Expression Of Glucocorticoid Receptor Target Genes Research Articles

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 .

Abstract 5728: A druggable FOXA1-glucocorticoid receptor transcriptional axis drives tumor growth in NSCLC

Abstract FOXA1 is a pioneer factor that is essential to steroid receptor function in hormone-dependent adenocarcinomas, including breast and prostate cancer. FOXA1 expression is also observed in other tumors of epithelial lineage such as non-small cell lung cancer (NSCLC), but whether and how it is required for tumor growth in these contexts is not known. Analyzing data from genome-scaled pooled loss-of-function screens, we identified and validated a subset of NSCLC cell lines with reduced cellular fitness following FOXA1 knockout. We performed a rapid immunoprecipitation and mass spectrometry of endogenous proteins (RIME) screen to nominate candidate factors that may cooperate with FOXA1 to mediate oncogenic transcription programs. This screen revealed a chromatin-localized interaction between FOXA1 and the glucocorticoid receptor (GR). Depletion of GR expression using shRNA or CRISPR/Cas9 suppressed the proliferation of FOXA1-dependent, but not FOXA1-independent NSCLC models, suggesting GR specifically contributes to tumor growth in the FOXA1-dependent context. To assess the potential therapeutic utility of targeting the FOXA1-GR signaling axis, we employed two structurally distinct ligand antagonists of GR from ORIC Pharmaceuticals: ORIC-101, a steroidal antagonist, and OP-4954, a non-steroidal antagonist. Both antagonists inhibited dexamethasone stimulated GR-transactivation of a glucocorticoid response element (GRE)-luciferase reporter with equal potency. However, proliferation and colony formation of the FOXA1/GR-dependent NSCLC cells were only inhibited upon treatment with ORIC-101, and not OP-4954. Gene expression analysis uncovered a discrete set of GR target genes, including SFTPB, TRAF4 and SGK1, whose expression was reduced by ORIC-101 but unaffected or even induced by OP-4954. The specific inhibition of GR target gene expression and attendant induction of apoptosis by ORIC-101 suggest a hierarchy of GR targets relevant to the FOXA1/GR-growth program in NSCLC. Finally, we evaluated the in vivo therapeutic potential of ORIC-101 using NSCLC xenografts and found that single-agent ORIC-101 exhibited potent antitumor effects in these models. Taken together, these data establish a model of FOXA1-GR-dependent tumor growth amongst a subset of NSCLCs and demonstrate that effective blockade of this complex is a rational therapeutic avenue to be explored in the clinic. Citation Format: Madeline Dorso. A druggable FOXA1-glucocorticoid receptor transcriptional axis drives tumor growth in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5728.

Abstract P041: Initial results from a phase 1b study of ORIC-101, a glucocorticoid receptor antagonist, in combination with enzalutamide in patients with metastatic prostate cancer

Abstract Background: Upregulation of the glucocorticoid receptor (GR) is a potential mechanism of resistance to enzalutamide and other androgen receptor (AR) modulators in prostate cancer. Preclinical studies have demonstrated that GR activation can bypass enzalutamide-mediated AR inhibition and support prostate cancer cell growth. Overexpression of GR is associated with poor outcomes in castration-resistant prostate cancer patients (CRPC) treated with enzalutamide. ORIC-101 is a potent and selective orally bioavailable, small molecule antagonist of GR. Mechanistically, ORIC-101 inhibits GR transcriptional activity and blocks the pro-survival signals mediated by the activated nuclear hormone receptor. Methods: A modified interval 3+3 (i3+3) design was used to assess safety, pharmacokinetics (PK), and pharmacodynamics (PD) to select the Recommended Phase 2 Dose (RP2D) of ORIC-101 in combination with enzalutamide in patients with metastatic CRPC progressing on enzalutamide 160 mg, dosed once daily (NCT04033328). ORIC-101, at doses ranging from 80 to 240 mg once daily, given in a continuous dosing regimen, was added to enzalutamide at the time of disease progression. Plasma PK and PD biomarkers were assessed on multiple days and times before and after dosing. PD modulation in blood-derived peripheral blood mononuclear cells (PBMCs) was assessed by RT-qPCR for GR target genes. Antitumor activity was assessed by Prostate Cancer Clinical Trials Working Group 3 (PCWG3) and RECIST 1.1. Results: 10 patients were treated in 3 cohorts in the dose escalation portion of the study. ORIC-101 exposure increased with dose and no drug-drug interaction (DDI) was observed that necessitated reduction from the standard enzalutamide dose of 160 mg. No dose limiting toxicities were observed at any dose level. Based upon plasma exposure and PD modulation, the RP2D was established as 240 mg ORIC-101 plus 160 mg enzalutamide, both dosed once daily continuously in 28-day cycles. All adverse events (AEs) were Grade 1 or 2 with the most common (>15%), treatment-related AEs being fatigue (40%), nausea (30%), constipation (20%), decreased appetite (20%), high aspartate aminotransferase (20%), high alkaline phosphatase (20%), and headache (20%). There were no Grade ≥3 treatment-related AEs. Biomarker data demonstrated ORIC-101 induced reduction in GR target gene expression in PBMCs, indicating PD modulation across dose levels of ORIC-101. Data will be updated at the time of the presentation. Conclusions: Preliminary evidence suggests that ORIC-101 effectively modulates GR and has an acceptable tolerability profile when combined with enzalutamide. Dose expansion is ongoing at the RP2D. Citation Format: Wassim Abida, Neeraj Agarwal, Andrew W. Hahn, Neal Shore, Paul Sieber, Tanya Dorff, Mathew Rettig, Mathew Smith, Paul Monk, Rongda Xu, Ann Johnson, Anneleen Daemen, Edna Chow Maneval, Pratik S. Multani, Rupal Patel, Michael J. Morris. Initial results from a phase 1b study of ORIC-101, a glucocorticoid receptor antagonist, in combination with enzalutamide in patients with metastatic prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P041.

