Targeting NR4A1/2 may represent a promising therapeutic strategy for overcoming resistance in rhabdomyosarcoma, and early CD71 induction combined with suppression of GPX4, SLC7A11, and FSP1 highlights a multi-level ferroptotic mechanism in RMS.
Abstract
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and adolescents, with poor outcomes in metastatic and treatment-resistant disease. The alveolar subtype is frequently driven by the PAX3-FOXO1 fusion oncoprotein, which promotes tumor progression and therapeutic resistance. Ferroptosis, an iron-dependent regulated cell death process characterized by lipid peroxidation and oxidative stress, represents a promising therapeutic vulnerability in RMS.
This study investigated whether bis-indole-derived dual NR4A1/2 ligands (C-DIM compounds) suppress PAX3-FOXO1 signaling and induce ferroptosis through regulation of iron metabolism and antioxidant defense pathways in RMS cells.
Human RMS cell lines (RD and RH30) were treated with C-DIM compounds. Cell viability was assessed using XTT assays. Intracellular ROS and lipid peroxidation were measured using H2DCFDA and BODIPY™ 581/591 staining, and oxidative damage was quantified by malondialdehyde (MDA) assays. Expression of ferroptosis-related markers, including CD71, GPX4, SLC7A11, and FSP1, was analyzed by qPCR and Western blotting. NR4A1, NR4A2, and Sp1, Sp4 were silenced using siRNA to determine their functional roles.
C-DIM treatment significantly reduced RMS cell viability and suppressed PAX3-FOXO1 expression and downstream oncogenic signaling. These effects were associated with increased ROS production, lipid peroxidation, and MDA accumulation, confirming ferroptosis induction. Mechanistically, dual NR4A1/2 ligands triggered early and dose-dependent induction of CD71, suggesting enhanced iron uptake as an initiating event. In parallel, key ferroptosis-protective regulators, including GPX4, SLC7A11, and FSP1, were significantly downregulated. Knockdown of NR4A1/2 or Sp1/4 down regulates GPX4, SLC7A11, and FSP1and reduced ferroptotic cell death, indicating a critical role for the NR4A1/2–Sp1/4 axis.
Dual NR4A1/2 ligands suppress PAX3-FOXO1 signaling and promote ferroptosis through coordinated modulation of iron uptake and antioxidant defense pathways. Early CD71 induction combined with suppression of GPX4, SLC7A11, and FSP1 highlights a multi-level ferroptotic mechanism in RMS. Further studies are needed to elucidate the precise regulatory mechanisms underlying the suppression of ferroptosis-related genes. Targeting NR4A1/2 may represent a promising therapeutic strategy for overcoming resistance in rhabdomyosarcoma.
Arafat Rahman Oany, Stephen Safe. Dual NR4A1/2 ligands inhibit PAX3-FOXO1 and promote ferroptosis in Rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Breaking Barriers in the Fight against Rare Cancers; 2026 Jul 18-20; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(14_Suppl):Abstract nr A025.
Glioblastoma (GBM) is one of the most aggressive and treatment-resistant tumors of the central nervous system, demanding innovative therapeutic strategies. Ferroptosis, an iron-driven lipid peroxidation-dependent cell death, has emerged as a potential means to overcome resistance, with glutathione peroxidase 4 (GPX4) as its key suppressor. MLN4924 (pevonedistat), a first-in-class inhibitor of neddylation currently in clinical trials, has shown strong antitumor activity across multiple malignancies. However, its role in ferroptosis regulation and the underlying molecular mechanisms remain poorly defined. Here, we show that MLN4924 induces ferroptosis, with GPX4 emerging as a central regulatory node. GPX4 expression is elevated in gliomas compared to normal brains, and its higher levels correlate with increased grades and worse patient prognosis. MLN4924 downregulates GPX4 mRNA by suppressing STAT3 signaling, whereas RSL3 directly inhibits GPX4 enzymatic activity. The combination of MLN4924 and RSL3 synergistically potentiates ferroptosis and, in vivo, suppresses subcutaneous tumor growth with a considerable biosafety. Collectively, these findings identify GPX4 as the principal mediator of MLN4924-induced ferroptosis and establish that dual targeting of GPX4 transcription and activity represents a promising therapeutic strategy for GBM.
