Non-Receptor Tyrosine Kinases: Their Structure and Mechanistic Role in Tumor Progression and Resistance.

Protein tyrosine kinases (PTKs) function as key molecules in the signaling pathways in addition to their impact as a therapeutic target for the treatment of many human diseases, including cancer. PTKs are characterized by their ability to phosphorylate serine, threonine, or tyrosine residues and can thereby rapidly and reversibly alter the function of their protein substrates in the form of significant changes in protein confirmation and affinity for their interaction with protein partners to drive cellular functions under normal and pathological conditions. PTKs are classified into two groups: one of which represents tyrosine kinases, while the other one includes the members of the serine/threonine kinases. The group of tyrosine kinases is subdivided into subgroups: one of them includes the member of receptor tyrosine kinases (RTKs), while the other subgroup includes the member of non-receptor tyrosine kinases (NRTKs). Both these kinase groups function as an "on" or "off" switch in many cellular functions. NRTKs are enzymes which are overexpressed and activated in many cancer types and regulate variable cellular functions in response to extracellular signaling-dependent mechanisms. NRTK-mediated different cellular functions are regulated by kinase-dependent and kinase-independent mechanisms either in the cytoplasm or in the nucleus. Thus, targeting NRTKs is of great interest to improve the treatment strategy of different tumor types. This review deals with the structure and mechanistic role of NRTKs in tumor progression and resistance and their importance as therapeutic targets in tumor therapy.

Acesulfame potassium triggers inflammatory bowel disease via the inhibition of focal adhesion pathway.

Acesulfame potassium (ACK) was generally regarded as innocuous and extensively ingested. Nevertheless, ACK has recently gained attention as a burgeoning pollutant that has the potential to induce a range of health hazards, particularly to the digestive system. Herein, we uncover that ACK initiates inflammatory bowel disease (IBD) in mice and zebrafish, as indicated by the aggregation of macrophages in the intestine and the inhibition of intestinal mucus secretion. Transcriptome analysis of mice and zebrafish guts revealed that exposure to ACK typically impacts the cell cycle, focal adhesion, and PI3K-Akt signaling pathways. Using pharmacological approaches, we demonstrate that the PI3K-Akt signaling pathway and the generation of reactive oxygen species (ROS) triggered by cell division are not significant factors in the initiation of IBD caused by ACK. Remarkably, inhibition of the focal adhesion pathway is responsible for the IBD onset induced by ACK. Our results indicate the detrimental impacts and possible underlying mechanisms of ACK on the gastrointestinal system and provide insights for making informed choices about everyday dietary habits.

Phosphorylation of Ack1 by the Receptor Tyrosine Kinase Mer.

Ack1 is a nonreceptor tyrosine kinase that is associated with cellular proliferation and survival. The receptor tyrosine kinase Mer, a member of the TAM family of receptors, has previously been reported to be an upstream activator of Ack1 kinase. The mechanism linking the two kinases, however, has not been investigated. We confirmed that Ack1 and Mer interact by co-immunoprecipitation experiments and found that Mer expression led to increased Ack1 activity. The effect on Ack1 was dependent on the kinase activity of Mer, whereas mutation of the Mer C-terminal tyrosines Y867 and Y924 did not significantly decrease the ability of Mer to activate Ack1. Ack1 possesses a Mig6 Homology Region (MHR) that contains adjacent regulatory tyrosines (Y859 and Y860). Using synthetic peptides, we showed that Mer preferentially binds and phosphorylates the MHR sequence containing phosphorylated pY860, as compared to the pY859 sequence. This suggested the possibility of sequential phosphorylation within the MHR of Ack1, as has been observed previously for other kinases. In cells co-expressing Mer and Ack1 MHR mutants, the Y859F mutant had higher activity than the Y860F mutant, consistent with this model. The interaction between Mer and Ack1 could play a role in immune cell signaling in normal physiology and could also contribute to the hyperactivation of Ack1 in prostate cancer and other tumors.

