Glucocorticoid Resistance In Patients Research Articles

The Transcription Factors NFATc1 and NFATc2 Control Glucocorticoid Resistance in Pediatric T-Cell Acute Lymphoblastic Leukemia

Background: The overall survival of pediatric T-cell Acute Lymphoblastic Leukemia (T-ALL) is significantly improved (˜75-80%) with the current therapeutic approaches. Nevertheless, for those patients that do not respond to conventional treatment and experience relapse, the prognosis is extremely poor. In this context, it is well defined that resistance to glucocorticoids (GCs), pillar drugs in the treatment protocol, can predispose pediatric T-ALL patients to a poor outcome. A pivotal role of LCK kinase has been elucidated in supporting GC resistance in T-ALL cells in our previous study and recently confirmed by other colleagues. However, to date, the biological processes modulated by LCK in this context are not yet defined. Aim: the identification of new mechanisms underlying GC resistance can lead to alternative therapeutic approaches to prevent or overcome GC resistance and ameliorate the outcome of this subgroup of patients. Methods and Results: we uncovered the involvement of NFATc1 and NFATc2 transcription factors, that act downstream LCK kinase, in guiding GC resistance in T-ALL cells. Of note, a high NFATc1 and NFATc2 transcriptional activity characterizes pediatric GC resistant T-ALL patients at diagnosis, that in turns show a low Glucocorticoid Receptor (GR) activity. In agreement, NFATc1 or NFATc2 specific gene silencing in 3 T-ALL GC resistant cell line models and primary cells increases dexamethasone response, by restoring GR canonical transcriptional activity through the increaseexpression of BIM GR targetgene (p value <0.05). Conversely, NFATc1 or NFATc2 overexpression in a murine T-ALL GC sensitive cell line confers resistance to dexamethasone treatment. Interestingly, by Gene Expression Profile (GEP) and Nuclear Magnetic Resonance (NMR) analysis, we observed that NFATc1 gene silencing in GC resistant cells significantly downregulates intracellular cholesterol abundance. Additionally, by Chromatin Immunoprecipitation (ChIP) we revealed that NFATc1 can directly control the transcription of HMGCS1, EBP and DHCR7, key enzymes of cholesterol biosynthesis process (n≥3, p value <0.05 for all the three genes). In agreement, exogenous cholesterol addition to NFATc1 knock-down cell lines rebuild dexamethasone resistance. Conversely, by GEP and flow cytometry analysis we observed that NFATc2 gene silencing in T-ALL GC resistant cells leads to a downregulation of the Wnt/β-catenin signaling pathway and to an increased T-cell differentiation. Furthermore, by ChIP analysis we revealed that in T-ALL GC resistant cells NFATc2 can directly affect the transcription of LRP6, a key Wnt/β-catenin signaling component (n=3, p value <0.05). In agreement, the Wnt/β-catenin signaling activation, by Wnt3a stimulation, restores dexamethasone resistance in NFATc2 knock-down T-ALL cells. Finally, we observed that the inhibition of cholesterol biosynthesis by simvastatin or of Wnt/β-catenin by PRI-724 increases GC sensitivity in T-ALL GC resistant cells by the High-Throughput drug synergism Screening (HTS) and the Highest Single Agent (HSA) approach. Conclusions: Overall, we revealed for the first time the involvement of NFATc1 and NFATc2 transcription factors in supporting GC resistance in T-ALL cells by the modulation of cholesterol biosynthesis and Wnt/β-catenin signaling, both processes well-described in sustaining chemotherapy resistance, paving the rationale to alternative therapeutic options for T-ALL GC resistant pediatric patients.

Correlation between nuclear expression of heat shock protein 90 in dermis and glucocorticoid resistance in bullous dermatosis

Backgroundbullous dermatosis is a group of skin diseases that occur on the skin and mucous membrane, with blister and bulla as basic damage, mainly including pemphigus and bullous pemphigoid. Glucocorticoid (GC) is still the preferred drug for its treatment, but some patients respond poorly to GC and even develop glucocorticoid resistance (GCR). However, at present about the disease the understanding of the mechanisms for GCR is limited. ObjectiveThis study attempted to investigate the molecular mechanism of GCR in bullous dermatosis with heat shock proteins 90 (HSP90) and glucocorticoid receptor (GR) as molecular targets. MethodsIn this study, flow cytometry was used to measure and analyze the expression of HSP90 and GR in the lesions of patients with glucocorticoid-resistant bullosa dermatosis. Immunohistochemistry and immunofluorescence were used to observe the expression distribution and cell localization of HSP90 and GR. ResultsThe expression of HSP90 in skin lesions of GCR group was significantly higher than that of glucocorticoid-sensitive (GCS) group, while the expression level of GR was lower than that of GCS group. In the epidermis, the expression and distribution of HSP90 were not different between the GCR group and the GCS group. And in the dermis, HSP90 and GR were more likely to be expressed in the nucleus in the GCR group. ConclusionThe overexpression and nuclear distribution of HSP90 may be related to the occurrence of GCR in patients with bullous dermatosis. And this correlation is more likely to occur in the dermis than in the epidermis.

