Receptor Kinase Activity Research Articles

Probing Plant Receptor Kinase Functions with Labeled Ligands.

Plant cells rely on numerous membrane-localized receptor kinases for communication with each other as well as with the environment to ensure coordinated growth, development and survival. Additionally, several families of small secreted peptides have been identified in plants that possibly regulate various developmental processes through activation of specific membrane receptor kinases. In research efforts to elucidate the underlying signaling processes in plants, it has become obvious that knowledge of both the extracellular ligand and its corresponding receptor(s) is a prerequisite to understand cell communication. Of more than 1,000 orphan peptides in plants, only a few have been functionally characterized and paired with their respective receptors. Here, we discuss recent advances made in the field of receptor-ligand pairing and visualization in plant cells by means of chemically labeled ligands. We summarize and compare various chemical strategies from the mammalian field that can be employed to extend our understanding of the plant receptor kinase functions in plants.

A role for two-component signaling elements in the Arabidopsis growth recovery response to ethylene.

Previous studies indicate that the ability of Arabidopsis seedlings to recover normal growth following an ethylene treatment involves histidine kinase activity of the ethylene receptors. As histidine kinases can function as inputs for a two‐component signaling system, we examined loss‐of‐function mutants involving two‐component signaling elements. We find that mutants of phosphotransfer proteins and type‐B response regulators exhibit a defect in their ethylene growth recovery response similar to that found with the loss‐of‐function ethylene receptor mutant etr1‐7. The ability of two‐component signaling elements to regulate the growth recovery response to ethylene functions independently from their well‐characterized role in cytokinin signaling, based on the analysis of cytokinin receptor mutants as well as following chemical inhibition of cytokinin biosynthesis. Histidine kinase activity of the receptor ETR1 also facilitates growth recovery in the ethylene hypersensitive response, which is characterized by a transient decrease in growth rate when seedlings are treated continuously with a low dose of ethylene; however, this response was found to operate independently of the type‐B response regulators. These results indicate that histidine kinase activity of the ethylene receptor ETR1 performs two independent functions: (a) regulating the growth recovery to ethylene through a two‐component signaling system involving phosphotransfer proteins and type‐B response regulators and (b) regulating the hypersensitive response to ethylene in a type‐B response regulator independent manner.

ScFvs as Allosteric Inhibitors of VEGFR-2: Novel Tools to Harness VEGF Signaling.

Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) is the main mediator of angiogenic signaling in endothelial cells and a primary responder to VEGF. VEGF dependent VEGFR-2 activation regulates endothelial cell migration and proliferation, as well as vessel permeability. VEGF is presented as an antiparallel homodimer, and its binding to VEGFR-2 brings two receptors in close proximity. Downstream signaling is triggered by receptor dimerization, kinase activation, and receptor internalization. Our aim was to further investigate allosteric inhibition using binders targeting extracellular subdomains 4–7 of VEGFR-2 as an alternative to existing anti-angiogenic therapies, which rely on neutralizing VEGF or blocking of the ligand-binding site on the receptor. We applied phage display technology to produce single chain antibody fragments (scFvs) targeting VEGFR-2. Selected antibody fragments were characterized using biophysical and biological assays. We characterized several antibody fragments, which exert their inhibitory effect of VEGFR-2 independent of ligand binding. These reagents led to rapid clearance of VEGFR-2 from the cell surface without kinase activation, followed by an increase in intracellular receptor-positive vesicles, suggesting receptor internalization. Our highly specific VEGFR-2 binders thus represent novel tools for anti-angiogenic therapy and diagnostic applications.

