Background/Objective: Coffee is the most highly consumed beverage worldwide, and coffee drinkers exhibit decreased mortality and protection from aging-related diseases. This study investigates the role of orphan nuclear receptor 4A1 (NR4A1) in mediating the effects of brewed coffee and the major polyphenolic and polyhydroxy compounds in brewed coffee and also in determining their binding to NR4A1. Methods: The interactions of brewed coffee and several of the major individual compounds in brewed coffee with the ligand-binding domain of NR4A1 were determined using a fluorescent binding assay. For specific compounds, binding was also carried out by surface plasmon resonance, and molecular docking studies were also performed. NR4A1-responsive Rh30 cancer cells were used as models to determine NR4A1-dependent transactivation, cell growth inhibition and inhibition of specific gene products, and in some studies, knockdown of NR4A1 by RNA interference was also determined. Inhibition of lipopolysaccharide-induced IkBα by key polyphenolics was also investigated in RAW264.7 macrophages. Results: Brewed coffee and several polyphenolics, including caffeic acid, ferulic acid, chlorogenic acid, p-coumaric acid, several cinnamic acid derivatives, kahweol, and cafestrol, bound NR4A1 in binding assays, and most Kd values were <10 µM. Brewed coffee and the major polyphenolics inhibited growth of NR4A1-responsive Rh30 cells, and this was attenuated in NR4A1-deficient Rh30 cells. These same compounds also exhibited NR4A1-dependent effects on transactivation and gene product responses in Rh30 and RAW264.7 macrophages and exhibited inverse NR4A1 agonist activity. In contrast, the NR4A1-dependent activity of caffeine and quinic acid was highly variable, suggesting that they are selective NR4A1 ligands. Conclusions: The results of this study demonstrate that brewed coffee and its major polyphenolics and polyhydroxy constituents are NR4A1 ligands and that NR4A1 may play an important role in the health-protective effects of coffee. These results, coupled with recent studies, indicate that NR4A1 and its ligands may play an important role in diet and health.

1,1-Bis(3’-indolyl)-1-(3,5-disubstitutedphenyl)methane (DIM-3,5) compounds in the presence or absence of a 4-hydroxylphenyl group bind both orphan nuclear receptor 4A1 (NR4A1) and NR4A2. In cancer cells, these compounds bind and inactivate pro-oncogenic NR4A1 and NR4A2 and downstream pathways acting as inverse agonists that inhibit cancer cell growth, survival, migration and invasion, and induce ferroptosis. Similar results are observed in endometriotic cells where the DIM-3,5 dual NR4A1/2 ligands inhibit NR4A1/NR4A2-mediated pro-endometriotic genes and pathways. The potency of these DIM-3,5 dual NR4A1/NR4A2 ligands is also observed in tumor infiltrating lymphocytes where both receptors are expressed and regulate comparable functions.

Background/Objectives: The orphan nuclear receptors 4A1 (NR4A1) and NR4A2 are overexpressed in multiple solid tumors, and both receptors exhibit tumor promoter-like activities. A recent study reported that luteolin, a flavonoid that binds NR4A1, decreased the expression of the pro-oncogenic receptor tyrosine kinase MerTK in colon cancer cells. Methods/Results: In this study, we observed that MerTK protein was expressed in human SW480 and HCT116 and mouse CT26 colon cancer cell lines, and was significantly downregulated after treatment with 1,1-bis(3'-indolyl)-1-(3,5-disubstitutedphenyl)methane (DIM-3,5) compounds, which are dual NR4A1/NR4A2 ligands. Moreover, knockdown of NR4A1 and NR4A2 also decreased MerTK protein expression and DIM-3,5 ligands, and receptor knockdown also decreased MerTK RNA levels expression. MerTK expression was also downregulated by knockdown of Sp1, Sp3, or Sp4 and by treatment with mithramycin. Subsequent studies using chromatin immunoprecipitation and transfection of a MERTK (promoter)-luciferase construct containing transcriptionally active GC-rich promoter elements indicated that MerTK expression in colon cancer cells was regulated by NR4A/Sp complexes, including NR4A1, NR4A2, Sp1, Sp3, and Sp4 transcription factors. Conclusions: The participation of NR4A1 and NR4A2 in the regulation of MerTK indicates that DIM-3,5 ligands represent a novel class of agents that can be used to inhibit MerTK expression in cancer cells by acting as dual NR4A1 and NR4A2 inverse agonists.

Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with poor prognosis and limited therapeutic options. Although epigenetic dysregulation is a hallmark of HCC, rational combinatorial targeting strategies remain incompletely defined. Here, we identify cooperative oncogenic functions of the chromatin modifiers enhancer of zeste homolog 2 (EZH2) and lysine-specific demethylase 1 (LSD1) in HCC. Analysis of the TCGA-LIHC cohort revealed that co-elevated EZH2 and LSD1 expressions are significantly associated with reduced overall survival. Gene set enrichment analysis demonstrated enrichment of Sonic Hedgehog (SHH) signaling and stress-responsive transcriptional programs in tumors with high EZH2/LSD1 expression. Functionally, dual pharmacological inhibition of EZH2 (GSK126) and LSD1 (SP2509) suppressed HCC cell proliferation, induced G1-phase arrest, and enhanced apoptosis, as evidenced by increased caspase-3/7 activity and decreased pro-caspase levels. Dual inhibition also impaired migration, invasion, tumor sphere formation, and stemness-associated gene expression. Mechanistically, co-targeting disrupted SHH signaling through the suppression of GLI1 expression. Chromatin immunoprecipitation revealed reduced EZH2, LSD1, and STAT3 occupancy at the GLI1 promoter following dual inhibition, leading to the repression of GLI1 and its downstream targets. Collectively, these findings demonstrate that EZH2 and LSD1 cooperatively sustain GLI1-dependent SHH signaling in HCC, and that dual epigenetic inhibition represents a mechanistically defined therapeutic strategy.

Gut microbial metabolism of dietary flavonoids leads to a diverse array of bioactive products that are closely associated with human health. Combining enzyme promiscuity prediction, metabolomics, and in vitro model systems, we identified a chalcone-synthase-like bacterial polyketide synthase that can initiate the metabolism of naringenin by catalyzing the C-ring cleavage. This was validated using a mutant strain of the model organism Bacillus subtilis (ATCC 23857). Our prediction–validation methodology could be used to systematically characterize the products of gut bacterial flavonoid metabolism and identify the responsible enzymes and species. In vitro experiments with Caco-2 cells revealed that naringenin and its bacterial metabolites differentially engage the aryl hydrocarbon receptor (AhR) and orphan nuclear receptor 4A (NR4A). These results suggest that metabolism by gut bacterial species could directly impact the profile of bioactive flavonoids and influence inflammatory responses in the intestine. These results are significant for understanding gut-microbiota-dependent physiological effects of dietary flavonoids.

