Protein Kinase CK2α Maintains Extracellular Signal-regulated Kinase (ERK) Activity in a CK2α Kinase-independent Manner to Promote Resistance to Inhibitors of RAF and MEK but Not ERK in BRAF Mutant Melanoma

The protein kinase casein kinase 2 (CK2) is a pleiotropic and constitutively active kinase that plays crucial roles in cellular proliferation and survival. Overexpression of CK2, particularly the α catalytic subunit (CK2α, CSNK2A1), has been implicated in a wide variety of cancers and is associated with poorer survival and resistance to both conventional and targeted anticancer therapies. Here, we found that CK2α protein is elevated in melanoma cell lines compared with normal human melanocytes. We then tested the involvement of CK2α in drug resistance to Food and Drug Administration-approved single agent targeted therapies for melanoma. In BRAF mutant melanoma cells, ectopic CK2α decreased sensitivity to vemurafenib (BRAF inhibitor), dabrafenib (BRAF inhibitor), and trametinib (MEK inhibitor) by a mechanism distinct from that of mutant NRAS. Conversely, knockdown of CK2α sensitized cells to inhibitor treatment. CK2α-mediated RAF-MEK kinase inhibitor resistance was tightly linked to its maintenance of ERK phosphorylation. We found that CK2α post-translationally regulates the ERK-specific phosphatase dual specificity phosphatase 6 (DUSP6) in a kinase dependent-manner, decreasing its abundance. However, we unexpectedly showed, by using a kinase-inactive mutant of CK2α, that RAF-MEK inhibitor resistance did not rely on CK2α kinase catalytic function, and both wild-type and kinase-inactive CK2α maintained ERK phosphorylation upon inhibition of BRAF or MEK. That both wild-type and kinase-inactive CK2α bound equally well to the RAF-MEK-ERK scaffold kinase suppressor of Ras 1 (KSR1) suggested that CK2α increases KSR facilitation of ERK phosphorylation. Accordingly, CK2α did not cause resistance to direct inhibition of ERK by the ERK1/2-selective inhibitor SCH772984. Our findings support a kinase-independent scaffolding function of CK2α that promotes resistance to RAF- and MEK-targeted therapies

Synthesis and Biological Evaluation of New Madecassic Acid Derivatives Targeting ERK Cascade Signaling

In the present study, a series of novel madecassic acid derivatives was synthesized and screened against the National Cancer Institute's 60 human cancer cell line panel. Among them, compounds 5, 12, and 17 displayed potent and highly differential antiproliferative activity against 80% of the tumor cells harboring the B-RafV600E mutation within the nanomolar range. Structure-activity analysis revealed that a 5-membered A ring containing an α,β-unsaturated aldehyde substituted at C-23 with a 2-furoyl group seems to be crucial to produce this particular growth inhibition signature. In silico analysis of the cytotoxicity pattern of these compounds identified two highly correlated clinically approved drugs with known B-RafV600E inhibitory activity. Follow-up analysis revealed inhibition of the ERK signaling pathway through the reduction of cellular Raf protein levels is a key mechanism of action of these compounds. In particular, 17 was the most potent compound in suppressing tumor growth of B-RafV600E-mutant cell lines and displayed the highest reduction of Raf protein levels among the tested compounds. Taken together, this study revealed that modifications of madecassic acid structure can provide molecules with potent anticancer activity against cell lines harboring the clinically relevant B-RafV600E mutation, with compound 17 identified as a promising lead for the development of new anticancer drugs

Fusion dual-tracer SPECT-based hepatic dosimetry predicts outcome after radioembolization for a wide range of tumour cell types

