Glucocorticoid Receptor Target Research Articles

6676 Exposure to Neighborhood Violence Rewires Glucocorticoid Receptor Binding and Drives Proliferation and Invasiveness in Lung Tumor Samples

Abstract Disclosure: H. Heath: None. J. Yoo: None. S. Akter: None. A. Jain: None. V. Sharma: None. H. McGee: None. A. Soliman: None. A. Mohamed: None. A.K. Matthews: None. R. Winn: None. Z. Madak Erdogan: None. S. Kim: None. Background: Despite the lower rates and frequency of smoking, Black men experience a higher incidence of lung cancer compared to white men. This health disparity is even more prevalent in Black men from Chicago, suggesting local neighborhood factors impacting tumorigenesis. Preliminary data indicate that, compared to white men, Black men are more likely to reside in neighborhoods with high levels of violence and have elevated levels of hair cortisol. Objective: To understand the link between lung tumorigenesis and exposure to neighborhood violence, our objective is to investigate the chromatin recruitment of the receptor for cortisol, called the glucocorticoid receptor (GR), and to correlate this recruitment with target gene expression levels. Methods: Utilizing CUT & RUN, we identified the gene binding sites of GR using 15 lung tumor and corresponding healthy tissue samples from lung cancer patients living in Chicago. GR recruitment to chromatin was correlated with the neighborhood homicide rate obtained using patients’ zip codes. Spatial transcriptome profiles for these 15 lung tumor samples were obtained from the Space Ranger pipeline and gene expression hot spots of GR target genes were visualized using Loupe Browser (10x Genomics). Results: GR recruitment to regulatory regions of chromatin increases with greater zip code level violence rates. In patients from high violence neighborhoods, tumor samples had higher magnitude of binding compared to normal neighboring tissue. In tumor samples from patients living in high violence compared to low violence neighborhoods, GR binding is seen in genes that are associated with increased tumor aggressiveness. Hot-spot analysis of these genes found strong co-expression of genes associated with proliferation and invasion in samples from high violence but not low violence neighborhoods. Conclusion: Exposure to neighborhood violence may impact tumor biology via increased GR recruitment to genes associated with proliferation and invasion. Presentation: 6/2/2024

8644 Dynamic Insights into Glucocorticoid Receptor Dysfunction: Novel patient-derived T437I Mutation and Its Impact on Glucocorticoid Receptor Function

Abstract Disclosure: T. Tettey: None. S. Kim: None. K. Wagh: None. Q. Lao: None. L.R. Schiltz: None. D.A. Stavreva: None. D.M. Presman: None. D.P. Merke: None. G.L. Hager: None. The glucocorticoid receptor (GR) is a hormone regulated transcription factor which binds chromatin to regulate many genes, including stress-response and inflammatory genes. Dexamethasone, a GR agonist, is widely used in the treatment of severe inflammation, including COVID-19. GR is vital for life and several GR mutations have been linked to severe pathophysiological conditions. Here we report a novel mutation in the DNA binding domain (DBD) of GR (T437I) in a patient with partial glucocorticoid resistance, a rare genetic disorder characterized by generalized insensitivity to glucocorticoids and a consequent hyperactivation of the hypothalamic pituitary adrenal axis. While the clinical manifestations of this hT437I GR mutation are apparent, its molecular mechanisms remain unexplored. Employing a comprehensive approach that integrates biophysical methods, molecular biology, and genomics, we investigate the molecular underpinnings of this genetic anomaly. Using CRISPR/Cas9 gene editing, we introduce GFP or Halo-tagged hT437I GR mutant into GR null mouse cells. Our findings indicate that the T437I GR variant exhibits normal hormone binding and nuclear translocation. Live-cell imaging using number and brightness analysis reveals that the mutant receptor maintains a wildtype-like oligomeric state but adopts a heterodimeric conformation in the presence of endogenous GR. Global transcriptomics analyses of the mouse hT437I mutant show that most dexamethasone-regulated genes are unresponsive following hormone treatment. We hypothesize that this mutation within the DBD compromises the binding of GR to regulatory sites of target genes. Leveraging fluorescence high-resolution microscopy, we employ single molecule tracking to study the dynamic behavior of Halo-tagged hT437I GR in mouse cells. Our results indicate that the GR mutant has a higher dwell-time on chromatin compared to the wild-type receptor. This suggests that the hT437I mutation impairs the receptor's binding to genomic sites, potentially leading to a failure in regulating GR target genes. This ongoing exploration of the molecular intricacies aims to deepen our understanding of the clinical phenotype observed in the patient. Presentation: 6/2/2024

