xCT Inhibition Research Articles

A search for food extracts inhibiting ferroptosis using immortalized mouse embryonic fibroblasts derived from xCT knockout mice.

Depleting glutathione by xCT inhibition induces iron-dependent ferroptotic cell death, which is suppressed by lipophilic antioxidants. We screened food extracts with xCTKO-MEFs, identifying garlic extracts as particularly potent in inhibiting ferroptosis among the food extracts examined in this study. xCTKO-MEFs can serve as a convenient tool for identifying food extracts that are effective in inhibiting ferroptosis.

Abstract 5962: Novel xCT-targeted biologics significantly suppress metastasis, improve survival, and sensitize primary tumors to chemotherapy in TNBC xenograft models

Abstract Background: Triple negative breast cancer (TNBC) is a highly heterogeneous subset of breast cancer, which has the worst prognosis and lacks specifically targeted therapeutic options. The TNBC subtype termed M/MSL is enriched with cells of high mesenchymal state and increased stemness, and is particularly resistant to current therapies1,2. But this cancer cell phenotype is susceptible to ferroptosis, hence targeting these aggressive tumors with ferroptosis inducers such as xCT inhibitors (xCTi) is an attractive alternative. The cystine/glutamate exchanger xCT/SLC7A11 plays a key role in maintaining redox homeostasis in cancer cells. xCT is expressed on subsets of most solid tumors, including stem/progenitor and metastatic tumor cells. xCT inhibition yields a variety of anti-proliferative phenotypes including death by ferroptosis3. xCT knockout mice exhibit normal development and lifespan. Altogether, targeting xCT outside of CNS may potentially have widespread clinical utility in cancer treatment. Results: We present preclinical data demonstrating development of novel, first-in-class xCT-targeted biotherapeutics (AX68c, AX76c) using studies in TNBC cell lines in vitro and in xenograft. We find that these agents have highly favorable pharmacokinetics, with FcRn-dependent serum half-lives greatly exceeding those of known small molecule xCTi. In vitro, AX68c or AX76c reduces glutathione levels in TNBC cells in accord with inhibition of xCT function. Analysis of anti-proliferative effects in a panel of TNBC cell lines revealed particular potency against those of M/MSL phenotype (IC50s 0.3-1uM). We further tested the susceptibility of the mesenchymal phenotype to AX68c and found that EMT induction in epithelial cancer cells significantly increased sensitivity in a ferroptosis-dependent manner. In wildtype and immunodeficient mice, AX68c is well tolerated at steady-state serum concentrations that are 15-300 fold of its in vitro IC50s in tested TNBC cells. In orthotopic MDA-MB-231 xenografts, AX68c treatment significantly reduced tumor metastasis and sensitized primary tumors to cisplatin and paclitaxel. Bulk RNAseq and GSEA analysis on primary tumors revealed treatment associated alterations in key cancer signaling pathways such as TGFβ, INFγ and TNFα/NFκB. In MDA-MB-436 orthotopic xenografts, AX68c alone led to significantly improved survival primarily through abrogating metastasis. Combining AX68c and cisplatin further prolonged survival through concurrent suppression of metastasis and primary tumor growth. Conclusions: We conclude that our anti-xCT biologics may provide novel targeted therapies for M/MSL-subtype TNBC and metastatic tumors with a large mesenchymal component. 1. Viswanathan, et al., Nature, 2017; 2. Wang, et al., npj Precision Oncology, 2021; 3. Koppula, et al., Protein and Cell, 2021 (review). Citation Format: He Gong, Jun Li, Cyrus Becker, Luika Timmerman, Shaheen Sikandar, Yong Huang. Novel xCT-targeted biologics significantly suppress metastasis, improve survival, and sensitize primary tumors to chemotherapy in TNBC xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5962.

The m6A reader YTHDC2 promotes the pathophysiology of temporal lobe epilepsy by modulating SLC7A11-dependent glutamate dysregulation in astrocytes.

