Negative Feedback Mechanism Research Articles

THE POTENTIAL USE OF INTRANASAL OXYTOCIN AS EARLY PREVENTIVE INTERVENTION FOR POST-TRAUMATIC STRESS DISORDER

IntroductionPost-traumatic stress disorder (PTSD) is defined by an exaggerated fear responses (FA) which fails to extinguish over time and cannot be inhibited in safe contexts. Studies report that traumatic experiences (TE) affect hormonal systems mediated by the hypothalamic-pituitary-adrenal (HPA) axis and the oxytocinergic system. Oxytocin (OXT) is a neurohormone produced in the hypothalamus that has social functions like the promotion of prosocial and affiliative behaviors, increased self-confidence and positive social memories. In PTSD there is a diminished inhibitory top-down control over the FA, which is characterized by amygdala hyperactivity, ventromedial prefrontal cortex (vmPFC) hypoactivity and diminished structural and functional connectivity between both areas, which results in anxiety increase and dysregulated autonomic and endocrine FA. In parallel, TE decrease the synthesis and release of OXT, resulting in the dysfunction of the negative feedback mechanism on the HPA, leading to hypercortisolemia and maximizing the response to a stressful stimulus. Previous studies report that the administration of OXT can reduce cortisol levels as well as attenuating amygdala hyperactivity and normalizing the connectivity of this structure with frontal areas, diminishing the FA. Therefore, OXT has been investigated as a potential therapeutic agent administered intranasally early after trauma as a strategy to prevent PTDS on individuals having high risk.ObjectivesThe aim of this work is to review the potential of intranasal OXT administration as early preventive intervention for PTSD.MethodsSystematic review of the literature published in Pubmed, using the terms “Oxytocin”, “Post-traumatic Stress Disorder”, “Stress”.ResultsStudies found significant associations between TE and OXT and report that TE and PTSD are strongly associated with reductions in OXT. Literature report that the acute effects of OXT administrations in individuals with TE tend to be anxiolytic only in less severe forms, by modulating the HPA axis and the autonomic nervous system. Moreover, in recent TE, OXT seems to increase the re-experience of traumas and restore the function of different networks associated with fear control in PTSD patients. FMRI studies indicate that intranasal OXT attenuates amygdala hyperactivity and enhances amygdala´s connectivity with vmPFC, resulting in increased control over the FA. Finally, studies report that a single oxytocin administration increases neuronal fear processing but repeated administration reduces PTSD symptoms up to 6 months post trauma in patients with high acute symptoms.ConclusionsRepeated administration of intranasal oxytocin early after trauma seems to diminish the acute symptoms in early stages of PTDS, being a potential pharmacological strategy to prevent PTDS in individuals at high risk by increasing the control of FA.Disclosure of InterestNone Declared

Distinctive modulation of hepcidin in cancer and its therapeutic relevance.

Hepcidin, a short peptide synthesized primarily by hepatocytes in response to increased body iron and inflammation, is a crucial iron-regulating factor. Hepcidin regulates intestinal iron absorption and releases iron from macrophages into plasma through a negative iron feedback mechanism. The discovery of hepcidin inspired a torrent of research into iron metabolism and related problems, which have radically altered our understanding of human diseases caused by an excess of iron, an iron deficiency, or an iron disparity. It is critical to decipher how tumor cells manage hepcidin expression for their metabolic requirements because iron is necessary for cell survival, particularly for highly active cells like tumor cells. Studies show that tumor and non-tumor cells express and control hepcidin differently. These variations should be explored to produce potential novel cancer treatments. The ability to regulate hepcidin expression to deprive cancer cells of iron may be a new weapon against cancer cells.

Discoveries Around the Hypothalamic-Pituitary-Thyroid Axis.

Many members of the American Thyroid Association played prominent roles in discovering the various aspects of the hypothalamic-pituitary-thyroid axis. This axis is fundamental for maintaining the normal serum levels of circulating thyroid hormones (THs) and thus the euthyroid state. The pituitary glycoprotein hormone, thyrotropin (TSH), controls the activity of the thyroid gland. Thyrotropin-releasing hormone and the negative feedback mechanism of circulating TH regulate the synthesis and the secretion of TSH. The dynamic interplay of these two dominant mechanisms has essential effects on TSH release. Therefore, the finding of abnormal serum levels of TSH often indicates the presence of a disorder of thyroid gland function. A summary of key historical discoveries in the understanding of the hypothalamic-pituitary axis is presented.