REVEALING THE MOLECULAR-GENETIC AND CLINICAL PREDICTORS OF GLUCOCORTICOID RESISTANCE IN PATIENTS WITH HAND ECZEMA.

The aim: To reveal the possible predictors of the glucocorticoid resistance in patients with hand eczema (HE) based on the demographic, clinical, and molecular-genetic data. Materials and methods: 143 patients with HE were included in the study. Demographic, clinical, biochemical (blood content of IgE, IL-17A, IL-2, 25(OH)D), and genetic (rs41423247 genotypes) data were obtained from all patients. Results: After 2 weeks of treatment by glucocorticoids, all subjects were divided into "responder" and "non-responder" groups according to change of the Hand Eczema Severity Index (HECSI). Statistical analysis was done using SPSS (version 22.0.). Binary logistic regression was used to identify predictors of glucocorticoid resistance. P-value 0.05). The results of the multivariate regression showed that Bcl-1 G-allele (OR =3.83; P = 0.033), and severe eczema (OR = 2.52; P = 0.023) are linked with an elevated risk of glucocorticoid resistance in patients with hand eczema. Conclusions: Insensitivity to glucocorticoids in HE patients is associated with NR3C1 gene Bcl-1 polymorphism, eczema severity and blood level of IL-17, IL-2, 25(OH)D. The final adjustment showed that minor C-allele of the Bcl-1 polymorphism and severe eczema are the strongest predictors of the glucocorticoid resistance.

Repression of the Orphan Nuclear Receptor Esrrb Results in Glucocorticoid Resistance in ALL

▪Acute lymphoblastic leukemia (ALL) is an aggressive hematological malignancy, in which 20-30% of pediatric patients relapse or undergo induction failure (Vrooman, 2009). ALL is treated with a combination of chemotherapeutic agents, including glucocorticoids (GCs). Pediatric ALL patients who exhibit resistance to GCs at diagnosis or relapse have a poor prognosis(Dördelmann, 1999). We performed an unbiased whole genome survival-based shRNA screen in primary mouse T-ALL cells to identify GC resistance genes. This shRNA screen identified known mediators of GC resistance as well as novel genes involved in chromatin remodeling, cell differentiation and metabolism.One of the top hits in the GC resistance screen were shRNAs targeting the orphan nuclear receptor ESRRB. ESRRB is essential for the maintenance of pluripotency in mouse embryonic stem cells and decreases in ESRRB expression stimulate differentiation (Zhang, 2008). We observe no effects of ESRRB deficiency on leukemic cell growth or differentiation. However, silencing of Esrrb prevents GC-induced apoptosis in mouse and human T-ALL cell lines in vitro . Although the screen was done in mouse T-ALL cells, we find that ESRRB-deficiency also results in GC resistance in human B-ALL cells. As many published GC resistance genes fail to translate in vivo, we transplanted mice with T-ALL cells transduced with shRNAs targeting Esrrb or empty vector. Dexamethasone treatment prevented disease progression in mice transplanted with control T-ALL cells however, dexamethasone treatment had no significant effect on disease development in mice transplanted with ESRRB deficient T-ALL cells, indicating that ESRRB deficiency confers GC resistance in vivo.As ESRRB has no established role in T-cells or the GC response, we performed RNA-sequencing on normal and ESRRB-deficient T-ALL cells in the absence and presence of GCs to uncover the mechanism of GC resistance. ESRRB deficient T-ALL cells fail to upregulate 65% of GC-induced genes, indicating that decreases in ESRRB expression prevents optimal GC-induced gene expression and apoptosis. Furthermore, we identified Esrrb as a GC-induced gene, supporting its role in the GC response. Due to the global repression of GC-induced gene expression in ESRRB-deficient leukemic cells, we examined GC receptor (GR) localization, but found that ESRRB had no detectable effects on GR localization.Collectively these data suggest that ESRRB may function as a novel co-activator of GR mediated transcription. Consistently, we found ESRRB expression decreased in a subset of relapse ALL patients, suggesting that ESRRB may contribute to GC resistance in patients. These findings identify ESRRB as a novel GR co-activator, whose expression is suppressed in a subset of relapse ALL patients. We demonstrate that treatment with the synthetic ESRRB agonist GSK4716 stimulates GC-induced apoptosis and gene expression in human ALL cell lines. These data raise the intriguing possibility that ESRRB agonists may re-sensitize GC resistant leukemia patients to GC therapy. DisclosuresNo relevant conflicts of interest to declare.

