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Recent reports have shown that Zika virus (ZIKV) has oncolytic potential against human glioblastoma (GBM); however, the mechanisms underlying its tropism and cell entry are not completely understood. The receptor tyrosine kinase AXL has been identified as an entry receptor for ZIKV in a cell-type-specific manner. Interestingly, AXL is frequently overexpressed in GBM patients. Using commercially available GBM cell lines, we first show that cells expressing AXL are permissive for ZIKV infection, while cells that do not express AXL are not. Furthermore, inhibition of AXL kinase using R428 and antibody blockade of AXL receptor strongly attenuated virus entry in GBM cell lines. Additionally, CRISPR knockout of the AXL gene in GBM cell lines completely abolished ZIKV infection, significantly inhibited viral replication, and significantly reduced apoptosis compared with parental lines. Lastly, introduction of AXL receptor into non-expressing cell lines renders the cells susceptible to ZIKV infection. Together, these findings demonstrate that ZIKV entry into GBM cells in vitro is mediated by the AXL receptor and that following cell entry, productive infection is cytotoxic. Thus, ZIKV is a potential oncolytic virus for GBM., Graphical abstract, Zika virus (ZIKV) can infect glioblastoma (GBM) cells. We show that ZIKV entry and infection is mediated by the tyrosine kinase receptor AXL. AXL antibody blockade, CRISPR knockout, and kinase inhibition all mitigate ZIKV entry and infection. This receptor-mediated tropism suggests ZIKV as a candidate oncolytic virus for GBM.

The roles of ubiquitin-related genes in hepatocellular carcinoma (HCC) have not been thoroughly investigated. This study aimed to systematically examine ubiquitin-related genes and identify subtypes and stratify prognosis of HCC by using ubiquitin-related signatures. Survival, biological processes, tumor microenvironment (TME), and genomic alterations of the HCC subtypes were investigated. Patients with HCC were classified into two subtypes (clusters 1 and 2) with distinct survival outcomes, pathways, and genomic alterations. Cluster 2 had better prognosis than did cluster 1. Hepatitis B, hepatitis C, Janus tyrosine kinase (JAK)-signal transducer and activator of transcription (STAT) pathway, and natural killer cell-mediated cytotoxicity were enriched in cluster 1. Moreover, cluster 2 had a higher immune score and immune cell infiltrations, whereas cluster 1 had a lower immune score and immune infiltrations. Additionally, mutations, amplifications, and deletions among the phosphatidylinositol 3-kinase (PI3K)-AKT, p53, and receptor tyrosine kinase (RTK)-RAS pathways more frequently occurred in cluster 1, while those among the Hippo, MYC, and Notch signaling pathways were found in cluster 2. Finally, a prognostic signature, consisting of eight ubiquitin-related genes, was established and validated. In brief, our study established a new classification and developed a prognostic signature for HCC., Graphical abstract, This study aimed to examine ubiquitin-related genes and identify subtypes and stratify prognosis of HCC by using ubiquitin-related signatures. Patients with HCC were classified into two subtypes with distinct survival outcomes, pathways, and genomic alterations. Additionally, a prognostic signature, consisting of eight ubiquitin-related genes, was established and validated for HCC.

Tropomyosin receptor kinase A, B and C (TRKA, TRKB and TRKC), which are well-known members of the cell surface receptor tyrosine kinase (RTK) family, are encoded by the neurotrophic receptor tyrosine kinase 1, 2 and 3 (NTRK1, NTRK2 and NTRK3) genes, respectively. TRKs can regulate cell proliferation, differentiation and even apoptosis through the RAS/MAPKs, PI3K/AKT and PLCγ pathways. Gene fusions involving NTRK act as oncogenic drivers of a broad diversity of adult and pediatric tumors, and TRKs have become promising antitumor targets. Therefore, achieving a comprehensive understanding of TRKs and relevant TRK inhibitors should be urgently pursued for the further development of novel TRK inhibitors for potential clinical applications. This review focuses on summarizing the biological functions of TRKs and NTRK fusion proteins, the development of small-molecule TRK inhibitors with different chemotypes and their activity and selectivity, and the potential therapeutic applications of these inhibitors for future cancer drug discovery efforts., Graphical abstract This review summarized the biological structure and function of tropomyosin receptor kinases (TRKs), as well as neurotrophic receptor tyrosine kinase (NTRK) fusion proteins. The discovery and development of typical TRK inhibitors were discussed by concentrating on their different chemotypes, activity, selectivity and cocrystal structures.Image 1

