Glucocorticoid Sensitivity Research Articles

Epigenetic modifications and glucocorticoid sensitivity in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)

BackgroundMyalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating idiopathic disease characterized by unexplained fatigue that fails to resolve with sufficient rest. Diagnosis is based on a list of symptoms and exclusion of other fatigue-related health conditions. Despite a heterogeneous patient population, immune and hypothalamic-pituitary-adrenal (HPA) axis function differences, such as enhanced negative feedback to glucocorticoids, are recurring findings in ME/CFS studies. Epigenetic modifications, such as CpG methylation, are known to regulate long-term phenotypic differences and previous work by our group found DNA methylome differences in ME/CFS, however the relationship between DNA methylome modifications, clinical and functional characteristics associated with ME/CFS has not been examined.MethodsWe examined the DNA methylome in peripheral blood mononuclear cells (PBMCs) of a larger cohort of female ME/CFS patients using the Illumina HumanMethylation450 BeadChip Array. In parallel to the DNA methylome analysis, we investigated in vitro glucocorticoid sensitivity differences by stimulating PBMCs with phytohaemagglutinin and suppressed growth with dexamethasone. We explored DNA methylation differences using bisulfite pyrosequencing and statistical permutation. Linear regression was implemented to discover epigenomic regions associated with self-reported quality of life and network analysis of gene ontology terms to biologically contextualize results.ResultsWe detected 12,608 differentially methylated sites between ME/CFS patients and healthy controls predominantly localized to cellular metabolism genes, some of which were also related to self-reported quality of life health scores. Among ME/CFS patients, glucocorticoid sensitivity was associated with differential methylation at 13 loci.ConclusionsOur results indicate DNA methylation modifications in cellular metabolism in ME/CFS despite a heterogeneous patient population, implicating these processes in immune and HPA axis dysfunction in ME/CFS. Modifications to epigenetic loci associated with differences in glucocorticoid sensitivity may be important as biomarkers for future clinical testing. Overall, these findings align with recent ME/CFS work that point towards impairment in cellular energy production in this patient population.

2-Deoxy-D-glucose Restore Glucocorticoid Sensitivity in Acute Lymphoblastic Leukemia via Modification of N-Linked Glycosylation in an Oxygen Tension-Independent Manner.

In childhood acute lymphoblastic leukemia, treatment failure is associated with resistance to glucocorticoid agents. Resistance to this class of drugs represents one of the strongest indicators of poor clinical outcome. We show that leukemic cells, which are resistant to the glucocorticoid drug methylprednisolone, display a higher demand of glucose associated with a deregulation of metabolic pathways, in comparison to sensitive cells. Interestingly, a combinatorial treatment of glucocorticoid and the glucose analog 2-deoxy-D-glucose displayed a synergistic effect in methylprednisolone-resistant cells, in an oxygen tension-independent manner. Unlike solid tumors, where 2-deoxy-D-glucose promotes inhibition of glycolysis by hexokinase II exclusively under hypoxic conditions, we were able to show that the antileukemic effects of 2-deoxy-D-glucose are far more complex in leukemia. We demonstrate a hexokinase II-independent cell viability decrease and apoptosis induction of the glucose analog in leukemia. Additionally, due to the structural similarity of 2-deoxy-D-glucose with mannose, we could confirm that the mechanism by which 2-deoxy-D-glucose predominantly acts in leukemia is via modification in N-linked glycosylation, leading to endoplasmic reticulum stress and consequently induction of the unfolded protein response.

