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Receptor tyrosine kinases (RTKs) can show extensive crosstalk, directly and indirectly. Elucidating RTK crosstalk remains an important goal in the clinical combination of anti-cancer therapies. Here, we present mass spectrometry and pharmacological approaches showing the hepatocyte growth factor receptor (MET)-promoting tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) and other membrane receptors in MET-amplified H1993 NSCLC cells. Conversely, in H292 wt-EGFR NSCLC cells, EGFR promotes the tyrosine phosphorylation of MET. Reciprocal regulation of the EGFR and insulin receptor (IR) was observed in the GEO CRC cells, where inhibition of the EGFR drives tyrosine phosphorylation of the insulin receptor. Similarly, in platelet-derived growth factor receptor (PDGFR)-amplified H1703 NSCLC cells, inhibition of the EGFR promotes the tyrosine phosphorylation of the PDGFR. These RTK interactions are used to illustrate basic principles applicable to other RTK signaling networks. More specifically, we focus on two types of RTK interaction: (1) co-option of one RTK by another and (2) reciprocal activation of one receptor following the inhibition of a distinct receptor.

HER2 belongs to the ErbB sub-family of receptor tyrosine kinases and regulates cellular proliferation and growth. Different from other ErbB receptors, HER2 has no known ligand. Activation occurs through heterodimerization with other ErbB receptors and their cognate ligands. This suggests several possible activation paths of HER2 with ligand-specific, differential response, which so far remained unexplored. Using single-molecule tracking and the diffusion profile of HER2 as a proxy for activity, we measured the activation strength and temporal profile in live cells. We found that HER2 is strongly activated by EGFR-targeting ligands EGF and TGFα, yet with a distinguishable temporal fingerprint. The HER4-targeting ligands EREG and NRGβ1 showed weaker activation of HER2, a preference for EREG, and a delayed response to NRGβ1. Our results indicate a selective ligand response of HER2 that may serve as a regulatory element. Our experimental approach is easily transferable to other membrane receptors targeted by multiple ligands.HighlightsHER2 exhibits heterogeneous motion in the plasma membraneThe fraction of immobile HER2 correlates with phosphorylation levelsDiffusion properties serve as proxies for HER2 activationHER2 exhibits ligand-specific activation strength and temporal profilesGraphical Abstract

Background MET-driven acquired resistance is emerging with unanticipated frequency in patients relapsing upon molecular therapy treatments. However, the determination of MET amplification remains challenging using both standard and next-generation sequencing-based methodologies. Liquid biopsy is an effective, non-invasive approach to define cancer genomic profiles, track tumor evolution over time, monitor treatment response and detect molecular resistance in advance. Circular RNAs (circRNAs), a family of RNA molecules that originate from a process of back-splicing, are attracting growing interest as potential novel biomarkers for their stability in body fluids. Methods We identified a circRNA encoded by the MET gene (circMET) and exploited blood-derived cell-free RNA (cfRNA) and matched tumor tissues to identify, stratify and monitor advanced cancer patients molecularly characterized by high MET activity, generally associated with genomic amplification. Results Using publicly available bioinformatic tools, we discovered that the MET locus transcribes several circRNA molecules, but only one candidate, circMET, was particularly abundant. Deeper molecular analysis revealed that circMET levels positively correlated with MET expression and activity, especially in MET-amplified cells. We developed a circMET-detection strategy and, in parallel, we performed standard FISH and IHC analyses in the same specimens to assess whether circMET quantification could identify patients displaying high MET activity. Longitudinal monitoring of circMET levels in the plasma of selected patients revealed the early emergence of MET amplification as a mechanism of acquired resistance to molecular therapies. Conclusions We found that measurement of circMET levels allows identification and tracking of patients characterized by high MET activity. Circulating circMET (ccMET) detection and analysis could be a simple, cost-effective, non-invasive approach to better implement patient stratification based on MET expression, as well as to dynamically monitor over time both therapy response and clonal evolution during treatment.

Cells must reliably respond to changes in transcription factor levels in order to execute cell state transitions in the correct time and place. These transitions are typically thought to be triggered by changes in the absolute nuclear concentrations of relevant transcription factors. We have identified a developmental context in which cell fate transitions depend on changes in the relative concentrations of two transcription factors. Here, we quantify the in vivo expression dynamics of Yan and Pointed, two essential E-twenty-six (ETS) proteins that regulate transcription during eye development in Drosophila. These two factors exert opposing influences; one impedes transcription of gene targets required for differentiation while the other promotes it. We show that both proteins are transiently co-expressed in eye progenitor cells and also during photoreceptor specification. To decide whether to undergo state transitions, cells respond to the ratio of the two protein concentrations rather than changes in the absolute abundance of either transcription factor. We show that a simple model based on the statistical physics of protein-DNA binding illustrates how this ratiometric sensing of transcription factor concentrations could occur. Gene dosage experiments reveal that progenitor cells stabilize the ratio against fluctuations in the absolute concentration of either protein. We further show that signaling inputs via the Notch and Receptor Tyrosine Kinase (RTK) pathways set the ratio in progenitor cells, priming them for either transit to differentiation or for continued multipotency. A sustained change in the ratio accompanies the transit to differentiation This novel mechanism allows for distributed control of developmental transitions by multiple transcription factors, making the system robust to fluctuating genetic or environmental conditions.