Fibroblasts as an in vitro model of circadian genetic and genomic studies

Bipolar disorder (BD) is a heritable disorder characterized by shifts in mood that manifest in manic or depressive episodes. Clinical studies have identified abnormalities of the circadian system in BD patients as a hallmark of underlying pathophysiology. Fibroblasts are a well-established in vitro model for measuring circadian patterns. We set out to examine the underlying genetic architecture of circadian rhythm in fibroblasts, with the goal to assess its contribution to the polygenic nature of BD disease risk. We collected, from primary cell lines of 6 healthy individuals, temporal genomic features over a 48 h period from transcriptomic data (RNA-seq) and open chromatin data (ATAC-seq). The RNA-seq data showed that only a limited number of genes, primarily the known core clock genes such as ARNTL, CRY1, PER3, NR1D2 and TEF display circadian patterns of expression consistently across cell cultures. The ATAC-seq data identified that distinct transcription factor families, like those with the basic helix-loop-helix motif, were associated with regions that were increasing in accessibility over time. Whereas known glucocorticoid receptor target motifs were identified in those regions that were decreasing in accessibility. Further evaluation of these regions using stratified linkage disequilibrium score regression analysis failed to identify a significant presence of them in the known genetic architecture of BD, and other psychiatric disorders or neurobehavioral traits in which the circadian rhythm is affected. In this study, we characterize the biological pathways that are activated in this in vitro circadian model, evaluating the relevance of these processes in the context of the genetic architecture of BD and other disorders, highlighting its limitations and future applications for circadian genomic studies.

Abstract 6275: Exposure to neighborhood violence rewires glucocorticoid receptor binding and drives proliferation and invasiveness in lung tumor samples

Abstract Background: Despite smoking less, Black men experience higher incidence of lung cancer compared to white men. This health disparity is even more prevalent in Black men from Chicago, suggesting local neighborhood factors impacting tumorigenesis. Preliminary data indicate that, compared to white men, Black men are more likely to reside in neighborhoods with high levels of violence and have elevated levels of hair cortisol. Objective: To understand the link between lung tumorigenesis and exposure to neighborhood violence, our objective is to investigate the chromatin recruitment of the receptor for cortisol, called the glucocorticoid receptor (GR), and to correlate this recruitment with target gene expression levels. Methods: Utilizing CUT & RUN, we identified the gene binding sites of GR using 15 lung tumor and corresponding healthy tissue samples from lung cancer patients living in Chicago. GR recruitment to chromatin was correlated with the neighborhood homicide rate obtained using patients’ zip codes. Spatial transcriptome profiles for these 15 lung tumor samples were obtained from the Space Ranger pipeline and gene expression hot spots of GR target genes were visualized using Loupe Browser (10x Genomics). Results: GR recruitment to regulatory regions of chromatin increases with greater zip code level violence rates. In patients from high violence neighborhoods, tumor samples had higher magnitude of binding compared to normal neighboring tissue. In tumor samples from patients living in high violence compared to low violence neighborhoods, GR binding is seen in genes that are associated with increased tumor aggressiveness. Hot-spot analysis of these genes found strong co-expression of genes associated with proliferation and invasion in samples from high violence but not low violence neighborhoods. Conclusion: Exposure to neighborhood violence may impact tumor biology via increased GR recruitment to genes associated with proliferation and invasion. Citation Format: Hannah Heath, Jin Young Yoo, Vani Sharma, Hannah McGee, Aiman Soliman, Abeer Mohamed, Alicia K. Matthews, Robert Winn, Zeynep Madak-Erdogan, Sage Kim. Exposure to neighborhood violence rewires glucocorticoid receptor binding and drives proliferation and invasiveness in lung tumor samples [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 6275.

Abstract P04: Role of glucocorticoid receptor in normal and malignant germinal center B cells

