A genome-wide CRISPR screen implicates plasma membrane asymmetry in exogenous C6-ceramide toxicity

The bioactive sphingolipid ceramide impacts diverse cellular processes (e.g., apoptosis and cell proliferation) through its effects on membrane dynamics and intracellular signalling pathways. The dysregulation of ceramide metabolism has been implicated in cancer evasion of apoptosis and targeting ceramide metabolism has potential therapeutic benefits as a strategy to kill cancer cells and slow tumor growth. However, the mechanisms of cancer cell resistance to ceramide-mediated cell death are vastly intertwined and incompletely understood. To shed light on this mystery, we performed a genome wide CRISPR-Cas9 screen to systematically identify regulators of cancer resistance to the soluble short chain ceramide, C6 ceramide (C6-Cer). Our results reveal a complex landscape of genetic modifiers of C6-Cer toxicity, including genes associated with ceramide and sphingolipid metabolism, vesicular trafficking, and membrane biology. Furthermore, we find that loss of the phospholipid flippase subunit TMEM30A impairs the plasma membrane trafficking of its binding partner the P4-type ATPase ATP11B, and depletion of TMEM30A or ATP11B disrupts plasma membrane asymmetry and promotes resistance to C6-Cer toxicity independent of alterations in C6-Cer uptake. Together, our findings provide a resource of genetic modifiers of C6-Cer toxicity and reveal an unexpected role of plasma membrane asymmetry in C6-Cer induced cell death.