Arginine Deprivation Inhibits the Warburg Effect and Upregulates Glutamine Anaplerosis and Serine Biosynthesis in ASS1-Deficient Cancers

Abstract

Targeting defects in metabolism is an underutilized strategy for the treatment of cancer. Arginine auxotrophy resulting from the silencing of argininosuccinate synthetase 1 (ASS1) is a common metabolic alteration reported in a broad range of aggressive cancers. To assess the metabolic effects that arise from acute and chronic arginine starvation in ASS1-deficient cell lines, we performed metabolite profiling. We found that pharmacologically induced arginine depletion causes increased serine biosynthesis, glutamine anaplerosis, oxidative phosphorylation, and decreased aerobic glycolysis, effectively inhibiting the Warburg effect. The reduction of glycolysis in cells otherwise dependent on aerobic glycolysis is correlated with reduced PKM2 expression and phosphorylation and upregulation of PHGDH. Concurrent arginine deprivation and glutaminase inhibition was found to be synthetic lethal across a spectrum of ASS1-deficient tumor cell lines and is sufficient to cause in vivo tumor regression in mice. These results identify two synthetic lethal therapeutic strategies exploiting metabolic vulnerabilities of ASS1-negative cancers.

Keywords: Warburg effect; arginine; arginine deprivation; argininosuccinate synthetase 1; cancer metabolism; glutamine; glutamine anaplerosis; sarcoma; serine; serine biosynthesis.

Figure 1. Effects of Acute and Chronic Arginine Deprivation on Cell Growth and Metabolism

(A) Immunoblot measuring ASS1 and c-Myc expression in untreated WT, short-term ADI-PEG20-treated, and LTAT samples (representative of n = 3). (B) Viable cell count normalized to plating density after short- and long-term ADI-PEG20 treatment (n = 3). Data are represented as mean ± SD. (C) Metabolite levels in SKLMS1 WT NT, WT + ADI-PEG20, and LTAT cells (n = 2). Data are represented as mean ± SD. G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; F1,6P, fructose-1,6,bisphosphate; 3-PG, 3-phosphoglycerate; 2-PG, 2-phosphoglycerate; α-KG, α-ketoglutarate. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S1 and S2 and Table S1.

Figure 2. Loss of PKM2 and Alterations in Glycolysis after Arginine Deprivation

(A) Protein expression in untreated WT, WT + ADI-PEG20, and LTAT cell lines. Densitometry was performed on p-PKM2/PKM2 and PKM2/tubulin, with relative expression normalized to untreated levels shown (representative of n = 3), (B) Normalized proliferation rates of cells grown in various glucose concentrations after 36 hr (n = 3). Data are represented as mean ± SD. (C) Changes in glucose uptake upon short- and long-term ADI-PEG20 treatment (n = 6). Data are represented as mean ± SD. (D) Oligomycin-induced cell death in WT and LTAT cell lines as assayed by propidium iodide (PI) fluorescence-activated cell sorting (FACS) (n = 3). Data are represented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S3.

Figure 3. Glucose and Serine Metabolism after Arginine Deprivation

(A) Average relative and normalized isotopomer abundance from U¹³C glucose in SKLMS1 WT NT, WT + ADI-PEG20, and SKLMS1 LTAT cell lines. Red arrows indicate increased ¹³C flux upon short-term treatment (n = 3). (B) Cell death induction in WT cell lines after treatment with ADI-PEG20 and/or CBR-5884 as measured by PI FACS (n = 3). Data are represented as mean ± SD. (C and D) PI FACS measurement of cell death in WT and LTAT cell lines after treatment with CBR-5884 (n = 3) (C) or treatment with MTX (n = 3) (D). Data are represented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S3.

Figure 4. Glutamine Metabolism after Arginine Deprivation

(A) Normalized viable cell counts of WT and LTAT cell lines grown in glutamine-free medium after 24, 48, or 72 hr (n = 3). Data are represented as mean ± SD. (B) Metabolite levels in SKLMS1 WT NT, WT + ADI-PEG20, and LTAT cell lines (n = 2). Data are represented as mean ± SD. (C) Normalized measurements of [³H]-Gln uptake after short- and long-term ADI-PEG20 treatment (n = 12). Data are represented as mean ± SEM. (D) Immunoblot analysis of enzymes involved in glutamine metabolism and asparagine biosynthesis upon treatment with ADI-PEG20 (representative of n = 3). (E) Average relative and normalized isotopomer abundance from stable isotope tracing of U¹³C glutamine in SKLMS1 WT NT, WT + ADI-PEG20, and SKLMS1 LTAT cells (n = 3). *p < 0.05, **,p < 0.01, ***p < 0.001. See also Figures S4 and S5.

Figure 5. Inhibition of the Warburg Effect after Arginine Deprivation

(A and B) Normalized measurements of ECAR (A) and OCR (B) upon ADI-PEG20 treatment (WT, n = 11; ADI, n = 12). (C) Percent of ATP generated from glycolysis in WT and LTAT cell lines (WT, n = 11; LTAT, n = 12). (D) Plot of ECAR versus OCR measurements upon short- and long-term ADI-PEG20 treatment (WT, n = 11; ADI, n = 12; LTAT, n = 12). All data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***,p < 0.001. See also Figure S6.

Figure 6. ADI-PEG20 Treatment and Glutamine Metabolism Inhibition Induce Synthetic Lethality

(A and B) Normalized viable cell counts (n = 3) (A) and PI FACS measurement of cell death (B) after treatment with ADI-PEG20 and/or BPTES (n = 3). Data are represented as mean ± SD. (C) Normalized viable cell counts after ADI-PEG20 and/or BPTES treatment in shGFP and shGLS cell lines (n = 3). Data are represented as mean ± SD. (D) Normalized viable cell count after treatment with ADI-PEG20 and/or BPTES in a variety of cancer types. Statistics represent significance between samples treated with ADI-PEG20 and BPTES and samples from other treatment conditions (n = 3). Data are represented as mean ± SD. (E) Tumor volume of SKMEL2 WT shGFP and shGLS xenografts left untreated and treated with ADI-PEG20. Data are represented as mean ± SEM. Statistics represent significance between shGFP and shGLS tumors treated with ADI-PEG20 (n = 5). (F) Immunoblot analysis of SKMEL2 WT shGFP and shGLS tumors harvested following treatment with ADI-PEG20 for 30 days. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S7.

Figure 7. Model of Arginine Deprivation-Induced Metabolic Changes in ASS1-Deficient Cancers

Model of metabolic changes upon ADI-PEG20-induced arginine deprivation causing the shift of glucose from aerobic glycolysis to glucose-dependent serine biosynthesis and the simultaneous upregulation of glutamine anaplerosis and aspartate and asparagine biosynthesis. THF, tetrahydrofolate; meTHF, 5,10-methylenetetrahydrofolate.