Mechanisms Of Glucocorticoid Resistance Research Articles

The Role of Glucocorticoid Receptor in the Pathophysiology of Pituitary Corticotroph Adenomas.

Adrenocorticotropic Hormone (ACTH)-secreting pituitary adenomas are rare tumors characterized by autonomous ACTH secretion with a consequent increase in circulating cortisol levels. The resulting clinical picture is called Cushing’s disease (CD), a severe condition burdened with high morbidity and mortality. Apart from increased cortisol levels, CD patients exhibit a partial resistance to the negative glucocorticoid (GC) feedback, which is of paramount clinical utility, as the lack of suppression after dexamethasone administration is one of the mainstays for the differential diagnosis of CD. Since the glucocorticoid receptor (GR) is the main regulator of negative feedback of the hypothalamic–pituitary–adrenal axis in normal conditions, its implication in the pathophysiology of ACTH-secreting pituitary tumors is highly plausible. In this paper, we review GR function and structure and the mechanisms of GC resistance in ACTH-secreting pituitary tumors and assess the effects of the available medical therapies targeting GR on tumor growth.

Research advances concerning the mechanism of glucocorticoid resistance in relation to traditional Chinese medicine for patients with chronic obstructive pulmonary disease

Research advances concerning the mechanism of glucocorticoid resistance in relation to traditional Chinese medicine for patients with chronic obstructive pulmonary disease

SAT-734 HSD3B1 Expression in the Human Immune System

3β-hydroxysteroid dehydrogenase-1 (3βHSD1), catalyzing conversion of dehydroepiandrosterone (DHEA) to Δ 4-androstenedione, is an essential enzyme in the pathway toward production of biologically active androgens such as dihydrotestosterone from the adrenally produced precursor DHEA sulfate, the most predominant steroid hormone in circulation. We previously identified, in the gene (HSD3B1) that encodes 3βHSD1, a germline gain-of-function missense-encoding variant that has now been validated in several studies as predicting more rapid progression in prostate cancer patients treated with gonadal testosterone deprivation. Production of androgens from adrenal precursors is important not just in the context of prostate cancer but in other physiologic and pathophysiologic processes, which could include asthma. Androgens are associated with better lung function in both asthma and healthy cohorts, and increasing circulating androgen levels in males help explain the switchover in asthma being more common in boys than girls but then more common in women than men. A main treatment for asthma, as well as other inflammatory processes, is administration of glucocorticoids, yet unresponsiveness to glucocorticoids in a subset of patients remains a major problem. Systemic glucocorticoid administration suppresses adrenally produced DHEA and DHEA-S, suggesting a depleted pool for biologically active androgen production as a mechanism for glucocorticoid resistance. Our surprising preliminary data support a link between glucocorticoid responsiveness and the more active HSD3B1 allele: patients homozygous for the adrenal-permissive HSD3B1(1245C) allele exhibit better response to oral glucocorticoids than those homozygous for the adrenal-restrictive HSD3B1(1245A), with heterozygous patients falling in the middle. This suggests a model in which patients with the more active (adrenal-permissive) form of 3βHSD1 produce sufficient androgens despite the depleted pool of precursor hormones whereas patients with the less active (adrenal-restrictive) form cannot. To further elucidate the link between 3βHSD1 activity and immune response, we assayed HSD3B1 expression in different types of white blood cells. Leukocyte subsets from asthma patients and healthy controls were purified using fluorescence-activated cell sorting, and HSD3B1 expression was analyzed using qPCR. White blood cells of several types expressed HSD3B1 at levels comparable to or greater than both prostate cancer and placental choriocarcinoma cell lines with very robust 3βHSD1 activity. Further determining the cell type specific expression and activity of this key enzyme is an important step in unraveling the link between the HSD3B1 polymorphism and asthma along with potentially many other immune processes.

Loss of glucocorticoid receptor expression mediates in vivo dexamethasone resistance in T-cell acute lymphoblastic leukemia.

Despite decades of clinical use, mechanisms of glucocorticoid resistance are poorly understood. We treated primary murine T lineage acute lymphoblastic leukemias (T-ALLs) with the glucocorticoid dexamethasone (DEX) alone and in combination with the pan-PI3 kinase inhibitor GDC-0941 and observed a robust response to DEX that was modestly enhanced by GDC-0941. Continuous in vivo treatment invariably resulted in outgrowth of drug-resistant clones, ~30% of which showed markedly reduced glucocorticoid receptor (GR) protein expression. A similar proportion of relapsed human T-ALLs also exhibited low GR protein levels. De novo or preexisting mutations in the gene encoding GR (Nr3c1) occurred in relapsed clones derived from multiple independent parental leukemias. CRISPR/Cas9 gene editing confirmed that loss of GR expression confers DEX resistance. Exposing drug-sensitive T-ALLs to DEX in vivo altered transcript levels of multiple genes, and this response was attenuated in relapsed T-ALLs. These data implicate reduced GR protein expression as a frequent cause of glucocorticoid resistance in T-ALL.

