P70 S6 Kinase Research Articles

Role of the mTOR Signalling Pathway During Extracellular Acidosis in Tumour Cells.

The metabolic microenvironment of solid tumours is often dominated by extracellular acidosis which results from glycolytic metabolism. Acidosis can modulate gene expression and foster the malignant progression. The aim of the study was to analyse the effects of extracellular acidosis on the mTOR signalling pathway, an important regulator of anabolic and catabolic processes like cell proliferation and autophagy. The study was performed in two tumour cell lines, AT-1 prostate and Walker-256 mammary carcinoma cells. Cells were incubated at pH7.4 or 6.6 for 3h and 24h. Then RNA and protein were extracted and analysed by qPCR and western blot. mTOR and P70-S6 kinase (P70-S6K), an important downstream target of mTOR, as well as the autophagic flux were studied. The effect of acidosis on P70S6K phosphorylation was compared to pharmacological mTOR inhibition with LY294002 and rapamycin. In both cell lines the total mTOR expression was not altered by acidosis, however, the mTOR phosphorylation was reduced after 3h but not after 24h. The P70S6K phosphorylation was reduced at both time points comparable to changes by pharmacological mTOR inhibitors. The autophagic flux, also a target of mTOR and measured by LC3-II expression, was increased in both cell lines after 24h of acidosis. The results of this study indicate that mTOR signalling is inhibited by extracellular acidosis which then lead to a reduced activity of the P70-S6 kinase (modulating gene expression) and increased autophagy possibly mediated by ULK1/2 activity. These finding may offer new perspectives for therapeutic interventions in acidic tumours.

MicroRNA-193b impairs muscle growth in mouse models of type 2 diabetes by targeting the PDK1/Akt signalling pathway

Aims/hypothesisType 2 diabetes is associated with a reduction in skeletal muscle mass; however, how the progression of sarcopenia is induced and regulated remains largely unknown. We aimed to find out whether a specific microRNA (miR) may contribute to skeletal muscle atrophy in type 2 diabetes.MethodsAdeno-associated virus (AAV)-mediated skeletal muscle miR-193b overexpression in C57BLKS/J mice, and skeletal muscle miR-193b deficiency in db/db mice were used to explore the function of miR-193b in muscle loss. In C57BL/6 J mice, tibialis anterior-specific deletion of 3-phosphoinositide-dependent protein kinase-1 (PDK1), mediated by in situ AAV injection, was used to confirm whether miR-193b regulates muscle growth through PDK1. Serum miR-193b levels were also analysed in healthy individuals (n = 20) and those with type 2 diabetes (n = 20), and correlations of miR-193b levels with HbA1c, fasting blood glucose (FBG), body composition, triacylglycerols and C-peptide were assessed.ResultsIn this study, we found that serum miR-193b levels increased in individuals with type 2 diabetes and negatively correlated with muscle mass in these participants. Functional studies further showed that AAV-mediated overexpression of miR-193b induced muscle loss and dysfunction in healthy mice. In contrast, suppression of miR-193b attenuated muscle loss and dysfunction in db/db mice. Mechanistic analysis revealed that miR-193b could target Pdk1 expression to inactivate the Akt/mammalian target of rapamycin (mTOR)/p70S6 kinase (S6K) pathway, thereby inhibiting protein synthesis. Therefore, knockdown of PDK1 in healthy mice blocked miR-193b-induced inactivation of the Akt/mTOR/S6K pathway and impairment of muscle growth.Conclusions/interpretationOur results identified a previously unrecognised role of miR-193b in muscle function and mass that could be a potential therapeutic target for treating sarcopenia.Graphical abstract

RSK Inhibition Induces Apoptosis by Downregulating Protein Synthesis in a Variety of Acute Myeloid Leukemia Cell Lines.

Fms-like tyrosine kinase 3 (FLT3) and isocitrate dehydrogenase 1/2 (IDH1/2) mutations drive malignancy in acute myeloid leukemia (AML), which accounts for approximately 40% of AML cases. Treatment with FLT3 or IDH1/2 inhibitors is used for such patients; however, it is not considered for most patients with AML who lack mutations on the respective genes. In this study, p90 ribosomal S6 kinase (RSK) was found to serve as a new therapeutic target in various AMLs with or without FLT3 mutations. BI-D1870, a potent inhibitor of RSK, significantly suppressed the proliferation of AML cell lines, among which three encoded wild-type FLT3 and three contained FLT3 driver mutations, compared with chronic myeloid leukemia K562 cells or other adherent cancer cells. BI-D1870 inhibited protein synthesis by dephosphorylating the p70 S6 kinase and eukaryotic initiation factor 4E-binding protein 1 in all AML cells except KG-1a cells. Meanwhile, the expression of microtubule-associated protein light chain 3B-I and -II increased in KG-1a cells treated with BI-D1870. BI-D1870 induced caspase-dependent apoptosis in all AML cells, including KG-1a cells. We next investigated the synergistic effect of BI-D1870 with cytarabine, a traditional anticancer drug used in AML. Synergistic effects of BI-D1870 and cytarabine were not observed in any of the cell lines. The findings suggested that BI-D1870 alone exerts an adequate antiproliferative effect on AML with or without FLT3 mutations and serves as a novel AML therapeutic agent.

Homeostatic scaling is driven by a translation-dependent degradation axis that recruits miRISC remodeling.

