The receptors at the heart of this work belong to the nuclear receptor superfamily, a large group of proteins that act as molecular switches controlling gene expression. Unlike their better-studied cousins such as the estrogen or glucocorticoid receptors, NR4A1 and NR4A2 are called orphan receptors because no natural ligand has been definitively identified for them. In most solid tumors, including colon cancer, both receptors behave as pro-oncogenic factors: they help cancer cells proliferate, resist apoptosis, invade surrounding tissue, and survive metabolic stress. Because they sit at the intersection of so many tumor-promoting pathways, they have long been considered attractive drug targets, but exploiting them therapeutically requires molecules that can bind and modulate their activity in a controlled way.

The Safe laboratory has spent years developing a class of synthetic ligands based on a chemical scaffold known as 1,2-bis(3′-indolyl)-1-(3,5-disubstitutedphenyl)methane, abbreviated DIM-3,5. These compounds are built around two indole rings, the same structural motif found in compounds derived from cruciferous vegetables, connected through a central methane carbon to a substituted phenyl ring. By varying the substituents on that phenyl ring, the chemists can tune how the molecule interacts with NR4A1 and NR4A2. In the new study, the researchers focused on dual NR4A1/2 ligands and asked a specific question: can these compounds push colon cancer cells into ferroptosis, an iron-dependent form of regulated cell death that has attracted enormous interest as a vulnerability in tumors resistant to conventional therapies?

Ferroptosis is mechanistically very different from apoptosis. Instead of the orderly cellular dismantling characteristic of programmed cell death, ferroptosis is driven by catastrophic oxidative damage to cell membranes. When the lipid bilayer is not adequately protected by antioxidant systems, polyunsaturated fatty acids in the membrane undergo peroxidation, generating reactive oxygen species and lipid peroxides that rupture the cell from the outside in. The central guardian against this process is the enzyme glutathione peroxidase 4, or GPX4, which detoxifies lipid hydroperoxides using glutathione as a cofactor. Closely linked to this defense is the cystine/glutamate antiporter SLC7A11, which imports cystine needed to replenish glutathione. Tumor cells, which already operate under high oxidative stress, are often exquisitely dependent on this GPX4-SLC7A11 axis, making it a promising therapeutic chokepoint.

The experimental results reported in the study show that the DIM-3,5 ligands do exactly what a ferroptosis-inducing agent should do. Treatment of colon cancer cells led to a marked enhancement of reactive oxygen species, detected using BODIPY fluorescence, a widely used probe whose signal shifts when lipid peroxidation accumulates in membranes. Consistent with this, the cells produced increased amounts of malondialdehyde, a well-established degradation product of lipid peroxides that serves as a chemical fingerprint of membrane damage. These biochemical changes were accompanied by a characteristic reprogramming of ferroptosis-related gene expression: the transferrin receptor, also known as TFR or CD71, was induced, while GPX4 and SLC7A11 were both decreased. Inducing the transferrin receptor increases iron uptake into the cell, feeding the Fenton chemistry that drives lipid peroxidation, while simultaneously suppressing the two main antioxidant safeguards. The net effect is a convergence of pressure on the membrane lipids from multiple directions at once.

One of the most technically significant contributions of the paper is the molecular dissection of how NR4A receptors control GPX4 expression in the first place. Using knockdown experiments, promoter analysis, and chromatin immunoprecipitation, the team demonstrated that GPX4 transcription is regulated by a partnership between NR4A2 and the transcription factor Sp4. Sp4 binds directly to specific DNA sequences in the GPX4 promoter, and NR4A2 functions as a ligand-dependent cofactor for the DNA-bound Sp4. In other words, NR4A2 does not simply float in the nucleus as an independent regulator; it is recruited to the promoter through its interaction with Sp4, and its activity there depends on the ligand occupying its binding pocket. When the DIM-3,5 ligands engage NR4A2, this cofactor arrangement is disrupted, and the GPX4 gene is no longer efficiently transcribed. This mechanism explains how a single compound can lower the cell’s antioxidant shield and provides a concrete structural and functional rationale for targeting NR4A2 in ferroptosis-based cancer therapy.