FKBP5 polymorphism is associated with insulin resistance in children and adolescents with obesity

Since metabolic syndrome shares several clinical features with hypercortisolism, it was hypothesised that genes altering individual glucocorticoid (GC) sensitivity might be implicated in pathogenesis of obesity and its adverse outcomes. FKBP5 gene encodes a chaperon protein in the GC receptor (GR) complex, which modulates steroid action upon target genes. Its functional variant, rs1360780, may enhance FKBP5 gene transcription, affect GR signalling and thereby influence the hypothalamo-pituitary-adrenal axis. We investigated the association of rs1360780 with obesity and metabolic characteristics in 250 obese children and adolescents (mean age 12.3±3.6years, BMI ≥95th percentile). Anthropometric measurements, body composition, biochemical and hormonal results were analysed. Genotyping of rs1360780 was compared with 568 lean controls. Impaired fasting glucose was present in 8.8%, glucose intolerance in 10.4%, diabetes in 2.8% and dyslipidemia in 28.8% obese individuals. Hypertension was diagnosed in 34 out of 143 patients. No difference was found in FKBP5 polymorphism distribution between subjects with obesity and controls (p>0.05). Stratification by rs1360780 revealed no differences in body mass and composition. However, carriers of the minor allele displayed enhanced insulin resistance (p=0.009) and elevated serum triglyceride (p=0.006), whereas cholesterol, HbA1c, and oral glucose challenge results were similar for all genotypes. Morning ACTH and cortisol did not differ but evening cortisol was higher in minor allele carriers (p=0.039), although this association was lost in logistic regression analysis. This study does not support the association of FKBP5 with obesity but demonstrates plausible implication of its variant in susceptibility to obesity-related insulin resistance and hypertriglyceridemia.

Early Different Downstream Target of Glucocorticoid Receptor Contributing to Glucocorticoids Sensitivity in Kasumi-1 Cells

The t(8;21) chromosome translocation frequently occurs in acute myeloid leukemia (AML), resulting in an in-frame fusion between the DNA-binding domain of AML1 and almost the entire of ETO gene. The fusion AML1-ETO protein is thought to play a critical role in the abnormal proliferation and differentiation of myeloid leukemia cells, such as Kasumi-1 and SKNO-1 cells. Glucocorticoids (GC) can induce apoptosis in these cells at low concentrations, whereas most other myeloid leukemia cell lines are resistant to glucocorticoid-induced apoptosis. To experimentally address possible sensitive mechanisms in leukemia cells with AML1-ETO translocation, we generated aGC-resistant Kasumi-1 cell line by induction of 10-6 M dexamethasone (Dex) for three weeks. The IC50 of Dex to cells is increased from 2.5×10-8 M for original GC-sensitive Kasumi-1 cell line ( K-S cell line) to more than 1×10-5 M for induced GC-resistant Kasumi-1 cell line (K-R cell line). Since GC resistance often results from mutations in the glucocorticoid receptor (GR), all the exons of GR gene were sequenced and no mutation was found in K-R cells. Comparing to those in K-S cells, the GR protein level didn’t decrease in K-R cells after 2h, 4h, 8h, 12h and 24h exposure to dexamethasone. Given that the difference of direct GR downstream genes between K-S and K-R cells may play a key role in the GC sensitivity, we systematically analyzed the changes of gene expression induced by Dex versus ethanol vehicle for 8h in K-S and K-R cells by high throughput RNA sequencing. The time point of 8h was selected according to the expression peaks of several foregone GR target genes after Dex induction. There were found 32 genes conversely regulated in K-S and K-R cells, including 14 mRNAs and 18 long non-coding RNAs. Pathway analysis indicated that the upregulated genes in K-S cells might promote the AML1-ETO fusion protein degradation by proteasomes, while the component genes of this pathway were downregulated in K-R cells. Further validation and function studies of these mRNAs and long non-coding RNAs are ongoing. Our data suggested that the downstream targets of GR among GC-sensitive and -resistant Kasumi-1 cells were significant different and they may contribute to the GC sensitivity and resistance by degradation or reservation of AML-ETO fusion protein and the regulation of apoptosis in t(8;21) leukemia cell subtype. DisclosuresNo relevant conflicts of interest to declare.