Zhou Jing, Fangyuan Wang, Hao Li et al.· Carcinogenesis· 0 citations
Background Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation (LPO), represents a promising therapeutic strategy. Magnolia officinalis, a traditional herb for resolving dampness and phlegm, is known to modulate cellular metabolism. While magnolol (MAG), a bioactive neolignan from M. officinalis, shows anti-TNBC activity, its role in inducing ferroptosis remains unexplored. Methods Anti-TNBC effects of MAG were assessed in MDA-MB-231 and 4T1 cells via viability, apoptosis, and ferroptosis assays (intracellular Fe2+, LPO, GSH). Target identification employed network pharmacology, RNA-seq, surface plasmon resonance, and pull-down assays. Mechanisms were validated using siRNA/overexpression, Co-IP, and immunofluorescence. In vivo efficacy was evaluated in xenograft models. Results MAG inhibited proliferation and induced apoptosis and ferroptosis in TNBC cells, evidenced by elevated Fe2+ and lipid peroxidation, depleted GSH, downregulated SLC7A11/GPX4, and mitochondrial shrinkage effects reversed by ferroptosis inhibitors. MAG directly bound and suppressed transcription factor ETS2. ETS2 knockdown sensitized cells to MAG-induced ferroptosis, while its overexpression restored SLC7A11/GPX4 expression and conferred resistance. Mechanistically, ETS2 transcriptionally regulated SLC7A11, and MAG enhanced ETS2-SLC7A11 protein interaction. In vivo, MAG significantly suppressed tumor growth with low toxicity and downregulated ETS2, SLC7A11, and GPX4. Conclusion MAG induces ferroptosis in MDA-MB-231 cells, which may be mediated by targeting the ETS2/SLC7A11/GPX4 signaling axis, providing mechanistic insights into its anti-TNBC activity.
Jing Zhu, Ke Zhong, Zhuang Yan et al.· Frontiers in Pharmacology· 0 citations
In vivo, METTL3 overexpression promoted tumor growth, reduced 4-HNE levels, and regulated ferroptosis and FAK/MAPK/ERK pathway proteins, all of which were reversed by COL5A2 knockdown.
Xinke Guo, Shuang Li, Yu-Ying Guo et al.· Technology in Cancer Researc...· 0 citations
Background PD‐1 blockade has yet to achieve broad clinical success in colorectal cancer (CRC), with microsatellite‐stable tumors proving especially resistant. At the same time, ferroptosis has emerged as a mechanistic link between redox control in tumor cells and the antitumor immune response. GPR39 is overexpressed in CRC, but its functional role in ferroptosis and immunotherapy resistance is currently unclear. Methods GPR39 expression was analyzed in human and mouse CRC cell lines. GPR39 knockdown, with or without the ferroptosis inhibitor liproxstatin‐1, was performed to determine whether GPR39 regulates CRC cell proliferation and migration through ferroptosis. Ferroptosis was evaluated by measuring lipid peroxidation, glutathione (GSH) levels, ferrous iron (Fe2+) accumulation, and reactive oxygen species (ROS). Mechanistic involvement of the nuclear factor erythroid 2–related factor 2 (Nrf2)/solute carrier family 7 member 11 (SLC7A11) signaling axis was examined using Nrf2 overexpression. Subcutaneous implantation of MC38 cells with stable GPR39 knockdown was performed to evaluate whether GPR39 regulates tumor sensitivity to anti‐PD‐1 treatment in vivo in a ferroptosis‐dependent manner. Results GPR39 was markedly upregulated in CRC cell lines. GPR39 knockdown induced ferroptosis, characterized by increased lipid peroxidation, Fe2+ accumulation, and oxidative stress and accompanied by suppression of the Nrf2/SLC7A11 pathway. Nrf2 overexpression reversed these changes. Functionally, silencing GPR39 inhibited the proliferative and migratory capacities of CRC cells, effects that were largely rescued by liproxstatin‐1. In vivo, GPR39 knockdown significantly potentiated the antitumor activity of PD‐1 blockade, as evidenced by suppressed tumor progression accompanied by enhanced infiltration of CD8+ T cells, whereas ferroptosis inhibition abrogated these effects. Conclusion GPR39 suppresses ferroptosis in CRC via the Nrf2/SLC7A11 axis, thereby limiting PD‐1 immunotherapy efficacy.
Cong Zhou, Xiaoling Fang, Jiaying Lin et al.· Canadian Journal of Gastroen...· 0 citations
Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive pediatric brainstem tumor with limited treatment options and dismal prognosis. Previously, we showed that Ogremorphin (OGM), a specific GPR68 inhibitor, induced ferroptosis in glioblastoma multiforme (GBM) cells. Given their shared originating cell type, we investigated OGM’s efficacy in inducing ferroptosis in DIPG cells, using normal human astrocytes (NHAs) for comparison.