Impaired glucocorticoid receptor expression in liver disrupts feeding-induced gene expression, glucose uptake, and glycogen storage.

The transition from a fasted to a fed state is associated with extensive transcriptional remodeling in hepatocytes facilitated by hormonal- and nutritional-regulated transcription factors. Here, we use a liver-specific glucocorticoid receptor (GR) knockout (L-GRKO) model to investigate the temporal hepatic expression of GR target genes in response to feeding. Interestingly, in addition to the well-described fasting-regulated genes, we identify a subset of hepatic feeding-induced genes that requires GR for full expression. This includes Gck, which is important for hepatic glucose uptake, utilization, and storage. We show that insulin and glucocorticoids cooperatively regulate hepatic Gck expression in a direct GR-dependent manner by a 4.6 kb upstream GR binding site operating as a Gck enhancer. L-GRKO blunts preprandial and early postprandial Gck expression, which ultimately affects early postprandial hepatic glucose uptake, phosphorylation, and glycogen storage. Thus, GR is positively involved in feeding-induced gene expression and important for postprandial glucose metabolism in the liver.

Initial results from a phase 1b study of ORIC-101, a glucocorticoid receptor antagonist, in combination with nab-paclitaxel in patients with advanced solid tumors.

2553 Background: ORIC-101 is a potent and selective, orally bioavailable, small molecule antagonist of the glucocorticoid receptor (GR). Preclinical studies have demonstrated that activation of GR signaling leads to decreased responsiveness to chemotherapeutics (eg, taxanes) and antiandrogens across multiple tumor types. Mechanistically, ORIC-101 inhibits GR transcriptional activity and blocks the prosurvival signals mediated by the activated nuclear hormone receptor. Methods: A 3+3 dose escalation design was used to assess safety, pharmacokinetics (PK), pharmacodynamics (PD), and select the Recommended Phase 2 Dose (RP2D) of ORIC-101 in combination with nab-paclitaxel (nab-pac; NCT03928314). ORIC-101 doses ranging from 80 to 240 mg once daily, given either intermittently or in a continuous dosing regimen, were evaluated in combination with weekly nab-pac at 75 or 100 mg/m2. Plasma PK and PD biomarkers were assessed on day 1 and after repeat dosing. PD modulation in blood-derived peripheral blood mononuclear cells (PBMCs) was assessed by RT-qPCR for GR target genes. Antitumor activity was assessed by RECIST v1.1. Results: 21 patients with 10 different solid tumors, with and without a prior taxane, were treated in 5 cohorts. ORIC-101 exposure increased with dose, with no evidence for drug-drug interaction with nab-pac. In the initial cohort at 240 mg ORIC-101 and 100 mg/m2 nab-pac, 2 patients experienced dose limiting toxicities (DLTs) of Grade 3 fatigue and Grade 4 neutropenia/thrombocytopenia, respectively. No further DLTs were observed in subsequent cohorts and the RP2D was established as 160 mg ORIC-101 dosed once daily continuously for 21 days with nab-pac 75 mg/m2 given on days 1, 8, and 15 of each 28-day cycle, without requirement for prophylactic granulocyte colony-stimulating factor (G-CSF). The most common (> 15%), all grade treatment-related adverse events (AEs) were nausea (38%), diarrhea (33%), fatigue (29%), leukopenia (29%), neutropenia (29%), anemia (24%), and 19% of patients had increased liver function tests and alopecia. Biomarker data demonstrated ORIC-101-induced reduction in GR target gene expression in PBMCs, indicating PD modulation at all dose levels of ORIC-101. Preliminary antitumor activity was observed in 3 taxane-refractory patients with breast, endometrial, and pancreatic cancers. Conclusions: The combination of ORIC-101 and nab-paclitaxel demonstrated an acceptable tolerability profile and does not require prophylactic G-CSF. PK and PD showed no evidence of drug-drug-interaction and demonstrated GR target inhibition. Preliminary antitumor activity was observed in patients with solid tumors that previously progressed on a taxane-containing regimen. Dose expansion is ongoing at the RP2D in dedicated pancreatic, ovarian, triple negative breast cancers, and tissue-agnostic cohorts. Clinical trial information: NCT03928314.