Background:Catechin is a phytochemical and is a major component of our daily use beverages, which has shown great potential in improving general health and fighting against several medical conditions. Clinical studies have confirmed its effectiveness in conditions ranging from acute upper respiratory tract infection, neuroprotection, to cardio-protection effects. Though most studies relate their potential to anti-oxidative action and radical scavenging action, still the mechanism of action is not clearly understood.Objective:The present review article is focused on addressing various pharmacological actions and underlying mechanisms of catechin. Additionally, we will try to figure out the major adverse effect and success in trials with catechin and lead to a conclusion for its effectiveness.Methods:This review article is based on the recent/ most cited papers of PubMed and Scopus databases.Description:Catechin can regulate Nrf2 and NFkB pathways in ways that impact oxidative stress and inflammation by influencing gene expression. Other pathways like MAPKs and COMT and receptor tyrosine kinase are also affected by catechin and EGCG that alter their action and barge the cellular activity. This review article explored the structural aspect of catechin and its different isomers and analogs. It also evaluated its various therapeutic and pharmacological arrays.Conclusion:Catechin and its stereo-isomers have shown their effectiveness as anti-inflammatory, anti-diabetic, anti-cancer, anti-neuroprotective, bactericidal, memory enhancer, anti-arthritis, and hepato-protective mainly through its activity to alter the pathway by NF-κB, Nrf-2, TLR4/NF-κB, COMT, and MAPKs.

Acute myeloid leukemia (AML) is the most common acute leukemia of adults, with a five-year survival that remains poor (approximately 25%). Knowledge and understanding of AML genomics have expanded tremendously over the past decade and are now included in AML prognostication and treatment decisions. FMS-like tyrosine kinase 3 (FLT3) is a Class III receptor tyrosine kinase (RTK) expressed primarily in the cell membranes of early hematopoietic progenitor cells, found in 28% of all patients with AML. FLT3 is the second most frequent mutation in adult AML following Nuclear-cytoplasmic shuttling phosphoprotein (NPM1), which is found in 50% of cases.1 FLT3 inhibitors are promising new molecular therapeutics increasingly becoming standard of care for both newly diagnosed and relapsed/refractory FLT3 positive AML. This review will focus on the clinical trials/evidence, similarities, differences, clinical toxicities, and drug interactions relevant to treating clinicians as pertains to 5 FLT3-inhibitors: midostaurin, sorafenib, gilteritinib, crenolanib, and quizartinib.

PURPOSE Insertion mutations in Erb-b2 receptor tyrosine kinase 2 gene ( ERBB2 or HER2) exon 20 occur in 2%-5% of non–small-cell lung cancers (NSCLCs) and function as an oncogenic driver. Poziotinib, a tyrosine kinase inhibitor, was evaluated in previously treated patients with NSCLC with HER2 exon 20 insertions. METHODS ZENITH20, a multicenter, multicohort, open-label phase II study, evaluated poziotinib in patients with advanced or metastatic NSCLC. In cohort 2, patients received poziotinib (16 mg) once daily. The primary end point was objective response rate evaluated by independent review committee (RECIST v1.1); secondary outcome measures were disease control rate, duration of response, progression-free survival, and safety and tolerability. Quality of life was assessed. RESULTS Between October 2017 and March 2021, 90 patients with a median of two prior lines of therapy (range, 1-6) were treated. With a median follow-up of 9.0 months, objective response rate was 27.8% (95% CI, 18.9 to 38.2); 25 of 90 patients achieved a partial response. Disease control rate was 70.0% (95% CI, 59.4 to 79.2). Most patients (74%) had tumor reduction (median reduction 22%). Median progression-free survival was 5.5 months (95% CI, 3.9 to 5.8); median duration of response was 5.1 months (95% CI, 4.2 to 5.5). Clinical benefit was seen regardless of lines and types of prior therapy, presence of central nervous system metastasis, and types of HER2 mutations. Grade 3 or higher treatment-related adverse events included rash (48.9%), diarrhea (25.6%), and stomatitis (24.4%). Most patients had poziotinib dose reductions (76.7%), with median relative dose intensity of 71.5%. Permanent treatment discontinuation because of treatment-related adverse events occurred in 13.3% of patients. CONCLUSION Poziotinib demonstrates antitumor activity in previously treated patients with HER2 exon 20 insertion NSCLC.