JAK/STAT Pathway Inhibition Reverts IL7-Induced Glucocorticoid Resistance in a Subset of Human T-Cell Acute Lymphoblastic Leukemia

Although outcomes for patients with T-cell acute lymphoblastic leukemia (T-ALL) have improved dramatically, survival rates for relapsed or refractory T-ALL remain less than 10%. While mechanisms mediating chemotherapy resistance in these patients remain incompletely understood, resistance to glucocorticoids (GC), a central component of therapy, may be particularly important. GC resistance occurs more commonly than resistance to other chemotherapeutic agents in ALL. Additionally, newly diagnosed patients that fail to rapidly clear their peripheral leukemic blasts during an upfront window of prednisone (prednisone poor response) have a poorer outcome, suggesting differences in GC sensitivity may exist at diagnosis.Here, we develop an in vitro assay to model the early prednisone response. Using primary, pre-treatment human T-ALL samples, we demonstrate that individual T-ALLs have distinct intrinsic GC sensitivity thresholds at diagnosis and that this threshold can predict end induction MRD. To interrogate potential mechanisms of GC resistance, we use a panel of patient-derived xenografts (PDX) generated from diagnostic T-ALLs. We find that intrinsic GC resistance is uniformly seen in T-ALLs that arise at the early thymic progenitor (ETP) stage as well as in a subset of non-ETP T-ALLs. Removal of IL7 from the media or inhibition of IL7/JAK/STAT signaling with the JAK1/2 inhibitor ruxolitinib or a novel JAK3 inhibitor, JAK3i, reverses GC resistance in ETP and a subset of the non-ETP T-ALL. This effect is drug specific, since IL7 does not offer protection from death induced by other agents. IL7-dependent GC resistance can be predicted by hyper-responsiveness to IL7 stimulation. Mechanistically, the combination of dexamethasone and ruxolitinib alters the balance between BCL2 and BIM in IL7-dependent, but not IL7-independent, GC resistant T-ALL samples.Together, these data support a model where IL7, a cytokine with leukemogenic properties that is normally present in lymphopoietic niches, contributes to intrinsic GC resistance in a subset of T-ALL samples. This environmentally induced GC resistance may be reversed with IL7/JAK/STAT pathway inhibition. This could result in an augmented leukemotoxic effect of GC treatment, potentially enhancing efficacy of glucocorticoids in a subset of patients and justifying exposure to the toxic side effects of GCs. DisclosuresTaunton:Global Blood Therapeutics: Equity Ownership; Kezar Life Sciences: Equity Ownership, Research Funding; Pfizer: Research Funding; Principia Biopharma: Consultancy, Equity Ownership; Circle Pharma: Consultancy, Equity Ownership; Cell Design Labs: Consultancy, Equity Ownership. Wood:Juno: Other: Laboratory Services Agreement; Amgen: Honoraria, Other: Laboratory Services Agreement; Pfizer: Honoraria, Other: Laboratory Services Agreement; Seattle Genetics: Honoraria, Other: Laboratory Services Agreement. Teachey:Novartis: Research Funding.

Development and validation of a novel bioassay to determine glucocorticoid sensitivity.

BackgroundGlucocorticoids (GCs) remain the first line treatment for almost all non-infectious inflammatory diseases, ranging from acute asthma to rheumatoid arthritis. However, across all conditions, patients have a variable response to GCs with approximately 30% being non-responders. This group of GC resistant patients is typically exposed to high-dose GCs and their side-effects before more appropriate immunotherapy is instituted. Hence, there is a pressing clinical need for a predictive biomarker of GC responsiveness. The availability of such a tool would also enable patient stratification for the conduct of smart clinical trials in GC resistance. Lymphocyte GC sensitivity has been shown to be closely associated with clinical GC sensitivity in a number of inflammatory diseases. However, the method for determining in vitro GC response is not standardized and requires the use of specialist equipment, including a radioisotope to quantify cellular proliferation, making it challenging to translate into clinical practice.ResultsHere we describe the optimization and validation of a novel non-radioactive in vitro bioassay based on measuring cellular proliferation by incorporation of bromodeoxyuridine (BrdU), termed the BrdU incorporation in lymphocyte steroid sensitivity assay (BLISS). In comparison to the current gold standard lymphocyte GC sensitivity assay in 101 healthy control samples, BLISS has an area under receiver operating characteristic of 0.82 and a sensitivity of 83% for correctly identifying GC resistant subjects.ConclusionsThe performance of the novel BLISS bioassay makes it a strong candidate biomarker for clinical application. It now requires validation in a prospective patient cohort.Electronic supplementary materialThe online version of this article (doi:10.1186/s40364-016-0079-y) contains supplementary material, which is available to authorized users.

