Glucosylceramide synthase is required for C6-ceramide nanoliposome-induced organelle stress and cell death.

Sphingolipids are bioactive lipids that regulate key signaling pathways both directly as ligands and through membrane re-organization. Ceramide sits at the center of this network and is considered pro-death in many contexts, making ceramide accumulation an attractive therapeutic strategy. Given the wide-ranging regulation of cellular responses this network exerts, better understanding ceramide metabolism may promote therapeutic efficacy of sphingolipid-based therapeutics. Ceramide glycosylation, catalyzed by glucosylceramide synthase (GCS; UGCG), is in turn widely regarded as a detoxification route thus limiting efficacy of ceramide-based therapeutics. Here we show the opposite. Delivery of short-chain C6-ceramide via a ceramide nanoliposome (CNL) induced organelle stress and cell death in chronic lymphocytic leukemia (CLL) via the accumulation of glycosphingolipids (GSLs), rather than through ceramide itself. Pharmacologic and genetic blockade of GCS protected B-cell leukemia, breast carcinoma, glioblastoma, lung adenocarcinoma, and non-malignant embryonic kidney cells from CNL-induced death. Conversely, exogenous C8-glucosylceramide was sufficient to kill cells that cannot degrade it. We show that GSL accumulation drives an ordered organelle response beginning with lysosomal deacidification, endoplasmic reticulum stress, followed by mitochondrial respiratory capacity decline, each attenuated by inhibition of GSL synthesis. These effects were accompanied by MLKL phosphorylation, increased activity of the stress sensor JNK and CHOP induction, with JNK inhibition partially protecting from death. These findings invert the prevailing view of ceramide glycosylation as a resistance mechanism and identify glycosphingolipid flux as a required effector arm of ceramide-directed therapy.