Recurrent alterations in receptor tyrosine kinase (RTK) and downstream effectors are described in infantile fibrosarcoma (IFS)/cellular congenital mesoblastic nephroma (cCMN) and a subset of spindle cell sarcomas, provisionally designated 'NTRK-rearranged' spindle cell neoplasms. These two groups of tumours demonstrate overlapping morphologies and harbour alterations in NTRK1/2/3, RET, MET, ABL1, ROS1, RAF1 and BRAF, although their relationship is not fully elucidated. We describe herein a cohort of paediatric tumours with clinicopathological features not typical for inflammatory myofibroblastic tumour, but rather with similarities to cCMN/IFS harbouring ALK fusions.Clinicopathological features were assessed and partner agnostic targeted RNA sequencing on clinically validated platforms were performed. Tumours occurred in patients aged from 2 to 10 years (median age 2 years) with a 2:2 male to female ratio and an average size of 8.4 cm. Two tumours arose in soft tissues and two in the kidney. Morphological features included spindle to ovoid cells arranged in long fascicles or haphazardly within a myxoid to collagenised stroma; a subset of cases had either dilated, ectatic vessels or focal perivascular hyalinosis. By immunohistochemistry, all cases tested showed cytoplasmic expression of anaplastic lymphoma kinase (ALK) and one case demonstrated co-expression of CD34 and S100.This series of ALK-rearranged IFS-like tumours expands the spectrum of targetable kinases altered in these tumours and reinforces the potential overlap between IFS/cCMN-like tumours and the provisional entity of 'NTRK-rearranged' spindle cell neoplasms.

Juvenile hormone (JH) and 20-hydroxyecdysone (20E) coordinately regulate development and metamorphosis in insects. Two JH intracellular receptors, methoprene-tolerant (Met) and germ-cell expressed (Gce), have been identified in the fruit fly Drosophila melanogaster. To investigate JH membrane signaling pathway without the interference from JH intracellular signaling, we characterized phosphoproteome profiles of the Met gce double mutant in the absence or presence of JH in both chronic and acute phases. Functioning through a potential receptor tyrosine kinase and phospholipase C pathway, JH membrane signaling activated protein kinase C (PKC) which phosphorylated ultraspiracle (USP) at Ser35, the PKC phosphorylation site required for the maximal action of 20E through its nuclear receptor complex EcR-USP. The uspS35A mutant, in which Ser was replaced with Ala at position 35 by genome editing, showed decreased expression of Halloween genes that are responsible for ecdysone biosynthesis and thus attenuated 20E signaling that delayed developmental timing. The uspS35A mutant also showed lower Yorkie activity that reduced body size. Altogether, JH membrane signaling phosphorylates USP at Ser35 and thus potentiates 20E action that regulates the normal fly development. This study helps better understand the complex JH signaling network.

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase widely expressed in many cancers such as non-small cell lung cancer (NSCLC), pancreatic cancer, breast cancer, and head and neck cancer. Mutations such as L858R in exon 21, exon 19 truncation (Del19), exon 20 insertions, and others are responsible for aberrant activation of EGFR in NSCLC. First-generation EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib have clinical benefits for EGFR-sensitive (L858R and Del19) NSCLC patients. However, after 10-12 months of treatment with these inhibitors, a secondary T790M mutation at the gatekeeper position in the kinase domain of EGFR was identified, which limited the clinical benefits. Second-generation EGFR irreversible inhibitors (afatinib and dacomitinib) were developed to overcome this T790M mutation. However, their lack of selectivity toward wild-type EGFR compromised their clinical benefits due to serious adverse events. Recently developed third-generation irreversible EGFR TKIs (osimertinib and lazertinib) are selective toward driving mutations and the T790M mutation, while sparing wild-type EGFR activity. The latest studies have concluded that their efficacy was also compromised by additional acquired mutations, including C797S, the key residue cysteine that forms covalent bonds with irreversible inhibitors. Because second- and third-generation EGFR TKIs are irreversible inhibitors, they are not effective against C797S containing EGFR triple mutations (Del19/T790M/C797S and L858R/T790M/C797S). Therefore, there is an urgent unmet medical need to develop next-generation EGFR TKIs that selectively inhibit EGFR triple mutations via a non-irreversible mechanism.

AZD3759 is a novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) on the basis of gefitinib and has been proven to enter the central nervous system. Although the promising antitumor effects of AZD3759 on non-small cell lung cancer (NSCLC) have been demonstrated in clinical trials, the regulatory effects of this inhibitor on the antitumor efficacy of radiation (RA) are unclear. The present study aimed to compare the effects of AZD3759 and osimertinib on RA efficacy in NSCLC and explore the potential mechanism of action of AZD3759. We found that the survival in RA-treated NSCLC cells was significantly decreased by treatment with 500 nM AZD3759 and osimertinib at the RA dosage of 8 Gy. The apoptotic rate, cell cycle arrest, and DNA damage in RA-treated NSCLC cells and brain metastasis in RA-treated xenograft nude mice were significantly enhanced by the co-administration of AZD3759 and osimertinib, respectively. In addition, AZD3759 showed a significantly stronger efficacy than osimertinib did. Mechanistically, the receptor tyrosine kinase signaling antibody array revealed that Janus kinase-1 (JAK1) was specifically inhibited by AZD3759, but not by osimertinib. The effects of AZD3759 on RA efficacy in PC-9 cells and in a brain metastasis animal model were significantly abolished by the overexpression of JAK1. Collectively, our results suggested that AZD3759 promoted RA antitumor effects in NSCLC by synergistic blockade of EGFR and JAK1.