Ginsenoside Rg1 attenuates ultraviolet B-induced glucocortisides resistance in keratinocytes via Nrf2/HDAC2 signalling.

Oxidative stress, which occurs after ultraviolet (UV) radiation, usually results in Glucocorticoid (GC) resistance and the subsequent development of skin inflammation. One approach to protecting the skin against UV radiation is the use of antioxidants. The ginsenoside Rg1 is a novel natural antioxidant isolated from the medicinal plant Panax ginseng C.A. Mey. We demonstrated that UVB exposure exacerbated inflammation and reduced both the level of the glucocorticoid receptor (GR) and the efficacy of dexamethasone (Dex) in human keratinocytes (HaCaT cells). Pretreatment with Rg1 increased the expression of GR and restored Dex responsiveness to inflammation in UVB-irradiated HaCaT cells. Mechanistically, Rg1 rescued UVB-induced HDAC2 degradation. HDAC2 knockdown partially abolished the Rg1-induced up-regulation of GR and the enhancement of GC sensitivity. In addition, Rg1 reduced the production of reactive oxygen species (ROS), which preceded the up-regulation of HDAC2, and consequent sensitization of cells to Dex. Moreover, Rg1 treatment promoted the translocation and activation of Nrf2. Nrf2 knockdown partially abolished the Rg1-induced decrease of ROS production and increase of HDAC2. Rg1 also potentiated the anti-inflammatory effects of Dex in UVB-irradiated mouse skin. In conclusion, we demonstrated that Rg1 attenuated UVB-induced GC insensitivity. Notably, these effects were partially mediated by the Nrf2/HDAC2 pathway.

Enhanced sensitivity to glucocorticoids in cytarabine-resistant AML

We sought to identify drugs that could counteract cytarabine resistance in acute myeloid leukemia (AML) by generating eight resistant variants from MOLM-13 and SHI-1 AML cell lines by long-term drug treatment. These cells were compared with 66 ex vivo chemorefractory samples from cytarabine-treated AML patients. The models and patient cells were subjected to genomic and transcriptomic profiling and high-throughput testing with 250 emerging and clinical oncology compounds. Genomic profiling uncovered deletion of the deoxycytidine kinase (DCK) gene in both MOLM-13- and SHI-1-derived cytarabine-resistant variants and in an AML patient sample. Cytarabine-resistant SHI-1 variants and a subset of chemorefractory AML patient samples showed increased sensitivity to glucocorticoids that are often used in treatment of lymphoid leukemia but not AML. Paired samples taken from AML patients before treatment and at relapse also showed acquisition of glucocorticoid sensitivity. Enhanced glucocorticoid sensitivity was only seen in AML patient samples that were negative for the FLT3 mutation (P=0.0006). Our study shows that development of cytarabine resistance is associated with increased sensitivity to glucocorticoids in a subset of AML, suggesting a new therapeutic strategy that should be explored in a clinical trial of chemorefractory AML patients carrying wild-type FLT3.

The evolution, structure and function of the ray finned fish (Actinopterygii) glucocorticoid receptors