Abstract We have reported that super-enhancers (SEs) are specifically hypermutated by Activation-Induced Deaminase (AID)-induced Aberrant Somatic Hypermutation in >90% of diffuse large B cell lymphoma (DLBCL) cases (Bal et al. Nature 2022). Analysis of 122 primary DLBCL biopsies and cell lines identified more than 80 SEs that are recurrently hypermutated, with an average of 12 hypermutated SE/case. Hypermutated SEs are predominantly linked to genes encoding regulators of B cell development and well-known lymphoma oncogenes, including BCL6, BCL2 and CXCR4. We showed that specific hotspot mutations prevent binding and transcriptional downregulation by transcriptional repressors leading to target gene dysregulation. Among these, hypermutation of the SEs linked to BCL2 and CXCR4 abrogates the binding of the glucocorticoid receptor (GR)/transcription factor encoded by NR3C1. As a result, BCL2 and CXCR4 escape GR-mediated transcriptional repression leading to their de-regulated expression in germinal center (GC) B cells. Together with the observation that the NR3C1 gene is genetically inactivated in a small fraction of DLBCL, and the relevance of glucocorticoid-based therapy in DLBCL, these results prompted a comprehensive analysis of the role of GR in normal and malignant B cells. We report that GR is detectable in the nucleus of the great majority of GC B cells, suggesting that it is active as a transcription factor. Similarly, DLBCL primary cases (including GCB-, ABC- and unclassified DLBCL) display GR nuclear expression. Analysis of a conditional GC-specific Nr3c1 knock-out (KO) mouse model showed that GC B cells form normally in the absence of Nr3c1. However, the transcriptional profile of Nr3c1-KO GC B cells was significantly altered compared to wild-type cells. By integrating the GR binding profile obtained in normal human GC B cells with the transcripts differentially expressed in Nr3c1-KO GC B cells, we identified a set of about 2,000 genes that are directly modulated by GR during the GC reaction. Pathway enrichment analysis showed that GR controls several signaling pathways, suggesting a modulatory role on the BCR and CXCR4 pathways. In addition, GR targets display a significant overlap with the BCL6 transcriptional network, suggesting a cooperative role in GC B cell development. Notably, GR was shown to regulate pathways involved in plasma cell differentiation, while repressing targets associated with memory B cell development. In DLBCL cases, several NR3C1 binding sites are recurrently targeted by mutations, suggesting that genetic impairment of GR activity is heterogeneous and extends beyond CXCR4 and BCL2. Together, these data support a role for the glucocorticoid pathway in GC physiology. Recurrent inactivation of the NR3C1 gene or part of its transcriptional regulatory program may contribute to DLBCL pathogenesis, with implications for presently unknown specific roles of glucocorticoids in the therapeutic regimens for DLBCL. Citation Format: Clarissa Corinaldesi, Antony B Holmes, Elodie Bal, Laura Pasqualucci, Katia Basso, Riccardo Dalla-Favera. Role of glucocorticoid receptor in normal and malignant germinal center B cells [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P04.

The glucocorticoid receptor as a master regulator of the Müller cell response to diabetic conditions in mice

Diabetic retinopathy (DR) is considered a primarily microvascular complication of diabetes. Müller glia cells are at the centre of the retinal neurovascular unit and play a critical role in DR. We therefore investigated Müller cell-specific signalling pathways that are altered in DR to identify novel targets for gene therapy. Using a multi-omics approach on purified Müller cells from diabetic db/db mice, we found the mRNA and protein expression of the glucocorticoid receptor (GR) to be significantly decreased, while its target gene cluster was down-regulated. Further, oPOSSUM TF analysis and ATAC- sequencing identified the GR as a master regulator of Müller cell response to diabetic conditions. Cortisol not only increased GR phosphorylation. It also induced changes in the expression of known GR target genes in retinal explants. Finally, retinal functionality was improved by AAV-mediated overexpression of GR in Müller cells. Our study demonstrates an important role of the glial GR in DR and implies that therapeutic approaches targeting this signalling pathway should be aimed at increasing GR expression rather than the addition of more ligand.Graphical

Restricted effects of androgens on glucocorticoid signaling in the mouse prefrontal cortex and midbrain.

Glucocorticoids are key executors of the physiological response to stress. Previous studies in mice showed that the androgen receptor (AR) influenced the transcriptional outcome of glucocorticoid treatment in white and brown adipocytes and in the liver. In the brain, we observed that chronic hypercorticism induced changes in gene expression that tended to be more pronounced in male mice. In the present study, we investigated if glucocorticoid signaling in the brain could be modulated by androgen. After chronic treatment with corticosterone, dihydrotestosterone, a combination of both, and corticosterone in combination with the AR antagonist enzalutamide, we compared the expression of glucocorticoid receptor (NR3C1, also abbreviated GR) target genes in brain regions where AR and GR are co-expressed, namely: prefrontal cortex, hypothalamus, hippocampus, ventral tegmental area and substantia nigra. We observed that androgen affected glucocorticoid signaling only in the prefrontal cortex and the substantia nigra. Dihydrotestosterone and corticosterone independently and inversely regulated expression of Sgk1 and Tsc22d3 in prefrontal cortex. AR antagonism with enzalutamide attenuated corticosterone-induced expression of Fkbp5 in the prefrontal cortex and of Fkbp5 and Sgk1 in the substantia nigra. Additionally, in the substantia nigra, AR antagonism increased expression of Th and Slc18a1, two genes coding for key components of the dopaminergic system. Our data indicate that androgen influence over glucocorticoid stimulation in the brain is not a dominant phenomenon in the context of high corticosterone levels, but can occur in the prefrontal cortex and substantia nigra.

FGF21 Induces Skeletal Muscle Atrophy and Increases Amino Acids in Female Mice: A Potential Role for Glucocorticoids.