The intracellular signaling pathway of growth hormone (GH)-stimulated DNA synthesis and proliferation was investigated in primary cultures of adult rat hepatocytes. DNA synthesis and cell proliferation were detected in hepatocyte parenchymal cells grown in serum-free, defined medium containing GH (100 ng/ml). GH-stimulated hepatocyte DNA synthesis and proliferation were almost completely blocked by TG101209 (10−6 M), a selective Janus kinase (JAK)2 inhibitor, U-73122 (10−6 M), a selective phospholipase C (PLC) inhibitor, and a monoclonal antibody to insulin-like growth factor-I (IGF-I) receptor (100 ng/ml) or anti-secretion agents such as somatostatin (10−6 M) and BAPTA/AM (10−7 M). In addition, blocking monoclonal antibodies to IGF-I, but not transforming growth factor-α, completely inhibited GH-induced hepatocyte DNA synthesis and proliferation. IGF-I levels in the culture medium increased rapidly versus baseline levels within 5 min in response to GH (100 ng/ml), and the maximum IGF-I level (100 pg/ml) was reached 20 min after GH stimulation. Autocrine secretion of IGF-I into the culture medium was inhibited by a growth-inhibitory dose of TG101209, U-73122, somatostatin, or BAPTA/AM. These data indicate that the proliferative mechanism of action of GH is mediated mainly through a GH receptor/JAK2/PLC-stimulated increase in the autocrine secretion of IGF-I by primary cultured hepatocytes, followed by stimulation of the 95 kDa IGF-I receptor tyrosine kinase signaling pathway., Article No.173753

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase which becomes oncogenic and drives tumor growth in multiple tumor types, including non-small cell lung cancer (NSCLC). We discuss the pathophysiology of ALK in NSCLC, which is driven predominantly by ALK gene rearrangements. We review the various methods of molecular testing for ALK gene rearrangements in NSCLC, and their limitations. Oral Tyrosine Kinase Inhibitors (TKIs) targeting the ALK pathway have been rapidly developed in the past 10 years. These drugs are well tolerated and offer prolonged disease control. We highlight the most up-to-date clinical data for each of the agents currently available which target the ALK pathway, and discuss adverse events associated with each agent. We discuss mechanisms of resistance to the ALK-targeted TKIs, and options for treatment at disease progression.

Many human malignancies require extracellular arginine (Arg) for survival because the key enzyme for de novo Arg biosynthesis, argininosuccinate synthetase 1 (ASS1), is silenced. Recombinant arginine deiminase (ADI-PEG20), which digests extracellular Arg, has been in clinical trials for treating ASS1-negative tumors. Reactivation of ASS1 is responsible for the treatment failure. We previously demonstrated that ASS1 reactivation is transcriptionally regulated by c-Myc via the upstream Gas6-Axl tyrosine kinase (RTK) signal. Here, we report that another RTK EphA2 is coactivated via PI3K-ERK/RSK1 pathway in a ligand-independent mechanism. EphA2 is also regulated by c-Myc. Moreover, we found that knockdown Axl upregulates EphA2 expression, demonstrating cross-talk between these RTKs. ADIR cell lines exhibits enhanced sensitivities to nutrient deprivation such as charcoal-stripped FBS and multiple RTK inhibitor foretinib but resistance to EGFR inhibitors. Knockdown EphA2, and to lesser extent, Axl, overcomes EGFRi resistance. c-Myc inhibitor JQ1 can also sensitize ADIR cells to ADI-PEG20. This study elucidates molecular interactions of multiple RTKs in Arg-stress response and offers approaches for developing strategies of overcoming ADI-PEG20 resistance.