Importantly, the researchers did not stop at cell culture experiments. To test whether the compounds could work in a living organism with an intact immune system, they used a syngeneic mouse xenograft model in which CT26 mouse colon cancer cells are implanted into immunocompetent mice. Syngeneic models are particularly valuable because, unlike human tumor cells grown in immunodeficient mice, they allow the full interplay between the tumor and the host immune system to unfold. The team treated the animals with the 3,5-dichloro analog of the DIM-3,5 series, referred to as DIM-3,5-Cl2, at a dose of 2.5 milligrams per kilogram per day. The result was a significant inhibition of colon tumor growth, confirming that the ferroptotic and gene-regulatory effects observed in vitro translate into tangible antitumor activity in vivo.

Perhaps the most striking findings came from the analysis of the tumor-infiltrating lymphocytes and other immune cells within the treated tumors. Flow cytometric profiling revealed that DIM-3,5-Cl2 treatment increased the population of CD8-positive T cells, the cytotoxic lymphocytes that directly kill tumor cells and are the primary mediators of successful cancer immunotherapy. At the same time, the treatment decreased the abundance of granulocytic myeloid-derived suppressor cells, or g-MDSC, a population of immature myeloid cells that potently suppresses antitumor immune responses and is associated with poor prognosis in many cancers. The ratio of M1 to M2 macrophages also shifted favorably, moving away from the immunosuppressive, tumor-promoting M2 phenotype and toward the inflammatory, tumoricidal M1 phenotype. In addition, the percentage of B cells within the tumor microenvironment increased, further supporting enhanced immune surveillance.

This dual mechanism, direct ferroptosis induction combined with immune reprogramming, is what makes the study noteworthy beyond the colon cancer field. Ferroptosis and immunity are increasingly recognized as deeply intertwined processes. When tumor cells die by ferroptosis, they release damage-associated molecular patterns and oxidized lipid mediators that can stimulate dendritic cells and promote cross-presentation of tumor antigens to T cells. Conversely, immunosuppressive myeloid cells within tumors secrete factors that help cancer cells resist lipid peroxidation. By simultaneously weakening the tumor’s antioxidant defenses and dismantling the suppressive immune circuitry that shields it, the DIM-3,5 ligands appear to attack the cancer from two complementary directions, a strategy that mirrors the logic of successful combination immunotherapies but achieves it with a single small molecule.

The work, which was supported by the Syd Kyle Chair Endowment, Texas A&M AgriLife, and the National Institutes of Health, is still at the preclinical stage, and the path from a mouse xenograft model to an approved human therapy is long and uncertain. Dose optimization, pharmacokinetics, toxicity profiling, and testing in additional tumor models will all be necessary before NR4A ligands could enter clinical trials. Nevertheless, the study establishes a clear mechanistic framework: NR4A1 and NR4A2, long appreciated as pro-oncogenic transcriptional regulators, can be pharmacologically exploited to collapse the GPX4-dependent antioxidant defense through the NR4A2-Sp4 cofactor mechanism, thereby triggering ferroptosis, while the same ligands reshape the tumor immune microenvironment in ways that favor immune-mediated tumor control. For a disease as stubborn as colon cancer, a molecule that turns the tumor’s own lipid chemistry against it and simultaneously invites the immune system to the scene represents a genuinely compelling new direction.