Rationale: Epilepsy affects over 70 million people globally, with temporal lobe epilepsy with hippocampal sclerosis (TLE-HS) often progressing to a drug-resistant state. Recent research has highlighted the role of reactive astrocytes and glutamate dysregulation in epilepsy pathophysiology. This study aims to investigate the involvement of astrocytic xCT, a glutamate-cystine antiporter, and its regulation by the m6A reader protein YTHDC2 in TLE-HS. Methods: A pilocarpine-induced epilepsy model in mice was used to study the role of xCT in reactive astrocytes. The expression of xCT and its regulation by YTHDC2 were assessed through various molecular and cellular techniques. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were used to measure mRNA and protein levels of xCT and YTHDC2, respectively; immunofluorescence was utilized to visualize their localization and expression in astrocytes. In vivo glutamate measurements were conducted using microdialysis to monitor extracellular glutamate levels in the hippocampus. RNA immunoprecipitation-qPCR (RIP-qPCR) was performed to investigate the binding of YTHDC2 to SLC7A11 mRNA, while methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) was performed to quantify m6A modifications on SLC7A11 mRNA. A dual-luciferase reporter assay was conducted to assess the effect of m6A modifications on SLC7A11 mRNA translation, and polysome profiling was employed to evaluate the translational efficiency of SLC7A11 mRNA. Inhibition experiments involved shRNA-mediated knockdown of SLC7A11 (commonly known as xCT) and YTHDC2 expression in astrocytes. Video-electroencephalogram (EEG) recordings were used to monitor seizure activity in mice. Results: The xCT transporter in reactive astrocytes significantly contributes to elevated extracellular glutamate levels, enhancing neuronal excitability and seizure activity. Increased xCT expression is influenced by the m6A reader protein YTHDC2, which regulates its expression through m6A methylation. Inhibition of xCT or YTHDC2 in astrocytes reduces glutamate levels and effectively controls seizures in a mouse model. Specifically, mice with SLC7A11- or YTHDC2-knockdown astrocytes showed decreased glutamate concentration in the hippocampus and reduced frequency and duration of epileptic seizures. Conclusions: This study highlights the therapeutic potential of targeting YTHDC2 and xCT in reactive astrocytes to mitigate epilepsy. The findings provide a novel perspective on the mechanisms of glutamate dysregulation and their implications in seizure pathophysiology, suggesting that modulation of YTHDC2 and xCT could be a promising strategy for treating TLE.

A comprehensive overview of recent developments on the mechanisms and pathways of ferroptosis in cancer: the potential implications for therapeutic strategies in ovarian cancer.

Cancer cells adapt to environmental changes and alter their metabolic pathways to promote survival and proliferation. Metabolic reprogramming not only allows tumor cells to maintain a reduction-oxidation balance by rewiring resources for survival, but also causes nutrient addiction or metabolic vulnerability. Ferroptosis is a form of regulated cell death characterized by the iron-dependent accumulation of lipid peroxides. Excess iron in ovarian cancer amplifies free oxidative radicals and drives the Fenton reaction, thereby inducing ferroptosis. However, ovarian cancer is characterized by ferroptosis resistance. Therefore, the induction of ferroptosis is an exciting new targeted therapy for ovarian cancer. In this review, potential metabolic pathways targeting ferroptosis were summarized to promote anticancer effects, and current knowledge and future perspectives on ferroptosis for ovarian cancer therapy were discussed. Two therapeutic strategies were highlighted in this review: directly inducing the ferroptosis pathway and targeting metabolic vulnerabilities that affect ferroptosis. The overexpression of SLC7A11, a cystine/glutamate antiporter SLC7A11 (also known as xCT), is involved in the suppression of ferroptosis. xCT inhibition by ferroptosis inducers (e.g., erastin) can promote cell death when carbon as an energy source of glucose, glutamine, or fatty acids is abundant. On the contrary, xCT regulation has been reported to be highly dependent on the metabolic vulnerability. Drugs that target intrinsic metabolic vulnerabilities (e.g., GLUT1 inhibitors, PDK4 inhibitors, or glutaminase inhibitors) predispose cancer cells to death, which is triggered by decreased nicotinamide adenine dinucleotide phosphate generation or increased reactive oxygen species accumulation. Therefore, therapeutic approaches that either directly inhibit the xCT pathway or target metabolic vulnerabilities may be effective in overcoming ferroptosis resistance. Real-time monitoring of changes in metabolic pathways may aid in selecting personalized treatment modalities. Despite the rapid development of ferroptosis-inducing agents, therapeutic strategies targeting metabolic vulnerability remain in their infancy. Thus, further studies must be conducted to comprehensively understand the precise mechanism linking metabolic rewiring with ferroptosis.