Homeostatic scaling in neurons has been attributed to the individual contribution of either translation or degradation; however, there remains limited insight toward understanding how the interplay between the two processes effectuates synaptic homeostasis. Here, we report that a codependence between protein synthesis and degradation mechanisms drives synaptic homeostasis, whereas abrogation of either prevents it. Coordination between the two processes is achieved through the formation of a tripartite complex between translation regulators, the 26S proteasome, and the miRNA-induced silencing complex (miRISC) components such as Argonaute, MOV10, and Trim32 on actively translating transcripts or polysomes. The components of this ternary complex directly interact with each other in an RNA-dependent manner. Disruption of polysomes abolishes this ternary interaction, suggesting that translating RNAs facilitate the combinatorial action of the proteasome and the translational apparatus. We identify that synaptic downscaling involves miRISC remodeling, which entails the mTORC1-dependent translation of Trim32, an E3 ligase, and the subsequent degradation of its target, MOV10 via the phosphorylation of p70 S6 kinase. We find that the E3 ligase Trim32 specifically polyubiquitinates MOV10 for its degradation during synaptic downscaling. MOV10 degradation alone is sufficient to invoke downscaling by enhancing Arc translation through its 3' UTR and causing the subsequent removal of postsynaptic AMPA receptors. Synaptic scaling was occluded when we depleted Trim32 and overexpressed MOV10 in neurons, suggesting that the Trim32-MOV10 axis is necessary for synaptic downscaling. We propose a mechanism that exploits a translation-driven protein degradation paradigm to invoke miRISC remodeling and induce homeostatic scaling during chronic network activity.

TAK-653, an AMPA receptor potentiator with minimal agonistic activity, produces an antidepressant-like effect with a favorable safety profile in rats

The N-methyl-d-aspartate receptor antagonist, ketamine, exhibits rapid and sustained antidepressant activity in patients with treatment-resistant depression (TRD), but its use is associated with psychotomimetic side effects. Evidence has suggested that the activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors followed by activation of the mechanistic target of rapamycin (mTOR) signaling pathway and production of brain derived neurotrophic factor (BDNF) protein may underlie the antidepressant efficacy of ketamine. In this study, we characterized the antidepressant-like effects of TAK-653, a novel AMPA receptor potentiator with virtually no agonistic activity. In rat primary cortical neurons, TAK-653 significantly increased phosphorylated and activated forms of mTOR and p70S6 kinase and their upstream regulators Akt and extracellular signal-regulated kinase (ERK). TAK-653 also significantly increased BDNF protein levels in rat primary cortical neurons. Ketamine at 30 mg/kg, i.p. produced antidepressant-like effects in the reduction of submissive behavior model (RSBM) in rats. Ketamine's antidepressant-like effect was blocked by pretreatment with the AMPA receptor antagonist NBQX at 10 mg/kg, i.p., indicating the essential role of AMPA receptor activation in the antidepressant-like effect of ketamine. Consistent with this finding, a sub-chronic administration of TAK-653 for 6 days produced significant antidepressant-like effect in the rat RSBM. Unlike ketamine, however, TAK-653 did not induce a hyperlocomotor response in rats, which is a behavioral index associated with psychotomimetic side effects in humans. TAK-653 may be a promising drug for the treatment of major depressive disorders including TRD with the potential for an improved safety profile compared with ketamine.

Azolla leaf meal at 5% of the diet improves growth performance, intestinal morphology and p70S6K1 activation, and affects cecal microbiota in broiler chicken

With growing concern about including unconventional dietary protein sources in poultry diets to substitute the protein sources that are essential for human consumption such as soybean meal, Azolla leaf meal (ALM) has grown in popularity. In our prior experiment, ALM was used at inclusion rates of 5 and 10%. Five per cent inclusion of ALM increased broiler chicken growth performance, the concentration of cecal propionic acid, and activation of skeletal muscle p70S6 Kinase1 (p70S6K1) without having detrimental effects on the meat quality. Those results prompted us to further evaluate the effect of the same inclusion rates of ALM on phase feeding and intestine and liver health of the broiler chicks. The current study hypothesis is that dietary ALM positively affects phase feeding, intestinal morphology and p70S6K1 activation, cecal microbial gene expression, and improves the liver energy status. For this, we enrolled 135 one-day-old broiler chicks and collected growth performance data (starter, grower, and finisher stages) and samples of the gastrointestinal tract to analyse the morphology of the villi, immune-related organs, mucin, and abundance of intestinal p70S6K1. Cecal bacterial species were analysed using qPCR and liver samples were collected to analyse adenosine monophosphate (AMP) and ATP content and selected oxidative stress biomarkers. ALM increased BW and feed intake during the starter and grower phases but did not affect the feed conversion ratio. Liver oxidative stress and AMP: ATP ratio increased in chickens fed on a diet containing 10% ALM (AZ10; P < 0.05). Jejunum villi length and abundance of duodenal neutral mucin increased but villi of the ileum decreased in chickens fed on a diet containing 5% ALM (AZ5), while lymphoid follicle areas of the cecal tonsils decreased with both doses of ALM. Activation of p70S6K1 increased with AZ10 in the duodenum and AZ5 in the jejunum. In the gut, the family of Enterobacteriaceae decreased with both ALM doses. In conclusion, our results indicate an overall positive effect of dietary inclusion of ALM in the broiler chicken diet via its positive effect on intestinal morphology and function; however, a negative effect on the liver was observed with 10% ALM.

Gymnema sylvestre Extract Restores the Autophagic Pathway in Human Glioblastoma Cells U87Mg.