Dual Anta-Inhibitors Targeting Protein Kinase CK1δ and A2A Adenosine Receptor Useful in Neurodegenerative Disorders.

Currently, the number of patients with neurodegenerative pathologies is estimated at over one million, with consequences also on the economic level. Several factors contribute to their development, including overexpression of A2A adenosine receptors (A2AAR) in microglial cells and up-regulation and post-translational alterations of some casein kinases (CK), among them, CK-1δ. The aim of the work was to study the activity of A2AAR and CK1δ in neurodegeneration using in-house synthesized A2A/CK1δ dual anta-inhibitors and to evaluate their intestinal absorption. Experiments were performed on N13 microglial cells, which were treated with a proinflammatory CK cocktail to simulate an inflammatory state typical of neurodegenerative diseases. Results showed that the dual anta-inhibitors have the ability to counteract the inflammatory state, even if compound 2 is more active than compound 1. In addition, compound 2 displayed an important antioxidant effect similar to the reference compound ZM241385. Since many known kinase inhibitors are very often unable to cross lipid bilayer membranes, the ability of A2A/CK1δ double anta-inhibitors to cross the intestinal barrier was investigated by an everted gut sac assay. HPLC analysis revealed that both compounds are able to cross the intestinal barrier, making them promising candidates for oral therapy.

A novel anti-apoptotic role for Cdc42/ACK-1 signaling in neurons.

Neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's and Parkinson's disease are caused by a progressive and aberrant destruction of neurons in the brain and spinal cord. These disorders lack effective long-term treatments that impact the underlying mechanisms of pathogenesis and as a result, existing options focus primarily on alleviating symptomology. Dysregulated programmed cell death (i.e., apoptosis) is a significant contributor to neurodegeneration, and is controlled by a number of different factors. Rho family GTPases are molecular switches with recognized importance in proper neuronal development and migration that have more recently emerged as central regulators of apoptosis and neuronal survival. Here, we investigated a role for the Rho GTPase family member, Cdc42, and its downstream effectors, in neuronal survival and apoptosis. We initially induced apoptosis in primary cultures of rat cerebellar granule neurons (CGNs) by removing both growth factor-containing serum and depolarizing potassium from the cell medium. We then utilized both chemical inhibitors and adenoviral shRNA targeted to Cdc42 to block the function of Cdc42 or its downstream effectors under either control or apoptotic conditions. Our in vitro studies demonstrate that functional inhibition of Cdc42 or its downstream effector, activated Cdc42-associated tyrosine kinase-1 (ACK-1), had no adverse effects on CGN survival under control conditions, but significantly sensitized neurons to cell death under apoptotic conditions. In conclusion, our results suggest a key pro-survival role for Cdc42/ACK-1 signaling in neurons, particularly in regulating neuronal susceptibility to pro-apoptotic stress such as that observed in neurodegenerative disorders.

Paris saponins â ¦ inhibits glycolysis of ovarian cancer via the RORC/ACK1 signaling pathway.

Rhizoma Paridis is a traditional Chinese medicine commonly used for treatment of malignant tumors. Paris saponins Ⅶ (PSⅦ) is one of the components of Rhizoma Paridis, but the role of PSⅦ in glucose metabolism in ovarian cancer remains elucidated. A series of experiments in the current study demonstrated that PSⅦ inhibites glycolysis and promotes cell apoptosis in ovarian cancer cells. Expression levels of glycolysis-related proteins and apoptosis-related proteins were significantly altered by upon treatment with PSⅦ, as determined from western blot analyses. Mechanistically, PSⅦ exerted its anti-tumor effects by targeting the RORC/ACK1 signaling pathway. These findings indicate that PSⅦ inhibits glycolysis-induced cell proliferation and apoptosis through the RORC/ACK1 pathway, supporting its potential development as a candidate chemotherapeutic agent for ovarian cancer.

