Targeted inhibition of tumor-specific glutaminase diminishes cell-autonomous tumorigenesis
Yan Xiang, … , Dean W. Felsher, Chi V. Dang
Yan Xiang, … , Dean W. Felsher, Chi V. Dang
Published April 27, 2015
Citation Information: J Clin Invest. 2015;125(6):2293-2306. https://doi.org/10.1172/JCI75836.
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Research Article Oncology

Targeted inhibition of tumor-specific glutaminase diminishes cell-autonomous tumorigenesis

Abstract

Glutaminase (GLS), which converts glutamine to glutamate, plays a key role in cancer cell metabolism, growth, and proliferation. GLS is being explored as a cancer therapeutic target, but whether GLS inhibitors affect cancer cell–autonomous growth or the host microenvironment or have off-target effects is unknown. Here, we report that loss of one copy of Gls blunted tumor progression in an immune-competent MYC-mediated mouse model of hepatocellular carcinoma. Compared with results in untreated animals with MYC-induced hepatocellular carcinoma, administration of the GLS-specific inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) prolonged survival without any apparent toxicities. BPTES also inhibited growth of a MYC-dependent human B cell lymphoma cell line (P493) by blocking DNA replication, leading to cell death and fragmentation. In mice harboring P493 tumor xenografts, BPTES treatment inhibited tumor cell growth; however, P493 xenografts expressing a BPTES-resistant GLS mutant (GLS-K325A) or overexpressing GLS were not affected by BPTES treatment. Moreover, a customized Vivo-Morpholino that targets human GLS mRNA markedly inhibited P493 xenograft growth without affecting mouse Gls expression. Conversely, a Vivo-Morpholino directed at mouse Gls had no antitumor activity in vivo. Collectively, our studies demonstrate that GLS is required for tumorigenesis and support small molecule and genetic inhibition of GLS as potential approaches for targeting the tumor cell–autonomous dependence on GLS for cancer therapy.

Authors

Yan Xiang, Zachary E. Stine, Jinsong Xia, Yunqi Lu, Roddy S. O’Connor, Brian J. Altman, Annie L. Hsieh, Arvin M. Gouw, Ajit G. Thomas, Ping Gao, Linchong Sun, Libing Song, Benedict Yan, Barbara S. Slusher, Jingli Zhuo, London L. Ooi, Caroline G.L. Lee, Anthony Mancuso, Andrew S. McCallion, Anne Le, Michael C. Milone, Stephen Rayport, Dean W. Felsher, Chi V. Dang

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Figure 7

Anti-GLS Vivo-Morpholino inhibits xenograft growth cell autonomously.

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Anti-GLS Vivo-Morpholino inhibits xenograft growth cell autonomously. (A...
(A) The GLS NMD Vivo-Morpholino (red bar) caused skipping of human GLS exon 4, resulting in a premature truncation codon (PTC) in exon 5. (B) 2 μM GLS NMD Vivo-Morpholino (Vivo-Mo) reduced GLS mRNA in P493 cells. Data are shown as mean ± SEM. n = 3. (C) 10 μM GLS NMD Vivo-Morpholino reduced GLS protein in human P493 cells but not mouse GLS in mHCC 3–4 cells. (D) 2 μM GLS NMD Vivo-Morpholino decreased glutaminase activity in P493 cells. P = 0.01. (E) GLS knockdown in P493 increased ROS as indicated by increased DCFDA staining. (F) 2 μM GLS NMD Vivo-Morpholino inhibited growth of P493 cells in vitro versus standard control morpholino. Growth is rescued with a combination of TCA cycle intermediate oxaloacetate (OAA) and the antioxidant NAC. Data are shown as mean ± SD. n = 3. GLS NMD Vivo-Morpholino vs. GLS NMD Vivo-Morpholino OAA + Nac, P < 0.01. (G) Mismatch between human and mouse sequence provides species specificity for human GLS NMD Vivo-Morpholino (exon, underlined; intron, not underlined). (H and I) The GLS NMD Vivo-Morpholino slowed growth of P493 xenografts when treatment was begun at a xenograft volume of (H) 100 mm3 (PBS and standard control, n = 5; GLS NMD, n = 6) or (H or I) 200 mm3 (PBS, n = 5; standard control, n = 4; GLS NMD Vivo-Morpholino, n = 6). SD of replicates. GLS NMD versus standard control final day for G and H. P ≤ 0.01. (J) GLS IHC of P493 xenografts treated with GLS NMD or standard control Vivo-Morpholino shows reduced GLS staining in GLS NMD Vivo-Morpholino–treated xenografts. Original magnification, ×100. Student’s t test was used.