Simple SummaryThe treatment of GBM is extremely difficult and complicated by the heterogeneous nature of neoplastic cells. The problems inherent in treating any central nervous system tumour are due to the anatomical complexity and the limited repair mechanisms of the surrounding unaffected tissues. The choice of the most suitable treatment for GBM depends on several factors: the location of the disease, the extent, and the nature of the tumour. The limit of this choice is mainly due to the degree of complexity of the disease and to the mechanisms of drug resistance that the neoplasm develops during the treatment. Herbal medicines and their derived phytocompounds are increasingly recognised as useful complementary treatments for cancer. Numerous clinical studies have reported the beneficial effects of herbal medicines on survival, immune modulation, and quality of life of cancer patients when used in combination with conventional therapies. In this study, we investigated all the mechanisms that control tumour cell growth after induction with Gymnema sylvestre (GS) extract and the key proteins that regulate these mechanisms in glioblastoma cells. The study is of great translational interest because the natural substances used could be proposed as natural adjuvant drugs for the treatment of glioblastoma, and therefore could act by modulating new molecular targets for the control of brain tumour cell growth.Glioblastoma is a brain tumour, characterised by recurrent or innate resistance to conventional chemoradiotherapy. Novel natural molecules and phyto-extracts have been proposed as adjuvants to sensitise the response to Temozolomide (TMZ). In this study, we investigated the effect of GS extract on human glioblastoma cells U87Mg. According to the IC50-values, GS extract displayed a significant cytotoxicity. This was confirmed by cell growth inhibition and alteration in metabolic activity evaluated by cell count and MTT assay. GS induced reduction in Pro-caspase 9, 3, but not PARP cleavage nor DNA fragmentation. Thus, in GS-induced cytotoxicity, cell death is not associated with apoptosis. In this context, short-term treatment of U87Mg cells with GS extract (1 mg/mL) reduced the phosphorylation levels of mTOR and of its downstream target P70 S6 kinase, highlighting the role of GS extract into autophagy induction. The activation of autophagic flux by GS extract was confirmed by Western blot analysis, which revealed the reduction in p62 and the concomitant increase in LC3B II/I ratio. Immunofluorescence evidenced the accumulation of LC3B puncta in U87Mg cells pretreated with autophagy inhibitor Bafilomycin A1. Furthermore, as main key regulators of type II programmed cell death, p53, p21 and CDK4 were also investigated and were inhibited by GS treatment. In conclusion, GS extract could be considered as an autophagy inducer in glioblastoma cells U87Mg.

Multidrug-Resistant Pseudomonas aeruginosa Triggers Differential Inflammatory Response in Patients With Endophthalmitis.

PurposeInfections with multidrug-resistant Pseudomonas aeruginosa (MDR-PA) lead to poor clinical outcomes in endophthalmitis patients, and its interactions with the host immune system remain largely unknown. The current study aimed to determine the association of MDR-PA infection with the cytokine expression profile in patients with endophthalmitis.MethodsVitreous of 12 patients with culture-proven MDR-PA along with 12 samples from antibiotic-susceptible P. aeruginosa (S-PA) and 20 non-infectious controls were included in the study. Expression patterns of IL-6, IL-10, IL-1α, IL-1β, IFN-γ, TNF-α, IL-8, and GM-CSF in the vitreous were analyzed by multiplex immunoassay and correlated with the clinical severity. We also assessed the phosphorylation level of different immune pathway molecules.ResultsIn the MDR-PA group, significantly (P < 0.05) increased expression of IL-6, IL-8, IL-10, IL-1β, and TNF-α was observed in comparison with the S-PA group. The increased inflammatory mediators in MDR-PA correlated with the disease severity. Additionally, the increased expression of inflammatory mediators was positively correlated to the activation levels of Akt, STAT3, JNK, p70 S6 kinase, and NF-кB (P < 0.05) in the MDR-PA group.ConclusionsThe current study shows the differential host immune response and phosphorylation levels of signaling molecules in MDR-PA endophthalmitis, thereby linking antibiotic resistance with distinct immune regulation.Translational RelevanceThis study provides evidence for the use of inflammatory mediator levels of IL-6, IL-8, IL-10, IL-1β, and TNF-α as potential diagnostic biomarkers of MDR endophthalmitis warranting prompt administration of immune modulators to avoid irreversible damage to the retina and vision loss.

Long-Term Protection of CHBP Against Combinational Renal Injury Induced by Both Ischemia-Reperfusion and Cyclosporine A in Mice.

Renal ischemia–reperfusion (IR) injury and cyclosporine A (CsA) nephrotoxicity affect allograft function and survival. The prolonged effects and underlying mechanisms of erythropoietin derived cyclic helix B peptide (CHBP) and/or caspase-3 small interfering RNA (CASP-3siRNA) were investigated in mouse kidneys, as well as kidney epithelial cells (TCMK-1), subjected to transplant-related injuries. Bilateral renal pedicles were clamped for 30 min followed by reperfusion for 2 and 8 weeks, with/without 35 mg/kg CsA gavage daily and/or 24 nmol/kg CHBP intraperitoneal injection every 3 days. The ratio of urinary albumin to creatinine was raised by IR injury, further increased by CsA and lowered by CHBP at 2, 4, 6 and 8 weeks, whereas the level of SCr was not significantly affected. Similar change trends were revealed in tubulointerstitial damage and fibrosis, HMGB1 and active CASP-3 protein. Increased apoptotic cells in IR kidneys were decreased by CsA and CHBP at 2 and/or 8 weeks. p70 S6 kinase and mTOR were reduced by CsA with/without CHBP at 2 weeks, so were S6 ribosomal protein and GSK-3β at 8 weeks, with reduced CASP-3 at both time points. CASP-3 was further decreased by CHBP in IR or IR + CsA kidneys at 2 or 8 weeks. Furthermore, in TCMK-1 cells CsA induced apoptosis was decreased by CHBP and/or CASP-3siRNA treatment. Taken together, CHBP predominantly protects kidneys against IR injury at 2 weeks and/or CsA nephrotoxicity at 8 weeks, with different underlying mechanisms. Urinary albumin/creatinine is a good biomarker in monitoring the progression of transplant-related injuries. CsA divergently affects apoptosis in kidneys and cultured kidney epithelial cells, in which CHBP and/or CASP-3siRNA reduces inflammation and apoptosis.