Domain Architecture of the Nonreceptor Tyrosine Kinase Ack1.

The nonreceptor tyrosine kinase (NRTK) Ack1 comprises a distinct arrangement of non-catalytic modules. Its SH3 domain has a C-terminal to the kinase domain (SH1), in contrast to the typical SH3-SH2-SH1 layout in NRTKs. The Ack1 is the only protein that shares a region of high homology to the tumor suppressor protein Mig6, a modulator of EGFR. The vertebrate Acks make up the only tyrosine kinase (TK) family known to carry a UBA domain. The GTPase binding and SAM domains are also uncommon in the NRTKs. In addition to being a downstream effector of receptor tyrosine kinases (RTKs) and integrins, Ack1 can act as an epigenetic regulator, modulate the degradation of the epidermal growth factor receptor (EGFR), confer drug resistance, and mediate the progression of hormone-sensitive tumors. In this review, we discuss the domain architecture of Ack1 in relation to other protein kinases that possess such defined regulatory domains.

Co-targeting of ACK1 and KIT triggers additive anti-proliferative and -migration effects in imatinib-resistant gastrointestinal stromal tumors.

Most gastrointestinal stromal tumors (GIST) harbor mutated receptor tyrosine kinase (RTK) KIT/PDGFRA, which provides an attractive therapeutic target. However, a majority of GISTs ultimately develop resistance to KIT/PDGFRA inhibitor imatinib, multiple therapeutic targets will be identified as a reasonable strategy in imatinib-resistant GISTs. Biological mechanisms of non-RTK activated CDC42 associated kinase 1 (ACK1) are still unclear, which has been found to be activated in GISTs. In the current report, ACK1 overexpression is demonstrated in GIST cell lines and biopsies. RNA-seq analysis and immunoblotting show that ACK1 expression is dependent on imatinib treatment time in GIST-T1 cell line. The colocalization/complex of KIT and ACK1 in GIST cells are observed, and ACK1 activation is in a partially KIT and CDC42 dependent manner. Treatment with a specific ACK1 inhibitor AIM-100 or ACK1 siRNA, mildly suppresses cell viability, but markedly inhibits cell migration in imatinib sensitive and in imatinib resistant GIST cell lines, which is associated with inactivation of PI3K/AKT/mTOR and RAF/MAPK signaling pathways, and inhibition of epithelial-mesenchymal transition, evidencing upregulation of E-cadherin and downregulation of ZEB1, N-cadherin, vimentin, snail, and/or ß-catenin after treatment with AIM-100 or ACK1/CDC42 shRNAs. Combination inhibition of ACK1 and KIT results in additive effects of anti-proliferation and pro-apoptosis as well as cell cycle arrest, and inhibition of invasiveness and migration in vitro and in vivo, compared to either intervention alone through dephosphorylation of KIT downstream intermediates (AKT, S6, and MAPK). Our data suggest that co-targeting of ACK1 and KIT might be a novel therapeutic strategy in imatinib-resistant GIST.

Identification of Activated Cdc42-Associated Kinase Inhibitors as Potential Anticancer Agents Using Pharmacoinformatic Approaches.

BACKGROUND: Activated Cdc42-associated kinase (ACK1) is essential for numerous cellular functions, such as growth, proliferation, and migration. ACK1 signaling occurs through multiple receptor tyrosine kinases; therefore, its inhibition can provide effective antiproliferative effects against multiple human cancers. A number of ACK1-specific inhibitors were designed and discovered in the previous decade, but none have reached the clinic. Potent and selective ACK1 inhibitors are urgently needed. METHODS: In the present investigation, the pharmacophore model (PM) was rationally built utilizing two distinct inhibitors coupled with ACK1 crystal structures. The generated PM was utilized to screen the drug-like database generated from the four chemical databases. The binding mode of pharmacophore-mapped compounds was predicted using a molecular docking (MD) study. The selected hit-protein complexes from MD were studied under all-atom molecular dynamics simulations (MDS) for 500 ns. The obtained trajectories were ranked using binding free energy calculations (ΔG kJ/mol) and Gibb's free energy landscape. RESULTS: Our results indicate that the three hit compounds displayed higher binding affinity toward ACK1 when compared with the known multi-kinase inhibitor dasatinib. The inter-molecular interactions of Hit1 and Hit3 reveal that compounds form desirable hydrogen bond interactions with gatekeeper T205, hinge region A208, and DFG motif D270. As a result, we anticipate that the proposed scaffolds might help in the design of promising selective ACK1 inhibitors.