Salvianolic acid A attenuates steroid resistant nephrotic syndrome through suPAR/uPAR-αvβ3 signaling Inhibition

Ethnopharmacological relevanceSalvianolic acid A (SAA) is extracted from traditional Chinese medicine Salvia miltiorrhiza and is the main water-soluble and the biologically active ingredient. SAA possesses a variety of pharmacological activities and has an excellent protective effect on kidney disease, especially steroid resistant nephrotic syndrome (SRNS), and has advantages in improving the efficacy of glucocorticoids, but its mechanism needs to be further explored. PurposeThe study was designed to explore the effect of suPAR and uPAR in SRNS patients and evaluate the potential effect of SAA in improving podocyte steroid resistance and explore its mechanism. Methods and materialsThe ELISA kits were used to detect the levels of suPAR in the blood and urine of subjects. The levels of uPAR, GRα, and GRβ expression in renal tissues of SRNS patients was detected by immunohistochemistry and analyzed using the Pearson method. In vitro studies, steroid resistance model was induced by the TNF-α and IFN-γ. The protein and mRNA expression of Nephrin, GR, GRα and GRβ were analyzed using western blot and qRT-PCR. The activity of GR-DNA binding was detected by using TransAM™ GR kits. Adriamycin further induced steroid resistance podocyte. Flow cytometry was used to detect the effect of SAA on podocyte apoptosis. ELISA assay was used to detect the suPAR expression in the podocyte supernatant. Western blot and qRT-PCR were used to detect the protein and mRNA expression of uPAR and Nephrin in podocytes. ResultsThe serum and urine levels of suPAR were conspicuously higher in SRNS patients than healthy volunteers and SSNS patients, and the expression of uPAR in renal tissue of SRNS patients is negatively correlated with GRα, but positively correlated with GRβ. The combination of TNF-α and IFN-γ could conspicuously increase the GRβ expression and reduce GRα/GRβ, and induce steroid resistance in podocytes. Moreover, we found that SAA could reduce the apoptosis of podocytes and suppress the expression of suPAR/uPAR, and increase the expression of Nephrin. ConclusionThe level of suPAR and uPAR expression may have important value in predicting glucocorticoids resistance in patients with idiopathic nephrotic syndrome (INS). The combination of TNF-α and IFN-γ induce podocytes can establish steroid resistance model in vitro. SAA could improve glucocorticoids resistance of podocyte which can be attributed in part to regulate the suPAR/uPAR-αvβ3 signaling pathway.

Infliximab for reversible dementia in acute onset of neuro-Behçet's disease: A case report and cytokine analysis

We describe a 49-year-old female patient with neuro-Behçet's disease (NBD) with acute onset of fever and symptoms of dementia. High-dose glucocorticoid was partially effective for cognitive impairment, and infliximab, an anti-TNF-α antibody, gradually improved the symptoms. An analysis of cytokines showed that IP-10 in the cerebrospinal fluid was higher than that in the peripheral blood, and both decreased after treatment. This is the first known case of NBD wherein the patient with acute onset of dementia responded to a treatment with infliximab. In glucocorticoid-resistant patients, it is important to consider the introduction of infliximab to prevent irreversible brain dysfunction.

RELATION BETWEEN GLUCOCORTICOID RESISTANCE AND GLUCOCORTICOID RECEPTOR GENE BclI-POLYMORPHISM AND SOME CYTOKINE PROFILE PARAMETERS IN PATIENTS WITH HAND ECZEMA