Akt2 Mediates Glucocorticoid Resistance in Acute Lymphoblastic Leukemia through FoxO3a/Bim Axis and Serves As a Direct Target for Resistance Reversal

Background: Glucocorticoids (GCs) are common components in chemotherapeutic protocols for acute lymphoblastic leukemia (ALL). A major obstacle in GC therapy, however, is the gradual acquisition of apoptotic resistance in ALL cells repeatedly treated with these hormones. Previous reports indicate that 15-30% of pediatric ALL samples are resistant to GCs, while in refractory childhood ALL, the prevalence of GC resistance is as high as 70%. Identification of specific molecular mechanisms driving resistance to GC and targeting downstream molecules may lead to the development of new therapeutic strategies.Results: FoxO transcription factor FoxO3a has been shown to regulate apoptosis in lymphocytes. Unphosphorylated FoxO3a can be upregulated by dexamethasone (DEX), which subsequently translocates into the nucleus, upregulates Bim expression and induces apoptosis. In our current study, we cultured a GC-sensitive T-ALL cell line CCRF-CEM with a specific concentration of DEX over multiple passages and obtained a highly resistant cell line, designated CEM-DR. We observed this T-ALL cell line acquires resistance to DEX-mediated killing through abnormal activation of Akt, resulting in inhibition of the FoxO3a/Bim pathway (A). Pharmacologic inhibition of Akt with Akt inhibitor IV effectively restores sensitivity to DEX of CEM-DR by enhancing the FoxO3a/Bim pathway, but shows significant hepatotoxicity with increased serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in vivo (B). Common hematologic toxicities and hepatic toxicities with increased AST and ALT of Akt inhibitor have also been reported in the treatment of solid tumors in humans, partially limiting their clinical applicability (Becher OJ et al, Pediatr Blood Cancer 2017). There are two closely related, highly conserved homologues of Akt: Akt-1 and -2, which differ in enzyme function. Our study aimed to investigate the potential role of Akt isoforms Akt1 and Akt2 in the mechanism of GC resistance and explore a more direct and specific target for resistance reversal.By western blot analysis we observed the expression of Akt2 in intrinsically resistant T-ALL cells Jurkat was significantly higher than that in sensitive CCRF-CEM cells, while Akt1 expression in aforementioned cell lines was similar. The expression of Akt2 also increased synchronously with the increase of half maximal inhibitory concentration (IC50) of DEX in CEM-DR cells, further indicating that Akt2 expression increases in secondarily resistant lymphocytes. A significantly elevated expression of Akt2 not Akt1 were shown in relapsed/refractory ALL patients when compared with the newly diagnosed patients. To detect if Akt2 was able to directly interact with FoxO3a, co-immunoprecipitation assay was employed. MYC-FoxO3a was co-transfected with Flag-Akt1 or Flag-Akt2 into HEK293T cells. The result demonstrated the more presence of Flag-AKT2 than Flag-Akt1 in MYC-FoxO3a immunoprecipitates, suggesting that AKT2 as the major regulator directly interacting with FoxO3a. Reciprocal immunoprecipitation experiments confirmed the closer association between Flag-Akt2 and MYC-FoxO3a (C). Then we used siRNA to down-regulate Akt1 or Akt2 expression in resistant T-ALL cells; we observed GC-induced apoptosis increased significantly (D) after down-regulation of Akt2 expression, along with the expression of p-FoxO3a decreased (E) .To examine the therapeutic role of Akt isoform specific inhibitors, we treated resistant T-ALL cell with A-674563 (Akt1 inhibitor), CCT128930 (Akt2 inhibitor) or Akti1/2 (Akt1/2 inhibitor). Selective inhibition of Akt2 with CCT128930 more significantly enhances the FoxO3a/Bim signaling pathway, increases DEX-mediated killing of resistant T-ALL cells (F) and effectively reverses GC resistance than Akt1 inhibitor in vitro and in vivo. When exploring the potential influence on viscera by Akt isoform inhibitors in vivo, we found Akt2 inhibitor did not significantly influence hematopoiesis, cardiac and renal functions. Notably, Akt2 inhibitor did not show hepatic toxicities of increased serum ALT, AST and total bilirubin which appear in Akt1 or Akt1/2 inhibition.Conclusions: Akt2 might serve as a more direct and specific kinase mediating GC resistance through FoxO3a/Bim-signaling pathway in ALL, and targeting Akt2 with CCT128930 may be explored as a promising therapeutic strategy for resistance reversal. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Research progress of signal transduction pathways associated with glucocorticoid resistance in acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the common malignant tumor. Despite the great improvements in clinical protocols, some patients relapse or suffer from failure of treatment. Glucocorticoid is the core drug in the treatment of ALL. However, the resistance of glucocorticoid in ALL is a common problem in clinical treatment and the main cause of failure of treatment. Mechanism of glucocorticoid resistance is very complex. The expression of drug resistance proteins and the production of drug resistance in tumor cells are regulated by multiple signal transduction pathways, which mainly include MAKP, PI3K/AKT/mTOR, JAK/STAT, Wnt/β-catenin signal transduction pathway. In this paper, the recent progresses of glucocorticoid resistance related signal transduction pathway in ALL was reviewed. Key words: Leukemia, lymphoid; Precursor cell lymphoblastic leukemia-lymphoma; Signal transduction; Glucocorticoid; Drug resistance

