Glutamine anaplerosis is required for amino acid biosynthesis in human meningiomas.

BACKGROUND: We postulate that meningiomas undergo distinct metabolic reprogramming in tumorigenesis and unraveling their metabolic phenotypes provide new therapeutic insights. Glutamine catabolism is key to the growth and proliferation of tumors. Here, we investigated the metabolomics of freshly resected meningiomas and glutamine metabolism in patient-derived meningioma cells. METHODS: 1H NMR spectroscopy of tumor tissues from meningioma patients was used to differentiate the metabolite profiles of grade-I and grade-II meningiomas. Glutamine metabolism was examined using 13C/15N glutamine tracer, in 5 patient-derived meningioma cells. RESULTS: Alanine, lactate, glutamate, glutamine, and glycine were predominantly elevated only in grade-II meningiomas by 74%, 76%, 35%, 75%, and 33%, respectively, with alanine and glutamine levels being statistically significant (P ≤ .02). 13C/15N glutamine tracer experiments revealed that both grade-I and -II meningiomas actively metabolize glutamine to generate various key carbon intermediates including alanine and proline that are necessary for the tumor growth. Also, it is shown that glutaminase (GLS1) inhibitor, CB-839 is highly effective in downregulating glutamine metabolism and decreasing proliferation in meningioma cells. CONCLUSION: Alanine and glutamine/glutamate are mainly elevated in grade-II meningiomas. Grade-I meningiomas possess relatively higher glutamine metabolism providing carbon/nitrogen for the biosynthesis of key nonessential amino acids. GLS1 inhibitor (CB-839) is very effective in downregulating glutamine metabolic pathways in grade-I meningiomas leading to decreased cellular proliferation.

Sensory substitution reveals a manipulation bias.

Sensory substitution is a promising therapeutic approach for replacing a missing or diseased sensory organ by translating inaccessible information into another sensory modality. However, many substitution systems are not well accepted by subjects. To explore the effect of sensory substitution on voluntary action repertoires and their associated affective valence, we study deaf songbirds to which we provide visual feedback as a substitute of auditory feedback. Surprisingly, deaf birds respond appetitively to song-contingent binary visual stimuli. They skillfully adapt their songs to increase the rate of visual stimuli, showing that auditory feedback is not required for making targeted changes to vocal repertoires. We find that visually instructed song learning is basal-ganglia dependent. Because hearing birds respond aversively to the same visual stimuli, sensory substitution reveals a preference for actions that elicit sensory feedback over actions that do not, suggesting that substitution systems should be designed to exploit the drive to manipulate.

Undirected singing rate as a non-invasive tool for welfare monitoring in isolated male zebra finches.

Research on the songbird zebra finch (Taeniopygia guttata) has advanced our behavioral, hormonal, neuronal, and genetic understanding of vocal learning. However, little is known about the impact of typical experimental manipulations on the welfare of these birds. Here we explore whether the undirected singing rate can be used as an indicator of welfare. We tested this idea by performing a post hoc analysis of singing behavior in isolated male zebra finches subjected to interactive white noise, to surgery, or to tethering. We find that the latter two experimental manipulations transiently but reliably decreased singing rates. By contraposition, we infer that a high-sustained singing rate is suggestive of successful coping or improved welfare in these experiments. Our analysis across more than 300 days of song data suggests that a singing rate above a threshold of several hundred song motifs per day implies an absence of an acute stressor or a successful coping with stress. Because singing rate can be measured in a completely automatic fashion, its observation can help to reduce experimenter bias in welfare monitoring. Because singing rate measurements are non-invasive, we expect this study to contribute to the refinement of the current welfare monitoring tools in zebra finches.

SRC-Family Kinases in Acute Myeloid Leukaemia and Mastocytosis.

Protein tyrosine kinases have been recognized as important actors of cell transformation and cancer progression, since their discovery as products of viral oncogenes. SRC-family kinases (SFKs) play crucial roles in normal hematopoiesis. Not surprisingly, they are hyperactivated and are essential for membrane receptor downstream signaling in hematological malignancies such as acute myeloid leukemia (AML) and mastocytosis. The precise roles of SFKs are difficult to delineate due to the number of substrates, the functional redundancy among members, and the use of tools that are not selective. Yet, a large num ber of studies have accumulated evidence to support that SFKs are rational therapeutic targets in AML and mastocytosis. These two pathologies are regulated by two related receptor tyrosine kinases, which are well known in the field of hematology: FLT3 and KIT. FLT3 is one of the most frequently mutated genes in AML, while KIT oncogenic mutations occur in 80-90% of mastocytosis. Studies on oncogenic FLT3 and KIT signaling have shed light on specific roles for members of the SFK family. This review highlights the central roles of SFKs in AML and mastocytosis, and their interconnection with FLT3 and KIT oncoproteins.

