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Discovery at the VICB







Targeting Cancer by Glutamine Transporter Blockade


By: Carol A. Rouzer, VICB Communications
Published: January 22, 2018


V-9302, a selective inhibitor of ASTC2 suppresses growth and induces apoptosis in glutamine-dependent cancers.    


An interesting characteristic of many cancer cells is an unusual dependency on glutamine for optimal survival and growth. Glutamine directly or indirectly (via its deamidation product, glutamate) serves as a precursor for the synthesis of proteins and glutathione and as a source of tricarboxylic acid cycle intermediates (Figure 1). Recent evidence also suggests that glutamine mediates activation of the MAP kinase signaling pathway. Thus, drugs that interfere with glutamine metabolism might be effective anti-tumor agents in the case of cancers that are highly glutamine-dependent. Indeed, early-phase clinical trials of CB-839, an inhibitor of mitochondrial glutaminase, are currently underway. However, CB-839 only affects glutamine's metabolism within the mitochondrion, leaving many functions unimpeded. This led Vanderbilt Institute of Chemical Biology member Charles Manning to hypothesize that blockade of the cell's ability to acquire glutamine might be a more effective approach. Cancer cells import glutamine via the alanine-serine-cysteine transporter 2 (ASCT2), a sodium-dependent carrier of neutral amino acids. A high level of ASCT2 expression is associated with poor prognosis for a number of human cancers, including lung, breast, and colon. Now Manning and his laboratory report that V-9302, a small molecule inhibitor of ASCT2, reduces viability and/or promotes the death of glutamine-dependent cancer cells [M. L. Schulte, et al. Nat. Medicine, (2018) published online January 15, DOI: 10.1038/nm.4464].



FIGURE 1. Example of the role of ASCT2-mediated glutamine transport in cellular function. In this example, LAT1 transports leucine into the cell in exchange for glutamine, but then glutamine is transported back in by ASCT2. Increased amino acids promote mTORC1 signaling, which blocks SIRT4, relieving inhibition of glutamate dehydrogenase (GDH). Glutamine is converted to glutamate, which is then converted to α-ketoglutarate (Alpha-KG), a key intermediate in the citric acid cycle. Glutamate is also transported out of the cell by xCT in exchange for cystine transported into the cell by CD98hc(4F2). Glutamate and cystine serve as precursors for glutathione (GSH) synthesis, which protects the cell from oxidant damage by reactive oxygen species (ROS). Figure reproduced under the Creative Commons Attribution 4.0 International License 4.0 from G. J. Yoshida,  J. Exp. Clin. Cancer Res., (2015) 34, 111.



V-9302 (Figure 2) is a lead compound that emerged from a series of glutamine transporter antagonists. The investigators showed that in HEK293 cells expressing ASCT2, V-9302 selectively blocked the transport of glutamine and leucine (both ASCT2 substrates) while having no effect on the transport of amino acids that are not ASCT2 substrates. They further demonstrated that V-9302 interacts directly with ASCT2 using the drug affinity responsive target stability approach, which measures the ability of a compound to protect the target protein from proteolysis. The same approach demonstrated no interaction between V-9302 and ASCT1, a close paralog of ASCT2.



FIGURE 2. Structure of V-9302.



The researchers explored the binding interaction between V-9302 and ASCT2 in silico using a homology model of the protein (Figure 3). They found that the compound binds in the transporter's orthosteric binding pocket and likely forms significant interactions with the side chains of Ser-353 and Asp-464. This approach also confirmed that V-9302 does not bind favorably to the orthosteric pocket of the L-type amino acid transporter 1 (LAT1), which also transports glutamine.



FIGURE 3. (a) Homology model of the ASCT2 trimer with V-9302 shown bound in the orthosteric binding site. (b) Close up of the binding site of V-9302. (c) Overlay of V-9302 and glutamine docked into the ASCT2 binding site. Figure reproduced by permission from Springer Nature from M. L. Schulte, et al. Nat. Medicine, (2018) published online January 15, DOI: 10.1038/nm.4464. Copyright 2018, Springer Nature.


