Seth J. Parker

The sodium-dependent neutral amino acid transporter 2 (SNAT2) is a plasma membrane transporter that facilitates the uptake of small, aliphatic amino acids. Restricting SNAT2-mediated alanine uptake may be a promising therapeutic strategy for various diseases, including pancreatic ductal adenocarcinoma (PDAC). The post-translational mechanisms regulating the distribution of SNAT2 to intracellular membranes and its turnover remain uncharacterized in the context of PDAC and may be useful to devise future strategies to inhibit SNAT2 function. Human SNAT2 contains three conserved extracellular N-linked glycosylation moieties at N254, N258, and N274. A screen of SNAT2 expression across several human and mouse PDAC cell lines revealed that plasma membrane SNAT2 is exclusively modified by N-linked glycosylation. Preventing SNAT2 N-glycosylation using pharmacological inhibitors or mutagenesis abolished its glycosylation and attenuated its plasma membrane, but not lysosomal, localization. Further, overexpressing glycosylation-deficient SNAT2 in knockout cell lines fails to restore cell proliferative capacity relative to wild-type SNAT2, despite partially rescuing the metabolomic defect associated with SNAT2-deficiency. Using an inducible expression system, we also demonstrate that N-linked glycosylation-deficient SNAT2 mutants exhibit altered degradation kinetics but use similar pathways as wild-type SNAT2 to coordinate its turnover. Our results highlight the importance of N-linked glycosylation for regulating the stability and cell surface expression of nascent SNAT2 in PDAC cells.

Supplementary Figure S2 shows that CAIX catalytic activity and the CAIX intracellular domain regulate Gln uptake by hypoxic cancer cells.

Supplementary Figure S1 shows data describing the characterization of the interaction between SLC1A5 and CAIX in cancer cells.

Supplementary Figure S3 shows heatmaps showing the drug combination responses for cytotoxicity of cancer cells in hypoxia induced by SLC-0111 in combination with inhibition of Gln metabolism.

Supplementary Figure S5 shows heatmaps demonstrating that combined inhibition of CAIX activity and GCLC or RSL3 synergize to increase cytotoxicity of hypoxic cancer cells.

Supplementary Figure S6 shows IHC staining for 4-HNE in SUM159PT tumors, demonstrating that co-targeting CAIX/XII activity and GSH synthesis increases lipid peroxidation in vivo.

Supplementary Table S1 lists the shRNA sequences used to knock down gene expression of SLC1A5, GLS1 and GCLC in cancer cells.

Supplementary Figure S4 shows that the combined loss of CAIX activity and the GSH synthesis axis of Gln metabolism increases cytotoxicity of hypoxic cancer cells.

The ability of tumor cells to alter their metabolism to support survival and growth presents a challenge to effectively treat cancers. Carbonic anhydrase IX (CAIX) is a hypoxia-induced, metabolic enzyme that plays a crucial role in pH regulation in tumor cells. Recently, through a synthetic lethal screen, we identified CAIX to play an important role in redox homeostasis. In this study, we show that CAIX interacts with the glutamine (Gln) transporter, solute carrier family 1 member 5 (SLC1A5), and coordinately functions to maintain redox homeostasis through the glutathione/glutathione peroxidase 4 (GSH/GPX4) axis. Inhibition of CAIX increases Gln uptake by SLC1A5 and concomitantly increases GSH levels. The combined inhibition of CAIX activity and Gln metabolism or the GSH/GPX4 axis results in an increase in lipid peroxidation and induces ferroptosis, both in vitro and in vivo. Thus, this study demonstrates cotargeting of CAIX and Gln metabolism as a potential strategy to induce ferroptosis in tumor cells.