BCOR modulates transcriptional activity of a subset of glucocorticoid receptor target genes involved in cell growth and mobility

Glucocorticoid (GC) receptor (GR) is a key transcription factor (TF) that regulates vital metabolic and anti-inflammatory processes. We have identified BCL6 corepressor (BCOR) as a dexamethasone-stimulated interaction partner of GR. BCOR is a component of non-canonical polycomb repressor complex 1.1 (ncPCR1.1) and linked to different developmental disorders and cancers, but the role of BCOR in GC signaling is poorly characterized. Here, using ChIP-seq we show that, GC induces genome-wide redistribution of BCOR chromatin binding towards GR-occupied enhancers in HEK293 cells. As assessed by RNA-seq, depletion of BCOR altered the expression of hundreds of GC-regulated genes, especially the ones linked to TNF signaling, GR signaling and cell migration pathways. Biotinylation-based proximity mapping revealed that GR and BCOR share several interacting partners, including nuclear receptor corepressor NCOR1. ChIP-seq showed that the NCOR1 co-occurs with both BCOR and GR on a subset of enhancers upon GC treatment. Simultaneous depletion of BCOR and NCOR1 influenced GR target gene expression in a combinatorial and gene-specific manner. Finally, we show using live cell imaging that the depletion of BCOR together with NCOR1 markedly enhances cell migration. Collectively, our data suggest BCOR as an important gene and pathway selective coregulator of GR transcriptional activity.

MLL2 regulates glucocorticoid receptor-mediated transcription of ENACα in human retinal pigment epithelial cells

Glucocorticoids require the glucocorticoid receptor (GR), a type of ligand-dependent nuclear receptor to transmit their downstream effects. Upon glucocorticoid binding, GR associates with glucocorticoid response elements (GREs) and recruits other transcriptional coregulators to activate or repress target gene transcription. Many SET-domain family proteins have been demonstrated to contribute to GR-mediated transcriptional activity. However, whether histone H3K4-specific methyltransferase plays a cell-type-specific role in GR transcriptional regulation remains poorly understood. In this report, we examined MLL2 (KMT2D), a histone-lysine methyltransferase that catalyzes histone H3 lysine 4 methylation (H3K4me). Furthermore, we demonstrated that MLL2 specifically regulates the transcription of some GR target genes (e.g., ENACα and FLJ20371) in ARPE-19 cells, but has no effect in A549 cells. Mechanistically, co-immunoprecipitation assays revealed that MLL2 is associated with GR in a ligand-independent manner in APRE-19 cells. Moreover, chromatin immunoprecipitation analyses demonstrated that MLL2 could co-occupy glucocorticoid response elements (GREs) of GR target genes along with GR following Dex stimulation. Finally, the FAIRE-qPCR results illustrated that MLL2 is pivotal in establishing chromatin structure accessibility at the GREs of ARPE-19 specific genes in the presence of Dex. Taken together, our study determined that MLL2 regulates GR-mediated transcription in a cell-type-specific manner, and we provide a molecular mechanism to explain the specific role of MLL2 in regulating GR target gene expression in ARPE-19 cells.

P38 MAPK and glucocorticoid receptor crosstalk in bronchial epithelial cells

p38 MAPK inhibition may have additive and synergistic anti-inflammatory effects when used with corticosteroids. We investigated crosstalk between p38 MAPK inhibitors and corticosteroids in bronchial epithelial cells to investigate synergistic effects on cytokine production and the molecular mechanisms involved. Effects of the p38 MAPK inhibitor BIRB-796 and dexamethasone alone and in combination on LPS, polyI:C or TNFα -induced IL-6, CXCL8 and RANTES were assessed in 16HBEs (human epithelial cell line) and on TNFα-induced IL-6 and CXCL8 in primary human epithelial cells from asthma patients and healthy controls. 16HBEs were used to assess effects of BIRB-796 alone and in combination with dexamethasone on glucocorticoid receptor (GR) activity by reporter gene assay, expression of GR target genes and nuclear localisation using Western blot. The effects of BIRB-796 on TNFα stimulated phosphorylation of p38 MAPK and GR at serine (S) 226 by Western blot. Epithelial levels of phosphorylated p38 MAPK and GR S226 were determined by immunohistochemistry in bronchial biopsies from asthma patients and healthy controls. BIRB-796 in combination with dexamethasone increased inhibition of cytokine production in a synergistic manner. Combination treatment significantly increased GR nuclear localisation compared to dexamethasone alone. BIRB-796 inhibited TNFα-induced p38 MAPK and GR S226 phosphorylation. Phosphorylated GR S226 and p38 MAPK levels were increased in bronchial epithelium of more severe asthma patients. Molecular crosstalk exists between p38 MAPK activation and GR function in human bronchial epithelial cells, which alters GR activity. Combining a p38 MAPK inhibitor and a corticosteroid may demonstrate therapeutic potential in severe asthma.Key messages• Combination of corticosteroid and p38 inhibitor in human bronchial epithelial cells• Combination increased cytokine inhibition synergistically and nuclear GR• p38 MAPK inhibition reduced TNFα-induced phosphorylation of GR at S226 but not S211• Phosphorylated GRS226 and p38 is increased in bronchial epithelium in severe asthma• Combining a p38 inhibitor and a corticosteroid may be effective in asthma treatment