Investigating Phosphorylation Differences in FFA4 Short and Long Isoforms

The G protein‐coupled receptor Free Fatty Acid Receptor 4 (FFA4) is an attractive drug target due to its profound influence on a variety of endocrine, inflammatory, and metabolic processes. Humans contain a unique long splice variant of FFA4, which contains an additional 16 amino acid insert within the 3rd intracellular loop, and has not been found to be expressed in any other mammals. Although the shorter FFA4 isoform (FFA4‐S) is ubiquitously expressed, human FFA4‐L has only been found in colorectal tissue. More interestingly, although both isoforms are agonized by the same ligands, upon agonism, FFA4‐L has been demonstrated to possess an intrinsic bias towards β‐arrestin signaling rather than coupling to G proteins. Since it is known that receptor phosphorylation is required for interaction with β‐arrestin, the purpose of the current study was to determine whether any differences exist between the long and short isoform of FFA4 in terms of basal, homologous, or heterologous phosphorylation.MethodsWhole cell phosphorylation assays using 32P‐labeled orthophosphate were performed in HEK293 cells transiently expressing FFA4 isoforms or mutants. Inquiries into G‐protein receptor kinase (GRK) activity were performed using small interfering RNA (siRNA) followed by whole cell phosphorylation assay. Inquiries into protein kinase A (PKA) or protein kinase C (PKC) activity utilized the adenylyl cyclase activator forskolin to induce PKA activity, PKA inhibitor H‐89, PKC activator phorbol‐myristol acetate (PMA), and PKC inhibitor BIMII. Arrestin recruitment assays were conducted using yellow fluorescence protein (YFP) tagged arrestin‐3 and visualized via confocal microscopy. Truncated mutants were generated by sequential deletions via overlap primers and PCR mutagenesis.ResultsSimilar to our previously reported results with FFA4‐S, homologous phosphorylation of FFA4‐L is mediated by GRK6, whereas heterologous phosphorylation is mediated by PKC. FFA4‐L has lower basal phosphorylation compared to FFA4‐S. Truncation of FFA4‐L at S356 fully inhibits receptor phosphorylation and abrogates arrestin recruitment, matching reported observations for FFA4‐S at corresponding S340.ConclusionThe same kinases are responsible for homologous and heterologous phosphorylation of FFA4‐S and FFA4‐L, while basal phosphorylation of FFA4‐L remains lower compared to FFA4‐S. The arrestin sensor for both isoforms is located on the C‐terminal tail of the receptor following S340 (FFA4‐S) or S356 (FFA4‐L).Support or Funding InformationNIH/NIDDK DK098730 to NHM and Diabetes Action Research and Education Foundation grants to NHMThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of germinated brown rice on the LPS‐induced inflammation in adipocytes

Germinated brown rice (GBR, Orysa sartiva L.) has been reported to have anti‐obesity and anti‐inflammatory effects. However, the mechanisms underlying the anti‐inflammatory effect of GBR on adipocytes are not fully understood. The aim of this study was to explore the anti‐inflammatory mechanisms of GBR on lipopolysaccharide (LPS)‐stimulated 3T3‐L1 adipocytes. 3T3‐L1 adipocytes were pretreated with GBR extracts (0–20 mg/ml) 1h before LPS stimulation. The mRNA expressions of adipokines and Toll‐like receptor 4 (TLR4) were measured by RT‐PCR. The protein expressions of TLR4‐related molecules were detected by western blotting and nuclear factor‐κB (NF‐κB) activation was measured. Our results showed that GBR extract dose‐dependently inhibited mRNA expressions of LPS‐induced tumor necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), and monocyte chemoattractant protein‐1 (MCP‐1). GBR extract was found to inhibit LPS‐induced mRNA expression of TLR4 and protein expressions of both myeloid differentiation factor 88 (MyD88) and TNF receptor‐associated factor 6 (TRAF6). Furthermore, GBR extract significantly inhibited extracellular receptor‐activated kinase (ERK) phosphorylation and NF‐κB activation. These results suggest that GBR extract has the anti‐inflammatory property on LPS‐induced inflammation via inhibiting TLR4 signaling including ERK and NF‐κB signaling pathways in adipocytes. (This research was supported by the Soonchunhyang University Research Fund.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanistic basis for the activation of plant membrane receptor kinases by SERK-family coreceptors