Though glucocorticoids are widely used in dermatological practice, some patients with hand eczema may have resistance to glucocorticoids, even when they are taken in heavy doses. Glucocorticoids mediate their actions through glucocorticoid receptors. Polymorphism of the glucocorticoid receptor gene (NR3C1) can inhibit the cellular response to glucocorticoids and lead to reduced response to the therapy. Some cytokines can affect the production of various glucocorticoid receptor subunits, modulating the cell response to glucocorticoids. However, there is still need in detailed study of pathogenetic mechanisms and the detection of highly specific predictors of glucocorticoid resistance. The aim of this work was to investigate the possible relation between glucocorticoid resistance in patients with hand eczema, rs41423247 SNP, and blood concentration of interleukin-17A and interleukin-2. The venous blood of 143 patients with hand eczema (42% of women and 58% of men) mean age 42.2 ± 11.1 years was taken for the study. During the patients examination the data on age, sex, body mass index (kg/m2), body mass index ≥ 25 (kg/m2(%)), the habit of smoking, concemtration of immunoglobulin E (iu/ml), interleukin-17A (pg/ml) and interleukin-2 (pg/ml) were obtained. The еczema аrea and severity index was assessed in each subject before the therapy and in two weeks since the therapy started. According to index value, all the patients were divided into three subgroups: mild eczema, moderate eczema, and severe eczema. Patients with mild and moderate eczema were prescribed to apply topical glucocorticoid 0.1% mometasone furoate cream twice a day for 2 weeks. The patients with severe hand eczema were prescribed to receive additional systemic corticosteroid, a solution of dexamethasone by intramuscular injection in a dose of 8 mg / day, then 4 mg / day for another 2 days. BclI SNP (rs41423247) of the glucocorticoid receptor gene (NR3C1) was determined using PCR-RFLP method. The quantitative variables were tested for normal distribution by the Shapiro-Wilk test. The comparisons of the means between the two subgroups were performed by Student's t-test for independent samples. The comparison of the frequencies distribution in the subgroups was calculated by using the Pearson test. The P value < 0.05 was considered as significant. Thus, the obtained results revealed that insensitivity to glucocorticoids in patients with hand eczema is related to NR3C1 gene Bcl-1 polymorphism, eczema severity and plasma level of interleukin-17, interleukin-2. The plasma content of interleukin-17 and interleukin-2 in patients with glucocorticoid resistance was significantly higher compared to hormone-sensitive subjects. There were significantly more people with the C/G- and G/G genotypes in the group that did not have a clinical response to therapy.

Abstract IA30: Using single-cell, high-dimensional approaches to unravel tumor heterogeneity in pediatric cancer

Abstract Introduction: Despite improving responses to up-front therapies for children with cancer, relapse remains a significant barrier to long-term remissions and cure. Curing children of cancer requires effectively targeting all cancer-initiating cell populations. Understanding what cellular populations have initiating capacity and lead to poor clinical outcomes requires examining tumors at the single-cell level. Here we discuss such an approach to predict relapse in B-cell precursor acute lymphoblastic leukemia and response to common ALL therapies including glucocorticoids and chimeric antigen receptor T cells. Methods: Primary diagnostic or relapse bone marrow or peripheral blood samples or patient-derived xenografts were obtained under informed consent and IRB approval. Samples were clinically annotated for relevant prognostic features and clinical outcomes. Cryopreserved samples and healthy control bone marrows were treated in vitro with short-term stimulations to reveal signaling states. Cells were stained with a 40-antibody panel including relevant B-cell developmental proteins and intracellular signaling proteins. Samples were analyzed by mass cytometry (CyTOF). Patient samples underwent developmental classification in which each leukemia cell was assigned its most similar healthy counterpart. Further analysis was performed using various analysis methods, including machine learning modeling of relapse outcomes. Results: Studying over eighty primary patient diagnostic samples, we first organized the heterogeneous single-cell data classifying leukemic cells to 12 different subpopulations of B lymphopoiesis. We identified the transitional populations between early pro-B cells and late pre-B cells are expanded across almost all patients with BCP ALL. Extracting all measured features in these populations, we applied an elastic net model to determine cell populations associated with future relapse. This resulted in identification of early pre-B cells characterized by basally activated pCREB, pS6, pSYK, and p4EBP1 as predictive of future relapse. Further, these cells are apparent also at the time of relapse. Taking a similar approach, we also examined how these cell populations respond to treatment with glucocorticoids, a keystone of BCP ALL therapy. Similarly, we identify the same network activation in glucocorticoid-resistant patients associated also with a differentiation process. Interestingly, we can identify similar resistant phenotype in primary cells from patient treated in vivo with glucocorticoids. Finally, using these tools and methods, we describe CD19neg cells identified prior to treatment with CD19-targeted CAR T cells in patients who go on to suffer CD19neg relapse. Conclusions: Together, we present an approach to organizing single-cell tumor heterogeneity that reveals relapse-associated phenotypes. This highlights the translational potential of such an approach that could be applied to other single-cell studies in diverse tumor types. Citation Format: Kara L. Davis. Using single-cell, high-dimensional approaches to unravel tumor heterogeneity in pediatric cancer [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr IA30.