Fibroblast growth factor-21 (FGF21) is an intercellular signaling molecule secreted by metabolic organs, including skeletal muscle, in response to intracellular stress. FGF21 crosses the blood-brain barrier and acts via the nervous system to coordinate aspects of the adaptive starvation response, including increased lipolysis, gluconeogenesis, fatty acid oxidation, and activation of the hypothalamic-pituitary-adrenocortical (HPA) axis. Given its beneficial effects for hepatic lipid metabolism, pharmaceutical FGF21 analogues are used in clinical trials treatment of fatty liver disease. We predicted pharmacologic treatment with FGF21 increases HPA axis activity and skeletal muscle glucocorticoid signaling and induces skeletal muscle atrophy in mice. Here we found a short course of systemic FGF21 treatment decreased muscle protein synthesis and reduced tibialis anterior weight; this was driven primarily by its effect in female mice. Similarly, intracerebroventricular FGF21 reduced tibialis anterior muscle fiber cross-sectional area; this was more apparent among female mice than male littermates. In agreement with the reduced muscle mass, the topmost enriched metabolic pathways in plasma collected from FGF21-treated females were related to amino acid metabolism, and the relative abundance of plasma proteinogenic amino acids was increased up to 3-fold. FGF21 treatment increased hypothalamic Crh mRNA, plasma corticosterone, and adrenal weight, and increased expression of glucocorticoid receptor target genes known to reduce muscle protein synthesis and/or promote degradation. Given the proposed use of FGF21 analogues for the treatment of metabolic disease, the study is both physiologically relevant and may have important clinical implications.

E47 as a novel glucocorticoid-dependent gene mediating lipid metabolism in patients with endogenous glucocorticoid excess.

E47 has been identified as a modulating transcription factor of glucocorticoid receptor target genes, its loss protecting mice from metabolic adverse effects of glucocorticoids. We aimed to analyze the role of E47 in patients with endogenous glucocorticoid excess [Cushing's syndrome (CS)] and its association with disorders of lipid and glucose metabolism. This is a prospective cohort study including 120 female patients with CS (ACTH-dependent = 79; ACTH-independent = 41) and 26 healthy female controls. Morning whole blood samples after an overnight fast were used to determine E47 mRNA expression levels in patients with overt CS before and 6-12 months after curative surgery. Expression levels were correlated with the clinical phenotype of the patients. Control subjects underwent ACTH stimulation tests and dexamethasone suppression tests to analyze short-term regulation of E47. E47 gene expression showed significant differences in patient cohorts with overt CS vs. patients in remission (p = 0.0474) and in direct intraindividual comparisons pre- vs. post-surgery (p = 0.0353). ACTH stimulation of controls resulted in a significant decrease of E47 mRNA expression 30min after i.v. injection compared to baseline measurements. Administration of 1 mg of dexamethasone overnight in controls did not change E47 mRNA expression. E47 gene expression showed a positive correlation with total serum cholesterol (p = 0.0036), low-density lipoprotein cholesterol (p = 0.0157), and waist-arm ratio (p = 0.0138) in patients with CS in remission. E47 is a GC-dependent gene that is upregulated in GC excess potentially aiming at reducing metabolic glucocorticoid side effects such asdyslipidemia.

Exposure to low-intensity noise exacerbates nonalcoholic fatty liver disease by activating hypothalamus pituitary adrenal axis

Noise exposure induces metabolic disorders, in a latent, chronic and complex way. However, there is no direct evidence elucidating the relationship between low-intensity noise exposure and nonalcoholic fatty liver disease (NAFLD). Male mice (n = 5) on high-fat diet (HFD) were exposed to an average of 75 dB SPL noise for 3 months to reveal the effect of noise exposure on NAFLD, where the potential mechanisms were explored. In vivo (n = 5) and in vitro models challenged with dexamethasone (DEX) were used to verify the role of hypothalamus pituitary adrenal (HPA) axis activation in hepatic lipid metabolism. Typical chronic-restraint stress (CRS, n = 8) was used to explore the role of depression in modifying activity of HPA axis. Finally, animal experiment (n = 8) was repeated to validate the roles of depression and HPA axis activation in NAFLD development. Chronic low-intensity noise exposure exacerbated NAFLD in mice on HFD characterized by hepatocyte steatosis, modified lipid metabolism and inflammation level. Plasma ACTH in H + N group was 1.5-fold higher than that in HFD group. Transcription of glucocorticoid receptor target genes was increased by chronic low-intensity noise exposure in HFD-treated mice. Excessive glucocorticoids mimicking HPA axis activation induced NAFLD in vivo and in vitro. Plasma ACTH increase and lipid storage also occurred in depressive mice stressed by CRS. More interestingly, the same noise exposure simultaneously induced depression in mice, disrupted the HPA axis homeostasis and exacerbated NAFLD in a repeated experiment. Thus, three-month exposure to 75 dB SPL noise was sufficient to exacerbate NAFLD progress in mice, where activation of HPA axis played a critical role. Depression played an intermediate role and contributed to HPA axis activation up-stream of the exacerbation.