Plant-unique membrane receptor kinases with leucine-rich repeat ectodomains (LRR-RKs) can sense small molecule, peptide, and protein ligands. Many LRR-RKs require SERK-family coreceptor kinases for high-affinity ligand binding and receptor activation. How one coreceptor can contribute to the specific binding of distinct ligands and activation of different LRR-RKs is poorly understood. Here we quantitatively analyze the contribution of SERK3 to ligand binding and activation of the brassinosteroid receptor BRI1 and the peptide hormone receptor HAESA. We show that while the isolated receptors sense their respective ligands with drastically different binding affinities, the SERK3 ectodomain binds the ligand-associated receptors with very similar binding kinetics. We identify residues in the SERK3 N-terminal capping domain, which allow for selective steroid and peptide hormone recognition. In contrast, residues in the SERK3 LRR core form a second, constitutive receptor-coreceptor interface. Genetic analyses of protein chimera between BRI1 and SERK3 define that signaling-competent complexes are formed by receptor-coreceptor heteromerization in planta. A functional BRI1-HAESA chimera suggests that the receptor activation mechanism is conserved among different LRR-RKs, and that their signaling specificity is encoded in the kinase domain of the receptor. Our work pinpoints the relative contributions of receptor, ligand, and coreceptor to the formation and activation of SERK-dependent LRR-RK signaling complexes regulating plant growth and development.

The effect of growth hormone on bioactive IGF in overweight/obese women

ObjectiveOverweight/obesity is characterized by decreased growth hormone (GH) secretion whereas circulating IGF-I levels are less severely reduced. Yet, the activity of the circulating IGF-system appears to be normal in overweight/obese subjects, as estimated by the ability of serum to activate the IGF-I receptor in vitro (bioactive IGF). We hypothesized that preservation of bioactive IGF in overweight/obese women is regulated by an insulin-mediated suppression of IGF-binding protein-1 (IGFBP-1) and IGFBP-2, and by suppression of IGFBP-3, mediated by low GH. We additionally hypothesized that increases in bioactive IGF would drive changes in body composition with low-dose GH administration. DesignCross-sectional analysis and 3-month interim analysis of a 6-month randomized, placebo-controlled study of GH administration in 50 overweight/obese women without diabetes mellitus. Bioactive IGF (kinase receptor activation assay) and body composition (DXA) were measured. ResultsPrior to treatment, IGFBP-3 (r = −0.33, p = 0.02), but neither IGFBP-1 nor IGFBP-2, associated inversely with bioactive IGF. In multivariate analysis, lower IGFBP-3 correlated with lower peak stimulated GH (r = 0.45, p = 0.05) and higher insulin sensitivity (r = −0.74, p = 0.003). GH administration resulted in an increase in mean serum IGF-I concentrations (144 ± 56 to 269 ± 66 μg/L, p < 0.0001) and bioactive IGF (1.29 ± 0.39 to 2.60 ± 1.12 μg/L, p < 0.0001). The treatment-related increase in bioactive IGF, but not total IGF-I concentration, predicted an increase in lean mass (r = 0.31, p = 0.03) and decrease in total adipose tissue/BMI (r = −0.43, p = 0.003). ConclusionsOur data suggest that in overweight/obesity, insulin sensitivity and GH have opposing effects on IGF bioactivity through effects on IGFBP-3. Furthermore, increases in bioactive IGF, rather than IGF-I concentration, predicted GH administration-related body composition changes.Clinical Trial Registration Number: NCT00131378.

Partial TrkB receptor activation suppresses cortical epileptogenesis through actions on parvalbumin interneurons