Recipe for pituitary organoids.

Generation of a variety of organs and tissues from human pluripotent stem cells (hPSCs) has been attempted in vitro. We here present a simple and efficient method for induction of hypothalamic and pituitary tissues from hPSCs. On provision of exogenous agents important for early hypothalamus-pituitary organogenesis, including bone morphogenetic protein 4 and activators of sonic hedgehog, in three-dimensional culture, hPSCs spontaneously form spherical organoids with two distinct tissues, hypothalamus and adenohypophysis. The pituitary tissues derived from hPSCs not only secrete adenocorticotropic hormone, but also retain both positive and negative feedback mechanisms, recapitulating mature endocrine organs in vivo. Furthermore, the results of ectopic transplantation with mouse models of hypopituitarism suggest that these hypothalamus-pituitary organoids have potential as engraftment organs. In addition to their use in transplantation for patients with hypopituitarism they will allow establishment of disease models in vitro and enable research impossible in humans. Hypothalamus-pituitary organoids promise to be a powerful tool in regenerative medicine, drug discovery, and basic research into pituitary development.

Functional basis for calmodulation of the TRPV5 calcium channel.

Within the transient receptor potential (TRP) superfamily of ion channels, TRPV5 is a highly Ca2+ -selective channel important for active reabsorption of Ca2+ in the kidney. Its channel activity is controlled by a negative feedback mechanism involving calmodulin (CaM) binding. Combining advanced microscopy techniques and biochemical assays, this study characterized the dynamic lobe-specific CaM regulation. We demonstrate for the first time that functional (full-length) TRPV5 interacts with CaM in the absence of Ca2+ , and this interaction is intensified at increasing Ca2+ concentrations sensed by the CaM C-lobe that achieves channel pore blocking. Channel inactivation occurs without requiring CaM N-lobe calcification. Moreover, we show a Ca2+ -dependent binding stoichiometry at the single channel level. In conclusion, our study proposes a new model for CaM-dependent regulation - calmodulation - of this uniquely Ca2+ -selective TRP channel TRPV5 that involves apoCaM interaction and lobe-specific actions, which may be of significant physiological relevance given its role as gatekeeper of Ca2+ transport in the kidney. KEY POINTS: The renal Ca2+ channel TRPV5 is an important player in maintenance of the body's Ca2+ homeostasis. Activity of TRPV5 is controlled by a negative feedback loop that involves calmodulin (CaM), a protein with two Ca2+ -binding lobes. We investigated the dynamics of the interaction between TRPV5 and CaM with advanced fluorescence microscopy techniques. Our data support a new model for CaM-dependent regulation of TRPV5 channel activity with CaM lobe-specific actions and demonstrates Ca2+ -dependent binding stoichiometries. This study improves our understanding of the mechanism underlying fast channel inactivation, which is physiologically relevant given the gatekeeper function of TRPV5 in Ca2+ reabsorption in the kidney.

Influence of key parameters of ice accretion model under coexisting rain and fog weather

Based on 30 complete wire icing processes lasted longer than 24 h observed from the Enshi, Jinsha, Dacaoping and Shennongding of Shennongjia in mountainous areas of Hubei province during the winter of 2008–2016, the macroscopic effects of rain–fog weather on the ice accretion process were analyzed. Furthermore, the distribution characteristics of key simulation parameters in supercooled fog (SF) and freezing rain (FR) were discussed according to the physical model of icing process. Finally, the evolution characteristics of the simulated ice thickness in rain–fog weather were proposed. Results showed that the duration of ice accretion in mountainous areas is the key factor affecting the maximum ice thickness; the freezing rain is most frequent during the glaze icing process, which leads to the substantial growth of ice thickness. The average growth rates of ice thickness with and without freezing rain are 1.26 mm h−1 and -0.11 mm h−1, respectively. Collision rate is the main parameter for inhibiting ice accretion of SF, with an average value of ∼ 0.1, while freezing rate is the main parameter for inhibiting ice accretion of FR, with an average value of ∼ 0.6. The ice accretion of SF shows the characteristics of periodic growth, while the ice accretion of FR shows the explosive growth of ice thickness, which makes the simulated values of icing closer to the observations. The ice formation efficiency of FR was more than twice that of SF, with a negative feedback mechanism to the ice accumulation of SF.