Selective Glucocorticoid Receptor Modulation Prevents and Reverses Nonalcoholic Fatty Liver Disease in Male Mice.

Medication for nonalcoholic fatty liver disease (NAFLD) is an unmet need. Glucocorticoid (GC) stress hormones drive fat metabolism in the liver, but both full blockade and full stimulation of GC signaling aggravate NAFLD pathology. We investigated the efficacy of selective glucocorticoid receptor (GR) modulator CORT118335, which recapitulates only a subset of GC actions, in reducing liver lipid accumulation in mice. Male C57BL/6J mice received a low-fat diet or high-fat diet mixed with vehicle or CORT118335. Livers were analyzed histologically and for genome-wide mRNA expression. Functionally, hepatic long-chain fatty acid (LCFA) composition was determined by gas chromatography. We determined very-low-density lipoprotein (VLDL) production by treatment with a lipoprotein lipase inhibitor after which blood was collected to isolate radiolabeled VLDL particles and apoB proteins. CORT118335 strongly prevented and reversed hepatic lipid accumulation. Liver transcriptome analysis showed increased expression of GR target genes involved in VLDL production. Accordingly, CORT118335 led to increased lipidation of VLDL particles, mimicking physiological GC action. Independent pathway analysis revealed that CORT118335 lacked induction of GC-responsive genes involved in cholesterol synthesis and LCFA uptake, which was indeed reflected in unaltered hepatic LCFA uptake in vivo. Our data thus reveal that the robust hepatic lipid-lowering effect of CORT118335 is due to a unique combination of GR-dependent stimulation of lipid (VLDL) efflux from the liver, with a lack of stimulation of GR-dependent hepatic fatty acid uptake. Our findings firmly demonstrate the potential use of CORT118335 in the treatment of NAFLD and underscore the potential of selective GR modulation in metabolic disease.

Increasing G9a automethylation sensitizes B acute lymphoblastic leukemia cells to glucocorticoid-induced death

Synthetic glucocorticoids (GCs) are used to treat lymphoid cancers, but many patients develop resistance to treatment, especially to GC. By identifying genes that influence sensitivity to GC-induced cell death, we found that histone methyltransferases G9a and G9a-like protein (GLP), two glucocorticoid receptor (GR) coactivators, are required for GC-induced cell death in acute lymphoblastic leukemia (B-ALL) cell line Nalm6. We previously established in a few selected genes that automethylated G9a and GLP recruit heterochromatin protein 1γ (HP1γ) as another required coactivator. Here, we used a genome-wide analysis to show that HP1γ is selectively required for GC-regulated expression of the great majority of GR target genes that require G9a and GLP. To further address the importance of G9a and GLP methylation in this process and in cell physiology, we found that JIB-04, a selective JmjC family lysine demethylase inhibitor, increased G9a methylation and thereby increased G9a binding to HP1γ. This led to increased expression of GR target genes regulated by G9a, GLP and HP1γ and enhanced Nalm6 cell death. Finally, the KDM4 lysine demethylase subfamily demethylates G9a in vitro, in contrast to other KDM enzymes tested. Thus, inhibiting G9a/GLP demethylation potentially represents a novel method to restore sensitivity of treatment-resistant B-ALL tumors to GC-induced cell death.