Purpose Fusion dual-tracer SPECT imaging enables physiological rather than morphological voxel-based partitioning and dosimetry for Y-90 hepatic radioembolization (RE). We evaluated its prognostic value in a large heterogeneous cohort of patients with extensive hepatic malignancy. Methods A total of 122 patients with primary or secondary liver malignancy (18 different cell types) underwent SPECT imaging after intraarterial injection of Tc-99m macroaggregated albumin (TcMAA) as a simulation of subsequent Y-90 microsphere distribution, followed by administration of an excess of intravenous Tc-99m-labelled sulphur colloid (TcSC) as a biomarker for functional liver, and a second SPECT scan. TcMAA distribution was used to estimate Y-90 radiation absorbed dose in tumour (D (T)) and in functional liver. Laboratory and clinical follow-up were recorded for 12 weeks after RE, and radiographic responses according to (m)RECIST were evaluated at 3 and 6 months. Dose-response relationships were determined for efficacy and toxicity. Results Patients were treated with a median of 1.73 GBq activity of resin microspheres (98 patients) or glass microspheres (24 patients), in a whole-liver approach (97 patients) or a lobar approach (25 patients). The objective response rate was 41 % at 3 months and 48 % at 6 months. Response was correlated with D (T) (P <0.01). Median overall survival was 10.1 months (95 % confidence interval 7.4 - 12.8 months). Responders lived for 36.0 months compared to 8.7 months for nonresponders (P <0.01). Stratified for tumour cell type, D (T) was independently associated with survival (P <0.01). Absorbed dose in functional liver was correlated with toxicity grade change (P <0.05) and RE-induced liver disease (P <0.05). Conclusion Fusion dual-tracer SPECT imaging offers a physiology-based functional imaging tool to predict efficacy and toxicity of RE. This technique can be refined to define dosing thresholds for specific tumour types and treatments, but appears generally predictive even in a heterogeneous cohort

Fluids and Electrolytes under Confinement in Single-Digit Nanopores

Confined fluids and electrolyte solutions in nanopores exhibit rich and surprising physics and chemistry that impact the mass transport and energy efficiency in many important natural systems and industrial applications. Existing theories often fail to predict the exotic effects observed in the narrowest of such pores, called single-digit nanopores (SDNs), which have diameters or conduit widths of less than 10 nm, and have only recently become accessible for experimental measurements. What SDNs reveal has been surprising, including a rapidly increasing number of examples such as extraordinarily fast water transport, distorted fluid-phase boundaries, strong ion-correlation and quantum effects, and dielectric anomalies that are not observed in larger pores. Exploiting these effects presents myriad opportunities in both basic and applied research that stand to impact a host of new technologies at the water-energy nexus, from new membranes for precise separations and water purification to new gas permeable materials for water electrolyzers and energy-storage devices. SDNs also present unique opportunities to achieve ultrasensitive and selective chemical sensing at the single-ion and single-molecule limit. In this review article, we summarize the progress on nanofluidics of SDNs, with a focus on the confinement effects that arise in these extremely narrow nanopores. The recent development of precision model systems, transformative experimental tools, and multiscale theories that have played enabling roles in advancing this frontier are reviewed. We also identify new knowledge gaps in our understanding of nanofluidic transport and provide an outlook for the future challenges and opportunities at this rapidly advancing frontier

Psychometric Curve and Behavioral Strategies for Whisker-Based Texture Discrimination in Rats

The rodent whisker system is a major model for understanding neural mechanisms for tactile sensation of surface texture (roughness). Rats discriminate surface texture using the whiskers, and several theories exist for how texture information is physically sensed by the long, moveable macrovibrissae and encoded in spiking of neurons in somatosensory cortex. However, evaluating these theories requires a psychometric curve for texture discrimination, which is lacking. Here we trained rats to discriminate rough vs. fine sandpapers and grooved vs. smooth surfaces. Rats intermixed trials at macrovibrissa contact distance (nose >2 mm from surface) with trials at shorter distance (nose <2 mm from surface). Macrovibrissae were required for distant contact trials, while microvibrissae and non-whisker tactile cues were used for short distance trials. A psychometric curve was measured for macrovibrissa-based sandpaper texture discrimination. Rats discriminated rough P150 from smoother P180, P280, and P400 sandpaper (100, 82, 52, and 35 µm mean grit size, respectively). Use of olfactory, visual, and auditory cues was ruled out. This is the highest reported resolution for rodent texture discrimination, and constrains models of neural coding of texture information