Dynamic process of dissipative soliton build-up in mode-locked fiber laser

We experimentally studied the dynamic process of dissipative soliton build-up by dispersive Fourier transform in net normal dispersion cavity. The oscillation of spectral bandwidth and pulse energy before the stabilization of dissipative soliton was observed, the build-up and termination of the double dissipative solitons accompanied with soliton explosion and energy interaction were observed with higher pump power. Simulation of dissipative soliton build-up process was demonstrated through the extended nonlinear Schrödinger equation, negative feedback mechanism was summarized as the reason of the pulse oscillation during the build-up process.

HSP70 mediates a crosstalk between the estrogen and the heat shock response pathways

Cells respond to multiple signals from the environment simultaneously, which often creates crosstalk between pathways affecting the capacity to adapt to the changing environment. Chaperones are an important component in the cellular integration of multiple responses to environmental signals, often implicated in negative feedback and inactivation mechanisms. These mechanisms include the stabilization of steroid hormone nuclear receptors in the cytoplasm in the absence of their ligand. Here, we show using immunofluorescence, chromatin immunoprecipitation, and nascent transcripts production that the heat shock protein 70 (HSP70) chaperone plays a central role in a new crosstalk mechanism between the steroid and heat shock response pathways. HSP70-dependent feedback mechanisms are required to inactivate the heat shock factor 1 (HSF1) after activation. Interestingly, a steroid stimulation leads to faster accumulation of HSF1 in inactive foci following heat shock. Our results further show that in the presence of estrogen, HSP70 accumulates at HSF1-regulated noncoding regions, leading to deactivation of HSF1 and the abrogation of the heat shock transcriptional response. Using an HSP70 inhibitor, we demonstrate that the crosstalk between both pathways is dependent on the chaperone activity. These results suggest that HSP70 availability is a key determinant in the transcriptional integration of multiple external signals. Overall, these results offer a better understanding of the crosstalk between the heat shock and steroid responses, which are salient in neurodegenerative disorders and cancers.

HSA-MIR-183-3P TARGETING ATAXIA-TELANGIECTASIA MUTATED PROTEIN REGULATION OF NF-ΚB SIGNALING PATHWAY AFFECTS CELLULAR SENESCENCE CAUSED BY DNA DAMAGE IN LUMBAR DISC DEGENERATION.

To test the effect of Hsa-miR-183-3p on cell aging and disc degeneration in lumbar intervertebral disc. This study combined clinical research with basic cell experiment, analyzing clinical data from patients with lumbar disc degeneration and traumatic lumbar spine fracture, as well as the differences in baseline data. The degree of lumbar disc injury in patients of different ages was also compared. Differentially expressed miRNAs were predicted via GEO database, and qPCR confirmation was determined by collecting cartilage endplates from two groups. ACAN, Col2A1, p16, p21, and p53 were detected by immunofluorescence, Western blot and qPCR in human nucleus pulposus cells. Changes of cell senescence were detected. The binding of Hsa-miR-183-3p to ataxia-telangiectasia mutated protein was confirmed by dual luciferase reporter assay. Degenerative discs showed elevated expression of hsa-miR-183-3p, which may be upregulated by TNF-α via NF-κB signaling pathway and target ataxia-telangiectasia mutated protein regulation. Degeneration of the intervertebral disc can be accelerated by TNF-α. Additionally, Hsa-miR-183-3p passed NF-κB signaling pathway is blocked via up-regulation of TNF-α to reduce inflammation via targeting ataxia-telangiectasia mutated protein. As a result, this negative feedback mechanism may assist in maintaining a low degenerative load and preserving chronic disc degeneration.