Dual protein kinase and nucleoside kinase modulators for rationally designed polypharmacology.

Masitinib, a highly selective protein kinase inhibitor, can sensitise gemcitabine-refractory cancer cell lines when used in combination with gemcitabine. Here we report a reverse proteomic approach that identifies the target responsible for this sensitisation: the deoxycytidine kinase (dCK). Masitinib, as well as other protein kinase inhibitors, such as imatinib, interact with dCK and provoke an unforeseen conformational-dependent activation of this nucleoside kinase, modulating phosphorylation of nucleoside analogue drugs. This phenomenon leads to an increase of prodrug phosphorylation of most of the chemotherapeutic drugs activated by this nucleoside kinase. The unforeseen dual activity of protein kinase inhibition/nucleoside kinase activation could be of great therapeutic benefit, through either reducing toxicity of therapeutic agents by maintaining effectiveness at lower doses or by counteracting drug resistance initiated via down modulation of dCK target.

Comparative oncogenomics identifies tyrosine kinase FES as a tumor suppressor in melanoma.

Identification and functional validation of oncogenic drivers are essential steps toward advancing cancer precision medicine. Here, we have presented a comprehensive analysis of the somatic genomic landscape of the widely used BRAFV600E- and NRASQ61K-driven mouse models of melanoma. By integrating the data with publically available genomic, epigenomic, and transcriptomic information from human clinical samples, we confirmed the importance of several genes and pathways previously implicated in human melanoma, including the tumor-suppressor genes phosphatase and tensin homolog (PTEN), cyclin dependent kinase inhibitor 2A (CDKN2A), LKB1, and others. Importantly, this approach also identified additional putative melanoma drivers with prognostic and therapeutic relevance. Surprisingly, one of these genes encodes the tyrosine kinase FES. Whereas FES is highly expressed in normal human melanocytes, FES expression is strongly decreased in over 30% of human melanomas. This downregulation correlates with poor overall survival. Correspondingly, engineered deletion of Fes accelerated tumor progression in a BRAFV600E-driven mouse model of melanoma. Together, these data implicate FES as a driver of melanoma progression and demonstrate the potential of cross-species oncogenomic approaches combined with mouse modeling to uncover impactful mutations and oncogenic driver alleles with clinical importance in the treatment of human cancer.

Insight on Mutation-Induced Resistance from Molecular Dynamics Simulations of the Native and Mutated CSF-1R and KIT.

The receptors tyrosine kinases (RTKs) for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors' sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V) and CSF-1R (mutation D802V) by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i) the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii) the electrostatic interactions are a decisive factor affecting the binding energy; (iii) the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R) and D816V (KIT) mutations; (iv) the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib.

An essential pathway links FLT3-ITD, HCK and CDK6 in acute myeloid leukemia.

CDK4/CDK6 and RB proteins drive the progression through the G1 phase of the cell cycle. In acute myeloid leukemia (AML), the activity of the CDK/Cyclin D complex is increased. The mechanism involved is unknown, as are the respective roles played by CDK4 or CDK6 in this process. Here, we report that AML cells carrying FLT3-ITD mutations are dependent on CDK6 for cell proliferation while CDK4 is not essential. We showed that FLT3-ITD signaling is responsible for CDK6 overexpression, through a pathway involving the SRC-family kinase HCK. Accordingly, FLT3-ITD failed to transform primary hematopoietic progenitor cells from Cdk6-/- mice. Our results demonstrate that CDK6 is the primary target of CDK4/CDK6 inhibitors in FLT3-ITD positive AML. Furthermore, we delineate an essential protein kinase pathway -FLT3/HCK/CDK6- in the context of AML with FLT3-ITD mutations.

Mechanisms of STAT protein activation by oncogenic KIT mutants in neoplastic mast cells.

Mutations in the c-kit gene occur in the vast majority of mastocytosis. In adult patients as well as in the cell line derived from mast cell neoplasms, the mutations occur almost exclusively at amino acid 816 within the kinase domain of KIT. Among the downstream effectors of KIT signaling, STAT3 and STAT5 have been shown to be critical for cell proliferation elicited by the KIT-Asp(816) mutant protein. However, little is known about the mechanisms of activation of STAT proteins. In this study, we identify and clarify the contribution of various STAT kinases in two widely used neoplastic mast cell lines, P815 and HMC-1. We show that STAT1, -3, and -5 proteins are activated downstream of the KIT-Asp(816) mutant. All three STAT proteins are located in the nucleus and are phosphorylated on serine residues. KIT-Asp(816) mutant can directly phosphorylate STATs on the activation-specific tyrosine residues in vitro. However, within cells, SRC family kinases and JAKs diversely contribute to tyrosine phosphorylation of STAT proteins downstream of the KIT mutant. Using a panel of inhibitors, we provide evidence for the implication or exclusion of serine/threonine kinases as responsible for serine phosphorylation of STAT1, -3, and -5 in the two cell lines. Finally, we show that only STAT5 is transcriptionally active in these cells. This suggests that the contribution of STAT1 and STAT3 downstream of KIT mutant is independent of their transcription factor function.

