Abstract 2015: Interplay of the pH regulator, carbonic anhydrase IX and the glutamine transporter, ASCT2 in hypoxic tumor microenvironment

Solid tumors are characterized by poor vasculature, thereby causing insufficient oxygen and nutrient supply within the tumor. To thrive in such conditions, hypoxic tumor cells stimulate the expression of nutrient transporters to support cell metabolism and pH regulators to buffer the intracellular pH changes caused by metabolic by-products. Carbonic Anhydrase IX (CAIX) is a membrane-bound enzyme that plays a vital role in the pH regulation of hypoxic tumor cells. CAIX is highly expressed in several solid tumors, and is an indicator of poor prognosis and response to therapy. Although the importance of CAIX9s role in mediating tumor progression is well-known, the underlying mechanism remains unclear. To identify the interactors of CAIX in the hypoxic tumor microenvironment, we performed a proteomic analysis using proximity dependent biotin identification (BioID) and found the Alanaine, Serine, Cysteine transporter (ASCT2) to be a high confidence interactor of CAIX from this analysis. ASCT2, which predominantly acts as a glutamine transporter is upregulated in several cancers and is shown to support tumor growth by facilitating increased glutamine uptake. In this study, using a panel of solid tumor cell lines, we validate the interaction of CAIX with ASCT2 in the hypoxic microenvironment. Functionally, loss of CAIX causes an increased glutamine uptake via ASCT2 and an increased capacity of cells to utilize glutamine for mitochondrial respiration. Concomitantly, ROS and glutathione (GSH) levels also increase upon CAIX inhibition. Cytotoxicity studies show a robust cell death in the absence of glutamine, following treatment with the CAIX/XII small molecule inhibitor SLC-0111. A similar effect on cell death is observed by blocking glutamine metabolism using inhibitors such as V-9302 (ASCT2 inhibitor) or CB-839 (Glutaminase-1 inhibitor) with SLC-0111. Furthermore, blocking GSH synthesis using BSO (Glutamylcysteine synthatase inhibitor) with SLC-0111 causes significant cell death. These results suggest that CAIX depletion causes increased cellular stress and to overcome this, cells increase glutamine uptake in part for GSH synthesis. In summary, we show that CAIX and ASCT2 coordinate their functions in hypoxic cancer cells to support tumor growth, and co-targeting them could be beneficial in treating solid tumors. Citation Format: Geetha Venkateswaran, Shawn Chafe, Shannon Awrey, Oksana Nemirovsky, Shoukat Dedhar. Interplay of the pH regulator, carbonic anhydrase IX and the glutamine transporter, ASCT2 in hypoxic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2015.