2015;125(6):2293C2306. markedly inhibited P493 xenograft growth without affecting mouse expression. Conversely, a Vivo-Morpholino directed at mouse had no antitumor activity in vivo. Collectively, our studies demonstrate that GLS is required for tumorigenesis and support small molecule and genetic inhibition of GLS as potential approaches for targeting the tumor cellCautonomous dependence on GLS for cancer therapy. and is expressed as a long mRNA splice variant, slowed the growth of several different cancer types (13, 17, 19, 25C27), suggesting that pharmacological inhibition of GLS offers a potential therapeutic approach for treating cancer. Glutamine analog inhibitors, such as azaserine and acivicin, can inhibit tumor growth, but they often have considerable off-target effects (6). The glutamine analog 6-diazo-5-oxo-l-norleucine (DON) inhibits a range of glutamine-dependent enzymes, such as glutamine fructose-6-phosphate amidotransferase and glutaminase, as well as other glutamine-dependent reactions (28, Rotigotine HCl 29). Similarly, amino-oxyacetate (AOA), a transaminase inhibitor, has also been used to target a part of glutamine metabolism by inhibiting the production of -ketoglutarate from glutamate, Rotigotine HCl which is in turn derived from glutamine (12, 30). AOA, however, has been documented to inhibit a wide range of other pyridoxal-dependent enzymes in addition to GOT and GPT (31). Hence, AOAs biological activity is also nonspecific. The identification of an allosteric GLS-selective inhibitor, bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), raised the possibility of specifically inhibiting glutamine metabolism with minimal off-target effects (32). The crystal structure of BPTES-bound GLS reveals that BPTES docks in the GLS tetramer interfaces, locking GLS in an off mode and disabling phosphate-dependent activation of the enzyme (24, 33, 34). Other GLS inhibitors have been developed, including the BPTES-like drug candidate CB-839 and compound 968, which has a different mechanism of GLS inhibition (16, 35). We and others have exhibited that pharmacological inhibition of GLS slowed proliferation in several malignancy cell types in vitro and in xenograft models (9, 17, 19, 26, 36). However, previous studies have not directly addressed the mechanism of growth inhibition or whether off-target effects of Rotigotine HCl BPTES, CB-839, or 968 could underlie their antitumor activity (9, 16, 19, 35). GLS inhibition in vivo has been restricted to Rotigotine HCl xenograft studies in immunocompromised mice, in which the potential negative effects on the immune system could not be measured. Activated T cells are known to use high levels of glutaminolysis for proliferation, and inhibition of glutaminase may hinder the natural immune response to the formation of new tumors (37). Whether or not GLS inhibition is effective in immunocompetent mice is not known, particularly since many metabolic pathways used by cancer cells are shared with normal activated T lymphocytes (38). Further, altered glutamine metabolism in the tumor stroma has been reported (39), raising EDNRB the possibility that nonCcell autonomous functions of GLS inhibition may underlie the effects of GLS inhibition in vivo. In this report, we used an immunocompetent MYC-dependent genetically designed model (in liver tumorigenesis. The animal age at the time of MYC activation in this model affects the biology, such that earlier MYC activation resulted in more aggressive tumors (40, 41). In utero MYC activation induces an aggressive hepatoblastoma-like disease upon birth, as compared with the HCCs induced when MYC is usually activated after birth (40). Activation of MYC 4 weeks after birth resulted in multinodular HCC and an overall mean survival time of 15 weeks (40). These tumors display increases in both glucose and glutamine metabolism (42, 43). Here, we report that animals derived from Rotigotine HCl crosses of mice with (< 0.0001) prolonged survival of animals wild-type for (mRNA decay and found that P493 tumor xenograft growth in mice could be markedly and specifically inhibited only by Vivo-Morpholino directed at human in an inducible is required for tumorigenesis and tumor progression has not been known. In this model, MYC expression is usually under the control of a tetracycline-off (Tet-off) system regulated by the Tet transactivating protein (tTA), which in turn is usually driven by the liver-activating protein (LAP) promoter in mice (40, 41). Multifocal tumors in adult animals with varying aggressiveness can be induced according to the age at which MYC is usually activated by doxycycline withdrawal. The earlier the age at which MYC is usually activated, the.