Enhancing the Efficacy of Glutamine Metabolism Inhibitors in Cancer Therapy

Abstract

Glutamine metabolism is reprogrammed during tumorigenesis and has been investigated as a promising target for cancer therapy. However, efforts to drug this process are confounded by the intrinsic metabolic heterogeneity and flexibility of tumors, as well as the risk of adverse effects on the anticancer immune response. Recent research has yielded important insights into the mechanisms that determine the tumor and the host immune responses to pharmacological perturbation of glutamine metabolism. Here, we discuss these findings and suggest that, collectively, they point toward patient stratification and drug combination strategies to maximize the efficacy of glutamine metabolism inhibitors as cancer therapeutics.

Keywords: CB-839; JHU083; cancer metabolism; glutaminase; glutamine; immunometabolism.

Figure 1.. Glutamine metabolism in quiescent and proliferating cells.

Proliferative metabolism is characterized by a broad upregulation of biosynthetic pathways. Glutamine is acquired either through uptake transporters such as SLC1A5 or, in nutrient-poor microenvironments, from extracellular protein via macropinocytosis followed by lysosomal degradation. Glutamine is an obligate nitrogen donor for nucleotide and asparagine biosynthesis and an exchange factor for some less abundant amino acids. Its catabolite glutamate is a precursor of α-KG for TCA cycle anaplerosis, a substrate for glutathione biosynthesis, a carbon and nitrogen source for NEAA biosynthesis, and also an exchange factor for other amino acids. Blue arrows indicate pathways in which glutamine/glutamate serve as a nitrogen source, green arrows indicate use of glutamine-derived carbon for anaplerosis, orange arrows represent direct incorporation of glutamate into biosynthetic pathways, and yellow arrows show exchange factor functions. Abbreviations: α-KG, α-ketoglutarate; Ala, alanine, Asp, aspartate; Cys, cystine or cysteine; GCL, glutamate-cysteine ligase; GGC, γ-glutamylcysteine; Gln, glutamine; Glu, glutamate; GLS, glutaminase; GLS2, glutaminase 2; GLUD1, glutamate dehydrogenase 1; Gly, glycine; GOT2, mitochondrial aspartate aminotransferase; GPT2, mitochondrial alanine transaminase; GS, glutathione synthetase; GSH, reduced glutathione; NEAA, non-essential amino acids; OAA, oxaloacetate; PRPP, 5-phosphoribosyl-1-pyrophosphate; P-Ser, phosphoserine; Pyr, pyruvate; Ser, serine; SLC1A5 var, SLC1A5 variant; TCA, tricarboxylic acid.

Figure 2.. Timeline and chemical structures of representative glutamine metabolism inhibitors.

Glutamine metabolism inhibitors include glutamine antimetabolites and their prodrug forms and allosteric glutaminase inhibitors. L-DON, azaserine, and acivicin are glutamine antimetabolites, and JHU083 is a prodrug form of L-DON that is selectively activated in the tumor microenvironment. These antimetabolite-based inhibitors are marked by a blue box. The two major classes of allosteric glutaminase inhibitors are based on the lead compounds BPTES and 968, marked by red and orange boxes, respectively. Currently, CB-839 is the only GLS inhibitor to have entered clinical trials. 968 is a pan-glutaminase inhibitor, with four-fold higher potency against GLS2 than GLS. Ardisianone (AV-1), is a natural alkyl benzoquinone, the only known molecular scaffold that potently and selectively targets GLS2, marked in a purple box. In addition to GLS inhibitors, the glutamine uptake inhibitors GPNA and V-9302 are marked by green box. Abbreviations: BPTES, bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide; GPNA, L-γ-glutamyl-p-nitroanilide; JHU083, ethyl 2-(2-Amino-4-methylpentanamido)-DON; L-DON, 6-diazo-5-oxo-L-norleucine.

Figure 3.. Determinants of tumor sensitivity to GLS inhibitors.

Diverse factors affect the sensitivity of tumors to GLS inhibitors, and specific oncogenotypes and metabolic stresses (shown in orange) can lock cancer cells into a GLS-dependent state. High activity of the NRF2-antioxidant response pathway has emerged as a conserved biomarker for tumor GLS dependence and can occur through genetic activation of the pathway (for example by loss-of-function mutations in KEAP1) or through factors that impose sustained oxidative stress, such as IDH1/2 mutations that lead to neomorphic 2-hydroxyglutarate-producing enzymes. Expression of GLS is regulated by c-Myc and c-Jun, and high levels of these transcription factors also favor cellular dependence on GLS. The nutrient environment also influences sensitivity to GLS inhibitors, and excessive levels of extracellular cystine can drive glutamine addiction and GLS dependence by forcing glutamate efflux through the cystine/glutamate antiporter xCT/SLC7A11. Other factors promote resistance to GLS inhibitors (shown in blue). These factors include compensatory metabolic pathways such as pyruvate carboxylation, fatty acid oxidation, and glutamine hydrolysis catalyzed by GLS2. mTORC1 can enhance metabolic flexibility by regulating a shift to glucose metabolism, and mTORC1 inhibitors synergize with GLS inhibitors. Abbreviations: α-KG, α-ketoglutarate; CPT1, carnitine palmitoyltransferase I; GCL, glutamate-cysteine ligase; GLS, glutaminase; GPNA, L-γ-glutamyl-p-nitroanilide; IDH, isocitrate dehydrogenase; KEAP1, Kelch-like ECH-associated protein 1; mTOR, mechanistic target of rapamycin; NRF2, nuclear factor erythroid 2-related factor 2; OAA, oxaloacetate; PC, pyruvate carboxylase; ROS, reactive oxygen species; TME, tumor microenvironment.

Figure 4.. Glutamine metabolism impacts the anticancer immune response.

Graphical representation of the effects of allosteric GLS inhibitors (1) and non-selective glutamine antagonists (2-7). Selective GLS inhibition has distinct effects on the differentiation of CD4⁺ and CD8⁺ immune cell subsets, favoring CD4⁺ Th1 and CD8⁺ CTLs but suppressing CD4⁺ Th17 differentiation (1). Broader-spectrum glutamine antagonism alters the nutrient composition of the TME (2) and blocks MDSC generation and recruitment (3). Instead, glutamine antagonism favors conversion of MDSCs to pro-inflammatory TAMs, which inhibit tumor growth (4) and further activates pro-inflammatory TAMs via tumor cell-generated DAMPs (5). Inhibiting glutamine metabolism also directly modulates CD8⁺ CTL metabolism to promote a long-lasting, activated, memory-like phenotype; enhances antigen presentation by pro-inflammatory TAMs to CD8⁺ CTLs (6); decreases production of the immunosuppressive metabolite kynurenine in the TME (7); and increases CD8⁺ CTL tumor infiltration. These cumulative effects promote the anticancer immune response. Abbreviations: ASNS, asparagine synthetase; BPTES, bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide; CPS2, carbamoyl phosphate synthetase II; CTL, cytotoxic T lymphocyte; DAMPs, damage/danger-associated molecular patterns; ECAR, extracellular acidification rate; GFAT, glutamine-fructose-6-phosphate transaminase; GLS, glutaminase; IDO, indoleamine-2,3-dioxygenase; IFNγ, interferon gamma; IL-17, interleukin 17; L-DON, 6-diazo-5-oxo-L-norleucine; MDSC, myeloid-derived suppressor cell; OXPHOS, oxidative phosphorylation; PPAT, phosphoribosyl pyrophosphate amidotransferase; TAMs, tumor-associated macrophages; Th1, T-helper 1; Th17, T-helper 17.