BACKGROUND: Alpha-type platelet-derived growth factor receptor (PDGFRα) is a cell surface tyrosine kinase receptor for members of the platelet-derived growth factor family. PDGFRα plays an important role in the regulation of several biological processes and contributes to the pathophysiology of a broad range of human cancers, including glioma. Here, we hypothesize that the genes directly or indirectly influenced by PDGFRα might be useful for prognosis in glioma. METHODS: By comparing the genome-wide gene expression pattern between PDGFRα+ and PDGFRα− cells from human oligodendrocyte progenitor, we defined the genes potentially influenced by PDGFRα. RESULTS: The PDGFRα-influenced genes are strongly associated with cancer-related pathways. We subsequently developed a prognostic gene signature derived from the PDGFRα-influenced genes. This gene signature is able to predict clinical outcome of glioma. This signature is also independent of traditional prognostic factors of glioma. Resampling tests indicate that the prognostic power of this gene signature outperforms random gene sets selected from human genome. More importantly, this signature is superior to the random gene signatures selected from glioma related genes. CONCLUSIONS: Despite the absence of clear elucidation of molecular mechanisms, this study suggests the vital role of PDGFRα in carcinogenesis. Furthermore, the PDGFRα-based gene signature provides a promising prognostic tool for glioma and validates PDGFRα as a novel and effective therapeutic target in human cancers.

Background & Aims The miR-34a gene is a direct target of p53 and is commonly silenced in colorectal cancer (CRC). Here we identified the receptor tyrosine kinase CSF1R as a direct miR-34a target and characterized CSF1R as an effector of p53/miR-34a-mediated CRC suppression. Methods Analyses of TCGA-COAD and three other CRC cohorts for association of mRNA expression and signatures with patient survival and molecular subtypes. Bioinformatics identification and experimental validation of miRNA and transcription factor targets. Functional analysis of factors/pathways in the regulation of epithelial-mesenchymal transition (EMT), invasion, migration, acquired chemo-resistance and metastasis. Analyses of protein expression and CpG methylation within primary human colon cancer samples. Results In primary CRCs increased CSF1R, CSF1 and IL34 expression was associated with poor patient survival and a mesenchymal-like subtype. CSF1R displayed an inverse correlation with miR-34a expression. This was explained by direct inhibition of CSF1R by miR-34a. Furthermore, p53 repressed CSF1R via inducing miR-34a, whereas SNAIL induced CSF1R both directly and indirectly via repressing miR-34a in a coherent feed-forward loop. Activation of CSF1R induced EMT, migration, invasion and metastasis of CRC cells via STAT3-mediated down-regulation of miR-34a. 5-FU resistance of CRC cells was mediated by CpG-methylation of miR-34a and the resulting elevated expression of CSF1R. In primary CRCs elevated expression of CSF1R was detected at the tumor invasion front and was associated with CpG methylation of the miR-34a promoter as well as distant metastasis. Conclusions The reciprocal inhibition between miR-34a and CSF1R and its loss in tumor cells may be relevant for therapeutic and prognostic approaches towards CRC management., Graphical abstract

Epoxyeicosatrienoic acids (EETs) are formed from the metabolism of arachidonic acid by cytochrome P450s. EETs promote angiogenesis linked to tumor growth in various cancer models that is attenuated in vivo by cyclooxygenase 2 (COX-2) inhibitors. This study further defines a role for COX-2 in mediating endothelial EET metabolism promoting angiogenesis. Using human aortic endothelial cells (HAECs), we quantified 8,9-EET-induced tube formation and cell migration as indicators of angiogenic potential in the presence and absence of a COX-2 inducer [phorbol 12,13-dibutyrate (PDBu)]. The angiogenic response to 8,9-EET in the presence of PDBu was 3-fold that elicited by 8,9-EET stabilized with a soluble epoxide hydrolase inhibitor (t-TUCB). Contributing to this response was the COX-2 metabolite of 8,9-EET, the 11-hydroxy-8,9-EET (8,9,11-EHET), which exogenously enhanced angiogenic responses in HAECs at levels comparable to those elicited by vascular endothelial growth factor (VEGF). In contrast, the 15-hydroxy-8,9-EET isomer was also formed but inactive. The 8,9,11-EHET also promoted expression of the VEGF family of tyrosine kinase receptors. These results indicate that 8,9-EET-stimulated angiogenesis is enhanced by COX-2 metabolism in the endothelium through the formation of 8,9,11-EHET. This alternative pathway for the metabolism of 8,9-EET may be particularly important in regulating angiogenesis under circumstances in which COX-2 is induced, such as in cancer tumor growth and inflammation.