Mechanisms underlying glucocorticoid resistance in chronic rhinosinusitis with nasal polyps

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease that occurs in the nasal and sinus mucosa, which is a common disease in otorhinolaryngology. At present, CRSwNP can be effectively treated by glucocorticoids (GC). GC binds to GC receptors in the nasal mucosa, affects the expression of inflammatory genes, inhibits the activation and action of eosinophils, T cell-associated inflammatory responses in nasal polyps, as well as tissue remodeling. However, there are some patients fall reponse to GC, so called GC resistance. The study suggests that the possible mechanism of CRSwNP GC resistance is mainly related to GC receptor abnormal, the role of cytokines and transcription factors, such as Th cells and IL-8. In addition, MAPK-related kinases and histone deacetylase in the GC signaling pathway also play important roles in the GC resistance process. This paper reviews the mechanism of GC treatment of CRSwNP, the mechanism of GC resistance and alternative treatment of GC.

Abstract 46: CDK4/CDK6 inhibition in childhood B-ALL: A new strategy to mediate glucocorticoid sensitivity

Abstract Dysregulation of the cyclin D1-CDK4/CDK6 complex is frequently observed in almost all human cancer and contributes to aberrant cell proliferation and consequent tumorigenesis. Among the CDKs that control cell cycle progression, cyclin D-dependent kinases CDK4 and CDK6 are considered important oncogenic drivers in many cancers. CDK6 is a direct target of MLL rearranged B-cell acute lymphoblastic leukemia (B-ALL), and this prompted us to investigate if pharmacologic inhibition of CDK4/CDK6 could increase the efficiency of chemotherapy and overcome drug resistance. Although many reports described CDK4/CDK6 inhibitors against a broad range of carcinomas, few studies have been performed on leukemia. Deletion and methylation of CDK4/CDK6 inhibitor CDKN2a are frequently observed in B-ALL and gene expression analysis performed in a cohort of childhood patients showed that cyclin D1, CDK4 and CDK6 are highly expressed. Moreover, Reverse Phase Protein Array (RPPA) analysis showed that cyclin D1 expression is higher in high risk-MRD patients. These results suggest specific inhibition of cyclin D/CDK4/CDK6 axis as an attractive strategy to improve the effect of common chemotherapy on B-ALL patients. Standard treatment for newly diagnosed childhood B-ALL patients include chemotherapy treatment, and although glucocorticoids (GC) are the most important drugs used in the treatment of acute lymphoblastic leukemia, the molecular basis of GC sensitivity and resistance remains largely unknown; understanding the molecular mechanisms related to GC cytotoxicity is crucial for modulating GC resistance. The aim of this study was to evaluate the effect of dual inhibition of CDK4/CDK6 in B-ALL and if this inhibition could enhance cytotoxic killing of leukemia cells after combination treatment with dexamethasone. We treated two B-ALL dexamethasone-resistant cell lines, SEM and RCH-ACV, and two B-ALL dexamethasone-sensitive cell lines, RS4;11 and NALM6, with ribociclib, a highly specific CDK4/6 dual inhibitor. As expected, treatment with ribociclib induced a strong cell cycle arrest in G1 phase in a time-dose dependent manner along with a dose-dependent decrease in phosphorylated retinoblastoma (Rb); we investigated whether treatment with ribociclib could induce growth inhibition of B-ALL cell lines. We observed, in SEM and RCH-ACV, a strong growth inhibition at all drug concentration after continuous treatment. Washout of the inhibitor from the medium after 48 hours of treatment showed that both cell lines did not restore cell growth. These results show that ribociclib could have an irreversible activity. Moreover, a strong dose-dependent reduction of clonogenic potential was observed in SEM cell line, by CFU assay. Ribociclib exposure strongly synergizes (CI<1) with dexamethasone in SEM and RCH-ACV resistant cell lines with a strong decrease of proliferation and a significant increase of apoptotic cell death. Immunoblot analysis showed a decrease in phosphorylated Rb and Cyclin D1 starting from 48 hours of cotreatment and an increase in p27. Preliminary experiments showed a modest increase in glucocorticoid receptor (GR) after ribociclib and combination treatment in SEM and RCH-ACV cells. To further confirm the synergistic effect we evaluated mRNA level of GR targets, and we found that GR, BTG1, and GILZ are overexpressed in combination treatment in SEM and RCH-ACV. The synergistic effect of ribociclib-dexamethasone combination was also confirmed on primary cultures derived from pediatric patients affected by B-acute lymphoblastic leukemia. Our findings support the concept that pharmacologic inhibition of CDK4/CDK6 may represent a useful therapeutic strategy to control cell proliferation in B-ALL and provide new insight in understanding potential mechanism of glucocorticoid resistance. Citation Format: Elena Mattiuzzo, Roberta Bortolozzi, Benedetta Accordi, Luca Trentin, Giuseppe Basso, Giampietro Viola. CDK4/CDK6 inhibition in childhood B-ALL: A new strategy to mediate glucocorticoid sensitivity [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 46.