Activation of E3 ubiquitin ligase WWP2 by non-receptor tyrosine kinase ACK1.

WW domain containing E3 ubiquitin protein ligase 2 (WWP2) is a member of the NEDD4 E3 ubiquitin ligase family. WWP2 ligase activity is regulated by the 2, 3-linker auto-inhibition. Tyrosine phosphorylation of the 2, 3-linker was identified as an activating means for releasing the auto-inhibition of WWP2. However, the tyrosine kinase (TK) for the phosphorylation and activation remains unknown. In this report, we have found that non-receptor TK ACK1 binds to the WW3 domain of WWP2 and phosphorylates WWP2. ACK1 phosphorylates WWP2 at the 2, 3-linker and partially activates the ubiquitination ligase activity. Unexpectedly, tyrosine phosphorylation of the 2, 3-linker seems not a major mode for activation of WWP2, as ACK1 causes much higher activation of the 2, 3-linker tyrosine phosphorylation defective mutants of WWP2 than that of wild-type WWP2. Furthermore, epidermal growth factor (EGF) stimulates tyrosine phosphorylation of WWP2 and this EGF-stimulated phosphorylation of WWP2 is mediated by ACK1. Finally, knockdown of WWP2 by shWWP2 inhibits the EGF-dependent cell proliferation of lung cancer A549 cells, suggesting that WWP2 may function in the EGFR signaling in lung cancer progression. Taken together, our findings have revealed a novel mechanism underlying activation of WWP2.

Improvement of ACK1-targeted therapy efficacy in lung adenocarcinoma using chloroquine or bafilomycin A1.

BACKGROUND: Activated Cdc42-associated kinase 1 (ACK1) is a promising druggable target for cancer, but its inhibitors only showed moderate effects in clinical trials. The study aimed to investigate the underlying mechanisms and improve the antitumor efficacy of ACK1 inhibitors. METHODS: RNA-seq was performed to determine the downstream pathways of ACK. Using Lasso Cox regression analysis, we built a risk signature with ACK1-related autophagy genes in the lung adenocarcinoma (LUAD) patients from The Cancer Genome Atlas (TCGA) project. The performance of the signature in predicting the tumor immune environment and response to immunotherapy and chemotherapy were assessed in LUAD. CCK8, mRFP-GFP-LC3 assay, western blot, colony formation, wound healing, and transwell migration assays were conducted to evaluate the effects of the ACK1 inhibitor on lung cancer cells. A subcutaneous NSCLC xenograft model was used for in vivo study. RESULTS: RNA-seq revealed the regulatory role of ACK1 in autophagy. Furthermore, the risk signature separated LUAD patients into low- and high-risk groups with significantly different prognoses. The two groups displayed different tumor immune environments regarding 28 immune cell subsets. The low-risk groups showed high immune scores, high CTLA4 expression levels, high immunophenoscore, and low DNA mismatch repair capacity, suggesting a better response to immunotherapy. This signature also predicted sensitivity to commonly used chemotherapy and targeted drugs. In vitro, the ACK1 inhibitors (AIM-100 and Dasatinib) appeared to trigger adaptive autophagy-like response to protect lung cancer cells from apoptosis and activated the AMPK/mTOR signaling pathway, partially explaining its moderate antitumor efficacy. However, blocking lysosomal degradation with chloroquine/Bafilamycine A1 or inhibiting AMPK signaling with compound C/shPRKAA1 enhanced the ACK1 inhibitor's cytotoxic effects on lung cancer cells. The efficacy of the combined therapy was also verified using a mouse xenograft model. CONCLUSIONS: The resulting signature from ACK1-related autophagy genes robustly predicted survival and drug sensitivity in LUAD. The lysosomal degradation inhibition improved the therapeutic effects of the ACK1 inhibitor, suggesting a potential role for autophagy in therapy evasion.