Manganese-Based Redox Homeostasis Disruptor for Inducing Intense Ferroptosis/Apoptosis Through xCT Inhibition And Oxidative Stress Injury.

Intracellular redox homeostasis plays an important role in promoting tumor progression, development and even treatment resistance. To this end, redox balance impairment may become a prospective therapeutic target of cancer. Herein, a manganese-based homeostasis modulator (MHS) is developed for inducing severe reactive oxygen species accumulation and glutathione (GSH) deprivation, where such redox dyshomeostasis brings about dramatic ferroptosis/apoptosis. Tumor-specific degradation of manganese oxide nanocarriers contributes to hypoxia alleviation and loaded cargo release, resulting in apoptosis by augmented sonodynamic therapy and chemodynamic therapy. On the other hand, regional oxygenation significantly downregulates the expression of activating transcription factor 4, which can synergize with the released sulfasalazine to inhibit the downstream cystine antiporter xCT. Biosynthesis of GSH is sufficiently interrupted by the xCT suppression, leading to the reduction of glutathione peroxidase 4 (GPx4) level. The resultant excessive lipid peroxides promote intense ferroptosis to motivate cell death. On this basis, splendid treatment outcome by MHS is substantiated both in vitro and in vivo, thanks to the synergy of antitumor immunity elicitation. Taken together, this paradigm provides an insightful strategy to evoke drastic ferroptosis/apoptosis toward therapeutics and may also expand the eligibility of manganese-derived nanoagents for medical applications.

Mechanism of Cell Death by Combined Treatment with an xCT Inhibitor and Paclitaxel: An Alternative Therapeutic Strategy for Patients with Ovarian Clear Cell Carcinoma.

Ovarian clear cell carcinoma (OCCC) is a rare subtype of epithelial ovarian carcinoma that responds poorly to chemotherapy. Glutathione (GSH) is a primary antioxidant, which protects cells against reactive oxygen species (ROS). High levels of GSH are related to chemotherapeutic resistance. The glutamine/cystine transporter xCT is essential for intracellular GSH synthesis. However, whether xCT inhibition can overcome the resistance to chemotherapeutic agents in OCCC remains unclear. This study demonstrated that combined treatment with paclitaxel (PTX) and the xCT inhibitor sulfasalazine (SAS) significantly enhanced cytotoxicity more than the individual drugs did in OCCC cells. Treatment with PTX and SAS induced apoptosis more effectively than did individual drug treatments in the cells with significant generation of ROS. Moreover, combined treatment with PTX and SAS induced ferroptosis in the cells with low expression of glutathione peroxidase (GPx4), high levels of intracellular iron and significant lipid ROS accumulation. Therefore, our findings provide valuable information that the xCT inhibitor might be a promising therapeutic target for drug-resistant OCCC. The strategy of combined administration of PTX and SAS can potentially be used to treat OCCC and help to develop novel therapeutic methods.

Inhibition of xCT by sulfasalazine alleviates the depression-like behavior of adult male mice subjected to maternal separation stress

Maternal separation (MS) can induce emotional disorders. Our previous study reported that MS resulted in depression-like behavior. In this study, we aimed to explore the role of xCT in depression-like behavior in adult mice subjected to MS stress. Pups were divided into the control group, the control + sulfasalazine (SSZ, 75 mg/kg/day, i.p.) group, the MS group, and the MS+SSZ group. After MS, all pups were raised until PD60. Then, the depression-like behavior was detected by the novelty suppressed feeding (NSF) test, the forced swimming test (FST), and the tail suspension test (TST). The synaptic plasticity was examined by electrophysiological recordings and molecular biotechnology. The data showed that, compared with the control group, the mice in the MS group presented depression-like behavior, impairment of long-term potentiation (LTP), a reduction in the number of astrocytes, and activation of the microglia. Moreover, the expression of xCT was increased in the prefrontal cortex of MS mice, the EAAT2 and the Group Ⅱ metabotropic glutamate receptors (mGluR2/3) were decreased, and the level of pro-inflammatory factors was increased in the prefrontal cortex. After the administration with SSZ, the depression-like behavior and the impairment of LTP were alleviated, the number of astrocytes was increased, and the microglial activation was inhibited. Moreover, the levels of EAAT2 and mGluR2/3 were ameliorated, the over-activation of the microglia was mitigated, and the levels of glutamate and pro-inflammatory factors were decreased. In conclusion, the inhibition of xCT by SSZ could alleviate depression-like behavior partly via modulating the homeostasis of the glutamate system and dampening neuroinflammation.