MO006INFLAMMASOME ACTIVATOR NLRP3 HYPOMETHYLATION IS ASSOCIATED WITH GLUCOCORTICOID RESISTANCE IN PATIENTS WITH IDIOPATHIC NEPHROTIC SYNDROME

Abstract Background and Aims We have previously shown that the Nod-like receptor protein-3 (NLRP3) gene is over-expressed in leukocytes of many patients with chronic kidney disease, and studies have also shown that hypomethylation and overexpression of NLRP3 are associated with glucocorticoid resistance in acute lymphoblastic leukemia. Therefore, we hypothesized that hypomethylation of the NLRP3 promoter my cause glucocorticoid resistance in patients with Idiopathic Nephrotic Syndrome (INS) associated with minimal change disease (MCD) or focal glomerulosclerosis (FSGS). Method We assessed NLRP3 and Caspase 1 (CASP1) promoter methylation by SNuPE reaction in germline DNA from leukocytes of 14 glucocorticoid-resistant and 18 glucocorticoid-sensitive adult INS patients (discovery cohort) and 7 glucocorticoid-resistant and 14 glucocorticoid-sensitive pediatric INS patients with MCD/FSGS (validation cohort). The effects of NLRP3 inflammasome on glucocorticoid resistance were validated in vitro in human monocytic cell lines (THP-1 and U937). Results Methylation of CpG islands in the CASP1 promoter was undetectable in both groups whereas NLRP3 promoter methylation was significantly lower in glucocorticoid-resistant compared to glucocorticoid-sensitive in both adults and children. The AUROC curve was 81.2% (p=0.0003) in adults and 73.5% (p=0.0002) in children demonstrating the capability of NLRP3 methylation to discriminate glucocorticoid-resistant versus glucocorticoid-sensitive. Activation of the NLRP3 inflammasome by LPS/ATP reduced glucocorticoid receptor expression and increased glucocorticoid resistance in U937. Consistently, knockdown of NLRP3 in THP-1 increased sensitivity to glucocorticoids. Conclusion Our findings demonstrate a novel mechanism whereby INS patients develop resistance to glucocorticoids via leukocyte epigenetic changes of NLRP3 and reveal a new potential clinical biomarker to early detect this condition.

Correlations of MIF polymorphism and serum levels of MIF with glucocorticoid sensitivity of sudden sensorineural hearing loss.

ObjectiveThis study explored the relationship between macrophage migration inhibitory factor (MIF) gene polymorphism (−173G/C) and glucocorticoid sensitivity in sudden sensorineural hearing loss (SSNHL).MethodsA total of 120 patients with SSNHL were divided into a glucocorticoid-sensitive group and a glucocorticoid-resistant group. A group of 93 healthy individuals served as the control group. Serum MIF levels of the participants were measured and MIF genotyping was performed.ResultsThe frequency of the MIF −173C allele was significantly higher in glucocorticoid-sensitive patients than in glucocorticoid-resistant patients. Serum MIF levels were significantly higher in SSNHL patients than in healthy controls, and higher in the glucocorticoid-sensitive group than in the glucocorticoid-resistant group of SSNHL patients, which was unexpected. Compared with patients with the GG genotype, patients with the −173C allele (GC and CC genotypes) had significantly higher levels of serum MIF and superoxide dismutase activity and lower levels of tumor necrosis factor-α and malondialdehyde.ConclusionThe MIF −173G/C polymorphism is associated with glucocorticoid sensitivity in SSNHL patients. The C allele can result in higher MIF production, reduced oxidative stress, and greater glucocorticoid sensitivity.

Influence of vitamin D receptor gene FokI and ApaI polymorphisms on glucocorticoid response in patients with asthma.

Glucocorticoid (GC)-resistant asthma, a complex disease phenotype, has a high morbidity and mortality and takes up a disproportionate share of healthcare costs. The aim of this work was to assess serum 25-hydroxyvitamin D (25(OH)VD) levels in steroid-resistant, steroid-sensitive patients with asthma and in healthy controls, and to investigate the association between the vitamin D receptor gene (VDR) FokI and ApaI polymorphisms and GC resistance in patients with asthma. This case-control study included 70 patients with severe bronchial asthma and 30 apparently healthy controls. Atopic status was determined by skin-prick test reaction to the most common locally-encountered allergens. A GC reversibility test was performed to differentiate between GC-sensitive and GC-resistant asthma. For all subjects, analysis of the VDR FokI and ApaI polymorphisms by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and measurement of serum 25(OH)VD levels by enzyme-linked immunosorbent assay (ELISA) were performed. The frequencies of FokI polymorphism genotypes and alleles differed significantly between patients with asthma and controls. The frequencies of the TT genotype and T allele carriers were significantly higher among patients with asthma than among controls, and also among GC-resistant patients with asthma than among GC-sensitive patients with asthma. Additionally, serum 25(OH)VD levels differed significantly among the 3 VDR FokI polymorphic genotypes in GC-resistant patients with asthma; the highest level was detected in the TT genotype. No significant differences in ApaI were found. We found a possible association between the FokI T allele and GC resistance in patients with asthma. Variations in VDR FokI might also play a role in 25(OH)VD levels.