TNK2/ACK1-mediated phosphorylation of ATP5F1A (ATP synthase F1 subunit alpha) selectively augments survival of prostate cancer while engendering mitochondrial vulnerability.

The challenge of rapid macromolecular synthesis enforces the energy-hungry cancer cell mitochondria to switch their metabolic phenotypes, accomplished by activation of oncogenic tyrosine kinases. Precisely how kinase activity is directly exploited by cancer cell mitochondria to meet high-energy demand, remains to be deciphered. Here we show that a non-receptor tyrosine kinase, TNK2/ACK1 (tyrosine kinase non receptor 2), phosphorylated ATP5F1A (ATP synthase F1 subunit alpha) at Tyr243 and Tyr246 (Tyr200 and 203 in the mature protein, respectively) that not only increased the stability of complex V, but also increased mitochondrial energy output in cancer cells. Further, phospho-ATP5F1A (p-Y-ATP5F1A) prevented its binding to its physiological inhibitor, ATP5IF1 (ATP synthase inhibitory factor subunit 1), causing sustained mitochondrial activity to promote cancer cell growth. TNK2 inhibitor, (R)-9b reversed this process and induced mitophagy-based autophagy to mitigate prostate tumor growth while sparing normal prostate cells. Further, depletion of p-Y-ATP5F1A was needed for (R)-9b-mediated mitophagic response and tumor growth. Moreover, Tnk2 transgenic mice displayed increased p-Y-ATP5F1A and loss of mitophagy and exhibited formation of prostatic intraepithelial neoplasia (PINs). Consistent with these data, a marked increase in p-Y-ATP5F1A was seen as prostate cancer progressed to the malignant stage. Overall, this study uncovered the molecular intricacy of tyrosine kinase-mediated mitochondrial energy regulation as a distinct cancer cell mitochondrial vulnerability and provided evidence that TNK2 inhibitors can act as "mitocans" to induce cancer-specific mitophagy.Abbreviations: ATP5F1A: ATP synthase F1 subunit alpha; ATP5IF1: ATP synthase inhibitory factor subunit 1; CRPC: castration-resistant prostate cancer; DNM1L: dynamin 1 like; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; Mdivi-1: mitochondrial division inhibitor 1; Mut-ATP5F1A: Y243,246A mutant of ATP5F1A; OXPHOS: oxidative phosphorylation; PC: prostate cancer; PINK1: PTEN induced kinase 1; p-Y-ATP5F1A: phosphorylated tyrosine 243 and 246 on ATP5F1A; TNK2/ACK1: tyrosine kinase non receptor 2; Ub: ubiquitin; WT: wild type.

