Multifunctional Antitumor Molecule 5’-triphosphate SiRNA Combining Glutaminase Silencing And RIG-ⅠActivation

Resisting cell death, reprogrammed metabolism and immune escape are fundamental traits of hard-to-treat cancers. Therapeutic improvement can be expected by designing drugs targeting all three aspects.5’-triphosphate RNA (ppp-RNA), a specific ligand of the pattern recognition receptor retinoic acid inducible gene I (RIG-I), has been shown to trigger intrinsic apoptosis of malignant cells and to activate antitumor immune responses via type I interferons (IFNs). In this study, we designed a ppp-modified siRNA specifically silencing glutaminase (ppp-GLS), a key enzyme of glutaminolysis that is indispensable for many cancer types. Bi-functional ppp-GLS induced more prominent antitumor responses than RNA molecules that contained either the RIG-I ligand motif or GLS silencing capability alone. The cytopathic effect was constrained to tumor cells as non-malignant cells were not affected. We then analyzed the mechanisms leading to the profound antitumor efficacy. Firstly, ppp-GLS effectively induced intrinsic pro-apoptotic signaling. In addition, GLS silencing sensitized malignant cells to RIG-I-induced apoptosis. Moreover, disturbed glutaminolysis by GLS silencing contributed to enhanced cytotoxicity. Finally, RIG-I activation blocked autophagic degradation leading to dysfunctional mitochondria and ROS generation, whereas GLS silencing severely impaired ROS scavenging systems, leading to a vicious circle of ROS-mediated cytotoxicity. Taken together, ppp-GLS combines cell death induction, immune activation and glutaminase inhibition in a single molecule and has high therapeutic efficacy against cancer cells.