A genome-wide shRNA screen uncovers a novel potential ligand for NK cell activating receptors

Paolo Romania¹Loredana Cifaldi²Paula Gragera¹Valerio D'Alicandro¹Matteo Caforio¹Valentina Folgiero¹Valeria Lucarini¹Ombretta Melaiu¹Roberto Bei²Franco Locatelli¹Doriana Fruci^1*

• ¹Oncohaematology, Bambino Gesù Children's Hospital (IRCCS), Rome, Italy
• ²Department of Clinical Sciences and Translational Medicine, University of Rome “Tor Vergata”, Rome, Sicily, Italy

Natural Killer (NK) cells play a key role in both innate and adaptive immune responses against viruses and tumor cells. Their function relies on the dynamic balance between activating and inhibitory signals, which are mediated by receptors that bind ligands expressed on target cells. While much is known about the function and expression patterns of NK cell activating receptors (NKARs), many of their ligands remain unidentified. To address this, we performed a short hairpin RNA (shRNA) screening targeting 15,000 genes to identify potential ligands for NKARs. We identified ten candidate genes whose downregulation in K562 target cells decreased NK cell-mediated cytotoxicity to levels comparable to silencing the MICA gene. The most promising candidates were functionally validated through single-target gene silencing and overexpression. Among them, the placenta-specific 1 (PLAC1) gene stood out, as its inhibition conferred the greatest protection to target cells from NK cell lysis, while overexpression of PLAC1 significantly increased NK cell degranulation. Importantly, PLAC1 was found to interact with NKAR fusion proteins, including NKG2D, DNAM1 NKp44 and NKp30, suggesting its potential involvement in NK cell function. PLAC1 is typically silent in normal tissues, with the exception of placental trophoblasts and testicular germ cells, but is markedly overexpressed in a wide range of tumors. Notably, its prognostic significance appears to be tumor-type specific, associating with either favorable or poor outcomes depending on the cancer context. Our study identifies PLAC1 as a novel potential ligand for NKARs, suggesting it could be a valuable target for pharmacological strategies aimed at enhancing NK cell recognition. This finding holds promise for improving the efficacy of NK cell-based immunotherapies and advancing their clinical application.