Summary: The Met/hepatocyte growth factor (HGF) receptor tyrosine kinase (RTK) is deregulated in many cancers and is a recognized target for cancer therapies. Following HGF stimulation, the signaling output of Met is tightly controlled by receptor internalization and sorting for degradation or recycling. Here, we uncover a role for autophagy in selective degradation of Met and regulation of Met-dependent cell migration and invasion. Met engagement with the autophagic pathway is dependent on complex formation with the mammalian ATG8 family member MAP1LC3C. LC3C deletion abrogates Met entry into the autophagy-dependent degradative pathway, allowing identification of LC3C domains required for rescue. Cancer cells with low LC3C levels show enhanced Met stability, signaling, and cell invasion. These findings provide mechanistic insight into RTK recruitment to autophagosomes and establish distinct roles for ATG8 proteins in this process, supporting that differential expression of ATG8 proteins can shape the functional consequences of autophagy in cancer development and progression. : The Met RTK regulates migratory invasive responses during development and cancer. Bell et al. describe a mechanism whereby MAP1LC3C selectively targets ligand-activated Met for degradation upon starvation-induced autophagy. Loss of the VHL tumor suppressor decreases LC3C, thereby uncoupling Met from autophagic regulation and enhancing HGF-stimulated invasion in cancer. Keywords: MET, autophagy, RTK, LC3C, VHL, cell migration, cell signaling, membrane trafficking, membrane trafficking, endocytosis, cancer

Summary: The mitogen-activated protein kinase (MAPK) pathway is a critical effector of oncogenic RAS signaling, and MAPK pathway inhibition may be an effective combination treatment strategy. We performed genome-scale loss-of-function CRISPR-Cas9 screens in the presence of a MEK1/2 inhibitor (MEKi) in KRAS-mutant pancreatic and lung cancer cell lines and identified genes that cooperate with MEK inhibition. While we observed heterogeneity in genetic modifiers of MEKi sensitivity across cell lines, several recurrent classes of synthetic lethal vulnerabilities emerged at the pathway level. Multiple members of receptor tyrosine kinase (RTK)-RAS-MAPK pathways scored as sensitizers to MEKi. In particular, we demonstrate that knockout, suppression, or degradation of SHOC2, a positive regulator of MAPK signaling, specifically cooperated with MEK inhibition to impair proliferation in RAS-driven cancer cells. The depletion of SHOC2 disrupted survival pathways triggered by feedback RTK signaling in response to MEK inhibition. Thus, these findings nominate SHOC2 as a potential target for combination therapy. : Sulahian, Kwon, and Walsh et al. performed several loss-of-function CRISPR-Cas9 screens in KRAS-mutant cancer cells treated with a MEK inhibitor and define the landscape of modifiers of MEK inhibitor sensitivity while highlighting that SHOC2 is a potent synthetic lethal target that serves as a critical signaling node to mediate MAP kinase pathway reactivation upon MEK inhibition. Keywords: Ras, KRAS, MEK inhibitor, synthetic lethal, SHOC2, CRISPR-Cas9 screen

