DR4-Ser424O-GlcNAcylation Promotes Sensitization of TRAIL-Tolerant Persisters and TRAIL-Resistant Cancer Cells to Death

Abstract TNF-related apoptosis-inducing ligand (TRAIL) resistance, including nongenetically acquired tolerance in cancer persister cells, is a major obstacle to translating TRAIL therapy into patients with cancer. However, the underlying mechanisms remain to be elucidated. Here, we show that DR4/TRAIL-R1 is O-GlcNAcylated at Ser424 in its death domain to mediate both apoptosis and necrosis upon TRAIL ligation. We found that DR4–Ser424 mutations, identified from our cell-based functional screen using a cancer patient–derived cDNA expression library and from The Cancer Genome Atlas, caused TRAIL resistance in various human cancer cell lines. Using O-GlcNAc transferase knockdown cells, DR4-preferred versus DR5-preferred cancer cells, and a DR5-neutralizing antibody, we evaluated the essential role of DR4-specific O-GlcNAc modification in TRAIL cytotoxicity. In contrast to DR4, DR5 was not O-GlcNAcylated by TRAIL treatment, discriminating DR4 from DR5-mediated signaling. Apart from genetic changes in DR4-Ser424, we further classified various cancer cell lines originated from stomach, colon, lung, and glioblastoma according to their sensitivity to and receptor preference upon TRAIL death signaling and generated TRAIL-tolerant persister-derived DLD-1PER cells. Among these, we discovered that DR4 was not modified by O-GlcNAc in most of the TRAIL-resistant cancer cells and DLD-1PER cells. Interestingly, promoting DR4 O-GlcNAcylation intentionally using 2-deoxy-d-glucose or a high concentration of glucose sensitized those resistant cancer cells to TRAIL. The O-GlcNAcylation–defective DR4 failed to form DISC/necrosome and could not translocate to aggregated platforms for receptor clustering. Our findings demonstrate that DR4 O-GlcNAcylation is crucial for TRAIL death signaling, providing new opportunities for TRAIL therapy overcoming TRAIL resistance in cancers. Significance: This study reports that a novel posttranslational modification by O-GlcNAcylation of one of the two human TRAIL receptors with a death domain, TRAIL-R1 (DR4), plays a crucial role in enabling both apoptotic and necroptotic cell death induction by TRAIL.