Rhabdomyosarcoma (RMS) is the most prevalent soft tissue sarcoma in children and adolescents, with a poor prognosis in metastatic or treatment-resistant cases. Conventional therapies often fail to eliminate aggressive tumor populations. Ferroptosis, a regulated form of cell death driven by iron accumulation and lipid peroxidation, has emerged as a promising therapeutic strategy for targeting RMS cells. This study investigates the potential of bis-indole-derived C-DIM compounds to induce ferroptosis in RMS cells by targeting the NR4A1/2–Sp1–CD71 axis, which regulates iron uptake and oxidative stress. Human RMS cell lines (RD and RH30) were treated with C-DIM compounds, and cell viability was assessed using the XTT assay. Intracellular ROS levels and lipid peroxidation were evaluated using H2DCFDA and BODIPY™ 581/591 staining, respectively, while malondialdehyde (MDA) assays quantified oxidative damage. CD71 mRNA and protein levels were measured by qPCR and Western blot. siRNA-mediated knockdowns of NR4A1, NR4A2, and Sp1 were performed to examine their roles in regulating CD71 expression and ferroptosis. A CD71 promoter luciferase assay was used to assess Sp1-dependent transcriptional activity. C-DIM treatment significantly reduced RMS cell viability by enhancing ferroptosis, as evidenced by increased ROS, MDA production, and lipid peroxidation. CD71 expression was markedly upregulated following treatment. Knockdown of NR4A1/2 or Sp1 led to a decrease in CD71 expression and protected RMS cells from ferroptosis. CD71 promoter assays confirmed that Sp1 regulates its transcription in response to C-DIMs. C-DIM compounds promote ferroptosis in RMS cells through an NR4A1/2–Sp1-mediated upregulation of CD71. These findings highlight a novel therapeutic strategy targeting the NR4A1/2–Sp1–CD71 axis to induce ferroptosis and overcome treatment resistance in rhabdomyosarcoma. Arafat Rahman Oany, Srijana Upadhaya, Stephen Safe. Dual NR4A1/2 ligands inhibit rhabdomyosarcoma cell growth and trigger ferroptosis by activating CD71 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C074.

The nuclear receptor (NR) superfamily of ligand-activated receptors plays a key role in maintaining cellular homeostasis and in pathophysiology. NRs can be subdivided into functional activities structural similarity and the existence of endogenous ligands. Most NRs are classified as those that are adopted orphan or orphan receptors which have only possible ligands or no identified endogenous ligands, respectively. In this review, the activities of the complete orphan receptor sub-family of transcription factors have been reviewed with a focus on the effects of possible endogenous (biochemicals), natural product-derived and synthetic ligands. Despite their lack of a bona-fide ligand, the orphan receptors bind structurally diverse compounds that exhibit tissue-specific agonist, antagonist and inverse agonist activities with potential for future development as clinical therapeutics for the treatment of multiple diseases.

Previous studies show that orphan nuclear receptor 4A1 (NR4A1) regulates endometriotic cell growth, survival, estrogen receptor β (ERβ), mechanistic target of rapamycin signaling and fibrosis. NR4A2 is also expressed in epithelial and stromal derived endometriotic cells, and in this study the effects of 1,1-bis(3'-indolyl)-(3,5-disubstitutedphenyl)methane (DIM-3,5) dual NR4A1/nuclear receptor 4A2 (NR4A2) ligands and knockdown of NR4A1 and NR4A2 were investigated. The dual NR4A1/2 DIM-3,5 analogs inhibited previously identified proendometriotic pathways and gene products, and they also inhibited TWIST1 and multiple markers associated with epithelial-to-mesenchymal transition (EMT). The results show that both NR4A1 and NR4A2 regulate the same pathways, including endometriotic cell growth, survival, and migration and also some of the same genes in endometriotic epithelial and stromal cells. For example, DIM-3,5 compounds downregulate ERβ in stromal but not epithelial endometriotic cells, and this response is NR4A1- and not NR4A2-dependent. Among the EMT-related markers, claudin-1 is induced by DIM-3,5 ligands and after knockdown of NR4A1 or NR4A2 in both epithelial and stromal cells. Most of the EMT markers are downregulated by DIM-3,5 ligands and are coregulated by NR4A1 and NR4A2. In vivo studies showed that DIM-3,5-Cl2 significantly reduced the growth of endometriotic lesions in a mouse model without inducing cytotoxicity during treatment. Thus, DIM-3,5 derivatives simultaneously suppress NR4A1- and NR4A2-dependent endometriosis progression effectively and represent a promising nonhormonal therapeutic strategy to replace current hormone-based treatments that can be associated with adverse effects.