Abstract 956: The HER2×HER3 bi-specific antibody Zenocutuzumab is effective at blocking growth of tumors driven by NRG1 gene fusions

Abstract Fusions involving the neuregulin 1 gene (NRG1) occur at low frequency in pancreatic, lung, and other cancers. NRG1 fusion oncoproteins bind to HER3, leading to heterodimerization with HER2 and potent activation of downstream signaling mainly via the PI3K-AKT pathway. Zenocutuzumab (Zeno, MCLA-128), an ADCC-enhanced anti-HER2×HER3 bi-specific antibody, uniquely ‘docks' on HER2, to position the antibody and subsequently ‘block' NRG1 from interacting with HER3, effectively preventing HER2:HER3 heterodimerization and downstream signaling. Our goal in this study was to evaluate the efficacy of Zeno in preclinical models of NRG1 fusion-positive cancers. We tested Zeno in a panel of isogenic and patient-derived cell line and xenograft (PDX) models of lung, breast and pancreatic cancers. Cell lines either expressed an NRG1 fusion endogenously (MDA-MB-175-VII, DOC4-NRG1) or by lentiviral transfer of cDNAs (ATP1B1-NRG1 and SLC3A2-NRG1 in H6c7 pancreatic ductal cell line; CD74-NRG1 and VAMP2-NRG1 in immortalized human bronchial epithelial cells; and DOC4-NRG1 in MCF7 breast cancer cells). PDX models were generated from NSCLC samples harboring CD74-NRG1 (ST3204) or SLC3A2-NRG1 (LUAD-0061AS3) fusions and from a high grade serous ovarian cancer harboring a CLU-NRG1 fusion (OV-10-0050). Zeno treatment of NRG1 fusion-expressing breast, pancreatic, and lung cancer cell lines resulted in dose-dependent reduction of growth and abrogated phosphorylation of HER3, HER4, AKT, p70S6 kinase and STAT3 in all cell lines tested. Phosphorylation of HER2, EGFR and MEK/ERK was inhibited, albeit with some variation, in a cell line-specific manner. Growth of isogenic control cell lines without NRG1 fusion was not significantly altered. In breast and lung cancer cell lines, Zeno treatment down-regulated cyclin D1 expression and induced expression of the negative cell cycle regulators P21 or P27. Evidence of apoptosis activation (cleaved PARP, expression of BIM and PUMA) was also observed in cells exposed to Zeno. Treatment of mice bearing LUAD-0061AS3, ST3204 and OV-10-0050 PDX tumors (2.5, 8, 25 mg/kg, QW) caused a dose-dependent inhibition of tumor growth, with tumor shrinkage observed at higher doses. Finally, we assessed the ability of Zeno to induce antibody-dependent cellular cytotoxicity using a chromium release assay and peripheral blood mononuclear cells. Zeno induced significant cytotoxicity in MDA-MB-175-VII cells while a non-ADCC enhanced, non-specific IgG had no effect. Here we show that Zeno effectively blocks the growth of NRG1 fusion-positive cell line and xenograft models of tumors arising from lung, pancreas and other organs, and these results support the continued development of Zeno to treat patients with this molecularly defined subset of cancers. Citation Format: Igor Odintsov, Inna Khodos, Madelyn Espinosa-Cotton, Allan J. Lui, Marissa Mattar, Alison M. Schram, Ron C. Schackmann, Jeroen Lammerts van Bueren, Cecile A. Geuijen, Elisa de Stanchina, Marc Ladanyi, Romel Somwar. The HER2×HER3 bi-specific antibody Zenocutuzumab is effective at blocking growth of tumors driven by NRG1 gene fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 956.

Abstract 2000: Different oncogenic KRAS mutations produce distinct heterogenous outcomes in signaling pathways of isogenic mouse embryonic fibroblasts

Abstract Background: Growing evidence points towards the concept that not all RAS allelic variances are equal. Different RAS mutations produce distinct variations in signal transduction to sustain their proliferative needs accordingly. This is an especially important concept to consider as medicine shifts towards a more personalized approach. In accordance with this notion, through this study, we aim to explore the network dynamics and pinpoint disparities in signaling events associated with seven common KRAS allelic variances in non-small cell lung carcinoma. Methods: We used Mouse Embryonic Fibroblasts (MEF) provided by the RAS initiative (https://www.cancer.gov/research/key-initiatives/ras/outreach/reference-reagents/cell-lines). MEF were engineered to selectively harbor and express either wild type KRAS (4A and 4B WT) or one of seven following mutations: 4B G12D, G12C, G12R, G12V, G13D, Q61R and Q61L. Using high throughput immunoassay, Reverse Phase Protein Microarray (RPPA), we explored broad changes in signaling dynamics across our models. Specifically, we captured expression and/or activation levels of 68 proteins and phosphoproteins including: Receptor Tyrosine Kinases, MAPK, AKT/mTOR, JNK/STAT. Data were analyzed using unsupervised hierarchical clustering analysis using the Ward methods and the Kruskal Wallis test. P values &amp;lt;0.05 were considered significant. Results: Based on the unsupervised clustering analysis, Q61R and Q61L mutations showed the least activated signaling when compared to all other models. Activation of Raf was increased in cells harboring mutations of the G12 residues compared to those with mutations of the G13 and Q61 residues. Furthermore, the five G12 mutations clustered together with an overall higher active signaling compared to other cell lines. However, among the group of G12 mutants, unique signal transduction profiles were identified. We report distinct significant differences that divide the clustered G12 mutants into high versus low activation of specific signaling proteins. For example, ERK T202/Y204 (p=0.011) and S6 Ribosomal Protein S235/236 (p=0.001) phosphorylation were significantly higher in G12R compared to G12C and G12D models. On the contrary, STAT1 and STAT3 protein phosphorylation at residues Y701 and Y705 respectively were decreased in the G12R model. G12C has the lowest activation levels of p90RSK S380 (p=0.029), NF-KappaB p65 S536 (p=0.027) and P70 S6 Kinase S371 (p=0.013). Conclusions: In alignment with other findings, we show that based on RAS mutation, individual signaling components are affected differently, producing extensive heterogeneity in signaling dynamics. Even within grouped mutations presenting an overall similar net effect on downstream signaling, each individual variant produced discrete alterations. Citation Format: Emna El Gazzah, Elisa Baldelli, Chamodya Ruhunsiri, Emanuel F. Petricoin, Mariaelena Pierobon. Different oncogenic KRAS mutations produce distinct heterogenous outcomes in signaling pathways of isogenic mouse embryonic fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2000.