The Changing Landscape for Stroke\ua0Prevention in AF: Findings From the GLORIA-AF Registry Phase 2

Background GLORIA-AF (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients with Atrial Fibrillation) is a prospective, global registry program describing antithrombotic treatment patterns in patients with newly diagnosed nonvalvular atrial fibrillation at risk of stroke. Phase 2 began when dabigatran, the first non\u2013vitamin K antagonist oral anticoagulant (NOAC), became available. Objectives This study sought to describe phase 2 baseline data and compare these with the pre-NOAC era collected during phase&nbsp;1. Methods During phase 2, 15,641 consenting patients were enrolled (November 2011 to December 2014); 15,092 were eligible. This pre-specified cross-sectional analysis describes eligible patients\u2019 baseline characteristics. Atrial fibrillation&nbsp;disease characteristics, medical outcomes, and concomitant diseases and medications were collected. Data were analyzed using descriptive statistics. Results Of the total patients, 45.5% were female; median age was 71 (interquartile range: 64, 78) years. Patients were from Europe (47.1%), North America (22.5%), Asia (20.3%), Latin America (6.0%), and the Middle East/Africa (4.0%). Most had high stroke risk (CHA2DS2-VASc [Congestive heart failure, Hypertension, Age&nbsp; 6575 years, Diabetes mellitus, previous Stroke, Vascular disease, Age 65 to 74 years, Sex category] score&nbsp; 652; 86.1%); 13.9% had moderate risk (CHA2DS2-VASc&nbsp;= 1). Overall, 79.9% received oral anticoagulants, of whom 47.6% received NOAC and 32.3% vitamin K antagonists (VKA); 12.1% received antiplatelet agents; 7.8% received no antithrombotic treatment. For comparison, the proportion of phase 1 patients (of N&nbsp;= 1,063 all eligible) prescribed VKA was 32.8%, acetylsalicylic acid 41.7%, and no therapy 20.2%. In Europe in phase 2, treatment with NOAC was more common than VKA (52.3% and 37.8%, respectively); 6.0% of patients received antiplatelet treatment; and 3.8% received no antithrombotic treatment. In North America, 52.1%, 26.2%, and 14.0% of patients received NOAC, VKA, and antiplatelet drugs, respectively; 7.5% received no antithrombotic treatment. NOAC use was less common in Asia (27.7%), where 27.5% of patients received VKA, 25.0% antiplatelet drugs, and 19.8% no antithrombotic treatment. Conclusions The baseline data from GLORIA-AF phase 2 demonstrate that in newly diagnosed nonvalvular atrial fibrillation patients, NOAC have been highly adopted into practice, becoming more frequently prescribed than VKA in&nbsp;Europe and North America. Worldwide, however, a large proportion of patients remain undertreated, particularly in&nbsp;Asia&nbsp;and North America. (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients With Atrial Fibrillation [GLORIA-AF]; NCT01468701

Impact of Feedback Phosphorylation and Raf Heterodimerization on Normal and Mutant B-Raf Signaling▿

The B-Raf kinase is a Ras pathway effector activated by mutation in numerous human cancers and certain developmental disorders. Here we report that normal and oncogenic B-Raf proteins are subject to a regulatory cycle of extracellular signal-regulated kinase (ERK)-dependent feedback phosphorylation, followed by PP2A- and Pin1-dependent dephosphorylation/recycling. We identify four S/TP sites of B-Raf phosphorylated by activated ERK and find that feedback phosphorylation of B-Raf inhibits binding to activated Ras and disrupts heterodimerization with C-Raf, which is dependent on the B-Raf pS729/14-3-3 binding site. Moreover, we find that events influencing Raf heterodimerization can alter the transforming potential of oncogenic B-Raf proteins possessing intermediate or impaired kinase activity but have no significant effect on proteins with high kinase activity, such as V600E B-Raf. Mutation of the feedback sites or overexpression of the Pin1 prolyl-isomerase, which facilitates B-Raf dephosphorylation/recycling, resulted in increased transformation, whereas mutation of the S729/14-3-3 binding site or expression of dominant negative Pin1 reduced transformation. Mutation of each feedback site caused increased transformation and correlated with enhanced heterodimerization and activation of C-Raf. Finally, we find that B-Raf and C-Raf proteins containing mutations identified in certain developmental disorders constitutively heterodimerize and that their signaling activity can also be modulated by feedback phosphorylation