Orphan nuclear receptor 4A1 (NR4A1) is a member of the NR4A subfamily that was initially discovered as an intermediate early gene expressed in response to stressors, including inflammatory agents. This review addresses the hypothesis that NR4A1 is a key nutrient sensor that contributes to the anti-aging and health-protective effects of receptor ligands, dietary phenolics, and other diet-derived compounds. There is evidence in animal models including humans that NR4A1 serves as an important gene that decreases the rate of aging and its associated diseases. For example, in humans and mice, NR4A1 expression decreases with age and loss of NR4A1 enhances disease susceptibility, and survival curves show that NR4A1-deficient mice live 4 months less than wild-type animals. An extensive comparison of inflammatory diseases, immune dysfunction, and fibrosis in multiple tissues shows that in NR4A1−/− mice and rats these diseases and injuries are enhanced compared to wild-type NR4A1−/− animals. There is evidence showing that structurally diverse NR4A1 ligands reverse the induced adverse effects in NR4A1 wild-type mice. This raises an important question regarding the mechanisms of NR4A1-dependent inhibition of the aging process and the potential for this receptor as a nutrient sensor. It has been well established that polyphenolics, including flavonoids, resveratrol, and other compounds in the diet, are health-protective and decrease the aging process. Recent studies show that resveratrol and flavonoids such as quercetin and kaempferol bind NR4A1 and exhibit protective NR4A1-dependent inhibition of endometriosis and cancer. These limited studies support a role for NR4A1 as a potential dietary sensor of nutrients that are known to be health-protective and a potential nutrient target for improving health.

Glioblastoma multiforme (GBM) is an aggressive brain tumor with a median survival of 15–18 months, driven by treatment resistance and intratumoral heterogeneity. A key driver of these challenges is epithelial-to-mesenchymal transition (EMT), which enhances glioma stemness, invasiveness, and DNA repair mechanisms. Although EMT regulation can be difficult, indirect targeting of key transcription factors driving EMT such as Sp1 and TWIST1 offers a novel strategy. We evaluated bis-indole-derived compounds (CDIMs), inverse agonists of NR4A1 and NR4A2, which have shown efficacy in downregulating TWIST1 and enhancing temozolomide (TMZ) sensitivity. Four patient-derived GBM cell lines were tested individually with TMZ and three CDIM compounds (DIM-3-Cl-5-CF3, DIM-3,5-Cl2, and DIM-3,5-Br2) as well as in combination. CDIMs significantly enhanced TMZ efficacy and exhibit strong synergistic effects (Bliss Synergy Score > 1) across all cell lines ranging from 2.7 to 15.5. DIM-3,5-Cl2 emerged as the lead compound, causing an increased TMZ-induced cytotoxicity in GBM4V cell line by an additional 30.9% (p=0.01), 36.8% (p=0.007), and 30.9% (p=0.03) when combined with 125 µM, 250 µM, and 500 µM of TMZ, respectively. DIM-3,5-Cl2 (12.5 µM) also reduced U87 cell migration by over 50% compared to controls (p<0.05). Additionally, western blot analysis showed that combinatorial treatment of DIM-3,5-Cl2 and TMZ more effectively suppressed TWIST1, Sp1, G9a, β1-integrin, and survivin compared to either compound across all cell lines. In vivo, intracranial xenograft models showed early signs of efficacy, with an average survival extension of 3.3 days in CDIM-treated animals and continued survival in TMZ and combination groups at the time of reporting. These findings support DIM-3,5-Cl2 as a promising therapeutic candidate that targets EMT-related resistance pathways, enhances TMZ cytotoxicity, and impairs GBM migration. Ongoing studies are assessing its effects on stemness and EMT gene expression, as well as its potential to improve TMZ efficacy in vivo.