Summary: Monoclonal antibodies (mAbs) targeting the oncogenic receptor tyrosine kinase ERBB2/HER2, such as Trastuzumab, are the standard of care therapy for breast cancers driven by ERBB2 overexpression and activation. However, a substantial proportion of patients exhibit de novo resistance. Here, by comparing matched Trastuzumab-naive and post-treatment patient samples from a neoadjuvant trial, we link resistance with elevation of H3K27me3, a repressive histone modification catalyzed by polycomb repressor complex 2 (PRC2). In ErbB2+ breast cancer models, PRC2 silences endogenous retroviruses (ERVs) to suppress anti-tumor type-I interferon (IFN) responses. In patients, elevated H3K27me3 in tumor cells following Trastuzumab treatment correlates with suppression of interferon-driven viral defense gene expression signatures and poor response. Using an immunocompetent model, we provide evidence that EZH2 inhibitors promote interferon-driven immune responses that enhance the efficacy of anti-ErbB2 mAbs, suggesting the potential clinical benefit of epigenomic reprogramming by H3K27me3 depletion in Trastuzumab-resistant disease. : Hirukawa et al. link Trastuzumab resistance in ErbB2+ breast cancers with activity of the methyltransferase EZH2, a key epigenetic regulator. By silencing retrotransposons, EZH2 suppresses type-I interferon signaling to limit immune surveillance. Retrotransposon de-repression following EZH2 inhibition triggers interferon responses and sensitizes immunocompetent in vivo models to ErbB2 antibody therapy. Keywords: ErbB2/HER2, breast cancer, Polycomb Repressor Complex 2, PRC2, epigenetics, Trastuzumab resistance, endogenous retroviruses, immune surveillance, type I interferon signaling, transcriptional silencing

Summary Neutrophil elastase (NE) promotes multiple stages of tumorigenesis. However, little is known regarding the molecular mechanisms underlying its stimulatory role. This study shows that NE triggers dose-dependent ERK signaling and cell migration in a panel of prostate cell lines representing the spectrum of prostate cell malignancy. All cell lines tested internalize NE; however, NE endocytosis is not required for ERK activation. Instead, NE acts extracellularly by stimulating the release of amphiregulin to initiate EGFR-dependent signaling. Inhibiting amphiregulin's biological activity with neutralizing antibodies, as well as gene silencing of amphiregulin or EGFR, attenuates NE-induced migration in normal and benign prostatic cells. Alternatively, in prostate cancer cells, knockdown of receptor tyrosine kinase AXL, but not EGFR, impairs both basal and NE-stimulated migration. When prostate cells progress to malignancy, the switch from EGFR-to AXL-dependence in NE-mediated migration implies the potential combined application of EGFR and AXL targeted therapy in prostate cancer treatment., Graphical abstract, Highlights • NE triggers the release of AREG to activate EGFR/ERK signaling in prostate cells • AREG-EGFR axis is critical in NE-induced migration of nonmalignant prostate cells • NE promotes prostate cancer cell migration via AXL rather than EGFR • Accessibility of NE in prostate cells may be associated with cell malignancy, Cancer; Cell biology

Activation of receptor tyrosine kinases needs tight control by tyrosine phosphatases to keep their normal function. In this study, we investigated the regulation of activation of the type III receptor tyrosine kinase KIT by protein tyrosine phosphatase receptor type E (PTPRE). We found that PTPRE can associate with wild-type KIT and inhibit KIT activation in a dose-dependent manner, although the activation of wild-type KIT is dramatically inhibited even when PTPRE is expressed at low level. The D816V mutation of KIT is the most frequently found oncogenic mutation in mastocytosis, and we found that PTPRE can associate and inhibit the activation of KIT/D816V in a dose dependent manner, but the inhibition is much weaker compared with wild-type KIT. Similar to mastocytosis, KIT mutations are the main oncogenic mutations in gastrointestinal stromal tumors (GISTs) although GISTs carry different types of KIT mutations. We further studied the regulation of the activation of GISTs-type KIT mutants and other mastocytosis-type KIT mutants by PTPRE. Indeed, PTPRE can almost block the activation of GISTs-type KIT mutants, while the activation of mastocytosis-type KIT mutants is more resistant to the inhibition of PTPRE. Taken together, our results suggest that PTPRE can associate with KIT, and inhibit the activation of both wild-type KIT and GISTs-type KIT mutants, while the activation of mastocytosis-type KIT mutants is more resistant to PTPRE., Highlights • PTPRE associates with wild-type KIT and KIT mutants. • PTPRE inhibits the activation of both wild-type KIT and GISTs-type KIT mutants dramatically. • The activation of mastocytosis-type KIT mutants are more resistant to the inhibition of PTPRE.

