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A small molecule selectively targeting the cell-surface glutamine transporter ASCT2 disrupts glutamine signaling and metabolism. This compound displays low toxicity and strong antitumor activity in preclinical in vitro and in vivo models, thus holding promise as a treatment for glutamine-dependent tumors in a clinical setting. The unique metabolic demands of cancer cells underscore potentially fruitful opportunities for drug discovery in the era of precision medicine. However, therapeutic targeting of cancer metabolism has led to surprisingly few new drugs to date. The neutral amino acid glutamine serves as a key intermediate in numerous metabolic processes leveraged by cancer cells, including biosynthesis, cell signaling, and oxidative protection. Herein we report the preclinical development of V-9302, a competitive small molecule antagonist of transmembrane glutamine flux that selectively and potently targets the amino acid transporter ASCT2. Pharmacological blockade of ASCT2 with V-9302 resulted in attenuated cancer cell growth and proliferation, increased cell death, and increased oxidative stress, which collectively contributed to antitumor responses in vitro and in vivo . This is the first study, to our knowledge, to demonstrate the utility of a pharmacological inhibitor of glutamine transport in oncology, representing a new class of targeted therapy and laying a framework for paradigm-shifting therapies targeting cancer cell metabolism.

Ferroptosis is a regulated form of cell death driven by small molecules or conditions that induce lipid-based reactive oxygen species (ROS) accumulation. This form of iron-dependent cell death is morphologically and genetically distinct from apoptosis, necroptosis, and autophagy. miRNAs are known to play crucial roles in diverse fundamental biological processes. However, to date no study has reported miRNA-mediated regulation of ferroptosis. Here we show that miR-137 negatively regulates ferroptosis by directly targeting glutamine transporter SLC1A5 in melanoma cells. Ectopic expression of miR-137 suppressed SLC1A5, resulting in decreased glutamine uptake and malondialdehyde (MDA) accumulation. Meanwhile, antagomir-mediated inactivation of endogenous miR-137 increased the sensitivity of melanoma cells to erastin- and RSL3-induced ferroptosis. Importantly, knockdown of miR-137 increased the antitumor activity of erastin by enhancing ferroptosis both in vitro and in vivo. Collectively, these data indicate that miR-137 plays a novel and indispensable role in ferroptosis by inhibiting glutaminolysis and suggest a potential therapeutic approach for melanoma.

Human ASCT2 belongs to the SLC1 family of secondary transporters and is specific for the transport of small neutral amino acids. ASCT2 is upregulated in cancer cells and serves as the receptor for many retroviruses; hence, it has importance as a potential drug target. Here we used single-particle cryo-EM to determine a structure of the functional and unmodified human ASCT2 at 3.85-Å resolution. ASCT2 forms a homotrimeric complex in which each subunit contains a transport and a scaffold domain. Prominent extracellular extensions on the scaffold domain form the predicted docking site for retroviruses. Relative to structures of other SLC1 members, ASCT2 is in the most extreme inward-oriented state, with the transport domain largely detached from the central scaffold domain on the cytoplasmic side. This domain detachment may be required for substrate binding and release on the intracellular side of the membrane.

Retinoblastoma (Rb) protein is a tumor suppressor that is dysregulated in a majority of human cancers. Rb functions to inhibit cell cycle progression in part by directly disabling the E2F family of cell cycle-promoting transcription factors. Because the de novo synthesis of multiple glutamine-derived anabolic precursors is required for cell cycle progression, we hypothesized that Rb also may directly regulate proteins involved in glutamine metabolism. We examined glutamine metabolism in mouse embryonic fibroblasts (MEFs) isolated from mice that have triple knock-outs (TKO) of all three Rb family members (Rb-1, Rbl1 and Rbl2) and found that loss of global Rb function caused a marked increase in 13 C-glutamine uptake and incorporation into glutamate and tricarboxylic acid cycle (TCA) intermediates in part via upregulated expression of the glutamine transporter ASCT2 and the activity of glutaminase 1 (GLS1). The Rb-controlled transcription factor E2F-3 altered glutamine uptake by direct regulation of ASCT2 mRNA and protein expression, and E2F-3 was observed to associate with the ASCT2 promoter. We next examined the functional consequences of the observed increase in glutamine uptake and utilization and found that glutamine exposure potently increased oxygen consumption, whereas glutamine deprivation selectively decreased ATP concentration in the Rb TKO MEFs but not the wild-type (WT) MEFs. In addition, TKO MEFs exhibited elevated production of glutathione from exogenous glutamine and had increased expression of gamma-glutamylcysteine ligase relative to WT MEFs. Importantly, this metabolic shift towards glutamine utilization was required for the proliferation of Rb TKO MEFs but not for the proliferation of the WT MEFs. Last, addition of the TCA cycle intermediate α-ketoglutarate to the Rb TKO MEFs reversed the inhibitory effects of glutamine deprivation on ATP, GSH levels and viability. Taken together, these studies demonstrate that the Rb/E2F cascade directly regulates a major energetic and anabolic pathway that is required for neoplastic growth.