Development of a new candidate vaccine against piglet diarrhea caused by Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is an important type of pathogenic bacteria that causes diarrhea in humans and young livestock. The pathogen has a high morbidity and mortality rate, resulting in significant economic losses in the pig industry. To effectively prevent piglet diarrhea, we developed a new tetravalent genetically engineered vaccine that specifically targets ETEC. To eliminate the natural toxin activity of ST1 enterotoxin and enhance the preventive effect of the vaccine, the mutated ST 1, K88ac, K99, and LT B genes were amplified by PCR and site-specific mutation techniques. The recombinant strain BL21(DE3)(pXKK3SL) was constructed and achieved high expression. Animal experiments showed that the inactivated vaccine had eliminated the natural toxin activity of ST1. The immune protection test demonstrated that the inclusion body and inactivated vaccine exhibited a positive immune effect. The protection rates of the inclusion body group and inactivated vaccine group were 96 and 98%, respectively, when challenged with 1 minimum lethal dose, indicating that the constructed K88ac-K99-3ST1-LTB vaccine achieved a strong immune effect. Additionally, the minimum immune doses for mice and pregnant sows were determined to be 0.2 and 2 mL, respectively. This study suggests that the novel K88ac-K99-3ST1-LTB vaccine has a wide immune spectrum and can prevent diarrhea caused by ETEC through enterotoxin and fimbrial pathways. The aforementioned research demonstrates that the K88ac-K99-3ST1-LTB vaccine offers a new genetically engineered vaccine that shows potential for preventing diarrhea in newborn piglets.

The noncatalytic regions of the tyrosine kinase Tnk1 are important for activity and substrate specificity.

Human Tnk1 (thirty-eight negative kinase 1) is a member of the Ack family of nonreceptor tyrosine kinases. Tnk1 contains a sterile alpha motif, a tyrosine kinase catalytic domain, an SH3 (Src homology 3) domain, and a large C-terminal region that contains a ubiquitin association domain. However, specific physiological roles for Tnk1 have not been characterized in depth. Here, we expressed and purified Tnk1 from Sf9 insect cells and established an in vitro assay system using a peptide substrate derived from the Wiskott-Aldrich Syndrome Protein (WASP). By Tnk1 expression in mammalian cells, we found that the N-terminal SAM domain is important for self-association and kinase activity. We also studied a fusion protein, originally discovered in a Hodgkin's Lymphoma cell line, that contains an unrelated sequence from the C17ORF61 gene fused to the C-terminus of Tnk1. Cells expressing the fusion protein showed increased tyrosine phosphorylation of cellular substrates relative to cells expressing WT Tnk1. A truncated Tnk1 construct (residues 1-465) also showed enhanced phosphorylation, indicating that the C17ORF61 sequence was dispensable for the effect. Additionally, in vitro kinase assays with the WASP peptide substrate showed no increase in intrinsic Tnk1 activity in C-terminally truncated constructs, suggesting that the truncations did not simply remove an autoinhibitory element. Fluorescence microscopy experiments demonstrated that the C-terminus of Tnk1 plays an important role in the subcellular localization of the kinase. Taken together, our data suggest that the noncatalytic regions of Tnk1 play important roles in governing activity and substrate phosphorylation.

The brain-specific splice variant of the CDC42 GTPase works together with the kinase ACK to downregulate the EGF receptor in promoting neurogenesis.

The small GTPase CDC42 plays essential roles in neurogenesis and brain development. Previously, we showed that a CDC42 splice variant that has a ubiquitous tissue distribution specifically stimulates the formation of neural progenitor cells, whereas a brain-specific CDC42 variant, CDC42b, is essential for promoting the transition of neural progenitor cells to neurons. These specific roles of CDC42 and CDC42b in neurogenesis are ascribed to their opposing effects on mTORC1 activity. Specifically, the ubiquitous form of CDC42 stimulates mTORC1 activity and thereby upregulates tissue-specific transcription factors that are essential for neuroprogenitor formation, whereas CDC42b works together with activated CDC42-associated kinase (ACK) to downregulate mTOR expression. Here, we demonstrate that the EGF receptor (EGFR) is an additional and important target of CDC42b and ACK, which is downregulated by their combined actions in promoting neurogenesis. The activation status of the EGFR determines the timing by which neural progenitor cells derived from P19 embryonal carcinoma terminally differentiate into neurons. By promoting EGFR degradation, we found that CDC42b and ACK stimulate autophagy, which protects emerging neurons from apoptosis and helps trigger neural progenitor cells to differentiate into neurons. Moreover, our results reveal that CDC42b is localized in phosphatidylinositol (3,4,5)-triphosphate-enriched microdomains on the plasma membrane, mediated through its polybasic sequence 185KRK187, which is essential for determining its distinct functions. Overall, these findings now highlight a molecular mechanism by which CDC42b and ACK regulate neuronal differentiation and provide new insights into the functional interplay between EGFR degradation and autophagy that occurs during embryonic neurogenesis.

