GCN2 protein kinase is required to activate amino acid deprivation responses in mice treated with the anti-cancer agent L-asparaginase

Abstract

Asparaginase depletes circulating asparagine and glutamine, activating amino acid deprivation responses (AADR) such as phosphorylation of eukaryotic initiation factor 2 (p-eIF2) leading to increased mRNA levels of asparagine synthetase and CCAAT/enhancer-binding protein beta homologous protein (CHOP) and decreased mammalian target of rapamycin complex 1 (mTORC1) signaling. The objectives of this study were to assess the role of the eIF2 kinases and protein kinase R-like endoplasmic reticulum resident kinase (PERK) in controlling AADR to asparaginase and to compare the effects of asparaginase on mTORC1 to that of rapamycin. In experiment 1, asparaginase increased hepatic p-eIF2 in wild-type mice and mice with a liver-specific PERK deletion but not in GCN2 null mice nor in GCN2-PERK double null livers. In experiment 2, wild-type and GCN2 null mice were treated with asparaginase (3 IU per g of body weight), rapamycin (2 mg per kg of body weight), or both. In wild-type mice, asparaginase but not rapamycin increased p-eIF2, p-ERK1/2, p-Akt, and mRNA levels of asparagine synthetase and CHOP in liver. Asparaginase and rapamycin each inhibited mTORC1 signaling in liver and pancreas but maximally together. In GCN2 null livers, all responses to asparaginase were precluded except CHOP mRNA expression, which remained partially elevated. Interestingly, rapamycin blocked CHOP induction by asparaginase in both wild-type and GCN2 null livers. These results indicate that GCN2 is required for activation of AADR to asparaginase in liver. Rapamycin modifies the hepatic AADR to asparaginase by preventing CHOP induction while maximizing inhibition of mTORC1.

FIGURE 1.

A, AlbCre-mediated knockdown of floxed PERK (AlbCre⁺, PERK^fl/fl) in liver (Liv) but not pancreas (Pan), spleen (Spl), or tail of GCN2^+/+ and GCN2^−/− mice (upper panel). GCN2 was deleted in the liver, pancreas, and spleen of GCN2^−/−-AlbCre⁺-PERK^fl/fl mice but not GCN2^+/+-AlbCre⁺-PERK^fl/fl mice (lower panel). WT, wild type; KO, knock-out. B, asparaginase increased phosphorylation of eIF2 in the liver of both wild-type (GCN2^+/+-AlbCre⁻-PERK^fl/fl) and PERK knockdown (GCN2^+/+-AlbCre⁺-PERK^fl/fl) mice. Means not sharing same lowercase letter are different from each other (by one-way ANOVA), p < 0.05. C, phosphorylation of eIF2 is reduced in the liver of GCN2 null mice both with and without AlbCre-mediated PERK knockdown following asparaginase injection. **, main effect of asparaginase to increase p-eIF2 (by two-way ANOVA), p < 0.05. D, hepatic ASNS mRNA expression was not significantly increased following asparaginase treatment in GCN2 null mice with or without AlbCre-mediated deletion of floxed PERK in liver. Abbreviations: S, saline; A, asparaginase.

FIGURE 2.

GCN2 is required to activate the integrated stress response in the liver but not pancreas of mice treated with asparaginase. A, phosphorylation of eIF2 in the liver of GCN2^+/+ and GCN2^−/− mice. **, asparaginase increased p-eIF2 in GCN2^+/+ mice, p < 0.05; ++, loss of GCN2 reduced p-eIF2 independent of drug treatment, p < 0.05; +, effect of asparaginase to increase p-eIF2 required GCN2, p < 0.05. B, phosphorylation of eIF2 in the pancreas of GCN2^+/+ and GCN2^−/− mice was not altered by either drug or genotype. C, hepatic ASNS mRNA expression in GCN2^+/+ and GCN2^−/− mice. **, asparaginase increased ASNS mRNA in GCN2^+/+ mice, p < 0.05; ++, loss of GCN2 reduced ASNS mRNA independent of drug treatment, p < 0.05; +, effect of asparaginase to increase ASNS mRNA depended on GCN2, p < 0.05. D, hepatic CHOP/GADD153 mRNA expression in GCN2^+/+ and GCN2^−/− mice. **, asparaginase increased CHOP mRNA in GCN2^+/+ mice, p < 0.05; ++, loss of GCN2 reduced CHOP mRNA independent of drug treatment, p < 0.05; +, effect of asparaginase to increase CHOP mRNA required GCN2, p < 0.05; *, rapamycin reduced CHOP mRNA independent of genotype, p < 0.05; #, effect of asparaginase was blunted by rapamycin, p < 0.05. Abbreviations: SS, saline then saline; SA, saline then asparaginase; RS, rapamycin then saline; RA, rapamycin then asparaginase.

FIGURE 3.

Asparaginase and rapamycin impart tissue-specific effects on the phosphorylation of S6K1, 4E-BP1, and Akt. A and B, reduced p-S6K1 in liver and pancreas by asparaginase required GCN2. **, asparaginase reduced p-S6K1 in GCN2^+/+ mice, p < 0.05; *, rapamycin reduced p-S6K1 independent of genotype, p < 0.05; +, effect of asparaginase to decrease p-S6K1 depended on GCN2, p < 0.05. C, hepatic 4E-BP1 phosphorylation is reduced by rapamycin independent of genotype. *, p < 0.05. D, 4E-BP1 phosphorylation in pancreas was reduced by asparaginase and rapamycin. **, asparaginase reduced p-4E-BP1 in GCN2^+/+ mice, p < 0.05; *, rapamycin reduced p-4E-BP1 independent of genotype, p < 0.05; +, effect of asparaginase to decrease p-4E-BP1 depended on GCN2, p < 0.05, E, deletion of GCN2 blocked induction of hepatic p-Akt/PKB by asparaginase. **, asparaginase increased p-Akt in GCN2^+/+ mice, p < 0.05; +, effect of asparaginase to increase p-Akt depended on GCN2, p < 0.05, F, phosphorylation of Akt/PKB in pancreas. ϕ, rapamycin increased p-Akt only when in combination with GCN2 deletion, p < 0.05. Abbreviations: SS, saline then saline; SA, saline then asparaginase; RS, rapamycin then saline; RA, rapamycin then asparaginase.

FIGURE 4.

GCN2 is required to increase phosphorylation of ERK1/2 in the liver (A) and pancreas (B) of mice treated with asparaginase. A, ++, loss of GCN2 reduced overall hepatic p-ERK1/2 independent of drug treatments, p < 0.05; **, asparaginase increased p-ERK1/2 in GCN2^+/+ mice, p < 0.05; +, effect of asparaginase to increase p-ERK1/2 depended on GCN2, p < 0.05. Abbreviations: SS, saline then saline; SA, saline then asparaginase; RS, rapamycin then saline; RA, rapamycin then asparaginase.