Abstract 2541: Combined androgen and glucocorticoid receptor (AR/GR) activity drives TNBC progression

Abstract Background: A recent effort to distinguish early-stage TNBC based on outcome and to expose driver pathways using gene profiling/cluster analysis divided TNBC into four subtypes- basal-like, mesenchymal, immunomodulatory, and luminal androgen receptor (LAR). The clinical AR+ TNBC subtype (>10% AR by IHC) comprises up to 35% of metastatic disease. The discovery of this TNBC subtype and the concomitant development of potent new anti-androgens for prostate cancer have led to several clinical trials evaluating AR antagonists in TNBC. In parallel, glucocorticoid receptor (GR) expression and activation have been shown to mediate anti-apoptotic gene expression in TNBC. However, to our knowledge, no one has examined the coordinate expression of GR and AR activity and the potential crosstalk downstream of dual GR and AR activation. We hypothesized that AR/GR cooperative gene expression pathways may induce TNBC therapy resistance and tumor progression. Methods and Results: We analyzed GR+ TNBC primary tumor gene expression from TCGA samples and found a wide range of AR expression. To examine whether GR-associated gene expression differed in high versus low AR+ TNBC, we divided TCGA TNBC primary tumors (N=123) at the AR median into “high” versus “low” AR expression. We then identified relative GR target gene expression levels in those genes identified from a previous TNBC meta-analysis. Invasion, motility and proliferation pathways were found to be most significantly expressed among GR-associated genes in AR high TNBC (top 5 significant pathways). Similarly, we found that GR and AR co-activation in AR+ MDA-MB-453 TNBC cells significantly increased cell migration compared to GR or AR activation alone. In addition, when MDA-MB-453 cells were exposed to the AR antagonist enzalutamide (enza) for more than 30 days (long-term, LT), the magnitude of increase in cell migration following GR activation increased, suggesting that increased GR activity may compensate for long-term AR blockade. Furthermore, long-term enza exposure (LTenza) in AR+ MFM-223 and MDA-MB-453 cells resulted in increased GR mRNA and protein levels. The magnitude of GR-mediated target gene expression was also significantly increased in LTEnza versus control-treated cells. In vivo, LTenza-treated MDA-MB-453 xenografts were relatively insensitive to paclitaxel chemotherapy treatment, consistent with increased GR activity compared to control cells. Conclusions: We conclude that coordinate GR and AR expression in TNBC contributes to a particularly poor outcome in TNBC. Downstream AR/GR gene expression favoring cell survival and invasive phenotypes may contribute to this outcome. Consideration of dual AR/GR inhibition in TNBC may increase the effectiveness of anti-androgen therapy. Citation Format: Deniz N. Dolcen, Eva Tonsing-Carter, Ryan Harkless, Caroline Kim, Kathleen Bowie, Gini Fleming, Suzanne Conzen. Combined androgen and glucocorticoid receptor (AR/GR) activity drives TNBC progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2541.

Knockout of USP19 Deubiquitinating Enzyme Prevents Muscle Wasting by Modulating Insulin and Glucocorticoid Signaling.

Muscle atrophy arises because of many chronic illnesses, as well as from prolonged glucocorticoid treatment and nutrient deprivation. We previously demonstrated that the USP19 deubiquitinating enzyme plays an important role in chronic glucocorticoid- and denervation-induced muscle wasting. However, the mechanisms by which USP19 exerts its effects remain unknown. To explore this further, we fasted mice for 48 hours to try to identify early differences in the response of wild-type and USP19 knockout (KO) mice that could yield insights into the mechanisms of USP19 action. USP19 KO mice manifested less myofiber atrophy in response to fasting due to increased rates of protein synthesis. Insulin signaling was enhanced in the KO mice, as revealed by lower circulating insulin levels, increased insulin-stimulated glucose disposal and phosphorylation of Akt and S6K in muscle, and improved overall glucose tolerance. Glucocorticoid signaling, which is essential in many conditions of atrophy, was decreased in KO muscle, as revealed by decreased expression of glucocorticoid receptor (GR) target genes upon both fasting and glucocorticoid treatment. This decreased GR signaling was associated with lower GR protein levels in the USP19 KO muscle. Restoring the GR levels in USP19-deficient muscle was sufficient to abolish the protection from myofiber atrophy. Expression of GR target genes also correlated with that of USP19 in human muscle samples. Thus, USP19 modulates GR levels and in so doing may modulate both insulin and glucocorticoid signaling, two critical pathways that control protein turnover in muscle and overall glucose homeostasis.

Histone deacetylase inhibition ameliorates hypertension and hyperglycemia in a model of Cushing's syndrome.