Diagnostic value of the dual-luciferase report assay for predicting response to glucocorticoid in children with acute lymphoblastic leukemia.

Resistance to glucocorticoid (GC) is a significant clinical problem in some cases of acute lymphoblastic leukemia (ALL). Current methods of assessing GC resistance are time consuming and have limited reproducibility; in this study, we sought to define a new method of evaluating GC sensitivity and resistance in vitro. Based on the mechanisms of GC resistance, we hypothesized that the dual-luciferase report (DLR) assay could reflect the transcription effects of GC downstream of the GC-glucocorticoid receptor signaling pathway, thereby allowing the evaluation of reactions to GC. Sixty-two patients with differential GC response were included in this study. The prednisone induction test was used to divide the children with ALL into two groups: GC sensitive (GCS) and GC resistant (GCR). DLR assay was later conducted on those patients to evaluate its value for diagnosis of the GC reactivity. Receiver operating characteristic curves were used to identify the optimal assay cutoff for identifying response to GC. Using the DLR assay analysis, we found that GCR subjects showed significantly lower reporter/control ratios for luciferase, as compared with GCS subjects. The optimal cutoff value for GC response was 0.67, with sensitivity of 77.1% and specificity of 93.3%. The DLR assay results were consistent with prednisone induction test results. Further, the DLR assay was simpler, more sensitive, and less time-consuming than the prednisone induction test. Our study showed that the DLR assay is relatively fast, simple, and sensitive. Accordingly, it could be useful for detecting GC response in children with ALL.

The Role and Mechanism Study of Lncrnas Involved in Glucocorticoid Resistance in Paediatric Acute Lymphoblastic Leukemia

Objective: Glucocorticoids (GCs) are key components in the treatment of childhood acute lymphoblastic leukemia (ALL) and most ALL therapeutic failures can be explained by cellular resistance to GCs. However, the mechanisms of GC resistance are poorly understood. LncRNAs are involved in normal hematopoiesis and leukemia development, whereas the roles of lncRNAs in GC resistance are still unknown. Our goal was to investigate the role of lncRNAs involved in glucocorticoid resistance in paediatric acute lymphoblastic leukemia, and also elucidate the mechanism preliminarily.Methods: In this study, lncRNA microarray was performed on GC-resistant cell line CEM-C1 and GC-sensitive cell line CEM-C7 to screen the differential expression of lncRNAs. Five up-regulated and five down-regulated lncRNAs were randomly chosen for validation by Real-time PCR. GO-Pathway analysis was done to investigate potential signaling pathways regulated by the lncRNAs.Results: The microarray revealed that 4286 lncRNAs differed (p<0.05 and fold change>2.0) in GC-sensitive cell from those in GC-resistant cell. 826 lncRNAs changed more than 5-fold, while 356 lncRNAs changed more than 10-fold. Among them, 203 lncRNAs were up-regulated and 153 lncRNAs were down-regulated. Expression of ten selected lncRNAs was validated by Real-time PCR. GO analysis indicated that differentially expressed lncRNAs were involved in apoptotic process related GO terms. Pathway analysis revealed that these lncRNAs were involved in apoptosis, cell cycle, mTOR and some other signaling pathways.Conclusions: Our study showed that lncRNA expression profile was altered in GC-sensitive and GC-resistant cells, indicating that differentially expressed lncRNAs may play important functional roles in GC resistance of ALL. And these lncRNAs may be involved in GC resistance by regulating signaling pathways associated with cell proliferation, differentiation and apoptosis. Our study provides new biological foundations for further mechanism study in GC resistance and also provides a new strategy for therapeutic development of ALL. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1297: CDK4/CDK6 inhibition in childhood B-acute lymphoblastic leukemia: a new strategy to mediate glucocorticoid sensitivity