Ywhae Located on Chromosome 17p13 Promotes Myeloma Cells Proliferation Via Induction of Anti-Apoptosis and Survival Signaling Pathways

Patients with multiple myeloma (MM) carrying 17p deletion (del17p) have poor outcome. Around a third of MM patients with del(17p) are also reported to have a TP53 mutation. The acquisition of TP53 mutations seems to occur after allelic loss of 17p, and confers worse clinical outcomes. However, a recent publication has raised possibility that other additional genes resident at 17p13 may play a role in the clinical outcome of this subgroup of MM patients.The YWHAE gene is located at 17p13.3 and encodes the 14-3-3ε protein, one of the mammalian 14-3-3 protein family members that are highly conserved in eukaryotes. Altered 14-3-3ε expression is associated with development and progression of cancer. It has a context dependent pleotropic effect both as oncogene or tumor suppressor gene. However, its precise role in tumorigenesis has yet to be fully discovered.Using a large cohort of MM patients (n=369) RNA sequencing and SNP array data with complete clinical annotation, we observed a significant positive correlation (r=0.7) between YWHAE and TP53 at gene and copy number levels. In MM patients with del17p, YWHAE expression was significantly lower compared to normal plasma cells. Importantly, low expression of YWHAE correlated with poor clinical outcome (both EFS and OS), suggesting that YWHAE may be important in this specific myeloma clinical setting. However, in 2 independent MM datasets, we observed that overall YWHAE expression is higher in CD138+ MM cells compared to normal plasma cells. Moreover, we confirmed high expression of 14-3-3ε at the protein level in a large panel of MM cell lines and primary MM cells.To clarify this apparent paradox and shed light on the role of 14-3-3ε in MM, we used gene-specific CRISPR screen as well as shRNA knock down (KD) to study the functional impact of perturbation of YWHAE in MM. For the loss-of-function studies, we selected a large panel of MM cell lines based on their p53 status (WT, HD, Mu). We found a significant inhibitory effect of YWHAE KD on MM cell growth and survival over time, and induction of apoptosis via PARP cleavage and BAX increase. The antiproliferative effects were even more pronounced in p53 WT myeloma cell lines, such as MM1S and H929.Gene expression profiling (GEP) analysis showed a significant number of differentially expressed genes in YWHAE KD cells compared to control cells. Gene enrichment analysis revealed a major impact of YWHAE KD on a large set of genes involved in the mTORC1 signaling pathway. WB analysis confirmed a reduction of p-mTOR along with a decreased of p-p70 S6 kinase and p-4E BP1 after YWHAE KD. Considering the crucial role of mTOR pathway in the regulation of autophagy, we are now in the process of analyzing the effect of YWHAE on the autophagic response. Moreover, we found that YWHAE KD exerted a significant suppression of known E2F1 gene targets. Notably, a significant decrease in the expression of genes involved in the unfolded protein response was observed upon silencing/knock-out of YWHAE. As a consequence, myeloma cell sensitivity to bortezomib was increased after 14-3-3ε KD.In conclusion, YWHAE and its coding protein 14-3-3ε are constitutively expressed in MM cells. Perturbation of 14-3-3ε expression impacts MM cell growth via induction of apoptosis and inhibition of mTOR pathway. 14-3-3ε KD increase cell sensitivity to bortezomib. Ongoing efforts will further elucidate the potential molecular mechanism of YWHAE/14-3-3ε action in MM. DisclosuresAnderson:Oncopep: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Role of Glucose Transporter 1 in Graft-Versus-Host Disease and Graft-Versus-Tumor Effect

Graft-versus-host disease (GVHD) is a major obstacle to allogeneic hematopoietic cell transplantation. GVHD is induced when donor mature T cells encounter host antigens and get activated. Activated T cells require high metabolic flux through growth-promoting pathways to support clonal expansion. It is generally thought that glycolysis is employed in this process. However, conflicting data have been reported by different groups regarding the role of glycolysis in GVHD. Since glucose transporter 1 (Glut1) is essential for T cell glycolysis, we attempted to define the role of glycolysis in GVHD using donor T cells that are deficient of Glut1.One million T cells from Glut1-knockout (CD4CreGlut1fl/fl) or wild-type (control) C57BL/6 donors were transplanted into lethally irradiated (9.5 Gy) C3H/HeJ mice along with 1×107 donor T-cell-depleted bone marrow (TCDBM) cells. While all recipients of control T cells developed lethal GVHD and died within 20 days after transplantation, eight out of ten CD4CreGlut1fl/fl T cell recipients survived more than 60 days after transplantation (Figure 1, P<0.01). Body weights were slightly lower and clinical scores were slightly higher (P=not significant) in the surviving CD4CreGlut1fl/fl T cell recipients at day +56 when compared to those in the TCDBM alone group, suggesting that CD4CreGlut1fl/fl T cell recipients developed very mild GVHD if any. We further studied the role of Glut1 in CD4 and CD8 T cell mediated GVHD separately using C57BL/6 into BALB/c model. Similar to the results from the total T cell experiment, all CD4CreGlut1fl/fl CD4 and CD8 T cell recipients survived more than 60 days after transplantation. By contrast, 100% control CD4 and 50% CD8 T cell recipients died of lethal GVHD within 60 days after transplantation. These data demonstrate that Glut1 is required for alloresponses and glycolysis is indispensasble in the development of lethal GVHD.To understand the mechanism by which CD4CreGlut1fl/fl T cells mediate less GVHD, we first studied their ability to respond to alloantigens in mixed lymphocyte reaction. The results indicated that CD4CreGlut1fl/fl T cells have dramatically decreased ability to respond to BALB/c antigens when compared with the control T cells. Consistent with the in vitro data, T cell expansion was 59.4% less in CD4CreGlut1fl/fl T cell recipients than that in control T cell recipients when measured at day +7 after GVHD induction. Apoptosis was increased in alloantigen-activated (CD69+) CD4 but not in CD8 T cells from CD4CreGlut1fl/fl mice when compared with that from control mice when measured 16 hours after the initiation of culture. However, more cell death was observed in both activated CD4 and CD8 T cells from CD4CreGlut1fl/fl mice than that from control mice. In addition, alloreactive CD4CreGlut1fl/fl T cells failed to sustain activated mTORC1 signaling, as assessed by phosphorylation of the downstream p70 S6 kinase target, small ribosomal subunit S6. These results indicate that, upon response to alloantigens, T cells are prone to apoptosis and cell death and lead to decreased clonal expansion in the absence of Glut1, suggesting a mechanism by which CD4CreGlut1fl/fl T cells have decreased ability to induce GVHD.To determine whether absence of Glut1 in T cells affects graft-versus-tumor (GVT) effects, we transplanted 1×106 T cells from Glut1-knockout (CD4CreGlutfl/fl) C57BL/6 donors into lethally irradiated (8.5 Gy) BALB/c mice along with 1×107 donor TCDBM cells. At the time of transplantation, the recipients were challenged with 1×105 BCL1 cells, a BALB/c-derived leukemia/lymphoma cell line. While all TCDBM alone recipients died of leukemia/lymphoma and all control T cell recipients died of lethal GVHD, all CD4CreGlut1fl/fl T cell recipients survived more than 100 days after transplantation without leukemia/lymphoma (Figure 2), indicating that T cells deficient of Glut1 preserve GVT effect.The results from this study definitely demonstrate that glycolysis is essential for the development of GVHD. Inhibition of glycolysis in T cells increases apoptosis and cell death and suppresses clonal expansion of allospecific T cells. Interestingly, GVT effect is preserved despite the inhibition of glycolysis. These data suggest that glycolysis could potentially be targeted for prevention and treatment of GVHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Combined Anticancer Effect of Plasma-Activated Infusion and Salinomycin by Targeting Autophagy and Mitochondrial Morphology.