Targeting the function of membrane transporters in cancer stemlike cells is a potential new therapeutic approach. Cystine‐glutamate antiporter xCT expressed in CD44 variant (CD44v)‐expressing cancer cells contributes to the resistance to oxidative stress as well as cancer therapy through promoting glutathione (GSH)‐mediated antioxidant defense. Amino acid transport by xCT might, thus, be a promising target for cancer treatment, whereas the determination factors for cancer cell sensitivity to xCT‐targeted therapy remain unclear. Here, we demonstrate that high expression of xCT and glutamine transporter ASCT2 is correlated with undifferentiated status and diminished along with cell differentiation in head and neck squamous cell carcinoma (HNSCC). The cytotoxicity of the xCT inhibitor sulfasalazine relies on ASCT2‐dependent glutamine uptake and glutamate dehydrogenase (GLUD)‐mediated α‐ketoglutarate (α‐KG) production. Metabolome analysis revealed that sulfasalazine treatment triggers the increase of glutamate‐derived tricarboxylic acid cycle intermediate α‐KG, in addition to the decrease of cysteine and GSH content. Furthermore, ablation of GLUD markedly reduced the sulfasalazine cytotoxicity in CD44v‐expressing stemlike HNSCC cells. Thus, xCT inhibition by sulfasalazine leads to the impairment of GSH synthesis and enhancement of mitochondrial metabolism, leading to reactive oxygen species (ROS) generation and, thereby, triggers oxidative damage. Our findings establish a rationale for the use of glutamine metabolism (glutaminolysis)‐related genes, including ASCT2 and GLUD, as biomarkers to predict the efficacy of xCT‐targeted therapy for heterogeneous HNSCC tumors. Competition exists between xCT‐mediated cystine uptake and GLUD‐mediated alpha‐KG generation.

Background Mitochondrial dynamics play a fundamental role in determining stem cell fate. However, the underlying mechanisms of mitochondrial dynamics in the stemness acquisition of cancer cells are incompletely understood. Methods Metabolomic profiling of cells were analyzed by MS/MS. The genomic distribution of H3K27me3 was measured by CUT&Tag. Oral squamous cell carcinoma (OSCC) cells depended on glucose or glutamine fueling TCA cycle were monitored by 13C-isotope tracing. Organoids and tumors from patients and mice were treated with DRP1 inhibitors mdivi-1, ferroptosis inducer erastin, or combination with mdivi-1 and erastin to evaluate treatment effects. Results Mitochondria of OSCC stem cells own fragment mitochondrial network and DRP1 is required for maintenance of their globular morphology. Imbalanced mitochondrial dynamics induced by DRP1 knockdown suppressed stemness of OSCC cells. Elongated mitochondria increased α-ketoglutarate levels and enhanced glutaminolysis to fuel the TCA cycle by increasing glutamine transporter ASCT2 expression. α-KG promoted the demethylation of histone H3K27me3, resulting in downregulation of SNAI2 associated with stemness and EMT. Significantly, suppressing DRP1 enhanced the anticancer effects of ferroptosis. Conclusion Our study reveals a novel mechanism underlying mitochondrial dynamics mediated cancer stemness acquisition and highlights the therapeutic potential of mitochondria elongation to increase the susceptibility of cancer cells to ferroptosis.

The mitochondrial glutamine transporter SLC1A5_var plays a central role in the metabolic reprogramming of cancer cells by facilitating glutamine import into mitochondria for energy production and redox homeostasis. Despite its critical function, the development of effective and selective inhibitors targeting SLC1A5_var has remained a significant challenge. Here, we introduce iMQT_020, a selective allosteric inhibitor identified through structure-based screening. iMQT_020 disrupts the trimeric assembly of SLC1A5_var, causing metabolic crisis in cancer cells and selectively suppressing their growth. Mechanistically, iMQT_020 reduces glutamine anaplerosis and oxidative phosphorylation, resulting in a broad disruption of cancer metabolism. Additionally, iMQT_020 treatment epigenetically upregulates PD-L1 expression, enhancing the efficacy of combination therapies with anti-PD-L1 immune checkpoint inhibitors. These findings highlight the therapeutic potential of targeting SLC1A5_var as a critical metabolic vulnerability in cancer and demonstrate that targeting allosteric interprotomer interactions is a novel and promising therapeutic strategy for cancer treatment. Glutamine addiction is a hallmark of many cancers. iMQT_020, a first-in-class allosteric inhibitor of the mitochondrial glutamine transporter SLC1A5_var, disrupts glutamine-dependent mitochondrial metabolism, selectively killing cancer cells and enhancing immune checkpoint inhibitor efficacy.

