Fig 1.
Sensitivity of ccRCC and pRCC cells to telaglenastat correlates with glutamine dependency and is associated with reduced metabolites downstream of glutamine.
(A) Relative cell growth or cell death across a panel of kidney cancer cell lines following incubation with telaglenastat (1 μM; left panel) or glutamine withdrawal (right panel) for 72 hours. (B) Correlation between telaglenastat sensitivity (1 μM) and response to glutamine withdrawal at 72 hours. Each data point on the bivariate plot depicts an individual cell line. (C) Immunohistochemical staining of glutaminase in primary ccRCC tumors. (D) Intracellular metabolite levels of glutamine and its downstream metabolites following 1 μM telaglenastat treatment for 4 hours. Graphs represent two independent experiments (left panel: ACHN cell line; right panel: TUHR10TKB cell line) performed in duplicate. (E) Schematic representation of glutamine metabolism showing experimentally observed changes to levels of glutamine-derived metabolites following treatment with telaglenastat. For (A) and (B), data represent result of 2 or 3 independent experiments, each performed in triplicate. Error bars represent standard error of the mean. For (D), statistical analyses were conducted using t tests: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.
Fig 2.
Telaglenastat inhibits the mTORC1 pathway in RCC cells.
(A) Western blot of phospho-S6, total S6, phospho-4E-BP1 and total 4E-BP1 in RCC cell lines after 24 hours of telaglenastat treatment (1 μM) or DMSO control. (B) Relative phospho-S6 and phospho-4E-BP1 levels normalized to total S6 and 4E-BP1, respectively, quantified by densitometry. Representative blots of at least two independent experiments are shown.
Fig 3.
Synergistic anti-proliferative activity and disruption of glutamine and glucose metabolism by telaglenastat and everolimus in RCC cells.
(A) Viability of ACHN cells treated with telaglenastat, everolimus, or a combination of both inhibitors for 72 hours. Dotted line indicates the baseline CellTiter-Glo signal at the time of compound addition. (B) Measurements of glucose or glutamine consumption from media of ACHN cells treated with 75 nM telaglenastat, 25 nM everolimus, or the combination of both inhibitors for 24 hours. (C) Measurements of ECAR and OCR in ACHN cell cultures. ACHN cells were treated with DMSO, telaglenastat (75 nM), everolimus (25 nM), or the combination of both inhibitors for 24 hours and analyzed using the Seahorse Metabolic Analyzer. All experiments were performed in triplicate or quadruplicate. Error bars represent standard error of the mean. Statistical analyses were conducted using Brown-Forsythe and Welch 1-way ANOVA with Dunnett’s test for multiple comparisons: ***P ≤ 0.001; ****P ≤ 0.0001; ns = nonsignificant.
Fig 4.
Synergistic anti-proliferative activity of telaglenastat and cabozantinib in ccRCC cells.
(A) Viability of Caki-1 cells treated with telaglenastat, cabozantinib, or a combination of both inhibitors for 72 hours. The dashed line indicates the baseline CellTiter-Glo signal at the time of compound addition. (B) Western blots of total Akt and phospho-Akt and total Erk and phospho-Erk. A representative blot of at least two independent experiments is shown. (C) Measurement of OCR in Caki-1 cells treated for 24 hours with the indicated compounds. (D) Glucose and glutamine consumption and lactate and glutamate production, collected in media from Caki-1 cells after 24 hours of treatment with the indicated compounds. For parts (B)-(D), Caki-1 cells were treated with DMSO, telaglenastat (1 μM), cabozantinib (6 μM) or the combination of both inhibitors for 24 hours. Sample sizes were n = 5 or 6 for each condition. Error bars represent standard deviations. Statistical analyses were conducted using Brown-Forsythe and Welch 1-way ANOVA with Dunnett’s test for multiple comparisons: *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001.
Fig 5.
Telaglenastat enhances the antitumor activity of mTOR, VEGFR, or receptor tyrosine kinase inhibitors in vivo.
Tumor volumes were measured in mice implanted subcutaneously with Caki-1 RCC cells and treated with either vehicle, telaglenastat (200 mg/kg, dosed orally BID), or (A) everolimus (1 mg/kg, dosed orally QD), (B) cabozantinib (1 mg/kg dosed orally QD), (C) sunitinib (20 mg/kg dosed orally QD), or (D) axitinib (25 mg/kg dosed orally QD) alone or combinations of telaglenastat with each. Statistical analyses were conducted using Ordinary 1-way ANOVA with Tukey’s multiple comparison tests: *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001.