Report of the Committee on Resolutions- Declaration

Pamphlet concerning a declaration made by the National Educational Association at the forty-fourth annual convention

TBK1 Kinase Addiction in Lung Cancer Cells Is Mediated via Autophagy of Tax1bp1/Ndp52 and Non-Canonical NF-kappa B Signalling

K-Ras dependent non-small cell lung cancer (NSCLC) cells are 'addicted' to basal autophagy that reprograms cellular metabolism in a lysosomal-sensitive manner. Here we demonstrate that the xenophagy-associated kinase TBK1 drives basal autophagy, consistent with its known requirement in K-Ras-dependent NSCLC proliferation. Furthermore, basal autophagy in this context is characterised by sequestration of the xenophagy cargo receptor Ndp52 and its paralogue Tax1bp1, which we demonstrate here to be a bona fide cargo receptor. Autophagy of these cargo receptors promotes non-canonical NF-κB signalling. We propose that this TBK1-dependent mechanism for NF-κB signalling contributes to autophagy addiction in K-Ras driven NSCLC

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96 p.; 21 cm

Novel and potent GPR35 agonists aid identification of residues important in ligand interaction

The orphan G protein coupled receptor GPR35 has emerged as a therapeutic target in a number of disease states including metabolic disorder, cardiovascular disease, inflammation and nociception. Efforts to characterize this receptor, however, have been complicated by marked differences in the pharmacology of GPR35 species orthologs. We have identified novel agonist compounds with which to dissect the pharmacology and function of GPR35. Selected compounds were chosen to illustrate the differences in ligand structure activity relationship between human, mouse and rat orthologs of GPR35. Sequence alignment and receptor homology modeling techniques guided site directed mutagenesis, which highlighted a number of residues involved in GPR35 ligand interaction and revealed differences between rodent and human orthologs. These findings will benefit future drug development programs and efforts to generate therapeutically relevant GPR35-specific pharmacological compounds

Novel cross-talk within the IKK family controls innate immunity

International audienceMembers of the IκB kinase (IKK) family play a central role in innate immunity by inducing NFκB- and IRF-dependent gene transcription programmes required for the production of pro-inflammatory cytokines and interferons. However, the molecular mechanisms that activate these protein kinases and their complement of physiological substrates remain poorly defined. Using MRT67307, a novel inhibitor of IKKε/TBK1 and BI605906, a novel inhibitor of IKKβ, we demonstrate that two different signalling pathways participate in the activation of the IKK-related protein kinases by ligands that activate the IL-1, TLR3 and TLR4 receptors. One signalling pathway is mediated by the canonical IKKs, which directly phosphorylate and activate IKKε and TBK1, whereas the second pathway appears to culminate in the autocatalytic activation of the IKK-related kinases. In contrast, the TNFα-induced activation of the IKK-related kinases is mediated solely by the canonical IKKs. In turn, the IKK-related kinases phosphorylate the catalytic subunits of the canonical IKKs and their regulatory subunit NEMO, which is associated with reduced IKKα/β activity and NFκB-dependent gene transcription. We also show that the canonical IKKs and the IKK-related kinases not only have unique physiological substrates, such as IκBα, p105 and RelA (IKKα and IKKβ) and IRF3 (IKKε and TBK1), but also have several substrates in common, including the catalytic and regulatory (NEMO and TANK) subunits of the IKKs themselves. Taken together, our studies reveal that the canonical IKKs and the IKK-related kinases regulate each other by an intricate network involving phosphorylation of their catalytic and regulatory (NEMO, TANK) subunits to balance their activities during innate immunity

High-throughput identification and characterization of novel, species-selective GPR35 agonists