Fine-tuning of nitrogen-containing bisphosphonate esters that potently induce degradation of HMG-CoA reductase

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is the rate-limiting enzyme in the cholesterol biosynthetic pathway, and competitive inhibitors targeting the catalytic domain of this enzyme, so-called statins, are widely used for the treatment of hyperlipidemia. The membrane domain mediates the sterol-accelerated degradation, a post-translational negative feedback mechanism, and small molecules triggering such degradation have been studied as an alternative therapeutic option. Such strategies are expected to provide benefits over catalytic site inhibitors, as the inhibition leads to transcriptional and post-translational upregulation of the enzyme, necessitating a higher dose of the inhibitors and concomitantly increasing the risk of serious adverse effects, including myopathies. Through our previous study on SR12813, a synthetic small molecule that induces degradation of HMG-CoA reductase, we identified a nitrogen-containing bisphosphonate ester SRP3042 as a highly potent HMG-CoA reductase degrader. Here, we performed a systematic structure-activity relationship study to optimize its activity and physicochemical properties, specifically focusing on the reduction of lipophilicity. Mono-fluorination of tert-butyl groups on the molecules was found to increase the HMG-CoA reductase degradation activity while reducing lipophilicity, suggesting the mono-fluorination of saturated alkyl groups as a useful strategy to balance potency and lipophilicity of the lead compounds.

Integrating orientation mechanisms, adrenocortical activity, and endurance flight in vagrancy behaviour

Avian migratory processes are typically precisely oriented, yet vagrants are frequently recorded outside their normal range. Wind displaced vagrants often show corrective behaviour, and as an appropriate response is likely adaptive. We investigated the physiological response to vagrancy in passerines. Activation of the emergency life-history stage (ELHS), assessed by high baseline plasma corticosterone, is a potential mechanism to elicit compensatory behaviour in response to challenges resulting from navigational error, coupled with response to fuel load and flight. We compared circulating plasma corticosterone concentrations and body condition between three migratory groups in autumn: (1) wind displaced southwest (SW) vagrants and (2) long range southeast (SE) vagrants on the remote Faroe Islands, and (3) birds within the expected SW migratory route (controls) on the Falsterbo peninsula, Sweden. Vagrants were further grouped by those sampled immediately upon termination of over-water migratory flight and those already on the island. In all groups there was no indication of the activation of the ELHS in response to vagrancy. We found limited support for an increased rate of corticosterone elevation within our 3 min sample interval in a single species, but this was driven by an individual ELHS outlier. Fat scores were negatively correlated with circulating corticosterone; this relationship may suggest that ELHS activation depends upon an individual’s energetic states. Interestingly, in individuals caught at the completion of an obligate long-distance flight, we found some evidence of corticosterone suppression. Although limited, data did support the induction of negative feedback mechanisms that suppress corticosterone during endurance exercise, even when fuel loads are low.

Dynamic feedback regulation for efficient membrane protein production using a small RNA-based genetic circuit in Escherichia coli

BackgroundMembrane proteins (MPs) are an important class of molecules with a wide array of cellular functions and are part of many metabolic pathways. Despite their great potential—as therapeutic drug targets or in microbial cell factory optimization—many challenges remain for efficient and functional expression in a host such as Escherichia coli.ResultsA dynamically regulated small RNA-based circuit was developed to counter membrane stress caused by overexpression of different MPs. The best performing small RNAs were able to enhance the maximum specific growth rate with 123%. On culture level, the total MP production was increased two-to three-fold compared to a system without dynamic control. This strategy not only improved cell growth and production of the studied MPs, it also suggested the potential use for countering metabolic burden in general.ConclusionsA dynamically regulated feedback circuit was developed that can sense metabolic stress caused by, in casu, the overexpression of an MP and responds to it by balancing the metabolic state of the cell and more specifically by downregulating the expression of the MP of interest. This negative feedback mechanism was established by implementing and optimizing simple-to-use genetic control elements based on post-transcriptional regulation: small non-coding RNAs. In addition to membrane-related stress when the MP accumulated in the cytoplasm as aggregates, the sRNA-based feedback control system was still effective for improving cell growth but resulted in a decreased total protein production. This result suggests promiscuity of the MP sensor for more than solely membrane stress.