An in vitro efficacy screen of 29 colon, breast, or lung cancer cell lines revealed that a 48 hour exposure to 25 μM V-9302 reduced the viability of >50% of the cell lines by at least 20%. Some cell lines exhibited high susceptibility to the suppressive effects of V-9302 (EC50 = 9 – 15 μM). Vulnerability to V-9302-mediated toxicity did not correlate with ASCT2 expression, but it did correlate with sensitivity to glutamine deprivation.


When they used shRNA to knock down ASCT2 expression in HCC1806 breast cancer cells, the investigators observed similar effects on the cells as those that were obtained upon exposure to V-9302. These included decreased levels of phospho-S6 (a marker of protein synthesis) and phospho-ERK (a marker of MAP kinase pathway signaling). Further studies using these cells and HT29 colorectal carcinoma cells revealed that V-9302 causes a conversion of reduced to oxidized glutathione and increased levels of reactive oxygen species (ROS). Elevated levels of LC3B, a marker of autophagy also characterized V-9302-treated cells. These effects were not observed in cells treated with CB-839, suggesting that V-9302 is the more effective of the two compounds in blocking the many metabolic effects of intracellular glutamine.

In vivo studies in mice demonstrated that a single dose of V-9302 had no effect on plasma glucose levels but caused a slight increase in the level of glutamine. Chronic treatment (21 days) with the compound similarly did not impact glucose but led to a slight decrease in glutamine levels in the blood. Positron emission tomography (PET) imaging of mice bearing an HCC1806 xenograft revealed that a single dose of V-9302 suppressed uptake of [4-18F]fluoroglutamine by the tumor but not surrounding muscle tissue (Figure 4). Dosing of mice bearing HCT-116 or HT29 xenografts with V-9302 daily for 21 days slowed tumor growth as did a 10-day treatment of mice bearing HCC1806 and COLO205 xenografts. A patient-derived xenograft was also sensitive to V-9302-mediated growth suppression (Figure 5). The absence of weight loss or liver pathology in the V-9302-treated mice suggested that the compound's toxicity is tumor-selective.



FIGURE 4. [4-18F]fluoroglutamine PET imaging of a mice bearing an HCC1806 xenograft (arrowhead) before (left) or after (right) a single does of V-9302. Figure reproduced by permission from Springer Nature from M. L. Schulte, et al. Nat. Medicine, (2018) published online January 15, DOI: 10.1038/nm.4464. Copyright 2018, Springer Nature.




FIGURE 5. Growth suppression of xenografts derived from a primary tumor in mice treated with V-9302 (bottom) as compared to vehicle controls (top). Figure reproduced by permission from Springer Nature from M. L. Schulte, et al. Nat. Medicine, (2018) published online January 15, DOI: 10.1038/nm.4464. Copyright 2018, Springer Nature.



To better assess the effects of V-9302 on cancer cells, the investigators conducted a metabolomics study of HT29 xenografts following 21 days of treatment. They identified 782 metabolites that mapped to 50 distinct pathways. Compared to xenografts from vehicle-treated mice, those exposed to V-9302 exhibited significant changes in 239 metabolites mapping to 7 pathways. The affected pathways were directly related to ASCT2 transport, glutamine metabolism, carbohydrate metabolism, glutathione metabolism, and membrane biosynthesis and integrity.

The results support the hypothesis that blockade of ASCT2 impacts all aspects of glutamine metabolism (Figure 6) and is therefore a promising approach for the treatment of glutamine-dependent cancers. They also demonstrate the effectiveness of V-9302 as a probe compound for exploration of this pathway and as a lead compound for the discovery of new drugs targeting ASCT2. We look forward to further developments in this novel approach to new therapies for cancer.



FIGURE 6. Effects of ASCT2 inhibition in glutamine-dependent cancer cells. (Left) ASCT2 transports glutamine into the cell, promoting metabolism, protein synthesis, and proliferation while suppressing autophagy, apoptosis, and cellular stress. Blockade of ASCT2 by V-9302 (V) prevents glutamine transport, reversing the beneficial effects of glutamine. The result is loss of viability and/or death by apoptosis.




View Nature Medicine article: Pharmacological blockade of ASCT2-dependent glutamine transport leads to antitumor efficacy in preclinical models








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