XCT-mediated glutamate excretion in white adipocytes stimulates interferon-γ production by natural killer cells in obesity

Obesity-mediated hypoxic stress underlies inflammation, including interferon (IFN)-γ production by natural killer (NK) cells in white adipose tissue. However, the effects of obesity on NK cell IFN-γ production remain obscure. Here, we show that hypoxia promotes xCT-mediated glutamate excretion and C-X-C motif chemokine ligand 12 (CXCL12) expression in white adipocytes, resulting in CXCR4+ NK cell recruitment. Interestingly, this spatial proximity between adipocytes and NK cells induces IFN-γ production in NK cells by stimulating metabotropic glutamate receptor 5 (mGluR5). IFN-γ then triggers inflammatory activation of macrophages and augments xCT and CXCL12 expression in adipocytes, forming a bidirectional pathway. Genetic or pharmacological inhibition of xCT, mGluR5, or IFN-γ receptor in adipocytes or NK cells alleviates obesity-related metabolic disorders in mice. Consistently, patients with obesity showed elevated levels of glutamate/mGluR5 and CXCL12/CXCR4 axes, suggesting that a bidirectional pathway between adipocytes and NK cells could be a viable therapeutic target in obesity-related metabolic disorders.

Repurposing Sulfasalazine as a Radiosensitizer in Hypoxic Human Colorectal Cancer.

xCT overexpression in cancer cells has been linked to tumor growth, metastasis and treatment resistance. Sulfasalazine (SSZ), an FDA-approved drug for the treatment of rheumatoid sarthritis, and inflammatory bowel diseases, has anticancer properties via inhibition of xCT, leading to the disruption of redox homeostasis. Since reactive oxygen species (ROS) are pivotal for the efficacy of radiotherapy (RT), elevated levels of ROS are associated with improved RT outcomes. In this study, the influence of SSZ treatment on the radiosensitivity of human colorectal cancer (CRC) cells was investigated. Our principal finding in human HCT116 and DLD-1 cells was that SSZ enhances the radiosensitivity of hypoxic CRC cells but does not alter the intrinsic radiosensitivity. The radiosensitizing effect was attributed to the depletion of glutathione and thioredoxin reductase levels. In turn, the reduction leads to excessive levels of ROS, increased DNA damage, and ferroptosis induction. Confirmation of these findings was performed in 3D models and in DLD-1 xenografts. Taken together, this study is a stepping stone for applying SSZ as a radiosensitizer in the clinic and confirms that xCT in cancer cells is a valid radiobiological target.

Immunotherapy against the Cystine/Glutamate Antiporter xCT Improves the Efficacy of APR-246 in Preclinical Breast Cancer Models.

Breast cancer is the most frequent cancer in women. Despite recent clinical advances, new therapeutic approaches are still required. The cystine-glutamate antiporter xCT, encoded by the SLC7A11 gene, which imports cystine in exchange with glutamate, is a potentially new target for breast cancer therapy, being involved in tumor cell redox balance and resistance to therapies. xCT expression is regulated by the oncosuppressor p53, which is mutated in many breast cancers. Indeed, mutant p53 (mut-p53) can induce xCT post-transcriptional down modulation, rendering mut-p53 tumors susceptible to oxidative damage. Interestingly, the drug APR-246, developed to restore the wild-type function of p53 in tumors harboring its mutation, alters the cell redox balance in a p53-independent way, possibly rendering the cells more sensitive to xCT inhibition. Here, we propose a combinatorial treatment based on xCT immunetargeting and APR-246 treatment as a strategy for tackling breast cancer. We demonstrate that combining the inhibition of xCT with the APR-246 drug significantly decreased breast cancer cell viability in vitro and induced apoptosis and affected cancer stem cells' self-renewal compared to the single treatments. Moreover, the immunetargeting of xCT through DNA vaccination in combination with APR-246 treatment synergistically hinders tumor progression and prevents lung metastasis formation in vivo. These effects can be mediated by the production of anti-xCT antibodies that are able to induce the antibody dependent cellular cytotoxicity of tumor cells. Overall, we demonstrate that DNA vaccination against xCT can synergize with APR-246 treatment and enhance its therapeutic effect. Thus, APR-246 treatment in combination with xCT immunetargeting may open new perspectives in the management of breast cancer.