Association between Intratumoral CD8+ T Cells with FoxP3+ and CD163+ Cells: A Potential Immune Intrinsic Negative Feedback Mechanism for Acquired Immune Resistance.

Acquired immune resistance (AIR) describes a situation in which cancer patients who initially responded clinically to immunotherapies, after a certain period of time, progress with their disease. Considering that AIR represents a feedback response of the tumor against the immune attack generated during the course of immunotherapies, it is conceivable that AIR may also occur before treatment initiation as a mechanism to escape endogenous adaptive antitumor immunity (EAAI). In the present study, we assessed the EAAI in paraffin-embedded breast primary tumor tissue samples and drew correlations with the clinical outcomes. In particular, we analyzed densities of CD8+ cells as elements mediating antitumor cytotoxicity, and of CD163+ and FoxP3+ cells as suppressor elements. We found a direct correlation between the densities of CD8+ cells and of CD163+ and/or FoxP3+ cells in the vast majority of patients' tumors. Importantly, the vast majority of patients whose tumors were overpopulated by CD8+ cells developed AIR, which was characterized by high intratumoral CD163+ and/or FoxP3+ cell densities and reduced overall survival (OS). We also showed that AIR depends on the levels of CD8+ cell-ratios in the tumor center to the invasive margin. Our data suggest that tumors develop AIR only when under a robust endogenous immune pressure.

Prenatal Hypoxia-Induced Adverse Reaction to Mild Stress is Associated with Depressive-Like Changes in the Glucocorticoid System of Rats.

The effects of prenatal hypoxia on neurodevelopment are predominantly associated with impaired maternal glucocorticoid stimulation of the fetus, which is "imprinted" in altered sensitivity of glucocorticoid reception in brain structures of offspring and can affect brain plasticity during lifespan. This study aimed to investigate response of the brain glucocorticoid system to mild stress (MS) in adult rats that survived prenatal severe hypoxia (PSH) on embryonic days 14-16. In response to MS the control (but not PSH) rats demonstrate increased corticosterone levels, a decrease in exploratory activity and increased anxiety. In the raphe nuclei of adult PSH rats the expression of glucocorticoid receptors (GR) is increased without changes in serotonin levels in comparison with the control. MS induces a decrease in GR expression accompanied by up-regulation of tryptophan hydroxylase 2 (tph2) and down-regulation of monoamine oxidase A (maoa) transcription in the raphe nuclei of both control and PSH groups. PSH also causes significant deviations in GR expression and GR-dependent transcription in the hippocampus, the medial prefrontal cortex, but not in the amygdala of rats. However, in response to MS, PSH rats demonstrate mild changes in their activity, while in control animals the MS-induced activity of the glucocorticoid system in these brain structures is similar to intact PSH animals. Impaired activity of the glucocorticoid system in the extrahypothalamic brain structures of PSH rats is accompanied by increase in the hypothalamic corticotropin-releasing hormone (CRH) levels in comparison with the control regardless of MS. Synthesis of proopiomelanocortin (POMC) and release of adrenocorticotropic hormone (ACTH) into the blood are decreased in response to MS in the pituitary of control rats, which demonstrates a negative glucocorticoid feedback mechanism. Meanwhile, in the pituitary of PSH rats reduced POMC levels were found regardless of MS. Thus, prenatal hypoxia causes depression-like patterns in the brain glucocorticoid system with adverse reaction to mild stressors.

Pannexin 1 activity in astroglia sets hippocampal neuronal network patterns.