Benzimidazole (BI)/triphenylamine (TPA)-based compounds (AL-1 to AL-7) were synthesized and evaluated for their cytotoxic activity in the different cell lines. Among them, AL-1 is demonstrated good cytotoxic activity in all the chosen cell lines, as observed from the MTT results. The antiproliferative activity of AL-1 against various cancer cells indicates that this BI-TPA-based compound may be applicable as a potential multi cancer inhibitor. AL-1 has a low IC50 value to kill H446, demonstrating the efficacy of the selected BI-TPA derivatives against the highly aggressive cancer cells (H446, small cell lung cancer - SCLC). The cytotoxicity performance is correlated with the presence of two redox peaks in the cyclic voltammetry (CV) analysis. It can be found in insilico analysis that, AL-1 has a good Gscore towards mutant receptor tyrosine kinase (RTK), PDGFRɑ. And it has a high stability for mutant EGFR (4RJ5) in the molecular dynamics simulation study. The small binding free energy from MM-GBSA is correlated with the formation of a stable complex structure. These studies suggest that BI-TPA-based compounds (e.g., AL-1) can be used as prospective anticancer agents to treat cancer cells and their mutant variants (resistant cancer cells).

Heart failure (HF) is classified according to the degree of reduction in left ventricular ejection fraction (EF) in HF with reduced, mildly reduced, and preserved EF. Biomarkers could behave differently depending on EF type. Here, we analyze the soluble form of the AXL receptor tyrosine kinase (sAXL) in HF patients with reduced and preserved EF. Two groups of HF patients with reduced (HFrEF; n = 134) and preserved ejection fraction (HFpEF; n = 134) were included in this prospective observational study, with measurements of candidate biomarkers and functional, clinical, and echocardiographic variables. A Cox regression model was used to determine predictors for clinical events: cardiovascular mortality and all-cause mortality. sAXL circulating values predicted outcome in HF: for a 1.0 ng/mL increase in serum sAXL, the mortality hazard ratio (HR) was 1.019 for HFrEF (95% CI 1.000 to 1.038) and 1.032 for HFpEF (95% CI 1.013 to 1.052). In a multivariable Cox regression analysis, sAXL and NT-proBNP were independent markers for all-cause and cardiovascular mortality in HFpEF. In contrast, only NT-proBNP remained significant in the HFrEF group. When analyzing the event-free survival at a mean follow-up of 3.6 years, HFrEF and HFpEF patients in the higher quartile of sAXL had a reduced survival time. Interestingly, sAXL is a reliable predictor for all-cause and cardiovascular mortality only in the HFpEF cohort. The results suggest an important role for AXL in HFpEF, supporting sAXL evaluation in larger clinical studies and pointing to AXL as a potential target for HF therapy.

Polypeptide growth factors have been intensively studied since their initial discovery as pivotal regulators of cell proliferation and differentiation in multicellular organisms more than 70 years ago. Despite their name, many elicit multiple cellular responses during embryogenesis and throughout adulthood; dysregulated signaling can also contribute to disease. Few factors illustrate these principles as thoroughly as hepatocyte growth factor (Hgf), also known as scatter factor (SF). As evident from its pseudonyms, Hgf pleiotropism has been a striking feature from its initial discovery more than two decades ago by several groups with interests in liver regeneration, cell growth control, motility and morphogenesis. Thousands of scientific publications now document the critical contributions of Hgf signaling to normal development, adult homeostasis and several forms of cancer. Hgf protein and the related macrophage stimulating protein (MSP; also known as Hgf-like protein) comprise a small but distinct growth factor subfamily related to plasminogen serine proteinases, though they are devoid of proteolytic activity. In humans, a single gene encodes five HGF isoforms produced through mRNA splicing: two full-length forms that differ by only five residues and three substantially shorter forms. The more abundant full-length forms are secreted as inactive single chain polypeptides that are proteolytically converted to biologically active disulfide-linked heterodimers at the target cell surface. All isoforms bind strongly to heparan sulfate proteoglycan, a feature that profoundly influences their local and systemic distribution, receptor binding, and biological impact. Hgf signaling is primarily paracrine: it is secreted by mesenchymal cells in many tissues and acts on a broad spectrum of cellular targets that express the receptor tyrosine kinase known as Met. Hgf-related research has identified molecular pathways important for tissue protection, tissue regeneration and oncogenesis, with the promise to advance tissue engineering, regenerative medicine as well as cancer diagnosis and treatment., This work was supported by National Institutes of Health (USA) grants Z01 BC005626 and ZIA BC011124 to D. P. Bottaro.

The scaffold protein IQGAP1 associates with over 150 interactors to influence multiple biological processes. The molecular mechanisms that underly spatial and temporal regulation of these interactions, which are crucial for proper cell functions, remain poorly understood. The receptor tyrosine kinase MET phosphorylates IQGAP1 on Tyr1510. Separately, Src homology 2 (SH2) domains mediate protein–protein interactions by binding specific phosphotyrosine residues. Here, we investigate whether MET-catalyzed phosphorylation of Tyr1510 of IQGAP1 regulates the docking of SH2-containing proteins. Using a peptide array, we identified SH2 domains from several proteins, including the non-receptor tyrosine kinases Abl1 and Abl2, that bind to the Tyr1510 of IQGAP1 in a phosphorylation-dependent manner. Using pure proteins, we validated that full-length Abl1 and Abl2 bind directly to phosphorylated Tyr1510 of IQGAP1. In cells, MET inhibition decreases endogenous IQGAP1 phosphorylation and interaction with endogenous Abl1 and Abl2, indicating that binding is regulated by MET-catalyzed phosphorylation of IQGAP1. Functionally, IQGAP1 modulates basal and HGF-stimulated Abl signaling. Moreover, IQGAP1 binds directly to MET, inhibiting its activation and signaling. Collectively, our study demonstrates that IQGAP1 is a phosphotyrosine-regulated scaffold for SH2-containing proteins, thereby uncovering a previously unidentified mechanism by which IQGAP1 coordinates intracellular signaling.