Overexpression of Axl, a TAM-family receptor tyrosine kinase, plays key roles in the formation, growth, and spread of tumors as well as resistance to targeted therapies and chemotherapies. We identified novel llama VHHs against human Axl using multiple complementary phage display selection strategies and characterized a subset of high-affinity VHHs. The VHHs targeted multiple sites in Ig-like domains 1 and 2 of the Axl extracellular domain, including an immunodominant epitope overlapping the site of Gas6 interaction and two additional non-Gas6 competitive epitopes recognized by murine monoclonal antibodies. Only a subset of VHHs cross-reacted with cynomolgus monkey Axl and none recognized mouse Axl. As fusions to human IgG1 Fc, VHH-Fcs bound Axl+ tumor cell lines and mertansine-loaded VHH-Fcs were cytotoxic in vitro against Axl+ cells in proportion to their binding affinities. Engineered biparatopic VHH-VHH heterodimers bound Axl avidly, and a subset of molecules showed dramatically enhanced association rates indicative of intramolecular binding. These VHHs may have applications as modular elements of biologic drugs such as antibody-drug conjugates.

This article contains supplemental datasets of the recently published related research article “Synthesis, Inverse Docking-Assisted Identification and in vitro Biological Characterization of Flavonol-based Analogs of Fisetin as c-Kit, CDK2 and mTOR Inhibitors against Melanoma and Non-melanoma Skin Cancers” by Roy et al., [1] . It provides in-depth data not included in the original co-submission on the biophysical, molecular docking, and biological characterization of newly synthesized flavonol-based analogs of fisetin, a natural dietary small molecule with anticancer and anti-inflammatory properties. These synthetic small molecules were investigated as new, potential single and/or multi-kinase inhibitors of the cyclin-dependent kinase-2 (CDK2), receptor tyrosine kinases (c-KITs), and mammalian targets of rapamycin (mTOR) targets, potentially active against melanoma or non-melanoma skin cancers. Furthermore, this data-in-brief article comprises additional sets of results on several aspects of the properties of the dual and multiple kinase inhibitor compounds’ effects that were not presented in the associated article, including the activated targets that are dysregulated in skin cancers; the effects on markers of apoptosis; on colony formation; and in scratch wound healing assays. The study has identified a panel of novel fisetin analogs that are either single- or multi-kinase inhibitors, which may be further developed as active for the treatment of melanoma and non-melanoma skin cancers. The dataset presented herein will be utilized for additional studies aiming to establish a biological platform to steer for predictive and experimental screening of novel flavonoids and analogs in relevant organoids, humanized animal models and in vivo disease models. The present results should also serve as a key stepping-stone towards enabling target-structure-based design, synthesis and initial testing of novel analogs or derivatives of fisetin. The current study may eventually lead to the development of safe, promising and preclinical candidate entities for treatment of skin and other forms of cancers as well as various other human diseases, which can possibly add to the general armamentarium of promising and safe drugs for health promotion.

The clinical trials employing neuroprotectants targeting single, early pathogenic mechanisms in stroke have so far been barely successful. We found in human postmortem stroke brains that in addition to apoptosis, necroptosis also contributed to neuronal damage. Thus, a new strategy targeting both mechanisms might be necessary. While brain-derived neurotrophic factor (BDNF) is a potent survival factor for neurons, its poor bioavailability including low diffusion rate and short half-life makes it unlikely a therapeutic agent. We recently developed a TrkB agonistic antibody (Ab4B19) that mimicked BDNF functionally but exhibited better physicochemical and pharmacological features. We showed that Ab4B19 halted neuronal death in vitro under multiple conditions that simulate ischemia/reperfusion injury, including oxygen-glucose deprivation (OGD), glutamate toxicity, oxidative stress and nutrient deprivation. In a rat model of ischemia/reperfusion, Ab4B19 suppressed both apoptosis and necroptosis, leading to a reduction in infarct volume and acceleration of functional recovery from sensorimotor impairments. In neurons derived from human embryonic stem cells (hESCs), Ab4B19 activated TrkB and its downstream signaling, and rescued neuronal death from OGD at a similar level as that in mouse neurons. Together, our study revealed necroptosis in human stroke brain, and demonstrated a BDNF-based strategy targeting both apoptosis and necroptosis for ischemic stroke treatment.

