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 May 1, 2017
Citation Information: J Clin Invest. 2017;127(5):1631-1645. doi:10.1172/JCI87800.
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Categories: 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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Biosynthesis of DNA pyrimidines from glutamine carbons in RCC cells. (A)...

Figure 1

Biosynthesis of DNA pyrimidines from glutamine carbons in RCC cells.

(A) Diagram depicting the contribution of glutamine and glucose to the formation of aspartate and downstream intermediates of pyrimidine nucleosides, and the inhibition of GLS1 by BPTES. (B) Contribution of the different source pathways to the formation of aspartate. One representative experiment is shown, in which an isogenic pair of VHL–/– and VHL+/+ UMRC2 cells was labeled with [U-13C6]glucose, [U-13C5]glutamine, or [1-13C1]glutamine for 24 hours; the M0 obtained using the [U-13C5]glutamine tracer was used to depict the “unlabeled” fraction of aspartate. (C and D) Using [U-13C5]glutamine, the time course (8 days) shows the 13C enrichment of thymine (C), and cytosine (D), determined from column-purified and formic acid–hydrolyzed DNA. (E and F) Using [1-13C1]glutamine, the time course (8 days) shows the incorporation of the 13C label on DNA-derived thymine (E) and cytosine (F). Error bars represent SEM (n = 3). Student’s t test with Bonferroni correction (to account for multiple comparisons) compared VHL–/– to VHL+/+ cells in E and F. **P < 0.01, ***P < 0.001. GLS1, glutaminase 1; CPSII, carbamoyl phosphate synthetase II; ACTase, aspartate transcarbamoylase; OMP, orotidine monophosphate; UMP, uridine monophosphate; PRPP, 5-phosphoribosyl pyrophosphate.