Pediatric mastocytosis-associated KIT extracellular domain mutations exhibit different functional and signaling properties compared with KIT-phosphotransferase domain mutations.

Compared with adults, pediatric mastocytosis has a relatively favorable prognosis. Interestingly, a difference was also observed in the status of c-kit mutations according to the age of onset. Although most adult patients have a D(816)V mutation in phosphotransferase domain (PTD), we have described that half of the children carry mutations in extracellular domain (ECD). KIT-ECD versus KIT-PTD mutants were introduced into rodent Ba/F3, EML, Rat2, and human TF1 cells to investigate their biologic effect. Both ECD and PTD mutations induced constitutive receptor autophosphorylation and ligand-independent proliferation of the 3 hematopoietic cells. Unlike ECD mutants, PTD mutants enhanced cluster formation and up-regulated several mast cell-related antigens in Ba/F3 cells. PTD mutants failed to support colony formation and erythropoietin-mediated erythroid differentiation. ECD and PTD mutants also displayed distinct whole-genome transcriptional profiles in EML cells. We observed differences in their signaling properties: they both activated STAT, whereas AKT was only activated by ECD mutants. Consistently, AKT inhibitor suppressed ECD mutant-dependent proliferation, clonogenicity, and erythroid differentiation. Expression of myristoylated AKT restored erythroid differentiation in EML-PTD cells, suggesting the differential role of AKT in those mutants. Overall, our study implied different pathogenesis of pediatric versus adult mastocytosis, which might explain their diverse phenotypes.

FES kinase participates in KIT-ligand induced chemotaxis.

FES is a cytoplasmic tyrosine kinase activated by several membrane receptors, originally identified as a viral oncogene product. We have recently identified FES as a crucial effector of oncogenic KIT mutant receptor. However, FES implication in wild-type KIT receptor function was not addressed. We report here that FES interacts with KIT and is phosphorylated following activation by its ligand SCF. Unlike in the context of oncogenic KIT mutant, FES is not involved in wild-type KIT proliferation signal, or in cell adhesion. Instead, FES is required for SCF-induced chemotaxis. In conclusion, FES kinase is a mediator of wild-type KIT signalling implicated in cell migration.

The tyrosine kinase FES is an essential effector of KITD816V proliferation signal.

KIT is a tyrosine kinase receptor that is aberrantly activated in several neoplasms. In human pathologies, the most frequent mutation of KIT occurs at codon 816. The resulting KIT mutant protein is activated in the absence of ligand and is resistant to the clinically available inhibitors of KIT. In this report, we provide evidence for an essential function of the cytoplasmic tyrosine kinase FES downstream of KIT(D816V). FES is phosphorylated on tyrosine residues in cells that carry KIT(D816V) mutation, and this phosphorylation is KIT dependent. Reduction of FES expression using RNA interference results in decreased cell proliferation in human or murine cells harboring KIT(D816V) or the homologous mouse mutation KIT(D814Y). The reduced cell growth can be rescued using another cytokine (granulocyte-macrophage colony-stimulating factor [GM-CSF]) and is not observed when the closely related fer gene is targeted. Finally, signaling downstream of KIT(D816V) is altered in cells lacking FES expression. This study shows a major function of FES downstream of activated KIT receptor and thereby points to FES as a novel target in KIT-related pathologies.

The E3 ubiquitin ligase HOIL-1 induces the polyubiquitination and degradation of SOCS6 associated proteins.

The suppressor of cytokine signaling (SOCS) proteins are thought to exert their function through the recruitment of interacting-proteins to the ubiquitin/proteasome degradation pathway. All SOCS proteins bind an Elongin BC E3 ubiquitin ligase complex through the common Socs-box. Here, we show that haem-oxidized IRP2 ubiquitin ligase-1 (HOIL-1), another E3 ubiquitin ligase, interacts with SOCS6. The Ubl domain of HOIL-1 and the SH2 and Socs-box domains of SOCS6 are required for the interaction. HOIL-1 expression stabilizes SOCS6 and induces the ubiquitination and degradation of proteins associated with SOCS6. These data suggest that SOCS proteins may interact with different E3 ubiquitin ligases in addition to a common Elongin BC E3 complex.

NACA is a positive regulator of human erythroid-cell differentiation.