Klf9 plays a critical role in GR –dependent metabolic adaptations in cardiomyocytes

Glucocorticoids through activation of the Glucocorticoid receptor (GR) play an essential role in cellular homeostasis during physiological variations and in response to stress. Our genomic GR binding and transcriptome data from Dexamethasone (Dex) treated cardiomyocytes showed an early differential regulation of mostly transcription factors, followed by sequential change in genes involved in downstream functional pathways. We examined the role of Krüppel-like factor 9 (Klf9), an early direct target of GR in cardiomyocytes. Klf9-ChIPseq identified 2150 genes that showed an increase in Klf9 binding in response to Dex. Transcriptome analysis of Dex treated cardiomyocytes with or without knockdown of Klf9 revealed differential regulation of 1777 genes, of which a reversal in expression is seen in 1640 genes with knockdown of Klf9 compared to Dex. Conversely, only 137 (∼8%) genes show further dysregulation in expression with siKLf9, as seen with Dex treated cardiomyocytes. Functional annotation identified genes of metabolic pathways on the top of differentially expressed genes, including those involved in glycolysis and oxidative phosphorylation. Knockdown of Klf9 in cardiomyocytes inhibited Dex induced increase in glycolytic function and mitochondrial spare respiratory capacity, as measured by glycolysis and mito stress tests, respectively. Thus, we conclude that cyclic, diurnal GR activation, through Klf9 -dependent feedforward signaling plays a central role in maintaining cellular homeostasis through metabolic adaptations in cardiomyocytes.

Abstract P1148: Glucocorticoid Receptor - Klf9 Axis Regulates Cardiomyocyte Metabolic Homeostasis

Glucocorticoids (GCs) through activation of the Glucocorticoid receptor (GR) plays an essential role in cellular homeostasis during physiological variations and in response to stress. GC-GR signaling is involved in regulating several cellular processes including metabolism, circadian rhythm and inflammation for diurnal adaptations. Our genomic GR binding (ChIP) and transcriptome (RNAseq) data from Dexamethasone (Dex) treatment in cardiomyocytes show an early (1hr) differential regulation of mostly transcription factors, followed by sequential change in downstream signaling pathways (6-12hr). Here, we examine the role of an early direct target of GR in cardiomyocytes, Krüppel-like factor 9 (Klf9) in metabolic homeostasis. Our Klf9-ChIPseq identified 4100 genes with change in promoter Klf9 binding in response to Dex. Functional annotation of these genes lists metabolic pathway on the top of KEGG pathway, along with genes regulating transcription and survival. Interestingly, integration of GR and Klf9 data show overlapping targets (1181), suggesting that Klf9 could be serving as feedback regulator for these genes. Further, our transcriptome analysis of Dex treated cardiomyocytes with knockdown of Klf9 reveal differential regulation of 1777 genes (Dex+siKLf9 vs Dex+siLUC), of which a reversal in expression is seen in 1640 (92%) genes with siKlf9 vs. Dex. Conversely, only 137 (8%) genes show further dysregulation in expression with siKLf9 as Dex. Gene ontology of these 1640 genes show metabolic pathway on the top, including genes involved in glycolysis and oxidative phosphorylation. Conversely, mostly immuno-regulatory genes are among those 137 genes. Expectedly, knockdown of Klf9 in cardiomyocytes inhibits Dex induced increase in glycolysis and glycolytic capacity by 24%, and spare respiratory capacity by 50%, as measured by ECAR and OCR with glycolysis and mito stress tests, respectively. Thus, we conclude that cyclic, diurnal GC mediated GR activation, through Klf9 -dependent feedforward signaling plays a central role in maintaining cellular metabolic homeostasis. With hypertrophic stress a decrease in cardiac GR and dysregulated Klf9 signaling might result in metabolic crisis, contributing to mitochondrial and cardiac dysfunction

REDD1 (regulated in development and DNA damage1) modulates the glucocorticoid receptor function in keratinocytes.

Glucocorticoids (GCs) are widely used for the treatment of inflammatory skin diseases despite significant adverse effects including skin atrophy. Effects of GCs are mediated by the glucocorticoid receptor (GR), a well-known transcription factor. Previously, we discovered that one of the GR target genes, REDD1, is causatively involved in skin atrophy. Here, we investigated its role in GR function using HaCaT REDD1 knockout (KO) keratinocytes. We found large differences in transcriptome of REDD1 KO and control Cas9 cells in response to glucocorticoid fluocinolone acetonide (FA): both the scope and amplitude of response were significantly decreased in REDD1 KO. The status of REDD1 did not affect GR stability/degradation during self-desensitization, and major steps in GR activation-its nuclear import and phosphorylation at activating Ser211. However, the amount of GR phosphorylated at Ser226 that may play negative role in GR signalling, was increased in the nuclei of REDD1 KO cells. GR nuclear import and transcriptional activity also depend on the composition of GR chaperone complex: exchange of chaperone FKBP51 (FK506-binding protein 5) for FKBP52 (FK506-binding protein 4) being a necessary step in GR activation. We found the increased expression and abnormal nuclear translocation of FKBP51 in both untreated and FA-treated REDD1 KO cells. Overall, our results suggest the existence of a feed-forward loop in GR signalling mediated by its target gene REDD1, which has translational potential for the development of safer GR-targeted therapies.