Background Gliomas are the most frequent and aggressive malignancies of the central nervous system. Decades of molecular analyses have demonstrated that gliomas accumulate genetic alterations that culminate in enhanced activity of receptor tyrosine kinases and downstream mediators. While the genetic alterations, like gene amplification or loss, have been well characterized, little information exists about changes in the proteome of gliomas of different grades. Methods We performed unbiased quantitative proteomics of human glioma biopsies by mass spectrometry followed by bioinformatic analysis. Findings Various pathways were found to be up- or downregulated. In particular, endocytosis as pathway was affected by a vast and concomitant reduction of multiple machinery components involved in initiation, formation, and scission of endocytic carriers. Both clathrin-dependent and -independent endocytosis were changed, since not only clathrin, AP-2 adaptins, and endophilins were downregulated, but also dynamin that is shared by both pathways. The reduction of endocytic machinery components caused increased receptor cell surface levels, a prominent phenotype of defective endocytosis. Analysis of additional biopsies revealed that depletion of endocytic machinery components was a common trait of various glioma grades and subclasses. Interpretation We propose that impaired endocytosis creates a selective advantage in glioma tumor progression due to prolonged receptor tyrosine kinase signaling from the cell surface. Fund This work was supported by Grants 316030-164105 (to P. Jeno), 31003A-162643 (to M. Spiess) and PP00P3-176974 (to G. Hutter) from the Swiss National Science Foundation. Further funding was received by the Department of Surgery from the University Hospital Basel.

Summary: Phosphatidylinositol 3,4,5-trisphosphate (PIP3) generation at the plasma membrane is a key event during activation of receptor tyrosine kinases such as the insulin receptor required for normal growth and metabolism. We report that in Drosophila, phosphatidylinositol 5 phosphate 4-kinase (PIP4K) is required to limit PIP3 levels during insulin receptor activation. Depletion of PIP4K increases the levels of PIP3 produced in response to insulin stimulation. We find that PIP4K function at the plasma membrane enhances class I phosphoinositide 3-kinase (PI3K) activity, although the catalytic ability of PIP4K to produce phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] at the plasma membrane is dispensable for this regulation. Animals lacking PIP4K show enhanced insulin signaling-dependent phenotypes and are resistant to the metabolic consequences of a high-sugar diet, highlighting the importance of PIP4K in normal metabolism and development. Thus, PIP4Ks are key regulators of receptor tyrosine kinase signaling with implications for growth factor-dependent processes including tumor growth, T cell activation, and metabolism. : PIP3 is a signaling lipid generated upon insulin receptor activation. Sharma et al. find that the lipid kinase PIP4K acts as negative regulator of PIP3 production at the plasma membrane. Loss of PIP4K enhances the sensitivity of cells to insulin signaling and can suppress insulin resistance phenotypes in Drosophila. Keywords: PIP4K, insulin, class I PI3K, PIP3, Drosophila

Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting motor neurons, is characterized by rapid decline of motor function and ultimately respiratory failure. As motor neuron death occurs late in the disease, therapeutics that prevent the initial disassembly of the neuromuscular junction may offer optimal functional benefit and delay disease progression. To test this hypothesis, we treated the SOD1G93A mouse model of ALS with an agonist antibody to muscle specific kinase (MuSK), a receptor tyrosine kinase required for the formation and maintenance of the neuromuscular junction. Chronic MuSK antibody treatment fully preserved innervation of the neuromuscular junction when compared with control-treated mice; however, no preservation of diaphragm function, motor neurons, or survival benefit was detected. These data show that anatomical preservation of neuromuscular junctions in the diaphragm via MuSK activation does not correlate with functional benefit in SOD1G93A mice, suggesting caution in employing MuSK activation as a therapeutic strategy for ALS patients.