We have previously identified the transcript encoding NACA (the alpha chain of the nascent-polypeptide-associated complex) as a cytokine-modulated specific transcript in the human TF-1 erythroleukemic cell line. This protein was already known to be a transcriptional co-activator that acts by potentiating AP-1 activity in osteoblasts, and is known to be involved in the targeting of nascent polypeptides. In this study, we investigate the role of NACA in human hematopoiesis. Protein distribution analyses indicate that NACA is expressed in undifferentiated TF-1 cells and in human-cord-blood-derived CD34(+) progenitor cells. Its expression is maintained during in vitro erythroid differentiation but, in marked contrast, its expression is suppressed during their megakaryocytic or granulocytic differentiation. Ectopic expression of NACA in CD34(+) cells under culture conditions that induce erythroid-lineage differentiation leads to a marked acceleration of erythroid-cell differentiation. Moreover, ectopic expression of NACA induces erythropoietin-independent differentiation of TF-1 cells, whereas downregulation of NACA by RNA interference abolishes the induction of hemoglobin production in these cells and diminishes glycophorin-A (GPA) expression by CD34(+) progenitors cultured under erythroid differentiation conditions. Altogether, these results characterize NACA as a new factor involved in the positive regulation of human erythroid-cell differentiation.

Modulation of PAI-1 and proMMP-9 syntheses by soluble TNFalpha and its receptors during differentiation of the human monocytic HL-60 cell line.

During phorbol ester-induced differentiation of HL-60 monocytic cells, tumor necrosis factoralpha (TNFalpha) synthesis and secretion are increased, which contributes to the autocrine regulation of TNFalpha-responsive genes. We investigated how, during phorbol ester-induced differentiation of HL-60 cells, the secreted TNFalpha modulated plasminogen activator inhibitor type I (PAI-1) and gelatinase B (MMP-9) syntheses, two proteins involved in pericellular proteolysis. The differentiation-induced release of TNFalpha, was abolished by the hydroxamate-based matrix metalloproteinase (MMP) inhibitor, RU36156. RU36156 or a neutralizing anti-TNFalpha significantly down-regulated PAI-1 synthesis exclusively during the early phases of differentiation (from promyelocyte to monocytic-like cells), which underlined the activating role of autocrine TNFalpha during this time range. As cells progressed to monocyte/macrophage phenotype, they still released TNFalpha, but RU36156 or anti-TNFalpha no longer had an effect on PAI-1 synthesis. This lack of effect was not due to a default of TNFalpha signaling since PAI-1 synthesis was still stimulated in response to exogenous TNFalpha. TNFalpha receptor RI was also actively released and was shown to reduce TNFalpha activity which may account for the inability of soluble TNFalpha to up-regulate PAI-1 synthesis. In later mature stage, cells became susceptible to exogenous TNFalpha-induced apoptosis and rapidly lost their ability to respond to TNFalpha. The MMP-9 synthesis followed similar regulation as PAI-1. Isolated human blood monocytes-derived macrophages behave like HL-60-derived macrophages. In conclusion, these results show that during leukocyte differentiation, time windows exist during which the autocrine TNFalpha is active and then down-regulated by RI, which may temper a continuous up-regulation of the synthesis of proteins involved in pericellular proteolysis.

Effect of atorvastatin on adhesive phenotype of human endothelial cells activated by tumor necrosis factor alpha.

We studied the effect of atorvastatin on the adhesive phenotype of human endothelial cells (HUVEC) stimulated by tumor necrosis factor (TNF)-alpha. Surface expression of adhesion molecules on HUVEC was examined by flow cytometry and confocal microscopy, and adhesion of monocytes (human THP-1 cell line) was measured in vitro under flow conditions. In TNF-alpha-activated HUVEC, atorvastatin significantly enhanced surface expression of vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, E-selectin, and fractalkine, when compared with TNF-alpha stimulation alone. This enhancement was reversed by mevalonate or geranylgeranyl pyrophosphate (GGPP) and was mimicked by an inhibitor of geranylgeranylation. The enhancing effect of atorvastatin was restricted to TNF-alpha-inducible adhesion molecule and was the reflect of an increased protein synthesis (mRNA and protein) and not of a reduced shedding. Confocal microscopy examination showed that atorvastatin also altered the surface distribution of adhesion molecules. Adhesion of human THP-1 cells on TNF-alpha-activated HUVEC was significantly reduced by atorvastatin (-42% at 1 microM). Mevalonate or GGPP restored the TNF-alpha-induced adhesive potential. These results show that atorvastatin, by inhibiting prenylation of G proteins, enhances the TNF-alpha-induced expression of adhesion molecules at the endothelial cell surface and also alters their surface distribution which may account for the reduced binding of monocytes.