Astroglial release of molecules is thought to actively modulate neuronal activity, but the nature, release pathway, and cellular targets of these neuroactive molecules are still unclear. Pannexin 1, expressed by neurons and astrocytes, form nonselective large pore channels that mediate extracellular exchange of molecules. The functional relevance of these channels has been mostly studied in brain tissues, without considering their specific role in different cell types, or in neurons. Thus, our knowledge of astroglial pannexin 1 regulation and its control of neuronal activity remains very limited, largely due to the lack of tools targeting these channels in a cell-specific way. We here show that astroglial pannexin 1 expression in mice is developmentally regulated and that its activation is activity-dependent. Using astrocyte-specific molecular tools, we found that astroglial-specific pannexin 1 channel activation, in contrast to pannexin 1 activation in all cell types, selectively and negatively regulates hippocampal networks, with their disruption inducing a drastic switch from bursts to paroxysmal activity. This decrease in neuronal excitability occurs via an unconventional astroglial mechanism whereby pannexin 1 channel activity drives purinergic signaling-mediated regulation of hyperpolarisation-activated cyclic nucleotide (HCN)-gated channels. Our findings suggest that astroglial pannexin 1 channel activation serves as a negative feedback mechanism crucial for the inhibition of hippocampal neuronal networks.

Understanding Long COVID; Mitochondrial Health and Adaptation-Old Pathways, New Problems.

Many people infected with the SARS-CoV-2 suffer long-term symptoms, such as "brain fog", fatigue and clotting problems. Explanations for "long COVID" include immune imbalance, incomplete viral clearance and potentially, mitochondrial dysfunction. As conditions with sub-optimal mitochondrial function are associated with initial severity of the disease, their prior health could be key in resistance to long COVID and recovery. The SARs virus redirects host metabolism towards replication; in response, the host can metabolically react to control the virus. Resolution is normally achieved after viral clearance as the initial stress activates a hormetic negative feedback mechanism. It is therefore possible that, in some individuals with prior sub-optimal mitochondrial function, the virus can "tip" the host into a chronic inflammatory cycle. This might explain the main symptoms, including platelet dysfunction. Long COVID could thus be described as a virally induced chronic and self-perpetuating metabolically imbalanced non-resolving state characterised by mitochondrial dysfunction, where reactive oxygen species continually drive inflammation and a shift towards glycolysis. This would suggest that a sufferer's metabolism needs to be "tipped" back using a stimulus, such as physical activity, calorie restriction, or chemical compounds that mimic these by enhancing mitochondrial function, perhaps in combination with inhibitors that quell the inflammatory response.

Modelling the effect of tourism disturbance on hatching rate of the Chinese giant salamander (Andrias davidianus) by using artificial neural network

The endangered Chinese giant salamander (Andrias davidianus) is an endangered species among the conserved Chinese amphibians. Tourism-related pressures have increased for this species recently. The effect of tourism on the hatching rate of the target species was determined by experimentally observing the influences of different intensities of tourism disturbance on salamander in the Zhangjiajie Chinese Giant Salamander National Nature Reserve. Water quality factors (e.g. total nitrogen, total phosphorus, dissolved oxygen and Escherichia coli abundance) were analysed, and hatching rate was estimated. Results showed that high levels of tourism disturbance (500,000–1,200,000 visitors per year) had active effects on the hatching time and negative effects on the hatching rate. The prediction performance of artificial neural network models was validated by the low root mean square error values of 2.2539 and 3.2612 for the training and testing data and high determination coefficient values of 0.9732 and 0.9508 for the training and testing data, respectively. The potential for positive or negative feedback mechanisms in such relationships between tourists and wildlife highlights the importance of considering both sides of the complex interaction to find a balance between the development of tourism and wild animal protection.

CLAVATA3/EMBRYO SURROUNDING REGION Genes Involved in Symbiotic Nodulation in Pisum sativum