Post-traumatic epilepsy is one of the most common and difficult to treat forms of acquired epilepsy worldwide. Currently, there is no effective way to prevent post-traumatic epileptogenesis. It is known that abnormalities of interneurons, particularly parvalbumin-containing interneurons, play a critical role in epileptogenesis following traumatic brain injury. Thus, enhancing the function of existing parvalbumin interneurons might provide a logical therapeutic approach to prevention of post-traumatic epilepsy. The known positive effects of brain-derived neurotrophic factor on interneuronal growth and function through activation of its receptor tropomyosin receptor kinase B, and its decrease after traumatic brain injury, led us to hypothesize that enhancing trophic support might improve parvalbumin interneuronal function and decrease epileptogenesis. To test this hypothesis, we used the partial neocortical isolation (‘undercut’, UC) model of posttraumatic epileptogenesis in mature rats that were treated for 2 weeks, beginning on the day of injury, with LM22A-4, a newly designed partial agonist at the tropomyosin receptor kinase B. Effects of treatment were assessed with Western blots to measure pAKT/AKT; immunocytochemistry and whole cell patch clamp recordings to examine functional and structural properties of GABAergic interneurons; field potential recordings of epileptiform discharges in vitro; and video-EEG recordings of PTZ-induced seizures in vivo. Results showed that LM22A-4 treatment 1) increased pyramidal cell perisomatic immunoreactivity for VGAT, GAD65 and parvalbumin; 2) increased the density of close appositions of VGAT/gephyrin immunoreactive puncta (putative inhibitory synapses) on pyramidal cell somata; 3) increased the frequency of mIPSCs in pyramidal cells; and 4) decreased the incidence of spontaneous and evoked epileptiform discharges in vitro. 5) Treatment of rats with PTX BD4–3, another partial TrkB receptor agonist, reduced the incidence of bicuculline-induced ictal episodes in vitro and PTZ induced electrographic and behavioral ictal episodes in vivo. 6) Inactivation of TrkB receptors in undercut TrkBF616A mice with 1NMPP1 abolished both LM22A-4-induced effects on mIPSCs and on increased perisomatic VGAT-IR. Results indicate that chronic activation of the tropomyosin receptor kinase B by a partial agonist after cortical injury can enhance structural and functional measures of GABAergic inhibition and suppress posttraumatic epileptogenesis. Although the full agonist effects of brain-derived neurotrophic factor and tropomyosin receptor kinase B activation in epilepsy models have been controversial, the present results indicate that such trophic activation by a partial agonist may potentially serve as an effective therapeutic option for prophylactic treatment of posttraumatic epileptogenesis, and treatment of other neurological and psychiatric disorders whose pathogenesis involves impaired parvalbumin interneuronal function.

Antitumor Activity of Entrectinib, a Pan-TRK, ROS1, and ALK Inhibitor, in ETV6-NTRK3-Positive Acute Myeloid Leukemia.

Activation of tropomyosin receptor kinase (TRK) family tyrosine kinases by chromosomal rearrangement has been shown to drive a wide range of solid tumors and hematologic malignancies. TRK fusions are actionable targets as evidenced by recent clinical trial results in solid tumors. Entrectinib (RXDX-101) is an investigational, orally available, CNS-active, highly potent, and selective kinase inhibitor against TRKA/B/C, ROS1, and ALK kinase activities. Here, we demonstrate that TRK kinase inhibition by entrectinib selectively targets preclinical models of TRK fusion-driven hematologic malignancies. In acute myelogenous leukemia (AML) cell lines with endogenous expression of the ETV6-NTRK3 fusion gene, entrectinib treatment blocked cell proliferation and induced apoptotic cell death in vitro with subnanomolar IC50 values. Phosphorylation of the ETV6-TRKC fusion protein and its downstream signaling effectors was inhibited by entrectinib treatment in a dose-dependent manner. In animal models, entrectinib treatment at clinically relevant doses resulted in tumor regression that was accompanied by elimination of residual cancer cells from the bone marrow. Our preclinical data demonstrate the potential of entrectinib as an effective treatment for patients with TRK fusion-driven AML and other hematologic malignancies. Mol Cancer Ther; 17(2); 455-63. ©2017 AACR.

Neurotensin speeds inhibition of dopamine neurons through temporal modulation of GABAA and GABAB receptor-mediated synaptic input

