Metabolic reprogramming in cancer cells can increase their dependence on metabolic substrates such as glucose. As such, the vulnerability of cancer cells to glucose deprivation creates an attractive opportunity for therapeutic intervention. Because it is not possible to starve tumors of glucose in vivo, here we sought to identify the mechanisms in glucose deprivation–induced cancer cell death and then designed inhibitor combinations to mimic glucose deprivation–induced cell death. Using metabolomic profiling, we found that cells undergoing glucose deprivation–induced cell death exhibited dramatic accumulation of intracellular l-cysteine and its oxidized dimer, l-cystine, and depletion of the antioxidant GSH. Building on this observation, we show that glucose deprivation–induced cell death is driven not by the lack of glucose, but rather by l-cystine import. Following glucose deprivation, the import of l-cystine and its subsequent reduction to l-cysteine depleted both NADPH and GSH pools, thereby allowing toxic accumulation of reactive oxygen species. Consistent with this model, we found that the glutamate/cystine antiporter (xCT) is required for increased sensitivity to glucose deprivation. We searched for glycolytic enzymes whose expression is essential for the survival of cancer cells with high xCT expression and identified glucose transporter type 1 (GLUT1). Testing a drug combination that co-targeted GLUT1 and GSH synthesis, we found that this combination induces synthetic lethal cell death in high xCT-expressing cell lines susceptible to glucose deprivation. These results indicate that co-targeting GLUT1 and GSH synthesis may offer a potential therapeutic approach for targeting tumors dependent on glucose for survival.

Nucleotide synthesis regulates epithelial cell senescence Supporting Fig. 1: Senescent HMEC are frequently multi-nucleated. Videos from 3D reconstructions of Z-stack confocal imaging for HMEC cells stained with Hoechst dye (blue) and plasma membrane dye (yellow) for HMEC at PD 10 (A) and HMEC at PD 37 (B, C). Nucleotide synthesis regulates epithelial cell senescenceNucleotide synthesis regulates epithelial cell senescence Supporting Fig. 2: Extracellular medium analysis of amino acid and TCA cycle metabolites, intracellular AMP/ATP levels, and MSEA analysis of senescent HMEC intracellular metabolite pools A) Extracellular metabolite secretion data for TCA cycle metabolites in proliferating and senescent HMEC. Metabolite extracts from blank and conditioned media were analyzed by LC-MS. Secretion or uptake values were normalized to integrated cell number. Secreted metabolites have positive values, and consumed metabolites have negative values. * denotes p-value less than 0.05 by FDR-corrected Student's t-test. See Supporting Table S1 for all measured metabolites. B) Same as in A for amino acids. * denotes p-value less than 0.05 by FDR-corrected Student's t-test. See Supporting Table S1 for all measured metabolites. C) Intracellular pool sizes of AMP and ATP of proliferating and senescent HMEC. * denotes p-value less than 0.05 by FDR-corrected Student's t-test. See Supporting Table S2 for all measured metabolites. D) Metabolite set enrichment analysis (MSEA) analysis for intracellular metabolites pool sizes of proliferating and senescent HMEC. Metabolites were ranked based on log2 fold change of senescent/proliferating. Shown are the mountain plot of nucleoside mono/di/tri-phosphates (left), and table of all tested metabolic pathways (right). Supporting Fig. 7: Glucose fractional incorporation analysis of luciferase-and hTERT-expressing HMEC at low and high population doublings. Volcano plots representing the average log2 fold change versus the FDR-corrected p-value of [U-13 C]glucose fractional contribution for: A) Senescent luciferase-(Day ~45) v. proliferating hTERT-expressing HMEC (Day ~45). B) Senescent luciferase-(Day ~45) v. proliferating luciferase-expressing HMEC (Day ~15). C) Proliferating hTERT-(Day ~45) v. proliferating hTERT-expressing HMEC (Day ~15). D) Proliferating luciferase-(Day ~15) v. proliferating hTERT-expressing HMEC (Day ~15). Days represent the time since completion of drug selection following viral infection. Nucleotide synthesis regulates epithelial cell senescence Supporting Fig. 8: Metabolite set enrichment analysis of hTERT immortalization. MSEA analysis for fractional contribution of [U-13 C]-glucose in hTERT immortalized HMEC. Metabolites were ranked based on log2 fold change of luciferase/hTERT. Nucleotide synthesis regulates epithelial cell senescence Nucleotide synthesis regulates epithelial cell senescence Supporting Fig. 9: Inhibition of RRM2 induces senescence in HMEC and MCF10A cells. A) Western blots of p21, PAI-1, and actin for triapine-treated HMEC. Blots represent five inde...

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Oncogenes can create metabolic vulnerabilities in cancer cells. We tested how AKT (herein referring to AKT1) and MYC affect the ability of cells to shift between respiration and glycolysis. Using immortalized mammary epithelial cells, we discovered that constitutively active AKT, but not MYC, induced cell death in galactose culture, where cells rely on oxidative phosphorylation for energy generation. However, the negative effects of AKT were temporary, and AKT-expressing cells recommenced growth after ∼15 days in galactose. To identify the mechanisms regulating AKT-mediated cell death, we used metabolomics and found that AKT-expressing cells that were dying in galactose culture had upregulated glutathione metabolism. Proteomic profiling revealed that AKT-expressing cells dying in galactose also upregulated nonsense-mediated mRNA decay, a marker of sensitivity to oxidative stress. We therefore measured levels of reactive oxygen species (ROS) and discovered that galactose-induced ROS exclusively in cells expressing AKT. Furthermore, ROS were required for galactoseinduced death of AKT-expressing cells. We then confirmed that galactose-induced ROS-mediated cell death in breast cancer cells with upregulated AKT signaling. These results demonstrate that AKT but not MYC restricts the flexibility of cancer cells to use oxidative phosphorylation. This article has an associated First Person interview with the first author of the paper.

Cellular senescence is a mechanism by which cells permanently withdraw from the cell cycle in response to stresses including telomere shortening, DNA damage, or oncogenic signaling. Senescent cells contribute to both age-related degeneration and hyperplastic pathologies, including cancer. In culture, normal human epithelial cells enter senescence after a limited number of cell divisions, known as replicative senescence. Here, to investigate how metabolic pathways regulate replicative senescence, we used LC-MS-based metabolomics to analyze senescent primary human mammary epithelial cells (HMECs). We did not observe significant changes in glucose uptake or lactate secretion in senescent HMECs. However, analysis of intracellular metabolite pool sizes indicated that senescent cells exhibit depletion of metabolites from nucleotide synthesis pathways. Furthermore, stable isotope tracing with 13 C-labeled glucose or glutamine revealed a dramatic blockage of flux of these two metabolites into nucleotide synthesis pathways in senescent HMECs. To test whether cellular immortalization would reverse these observations, we expressed telomerase in HMECs. In addition to