The platelet-derived growth factor receptors (PDGFRs) play important roles in multiple cellular processes including cell survival, cell growth and cell migration. Dysregulation of PDGFRs causes aberrant PDGF signaling, which leads to various diseases. Insights from the research about PDGF/PDGFR signaling have enlightened new opportunities to understand the molecular mechanisms of cancer and other diseases. The goal of this thesis is to further investigate the mechanisms of the modulation of PDGF/PDGFR signaling to identify new ways of controlling aberrant signaling of these RTKs in various diseases, including cancer. Ubiquitination is an important post-translational modification related to protein degradation, receptor internalization, intracellular trafficking, cell proliferation, and other cellular processes. It can be reversed by DUBs, and the overexpression of DUBs is involved in various diseases including cancers. In paper I, we identified two main DUBs working on PDGFRβ, USP17 and USP4. They affected the timing of STAT3 activation and trafficking via different mechanisms, thus fine-tuning its transcriptional activity, which further regulated the proliferative response induced by PDGF-BB. SUMOylation is another post-translational modification that is important for the regulation of protein subcellular localization, protein stability, protein-DNA interactions, protein-protein interactions, genome organization, DNA repair and transcriptional regulation. In paper II, we have identified PDGFRα as a SUMOylation substrate and performed a characterization of the functional role of SUMOylation in PDGFRα signaling and cell proliferation. Proteolytic cleavage of RTKs regulates their downstream signaling pathways by affecting their structure, stability, subcellular localization and interaction with other proteins. In paper III, we have identified that PDGFRβ is cleaved in the region Y579-Y857 upon ligand stimulation by a Ca2+-dependent protease, which is dependent on its internalization. The proteasomal inhibitor bortezomib blocked the internalization, as well as the cleavage of PDGFRβ, and also affected its downstream signaling. Apart from the classic degradation in lysosomes and proteasomes, several RTKs have also been found to undergo autophagy and to be targeted in autophagosomes which further fuse with lysosomes. In liver hepatocytes (LX2) cells, it has been reported that PDGFRα, but not PDGFRβ, undergo selective autophagy. In paper IV, we identified that in certain types of cells, PDGFRβ may be involved in the autophagy pathway, which may affect the synthesis of new PDGFRβ. To conclude, in this study, we investigated how the turnover and signaling of the RTKs PDGFR isoforms α and β are modulated by ubiquitination, SUMOylation, proteolytic cleavage and degradation.

The interplay between membrane subregions and receptor tyrosine kinases (RTK) will influence signaling in both normal and pathological RTK conditions. In this study, epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor β (PDGFR-β) internalizations were investigated by immunofluorescent microscopy following simultaneous treatment with EGF and PDGF-BB. We found that the two receptors utilize separate routes of internalization, which merges in a common perinuclear endosomal compartment after 45 min of stimulation. This is further strengthened when contrasting the recruitment of either EGFR or PDGFR-β to either clathrin or caveolin-1: PDGFR-β dissociates from caveolin-1 upon stimulation, and engages clathrin, whilst an increased recruitment of EGFR, to both clathrin and caveolin-1, was observed upon EGF stimulation. The association between EGFR and caveolin-1 is supported by the observation that EGFR was localized in lipid raft associated fractions, whereas PDGFR-β was not. We also found that disruption of lipid rafts using MβCD led to an increased EGFR dimerization and phosphorylation in response to ligand, as well as a dramatic decrease in AKT- and a smaller but robust decrease in ERK1/2 phosphorylation. This suggest that lipid rafts may be important to effectively connect the EGFR with downstream proteins to facilitate signaling. Our data implies that cholesterol depletion of the plasma membrane affect the signaling of EGFR and PDGFRβ differently.