Basal ray-finned fish (Actinopterygii) possess a single glucocorticoid receptor (GR) and when compared to the lobe-finned vertebrate (Sarcopterygii) GR possess nine additional amino acids between the zinc-finger of the DNA binding domain. A whole genome duplication event which occurred between 320 and 350MYA in the teleost lineage following the split from the basal ray-finned fish resulted in 2 GRs: one GR group, GR1, has retained the 9 amino acids insert whereas the other group, GR2, has not. The exception to this is the zebrafish, that have lost one of the GRs, but they do possess 2 GRs with a splice variant that lacks the C-terminal portion of the GR to form GRβ which acts as a dominant-repressor of the wildtype GR. Another splice variant sees the basal ray-finned GR and teleost GR1 without the 9 amino acids insert. The molecular basis for GRs retention is beginning to be unravelled. In Pantadon buchholzi, rainbow trout, carp, marine and Japanese medaka GR2 is more sensitive to glucocorticoids (GC), thus potentially playing a more significant role in regulating gene expression at basal circulatory GC concentrations. However, this division in GC sensitivity is not seen in other species. The few studies to evaluate the significance of the 9 amino acid insert have shown that it affect maximal transactivational activity the extent to which is dependent on the number of glucocorticoid response elements (GREs) present in the reporter plasmid. The retention of these GRs would suggest there was an evolutionary advantage, which saw the development of a complex regulatory process to mediate the actions of the glucocorticoids.

Network-based expression analysis reveals key genes related to glucocorticoid resistance in infant acute lymphoblastic leukemia.

Despite vast improvements that have been made in the treatment of children with acute lymphoblastic leukemia (ALL), the majority of infant ALL patients (~80%, < 1year of age) that carry a chromosomal translocation involving the mixed lineage leukemia (MLL) gene shows a poor response to chemotherapeutic drugs, especially glucocorticoids (GCs), which are essential components of all current treatment regimens. Although addressed in several studies, the mechanism(s) underlying this phenomenon have remained largely unknown. A major drawback of most previous studies is their primary focus on individual genes, thereby neglectingthe putative significance of inter-gene correlations. Here, we aimed at studying GC resistance in MLL-rearranged infant ALL patients by inferring an associated module of genes using co-expression network analysis. The implications of newly identified candidate genes with associations to other well-known relevant genes from the same module, or with associations to known transcription factor or microRNA interactions, were substantiated using literature data. A weighted gene co-expression network was constructed to identify gene modules associated with GC resistance in MLL-rearranged infant ALL patients. Significant gene ontology (GO) terms and signaling pathways enriched in relevant modules were used to provide guidance towards which module(s) consisted of promising candidates suitable for further analysis. Through gene co-expression network analysis a novel set of genes (module) related to GC-resistance was identified. The presence in this module of the S100 and ANXA genes, both well-known biomarkers for GC resistance in MLL-rearranged infant ALL, supports its validity. Subsequent gene set net correlation analyses of the novel module provided further support for its validity by showing that the S100 and ANXA genes act as 'hub' genes with potentially major regulatory roles in GC sensitivity, but having lost this role in the GC resistant phenotype. The detected module implicates new genes as being candidates for further analysis through associations with known GC resistance-related genes. From our data we conclude that available systems biology approaches can be employed to detect new candidate genes that may provide further insights into drug resistance of MLL-rearranged infant ALL cases. Such approaches complement conventional gene-wise approaches by taking putative functional interactions between genes into account.

LUMAN/CREB3 is a key regulator of glucocorticoid-mediated stress responses

Altered glucocorticoid sensitivity is believed to contribute to a number of human diseases, including inflammatory and autoimmune conditions as well as disorders characterized by abnormal hypothalamic-pituitary-adrenal axis (HPA) function. LUMAN (or CREB3), originally identified through its interaction with a cell cycle regulator HCFC1, is an endoplasmic reticulum membrane-bound transcription factor that is involved in the unfolded protein response. Here we demonstrate that LUMAN changes the glucocorticoid response by modulating the expression of the glucocorticoid receptor leading to an overall increase in GR activity. Luman-deficient mice exhibited a blunted stress response characterized by low levels of both anxiety and depressive-like behaviour in addition to low circulating corticosterone levels. These mice also have reduced dendritic branching in the CA3 region of the hippocampus, consistent with increased GR responses. These findings are consistent with the notion that elevated GR activities are the primary cause of the observed phenotype in these LUMAN-deficient mice. We thus postulate that LUMAN is a key regulator of GR-mediated signaling and modulates HPA axis reactivity.

Immunopathology alters Th17 cell glucocorticoid sensitivity.