Cushing's syndrome (CS) caused by hypercortisolism is occasionally accompanied by metabolic disorders such as hypertension, diabetes mellitus (DM), dyslipidemia, and central obesity. Thus morbidity and mortality, observed in cardiovascular disease, are elevated in patients with CS. We hypothesized that HDAC inhibition (HDACi) decreased transcriptional activity of glucocorticoid receptor (GR), which ameliorates hypertension and hyperglycemia in patients with CS. To establish an animal model of hypercortisolism, Sprague-Dawley rats were infused with adrenocorticotropic hormone (ACTH, 40 ng/day) or dexamethasone (Dex, 10 μg/day) via osmotic minipumps for 4 wk. Expression of GR target genes was determined by quantitative real-time PCR (qRT-PCR). GR enrichment on specific loci, and across the whole genome, was analyzed by chromatin immunoprecipitation (ChIP) and ChIPseq, respectively. HDACi decreased blood pressure and expression of ion regulators in the kidneys of ACTH-infused rats. Additionally, HDACi reduced deposition of polysaccharide, fasting blood glucose level, glucose intolerance, and expression of gluconeogenesis genes in the livers and kidneys of ACTH- and Dex-infused rats. Among class I HDACs, HDAC1 and HDAC3 interacted with GR. HDAC1 knockdown resulted in increased level of acetylation and decreased transcriptional activity of GR. GR recruitment on the promoters of 2,754 genes, which include ion transporters, channels, and gluconeogenic genes, was significantly decreased by MS-275, a class I HDAC inhibitor. These results indicate that HDACi ameliorates hypertension and hyperglycemia in a model of CS by decreasing the transcriptional activity of GR via elevating its level of acetylation.

Compound A influences gene regulation of the Dexamethasone-activated glucocorticoid receptor by alternative cofactor recruitment

The glucocorticoid receptor (GR) is a transcription factor of which the underlying gene regulatory mechanisms are complex and incompletely understood. The non-steroidal anti-inflammatory Compound A (CpdA), a selective GR modulating compound in various cell models, has been shown to favour GR-mediated gene repression but not GR-mediated gene activation. Shifting balances towards only a particular subset of GR gene regulatory events may be of benefit in the treatment of inflammatory diseases. We present evidence to support that the combination of CpdA with Dexamethasone (DEX), a classic steroidal GR ligand, can shape GR function towards a unique gene regulatory profile in a cell type-dependent manner. The molecular basis hereof is a changed GR phosphorylation status concomitant with a change in the GR cofactor recruitment profile. We subsequently identified and confirmed the orphan nuclear receptor SHP as a coregulator that is specifically enriched at GR when CpdA and DEX are combined. Combining CpdA with DEX not only leads to stronger suppression of pro-inflammatory gene expression, but also enhanced anti-inflammatory GR target gene expression in epithelial cells, making ligand combination strategies in future a potentially attractive alternative manner of skewing and fine-tuning GR effects towards an improved therapeutic benefit.

Agonist-specific Protein Interactomes of Glucocorticoid and Androgen Receptor as Revealed by Proximity Mapping

Glucocorticoid receptor (GR) and androgen receptor (AR) are steroid-inducible transcription factors (TFs). The GR and the AR are central regulators of various metabolic, homeostatic and differentiation processes and hence important therapeutic targets, especially in inflammation and prostate cancer, respectively. Hormone binding to these steroid receptors (SRs) leads to DNA binding and activation or repression of their target genes with the aid of interacting proteins, coregulators. However, protein interactomes of these important drug targets have remained poorly defined. We used proximity-dependent biotin identification to map the protein interaction landscapes of GR and AR in the presence and absence of their cognate agonist (dexamethasone, 5α-dihydrotestosterone) and antagonist (RU486, enzalutamide) in intact human cells. We reproducibly identified more than 30 proteins that interacted with the GR in an agonist-specific manner and whose interactions were significantly influenced by the DNA-binding function of the receptor. Interestingly, the agonist-dependent interactome of the GR overlapped considerably with that of the AR. In addition to known coactivators, corepressors and components of BAF (SWI/SNF) chromatin-remodeling complex, we identified a number of proteins, including lysine methyltransferases and demethylases that have not been previously linked to glucocorticoid or androgen signaling. A substantial number of these novel agonist-dependent GR/AR-interacting proteins, e.g. BCOR, IRF2BP2, RCOR1, and TLE3, have previously been implicated in transcription repression. This together with our data on the effect of BCOR, IRF2BP2, and RCOR1 on GR target gene expression suggests multifaceted functions and roles for SR coregulators. These first high confidence SR interactomes will aid in therapeutic targeting of the GR and the AR.

Flightless-I homolog regulates glucocorticoid receptor-mediated transcription via direct interaction of the leucine-rich repeat domain.

Flightless-I homolog (FLII) is a member of the gelsolin family of proteins, and has been identified as a coactivator of estrogen receptor-mediated transcription. Here, we investigate the role of FLII in the glucocorticoid receptor (GR) signaling pathway. Reporter gene assay and real-time quantitative PCR in A549 were performed to investigate the function of FLII in the expression of GR target genes. Co-immunoprecipitation assay and in vitro binding assay were used to identify binding domain of FLII. Chromatin immunoprecipitation assay were carried out with FLII-depleted A549 cells to determine the role of FLII at GR binding sites. We demonstrate that FLII potentiates GR-mediated reporter gene activity synergistically with CARM1 and p300 to enhance GR transcriptional activity in the presence of dexamethasone (Dex) in A549 cells. Depletion of endogenous FLII inhibited the expression of Dex-regulated GR target genes in A549 cells, indicating that FLII is required for GR-mediated transcription. Further, we observed that FLII binds to GR via its N-terminal leucine-rich repeat (LRR) region, suggesting that the enhancement of GR activation may occur through the interaction of GR and FLII. Moreover, chromatin immunoprecipitation analysis demonstrated that FLII is recruited to the GR binding sites. In addition, depletion of endogenous FLII decreased the recruitment of p300, and subsequently RNA polymerase II, to specific sites of GR target genes. Taken together, these studies reveal a functional involvement of FLII in activating transcription of GR target genes, suggesting a physiological role for FLII in the GR signaling pathway.