Abstract IA022: Arginine, serine, xCT and ME, the therapeutic metabolism of different sarcomas

Abstract The absolute diversity of the biology of the sarcomas makes therapeutic development challenging. As most of tumor metabolism is composed of transporters and enzymes, finding metabolic dependencies should allow for small molecule targeting. Due to the rapid metabolic evolution that tumors undergo in response to the targeting of any one metabolic pathway, a deep understanding of sarcoma metabolism is needed. First, the most common metabolic adaptation that sarcomas undergo is loss of expression of argininosuccinate synthetase 1 (ASS1), which is silenced in ~90% of cases by methylation. This results in sarcomas being arginine auxotrophic and sensitive to arginine starvation therapies, such as with arginine deiminase (ADI-PEG20). Not only does arginine starvation alter the Warburgian biology of sarcomas making them dependent of glutamine, but it can be used to upregulate the expression of cell surface transports such as hENT, which allows to gemcitabine internalization. In addition, mouse modeling has demonstrated that vesicular trafficking is key to overcoming initial arginine starvation in vivo, a process that can be blocked with chloroquine. This arginine dependency is likely related to the mesenchymal origin of sarcomas, which would explain its high recurrence rate of ASS1 silencing across histologies. Next, due to the upregulation of 3-phosphoglycerate dehydrogenase (PHGDH), osteosarcoma preferentially utilizes glucose to make the serine that enters the folate cycle, as opposed to completing lower glycolysis. In addition, as osteosarcoma does not seem to depend on extracellular serine import, inhibition of PHGDH leads to pro-survival compensation by the mTORC pathway. This can be targeted on both the AMP Kinase and the AKT dependent parts of the MTORC pathway that converge at FOXO3. When both are inhibited approach demonstrates unique triple synergy and therapeutic strategy for osteosarcoma due to its unique serine biology. Finally, synovial sarcoma lack the expression of malic enzyme 1 (ME1), also likely due to its cell of origin. This leads to reduced glucose oxidation, enhanced glycolysis, and compensatory increased flux through the pentose phosphate pathway for cytoplasmic NADPH production. Additionally, absence of ME1 in SS results in significant reductions in the GSH/GSSG ratio as well as a reduced glutathione synthesis. Sensitivity to GSH pathway inhibition is also reduced while sensitivity to inhibition of the thioredoxin system is significantly increased. ME1 absence results in increases in the labile iron pool that sensitizes synovial sarcoma to ferroptosis. Therefore, ME1 null SS is exquisitely sensitive to induction of ferroptosis with xCT inhibition. This can be accomplished by erastin analogs in vitro and ACXT-3102 (a tumor targeted erastin) in vivo. As we learn more about each sarcoma, we must understand the underlying metabolism of each subtype and their cell of origin. Given the targetable nature of metabolic enzymes and transporters, an ever deeper understanding of sarcoma metabolism is warranted. Citation Format: Brian A. Van Tine. Arginine, serine, xCT and ME, the therapeutic metabolism of different sarcomas [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA022.

Liposomes embedded with PEGylated iron oxide nanoparticles enable ferroptosis and combination therapy in cancer.