Non-thermal atmospheric pressure plasma (NTAPP)-activated liquids have emerged as new promising anticancer agents because they preferentially injure malignant cells. Here, we report plasma-activated infusion (PAI) as a novel NTAPP-based anti-neoplastic agent. PAI was prepared by irradiating helium NTAP to form a clinically approved infusion fluid. PAI dose-dependently killed malignant melanoma and osteosarcoma cell lines while showing much lower cytotoxic effects on dermal and lung fibroblasts. We found that PAI and salinomycin (Sal), an emerging anticancer stem cell agent, mutually operated as adjuvants. The combined administration of PAI and Sal was much more effective than single-agent application in reducing the growth and lung metastasis of osteosarcoma allografts with minimal adverse effects. Mechanistically, PAI explicitly induced necroptosis and increased the phosphorylation of receptor-interacting protein 1/3 rapidly and transiently. PAI also suppressed the ambient autophagic flux by activating the mammalian target of the rapamycin pathway. PAI increased the phosphorylation of Raptor, Rictor, and p70-S6 kinase, along with decreased LC3-I/II expression. In contrast, Sal promoted autophagy. Moreover, Sal exacerbated the mitochondrial network collapse caused by PAI, resulting in aberrant clustering of fragmented mitochondrial in a tumor-specific manner. Our findings suggest that combined administration of PAI and Sal is a promising approach for treating these apoptosis-resistant cancers.

The involvement of Sestrin2 in the effect of IGF-I and leucine on mTROC1 activity in C2C12 and L6 myocytes

ObjectiveIGF-I and branched-chain amino acids have been reported to promote muscle hypertrophy via the stimulation of protein synthesis. Sestrin2, the function of which is regulated by leucine, has been reported to attenuate the activity of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) that stimulates protein synthesis. The objective of this study was to examine whether IGF-I modulates Sestrin2 abundance and to clarify the involvement of Sestrin2 in the effect of IGF-I and leucine on mTROC1. DesignC2C12 and L6 myocytes were stimulated by leucine (1 mM) with or without pretreatment with IGF-I (100 ng/mL). Phosphorylation of p70 S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1), both of which are targets of the mTORC1, was examined by western blotting. Effects of Sestrin2 small interfering RNA (siRNA) on the actions of leucine and IGF-I were examined. Sestrin2 mRNA and protein levels were also determined after Sestrin2 siRNA. ResultsLeucine increased the phosphorylation of S6K and 4E-BP1 in a dose-dependent manner. Pretreatment with IGF-I for 5 h further increased the stimulatory effect of leucine on the phosphorylation of S6K and 4E-BP1 in C2C12 cells. IGF-I increased Sestrin2 protein and messenger RNA levels. Sestrin2 siRNA increased or tended to increase basal phosphorylation of 4E-BP1 and decreased the leucine-induced phosphorylation in C2C12 and L6 cells, in particular after IGF-I treatment, suggesting the involvement of Sestrin2 in the action of leucine and IGF-I. The net increase in leucine-induced 4E-BP1 phosphorylation appeared to be attenuated by Sestrin2 siRNA. Likewise, Sestrin2 siRNA attenuated leucine-induced S6K phosphorylation in L6 cells. However, Sestrin2 siRNA did not influence leucine-induced S6K phosphorylation in C2C12 cells. ConclusionsIGF-I and leucine cooperatively increased mTORC1 activity in C2C12 cells. IGF-I increased Sestrin2. Sestrin2 siRNA experiments showed that Sestrin2 was involved in the effect of leucine and IGF-I on mTORC1 activity in C2C12 and L6 cells, and suggested that increased Sestrin2 by IGF-I pretreatment might play a role in enhancing the effect of leucine on mTORC1.

