Abstract P1160: Genome-wide CRISPR Screen Reveals Genetic And Small Molecule Modifiers Of Ca 2+ -mediated Cell Death

Abstract

Ca 2+ is a ubiquitous and highly regulated signaling ion in living cells. Thus, Ca 2+ is typically maintained at a low intracellular concentration (10-100 nM), tens of thousands of times lower than the outside space (1-2 mM). Failure to maintain this homeostasis—due to membrane instability or energetic failure—allows supraphysiologic concentrations to enter cells and trigger cell death pathways including mitochondrial permeability transition, apoptosis, and autophagy. This mechanism of cell death is shared by many conditions including myocardial infarction, stroke, and sepsis. Targeting of known protein effectors downstream of Ca 2+ overload, such as caspases and calpains, has not yet translated into therapies due to specificity concerns. Therefore, there is still a pressing need for a comprehensive examination of genetic effectors involved in Ca 2+ -induced cell death. Here, we performed genome-wide CRISPR knockout screening in human cells and identified genes and pathways previously unknown to mediate cell toxicity after Ca 2+ overload. We reveal 115 gene knockouts that protected cells and 9 knockouts that sensitized cells to intracellular Ca 2+ . Notably, genetic ablation of members from the store operated calcium entry, very long-chain fatty acid synthesis, and SWItch/Sucrose Non-Fermentable (SWI/SNF) pathways provided marked protection. Reassuringly, genes known to maintain Ca 2+ homeostasis (e.g., STIM1, ATP2B4, ATP2B1) were recovered by the screen. By comparing against the essential and druggable genomes, we determined that 82 of the protective knockouts are non-essential genes and 21 are targetable with small molecules. Therefore, a significant portion of our hits represent viable but unexplored targets for conditions that feature Ca 2+ overload. We tested this hypothesis using small molecule blockade of hits overlapping with the druggable genome. We found several antagonists that resulted in a reduction of ionomycin-induced cell death. Altogether, our results reveal numerous novel effectors of Ca 2+ -mediated cell death that will serve as a basis for improved mechanistic understanding of cell death and as targets for pharmacotherapeutic design.