Abstract Unrestrained cell proliferation and cell cycle deregulation are common features in almost all human cancers. Among the CDKs that tightly control cell cycle progression, cyclin D-dependent kinases, CDK4 and CDK6 are considered important oncogenic drivers in many cancers. Although many reports successfully described CDK4/CDK6 inhibitors against a broad range of carcinomas, few studies have been performed on leukemia. Deletion and methylation of CDK4/CDK6 inhibitor CDKN2A, are frequently observed in B-acute lymphoblastic leukemia (B-ALL) and gene expression analysis performed in a cohort of childhood patients showed that cyclin D1, CDK4 and CDK6 are highly expressed. Moreover, Reverse Phase Protein Arrays (RPPA) analysis showed that cyclin D1 expression is higher in High Risk-MRD patients. These results suggest specific inhibition of cyclin D/CDK4/CDK6 axis as an attractive strategy to improve the effects of standard chemotherapy on B-ALL patients. The aim of this study was to evaluate the effect of dual inhibition of CDK4/CDK6 in B-ALL. To this purpose we treated two B-ALL dexamethasone resistant cell lines, SEM and RCH-ACV, and two B-ALL dexamethasone sensitive cell lines, RS4;11 and NALM6, with ribociclib, a highly specific CDK4/6 inhibitor. As expected, treatment with ribociclib induced a strong cell cycle arrest in G1 phase in a time-dose dependent manner along with a dose-dependent decrease in phosphorylated Rb and increase of the expression of cell cycle inhibitors p21 and p27. Moreover, a strong dose-dependent reduction of clonogenic potential was observed in SEM cell line, by CFU assay. No significant reduction in cell viability was observed. However, ribociclib exposure strongly synergized (CI&amp;lt;1) with dexamethasone in SEM and RCH-ACV resistant cell lines with a strong decrease of proliferation and a significant increase of apoptotic cell death. Immunoblot analysis showed a decrease in phosphorylated Rb, the activation of caspase-9 and the cleavage of the effector caspase-3 starting from 48 h of co-treatment, in agreement with the appearance of annexin-V positive cells. The synergistic effect of ribociclib-dexamethasone combination was confirmed on primary cultures derived from pediatric patients affected by B-ALL. We are actually investigating on the mechanism of this synergistic activity, and the effect of CDK4, CDK6 and cyclin D1 silencing will be presented. Preliminary experiments show a modest increase in glucocorticoid receptor expression after ribociclib treatment or CDK6 silencing in RCH-ACV cells. Our findings support the concept that pharmacologic inhibition of CDK4/CDK6 may represent a useful therapeutic strategy to control cell proliferation in B-ALL and provide new insight in understanding potential mechanisms of glucocorticoid resistance. Citation Format: Roberta Bortolozzi, Elena Mattiuzzo, Elena Mariotto, Benedetta Accordi, Luca Trentin, Giuseppe Basso, Giampietro Viola. CDK4/CDK6 inhibition in childhood B-acute lymphoblastic leukemia: a new strategy to mediate glucocorticoid sensitivity. [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 1297.

Oxidative Stress Impairs Glucocorticoids Sensitivity of Allergic Asthma Animal Model