Activation of platelet-derived growth factor (PDGF) receptors α and β (PDGFRα and PDGFRβ) at the cell surface by binding of PDGF isoforms leads to internalization of receptors, which affects the amplitude and kinetics of signaling. Ubiquitination of PDGF receptors in response to ligand stimulation is mediated by the Casitas b-lineage lymphoma (Cbl) family of ubiquitin ligases, promoting internalization and serving as a sorting signal for vesicular trafficking of receptors. We report here that another E3 ligase, i.e., tripartite motif-containing protein 21 (TRIM21), contributes to the ubiquitination of PDGFRβ in human primary fibroblasts AG1523 and the osteosarcoma cell line U2OS and regulates basal levels of PDGFRβ. We found that siRNA-mediated depletion of TRIM21 led to decreased ubiquitination of PDGFRβ in response to PDGF-BB stimulation, while internalization from the cell surface and the rate of ligand-induced degradation of the receptor were not affected. Moreover, induction of TRIM21 decreased the levels of PDGFRβ in serum-starved cells, and even more in growing cells, in the absence of PDGF stimulation. Consistently, siRNA knockdown of TRIM21 caused accumulation of the total amount of PDGFRβ, both in the cytoplasm and on the cell surface, without affecting mRNA levels of the receptor. We conclude that TRIM21 acts post-translationally and maintains basal levels of PDGFRβ, thus suggesting that ubiquitination of PDGFRβ by TRIM21 may direct a portion of receptor for degradation in growing cells in a ligand-independent manner. Title in the list of papers of Niki Sarri's thesis: The E3 ubiquitin ligase TRIM21 modulates the basal levels of PDGFRβTitle in Web of Science: The E3 Ubiquitin Ligase TRIM21 Regulates Basal Levels of PDGFR beta

Glioblastoma Multiforme (GBM) is a poorly curable brain tumor because of its extremely invasive nature. Curcuminoids, as potential phytochemicals extracted from Curcuma Longa L., have been documented for their chemopreventive and antitumor activities against several types of malignancies. These compounds exert these effects via modulation of multiple signaling pathways and molecular targets at different stages of tumor progression, proliferation, and metastasis. In experimental studies, curcuminoids have demonstrated promising therapeutic benefits to overcome GBM. Curcuminoids have been shown to exert their anti-GBM effects through regulation of angiogenesis, apoptosis, autophagy, metastasis, invasion, as well as potential molecular targets, including receptor tyrosine kinases, Sonic Hedgehog, and NF-κB. This study reviews the observations regarding the impact of curcumin and its derivatives on GBM and the potential of translating the research findings into the clinic.

Neuronal remodeling after brain injury is essential for functional recovery. After unilateral cortical lesion, axons from the intact cortex ectopically project to the denervated midbrain, but the molecular mechanisms remain largely unknown. To address this issue, we examined gene expression profiles in denervated and intact mouse midbrains after hemispherectomy at early developmental stages using mice of either sex, when ectopic contralateral projection occurs robustly. The analysis showed that various axon growth-related genes were upregulated in the denervated midbrain, and most of these genes are reportedly expressed by glial cells. To identify the underlying molecules, the receptors for candidate upregulated molecules were knocked out in layer 5 projection neurons in the intact cortex, using the CRISPR/Cas9-mediated method, and axonal projection from the knocked-out cortical neurons was examined after hemispherectomy. We found that the ectopic projection was significantly reduced when integrin subunit β three or neurotrophic receptor tyrosine kinase 2 (also known as TrkB) was knocked out. Overall, the present study suggests that denervated midbrain-derived glial factors contribute to lesion-induced remodeling of the cortico-mesencephalic projection via these receptors.SIGNIFICANCE STATEMENTAfter brain injury, compensatory neural circuits are established that contribute to functional recovery. However, little is known about the intrinsic mechanism that underlies the injury-induced remodeling. We found that after unilateral cortical ablation expression of axon-growth promoting factors is elevated in the denervated midbrain and is involved in the formation of ectopic axonal projection from the intact cortex. Evidence further demonstrated that these factors are expressed by astrocytes and microglia, which are activated in the denervated midbrain. Thus, our present study provides a new insight into the mechanism of lesion-induced axonal remodeling and further therapeutic strategies after brain injury.

Internal tandem duplication within FLT3 (FLT3-ITD) is one of the most frequent mutations in acute myeloid leukemia (AML) and correlates with a poor prognosis. Whereas the FLT3 receptor tyrosine kinase is activated at the plasma membrane to transduce PI3K/AKT and RAS/MAPK signaling, FLT3-ITD resides in the endoplasmic reticulum and triggers constitutive STAT5 phosphorylation. Mechanisms underlying this aberrant FLT3-ITD subcellular localization or its impact on leukemogenesis remain poorly established. In this study, we discovered that FLT3-ITD is S-palmitoylated by the palmitoyl acyltransferase ZDHHC6. Disruption of palmitoylation redirected FLT3-ITD to the plasma membrane and rewired its downstream signaling by activating AKT and extracellular signal-regulated kinase pathways in addition to STAT5. Consequently, abrogation of palmitoylation increased FLT3-ITD–mediated progression of leukemia in xenotransplant-recipient mouse models. We further demonstrate that FLT3 proteins were palmitoylated in primary human AML cells. ZDHHC6-mediated palmitoylation restrained FLT3-ITD surface expression, signaling, and colonogenic growth of primary FLT3-ITD+ AML. More important, pharmacological inhibition of FLT3-ITD depalmitoylation synergized with the US Food and Drug Administration–approved FLT3 kinase inhibitor gilteritinib in abrogating the growth of primary FLT3-ITD+ AML cells. These findings provide novel insights into lipid-dependent compartmentalization of FLT3-ITD signaling in AML and suggest targeting depalmitoylation as a new therapeutic strategy to treat FLT3-ITD+ leukemias.

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.