Генетические факторы развития резистентности к глюкокортикоидам у больных эндокринной офтальмопатией

Генетические факторы развития резистентности к глюкокортикоидам у больных эндокринной офтальмопатией

Application of miR-182 for Determination of Glucocorticoid-Resistant Patients with Lymphoid Malignancy

To validate the clinical relationship between miR-182 and glucocorticoid-resistance in patients with lymphoid malignancy. Real-time quantitative PCR(qRT-PCR) was employed to detect the expression of miR-182 in lymphoma patients (68 cases, the specimens indluded bone marrow of 20 cases, and plasma of 48 cases), multiple myeloma patients (24 cases, the specimens included bone marrow of 14 cases and plasma of 10 cases), ALL patients (3 cases, specimen was plasma of 3 cases) and non-lymphotic system disorder patients (18 cases, specimens included bone marrow of 8 cases and plasma of 10 cases). The expression of miR-182 in refractory lymphoblastic tumor patients was significantly higher than that in initial treatment group (P<0.05); the expression of miR-182 in initial treatment patients was not significantly different from the controls. The expression level of miR-182 in plasma of lymphoid malignancy patient significantly correlated with their used dosage of corticosteroids (P<0.05). When the expression level of miR-182 in bone marrow cells was 10.09, its sensitivity and specificity for diagnosis were 100% and 88.2% respectively; when the expression level of miR-182 in plasma was 1.393, its sensitivity and specificity for diagnosis of glucocorticoid resistance of lymphoblastic tumor cells were 90.9% and 51.3% respectively. The expression of miR-182 in lymphoblastic tumor with glucocorticoid resistance is significantly up-regulated, suggesting that the glucocorticoid resistance of lymphoblastic tumor is related with the over-expression of miR-182. The miR-182 is expected as a new biomarker for the diagnosis of glucocorticoid resistant lymphoblastic tumor.

The effects of microRNAs on glucocorticoid responsiveness.

Glucocorticoids (GCs) are of wide usage in the clinical treatment of lymphoblastic malignancies such as acute lymphoblastic leukemia. However, individually distinctive responsiveness to the GC therapy may attenuate their clinical efficacy, and more reliable predictor for GC resistance is still eagerly needed. Recent studies indicate that microRNAs (miRNAs), which demonstrate regulatory functions targeting mRNAs during the post-transcription, involved in the regulation of GCs sensitivity through several mechanisms, especially adjusting the magnitude of GC receptors (GRs), which mediates the cellular effects of GCs and plays a pivotal role in GCs sensitivity, inspiring that special miRNAs pattern could serve as the biomarkers to predict GC sensitivity and bring forth potential strategies for overcoming drug resistance. In this review, we discuss related miRNAs and their diverse effects exerted on multifaceted complexity of GCs responsiveness for further exploiting the molecular mechanism of GC resistance and future construction of the molecular diagnostic method and reverse GC resistance. We have reviewed and searched for eligible literature relating to the effects of microRNAs on GC responsiveness from systematic PubMed searches. GC response can be mediated by miRNAs through influence on GC signaling pathway, leading to diverse glucocorticoid responsiveness. Mutations in miRNA gene also influence GC response. As well, GCs regulate the function of several miRNAs, and suggesting a bidirectional influence among them. It is possible and necessary that miRNAs serve as stable biomarkers and GC resistant patients would benefit from an effective and early screening test.