In-feed antibiotics have been commonly used to promote the growth performance of piglets. The antibiotics can increase protein utilization, but the underlying mechanism is largely unknown. The present study investigated the effects of in-feed antibiotics on intestinal AA transporters and receptors to test the hypothesis that the alteration of circulating AA profiles may be concomitant with the change of intestinal AA transporters and receptors. Sixteen litters of piglets at day 7 started to receive creep feed with (Antibiotic) or without (Control) antibiotic. Piglets were weaned at day 23 after birth, and fed the same diets until day 42. In-feed antibiotics did not affect the BW of 23-day-old ( P  = 0.248), or 42-day-old piglets ( P  = 0.089), but increased the weight gain to feed ratio from day 23 to 42 ( P  = 0.020). At day 42 after birth, antibiotic treatment increased the concentrations of most AAs in serum ( P  < 0.05), and decreased the concentrations of most AAs in jejunal and ileal digesta. Antibiotics upregulated ( P  < 0.05) the mRNA expression levels for jejunal AAs transporters (CAT1, EAAC1, ASCT2, y + LAT1), peptide transporters (PepT1), and Na + –K + –ATPase (ATP1A1), and ileal AA transporters (ASCT2, y + LAT1, b 0,+ AT, and B 0 AT1), and ATP1A1. The antibiotics also upregulated the mRNA expression of jejunal AAs receptors T1R3 and CaSR, and ileal T1R3. Protein expression levels for jejunal AA transporters (EAAC1, b 0,+ AT, and ASCT2) and PepT1 were also upregulated. Correlation analysis revealed that the alterations of AA profiles in serum after the in-feed antibiotics were correlated with the upregulations of mRNA expression levels for key AA transporters and receptors in the small intestine. In conclusion, the in-feed antibiotics increased serum level of most AAs and decreased most AAs in the small intestine. These changes correlated with the upregulations of mRNA expression levels for key AA transporters and receptors in the small intestine. The findings provide further insights into the mechanism of in-feed antibiotics, which may provide new framework for designing alternatives to antibiotics in animal feed in the future.

D-serine is a physiologic coagonist of NMDA receptors, but little is known about the regulation of its synthesis and synaptic turnover. The amino acid exchangers ASCT1 (Slc1a4) and ASCT2 (Slc1a5) are candidates for regulating D-serine levels. Using ASCT1 and ASCT2 KO mice, we report that ASCT1, rather than ASCT2, is a physiologic regulator of D-serine metabolism. ASCT1 is a major D-serine uptake system in astrocytes and can also export L-serine via heteroexchange, supplying neurons with the substrate for D-serine synthesis. ASCT1-KO mice display lower levels of brain D-serine along with higher levels of L-alanine, L-threonine, and glycine. Deletion of ASCT1 was associated with neurodevelopmental alterations including lower hippocampal and striatal volumes and changes in the expression of neurodevelopmental-relevant genes. Furthermore, ASCT1-KO mice exhibited deficits in motor function, spatial learning, and affective behavior, along with changes in the relative contributions of D-serine vs. glycine in mediating NMDA receptor activity. In vivo microdialysis demonstrated lower levels of extracellular D-serine in ASCT1-KO mice, confirming altered D-serine metabolism. These alterations are reminiscent of some of the neurodevelopmental phenotypes exhibited by patients with ASCT1 mutations. ASCT1-KO mice provide a useful model for potential therapeutic interventions aimed at correcting the metabolic impairments in patients with ASCT1 mutations.

Pancreatic ductal adenocarcinoma (PDAC) shows great cellular heterogeneity, with pronounced epithelial and mesenchymal cancer cell populations. However, the cellular hierarchy underlying PDAC cell diversity is unknown. Here we identify the tetraspanin CD9 as a marker of PDAC tumour-initiating cells. CD9 high cells had increased organoid formation capability, and generated tumour grafts in vivo at limiting dilutions. Tumours initiated from CD9 high cells recapitulated the cellular heterogeneity of primary PDAC, whereas CD9 low cells produced only duct-like epithelial progeny. CD9 knockdown decreased the growth of PDAC organoids, and heterozygous CD9 deletion in Pdx1-Cre; LSL-KRas G12D ; p53 F/F mice prolonged overall survival. Mechanistically, CD9 promoted the plasma membrane localization of the glutamine transporter ASCT2, enhancing glutamine uptake in PDAC cells. Thus, our study identifies a PDAC subpopulation capable of initiating PDAC and giving rise to PDAC heterogeneity, suggesting that the cellular diversity of PDAC is generated by PDAC stem cell differentiation. Wang et al. demonstrate that CD9 marks tumour-initiating cells in pancreatic ductal adenocarcinoma (PDAC), and enhances glutamine uptake to promote tumour development in vivo that recapitulates the cellular heterogeneity of primary PDAC.