Abstract 3972: Novel renal cell carcinoma therapy targeting glutaminolysis
Authors: Muto Yoshinari , Takeuchi Akihito , Zennami Kenji , Sugihara Eiji , Shiroki Ryoichi , Saya Hideyuki , Sumitomo Makoto 
Published: April 2023

Abstract
Objective: Metabolic reprogramming of cancer cells contributes to tumor development and provides a target for cancer therapy ASCT2 is a glutamine transporter on the plasma membrane. Glutamine is converted into the cell to glutamate by glutaminase (GLS) and utilized by the TCA cycle. This glutaminolysis has been implicated in tumor progression in various malignancies, including renal cell carcinoma. In the present study, we investigated whether the unique strategy that effectively targets glutamine addiction would be effective as a treatment for RCC.
Methods: Tumor tissue (T) and normal renal tissue (N) from surgical specimens of 66 patients with RCC were collected, and metabolomic analysis using liquid chromatography-mass spectrometry (LC/MS) to calculate the T/N ratio of Gln and Glu, respectively, were performed. In addition, immunohistological (IHC) staining of paraffin-embedded sections of 106 metastatic RCC cases was used to compare GLS and ASCT2 expression in tumor and normal tissue. In vitro, cell viability of the human RCC cell lines 786-O and 769-P was assessed by WST-8 assays. The protein expression was detected by Western blotting. Intracellular concentrations of glutamine and glutamate were analyzed using LC/MS. V9302 was used as the specific ASCT2 inhibitor, and CB839 as the specific GLS inhibitor. As the xenograft model in vivo, athymic nude mice were injected subcutaneously with 786-O cells and given various doses of V9302 and CB839 intraperitoneally.
Results: LC/MS assays using surgical specimens showed a notable increase in glutamine concentration in tumor tissue. IHC assays confirmed high expression of ASCT2 in tumor tissue, and patients with high expression of ASCT2 had a poorer prognosis than those with low expression. Treatment of V9302 with 786-O and 769-P resulted in a limited decrease in cell viability. LC/MS analysis confirmed that V9302 treatment reduced glutathione metabolites. V9302 administration was shown to increase intracellular glutamine levels, presumably by compensatory mechanisms, whereas the combination of V9302 and CB839 significantly decreased glutamine levels in 786-O cells. Western blot analysis showed that the expression levels of ASCT2 and GLS1 decreased after the combination therapy. The xenograft study confirmed that the combination of V9302 and CB839 significantly inhibited 786-O tumor growth compared with controls within three weeks after treatment (p &lt;0.001).
Conclusion: It has been shown that ASCT2 expression is a marker of poor prognosis in RCC and that suppression of ASCT2 has an antitumor effect by affecting glutathione metabolism. On the other hand, the administration of V9302 raised the issue that a compensatory mechanism increases intracellular glutamine levels. Our results suggest that a novel combination strategy using CB839 in addition to V9302 could overcome the tolerance for the monotherapy and effectively target glutamine addiction in RCC.
Citation Format: Yoshinari Muto, Akihito Takeuchi, Kenji Zennami, Eiji Sugihara, Ryoichi Shiroki, Hideyuki Saya, Makoto Sumitomo. Novel renal cell carcinoma therapy targeting glutaminolysis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3972.