Ferroptosis, an iron-dependent regulated cell death process driven by excessive lipid peroxides, can enhance cancer vulnerability to chemotherapy, targeted therapy and immunotherapy. As an essential upstream process for ferroptosis activation, lipid peroxidation of biological membranes is expected to be primarily induced by intrabilayer reactive oxygen species (ROS), indicating a promising strategy to initiate peroxidation by improving the local content of diffusion-limited ROS in the lipid bilayer. Herein, liposomes embedded with PEG-coated 3nm γ-Fe2O3 nanoparticles in the bilayer (abbreviated as Lp-IO) were constructed to promote the intrabilayer generation of hydroxyl radicals (•OH) from hydrogen peroxide (H2O2), and the integration of amphiphilic PEG moieties with liposomal bilayer improved lipid membrane permeability to H2O2 and •OH, resulting in efficient initiation of lipid peroxidation and thus ferroptosis in cancer cells. Additionally, Lp-IO enabled traceable magnetic resonance imaging and pH/ROS dual-responsive drug delivery. Synergistic antineoplastic effects of chemotherapy and ferroptosis, and alleviated chemotherapeutic toxicity, were achieved by delivering doxorubicin (capable of xCT and glutathione peroxidase inhibition) with Lp-IO. This work provides an efficient alternative for triggering therapeutic lipid peroxidation and a ferroptosis-activating drug delivery vehicle for combination cancer therapies.

Codon Usage and mRNA Stability are Translational Determinants of Cellular Response to Canonical Ferroptosis Inducers

Ferroptosis is a non-apoptotic cell death mechanism characterized by the generation of lipid peroxides. While many effectors in the ferroptosis pathway have been mapped, its epitranscriptional regulation is not yet fully understood. Ferroptosis can be induced via system xCT inhibition (Class I) or GPX4 inhibition (Class II). Previous works have revealed important differences in cellular response to different ferroptosis inducers. Importantly, blocking mRNA transcription or translation appears to protect cells against Class I ferroptosis inducing agents but not Class II. In this work, we examined the impact of blocking transcription (via Actinomycin D) or translation (via Cycloheximide) on Erastin (Class I) or RSL3 (Class II) induced ferroptosis. Blocking transcription or translation protected cells against Erastin but was detrimental against RSL3. Cycloheximide led to increased levels of GSH alone or when co-treated with Erastin via the activation of the reverse transsulfuration pathway. RNA sequencing analysis revealed early activation of a strong alternative splice program before observed changes in transcription. mRNA stability analysis revealed divergent mRNA stability changes in cellular response to Erastin or RSL3. Importantly, codon optimality biases were drastically different in either condition. Our data also implicated translation repression and rate as an important determinant of the cellular response to ferroptosis inducers. Given that mRNA stability and codon usage can be influenced via the tRNA epitranscriptome, we evaluated the role of a tRNA modifying enzyme in ferroptosis stress response. Alkbh1, a tRNA demethylase, led to translation repression and increased the resistance to Erastin but made cells more sensitive to RSL3.

Redox-dependent AMPK inactivation disrupts metabolic adaptation to glucose starvation in xCT-overexpressing cancer cells.

Accelerated aerobic glycolysis is a distinctive metabolic property of cancer cells that confers dependency on glucose for survival. However, the therapeutic strategies targeting this vulnerability are still inefficient and have unacceptable side effects in clinical trials. Therefore, developing biomarkers to predict therapeutic efficacy would be essential to improve the selective targeting of cancer cells. Here, we found that cell lines that are sensitive to glucose deprivation have high expression of cystine/glutamate antiporter xCT (also known as SLC7A11). We found that cystine uptake and glutamate export through xCT contributed to rapid NADPH depletion under glucose deprivation. This collapse of the redox system oxidized and inactivated AMP-activated protein kinase (AMPK), a major regulator of metabolic adaptation, resulting in a metabolic catastrophe and cell death. Although this phenomenon was prevented by pharmacological or genetic inhibition of xCT, overexpression of xCT sensitized resistant cancer cells to glucose deprivation. Taken together, these findings suggest a novel crosstalk between AMPK and xCT that links metabolism and signal transduction, and reveal a metabolic vulnerability to glucose deprivation in cancer cells expressing high levels of xCT.