Th17 cells contribute to several inflammatory conditions and increasing evidence supports that Th17 cells are glucocorticoid resistant. However, Th17 cells in psoriasis and related diseases are glucocorticoid sensitive. We compare glucocorticoid sensitive and resistant immunological diseases and suggest that several aspects in Th17-related diseases alter glucocorticoid sensitivity of Th17 cells. We identify molecular pathways that are implicated in glucocorticoid sensitivity of Th17 cells in the literature, as this information is useful for developing approaches to overcome glucocorticoid-resistant immunopathology.

Rooibos (Aspalathus linearis) facilitates an anti-inflammatory state, modulating IL-6 and IL-10 while not inhibiting the acute glucocorticoid response to a mild novel stressor in vivo

Rooibos is rich in polyphenols with a high anti-oxidant capacity, modulating adrenal steroidogenesis while also influencing cytokine levels. The aim of this study was to investigate Rooibos' immuno-modulating properties, given the interdependence of inflammatory and oxidative stress systems, together with the regulatory function of cytokines in steroidogenesis. Rooibos did not prevent an adequate corticosterone response in rats, with corticosterone and deoxycorticosterone levels significantly increased after 1 hr restraint. IL-6 levels decreased in Rooibos-treated rats while IL-10 (P < 0.05) and IL-10:IL-6 ratios (P < 0.01) increased. Rooibos inhibited glucocorticoid production more significantly in forskolin-stimulated than in basal H295R cells (corticosterone and deoxycorticosterone, P < 0.00001; 11-dehydrocorticosterone, P < 0.01), independent of IL-6 and IL-10. Rooibos therefore modulated the inflammatory response basally and during acute stress. While Rooibos affects steroid hormone biosynthesis, maintaining beneficial glucocorticoid levels and possibly achieving better long-term outcome in terms of glucocorticoid sensitivity, the ability to respond to acute stressors is unlikely to be compromised.

Glucocorticoid receptor haplotypes conferring increased sensitivity (BclI and N363S) are associated with faster progression of multiple sclerosis

As high cortisol levels are implicated in suppressed disease activity of multiple sclerosis (MS), glucocorticoid receptor (GR) polymorphisms that affect glucocorticoid (GC) sensitivity may impact on this by changing local immunomodulation or regulation of the hypothalamus–pituitary–adrenal (HPA)-axis. In this post-mortem study, we investigated whether GR haplotypes affect MS disease course and production of cortisol and soluble CD163 (sCD163), a molecule induced by GC on microglia/macrophages. We found that GR haplotypes that confer high GC sensitivity are associated with more aggressive MS but do not affect levels of cortisol secreted by the HPA-axis or shedding of CD163.

Abstract # 1824 Healthy smokers’ peripheral blood lymphocytes exhibit differential inflammatory gene expression to glucocorticoid and lipopolysaccharide exposure

Cigarette smoking is linked to elevated inflammation and development of chronic inflammation-related conditions such as asthma, autoimmune diseases, and chronic bronchitis. Among these patient populations, those who smoke often have poorer symptom management/relief via usual treatments, including glucocorticoids, suggesting glucocorticoid resistance. Even less is understood about the early changes in smokers’ immune function especially those who have not yet developed a clinical disease. Thus, we recruited healthy smokers (e.g., no clinical diagnoses or current medication use) and age-, sex-, and BMI-matched never smokers to examine glucocorticoid sensitivity. Following a 12-h fast, 47 adults (30.5 +/−8.1 yrs; 27 males) provided a blood sample and answered several psychosocial questionnaires. Using a repeated-measures ANOVA, dexamethasone significantly reduced all peripheral blood lymphocytes’ (PBLs) IL-6, TNF-alpha, and IFN-gamma mRNA expression response to lipopolysaccharide (LPS; p p p = .07) compared to smokers. Thus, our data suggest that even prior to chronic disease development and elevated systemic inflammation (i.e., CRP levels

Social role conflict predicts stimulated cytokine production among men, not women