Midbrain dopamine neurons play physiological roles in many processes including reward learning and motivated behavior, and are tonically inhibited by γ-aminobutyric acid (GABA)ergic input from multiple brain regions. Neurotensin (NT) is a neuropeptide which acutely modulates midbrain dopamine neuron excitability through multiple mechanisms, one of which is a decrease of GABA-mediated inhibition. However, the mechanisms through which NT depresses GABA signaling are not known. Here we used whole cell patch-clamp electrophysiology of dopamine neurons in mouse brain slices to show that NT acts both presynaptically to increase GABAA and postsynaptically to decrease GABAB receptor-mediated currents in the substantia nigra. The active peptide fragment NT8-13 enhanced GABAA signaling presynaptically by causing an increase in the size of the readily releasable pool of GABA via activation of the NT type-1 receptor and protein kinase A. Conversely, NT8-13 depressed GABAB signaling postsynaptically via the NT type-2 receptor in a process that was modulated by protein kinase C. Both forms of plasticity could be observed simultaneously in single dopamine neurons. Thus, as the kinetics of GABAA signaling are significantly faster than those of GABAB signaling, NT functionally speeds GABAergic input to midbrain dopamine neurons. This finding contributes to our understanding of how neuropeptide-induced plasticity can simultaneously differentiate and integrate signaling by a single neurotransmitter in a single cell and provides a basis for understanding how neuropeptides use temporal shifts in synaptic strength to encode information.

Structure based design and anti-breast cancer evaluation of some novel 4-anilinoquinazoline derivatives as potential epidermal growth factor receptor inhibitors

Quinazoline is one of the most widespread scaffolds amongst natural and synthetic bioactive compounds. Recently the quinazoline derivatives and in particular the 4-anilinoquinazolines have attracted much attention for their anticancer properties due to their capability to stabilize the kinase activity of epidermal growth factor receptor (EGFR). A series of fifteen previously designed and synthesized 4-anilinoquinazoline analogs (4-18) were evaluated for cytotoxic activity on two breast cancer cell lines (MCF-7 and MDA-MB-468). Ligand efficiency and binding mode studies were also done and evaluated for their potentially EGFR inhibitory effects in comparison with imatinib and erlotinib as reference drugs. Among the tested 4-anilinoquinazolines, compound 11, which contains diethoxy at phenyl ring and morpholino pendants at positions 5 and 7 of the quinazoline ring, demonstrated the most potent biological activity on both cell lines. Our new quinazoline derivatives with different substituents such as cyclic or linear ethers and flour groups may be a promising cytotoxic lead compounds for further anti-breast cancer research.

The Human Egg's Zona Pellucida.

Human zona pellucida (ZP) matrix, a delicate network of thin interconnected filaments, is primarily composed of four glycoproteins, namely, ZP1, ZP2, ZP3, and ZP4. All four zona proteins share common structural elements such as signal peptide, "ZP domain," consensus furin cleavage site, transmembrane-like domain, and short cytoplasmic tail. In addition, ZP1 and ZP4 also have "Trefoil domain." Recombinant/native human zona proteins have been used to investigate their binding characteristics to the capacitated and/or acrosome-reacted spermatozoa. These investigations revealed that ZP1, ZP3, and ZP4 primarily bind to the head region of the capacitated human spermatozoa, whereas ZP2 binds to the acrosome-reacted sperm. However, using transgenic mice, N-terminal region of human ZP2 has also been shown to play an important role in binding of sperm to the egg. ZP1, ZP3, and ZP4 lead to dose-dependent increase in acrosome reaction, suggesting that in humans more than one ZP glycoprotein is responsible for induction of acrosome reaction. Glycosylation of these proteins, in particular, N-linked glycosylation as well as sialyl-Lewisx, is essential for inducing acrosome reaction. Studies delineating downstream signaling events associated with induction of acrosome reaction reveal subtle differences between ZP3 and ZP1/ZP4 with respect to activation of Gi protein-coupled receptor and protein kinase A. The role of mutations in the zona proteins and ZP autoantibodies leading to infertility in women is suggestive and needs more rigorous experimentations for confirming their role in female infertility. The above-mentioned aspects of the human ZP glycoproteins have been discussed in this review.