Pharmacological Inhibition of Lck is Able to Revert Glucocorticoid Resistance in Pediatric T-ALL

The AIEOP-BFM group has traditionally used the peripheral blood blast cells count after a 7-day glucocorticoids (GC) prephase to classify patients as Prednisone Good Responders (PGR) or Prednisone Poor Responders (PPR). As described by Schrappe M. in 2011, PPR patients tend to have a worse prognosis, despite the fact that all of them are assigned to the High Risk protocol. Little is known about the molecular mechanisms that lead to GC resistance, guiding our research to the identification of new specific molecular targets in order to develop new approaches to improve therapy efficacy in these patients.To this end, we performed a Reverse Phase Protein Analysis (RPPA) of 54 PGR and 33 PPR pediatric T-ALL patients at diagnosis, and studied the activation or expression of 87 proteins involved in key cellular signaling pathways. Interestingly, we found a higher expression of LCK phosphorylated at Y505 (inhibited form) in PGR patients (p=0.001), together with a higher phosphorylation of SRC Y416 (active form) in PPR patients (p=0.01). Total LCK and LCK RNA expression were not differentially expressed in the two subgroups of patients, suggesting an increased activation of LCK in PPR patients. Indeed, in agreement with these results, also LCK downstream target PLCγ, phosphorylated at Y783, resulted hyperactivated in PPR compared to PGR patients (p=0.05), confirmed also by a positive correlation between PLCγ Y783 and SRC Y416 (r=0.51, p=0.01). Taken together, these results indicate a hyperactivation of the LCK pathway in PPR patients compared to PGR ones. LCK is part of the TCR multiprotein complex together with the GC receptor. In normal T lymphocytes, after GC treatment the complex is disrupted, LCK activation is decreased and downstream prosurvival signaling inhibited, thus leading to cell death. In this light, in GC resistant patients hyperactivated LCK might sustain cell survival regardless of GC activity.We then tested if FDA-approved or recently developed LCK inhibitors would revert GC resistance in T-ALL cells. GC resistant cell lines ALL-SIL, T-ALL1 and CEM were treated with Dasatinib, Bosutinib, Nintedanib and WH-4-023 alone or in combination with Dexamethasone (Dex). All four inhibitors alone are able to decrease cell survival, and all of them strongly synergize with Dex, bringing to the sensitization of these cells to GC treatment. We also tested these compounds alone or in combination with Dex in 4 PPR T-ALL patients cells derived from xenograft mice. Also in these cases we observed an enhanced sensitization of cells to GC treatment. Finally, corroborating the crucial role of LCK in GC resistance, we observed a strong decrease in cell viability after specific LCK gene silencing and Dex treatment in ALL-SIL cells, together with an increased GC resistance following LCK hyperactivation in P12-ICHIKAWA GC sensitive cells.Thus, our results strongly suggest that the inhibition of LCK using clinically approved drugs could represent a promising new additional therapeutic strategy to revert drug resistance in high-risk pediatric T-ALL patients. DisclosuresIndraccolo:OncoMed Pharmaceuticals, Inc.: 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.

The prognostic value of glucocorticoid receptors for adult acute lymphoblastic leukemia.

BackgroundTherapeutic protocols used in adult acute lymphoblastic leukemia (ALL) are widely variable, and glucocorticoids (GCs) are essential components in ALL treatment. Therefore, this study aimed to evaluate the distribution of prominent glucocorticoid receptor (GR) gene polymorphic variants among adult ALL patients. We also investigated the association between GR messenger ribonucleic acid (mRNA) isoform expressions and the response to chemotherapy.MethodsFifty-two newly diagnosed Philadelphia-negative adult ALL patients and 30 healthy control subjects were enrolled in this study. Genotyping was carried out using a polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis. GR mRNA isoform expressions were assayed by quantitative real-time PCR.ResultsALL patients in this study had a median age of 34 years (range, 18-75). GRα expression was associated with complete remission (P=0.03), while GRγ mRNA expression was significantly higher in GC resistant patients (P=0.032) and in non-responders (P=0.019). However, there were no significant associations with GC resistance. The BclI polymorphic variant of the GR gene was the most frequent in adult ALL patients and was not associated with the GC response. Both higher GRα expression and lower GRγ expression were associated with achievement of complete remission, while higher GRγ expression was associated with GC-resistance.ConclusionOur data suggest that the level of GR isoform expression may be useful in predicting GC response, achievement of complete remission, and better event-free survival in ALL patients. However, further evaluation with a larger cohort of patients is warranted.