Abstract 334: Novel selective glucocorticoid receptor modulators (SGRMs) in castration-resistant prostate cancer

Abstract (A) Introduction: Because of the central role of androgen receptor (AR) signaling in prostate cancer (PC), AR inhibition is the primary modality of initial PC treatment. While AR-targeted therapies are often initially effective in castration-resistant prostate cancer (CRPC), disease progression is common. AR and glucocorticoid receptor (GR) are structurally similar nuclear receptors, bind the same DNA response elements, and regulate common genes. Previous work from our lab demonstrated that GR expression is upregulated following AR antagonism, GR expression decreased the effectiveness of AR blockade, and inhibiting GR activity reversed these effects. However, the only FDA-approved GR antagonist, mifepristone, is not GR-specific and has significant drug-drug interactions; therefore, we report our initial data with a new generation of selective GR-antagonists (SGRMs) for PC treatment using models of CRPC. (B) Experimental Procedures: The LAPC4, CWR-22Rv1 and VCAP PC cell lines were utilized. GR target gene expression analysis was performed at various time-points by qRT-PCR and protein immunoblots subsequent to treatment with AR agonist (R1881, 1nM), AR antagonist (enzalutamide, 10 μM), GR agonist (dexamethasone, 100nM) and the new SGRMs CORT108297 and CORT118335(1μM). Quantification of cell numbers in vitro in response to these treatments was determined by trypan blue exclusion/live cell counting and the CellTiter Glo 2.0 ATP-luciferase assay. For in vivo studies, PC cells were injected subcutaneously into male nude mice and grown to a predetermined tumor size, mice were then castrated, and fifteen days later treated with mifepristone (12mg/kg), SGRM (16mg/kg, 20mg/kg), or vehicle by daily intra-peritoneal injection to assess time to CRPC. (C) Results: AR antagonism with concurrent GR activation resulted in increased expression of canonical AR/GR-target genes and SGRMs (1μM) effectively inhibited expression of glucocorticoid-mediated genes at the RNA and protein levels. SGRMs did not affect AR-mediated gene expression. When AR was antagonized, activated GR promoted cell survival, which was inhibited by SGRM treatment to varying degrees. Preliminary studies support the hypothesis that these novel SGRMs delay CRPC tumor xenograft progression in vivo. (D) Conclusion: In vitro, new generation SGRMs inhibit GR-mediated, but not AR-selective gene expression and result in decreased tumor cell number. SGRM administration to nude mice bearing PC xenografts can delay castration-resistant tumor growth. Therefore, new SGRMs may be an effective therapy for CRPC treatment worthy of further exploration. Studies are ongoing to determine how SGRMs modulate GR target gene expression globally and whether they decrease expression of tumorigenic pro-survival genes in vivo. Citation Format: Jacob Kach, Phillip Selman, Diana C. West, Donald J. Vander Griend, Suzanne D. Conzen, Russell Z. Szmulewitz. Novel selective glucocorticoid receptor modulators (SGRMs) in castration-resistant prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 334.