Drugs targeting the orphan receptor GPR35 have potential therapeutic application in a number of disease areas, including inflammation, metabolic disorders, nociception, and cardiovascular disease. Currently available surrogate GPR35 agonists identified from pharmacologically relevant compound libraries have limited utility due to the likelihood of off-target effects in vitro and in vivo and the variable potency that such ligands exhibit across species. We sought to identify and characterize novel GPR35 agonists to facilitate studies aimed at defining the physiologic role of GPR35. PathHunter β-arrestin recruitment technology was validated as a human GPR35 screening assay, and a high-throughput screen of 100,000 diverse low molecular weight compounds was conducted. Confirmed GPR35 agonists from five distinct chemotypes were selected for detailed characterization using both β-arrestin recruitment and G protein-dependent assays and each of the human, mouse, and rat GPR35 orthologs. These studies identified 4-{(Z)-[(2Z)-2-(2-fluorobenzylidene)-4-oxo-1,3-thiazolidin-5-ylidene]methyl}benzoic acid (compound 1) as the highest potency full agonist of human GPR35 yet described. As with certain other GPR35 agonists, compound 1 was markedly selective for human GPR35, but displayed elements of signal bias between β-arrestin-2 and G protein-dependent assays. Compound 1 also displayed competitive behavior when assessed against the human GPR35 antagonist, ML-145 (2-hydroxy-4-[4-(5Z)-5-[(E)-2-methyl-3-phenylprop-2-enylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]butanoylamino]benzoic acid). Of the other chemotypes studied, compounds 2 and 3 were selective for the human receptor, but compounds 4 and 5 demonstrated similar activity at human, rat, and mouse GPR35 orthologs. Further characterization of these compounds and related analogs is likely to facilitate a better understanding of GPR35 in health and disease

TBK1, autophagy and the Tax1bp1/Ndp52 cargo receptors maintain pro-survival/proliferation non-canonical NF-κB signalling in A549 NSCLC cells. a–c)

<p>A549 cells were treated overnight with DMSO or 10 µM MRT68601 (TBKi) and a) stained for RelB (scale bar = 50 µm), nuclear localisation quantified in b) (n = 3, ± S.E.M., ** = p<0.01) or c) nuclear extracts prepared and blotted for indicated proteins. <b>d–f)</b> A549 cells were transfected with indicated siRNA (<i>NTC</i> = non-targeting control) for 72 h and e) cell extracts blotted for indicated proteins or d,f) cells were stained and quantified for nuclear RelB (n = 3, ± S.E.M., * = p<0.05). <b>g)</b> A549 cells were treated with PBS or 10 mM 3-methyladenine (3-MA) for 24 hours and then stained and quantified for nuclear RelB (n = 3, ± S.E.M., *** = p<0.005). <b>h,i)</b> A549 cells were infected with indicated lentivirus and, at 72 h post infection, h) cell extracts blotted for indicated proteins or i) total RNA quantified for <i>BIRC3</i> mRNA (n = 3; ± S.D.). <b>j)</b> A549 cells were transfected with indicated siRNA for 72 h and total RNA extracts were subjected to qRT-PCR for <i>BIRC3</i> mRNA (n = 3; ± S.D.). <b>k)</b> A549 cells were infected with indicated lentivirus and at 120 h post infection cell number counted as described in Materials and Methods (n = 3, ± S.E.M., * = p<0.05).</p

TBK1 kinase activity engages basal autophagy in A549 NSCLC cells. a)

<p>A549 GFP-LC3B or GFP-LC3BΔG->A cells were treated with PBS or 5 µM chloroquine (CQ) for 8 h and cells imaged for GFP. <b>b, c)</b> A549 GFP-LC3B cells were transfected with indicated siRNA for 72 h (<i>NTC</i> = non-targeting control) and b) cell extracts blotted for indicated proteins (α-tub = α-tubulin) or cells were c) imaged for GFP-LC3B puncta. <b>d)</b> A549 FLAG-HA-LC3B cells were transfected with indicated siRNA and HA-positive puncta immunostained, imaged and counted at 72 h (n = 3, ± S.E.M., * = p<0.05, ** = p<0.01). <b>e,f)</b> A549 tandem-fluorescent-LC3B (tfLC3B) cells were treated with DMSO or 1 µM MRT68601 (TBKi) for 24 h and e) imaged and f) quantified for total number of autophagic puncta (green+red) and the subset of these puncta with undetectable GFP signal (red), as described in detail in Materials and Methods. <b>g)</b> A549 cells were treated with DMSO or 10 µM MRT68601 (TBKi) for 24 h in the presence or absence of PBS or 5 µM (CQ) chloroquine and cell extracts blotted for indicated proteins. Scale bars = 50 µm in all panels.</p