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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.

The Solute Carrier 1A (SLC1A) family includes two major mammalian transport systems—the alanine serine cysteine transporters (ASCT1-2) and the human glutamate transporters otherwise known as the excitatory amino acid transporters (EAAT1-5). The EAATs play a critical role in maintaining low synaptic concentrations of the major excitatory neurotransmitter glutamate, and hence they have been widely researched over a number of years. More recently, the neutral amino acid exchanger, ASCT2 has garnered attention for its important role in cancer biology and potential as a molecular target for cancer therapy. The nature of this role is still being explored, and several classes of ASCT2 inhibitors have been developed. However none have reached sufficient potency or selectivity for clinical use. Despite their distinct functions in biology, the members of the SLC1A family display structural and functional similarity. Since 2004, available structures of the archaeal homologues Glt Ph and Glt Tk have elucidated mechanisms of transport and inhibition common to the family. The recent determination of EAAT1 and ASCT2 structures may be of assistance in future efforts to design efficacious ASCT2 inhibitors. This review will focus on ASCT2, the present state of knowledge on its roles in tumour biology, and how structural biology is being used to progress the development of inhibitors.

Muscle regeneration is sustained by infiltrating macrophages and the consequent activation of satellite cells 1 – 4 . Macrophages and satellite cells communicate in different ways 1 – 5 , but their metabolic interplay has not been investigated. Here we show, in a mouse model, that muscle injuries and ageing are characterized by intra-tissue restrictions of glutamine. Low levels of glutamine endow macrophages with the metabolic ability to secrete glutamine via enhanced glutamine synthetase (GS) activity, at the expense of glutamine oxidation mediated by glutamate dehydrogenase 1 (GLUD1). Glud1 -knockout macrophages display constitutively high GS activity, which prevents glutamine shortages. The uptake of macrophage-derived glutamine by satellite cells through the glutamine transporter SLC1A5 activates mTOR and promotes the proliferation and differentiation of satellite cells. Consequently, macrophage-specific deletion or pharmacological inhibition of GLUD1 improves muscle regeneration and functional recovery in response to acute injury, ischaemia or ageing. Conversely, SLC1A5 blockade in satellite cells or GS inactivation in macrophages negatively affects satellite cell functions and muscle regeneration. These results highlight the metabolic crosstalk between satellite cells and macrophages, in which macrophage-derived glutamine sustains the functions of satellite cells. Thus, the targeting of GLUD1 may offer therapeutic opportunities for the regeneration of injured or aged muscles. Mouse models of muscle injuries and ageing characterized by low levels of intra-tissue glutamine are ameliorated by macrophage-specific deletion or systemic pharmacological inhibition of glutamate dehydrogenase 1, which results in constitutively high activity of glutamine synthetase.

Oncogenic KRAS mutations and inactivation of the APC tumor suppressor co-occur in colorectal cancer (CRC). Despite efforts to target mutant KRAS directly, most therapeutic approaches focus on downstream pathways, albeit with limited efficacy. Moreover, mutant KRAS alters the basal metabolism of cancer cells, increasing glutamine utilization to support proliferation. We show that concomitant mutation of Apc and Kras in the mouse intestinal epithelium profoundly rewires metabolism, increasing glutamine consumption. Furthermore, SLC7A5, a glutamine antiporter, is critical for colorectal tumorigenesis in models of both early- and late-stage metastatic disease. Mechanistically, SLC7A5 maintains intracellular amino acid levels following KRAS activation through transcriptional and metabolic reprogramming. This supports the increased demand for bulk protein synthesis that underpins the enhanced proliferation of KRAS-mutant cells. Moreover, targeting protein synthesis, via inhibition of the mTORC1 regulator, together with Slc7a5 deletion abrogates the growth of established Kras -mutant tumors. Together, these data suggest SLC7A5 as an attractive target for therapy-resistant KRAS-mutant CRC. Colorectal tumors with mutated KRAS and APC require the amino acid transporter SLC7A5 to drive tumorigenesis. Mechanistically, SLC7A5 drives transcriptional and metabolic reprogramming by maintaining intracellular amino acid levels, leading to enhanced protein synthesis.

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.

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.

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.

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.

ASCT2 (SLC1A5) is a sodium-dependent neutral amino acid transporter that controls amino acid homeostasis in peripheral tissues. In cancer, ASCT2 is up-regulated where it modulates intracellular glutamine levels, fueling cell proliferation. Nutrient deprivation via ASCT2 inhibition provides a potential strategy for cancer therapy. Here, we rationally designed stereospecific inhibitors exploiting specific subpockets in the substrate binding site using computational modeling and cryo-electron microscopy (cryo-EM). The final structures combined with molecular dynamics simulations reveal multiple pharmacologically relevant conformations in the ASCT2 binding site as well as a previously unknown mechanism of stereospecific inhibition. Furthermore, this integrated analysis guided the design of a series of unique ASCT2 inhibitors. Our results provide a framework for future development of cancer therapeutics targeting nutrient transport via ASCT2, as well as demonstrate the utility of combining computational modeling and cryo-EM for solute carrier ligand discovery.