A new promising quinazoline-derived Pan-KIT mutant inhibitor for the gastrointestinal stromal tumors (GIST) management

Gastrointestinalstromal tumors (GISTs) are the most common mesenchymal tumors commonly locatedin stomach (60%) and small intestine (25%), but they can also be found in otherparts of the gastrointestinal tract (1). GISTs are characterized by hotspotmutations in KIT and PDGFRA oncogenes. These oncogenes codifytype III receptor tyrosine kinase which, after binding to the ligand [stem cellfactor (SCF) and platelet-derived growth factor (PDGF), respectively], resultsin receptor homodimerization and kinase activation (2,3). The presence ofactivating mutations of KIT and PDGFRA causes ligand-independentkinase and downstream signaling pathways activation, including MAPK, PI3k/AKT,and STAT3 pathways (1,4).

Piperlongumine decreases cell proliferation and the expression of cell cycle-associated proteins by inhibiting Akt pathway in human lung cancer cells

Piperlongumine (PL) is an alkaloid of a pepper plant found in Southeast Asia. PL is known to induce selective toxicity towards a variety of cancer cell types. To explore the possible anti-lung cancer effects of PL, A549 cells were treated with PL (0–40 μM) for 24 h. Alterations in the expression of cell cycle-associated proteins (cyclin D1, cyclin-dependent kinase 4 (CDK4), CDK6 and retinoblastoma (Rb)) and intracellular signaling molecules (extracellular signal receptor-activated kinase 1/2 (ERK1/2), Akt, p38 and nuclear factor-κB (NF-κB)) were examined in cells following treatment of PL using Western blot analysis. Results showed that proliferation of cells were significantly decreased by PL in a dose-dependent manner. Flow cytometry results demonstrated increased number of cells in G1 phase in PL (40 μM)-treated group. Reactive oxygen species was significantly increased in cells treated with PL at 20–40 μM. The expression of cyclin D1, CDK4, CDK6 and p-Rb were markedly decreased in cells treated with PL at 40 μM. Treatment of cells with PL suppressed phosphorylation of Akt but increased ERK1/2 phosphorylation. Treatment of PL significantly decreased nuclear translocation of NF-κB p65 in cells. These results suggest that PL possesses antiproliferative properties in A549 cells.

Oxidative stress-induced inflammatory responses and effects of N-acetylcysteine in bovine mammary alveolar cells.

Bovine mastitis, an inflammation of the udder, results in reduced milk production and poor milk quality. Mastitis is usually, but not always, a response to pathogen infection. High milk yield can produce oxidative stress in the mammary tissue. High milk yield is also known to be associated with bovine mastitis. Thus, in the current study, we hypothesised that oxidative stress increases inflammatory responses in bovine mammary cells. To examine the hypothesis, we produced cellular oxidative stress and investigated resulting inflammatory responses in bovine mammary alveolar cells (MAC-T). To produce oxidative stress, cells were treated with the reactive oxygen species (ROS; e.g., superoxide anion)-producing agent, menadione (MD; 0-10 µm; 6 h). To ensure the ROS-induced responses, cells were pretreated with an antioxidant NAC (0-10 mm; 1 h). Results showed that MD elevated intracellular ROS levels and protein expression of cyclooxygenase-2 (COX-2), a biomarker of inflammation. Pretreatment of cells with NAC attenuated MD-induced COX-2 expression by scavenging intracellular ROS and enhancing intracellular glutathione levels. MD-induced COX-2 expression was mediated by activation of extracellular signal receptor-activated kinase 1/2 (ERK1/2), Akt, and nuclear factor-kappa B (NF-κB). NAC attenuated activation of these intracellular signalling molecules. Treatment of cells with pharmacological inhibitors for ERK1/2, Akt, and NF-κB confirmed the association of these signalling pathways in MD-induced COX-2 expression. These results support our hypothesis that oxidative stress, which is found in high-yielding dairy cows, can produce cellular inflammation in bovine mammary alveolar cells and prevention of oxidative stress can attenuate such pathological responses. This may be relevant for cases of clinical mastitis for which no pathogen can be isolated.