Non-small cell lung carcinoma patients with epidermal growth factor receptor (EGFR) mutations are offered EGFR tyrosine kinase inhibitors (TKI) as first line treatment, but 20-40% of these patients do not respond. High expression of alternative receptor tyrosine kinases, such as Fibroblast growth factor receptor 1 (FGFR1), potentially mediates intrinsic EGFR TKI resistance. To study this in molecular detail, we used CRISPR-dCas9 Synergistic Activation Mediator (SAM) for up-regulation of FGFR1 in physiological relevant levels in the EGFR mutated NSCLC cell lines HCC827 and PC9 thereby generating HCC827gFGFR1 and PC9gFGFR1. The sensitivity to the TKI erlotinib was investigated in vitro and in a BALBc nu/nu mouse xenograft model. FGFR1 up-regulation decreased TKI-sensitivity in both NSCLC cell lines in the presence of the ligand fibroblast growth factor 2 (FGF2). Xenografts were established with PC9gFGFR1 cells and it was demonstrated that there was no significant difference in tumor size between TKI- and vehicle-treated PC9gFGFR1 tumors. This supports decreased TKI-sensitivity in NSCLC cells with FGFR1 up-regulation. Our study points to FGFR1 signaling being an intrinsic resistance mechanism abolishing TKI response in EGFR mutated NSCLC.

Genomic alterations are commonly found in the signaling pathways of fibroblast growth factor receptors (FGFRs). Although there is no selective FGFR inhibitors in market, several promising inhibitors have been investigated in clinical trials, and showed encouraging efficacies in patients. By designing a hybrid between the FGFR-selectivity-enhancing motif dimethoxybenzene group and our previously identified novel scaffold, we discovered a new series of potent FGFR inhibitors, with the best one showing sub-nanomolar enzymatic activity. After several round of optimization and with the solved crystal structure, detailed structure—activity relationship was elaborated. Together with in vitro metabolic stability tests and in vivo pharmacokinetic profiling, a representative compound (35) was selected and tested in xenograft mouse model, and the result demonstrated that inhibitor 35 was effective against tumors with FGFR genetic alterations, exhibiting potential for further development., Graphical abstract With the guidance of the crystal structure, we discovered a series of selective FGFR inhibitors bearing 5H-pyrrolo[2,3-b]pyrazine scaffold. After considerable efforts to improve the metabolic stability and pharmacokinetic properties, finally, we obtained a potent and active compound 35 showing in vivo efficacy in xenograft mouse model.fx1

HER3, a member of the receptor tyrosine kinase super family, is overexpressed in a number of cancers, and is associated with malignant phenotypes. Control of the protein stability of the membrane, as well as nuclear receptors, has been known to be an important process affecting tumor cells; however, their relationships have yet to be elucidated. In this study, we demonstrate that estradiol promotes rapid degradation of HER3 via the proteasome pathway in ER-positive breast cancer, MCF-7. ER prevented HER3 degradation, and knockdown of ER expression by si-RNA promoted rapid degradation of HER3. Breakdown of HER3 and ER were regulated by a ubiquitin ligase Nedd4-1 in the presence of estradiol stimulation. We speculate that estradiol quickly degrades ER, making HER3 accessible by Nedd4-1, and leads to the rapid degradation of HER3. In addition, knockdown of ubiquitin ligase Nedd4-1 enhances estradiol induced cell proliferation. These results indicate that HER3 and Nedd4-1 in ER-positive breast cancers might be an important therapeutic target., Highlights • First time we found that estradiol promotes rapid degradation of HER3 in ER-positive breast cancer MCF-7 cells. • Knockdown of ubiquitin ligase Nedd4-1 decreased the degradation efficiency of HER3 in the presence of estradiol. • Knockdown of Nedd4-1 enhanced estradiol induced tumor cell proliferation.

Esophageal adenocarcinoma (EAC) is a highly aggressive malignancy that is characterized by resistance to chemotherapy and a poor clinical outcome. The overexpression of the receptor tyrosine kinase AXL is frequently associated with unfavorable prognosis in EAC. Although it is well documented that AXL mediates cancer cell invasion as a downstream effector of epithelial-to-mesenchymal transition, the precise molecular mechanism underlying this process is not completely understood. Herein, we demonstrate for the first time that AXL mediates cell invasion through the regulation of lysosomes peripheral distribution and cathepsin B secretion in EAC cell lines. Furthermore, we show that AXL-dependent peripheral distribution of lysosomes and cell invasion are mediated by extracellular acidification, which is potentiated by AXL-induced secretion of lactate through AKT-NF-κB-dependent MCT-1 regulation. Our novel mechanistic findings support future clinical studies to evaluate the therapeutic potential of the AXL inhibitor R428 (BGB324) in highly invasive EAC.