Open Access

Xiaoxuan Zhuang, James Woods, Yanlong Ji, Sebastian Scheich, Fei Mo, Sumati Rajagopalan, Zana A. Coulibaly, Matthias Voss, Henning Urlaub, Louis M. Staudt, Kuan-Ting Pan, Eric O. Long

• Highlights • Deletion of SPPL3 promotes resistance of malignant B cells to NK cell cytotoxicity • Loss of SPPL3 blocks ligand binding to NK receptors via increased N-glycosylation • B3GNT2 deletion reduces LacNAc addition and restores SPPL3-KO cell sensitivity to NK cells • SPPL3-deficient cells are enriched in tetra-antennary N-glycans with LacNAc elongations Summary Natural killer (NK) cells are primary defenders against cancer precursors, but cancer cells can persist by evading immune surveillance. To investigate the genetic mechanisms underlying this evasion, we perform a genome-wide CRISPR screen using B lymphoblastoid cells. SPPL3, a peptidase that cleaves glycosyltransferases in the Golgi, emerges as a top hit facilitating evasion from NK cytotoxicity. SPPL3-deleted cells accumulate glycosyltransferases and complex N-glycans, disrupting not only binding of ligands to NK receptors but also binding of rituximab, a CD20 antibody approved for treating B cell cancers. Notably, inhibiting N-glycan maturation restores receptor binding and sensitivity to NK cells. A secondary CRISPR screen in SPPL3-deficient cells identifies B3GNT2, a transferase-mediating poly-LacNAc extension, as crucial for resistance. Mass spectrometry confirms enrichment of N-glycans bearing poly-LacNAc upon SPPL3 loss. Collectively, our study shows the essential role of SPPL3 and poly-LacNAc in cancer immune evasion, suggesting a promising target for cancer treatment.