Extracellular Heat Shock Protein 70 Increases the Glucocorticoid Receptor and Dual-Specificity Phosphatase 1 via Toll-like Receptor 4 and Attenuates Inflammation in Airway Epithelial Cells.

Heat shock protein 70 (HSP70) regulates the ligand binding of the glucocorticoid receptor (GR). In asthma patients, heat treatment increased both the GR expression and secretion of extracellular HSP70 (eHSP70) by bronchial epithelial cells (EC). The objective of this study was to assess the effects of eHSP70 on GR expression and the GR-dependent regulation of immune response in human bronchial ECs. Cells were treated with either eHSP70 or transfected with an expression vector for intracellular HSP70 (iHSP70). Ribonucleic acid (RNA) and protein levels were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. Interleukin (IL-6 and IL-8) secretion was determined by enzyme linked immunosorbent assay (ELISA). The overexpression of iHSP70 decreased, while eHSP70 increased GR expression. In addition, eHSP70 increased the expression of the GR target dual-specificity phosphatase 1 (DUSP-1). In doing so, eHSP70 reduced the tumor growth factor (TGF)-β1-dependent activation of extracellular signal-regulated kinase (Erk)-1/2 and cyclic AMP response element binding protein (CREB) and the secretion of IL-6 and IL-8. Blocking the GR or Toll-like receptor 4 (TLR4) counteracted all eHSP70-induced effects. This study demonstrates a novel anti-inflammatory effect of eHSP70 by the signaling cascade of TLR4-GR-DUSP1, which inhibits TGF-β1-activated pro-inflammatory ERK1/2-CREB signaling and cytokine secretion. The findings suggest that eHSP70 might present a novel non-steroidal therapeutic strategy to control airway inflammation in asthma.

Identification of Glucocorticoid Receptor Target Genes That Potentially Inhibit Collagen Synthesis in Human Dermal Fibroblasts.

Over several decades, excess glucocorticoids (GCs) of endogenous or exogenous origin have been recognized to significantly inhibit collagen synthesis and accelerate skin aging. However, little is known regarding their molecular mechanisms. We hypothesized that the action of GCs on collagen production is at least partially through the glucocorticoid receptor (GR) and its target genes, and therefore aimed to identify GR target genes that potentially inhibit collagen synthesis in Hs68 human dermal fibroblasts. We first confirmed that dexamethasone, a synthetic GC, induced canonical GR signaling in dermal fibroblasts. We then collected 108 candidates for GR target genes reported in previous studies on GR target genes and verified that 17 genes were transcriptionally upregulated in dexamethasone-treated dermal fibroblasts. Subsequently, by individual knockdown of the 17 genes, we identified that six genes, AT-rich interaction domain 5B, FK506 binding protein 5, lysyl oxidase, methylenetetrahydrofolate dehydrogenase (NADP + dependent) 2, zinc finger protein 36, and zinc fingers and homeoboxes 3, are potentially involved in GC-mediated inhibition of collagen synthesis. The present study sheds light on the molecular mechanisms of GC-mediated skin aging and provides a basis for further research on the biological characteristics of individual GR target genes.

Folate and Cobalamin Deficiencies during Pregnancy Disrupt the Glucocorticoid Response in Hypothalamus through N-Homocysteinilation of the Glucocorticoid Receptor.

Vitamin B9 (folate)/B12 (cobalamin) deficiency is known to induce brain structural and/or functional retardations. In many countries, folate supplementation, targeting the most severe outcomes such as neural tube defects, is discontinued after the first trimester. However, adverse effects may occur after birth because of some mild misregulations. Various hormonal receptors were shown to be deregulated in brain tissue under these conditions. The glucocorticoid receptor (GR) is particularly sensitive to epigenetic regulation and post-translational modifications. In a mother-offspring rat model of vitamin B9/B12 deficiency, we investigated whether a prolonged folate supplementation could restore the GR signaling in the hypothalamus. Our data showed that a deficiency of folate and vitamin B12 during the in-utero and early postnatal periods was associated with reduced GR expression in the hypothalamus. We also described for the first time a novel post-translational modification of GR that impaired ligand binding and GR activation, leading to decrease expression of one of the GR targets in the hypothalamus, AgRP. Moreover, this brain-impaired GR signaling pathway was associated with behavioral perturbations during offspring growth. Importantly, perinatal and postnatal supplementation with folic acid helped restore GR mRNA levels and activity in hypothalamus cells and improved behavioral deficits.

Phase I trial of enzalutamide (Enz) plus the glucocorticoid receptor antagonist relacorilant (Rela) for patients with metastatic castration resistant prostate cancer.

