Glutaminase and poly(ADP-ribose) polymerase inhibitors suppress pyrimidine synthesis and VHL-deficient renal cancers
Arimichi Okazaki, … , Lee Zou, Othon Iliopoulos
Arimichi Okazaki, … , Lee Zou, Othon Iliopoulos
Published March 27, 2017
Citation Information: J Clin Invest. 2017;127(5):1631-1645. https://doi.org/10.1172/JCI87800.
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Research Article Metabolism Oncology

Glutaminase and poly(ADP-ribose) polymerase inhibitors suppress pyrimidine synthesis and VHL-deficient renal cancers

Abstract

Many cancer-associated mutations that deregulate cellular metabolic responses to hypoxia also reprogram carbon metabolism to promote utilization of glutamine. In renal cell carcinoma (RCC), cells deficient in the von Hippel–Lindau (VHL) tumor suppressor gene use glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate (αKG). Glutamine can also generate aspartate, the carbon source for pyrimidine biosynthesis, and glutathione for redox balance. Here we have shown that VHL–/– RCC cells rely on RC-derived aspartate to maintain de novo pyrimidine biosynthesis. Glutaminase 1 (GLS1) inhibitors depleted pyrimidines and increased ROS in VHL–/– cells but not in VHL+/+ cells, which utilized glucose oxidation for glutamate and aspartate production. GLS1 inhibitor–induced nucleoside depletion and ROS enhancement led to DNA replication stress and activation of an intra–S phase checkpoint, and suppressed the growth of VHL–/– RCC cells. These effects were rescued by administration of glutamate, αKG, or nucleobases with N-acetylcysteine. Further, we observed that the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib synergizes with GLS1 inhibitors to suppress the growth of VHL–/– cells in vitro and in vivo. This work describes a mechanism that explains the sensitivity of RCC tumor growth to GLS1 inhibitors and supports the development of therapeutic strategies for targeting VHL-deficient RCC.

Authors

Arimichi Okazaki, Paulo A. Gameiro, Danos Christodoulou, Laura Laviollette, Meike Schneider, Frances Chaves, Anat Stemmer-Rachamimov, Stephanie A. Yazinski, Richard Lee, Gregory Stephanopoulos, Lee Zou, Othon Iliopoulos

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Figure 2

Inhibition of GLS1 decreases intracellular aspartate and selectively suppresses de novo pyrimidine synthesis in VHL–/– cells.

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Inhibition of GLS1 decreases intracellular aspartate and selectively sup...
(A and B) Isogenic VHL–/– and VHL+/+ UMRC2 cells were cultured in the absence or presence of 1.5 μM BPTES for 48 hours, and the metabolites were analyzed by LC-MS/MS. (A) Effect of BPTES on the levels of pyrimidine nucleotides and their intermediates. (B) Effect of BPTES on the levels of glutamine and glutamate in the isogenic pair of UMRC2 cells. Metabolite levels were normalized to the sum of 288 metabolites obtained from extracts in the corresponding cell type. (C and D) The pair of UMRC2 cells was labeled with [U-13C5]glutamine in the absence or presence of 2 μM BPTES for 48 hours, and the metabolite enrichment was measured by LC-MS/MS. Effect of BPTES on the 13C enrichment of UMP (C) and CMP (D) is shown. (E–G) The pair of UMRC2 cells were labeled with [U-13C6]glucose with or without 2 μM BPTES for 48 hours, and the metabolite enrichment was measured by GC-MS. Effect of BPTES on the contribution of glucose oxidation, determined by the level of M2-enriched (E) and M3-enriched (F) TCA cycle intermediates, and of citrate enrichment (G). Error bars represent SEM (n = 3). Student’s t test compared BPTES-treated with corresponding control cells in B–G. *P < 0.05, **P < 0.01, ***P < 0.001. MID, Mass Isotopomer Distribution; Suc, succinate; Fum, fumarate; αKG, α-ketoglutarate; Mal, malate; Asp, aspartate; Glu, glutamate.