Activity of the nonreceptor tyrosine kinase Ack1 is regulated by tyrosine phosphorylation of its Mig6 homology region.

Ack1 is a proto-oncogenic tyrosine kinase with homology to the tumour suppressor Mig6, an inhibitor of the epidermal growth factor receptor (EGFR). The residues critical for binding of Mig6 to EGFR are conserved within the Mig6 homology region (MHR) of Ack1. We tested whether intramolecular interactions between the Ack1 MHR and kinase domain (KD) are regulated by phosphorylation. We identified two Src phosphorylation sites within the MHR (Y859, Y860). Addition of Src-phosphorylated MHR to the Ack1 KD enhanced enzymatic activity. Co-expression of Src in cells led to increased Ack1 activity; mutation of Y859/Y860 blocked this increase. Collectively, the data suggest that phosphorylation of the Ack1 MHR regulates its kinase activity. Phosphorylation of Y859/Y860 occurs in cancers of the brain, breast, colon, and prostate, where genomic amplification or somatic mutations of Ack1 play a role in disease progression. Our findings suggest that MHR phosphorylation could contribute to Ack1 dysregulation in tumours.

ACK1 Contributes to the Pathogenesis of Inflammation and Autoimmunity by Promoting the Activation of TLR Signaling Pathways.

Toll-like receptors (TLRs) are the first line of defense in the immune system, whose activation plays a key role in the pathogenesis of inflammation and autoimmunity. TLRs can activate a variety of immune cells such as macrophages and dendritic cells, which produce proinflammatory cytokines, chemokines, and co-stimulatory molecules that lead to the development of inflammation and autoimmune diseases. As a nonreceptor tyrosine kinase, ACK1 is involved in multiple signaling pathways and physiological processes. However, the roles of ACK1 in the activation of TLR pathways and in the pathogenesis of inflammation and autoimmune diseases have not yet been reported. We found that the expression of ACK1 could be upregulated by TLR pathways in vivo and in vitro. Intriguingly, overexpression of ACK1 significantly promoted the activation of TLR4, TLR7, and TLR9 pathways, while knockdown of ACK1 or the use of the ACK1 inhibitor AIM-100 significantly inhibited the activation of TLR4, TLR7, and TLR9 pathways. In vivo studies showed that the inhibition of ACK1 activity by AIM-100 could significantly protect mice from the TLR4 agonist lipopolysaccharide (LPS)-mediated endotoxin shock and alleviate the condition of imiquimod-mediated lupus-prone mice and MRL/lpr mice. In summary, ACK1 participates in TLR-mediated inflammation and autoimmunity and has great potential in controlling inflammation and alleviating autoimmune diseases.

Ceritinib is a novel triple negative breast cancer therapeutic agent.

