Nucleoside diphosphate kinase-3 (NME3) enhances TLR5-induced NFkB activation

Bacterial flagellin is a potent activator of NFkB signaling, inflammation, and host innate immunity, and recent data indicate that flagellin represents a novel antitumor ligand acting through toll-like receptor 5 (TLR5) and the NFkB pathway to induce host immunity and aid in the clearance of tumor xenografts. To identify innate signaling components of TLR5 responsible for these antitumor effects, a loss-of-function highthroughput screen was employed utilizing carcinoma cells expressing a dynamic NFkB bioluminescent reporter stimulated by Salmonella typhimurium expressing flagellin. A live cell screen of a siRNA library targeting 691 known and predicted human kinases to identify novel tumor cell modulators of TLR5-induced NFkB activation uncovered several interesting positive and negative candidate regulators not previously recognized, including nucleoside diphosphate kinase 3 (NME3), characterized as an enhancer of signaling responses to flagellin. Targeted knockdown and overexpression assays confirmed the regulatory contribution TLR5 expression in breast, lung, ovarian, and gastric cancers, and furthermore, high-level expression of NME3 increased overall survival for patients with breast, lung, and ovarian cancer, but the opposite in of NME3 to TLR5-mediated NFkB signaling, mechanistically downstream of MyD88. Furthermore, Kaplan-Meier survival analysis showed that NME3 expression correlated highly with gastric cancer. Together, these data identify a previously unrecognized proinflammatory role for NME3 in signaling downstream of TLR5 that may potentiate cancer immunotherapies. Implications: Proinflammatory signaling mediated by innate immunity engagement of flagellin-activated TLR5 in tumor cells results in antitumor effects through NME3 kinase, a positive downstream regulator of flagellin-mediated NFkB signaling, enhancing survival for several human cancers. Mol Cancer Res; 16(6); 986-99.