Design and synthesis of small molecule agonists of EphA2 receptor

Ligand-independent activation of EphA2 receptor kinase promotes cancer metastasis and invasion. Activating EphA2 receptor tyrosine kinase with small molecule agonist is a novel strategy to treat EphA2 overexpressing cancer. In this study, we performed a lead optimization of a small molecule Doxazosin that was identified as an EphA2 receptor agonist. 33 new analogs were developed and evaluated; a structure−activity relationship was summarized based on the EphA2 activation of these derivatives. Two new derivative compounds 24 and 27 showed much improved activity compared to Doxazosin. Compound 24 possesses a bulky amide moiety, and compound 27 has a dimeric structure that is very different to the parental compound. Compound 27 with a twelve-carbon linker of the dimer activated the kinase and induced receptor internalization and cell death with the best potency. Another dimer with a six-carbon linker has significantly reduced potency compared to the dimer with a longer linker, suggesting that the length of the linker is critical for the activity of the dimeric agonist. To explore the receptor binding characteristics of the new molecules, we applied a docking study to examine how the small molecule binds to the EphA2 receptor. The results reveal that compounds 24 and 27 form more hydrogen bonds to EphA2 than Doxazosin, suggesting that they may have higher binding affinity to the receptor.

Identification of key pathways and genes with aberrant methylation in prostate cancer using bioinformatics analysis.

Prostate cancer (PCa), a multifocal clinically heterogeneous disease, is the most commonly diagnosed non-cutaneous neoplasm in men worldwide. The epigenome of PCa is a typical representation of catastrophic model of epigenetic alterations during tumorigenesis and its progression. Alterations in methylation patterns in tumor suppressors, cell cycle, oncogenes and metabolism-related genes are the most commonly observed epigenetic alterations in PCa. In this study, we have developed a computational strategy to identify methylated biomarker signature panels as potential targets of PCa by screening >160 genes reported to be epigenetically dysregulated, and shortlisted 26 differentially methylated genes (DMGs) that significantly contribute to oncogenesis. The gene ontology and functional enrichment analysis were performed, which showed that identified DMGs contribute to cellular and metabolic processes such as cell communication, cell cycle, response to drugs, apoptosis and p53 signaling. The top hub genes AR, CDH13, CDKN2A, DAPK1, GSTP1, CD44 and RASSF1 identified from protein–protein interaction network construction using Search Tool for the Retrieval of Interacting Genes contributed to hormonal response, inflammatory response, cell cycle, reactive oxygen species activity and receptor kinase activity, which are related to hallmarks of cancer as revealed by their functional enrichment analysis by Cytoscape. These genes were further scrutinized for CpG islands, transcription start sites and positions of methylated cytosines to study their methylation profiles. Our analysis revealed high negative correlation values between methylation frequencies and gene expressions of the hub genes, namely, AR, CDH13, CDKN2A, DAPK1, CD44, GSTP1 and RASSF1, which can be used as potential diagnostic biomarkers for PCa.

The Axl kinase domain in complex with a macrocyclic inhibitor offers first structural insights into an active TAM receptor kinase

The receptor tyrosine kinase family consisting of Tyro3, Axl, and Mer (TAM) is one of the most recently identified receptor tyrosine kinase families. TAM receptors are up-regulated postnatally and maintained at high levels in adults. They all play an important role in immunity, but Axl has also been implicated in cancer and therefore is a target in the discovery and development of novel therapeutics. However, of the three members of the TAM family, the Axl kinase domain is the only one that has so far eluded structure determination. To this end, using differential scanning fluorimetry and hydrogen-deuterium exchange mass spectrometry, we show here that a lower stability and greater dynamic nature of the Axl kinase domain may account for its poor crystallizability. We present the first structural characterization of the Axl kinase domain in complex with a small-molecule macrocyclic inhibitor. The Axl crystal structure revealed two distinct conformational states of the enzyme, providing a first glimpse of what an active TAM receptor kinase may look like and suggesting a potential role for the juxtamembrane region in enzyme activity. We noted that the ATP/inhibitor-binding sites of the TAM members closely resemble each other, posing a challenge for the design of a selective inhibitor. We propose that the differences in the conformational dynamics among the TAM family members could potentially be exploited to achieve inhibitor selectivity for targeted receptors.

