Activation of Oxidative Stress Response in Cancer Generates a Druggable Dependency on Exogenous Non-essential Amino Acids

Abstract

Rewiring of metabolic pathways is a hallmark of tumorigenesis as cancer cells acquire novel nutrient dependencies to support oncogenic growth. A major genetic subtype of lung adenocarcinoma with KEAP1/NRF2 mutations, which activates the endogenous oxidative stress response, undergoes significant metabolic rewiring to support enhanced antioxidant production. We demonstrate that cancers with high antioxidant capacity exhibit a general dependency on exogenous non-essential amino acids (NEAAs) that is driven by the Nrf2-dependent secretion of glutamate through system x_c^- (XCT), which limits intracellular glutamate pools that are required for NEAA synthesis. This dependency can be therapeutically targeted by dietary restriction or enzymatic depletion of individual NEAAs. Importantly, limiting endogenous glutamate levels by glutaminase inhibition can sensitize tumors without alterations in the Keap1/Nrf2 pathway to dietary restriction of NEAAs. Our findings identify a metabolic strategy to therapeutically target cancers with genetic or pharmacologic activation of the Nrf2 antioxidant response pathway by restricting exogenous sources of NEAAs.

Keywords: Keap1; NRF2; amino acid synthesis; asparaginase; glutamate; glutaminase; lung cancer; metabolism; non-essential amino acids; oxidative stress; system x(c)(−).

Figure 1:. Keap1 loss increases dependency on exogenous supply of NEAAs

a) In vitro uptake assay of amino acids after 24 hours b) Serum levels of asparagine, glycine and serine in mice bearing subcutaneous tumors. c) Proliferation in media lacking specified amino acid. d) Relative viability of cells cultured treated with L-asparaginase for 3 days. e) Proliferation of wildtype (Wt) or Keap1 mutant (Mut) LKR (Kras^G12D/+;p53^+/+) cell lines in media lacking serine or asparagine. f) Proliferation of cells expressing an empty vector or WT Keap1 in RPMI lacking serine or asparagine. g) Schematic depicting synthesis of serine from glucose (top) and asparagine from glutamine (bottom). Filled blue circles represent ¹³C atoms derived from [U¹³C]-D-glucose or [U¹³C]-L-glutamine. h) Mass isotopomer analysis of serine and asparagine in cells cultured in complete or amino acid deprived conditions. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 2:. Acute activation of Nrf2 induces dependency on NEAAs

a) Proliferation of wildtype (Kras^G12D/+; p53^−/−) murine LUAD tumor cell lines in the absence of serine or asparagine. b) Proliferation of Keap1 wildtype LKR (Kras^G12D/+;p53^+/+) cell lines in media lacking serine or asparagine. c) Proliferation of wildtype murine Kras^G12D/+;p53^−/− pancreatic cancer cell line in media lacking serine. d) Relative viability of Keap1 wildtype murine Kras^G12D/+;p53^−/− pancreatic cancer cell line treated with L-asparaginase. e) Schematic depicting mechanism of action of oxidants used to activate Nrf2. f) Proliferation of wildtype cells in media lacking serine or asparagine after oxidant treatment. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3:. Low intracellular glutamate levels in cells with Nrf2 activation generates a dependency on exogenous NEAAs

a) Schematic depicting modulation of intracellular glutamate levels. b and c) Relative intracellular abundance of glutamate in cells supplemented with glutamate or treated with Erastin. d and e) Proliferation of cells in media lacking serine or asparagine and supplemented with glutamate or Erastin. f) Relative metabolite flux of glutamate in cells expressing Slc1a3 or an empty vector. g and h) Proliferation of cells expressing Slc1a3 or an empty vector control in media lacking serine or asparagine. i and j) Relative viability of cells cultured in 0.1U/mL of L-Asparaginase or 10% the normal concentration of serine with increasing doses of CB-839. Data is represented as response to CB-839 treatment alone in media with 100% serine for each cell line. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 4:. Glutamate availability restricts serine biosynthesis

a) Proliferation of Keap1 mutant cells in RPMI lacking serine and supplemented with antioxidants or downstream products of serine metabolism. b) Proliferation Keap1 mutant cells expressing an sgRNA against PSAT in media lacking serine supplemented with formate, glutamate or Erastin. c) Schematic depicting serine synthesis from glucose. Filled blue circles represent ¹³C atoms derived from [U¹³C]-D-glucose. d) Mass isotopomer analysis of serine and glycine in cells cultured without serine supplemented with glutamate or treated with CB-839. e) Schematic depicting serine synthesis from glucose utilizing an amino group from glutamate. Filled orange hexagons represent ¹⁵N atoms derived from [α¹⁵N]-L-glutamine. f) Mass isotopomer analysis of serine and glycine in cells cultured in RPMI lacking serine. *p<0.05, **p<0.01, ****p<0.0001

Figure 5:. Keap1 mutant tumors require exogenous NEAAs in vivo.

a) Relative tumor growth of subcutaneous tumors in animals receiving either -SG or AA CTL diet. b) Relative tumor growth of subcutaneous tumors in animals treated with either L-Asparaginase. c and d) Relative tumor growth of subcutaneous tumors in animals receiving -N or AA CTL diet in combination with L-asparaginase treatment. e and f) Relative tumor growth of subcutaneous Keap1 mutant (e) or wildtype (f) tumors in animals receiving either -SG or AA CTL diet and either treated with CB-839 or vehicle. *p<0.05, **p<0.01, ****p<0.0001

Figure 6:. Activation of oxidative stress response depletes intracellular glutamate and generates a dependency on exogenous amino acids

a) Schematic depicting how activation of the oxidative stress response via genetic, pharmacologic or physiological ROS stress to stabilize Nrf2, depletes intracellular glutamate. In cells with activated Nrf2 glutamate is shuttled into glutathione (GSH) biosynthesis or exported through the system x_c⁻ antiporter (xCT) to import cystine. This depletes intracellular glutamate levels and limits its availability for other biosynthetic reactions. Reduced availability of intracellular glutamate restricts its use in transamination reactions for synthesis of nonessential amino acids (NEAA) rendering cells dependent on uptake of NEAAs from the microenvironment.