ObjectiveTo assess whether perceived role conflict is associated with stimulated pro-inflammatory cytokine production and glucocorticoid sensitivity, and whether these associations are moderated by sex. Methods153 healthy adults (aged 45.8±5.5years, 78% female) listed their 3 main social roles and indicated the amount of role conflict they perceived between each pair of social roles. Subsequently, participants underwent blood draws and leukocyte response to microbial challenge and glucocorticoid sensitivity were assessed by incubating whole blood with lipopolysaccharide (LPS) in the presence or absence of hydrocortisone. Stimulated levels of Interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor alpha (TNFα) were measured. ResultsMultiple regression analyses controlling for sociodemographics revealed significant sex×role conflict interactions for LPS-stimulated production of IL-1β, IL-6, and TNFα (all interaction ps<0.05), and a marginal interaction on LPS-stimulated IL-8 production (interaction p<0.10). Greater perceived role conflict was associated with greater pro-inflammatory cytokine production in response to microbial stimulation only among men, not women. There also were significant sex×role conflict interactions with respect to glucocorticoid sensitivity for IL-1β, IL-6, and TNFα production (all interaction ps<0.05) and a marginal interaction for IL-8 (interaction p<0.10). Greater perceived role conflict was unrelated to glucocorticoid sensitivity among women, but associated with less sensitivity to glucocorticoid signaling among men. ConclusionsPerceived social role conflict, indicating greater perceived demand across multiple social roles, may take a greater toll on the regulation of inflammatory processes among men compared to women.

Abstract 4679: Acquisition of cytarabine resistance leads to increased glucocorticoid sensitivity in AML

Abstract Acquired resistance to standard chemotherapeutic agents, such as cytarabine, is a major challenge in the treatment of acute myeloid leukemia (AML). Here, we hypothesized that development of resistance to one chemotherapeutic agent may lead to increased sensitivity to other drugs. Hence, we sought to identify novel drug vulnerabilities that arise during the development of cytarabine resistance using both cytarabine resistant AML cell lines and samples from AML patients who had relapsed during cytarabine containing chemotherapy. We developed resistant variants of AML cell lines MOLM-13 and SHI-1 by long-term drug treatment with increasing doses of cytarabine. Profiling data from the in vitro generated cytarabine resistant cell line variants were systematically compared with corresponding data from 31 chemorefractory AML patient samples. All samples were subjected to genomic and transcriptomic profiling and high-throughput drug sensitivity and resistance testing with a panel of 250 chemical compounds (each in five doses). Cytarabine resistant AML cell line variants and patient samples showed co-resistance to other nucleoside analogues, such as cladribine, clofarabine and gemcitabine. Genomic profiling showed deletion of the deoxycytidine kinase gene DCK, a well-known genetic lesion related to cytarabine resistance, in both MOLM-13 and SHI-1 cytarabine resistant cell lines and in one chemorefractory AML patient. Importantly, comprehensive drug testing revealed that cytarabine resistant SHI-1 cell variants developed increased sensitivity to glucocorticoids, such as dexamethasone, methylprednisolone and prednisolone when compared to parental cells. This was accompanied by up-regulation of the glucocorticoid receptor NR3C1. We also observed acquisition of glucocorticoid sensitivity in paired samples from two AML patient cases who had relapsed after cytarabine containing chemotherapy. Systematic ex vivo drug testing of 31 relapsed and chemorefractory AML patient samples showed high sensitivity to dexamethasone in five (20%) and to prednisolone and methylprednisolone in four (13%) patient samples. In conclusion, our results from both cytarabine resistant AML cell lines and chemorefractory patient samples indicate that a subset of AML samples develop sensitivity to glucocorticoids. This novel finding indicates the need of detailed investigation of glucocorticoid efficacy in the clinic. Citation Format: Disha Malani, Astrid Murumägi, Bhagwan Yadav, Mika Kontro, Samuli Eldfors, Ashwini Kumar, Krister Wennerberg, Caroline Heckman, Kimmo Porkka, Maija Wolf, Tero Aittokallio, Olli Kallioniemi. Acquisition of cytarabine resistance leads to increased glucocorticoid sensitivity in AML. [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 4679.

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.

