey, Lauren Brady, Carlo Salas Salinas, Jerome Lin, Giorgos Skoufos, Ioannis Verginadis, Artemis Chatzigeorgiou, Constantinos Koumenis. Survivin, a novel mediator of the UPR identified by a functional genome wide CRISPR/Cas9 based knock out screen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 144.