Transglutaminase 2 (TG2) is a key epidermal squamous cell carcinoma cancer cell survival protein. However, how TG2 maintains the aggressive cancer phenotype is not well understood. The present studies show that TG2, which is highly expressed in epidermal cancer stem–like cells (ECS cells), maintains hepatocyte growth factor (HGF) signaling to drive an aggressive ECS cell cancer phenotype. Inhibiting TG2 reduces MET tyrosine kinase receptor expression and activity and attenuates the cancer cell phenotype. Moreover, inhibition of TG2 or HGF/MET function reduces downstream MEK1/2 and ERK1/2 activity, and this is associated with reduced cancer cell spheroid formation, invasion, and migration, and reduced stem and EMT marker expression. Treatment of TG2 knockdown cells with HGF partially restores the aggressive cancer phenotype, confirming that MET signaling is downstream of TG2. MET knockout reduces ERK1/2 signaling, doubles the time to initial tumor appearance, and reduces overall tumor growth. These findings suggest that TG2 maintains HGF/MET and MAPK (MEK1/2 and ERK1/2) signaling to drive the aggressive ECS cell cancer phenotype and tumor formation, and that TG2-dependent MET signaling may be a useful anti-cancer target. Implications: TG2 is an important epidermal squamous cell carcinoma stem cell survival protein. We show that TG2 activates an HGF/MET, MEK1/2 ERK1/2 signaling cascade that maintains the aggressive cancer phenotype.

Gastric cancer is among the most prevalent and deadliest of cancers globally. To derive mechanistic insight into the pathways governing this disease, we generated a Claudin18-IRES-CreERT2 allele to selectively drive conditional dysregulation of the Wnt, Receptor Tyrosine Kinase and Trp53 pathways within the gastric epithelium. This resulted in highly reproducible metastatic, chromosomal-instable-type gastric cancer. In parallel, we developed orthotopic cancer organoid transplantation models to evaluate tumour-resident Lgr5+ populations as functional cancer stem cells via in vivo ablation. We show that Cldn18 tumours accurately recapitulate advanced human gastric cancer in terms of disease morphology, aberrant gene expression, molecular markers and sites of distant metastases. Importantly, we establish that tumour-resident Lgr5+ stem-like cells are critical to the initiation and maintenance of tumour burden and are obligatory for the establishment of metastases. These models will be invaluable for deriving clinically relevant mechanistic insights into cancer progression and as preclinical models for evaluating therapeutic targets. Fatehullah et al. develop transgenic and orthotopic mouse models to recapitulate advanced human gastric cancer and uncover a mechanistic role for Lgr5+ stem-like cells in promoting disease initiation and progression.

Met tyrosine kinase, a receptor for a hepatocyte growth factor (HGF), plays a critical role in tumor growth, metastasis, and drug resistance. Mitochondria are highly dynamic and undergo fission and fusion to maintain a functional mitochondrial network. Dysregulated mitochondrial dynamics are responsible for the progression and metastasis of many cancers. Here, using structured illumination microscopy (SIM) and high spatial and temporal resolution live cell imaging, we identified mitochondrial trafficking of receptor tyrosine kinase Met. The contacts between activated Met kinase and mitochondria formed dramatically, and an intact HGF/Met axis was necessary for dysregulated mitochondrial fission and cancer cell movements. Mechanically, we found that Met directly phosphorylated outer mitochondrial membrane protein Fis1 at Tyr38 (Fis1 pY38). Fis1 pY38 promoted mitochondrial fission by recruiting the mitochondrial fission GTPase dynamin-related protein-1 (Drp1) to mitochondria. Fragmented mitochondria fueled actin filament remodeling and lamellipodia or invadopodia formation to facilitate cell metastasis in hepatocellular carcinoma (HCC) cells both in vitro and in vivo. These findings reveal a novel and noncanonical pathway of Met receptor tyrosine kinase in the regulation of mitochondrial activities, which may provide a therapeutic target for metastatic HCC.

Primary cilia are solitary, microtubule-based structures emanating from the surface of most vertebrate cells. Although it is understood that ciliary assembly and disassembly both depend upon and impact cell cycle progression, critical mechanistic details of these links remain unresolved. Accumulating evidence shows that the signaling pathways downstream of receptor tyrosine kinases and lysophosphatidic acid receptors control the dynamics of primary cilia. It has also become clear that primary cilia not only serve as signaling hubs but also regulate the composition of the surrounding membrane, which is likely to affect the response to growth factors. Here, we overview recent advances in understanding the interplay between primary cilia and the cell cycle, with a focus on growth factor signaling pathways.