5062 Background: Resistance to androgen receptor (AR) targeted therapies is common in mCRPC. Glucocorticoid receptor (GR) expression increases with AR inhibition in patients (pts) and blockade of GR signaling inhibits CRPC growth in preclinical models when combined with AR blockade. We thus conducted a phase I study of the combination of rela and enz in patients with mCRPC. Methods: The study used a 6+3 design to assess safety and pharmacokinetics (PK) of the 2-drug combination. The starting dose of rela 100mg/day was chosen based on current clinical studies indicating this dose was both safe for daily dosing and pharmacologically active and the starting dose for Enz was 120mg based on anticipated drug-drug interactions. Escalating doses of rela were combined with Enz 120 using pre-specified dose escalation rules. An additional 12 patients were to be assessed at the MTD to refine safety and PK profiles. Primary endpoint was safety of the combination and to establish safe and pharmacologically active doses of both agents. Key secondary endpoints were tumor response per RECIST 1.1 criteria and progression-free survival (PFS; defined as either PSA progression, radiographic progression or death). PSA was measured after 12 weeks or at the last time at which PSA data was available, including times <12 weeks. Results: 35 pts were enrolled. Pts had a median age of 74 (range 62-85) and baseline PSA of 89.5 (range 7.3-2391). 100% had prior potent AR signaling inhibitor (abi or enz) with 89% receiving prior enz, 94% prior abi, 83% prior both. 69% of pts received prior docetaxel. Median PSA change was 0.1%. Enz 120mg + Rela 100mg and Enz 120+Rela 150 were found to be safe and tolerable and achieved desirable Enz PK. DLTs were noted in 3 pts at the 200mg Rela dose, which was deemed to have exceeded the MTD, which was thus defined as 150mg Rela+120mg Enz. Most common ≥ grade 3 treatment related adverse events (TRAEs) were fatigue (11% of pts), anemia (9%), abdominal pain (6%) and pericardial effusion (6%). Median PFS was 2.5 months for 21 of 35 evaluable pts. Best response per RECIST 1.1 were Not Evaluable (40%), SD (34%) & PD (26%). No CR or PR were noted. Most common reasons for discontinuation from trial included progressive disease/hospice (82%), AEs (12%), physician discretion (3%) & death (3%). 2 pts remain on trial as of data cutoff (12/31/22). Conclusions: The combination of enzalutamide and relacorilant was safe & largely well tolerated. The majority of pts were discontinued from trial due to progressive disease or hospice. Further analyses of secondary & correlative endpoints such as translational/endocrine biomarkers, GR target gene expression and circulating tumor cells are ongoing. Clinical trial information: NCT03674814 .

EJE Prize 2023: genes on steroids-genomic control of hepatic metabolism by the glucocorticoid receptor.

Glucocorticoids are essential hormones produced by the adrenal cortex with prominent circadian rhythmicity and in times of stress. Glucocorticoids maintain liver homeostasis through coordinated activities that control the major pathways of energy metabolism. Glucocorticoids activate the glucocorticoid receptor (GR), a nuclear hormone receptor that regulates the transcription of hundreds of genes in response to ligand. This review aims to provide a comprehensive overview of glucocorticoid receptor signaling impact on glucose, amino acid, and lipid metabolism in the liver. We integrate fundamental and current findings elucidating key GR-regulated pathways from a physiologic, biochemical, and molecular point-of-view. Here, we focus on the transcriptional regulation of well-characterized hepatic GR target genes, and on those GR co-factors that coordinate nutritional and hormonal signals.

Abstract 3434: Selective glucocorticoid receptor modulation reveals a new role for CLEC10A in patients with solid tumors