1244-P: The Protein Phosphatase PPm1k Regulates Ribosomal Protein S6 Phosphorylation in Beta Cells

Phosphorylation of ribosomal protein S6 (RPS6) on ser235/236 and ser240/244 in the pancreatic beta cell increases cell size and insulin content and influences systemic glucose homeostasis. Our phosphoproteomic analysis identified the protein phosphatase, PPm1K, as a novel regulator of RPS6 phosphorylation. We find that beta cells account for most PPm1K expression within pancreatic islets and that exposure of INS1 832/13 beta cells to increasing concentrations of glucose results in suppression of PPm1K mRNA expression. siRNA mediated knockdown of PPm1K in INS1 832/13 cells, to levels achieved by glucose, increased insulin content and secretion in basal and glucose stimulated conditions that was accompanied by robust phosphorylation of RPS6 on ser235/236 and ser240/244. PPm1K knockdown did not increase phosphorylation of p70 S6 Kinase (S6K) or the mammalian target of rapamycin (mTOR) upstream of RPS6 suggesting that PPm1K regulates RPS6 phosphorylation in an mTOR-S6K independent manner. Accordingly, treatment with the mTORC1 inhibitor, Rapamycin, lowered S6K and RPS6 phosphorylation but did not override the effect of PPm1K knockdown to increase RPS6 phosphorylation compared to control siRNA treated cells. Lowering phosphorylation of PPm1K’s classical target, ser293 of the branched-chain keto acid dehydrogenase (BCKDH) with the small molecule, BT2, also did not impact the effect of PPm1K knockdown to increase RPS6 phosphorylation. Thus PPm1K exerts its effects on RPS6 phosphorylation in a BCKDH and mTOR/S6K independent manner. The effects of siRNA-mediated PPm1K knockdown on RPS6 phosphorylation were accompanied by a 1.5 fold increase in protein translation and the induction of an unfolded protein response as determined by an increase in XBP-1 splicing, and BiP expression. Together our data identify a new role for the phosphatase PPm1K as a glucose sensitive modulator of RPS6 phosphorylation and protein translation that impacts beta cell insulin content and secretion. Disclosure Y. Deleye: None. J. Herring: None. K. H. Hess: None. B. Leininger: None. R. W. Mcgarrah: None. J. S. Tessem: None. P. J. White: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-16-INI-17 to P.J.W.)

Elimination of endogenous high molecular weight FGF2 prevents pressure-overload-induced systolic dysfunction, linked to increased FGFR1 activity and NR1D1 expression.

Fibroblast growth factor 2 (FGF2), produced as high (Hi-) and low (Lo-) molecular weight isoforms, is implicated in cardiac response to injury. The role of endogenous FGF2 isoforms during chronic stress is not well defined. We investigated the effects of endogenous Hi-FGF2 in a mouse model of simulated pressure-overload stress achieved by transverse aortic constriction (TAC) surgery. Hi-FGF2 knockout mice, expressing only Lo-FGF2, FGF2(Lo), and wild-type mice, FGF2(WT), expressing both Hi-FGF2 and Lo-FGF2, were used. By echocardiography, a decline in systolic function was observed in FGF2(WT) but not FGF2(Lo) mice compared to corresponding sham-operated animals at 4-8weeks post-TAC surgery. TAC surgery increased markers of myocardial stress/damage including B-type natriuretic peptide (BNP) and the pro-cell death protein BCL2/adenovirus E1B 19kDa protein-interacting protein-3 (Bnip3) in FGF2(WT) but not FGF2(Lo) mice. In FGF2(Lo) mice, cardiac levels of activated FGF receptor 1 (FGFR1), and downstream signals, including phosphorylated mTOR and p70S6 kinase, were elevated post-TAC. Finally, NR1D1 (nuclear receptor subfamily 1 group D member 1), implicated in cardioprotection from pressure-overload stress, was downregulated or upregulated in the presence or absence, respectively, of Hi-FGF2 expression, post-TAC surgery. In wild-type cardiomyocyte cultures, endothelin-1 (added to simulate pressure-overload signals) caused NR1D1 downregulation and BNP upregulation, similar to the effect of TAC surgery on the FGF2(WT) mice. The NR1D1 agonist SR9009 prevented BNP upregulation, simulating post-TAC findings in FGF2(Lo) mice. We propose that elimination of Hi-FGF2 is cardioprotective during pressure-overload by increasing FGFR1-associated signaling and NR1D1 expression.