Abstract 130: Pharmacological effects of selective xCT inhibition in ARID1A mutated cancer models

Abstract xCT is a cystine/glutamate antiporter that exports intracellular glutamate for extracellular cystine. In the intracellular space, cystine is converted into cysteine which is subsequently used for glutathione (GSH) synthesis (a major antioxidant species). ARID1A is part of the SWI-SNF remodeling complex that binds on the xCT promoter and as such controls its gene expression. ARID1A deficiency which is highly prevalent in many cancers results in downregulation of xCT, impaired GSH biosynthesis and subsequent ROS induction, raising potential rationale to target xCT in ARID1A deficient cancers. By using a selective nanomolar range xCT inhibitor, we first showed that cell lines deficient for ARID1A were significantly more sensitive to the inhibition of xCT. Effects on proliferation could be reversed by N-acetylcysteine supplementation in the culture medium. In vivo, the xCT inhibitor confirmed its drug-like properties showing significant target engagement in an ARID1A deficient ovarian cell line xenograft model together with good PK and tolerability profiles. Altogether these studies have provided evidence that a specific drug-like inhibitor of xCT can be developed. This proprietary compound was used to confirm that in vitro the deficiency of ARID1A in ovarian cancer predicts sensitivity to xCT inhibition. Further in vivo pharmacological studies are required to confirm a specific molecular context predicting sensitivity towards xCT inhibition. Citation Format: Christophe Henry, Philippe Péron, Anne-Marie Blanchet, Arielle Genevois-Borella, Marc Trellu, Dorothée Bourges, Philippe Lienard, Alexandra Basset, Erwan Jouannot, Christophe Philippo, Laurent Debussche, Jürgen Moll. Pharmacological effects of selective xCT inhibition in ARID1A mutated cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 130.

MO-0141 Sulfasalazine radiosensitizes hypoxic colorectal cancer cells through the inhibition of xCT.

MO-0141 Sulfasalazine radiosensitizes hypoxic colorectal cancer cells through the inhibition of xCT.

Cystine uptake inhibition potentiates front-line therapies in acute myeloid leukemia

By querying metabolic pathways associated with leukemic stemness and survival in multiple AML datasets, we nominated SLC7A11 encoding the xCT cystine importer as a putative AML dependency. Genetic and chemical inhibition of SLC7A11 impaired the viability and clonogenic capacity of AML cell lines in a cysteine-dependent manner. Sulfasalazine, a broadly available drug with xCT inhibitory activity, had anti-leukemic activity against primary AML samples in ex vivo cultures. Multiple metabolic pathways were impacted upon xCT inhibition, resulting in depletion of glutathione pools in leukemic cells and oxidative stress-dependent cell death, only in part through ferroptosis. Higher expression of cysteine metabolism genes and greater cystine dependency was noted in NPM1-mutated AMLs. Among eight anti-leukemic drugs, the anthracycline daunorubicin was identified as the top synergistic agent in combination with sulfasalazine in vitro. Addition of sulfasalazine at a clinically relevant concentration significantly augmented the anti-leukemic activity of a daunorubicin-cytarabine combination in a panel of 45 primary samples enriched in NPM1-mutated AML. These results were confirmed in vivo in a patient-derived xenograft model. Collectively, our results nominate cystine import as a druggable target in AML and raise the possibility to repurpose sulfasalazine for the treatment of AML, notably in combination with chemotherapy.

Combined xCT and mTOR Inhibition as a New Therapeutic Option for Pancreatic Adenocarcinoma

Introduction: ACXT-3102 is an orally available cancer therapeutic that selectively targets the xCT transporter in tumor cells without causing significant toxicity. Inactivation of xCT results in oxidative stress and cell death, recently identified as ferroptosis. Furthermore, mTOR has been shown to modulate the xCT pathway. Therefore, we hypothesized that combination of xCT with an mTOR inhibitor would result in improved antitumor effects. Methods: Human and murine pancreatic cancer cell lines were treated in vitro with monotherapy (ACXT-3102 or the mTOR inhibitor Torin 1) or combination therapy for 48h. Cell viability was assessed by flow cytometry using a dye exclusion assay (7-AAD). C57BL/6 mice were implanted subcutaneously with syngeneic pancreatic cancer cells derived from the KPC tumor model. Athymic nude mice were implanted with human pancreatic cancer lines. All mice were treated for 14 days with ACXT-3102, Torin 1, ACXT-3102 + Torin 1 combination, or vehicle as a control. Results: The combination of ACXT-3102 and Torin 1 induced significantly more cell death in vitro than either compound alone (Fig. 1, p = 0.014, 0.0096). Also, combination treatment demonstrated significant tumor control in vivo (p < 0.05, data not shown). Conclusion: The enhanced therapeutic effect of a combined xCT (ACXT-3102) and mTOR inhibition (Torin 1) has been observed in vitro and in mouse models of PDAC. These results warrant additional evaluation as a novel therapy concept for PDAC patients.