Tuberculosis (TB), a significant global health issue, needs novel therapeutic approaches to reduce its burden. Studying host-pathogen interactions provides new targets for host-directed therapeutics (HDTs). Nuclear receptors (NRs) are important master regulators of cellular function and bona fide drug targets. Herein, we identify high basal expression of the NR4A NR family in human alveolar macrophages and determine that all 3 members (NR4A1, NR4A2, and NR4A3) are upregulated in response to Mycobacterium tuberculosis (M.tb) infection. NR4A expression was also increased in our recently developed human alveolar macrophage-like (AML) cell model compared to monocyte-derived macrophages. We investigated the role of the NR4As in apoptosis given its importance in controlling M.tb growth. NR4A small interfering RNA knockdown in AML cells prior to their treatment with apoptosis-inducing compounds resulted in reduced caspase-3/7 activity, indicating reduced apoptosis. Additionally, knockdown prior to M.tb infection resulted in reduced apoptosis of AML cells and increased M.tb growth. Treatment of AML cells with NR4A ligands significantly reduced M.tb growth while treatment with an NR4A antagonist significantly increased it. In conclusion, we identify the expression, location, and apoptotic activity of NR4A NRs in human macrophages and their potential as new TB HDT therapeutic targets.

Polyfluoroalkyl substances (PFAS) are widely used industrial compounds that have been identified as contaminants in almost every component of the global ecosystem, and in human studies, higher levels of PFAS have been correlated with increased incidence of multiple diseases. Based on the results of human and laboratory animal studies, we hypothesize that the orphan nuclear receptor 4A1 (NR4A1) may be a critical target for some PFAS such as the legacy linear polyfluorooctanesulfonate (PFOS) and other sulfonates. We show that PFOS and related compounds bound the ligand binding domain (LBD) of NR4A1 and induced the growth of several cancer cell lines and enhanced tumor growth in an athymic nude mouse model. Using NR4A1-responsive rhabdomyosarcoma Rh30 cells as a model, PFOS induced NR4A1-dependent cell proliferation and Rh30 cell migration and invasion. Moreover, in Rh30 cells, PFOS also induces several NR4A1-regulated genes including the PAX3-FOXO1 oncogene and downstream gene products, and in a chromatin immunoprecipitation assay, PFOS does not decrease NR4A1 binding to the promoter. These results demonstrate that PFOS is an NR4A1 ligand and enhances tumorigenesis through the activation of this receptor.

Ferroptosis is an iron-dependent cell death pathway that involves multiple genes, including the transferrin receptor (TFRC/CD71), glutathione peroxidase 4 (GPX4) and cystine-glutamate antiporter (SLC7A11). This study is based on the hypothesis that orphan nuclear receptor 4A1 (NR4A1) and NR4A2 maintain low levels of ferroptosis in triple negative breast cancer (TNBC) cells and bis-indole derived (CDIM) compounds act as NR4A1/2 ligands that induce ferroptosis by enhancing CD71 expression. 1,1-Bis(3′-indolyl)-1-(3,5-disubstitutedphenyl)methane (DIM-3,5) analogs were investigated for their cytotoxicity and effects on NR4A1 and NR4A2 regulated genes and induction of ferroptosis. Several assays also determined enhanced lipoperoxidation, reactive oxygen species and malondialdehyde formation in TNBC cells. Knockdown of NR4A1, NR4A2, Sp1 and Sp4 was carried out by RNA interference. Molecular mechanisms of NR4A1/2-mediated regulation of CD71 expression were determined using CD71-luciferase promoter constructs, overexpression of Sp1 and chromatin immunoprecipitation (ChIP) assays. Initial studies show that DIM-3,5 analogs act as an inverse NR4A1/NR4A2 agonists that downregulate the pro-oncogenic responses/gene products regulated by both receptors in TNBC cells. DIM-3,5 analogs also induced ROS, malondialdehyde and lipoperoxide formation in TNBC cells, and this was accompanied by decreased expression of GPX4 and SLC7A11 and induction of CD71. Induction of CD71, an important biomarker of ferroptosis was observed after treatment of TNBC cells with DIM-3,5 analogs, knockdown of NR4A1, NR4A2, Sp1 or Sp4 demonstrating that induction of CD71 was coregulated by both receptors. Moreover, both promoter and ChIP analysis indicated that NR4A1 and NR4A2 acted as ligand-dependent cofactors of Sp1/4-mediated expression of CD71 in TNBC cells. Thus, CD71, a key biomarker of ferroptosis is an NR4A1/2/Sp regulated gene that can be directly targeted by DIM-3,5 inverse NR4A1/2 agonists to induce ferroptosis in TNBC cells.