Correlation of multidrug resistance genes and clinical risk factors with glucocorticoid response in patients with inflammatory bowel disease

Objective To investigate the correlation of multidrug resistance gene 1 (MDR1),NR3C1 gene polymorphisms and clinical risk factors with efficacy,dependence,and resistance of glucocorticoid (GC) in patients with inflammatory bowel disease (IBD).Methods Anti coagulation blood samples of 196 healthy controls and 105 IBD patients received GC therapy were collected.There were 62 ulcerative colitis (UC) and 43 Crohn's disease (CD) in the IBD patients.The number of GC sensitive,GC dependent and GC resistant of UC patients were 36,13 and 13,respectively,and those of CD patients were 24,11 and eight.GC refractoriness included GC dependence and resistance.The genotype of MDR1 C3435T and NR3C1 Bcl Ⅰ of all the subjects was detected by the restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR).The correlation between each genotype frequency,clinical features of patients with IBD and the efficacy of GC treatment was analyzed by Chisquare test,Fisher exact probability method or t test.Results Among UC patients,the disease course of GC refractory group and GC resistant group was longer than that of GC sensitive group ((6.660±1.523)years,(6.500±1.111) yearsvs (3.350±0.697) years,t=2.211,P=0.031; t=2.930,P=0.005).The serum level of C reaction protein (CRP) of GC refractory group was higher than that of GC sensitive group ((47.628±13.913) mg/Lvs (16.854±4.121) mg/L,t=2.121,P=0.047).The chronic relapse type was more common in GC refractory UC patients (Fisher exact probability method,P=0.035),and severe patients were more common in UC with GC resistance (Fisher exact probability method,P=0.021).The white blood cell count of GC resistant and GC refractory CD patient was lower than that of GC sensitive CD patients ((5.710 ± 0.604) ×109/L,(5.878±0.405) × 109/L vs (7.814 ±0.670) × 109/L,t=2.334,P=0.028; t=2.045,P=0.018).Patients with extraqntestinal manifestations was more common in CD with GC resistance (Fisher exact probability method,P=0.035).There was no statistically significant difference in the frequencies of MDR1 C3435T,NR3C1 Bcl Ⅰ genotypes,allelic genes and gene carrier among control group and GC sensitive dependent and resistant group of IBD patients.However,the frequency of MDR1 C3435T gene carrier was significantly different between GC sensitive group and GC refractory group,especially between GC sensitive group and GC resistance group (68.33％ vs 48.89％,x2 =4.051,P=0.044; 68.33％ vs 42.86％,x2 =4.274,P =0.039).Conclusions GC sensitivity of IBD patients with MDR1 C3435T loci T gene carrier was higher than that of IBD patients without T gene carrier.NR3C1 gene polymorphisms was not related with GC resistance and GC dependence.Compared with GC sensitive IBD patients,in GC resistant and GC dependent IBD pantient UC patients with long disease course,chronic relapse type,severe type,high level of CRP and CD patients with low white blood cell count and extra-intestinal manifestations were more common. Key words: Inflammatory bowel diseases; MDR1 gene; Receptors, glucocorticoid; Polymorphism,genetic

Small Molecule that Reverses Dexamethasone Resistance in T-cell Acute Lymphoblastic Leukemia (T-ALL).

Glucocorticoids are one of the most utilized and effective therapies in treating T-cell acute lymphoblastic leukemia. However, patients often develop resistance to glucocorticoids, rendering these therapies ineffective. We screened 9517 compounds, selected for their lead-like properties, chosen from among 3 372 615 compounds, against a dexamethasone-resistant T-ALL cell line to identify small molecules that reverse glucocorticoid resistance. We synthesized analogues of the most effective compound, termed J9, from the screen in order to define the scaffold's structure-activity relationship. Active compounds restored sensitivity to glucocorticoids through upregulation of the glucocorticoid receptor. This compound and mechanism may provide a strategy for overcoming glucocorticoid resistance in patients with T-ALL.

Glucocorticoid resistance in dialysis patients reduces long-term graft survival after kidney transplantation

Glucocorticoid (GC) resistance has been observed in chronic kidney disease (CKD) patients on dialysis. It can be evaluated by binding assays based on the dissociation constant (Kd), which is inversely proportional to ligand affinity. CKD patients with GC resistance had increased number of acute rejection episodes. We followed up 26 patients that underwent kidney transplantation to observe whether GC resistance could affect the response to acute rejection episode pulse therapy and the long-term allograft outcome. Using Kaplan–Meier survival curve, GC resistant patients showed lower acute rejection-free survival (p=0.03) and lower kidney allograft survival (p=0.008). No difference was found regarding number of deaths. Multivariate logistic regression showed that high Kd value was an independent predictor of lower kidney allograft survival (p=0.001). There was a negative Spearman correlation between Kd and kidney allograft survival (r=−0.88, p=0.03). In conclusion, our findings indicate the usefulness of binding assay performed previously to kidney transplantation to define GC resistance. In addition, the dissociation constant (Kd) is a reliable and independent predictive marker of higher frequency of acute rejection episodes, lower rejection-free graft survival, poor response of acute rejection episodes to methylprednisolone pulse therapy, and lower kidney allograft survival in a long-term follow-up.