SESSION TITLE: Asthma III SESSION TYPE: Original Investigation Poster PRESENTED ON: Saturday, April 16, 2016 at 11:45 AM - 12:45 PM PURPOSE: To observe the influence of ozone inhalation on the typical therapeutic effects of glucocorticoids and further to explore the involvements of p38MAPK activation and MKP-1 expression upon a mouse asthma model. METHODS: Mice were sensitized and challenged with ovalbumin (OVA), dexamethasone were administrated ip 1 hr before each challenge (day24,25,26). Ozone expsoure (1.0 ppm 2hr) were performed on day 23, 25 and 27, and SB239063 were injected 1hr before and 4hr after each exposure. Enhanced pause (Penh) were measured at day 28 and followed by bronchoalveolar lavage (BAL). Lung tissues were collected for analysis of expression of inflammatory genes, MKP-1 and p38MAPK. RESULTS: Ozone inhalation significantly increased the AHR and airway inflammation of OVA model. Dexamethasone significantly decreased the AHR and airway inflammation of mice from OVA model. These inhibitory effects were significantly decreased after the ozone exposure. Administration of SB239063 partly restored the impaired effects of dexamethasone, parallel with inhibitions of the AHR and airway inflammation, especially the lung expression of IL-17 mRNA, in mice of O3-exposed OVA model. The activation of p38MAPK was promoted by ozone inhalation and inhibited by SB239063. There was no striking effect of dexamethasone on activation of p38MAPK except in O3-exposed OVA model group. Dexamethasone increased the mRNA and protein expression of MKP-1 on lungs of mice only from OVA group. This effects were diminished by ozone inhalation regardless of SB239063 administration. O3 inhalation promoted the expression of MKP-1 on mRNA but not on protein level. CONCLUSIONS: Ozone inhalation disturbs the inhibitory effects of glucocorticoids on allergic airway inflammation. This disturbance may involve the activation of p38 MAPK, induction of Th17 cytokines and negative regulation on MKP-1 protein expression. CLINICAL IMPLICATIONS: Glucocorticoid resistance is a common and hard-to-treat problem in chronic severe asthmatic patients. This study explored the underlying involvements of p38 MAPK and MKP-1 in the oxidative stress induced steroids resistance in a mouse asthma model, which may contribute to the knowledge accumulation to help understand the underlying mechanisms of Glucocorticoid resistance. DISCLOSURE: The following authors have nothing to disclose: Aihua Bao, Xin Zhou No Product/Research Disclosure Information

P-glycoprotein transporter expression on a549 respiratory epithelial cells is positively correlated with intracellular dexamethasone levels.

Several mechanisms of glucocorticoid resistance in asthma have been proposed. P-glycoprotein (P-gp), a ubiquitous efflux transport protein, is associated with variability in the disposition of many drugs and interindividual variability in drug treatment response. This study was undertaken to determine the effect of P-gp expression on glucocorticoid efflux from airway epithelial cells. Decreasing respiratory epithelial P-gp expression in dexamethasone-exposed airway epithelial cells in vitro will increase intracellular dexamethasone concentration. A549 lung epithelial cells, transfected with small interfering RNA (siRNA) targeted at messenger RNA for the gene encoding P-gp, were exposed to 100-nM dexamethasone for 15 minutes. Transfection efficiency of siRNA, P-gp expression, and intracellular dexamethasone were measured with flow cytometry. Cells transfected with both negative siRNA and siRNA targeted at P-gp exhibited a positive correlation of P-gp expression with intracellular dexamethasone. The mean ± SEM correlation coefficients were 0.78 ± 0.07 for cells transfected with negative siRNA and 0.79 ± 0.08 for cells transfected with siRNA targeted at P-gp. Contrary to our hypothesis, the positive correlation between P-gp expression and intracellular dexamethasone suggests that P-gp is not a primary transporter of glucocorticoids from airway epithelial cells. Increased P-gp expression is unlikely to be an important mechanism of glucocorticoid resistance in asthma.

Inflammasome-mediated glucocorticoid resistance: The receptor rheostat

ABSTRACTIn primary acute lymphoblastic leukemia cells exhibiting de novo resistance to glucocorticoids, we recently discovered decreased promoter methylation of caspase 1 (CASP1) and NLR family, pyrin domain containing 3 (NLRP3), which resulted in increased transcription, constitutive NALP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome activation, and caspase 1-mediated cleavage of the glucocorticoid receptor. This revealed a novel mechanism of glucocorticoid resistance that was recapitulated in model systems.

PI3K inhibition synergizes with glucocorticoids but antagonizes with methotrexate in T-cell acute lymphoblastic leukemia.

The PI3K pathway is frequently hyperactivated in primary T-cell acute lymphoblastic leukemia (T-ALL) cells. Activation of the PI3K pathway has been suggested as one mechanism of glucocorticoid resistance in T-ALL, and patients harboring mutations in the PI3K negative regulator PTEN may be at increased risk of induction failure and relapse. By gene expression microarray analysis of T-ALL cells treated with the PI3K inhibitor AS605240, we identified Myc as a prominent downstream target of the PI3K pathway. A significant association was found between the AS605240 gene expression signature and that of glucocorticoid resistance and relapse in T-ALL. AS605240 showed anti-leukemic activity and strong synergism with glucocorticoids both in vitro and in a NOD/SCID xenograft model of T-ALL. In contrast, PI3K inhibition showed antagonism with methotrexate and daunorubicin, drugs that preferentially target dividing cells. This antagonistic interaction, however, could be circumvented by the use of correct drug scheduling schemes. Our data indicate the potential benefits and difficulties for the incorporation of PI3K inhibitors in T-ALL therapy.