Functional analysis of C-TAK1 substrate binding and identification of PKP2 as a new C-TAK1 substrate

Cdc25C-associated kinase 1 (C-TAK1) has been implicated in cell cycle regulation and Ras signaling through its interactions with two putative substrates, the Cdc25C phosphatase and the MAPK scaffold KSR1. Here, we identify sequence motifs required for stable C-TAK1 association and substrate phosphorylation. Using a mutational approach to disrupt binding of C-TAK1 to KSR1 and Cdc25C, we demonstrate that C-TAK1 contributes to the regulation of these proteins in vivo through the generation of 14-3-3-binding sites. KSR1 proteins defective in C-TAK1 binding had severely reduced phosphorylation at the 14-3-3-binding site in vivo, were constitutively localized to the plasma membrane and had increased biological activity. Disruption of the Cdc25C–C-TAK1 interaction resulted in reduced 14-3-3-binding site phosphorylation and nuclear accumulation of Cdc25C in interphase cells. Finally, utilizing the acquired C-TAK1 binding and substrate phosphorylation data, we identify plakophilin 2 (PKP2) as a novel C-TAK1 substrate. Phosphorylation of PKP2 by C-TAK1 also generates a 14-3-3-binding site that influences PKP2 localization. These findings underscore the importance of C-TAK1 as a regulator of 14-3-3 binding and protein localization

KSR2 is a Calcineurin Substrate that Promotes ERK Cascade Activation in Response to Calcium Signals

Protein scaffolds have emerged as important regulators of MAPK cascades, facilitating kinase activation and providing crucial spatio/temporal control to their signaling outputs. Using a proteomics approach to compare the binding partners of the two mammalian KSR scaffolds, we find that both KSR1 and KSR2 interact with the kinase components of the ERK cascade and have a common function in promoting RTK-mediated ERK signaling. Strikingly, we find that the protein phosphatase calcineurin selectively interacts with KSR2 and that KSR2 uniquely contributes to Ca2+-mediated ERK signaling. Calcineurin dephosphorylates KSR2 on specific sites in response to Ca2+ signals, thus regulating KSR2 localization and activity. Moreover, we find that depletion of endogenous KSR2 impairs Ca2+-mediated ERK activation and ERK-dependent signaling responses in INS1 pancreatic beta-cells and NG108 neuroblastoma cells. These findings identify KSR2 as a Ca2+-regulated ERK scaffold and reveal a new mechanism whereby Ca2+ impacts Ras to ERK pathway signaling

CK2 Is a Component of the KSR1 Scaffold Complex that Contributes to Raf Kinase Activation

Kinase Suppressor of Ras (KSR) is a molecular scaffold that interacts with the core kinase components of the ERK cascade, Raf, MEK, and ERK and provides spatial and temporal regulation of Ras-dependent ERK cascade signaling. In this report, we identify the heterotetrameric protein kinase, casein kinase 2 (CK2), as a new KSR1-binding partner. Moreover, we find that the KSR1/CK2 interaction is required for KSR1 to maximally facilitate ERK cascade signaling and contributes to the regulation of Raf kinase activity. Binding of the CK2 holoenzyme is constitutive and requires the basic surface region of the KSR1 atypical C1 domain. Loss of CK2 binding does not alter the membrane translocation of KSR1 or its interaction with ERK cascade components; however, disruption of the KSR1/CK2 interaction or inhibition of CK2 activity significantly reduces the growth-factor-induced phosphorylation of C-Raf and B-Raf on the activating serine site in the negative-charge regulatory region (N-region). This decrease in Raf N-region phosphorylation further correlates with impaired Raf, MEK, and ERK activation. These findings identify CK2 as a novel component of the KSR1 scaffolding complex that facilitates ERK cascade signaling by functioning as a Raf family N-Region kinase