Transcriptome Profiling of the Shoot and Root Tips of S562L, a Soybean GmCLAVATA1A Mutant

Plant shoot apical meristems (SAM) and root apical meristems (RAM) contain stem cells that form overall-plant architecture. Mechanisms acting in these regions keep a balance between the stem cell population and differentiation. These mechanisms are well-studied in Arabidopsis, but little is known in the legume soybean (Glycine max (L.) Merr.). In Arabidopsis, the Leucine-rich repeat (LRR) Receptor kinase CLAVATA1 (CLV1) is a crucial regulator of this process in the SAM. In soybean, the receptor most similar to ATCLV1 is GmNARK, which is involved in nodulation control. In contrast, the homeologous partner of GmNARK in soybean, called GmCLV1A, appears to have no function in ‘Autoregulation of nodulation’ (AON) a role in regulating shoot architecture in the SAM. Here, the transcriptome of the shoot and root tip areas of a chemically induced and TILLING-selected GmCLV1A missense mutant, S562L, and its wild type, cultivar Forrest, were analysed to identify genes which are affected by impaired function of GmCLV1A. Among the differentially expressed genes identified, many were categorised as having a role in receptor kinase activity, transcription or defense/stress-response. Molecular categories over-represented in the shoot tip of the mutant include those involved in hormone biosynthesis/activity and secondary metabolism, signalling, photosynthesis, and transport. Functional categories including those involved in polyamine metabolism, nucleotide metabolism, RNA regulation, protein targeting and protein degradation were under-represented in the shoot tip of the mutant. In the root tip, categories associated with signal ling, transport, protein synthesis and metabolism were over-represented in the mutant, while categories associated with cell wall degradation, stress, RNA regulation, protein degradation and targeting were under-represented in the mutant. Factors similar to Arabidopsis regulatory components are most likely functioning in specialised shoot structures in legumes. Furthermore, GmCLV1A may have an unexpected role in the regulation of flavonoid biosynthesis in soybean.

Ado-trastuzumab emtansine in patients with HER2 mutant lung cancers: Results from a phase II basket trial.

8510 Background: Human epidermal growth factor receptor 2 ( HER2, ERBB2) mutations occur in 2% of lung cancers, resulting in receptor dimerization and kinase activation with in vitro sensitivity to trastuzumab. Ado-trastuzumab emtansine is a HER2 targeted antibody drug conjugate linking trastuzumab with the anti-microtubule agent emtansine. Methods: Patients (pts) with HER2 mutant lung cancers were enrolled into a cohort of the basket trial of ado-trastuzumab emtansine in HER2amplified or mutant cancers, treated at 3.6mg/kg IV every 3 weeks. The primary endpoint was overall response rate (ORR) using RECIST v1.1. A Simon two stage optimal design was used with type I error rate under 2.7% (and a family wise error rate across baskets under 10%), power of 89%, H0 10%, H1 40%; the H0 will be rejected if 5 or more responses are observed in 18 pts. Other endpoints include duration of response (DOR), progression-free survival (PFS) and toxicity. HER2 testing was performed on tumor tissue by next generation sequencing (NGS), fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Results: The cohort completed accrual with 18 pts treated. The median age was 63 (range 47-74 years), 72% were female, 39% were never smokers and all had adenocarcinomas. The median lines of prior systemic therapy was 2 (range 0-4). ORR was 33% (5/15 confirmed, 95% CI 12-62%) not including a partial response awaiting confirmation and 3 pts pending response evaluation. Median DOR was not reached (range 3 to 7+ mo), median PFS was 4mo (95% CI 3mo-not reached). Toxicities were mainly grade 1 or 2 including infusion reaction, thrombocytopenia and transaminitis, there were no dose reductions or treatment related deaths. There were 10 (56%) exon 20 insertions and 8 (44%) point mutations; responders were seen across mutation subtypes (A775_G776insYVMA, G776delinsVC, V659E, S310F). HER2amplification was negative for all pts by NGS and positive for 1 of 12 pts by FISH. There was no IHC3+ in 10 pts tested. Conclusions: Ado-trastuzumab emtansine is active and well tolerated in pts with HER2 mutant lung cancers. This study has met its primary endpoint. Further development in a multicenter study is warranted. Clinical trial information: NCT02675829.