Inhibition of autophagy overcomes glucocorticoid resistance in lymphoid malignant cells

Glucocorticoid (GC) resistance remains a major obstacle to successful treatment of lymphoid malignancies. Till now, the precise mechanism of GC resistance remains unclear. In the present study, dexamethasone (Dex) inhibited cell proliferation, arrested cell cycle in G0/G1-phase, and induced apoptosis in Dex-sensitive acute lymphoblastic leukemia cells. However, Dex failed to cause cell death in Dex-resistant lymphoid malignant cells. Intriguingly, we found that autophagy was induced by Dex in resistant cells, as indicated by autophagosomes formation, LC3-I to LC3-II conversion, p62 degradation, and formation of acidic autophagic vacuoles. Moreover, the results showed that Dex reduced the activity of mTOR pathway, as determined by decreased phosphorylation levels of mTOR, Akt, P70S6K and 4E-BP1 in resistant cells. Inhibition of autophagy by either chloroquine (CQ) or 3-methyladenine (3-MA) overcame Dex-resistance in lymphoid malignant cells by increasing apoptotic cell death in vitro. Consistently, inhibition of autophagy by stably knockdown of Beclin1 sensitized Dex-resistant lymphoid malignant cells to induction of apoptosis in vivo. Thus, inhibition of autophagy has the potential to improve lymphoid malignancy treatment by overcoming GC resistance.

Abstract 3964: NVP-BEZ235 enhances dexamethasone-induced BIM expression and apoptosis in models of T-ALL with PTEN dysfunction and increased PI3K/AKT activity

Abstract Introduction: Glucocorticoid (GC) resistance presents a major challenge in pediatric acute lymphoblastic leukemia (ALL) treatment. Increased activation of the PI3K/AKT pathway, which may occur following loss of PTEN function, may be a possible mechanism of GC resistance. We therefore investigated the effects of inhibiting the PI3K/AKT pathway with the dual PI3K/mTOR inhibitor NVP-BEZ235 on the enhancement of dexamethasone activity in in vitro and in vivo models of ALL and the role the PI3K/AKT pathway plays in resistance to GCs. Methods: Cytotoxicity of NVP-BEZ235, dexamethasone, and the combination were evaluated using DIMSCAN in in vitro models of T- and B-ALL and combination indices (CI) were calculated using CalcuSyn. Apoptotic activity for each single agent and the combination was determined by flow cytometry. Protein and mRNA expression were measured by immunoblotting and RT-PCR, respectively. Knockdown of BIM was accomplished using siRNA. In vivo effects were assessed following 28 days of treatment using NOD/SCID mice systemically engrafted with T-ALL model COG-LL-317h. Results: The combination of dexamethasone plus NVP-BEZ235 showed strong synergistic activity (CI &amp;lt; 0.3) in 6/6 T-ALL models tested, while such activity was seen in only 1/6 B-ALL models. Synergistic cytotoxicity corresponded with increased AKT phosphorylation. A systemic xenograft model of COG-LL-317h also showed significant increase in event-free survival in combination treated mice compared to vehicle control mice (P &amp;lt; 0.01), while there was no significant increase in survival with mice treated with either agent alone. In COG-LL-317h, the percentage of apoptotic cells were significantly greater in cells treated with the combination (24.8 ± 1.0%) compared with vehicle control (2.1 ± 0.2%, P &amp;lt; 0.0001), dexamethasone (4.4 ± 0.3%, P &amp;lt; 0.0001), or NVP-BEZ235 (2.0 ± 0.2%, P &amp;lt; 0.0001). In COG-LL-317h, the pro-apoptotic Bcl-2 family protein BIM was substantially increased at both the mRNA and protein levels when treated with the combination compared with either agent alone, while the B-cell ALL RS4;11 did not show any enhancement with the combination. Compared with non-targeting siRNA, transfection with siRNA against BIM resulted in significant reduction of cells undergoing apoptosis (39.3 ± 3.3% vs. 20.3 ± 0.7%, P=0.0007) and reduced caspase activation in response to the combination. Conclusion: These data suggest activation of the PI3K/AKT pathway following PTEN loss plays a significant role in reducing GC responses and use of the combination may offset that effect by increasing BIM expression and apoptosis. Our results show that NVP-BEZ235 may provide a new adjunct for ALL patients with hyperactivated PI3K/AKT pathway by enhancing dexamethasone activity. Citation Format: Connor Hall, Min Kang. NVP-BEZ235 enhances dexamethasone-induced BIM expression and apoptosis in models of T-ALL with PTEN dysfunction and increased PI3K/AKT activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3964. doi:10.1158/1538-7445.AM2014-3964