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor marked by resistance to standard therapies, largely due to a profoundly immunosuppressive tumor microenvironment (TME). The nuclear orphan receptors NR4A1 and NR4A2 are overexpressed in GBM and act as key drivers of immune dysfunction and epithelial–mesenchymal transition (EMT). Novel dual NR4A1/2 inverse agonists, termed CDIMs, have been shown to inhibit PD-L1 and TWIST1 while reactivating S1PR1, restoring T cell trafficking to the tumor. Three CDIM compounds (DIM-3,5-Cl₂, DIM-3-Cl-5-CF₃, and DIM-4-OH-3,5-(tBu)₂) were administered at low (8-10 mg/kg) and high (20-25 mg/kg) dose for 12 consecutive days in healthy immunocompetent mice to evaluate tolerability and effects on immune cell distribution. Splenocytes, bone marrow, and peripheral blood mononuclear cells (PBMCs) were then isolated and analyzed via FACS. Notably, DIM-3,5-Cl₂ and DIM-3-Cl-5-CF₃ reduced CD4⁺ and CD8⁺ T cells in the bone marrow, with a corresponding increase in PBMCs, supporting reversal of T cell sequestration. To assess therapeutic relevance, the same regimen was applied in a syngeneic orthotopic CT2A glioma model. DIM-3,5-Cl₂ maintained elevated CD4⁺ and CD8⁺ T cells in PBMCs, further supporting its role in restoring systemic T cell circulation and availability for tumor infiltration. In contrast, DIM-3-Cl-5-CF₃ and DIM-4-OH-3,5-(tBu)₂ induced pronounced immune remodeling, including reduced CD19⁺ B cells and expansion of CD11b⁺ and triple-negative (CD11b⁻CD4⁻CD8⁻) populations, thus reshaping adaptive immunity. Ongoing studies will evaluate survival benefit, systemic cytokine responses, and local immune changes within the tumor microenvironment. Brains from treated animals will be analyzed by immunohistochemistry to assess changes in key immune markers to clarify the impact of CDIMs on intratumoral immune composition. Overall, the presented findings position DIM-3,5-Cl₂ as a promising candidate for reversing immune suppression in GBM and enhancing response to immune checkpoint therapies.

The human nuclear receptor (NR) superfamily consists of 48 genes that are ligand-activated transcription factors that play a key role in maintaining cellular homeostasis and in pathophysiology. NRs are important drug targets for both cancer and non-cancer endpoints as ligands for these receptors can act as agonists, antagonists or inverse agonists to modulate gene expression. With two exceptions, the classical mechanism of action of NRs involves their interactions as monomers, dimers or heterodimers with their cognate response elements (cis-elements) in target gene promoters. Several studies showed that a number of NR-regulated genes did not directly bind their corresponding cis-elements and promoter analysis identified that NR-responsive gene promoters contained GC-rich sequences that bind specificity protein 1 (Sp1), Sp3 and Sp4 transcription factors (TFs). This review is focused on identifying an important sub-set of Sp-regulated genes that are indirectly coregulated through interactions with NRs. Subsequent studies showed that many NRs directly bind Sp1 (or Sp3 and Sp4), the NR/Sp complexes bind GC-rich sites to regulate gene expression and the NR acts as a ligand-modulated nuclear cofactor. In addition, several reports show that NR-responsive genes contain cis-elements that bind both Sp TFs and NRs, and mutation of either cis-element results in loss of NR-responsive (inducible and/or basal). Regulation of these genes involves interactions between DNA-bound Sp TFs with proximal or distal DNA-bound NRs, and, in some cases, other nuclear cofactors are required for gene expression. Thus, many NR-responsive genes are regulated by NR/Sp complexes, and these genes can be targeted by ligands that target NRs and also by drugs that induce degradation of Sp1, Sp3 and Sp4.