DIPG cell lines (SF7761, DIPG36, DIPG007) and NHAs were evaluated. OGM sensitivity was assessed via CellTiter-Glo viability assays. Ferroptosis markers including glutathione (GSH) levels, lipid peroxidation (BODIPY C11), and qRT-PCR of ATF4 and SLC7A11 were measured. Rescue experiments used liproxstatin-1 for ferroptosis and Z-VAD-FMK for apoptosis. Genetic validation used shRNA knockdown and overexpression of GPR68/ATF4, plus an ATF4 luciferase reporter assay.
OGM treatment in N5 media revealed that NHAs were more resistant (LD50 = 3.84 µM) than DIPG cells (LD50 ≤ 0.79 µM). DIPG sensitivity to OGM was comparable to GBM. Knockdown of GPR68 via shRNA confirmed that GPR68 inhibition induces cell death in DIPG cells. OGM robustly triggered ferroptosis marked by elevated lipid radicals, reduced GSH, and increased ATF4 and SLC7A11 expression in DIPG and NHA cells, except SF7761, which lacked SLC7A11 induction. Furthermore, liproxstatin-1 rescued the loss of cell viability, whereas Z-VAD-FMK had no effect. ATF4 overexpression caused cell death, while its knockdown rescued OGM-mediated effects.
These findings suggest that GPR68 inhibition is an attractive therapeutic target for DIPG. Notably, GPR68 inhibition mirrors the effects observed in GBM, inducing ATF4-dependent ferroptotic cell death.
Leif R. Neitzel, Charles C. Hong. Inhibition of GPR68 induces ferroptosis in diffuse intrinsic pontine gliomas [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Breaking Barriers in the Fight against Rare Cancers; 2026 Jul 18-20; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(14_Suppl):Abstract nr A023.
L. Neitzel, Charles C Hong· Cancer Research· 0 citations
Aplastic anemia (AA) is a severe hematological disorder caused by hyperactivated T cell-mediated hematopoietic failure. It is characterized by hematopoietic stem cell deficiency and hypocellular hematopoiesis in the bone marrow (BM). Ferroptosis, a specific type of programmed cell death, is defined by iron-dependent lipid peroxidation, which has garnered the attention of researchers due to its unique role and biological importance in various diseases. However, the correlation between immunological imbalance-induced ferroptosis and the mortality of hematopoietic stem cells within the BM microenvironment in AA is unclear. Resveratrol (RSV) is crucial in activating NRF2, hence influencing the onset and progression of diseases by regulating ferroptosis. This study aimed to investigate the roles and molecular mechanisms of RSV in the hematopoietic recovery of AA regarding ferroptosis. We measured some biomarkers in AA mice and IFN-gamma-treated 32D cells, representing AA syndromes and ferroptosis features. Furthermore, in RSV-treated 32D AA cells, cell activity, cell apoptosis, mitochondrial membrane potential, mitochondrial membrane permeability, and intracellular levels of MDA, ferrous iron, 4-HNE, GSH, ROS, and lipid peroxidation were assessed. Subsequently, additional in vitro and in vivo experiments were performed to investigate the mechanisms by which RSV effectively regulates NRF2 stability, further inhibiting ferroptosis in AA. We demonstrated that ferroptosis contributes to the occurrence and development of AA disease. RSV dose-dependently inhibits ferroptosis in 32D AA cells by targeting GPX4 expression, enhancing the cell activity of hematopoietic BM cells. Mechanically, RSV significantly increases NRF2 phosphorylation through the PI3K/AKT/mTOR signaling pathway, which maintains NRF2 stability to promote the GPX4 metabolic pathway in AA. Protein levels of p-AKT, p-mTOR, p-NRF2, and GPX4 were markedly increased in BM cells in RSV-treated AA mice, resulting in the suppression of AA development. Our data demonstrate that RSV effectively inhibits ferroptosis in AA or IFN-gamma-treated 32D cells by targeting the PI3K/AKT/mTOR signaling pathway, which enhances NRF2-mediated GPX4 transcription, thereby ameliorating AA symptoms in vitro and in vivo. This study concludes that RSV is a potential therapeutic drug for ferroptosis-related AA treatment.
Erhao Zhang, Jianan Cui, Xinran Zhu et al.· Chinese Medicine· 0 citations