Subject of Research: NR4A nuclear receptor ligands as inducers of ferroptosis and immune modulation in colon cancer

Article Title: Orphan nuclear receptor 4A (NR4A) ligands induce ferroptosis and modify immune function in colon cancer

Article References: Oany, A. R., Upadhyay, S., Wright, G. A., Farkas, E., Sivaram, G., Tsui, W. N. T., Hailemariam, A., Chen, S.-H., & Safe, S. (2026). Orphan nuclear receptor 4A (NR4A) ligands induce ferroptosis and modify immune function in colon cancer. Cell Death Discovery. https://doi.org/10.1038/s41420-026-03369-4

Image Credits: AI Generated

DOI: 10.1038/s41420-026-03369-4

Keywords: NR4A1, NR4A2, ferroptosis, colon cancer, GPX4, DIM-3,5 ligands, reactive oxygen species, tumor microenvironment, CD8 T cells, myeloid-derived suppressor cells, immunotherapy, nuclear receptors

The team began by isolating so-called red blood cell ghosts, the hemoglobin-free membrane remnants left behind after erythrocytes are lysed under controlled laboratory conditions. When human cardiac microvascular endothelial cells were exposed to these ghosts for just two hours, bulk RNA sequencing revealed 113 differentially expressed genes, the vast majority of them upregulated. Among the most enriched biological pathways were inflammatory response and blood vessel morphogenesis, with prominent induction of genes such as VEGF, TNFA, PTGS2 and NFKBIA. Bioinformatic mapping also linked the altered genes to disease pathways including ischemia, peripheral vascular disease, arteriosclerosis and diabetic retinopathy, hinting that the response is directly relevant to cardiovascular pathology.

Chromatin immunoprecipitation sequencing added an epigenetic dimension to the picture. The researchers detected 77 sites of altered histone 3 lysine 27 acetylation, a mark of active enhancers, with the overwhelming majority gaining acetylation after ghost treatment. Motif analysis of these newly opened genomic regions showed a striking predominance of binding sites for the activator protein-1, or AP-1, family of transcription factors, which accounted for 55 percent of the target sequences. Immunofluorescence and cellular fractionation confirmed that JUNB, a key AP-1 member, accumulated in the nuclei of treated endothelial cells, indicating that the ghost signal rewires the transcriptional landscape rather than merely nudging existing programs.

On the inflammatory side, the membrane fraction triggered robust activation of the transcription factor NFκB. Within one to two hours, endothelial cells showed increased phosphorylation of the NFκB p65 subunit at serine 536, followed by phosphorylation at serine 276, a site targeted by cyclic AMP-dependent protein kinase and critical for transcriptional activity. The inflammatory enzyme COX2 rose in parallel. Blocking NFκB activation with the IκBα inhibitor BAY11-7082 abolished both the COX2 increase and, crucially, the angiogenic response, establishing NFκB as the central gatekeeper of the ghost-induced program.

Functionally, the treated endothelial cells behaved like cells primed for vessel building. In spheroid angiogenesis assays, exposure to red blood cell ghosts increased the number of sprouts per spheroid and the total endothelial network length, an effect comparable to recombinant VEGF. The cells also proliferated more readily, as measured by Ki-67 staining, and migrated faster in scratch-wound assays. Notably, the hemoglobin-free membrane fraction alone was sufficient to drive these effects, while isolated heme had no impact, ruling out the iron-containing pigment as the responsible agent and pointing instead to components retained in the membrane itself.

Metabolic profiling revealed the engine behind this angiogenic conversion. Using Seahorse extracellular flux analysis, the researchers observed that ghost addition boosted endothelial glycolysis within minutes, with elevated proton efflux and extracellular acidification sustained for roughly five hours. Mass spectrometry-based metabolomics confirmed accumulation of glycolytic intermediates, activation of the pentose phosphate pathway, and increased acetyl-CoA and TCA cycle metabolites. Isotope tracing showed glucose-derived ribose feeding purine synthesis and glutamine carbon flowing into pyrimidine synthesis, a classic anabolic profile. The glycolytic regulator PFKFB3, previously established as a hub of vessel sprouting metabolism, rose significantly at the protein level, and blocking glycolysis with the glucose analog 2-deoxy-D-glucose or inhibiting glutaminolysis with BPTES abolished the sprouting response.