POS1001 CLINICAL AND SURROGATE CARDIOVASCULAR RISK ASSESSMENT AND ITS RELATIONSHIP WITH PSORIATIC ARTHRITIS PATHOGENESIS

Background:Psoriatic arthritis (PsA) is a chronic inflammatory disease associated with an increased prevalence of cardiovascular (CV) events. Traditional CV risk factors do not account for the increased CV disease mortality in PsA. Inflammation seems to have a key role in the development of this comorbidity, however the specific molecular mechanisms involved are not defined yet.Objectives:To evaluate clinical CV risk factors and surrogate markers and their relationship with inflammation, disease activity and metabolic comorbidities in PsA patientsMethods:This is a cross-sectional study including 100 PsA patients without CV disease recruited in the routine clinical practice at the Rheumatology Department, Reina Sofia Hospital of Cordoba and 100 age-matched healthy donors (HDs). Different parameters related to the cardiometabolic risk were analyzed. Clinical and analytical parameters were collected: lipid profile (cholesterol, HDL, LDL, TG, ApoA and ApoB), glucose and insulin, body surface area (BSA) affected by psoriasis, number of tender and swollen joints, DAPSA, VAS, CRP and ESR. To measure the persistence of inflammation, CRP levels were recorded retrospectively once, twice, or three times during the 5 years prior to study and at the moment of the study. Increased levels of CRP in at least 50% of the determinations was considered as persistent inflammation. Plasma and peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of patients and HDs. A panel of 92 proteins involved in CV disease and an adipocytokine profile was measured in plasma and PBMCs. In addition, activation of 18 intracellular pathways involved in cell activation was also measured in PBMCs. In vitro experiments in adipocytes treated with serum from PsA patients were also carried out.Results:Traditional CV risk factors including atherogenic risk, insulin resistance (IR), metabolic syndrome, smoking, obesity, arterial hypertension, apolipoprotein B/A risk, type 2 diabetes mellitus and the levels of SCORE were significantly increased in PsA patients. The presence of IR was associated with disease activity markers (DAPSA, ESR and CRP). In fact, the HOMA-IR index was related to the CRP persistence. PsA patients with obesity had significantly increased the number of tender and swollen joints, the levels of DAPSA and CRP. Twenty-eight proteins involved in CV disease and six adipocytokines were significantly elevated in the plasma of PsA patients. Several of these cardiovascular molecules were associated with higher levels of DAPSA (CTSD, GAL3, CD163, FABP4, IL6 and IL1RT2), acute phase reactants (GAL3, TNFα, Adiponectin, TNFR1 and IL6), affected body surface area (IL2RA, GAL3, CCL15, TRAP, CSTB, CD163, OPG and CNTN1) and onychopaty (TRAP, VWF, MCP-1, GAL3, LTBR, TFPI, CHI3L1, CTSZ and JAM-A). In addition, the mRNA expression of most of those 28 CV molecules were significantly increased in PBMCs from PsA patients. At intracellular level, the activation of 11 kinases (ERK1/2, AKT, S6 Ribosomal, mTOR, HSP27, Bad, p70 S6 kinase, PRAS40, p53 and caspase-3) involved in insulin signaling, inflammation, cell survival and apoptosis were altered in PBMCs. Finally, serum from PsA patients was able to modify the expression of these molecules in adipocytes.Conclusion:1) Disease activity and inflammatory burden are closely associated with the presence of metabolic alterations, specifically obesity and IR in patients with PsA. 2) The development of IR is extremely related to the persistence of CRP levels in the previous 5 years. 3) Inflammation is closely associated to the adipose tissue dysfunction in PsA and 4) FABP4, CD163 and GAL3 are surrogate CV markers commonly associated with clinical features of PsA, suggesting the role of these molecules linking CVD and PsA pathogenesis.Funded by ISCIII (PI17/01316 and RIER RD16/0012/0015) co-funded with FEDER.Disclosure of Interests:None declared.

PI3K Inhibition by BKM120 Results in Antiproliferative Effects on Corticotroph Tumor Cells

Purpose: Cushing’s disease is associated with significant morbidity, thus additional tumor-directed drugs with the potential to exert antineoplastic effects on corticotroph adenoma cells are desired. The PI3K (phosphoinositide-3-kinase)/AKT (protein kinase B) pathway, which plays regulatory roles in cell survival and proliferation, is activated in pituitary adenomas. The present study evaluated the effects of BKM120 (Buparlisib), an oral PI3K inhibitor, in corticotroph tumor cells. Methods: AtT-20/D16v-F2 mouse pituitary corticotroph tumor cells were treated with increasing concentrations of BKM120 or vehicle. Cell viability was measured using MTS-based assay. Apoptosis was evaluated by Annexin V staining. ACTH levels were measured in the culture supernatants by chemiluminescent immunometric assay. Cell cycle analysis was performed by propidium iodide DNA staining and flow cytometry. Gene expression of cell cycle regulators (Cdkn1b, Rb1, Ccnd1, Cdk4, Cdk2, and Myc) was assessed by qPCR. Protein expression of p27, p70 S6 Kinase, p85 S6 Kinase, and phosphorylated AKT was assessed by Western blot. Results: Treatment with BKM120 decreased AtT-20/D16v-F2 cell proliferation and ACTH levels in the cell culture supernatants. Furthermore, BKM120 treatment diminished the phosphorylation of AKT at residue 473, increased p27 expression and induced a G0/G1 cell cycle arrest. Conclusion:In vitro inhibition of PI3K/AKT pathway by BKM120 resulted in antiproliferative effects on corticotroph tumor cells, decreasing cell viability and ACTH production. These encouraging findings shape the path for further experiments with the inhibition of PI3K/AKT pathway in Cushing’s disease.

The Effects of Severe Psychological Stress Coupled with Binge Drinking on Markers of Skeletal Muscle Protein Synthesis

Acute exposure to a severe stressor can cause an increase in circulating glucocorticoid (GC) concentrations by approximately ten-fold, which can remain elevated for as long as ten days. Elevated GCs can promote skeletal muscle atrophy and diminish muscle strength. Unfortunately, a common coping mechanism in response to severe stress is the use and abuse of alcohol. Alcohol abuse also suppresses muscle protein synthesis; thus, the combination of stress and alcohol may exacerbate skeletal muscle impairments. The aim of this current study was to examine the combined effects of stress and alcohol on muscle protein synthesis signaling in skeletal muscle. Our hypothesis was that voluntary binge-patterned drinking preceded by acute stress would impair signaling of muscle protein synthesis compared to binge drinking alone. Male C57BL/6 mice (7 weeks old) were exposed to a single episode of 100 inescapable tail shocks. Following acute stress, mice were randomized into groups with temporary access to either water or alcohol during their active period daily for 5 days, a voluntary binge-patterned drinking protocol referred to as Drinking in the Dark (DID). On the fifth day, following DID, mice were euthanized, and muscles of interest were rapidly collected. The experimental set up resulted in 2 groups: Stress Alcohol (SA) (n=7) and Stress Water (SW) (n=8). Gastrocnemius was analyzed via western blotting and the canonical Akt-mTORC1 pathway of protein synthesis was assessed for changes in signaling. The SA group tended to have less phosphorylation of Akt (p=0.068), but there was no difference in the phosphorylation of mTOR between groups (p=0.838). The SA group tended to have less phosphorylation of p70S6 kinase (p=0.058), a downstream target of mTORC1. Similarly, phosphorylation of 4E Binding Protein 1 (p4EBP1) was significantly lower in SA (p=0.017). Our results suggest binge drinking in the context of stress may impair muscle protein synthesis along the Akt-mTORC1 pathway.