Abstract CT115: Pharmacokinetics of standard versus short high-dose dexamethasone therapy in childhood acute lymphoblastic leukemia: results from the UKALL 2011 trial

Abstract Dexamethasone (Dex) is a key component of ALL therapy, with glucocorticoid sensitivity strongly linked to prognosis. However, it also contributes to life threatening toxicities. The ongoing UKALL 2011 trial is investigating a new schedule of Dex (10mg/m2 × 14 days vs 6mg/m2 × 28 days), in an attempt to bring about a more rapid cytoreduction whilst limiting toxicities associated with long term steroid exposure. There are limited data regarding Dex pharmacokinetics, however large variability has been reported in children with ALL (Yang et al. 2008). For Dex pharmacokinetic studies, blood samples were collected up to 8h post oral administration on one of the first and last three days of induction chemotherapy. Plasma Dex levels were analysed using a validated LC-MS method, with a range of 5-250ng/ml, and non-compartmental pharmacokinetic analysis was performed. Pharmacokinetic parameters from day one sampling are shown below. Exposure, as defined by AUC0-12h, and Cmax were significantly higher on the short arm (p = 0.0002 and 0.0007, respectively). However there was substantial overlap between the two arms, with a number of patients on the standard arm exhibiting higher exposures than those on short therapy, despite having a longer duration of treatment. This is reflected in the AUC0-12h ranges observed on the two arms (short: 202-1606; standard: 38-1009 hr*ng/mL). Pharmacokinetic profiles also differed between the two days of treatment, with AUC0-12h being significantly higher at the end of induction chemotherapy (n = 37, study day 1: 655 ± 322; study day 2: 894 ± 496 hr*ng/mL, p = 0.003). Based on these preliminary data it will be important to consider pharmacokinetic variability when analysing the results generated from the UKALL 2011 trial. Further evaluation of relationships between pharmacokinetic variation, dose scheduling and clinical outcome may enable better stratification of Dex therapy for future patients. Dexamethasone pharmacokinetics in short and standard therapy arms following day one samplingPharmacokinetic parameters (mean ± SD)Half life (hr)Tmax (hr)Cmax(ng/mL)Clearance(L/hr/m2)AUC0-12h(hr*ng/mL)Short (n=43)3.96 ± 3.21.9 ± 1.0128 ± 558.1 ± 5.3692 ± 325Standard (n=33)3.68 ± 2.11.7 ± 1.187 ± 419.2 ± 12.7443 ± 202 Citation Format: Rosanna K. Jackson, Julie AE Irving, Gareth J. Veal. Pharmacokinetics of standard versus short high-dose dexamethasone therapy in childhood acute lymphoblastic leukemia: results from the UKALL 2011 trial. [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 CT115.

MECHANISMS IN ENDOCRINOLOGY: Endogenous subclinical hypercortisolism and bone: a clinical review.

In recent years, the condition of subclinical hypercortisolism (SH) has become a topic of growing interest. This is due to the fact that SH prevalence is not negligible (0.8-2% in the general population) and that, although asymptomatic, this subtle cortisol excess is not harmless, being associated with an increased risk of complications, in particular of osteoporosis and fragility fractures. As specific symptoms of hypercortisolism are absent in SH, the SH diagnosis relies only on biochemical tests and it is a challenge for physicians. As a consequence, even the indications for the evaluation of bone involvement in SH patients are debatable and guidelines are not available. Finally, the relative importance of bone density, bone quality and glucocorticoid sensitivity in SH is a recent field of research. On the other hand, SH prevalence seems to be increased in osteoporotic patients, in whom a vertebral fracture may be the presenting symptom of an otherwise asymptomatic cortisol excess. Therefore, the issue of who and how to screen for SH among the osteoporotic patients is widely debated. The present review will summarize the available data regarding the bone turnover, bone mineral density, bone quality and risk of fracture in patients with endogenous SH. In addition, the role of the individual glucocorticoid sensitivity in SH-related bone damage and the problem of diagnosing and managing the bone consequences of SH will be reviewed. Finally, the issue of suspecting and screening for SH patients with apparent primary osteoporosis will be addressed.