The hunt for the transcriptional conductor of this program led to NR4A1, an orphan nuclear receptor also known as NUR77, which was among the most strongly induced genes in both the RNA and ChIP sequencing datasets. NR4A1 protein, nearly undetectable in resting cells, surged after two hours of ghost exposure and accumulated in the nucleus in an NFκB-dependent manner. Silencing NR4A1 with siRNA or blocking it pharmacologically prevented the induction of JUNB, PFKFB3 and VEGF and significantly reduced sprout formation. Intriguingly, NR4A1 knockdown also increased basal NFκB phosphorylation, revealing a built-in negative feedback loop: the receptor dampens inflammation while simultaneously switching on the metabolic and angiogenic machinery.

The causal trigger turned out to be adenosine triphosphate stored within the erythrocyte membrane. Fluorescent ATP analog labeling confirmed ATP binding sites on red cell membranes, and extracellular ATP at micromolar concentrations reproduced the full ghost response, from NR4A1 induction to sprouting. Degrading extracellular ATP with apyrase blunted the angiogenic effect. Pharmacological and genetic experiments identified two purinergic receptors as essential: the fast-acting ionotropic P2X7 receptor, which drives the inflammatory NFκB activation, and the metabotropic P2Y11 receptor, which couples to adenylyl cyclase and cyclic AMP production. Real-time FRET imaging with a genetically encoded cAMP biosensor captured robust cytosolic cAMP surges in single endothelial cells exposed to either ATP or ghosts, and inhibiting membrane-bound adenylyl cyclase or preventing cAMP degradation with PDE4 inhibitors modulated the response accordingly.

The translational payoff came from patients with severe peripheral artery disease, a condition defined by defective vessel growth. Immunostaining of ischemic muscle from mice and patients confirmed that erythrocyte extravasation accompanies ischemia in vivo. When endothelial cells were treated with ghosts prepared from PAD patients, the inflammatory activation persisted, but the upregulation of PFKFB3, VEGF and the cAMP sensor EPAC1 was significantly blunted, and the sprouting response was markedly weaker than with ghosts from age- and sex-matched healthy controls. Remarkably, inhibiting phosphodiesterase 4 with rolipram to prevent cAMP degradation restored the patient ghost response to healthy levels, suggesting that the ATP–P2Y11–cAMP axis fails in disease and can be pharmacologically rescued.

The authors caution that their patient cohort was small and exploratory, that P2Y11 receptors are absent in mice, limiting animal validation, and that other red cell membrane factors may contribute alongside ATP. Even so, the study fundamentally repositions erythrolysis as an instructive event in vascular repair. The same mechanism that helps restore perfusion after hemorrhage could be exploited therapeutically to boost angiogenesis in ischemic limbs, or conversely suppressed in settings such as cancer or retinal disease, where erythrocyte leakage and pathological vessel growth feed each other. Targeting endothelial cAMP metabolism, the work suggests, may be a promising lever for tipping that balance in either direction.

Subject of Research: How lysed erythrocyte membranes and their ATP content metabolically prime endothelial cells for angiogenesis

Article Title: Lysed erythrocyte membranes metabolically prime endothelial cells for angiogenesis

Article References: Gogiraju, R., Moiko, K., Bochenek, M. L., Greulich, F., Witzler, C., Schmitz, W., Zifkos, K., Derieux, C., Ghasemi, I., Guliani, P., Sun, B., Espinola-Klein, C., Uhlenhaut, H. N., Bock, A., Ruf, W., Madhusudhan, T., Lurz, P., & Schäfer, K. (2026). Lysed erythrocyte membranes metabolically prime endothelial cells for angiogenesis. Angiogenesis, 29(4), Article 74. https://doi.org/10.1007/s10456-026-10096-y

Image Credits: AI Generated

DOI: 10.1007/s10456-026-10096-y

Keywords: angiogenesis, endothelial cells, erythrocytes, hemolysis, ATP signaling, NFκB, NR4A1, PFKFB3, cAMP, P2X7 receptor, P2Y11 receptor, peripheral artery disease