D-Cysteine supplementation partially protects against ferroptosis induced by xCT dysfunction via increasing the availability of glutathione.

Glutathione (GSH) is synthesized from three amino acids and the overall process is highly dependent on the availability of l-cysteine (l-Cys). GSH serves as an essential cofactor for glutathione peroxidase 4 (Gpx4), which reduces phospholipid hydroperoxides. The inactivation of Gpx4 or an insufficient supply of l-Cys results in the accumulation of lipid hydroperoxides, eventually leading to iron-dependent cell death, ferroptosis. In this study, we investigated the anti-ferroptotic properties of d-cysteine (d-Cys) under conditions of dysfunction in cystine transporter, xCT. l-Cys supplementation completely rescued ferroptosis that had been induced by the erastin-mediated inhibition of xCT in Hepa 1-6 cells. Upon d-Cys supplementation, the erastin-treated cells remained completely viable for periods of up to 24 h but eventually died after 48 h. d-Cys supplementation suppressed the production of lipid peroxides, thereby ferroptosis. The addition of d-Cys sustained intracellular Cys and GSH levels to a certain extent. When Hepa 1-6 cells were treated with a combination of buthionine sulfoximine and erastin, the anti-ferroptotic effect of d-Cys was diminished. These collective results indicate that, although d-Cys is not the direct source of GSH, d-Cys supplementation protects cells from ferroptosis in a manner that is dependent on GSH synthesis via stimulating the uptake of l-Cys.

Overexpression of Cystine/Glutamate Antiporter xCT Correlates with Nutrient Flexibility and ZEB1 Expression in Highly Clonogenic Glioblastoma Stem-like Cells (GSCs)

Simple SummaryGlioblastoma (GBM) is the most aggressive form of glioma (WHO grade IV), and mounting evidence suggests that glioblastoma stem-like cells (GSCs) play an important role in tumor growth and response to therapy. In the current study, we sought to understand the metabolic dependencies of GSCs using high-resolution proton magnetic resonance spectroscopy (1H-NMR). In a defined experimental setting, we stratified in vitro GSC models into two subtypes (Gln/GluHigh, Gln/GluLow) and used diverse molecular approaches to perform comprehensive analyses in GSC neurosphere cultures and primary GBM samples.Cancer stem-like cells mediate tumor initiation, progression, and therapy resistance; however, their identification and selective eradication remain challenging. Herein, we analyze the metabolic dependencies of glioblastoma stem-like cells (GSCs) with high-resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy. We stratify our in vitro GSC models into two subtypes primarily based on their relative amount of glutamine in relationship to glutamate (Gln/Glu). Gln/GluHigh GSCs were found to be resistant to glutamine deprivation, whereas Gln/GluLow GSCs respond with significantly decreased in vitro clonogenicity and impaired cell growth. The starvation resistance appeared to be mediated by an increased expression of the glutamate/cystine antiporter SLC7A11/xCT and efficient cellular clearance of reactive oxygen species (ROS). Moreover, we were able to directly correlate xCT-dependent starvation resistance and high Gln/Glu ratios with in vitro clonogenicity, since targeted differentiation of GSCs with bone morphogenic protein 4 (BMP4) impaired xCT expression, decreased the Gln/Glu ratio, and restored the sensitivity to glutamine starvation. Moreover, significantly reduced levels of the oncometabolites lactate (Lac), phosphocholine (PC), total choline (tCho), myo-inositol (Myo-I), and glycine (Gly) were observed in differentiated GSCs. Furthermore, we found a strong association between high Gln/Glu ratios and increased expression of Zinc finger E-box-binding homeobox 1 (ZEB1) and xCT in primary GBM tumor tissues. Our analyses suggest that the inhibition of xCT represents a potential therapeutic target in glioblastoma; thus, we could further extend its importance in GSC biology and stress responses. We also propose that monitoring of the intracellular Gln/Glu ratio can be used to predict nutrient stress resistance.