Abstract Selective glucocorticoid receptor (GR) modulators (SGRMs) represent a clinically validated mechanism of enhancing chemosensitivity and ameliorating the sequelae of hypercortisolism. While GRs reportedly control the expression of up to 20% of the human genome, specific targets of systemic GR antagonism in humans have yet to be identified. To address this question, we established a robust NanoString assay to measure candidate GR target genes in human blood and assessed them before and after therapy with the oral SGRM relacorilant, which selectively antagonizes the GR. In a randomized, controlled phase 2 study in patients with recurrent, platinum-resistant ovarian cancer (NCT03776812), gene changes were assessed in 139 patients after treatment with relacorilant + nab-paclitaxel (NP) or NP alone. Using cross-validated random forest methods, a gene panel that accurately identified patients who had received relacorilant was established (ROC AUC 0.945 +/- 0.038). In 13 patients who crossed over from NP alone to relacorilant + NP, the genes in the panel were modified only after crossover (paired t-test P=0.0003), validating the gene panel’s ability to detect GR activity. The 4 most induced (LILRB4, FPR3, CLEC10A, CCR2) and 4 most suppressed (CDKN1C, TNFRSF17, BRIP1, PDK1) genes by relacorilant + NP vs. NP alone were selected and shown to be reliable indicators of GR activity across clinical studies in patients with pancreatic, prostate, and other solid tumors (NCT04329949, NCT03674814, NCT02762981), independent of concomitant medications or other confounders. CLEC10A (C-type lectin domain containing 10A) was identified as a particularly sensitive marker of GR activity as its expression was strongly suppressed by the GR agonist prednisone in healthy volunteers (paired t-test P<0.00010, NCT03335956) and strongly induced by relacorilant in patients with solid tumors (paired t-test P<0.00010). CLEC10A induction in blood by relacorilant was associated with longer overall survival in patients with ovarian cancer treated with relacorilant + NP (HR=0.39, Cox PH P=0.0135). High baseline tumor CLEC10A expression was also associated with longer overall survival in a large tumor ‘omics’ database (N=1656 ovarian tumors; HR 0.8, log-rank P=0.0008). CLEC10A and the genes correlated with its expression were found to be primarily expressed by a specific subset of dendritic cells, supporting previous reports that GR agonism can suppress dendritic cells. This analysis of CLEC10A and other identified GR target genes confirmed that systemic GR activity in patients with a range of solid tumors can be modulated pharmacologically, indicates that GR modulation may be associated with survival in patients with ovarian cancer, and provides new insights into the systemic functions of the GR in humans. Citation Format: Nicoletta Colombo, Toon Van Gorp, Ursula A. Matulonis, Ana Oaknin, Rachel N. Grisham, Diane Provencher, Gini F. Fleming, Alexander B. Olawaiye, Erkut H. Borazanci, Russell Z. Szmulewitz, Subhagya A. Wadekar, Grace Mann, Hazel J. Hunt, Andrew E. Greenstein, Domenica Lorusso. Selective glucocorticoid receptor modulation reveals a new role for CLEC10A in patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3434.

Phase 1 efficacy and pharmacodynamic results of exicorilant + enzalutamide in patients with metastatic castration-resistant prostate cancer.

145 Background: Metastatic castration-resistant prostate cancer (mCRPC) remains an incurable disease with significant morbidity. Androgen receptor (AR) signaling is a key driver of tumor growth in mCRPC, and AR-targeted therapies are the mainstay for patients (pts) with locally advanced or metastatic disease. Enzalutamide (ENZA) is commonly used, but resistance typically develops within 1–2 years. The glucocorticoid receptor (GR) can substitute for the AR, providing a tumor escape pathway (Arora et al. Cell 2013). In the 22Rv1 CRPC xenograft model, the selective GR modulator exicorilant (EXI) combined with ENZA reduced tumor growth, supporting the hypothesis that dual antagonism of GR + AR may block this escape pathway. Safety and pharmacokinetics from the first study of EXI + ENZA in pts with mCRPC (NCT03437941) were previously presented (Linch et al. ESMO 2022). Here, we report efficacy and pharmacodynamic (PD) results from the same study. Methods: Segment 1 (Seg 1) of this phase 1 study evaluated open-label, fasting, BID dosing of EXI (140 or 180 mg) + ENZA 160 mg QD. Segment 2 (Seg 2) tested QD dosing of EXI with food in a double-blind design: All pts received EXI 240 mg + ENZA and were randomized 3:1 to EXI titration (to 280 mg followed by 320 mg) or to remain on EXI 240 mg + placebo. Efficacy assessments included radiographic response and changes in prostate-specific antigen (PSA) levels. PSA was collected prior to study entry and PSA doubling times were calculated before and during treatment. PD analyses included baseline (BL) tumor GR expression and modulation of GR target genes in whole blood. As not all pts enrolled in Seg 1 were ENZA naïve, efficacy data are reported for Seg 2 only. Data cutoff date: July 7, 2022. Results: 39 pts were enrolled (Seg 1: 14, irrespective of prior ENZA exposure; Seg 2: 25, on a stable ENZA dose with rising PSA, defined as a 25% increase over nadir and absolute value >1 ng/mL). In Seg 2, there were no radiographic responses, 18 pts had a best overall response of stable disease per PCWG3, and 1 pt achieved a PSA response (≥50% PSA reduction from BL). BL tumor GR expression was detectable in all assessed tumors. PD analyses demonstrated EXI modulation of GR target genes, such as CDKN1C. Comparable PD effects were observed across EXI doses (240–320 mg QD). While BL 24-h urinary free cortisol (UFC) for most pts was within the normal range, improvements in PSA doubling times after treatment with EXI + ENZA were predominantly observed in pts with higher BL UFC ( P<0.05). Conclusions: This study did not include an ENZA-alone arm and was thus not designed to assess the contribution of EXI to the efficacy of the EXI + ENZA combination. In heavily pretreated pts with prior ENZA exposure, 1 PSA response and no radiographic responses were observed. PD analysis confirmed systemic GR modulation. Instances of PSA doubling time improvements were observed in pts with relatively higher UFC. Clinical trial information: NCT03437941 .