GLS2 is a tumor suppressor and a regulator of ferroptosis 1 in hepatocellular carcinoma

47 Glutamine synthase 2 (GLS2) is a key regulator of glutaminolysis and has been 48 previously implicated in activities consistent with tumor suppression. Here we generated 49 Gls2 knockout (KO) mice that develop late-occurring B cell lymphomas and 50 hepatocellular carcinomas (HCC). Further, Gls2 KO mice subjected to the 51 hepatocarcinogenic Stelic Animal Model (STAM) protocol produce larger HCC tumors 52 than seen in wild-type mice. GLS2 has been shown to promote ferroptosis, a form of cell 53 death characterized by iron-dependent accumulation of lipid peroxides. In line with this, 54 GLS2 deficiency, either in cells derived from Gls2 KO mice or in human cancer cells 55 depleted of GLS2, conferred significant resistance to ferroptosis. Mechanistically, GLS2, 56 but not GLS1, increased lipid ROS production by facilitating the conversion of glutamate 57 to α-ketoglutarate, thereby promoting ferroptosis. Ectopic expression of wild-type GLS2 58 in a human hepatic adenocarcinoma xenograft model significantly reduced tumor size; 59 this effect was nullified by either expressing a catalytically inactive form of GLS2 or by 60 blocking ferroptosis. Furthermore, analysis of cancer patient datasets supported a role 61 for GLS2-mediated regulation of ferroptosis in human tumor suppression. These data 62 suggest that GLS2 is a bona fide tumor suppressor and that its ability to favor 63 ferroptosis by regulating glutaminolysis contributes to its tumor suppressive function. study demonstrates the key regulator of glutaminolysis, GLS2, can limit hepatocellular carcinoma in vivo by promoting ferroptosis through α-ketoglutarate dependent lipid ROS, in foundation for a novel therapeutic approach. when These findings extend to human we demonstrate that modulation of GLS2 levels leads to correspondingly altered extents of ferroptosis. Most importantly, ferroptosis is required to suppress tumors derived from cells expressing wild-type GLS2 from forming in mice. Finally we show that human HCC cancer patients’ tumors as well as HCCs from mice display reduced levels of GLS2. Taken together these data strongly support a role for GLS2 in ferroptosis and consequent tumor suppression.