Abstract PD3-02: Second-generation selective glucocorticoid receptor modulators in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) lacks expression of the estrogen receptor (ER), progesterone receptor (PR), and HER2. A subset of primary TNBCs (at least 30%) expresses moderate to strong glucocorticoid receptor (GR) in greater than 10% of invasive tumor cells; in addition, we have reported that increased GR signaling promotes TNBC cell survival. Furthermore, our laboratory recently observed that patients with early stage/high-GR-expressing TNBCs have a relatively poor prognosis. Therefore, GR is being explored as a target for improving outcome in TNBC. Indeed, mifepristone, a well-characterized non-selective steroidal GR/PR antagonist, has shown promise in reversing GR-mediated tumor cell-survival signaling in TNBC. In vivo, increased TNBC xenograft chemosensitivity was observed when mifepristone was added to chemotherapy treatment. Most recently, a Phase I clinical trial demonstrated the safety and potential efficacy of mifepristone added to nab-paclitaxel chemotherapy in Stage IV TNBC patients. Three highly selective non-steroidal GR antagonists (GRAs) have been investigated, CORT108297. CORT125134 (two aryl pyrazole-fused azadecalins) and CORT118335 (a pyrimidine dione). These new GRAs have 1) far less cross-reactivity than mifepristone with other nuclear receptor family members and 2) lower interaction profiles for drug metabolizing CYP enzymes. We hypothesized that selective GRAs would not only inhibit GR-induced pro-survival gene expression, but also increase TNBC chemosensitivity, and may eventually be a valuable adjunct therapy for treating chemotherapy-resistant TNBC. To understand how GR modulators interact with the GR ligand-binding domain (LBD), we are employing two approaches: 1) computational modeling of the GRA-GR LBD based on published crystal structures of the GR LBD with mifepristone (in collaboration with UIUC), and 2) crystal structure analysis of the GR LBD with GR modulators. Preliminary computational results indicate that CORT108297 docks into the LBD and antagonizes GR activity by indirectly disordering Helix 12. Experiments are ongoing using various buffer conditions to produce crystals of the GR LBD with CORT108297 and CORT118335 for x-ray crystallography. Secondly, we are functionally characterizing CORT108297, CORT125134, and CORT118335 for their ability to inhibit glucocorticoid induction of key GR-mediated target genes. Over the course of eight hours, we observed that the compounds exhibited temporal antagonism of the expression of GR target genes encoding anti-apoptotic proteins such as SGK-1, MCL-1, and MKP-1/DUSP1. Lastly, we are examining the selective GRAs' ability to enhance tumor cell cytotoxicity from paclitaxel in cell culture and in TNBC xenograft models. Preliminary results indicate that CORT125134 and CORT108297 significantly increase GR+ TNBC sensitivity to paclitaxel, and are well-tolerated in our in vivo models. Citation Format: West DC, Hosfield DJ, Mayne CG, Skor MN, Styke SC, Pierce CF, Kocherginsky M, Hunt H, Fleming GF, Szmulewitz RZ, Tajkhorshid E, Greene GL, Conzen SD. Second-generation selective glucocorticoid receptor modulators in triple-negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD3-02.

Electrophilic lipid mediator 15-deoxy-Δ12,14-prostaglandin j2 modifies glucocorticoid signaling via receptor SUMOylation.

Cortisol, the central stress hormone in humans, activates the glucocorticoid receptor (GR). Anti-inflammatory effects are the most important pharmaceutical effects mediated by the GR. Inasmuch as electrophilic cyclopentenone prostaglandin 15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) has potent anti-inflammatory properties and activates the SUMOylation pathway, we have investigated the effect of 15d-PGJ2 on glucocorticoid signaling and receptor SUMOylation. To this end, we studied isogenic HEK293 cells expressing either wild-type GR or SUMOylation-defective GR. Interestingly, 15d-PGJ2 triggered SUMO-2 and -3 (SUMO-2/3) modification in the primary SUMOylation sites of the GR. Gene expression profiling and pathway analyses indicate that 15d-PGJ2 inhibits GR signaling in a genome-wide fashion that is significantly dependent on the GR SUMOylation sites. Chromatin immunoprecipitation assays showed that the repressive effect of 15d-PGJ2 on GR target gene expression occurs in parallel with the inhibition of receptor binding to the target gene chromatin. Furthermore, depletion of UBC9, the sole SUMO E2 conjugase, from HEK293 cells confirmed the involvement of active SUMOylation in the regulatory process. Taken together, our data indicate that GR SUMOylation modulates the glucocorticoid signaling during acute cell stress. Our data also suggest that GR SUMOylation modulates cross talk of the glucocorticoid signaling with other transcription factors that are responsive to cell stress.

11β-Hydroxysteroid dehydrogenase-1 is involved in bile acid homeostasis by modulating fatty acid transport protein-5 in the liver of mice.

11β-Hydroxysteroid dehydrogenase-1 (11β-HSD1) plays a key role in glucocorticoid receptor (GR) activation. Besides, it metabolizes some oxysterols and bile acids (BAs). The GR regulates BA homeostasis; however, the impact of impaired 11β-HSD1 activity remained unknown. We profiled plasma and liver BAs in liver-specific and global 11β-HSD1-deficient mice. 11β-HSD1-deficiency resulted in elevated circulating unconjugated BAs, an effect more pronounced in global than liver-specific knockout mice. Gene expression analyses revealed decreased expression of the BA-CoA ligase Fatp5, suggesting impaired BA amidation. Reduced organic anion-transporting polypeptide-1A1 (Oatp1a1) and enhanced organic solute-transporter-β (Ostb) mRNA expression were observed in livers from global 11β-HSD1-deficient mice. The impact of 11β-HSD1-deficiency on BA homeostasis seems to be GR-independent because intrahepatic corticosterone and GR target gene expression were not substantially decreased in livers from global knockout mice. Moreover, Fatp5 expression in livers from hepatocyte-specific GR knockout mice was unchanged. The results revealed a role for 11β-HSD1 in BA homeostasis.