CLE (CLAVATA3/Embryo Surrounding Region) peptides regulate different aspects of plant development. In legumes, CLE peptides are known as key components of autoregulation of nodulation (AON), which systemically controls the number of nitrogen-fixing nodules formed on the root upon symbiotic interaction with soil bacteria rhizobia. CLE peptides are produced in the root in response to rhizobia inoculation and are transported via xylem to the shoot, where they are recognized by a specific receptor. As a result, a subsequent nodule development is suppressed by a negative feedback mechanism. In addition, nitrate-induced CLE genes have been identified in model legumes, which mediate nitrate-dependent inhibition of nodulation. However, little is known about the functions of nodulation-related CLE peptides, which have not been studied in Pisum sativum. Here, we studied four homologues of CLE genes in Pisum sativum, which are closely related to nodulation-suppressing CLEs from other legumes. The expression levels of these genes were increased in developing nodules. Among them, PsCLE13, PsCLE12, and the PsNIC-like genes were upregulated in response to nitrate treatment. Moreover, we found that overexpression of the PsCLE13 and PsCLE12 genes resulted in the decreased nodule number on transgenic roots. The expression levels of pea homologues of the TOO MUCH LOVE (TML) genes were upregulated in PsCLE13- and PsCLE12-overexpressing roots in comparison with the control (GUS-overexpressing) roots, suggesting that inhibitory effect of PsCLE13 and PsCLE12 is mediated through the induction of the PsTML genes.

A new negative feedback mechanism for MAPK pathway inactivation through Srk1 MAPKAP kinase

The fission yeast mitogen-activated kinase (MAPK) Sty1 is essential for cell survival in response to different environmental insults. In unstimulated cells, Sty1 forms an inactive ternary cytoplasmatic complex with the MAPKK Wis1 and the MAPKAP kinase Srk1. Wis1 phosphorylates and activates Sty1, inducing the nuclear translocation of the complex. Once in the nucleus, Sty1 phosphorylates and activates Srk1, which in turns inhibits Cdc25 and cell cycle progression, before being degraded in a proteasome-dependent manner. In parallel, active nuclear Sty1 activates the transcription factor Atf1, which results in the expression of stress response genes including pyp2 (a MAPK phosphatase) and srk1. Despite its essentiality in response to stress, persistent activation of the MAPK pathway can be deleterious and induces cell death. Thus, timely pathway inactivation is essential to ensure an appropriate response and cell viability. Here, uncover a role for the MAPKAP kinase Srk1 as an essential component of a negative feedback loop regulating the Sty1 pathway through phosphorylation and inhibition of the Wis1 MAPKK. This feedback regulation by a downstream kinase in the pathway highlights an additional mechanism for fine-tuning of MAPK signaling. Thus, our results indicate that Srk1 not only facilitates the adaptation to stress conditions by preventing cell cycle progression, but also plays an instrumental role regulating the upstream kinases in the stress MAPK pathway.

EXTH-03. HSP90 INHIBITION OVERCOMES RESISTANCE TO MOLECULAR TARGETED THERAPY IN BRAFV600E MUTANT HIGH-GRADE GLIOMA

Abstract INTRODUCTION Molecular targeted therapy using BRAF and/or MEK inhibitors has been applied to BRAFV600E mutant high-grade gliomas (HGGs); however, the therapeutic effect is limited by the emergence of drug resistance. METHODS We established multiple paired BRAFV600E mutant HGG patient-derived xenograft (PDX) models based on tissues collected prior to and at relapse after molecular targeted therapy. Using these models, we dissected treatment resistant mechanisms for molecular targeted therapy and explored therapeutic targets to overcome resistance in BRAFV600E HGG models in vitro and in vivo. RESULTS We found that, despite causing no major genetic and epigenetic changes, BRAF and/or MEK inhibitor treatment deregulated multiple negative feedback mechanisms, which led to the re-activation of the MAPK pathway through c-Raf and AKT signaling. This altered oncogenic signaling primarily mediated resistance to molecular targeted therapy in BRAFV600E mutant HGG. To overcome this resistance mechanism, we performed a high-throughput drug screening to identify therapeutic agents that potently induce additive cytotoxicity with BRAF and MEK inhibitors. We discovered that HSP90 inhibition combined with BRAF/MEK inhibition coordinately deactivated the MAPK and AKT/mTOR pathways, and subsequently induced apoptosis via dephosphorylation of GSK3β (Ser9) and inhibition of Bcl-2 family proteins. This mediated potent cytotoxicity in vitro and in vivo in refractory models with acquired resistance to molecular-targeted therapy. CONCLUSIONS The combination of an HSP90 inhibitor with BRAF or MEK inhibitors can overcome the limitations of the current therapeutic strategies for BRAFV600E mutant HGG.