Introduction: The glutamine-dependent nature of cancer cells could be used as a new strategy for treating cancers that do not have drug-driver genes, such as renal cell carcinoma (RCC). We recently reported that Alanine-serine-cysteine transporter 2 (ASCT2) is associated with metastatic progression and survival in RCC. However, the administration of V9302, a specific inhibitor of ASCT2, raised the issue that intracellular glutamine levels are increased by a compensatory mechanism (presented at 2021 AUA annual meeting). In the present study, we investigated whether the combination strategy that effectively targets glutamine addiction would be effective as a treatment for RCC. Methods: The human RCC cell lines, 786-O and 769-P were used. The protein expression was detected by Western blotting. Cell viability was assessed using WST-8 assay. Intracellular concentrations of glutamine and glutamate were analyzed using liquid chromatography-mass spectrometry (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 showed that intracellular glutamine levels in the RCC cell line increased after 48 hours of treatment with V9302 alone, but that glutamine levels were significantly reduced by the combination of V9302 and CB839. Western blot analysis showed that the expression levels of ASCT2 and GLS1 were both decreased after the combination therapy. The tumor xenograft study confirmed that the combination of V9302 (30 mg/kg, intraperitoneal injection) and CB839 (20 mg/kg, intraperitoneal injection) significantly inhibited 786-O tumor growth compared with controls within 3 weeks after treatment (p <0.001). Conclusions: Our results suggest that V9302 monotherapy is insufficient for the treatment of RCC but 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. SOURCE OF Funding: None