BACKGROUND: Triple-negative breast cancers (TNBCs) are clinically aggressive subtypes of breast cancer. TNBC is difficult to treat with targeted agents due to the lack of commonly targeted therapies within this subtype. Androgen receptor (AR) has been detected in 12-55% of TNBCs. AR stimulates breast tumor growth in the absence of estrogen receptor (ER), and it has become an emerging molecular target in TNBC treatment. METHODS: Ceritinib is a small molecule inhibitor of tyrosine kinase and it is used in the therapy of non-small lung cancer patients. Enzalutamide is a small molecule compound targeting the androgen receptor and it is used to treat prostate cancer. Combination therapy of these drugs were investigated using AR positive breast cancer mouse xenograft models. Also, combination treatment of ceritinib and paclitaxel investigated using AR- and AR low mouse xenograft and patient derived xenograft models. RESULTS: We screened 133 FDA approved drugs that have a therapeutic effect of AR+ TNBC cells. From the screen, we identified two drugs, ceritinib and crizotinib. Since ceritinib has a well- defined role in androgen independent AR signaling pathways, we further investigated the effect of ceritinib. Ceritinib treatment inhibited RTK/ACK/AR pathway and other downstream pathways in AR+ TNBC cells. The combination of ceritinib and enzalutamide showed a robust inhibitory effect on cell growth of AR+ TNBC cells in vitro and in vivo. Interestingly Ceritinib inhibits FAK-YB-1 signaling pathway that leads to paclitaxel resistance in all types of TNBC cells. The combination of paclitaxel and ceritinib showed drastic inhibition of tumor growth compared to a single drug alone. CONCLUSIONS: To improve the response of AR antagonist in AR positive TNBC, we designed a novel combinational strategy comprised of enzalutamide and ceritinib to treat AR+ TNBC tumors through the dual blockade of androgen-dependent and androgen-independent AR signaling pathways. Furthermore, we introduced a novel therapeutic combination of ceritinib and paclitaxel for AR negative or AR-low TNBCs and this combination inhibited tumor growth to a great extent. All agents used in our study are FDA-approved, and thus the proposed combination therapy will likely be useful in the clinic.

Chemical Reactivity and Optical and Pharmacokinetics Studies of 14 Multikinase Inhibitors and Their Docking Interactions Toward ACK1 for Precision Oncology.

Activated Cdc42-associated kinase 1 (ACK1/TNK2) has a significant role in cell endocytosis, survival, proliferation, and migration. Mutations in ACK1 are closely associated with the occurrence and development of cancers. In this work, a conceptual density functional theory (CDFT)-based computational peptidology (CDFT-CP) method is used to study the chemical reactivity of 14 multikinase inhibitors. Optical properties of these inhibitors are studied by time-dependent density functional theory (TDDFT). Various biological and pharmacokinetic parameters are studied by Osiris, Molinspiration, and BOILED-Egg in SwissADME software tools. Physicochemical and biopharmaceutical (PCB), Salmonella typhimurium reverse mutation assay (AMES) mutagenicity, toxicity, and risk prediction are estimated by Simulations plus ADMET Predictor 10.2 software. MD simulations for an active model of ACK1 is carried out by the CABS-flex 2.0 web server, and potential binding pockets for ACK1 are searched using the PrankWeb server. SwissTargetPrediction is used to predict the potential targets for the multikinase inhibitors. Docking studies are carried out for ACK1-multikinase inhibitors using Autodock 4.2 software. Noncovalent interactions for ACK1-multikinase inhibitor complexes are studied using the Protein-Ligand Interaction Profiler (PLIP) server. Results indicated higher binding affinities and strong noncovalent interactions in ACK1-multikinase inhibitor complexes.

Energy Consumption Model of SCHC Packet Fragmentation over Sigfox LPWAN.

The Internet Engineering Task Force (IETF) has standardized a new framework, called Static Context Header Compression and fragmentation (SCHC), which offers adaptation layer functionality designed to support IPv6 over Low Power Wide Area Networks (LPWANs). The IETF is currently profiling SCHC, and in particular its packet fragmentation and reassembly functionality, for its optimal use over certain LPWAN technologies. Considering the energy constraints of LPWAN devices, it is crucial to determine the energy performance of SCHC packet transfer. In this paper, we present a current and energy consumption model of SCHC packet transfer over Sigfox, a flagship LPWAN technology. The model, which is based on real hardware measurements, allows to determine the impact of several parameters and fragment transmission strategies on the energy performance of SCHC packet transfer over Sigfox. Among other results, we have found that the lifetime of a device powered by a 2000 mAh battery, transmitting packets every 5 days, is 168 days for 2250-byte packets, while it increases to 1464 days for 77-byte packets.