Glucocorticoid-resistant asthma: more than meets the eye

Introduction: For decades glucocorticoids have been considered as the gold standard for the treatment of asthma. We present a case report of typical glucocorticoid-resistant asthma and current consensus in definitions of “severe refractory”, “difficult” and “glucocorticoid-resistant” asthma. Methods: Full-text papers and abstracts were identified on the basis of a comprehensive literature search primarily in MEDLINE (1966 to June 2012) but also in the Cochrane Central Register of Controlled Trials database. Results: Glucocorticoid-resistant asthmatics are a small subset of patients who pose noteworthy diagnostic challenges while contributing disproportionately to health care costs. Recognition of various asthma phenotypes has aided in characterizing groups with severe asthma and given a better understanding of its pathophysiological process. The molecular mechanism of glucocorticoid action is complicated and several pathways have been identified to explain drug resistance, which in turn is crucial for drug development. Tobacco smoking appears to be the single most important contributor of glucocorticoid resistance. We present the emerging and promising concepts in the management of glucocorticoid-resistant asthma, which mainly include drugs targeting specific molecules, receptors, inflammatory cells or immune processes. Conclusion: The challenges in making a diagnosis of glucocorticoid-resistant asthma may contribute to underestimating its prevalence and impact on patient care. Considerable progress has been made in identifying distinct phenotypes and mechanisms of glucocorticoid resistance; therefore the future of new drug development in management of asthma is promising.

Emerging roles of pulmonary macrophages in driving the development of severe asthma

Asthma is recognized as a heterogeneous disorder, although in most patients, the clinical manifestations are effectively managed with established combination therapies. However, 5-10% of asthmatics have severe asthma, which does not respond to treatment, and these patients account for >50% of asthma-related healthcare costs. New investigations into the pathogenesis of glucocorticoid resistance in severe asthma indicate that pulmonary macrophages may play central roles in promoting airway inflammation, particularly in asthma that is resistant to steroid therapy. Importantly, factors that are linked to the activation of pulmonary macrophages may contribute to glucocorticoid resistance and severe asthma. Here, we review recent advances in understanding the roles of pulmonary macrophages in the mechanisms of glucocorticoid resistance and the pathogenesis of severe asthma. We discuss the role of macrophage phenotype, infection, IFN-γ, LPS, associated signaling pathways, TNF-α, MIF, and other macrophage-associated factors. Understanding the pathogenesis of steroid-resistant severe asthma will contribute to the identification of optimal therapeutic strategies for the effective management of the disease.

Glucocorticoid Sensitivity and Proinflammatory Cytokines Pattern in Pemphigus

Glucocorticoids (GC) represent the main treatment for pemphigus; however, some patients show GC resistance. GC sensitivity was evaluated in 19 pemphigus patients and 41 controls by the number of binding sites [B(max) (fmol/mg protein)] and the affinity of GC receptor [Kd (nM)] to dexamethasone (DEX) as well as by the pattern of cytokine by DEX-mediated inhibition of concanavalin-A (Con-A)-stimulated PBMC proliferation. The Kd (15.7 ± 2.8 vs.8.1 ± 1.3) and Bmax (6.5 ± 0.9 vs. 3.9 ± 0.3) were higher in pemphigus than controls (p = 0.002). Considering the values above the 95th percentile of normal group as a cut-off (K(d) > 24.9nM and B(max) > 8.1fmol/mg protein), elevated K(d) and B(max) were observed in 9.8% and 2.4% of controls and 15.8% and 36.8% of patients (p = 0.02). PBMC proliferation was stimulated by Con-A and inhibited by DEX (p < 0.001) in both pemphigus and control groups. IL-6 and TNFα (pg/mL) basal production were higher in patients than controls. There was an increment of these cytokines after Con-A stimulation, and they were inhibited by DEX (p = 0.002) in controls and remained elevated in pemphigus (p < 0.02). Patients and controls showed no difference in basal and stimulated production of IL-8 and IL-10. There is an alteration on GC sensitivity in pemphigus patients and a higher production of proinflammatory cytokines. Therefore, in pemphigus patients, proinflammatory cytokines might be involved in the mechanism of GC resistance and/or in its maintenance.