HER2 belongs to the ErbB sub-family of receptor tyrosine kinases and regulates cellular proliferation and growth. Different from other ErbB receptors, HER2 has no known ligand. Activation occurs through heterodimerization with other ErbB receptors and their cognate ligands. This suggests several possible activation paths of HER2 with ligand-specific, differential response, which so far remained unexplored. Using single-molecule tracking and the diffusion profile of HER2 as a proxy for activity, we measured the activation strength and temporal profile in live cells. We found that HER2 is strongly activated by EGFR-targeting ligands EGF and TGF$, yet with a distinguishable temporal fingerprint. The HER4-targeting ligands EREG and NRG$1 showed weaker activation of HER2, a preference for EREG and a delayed response to NRG$1. Our results indicate a selective ligand response of HER2 that may serve as a regulatory element. Our experimental approach is easily transferable to other membrane receptors targeted by multiple ligands. Highlights ![Figure][1]textless/imgtextgreater Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

Introduction c-Met tyrosine kinase receptor is a high-affinity ligand of hepatocyte growth factor (HGF). c-Met is widely expressed in a variety of normal human tissues, but it exhibits abnormally high expression, amplification or mutation in tumor tissues such as lung cancer, gastric cancer, and breast cancer. Therefore, the use of c-Met as a target can achieve the inhibition of a series of abnormal physiological processes such as tumourigenesis, development and metastasis. A number of small molecule tyrosine kinase inhibitors targeting c-Met have been successfully marketed. Areas covered This article reviews recent advances in patented c-Met small molecule inhibitors and their inhibitory activity against various cancer cells from 2018 to date. Expert opinion To date, small molecule inhibitors targeting c-Met have demonstrated impressive therapeutic efficacy in the clinical setting. Most recent patents have focused on addressing the direction of c-Met amplification and overexpression. Despite the great success in the development of selective c-Met inhibitors, the effects of bypass secretion and mutagenesis have led to a need for new c-Met small molecule inhibitors that are safe, efficient, selective and less toxic with novel structures and effective against other targets.

The proto-oncogene ALK encodes anaplastic lymphoma kinase, a receptor tyrosine kinase that is expressed primarily in the developing nervous system. After development, ALK activity is associated with learning and memory1 and controls energy expenditure, and inhibition of ALK can prevent diet-induced obesity2. Aberrant ALK signalling causes numerous cancers3. In particular, full-length ALK is an important driver in paediatric neuroblastoma4,5, in which it is either mutated6 or activated by ligand7. Here we report crystal structures of the extracellular glycine-rich domain (GRD) of ALK, which regulates receptor activity by binding to activating peptides8,9. Fusing the ALK GRD to its ligand enabled us to capture a dimeric receptor complex that reveals how ALK responds to its regulatory ligands. We show that repetitive glycines in the GRD form rigid helices that separate the major ligand-binding site from a distal polyglycine extension loop (PXL) that mediates ALK dimerization. The PXL of one receptor acts as a sensor for the complex by interacting with a ligand-bound second receptor. ALK activation can be abolished through PXL mutation or with PXL-targeting antibodies. Together, these results explain how ALK uses its atypical architecture for its regulation, and suggest new therapeutic opportunities for ALK-expressing cancers such as paediatric neuroblastoma. Analysis of crystal structures of anaplastic lymphoma kinase elucidate the mechanism by which ligand binding and the glycine-rich domain regulate its activity.

Ras genes are frequently mutated in many cancer types; however, there are currently no conclusively effective anticancer drugs against Ras-induced cancer. Therefore, the downstream effectors of Ras signaling need to be identified for the development of promising novel therapeutic approaches. We previously reported that oncogenic Ras induced the expression of NF-HEV/IL-33, a member of the interleukin-1 family, and showed that intracellular IL-33 was required for oncogenic Ras-induced cellular transformation. In the present study, we demonstrated that the c-Mer proto-oncogene tyrosine kinase (MerTK), a receptor tyrosine kinase, played essential roles in oncogenic Ras/IL-33 signaling. The expression of MerTK was enhanced in transformed NIH-3T3 cells by the expression of oncogenic Ras, H-Ras (G12V), in an IL-33-dependent manner. In human colorectal cancer tissues, MerTK expression also correlated with IL-33 expression. The knockdown of IL-33 or MerTK effectively attenuated the migration of NIH-3T3 cells transformed by H-Ras (G12V) and A549, LoVo, and HCT116 cells harboring an oncogenic K-Ras mutation. Furthermore, the suppression of Ras-induced cell migration by the knockdown of IL-33 was rescued by the enforced expression of MerTK. The present results also revealed that MerTK was effectively phosphorylated in NIH-3T3 cells transformed by Ras (G12V). Ras signaling was essential for the tyrosine phosphorylation of MerTK, and the kinase activity of MerTK was indispensable for accelerating cell migration. Collectively, the present results reveal a novel role for MerTK in cancer malignancy, which may be utilized to develop novel therapeutic strategies that target Ras-transformed cells.

Penttinen type of premature aging syndrome is an extremely rare progeroid disorder, caused by activating variants in the receptor tyrosine kinase domain of the PDGFRB gene. Only eight individuals have been previously reported worldwide, with a consistent phenotype of prematurely aged appearance, lipoatrophy, hypertrophic skin lesions, proptosis, malar hypoplasia, and marked acro-osteolysis. We report the first patient of Penttinen syndrome from India, with novel radiographic findings of terminal phalangeal tufting, thereby expanding the phenotypic spectrum of Penttinen syndrome.

The HER3/ERBB3 receptor is an oncogenic receptor tyrosine kinase that forms heterodimers with EGFR family members and is overexpressed in numerous cancers. HER3 overexpression associates with reduced survival and acquired resistance to targeted therapies, making it a potential therapeutic target in multiple cancer types. Here, we report on immunogenic, promiscuous MHC class II–binding HER3 peptides, which can generate HER3-specific CD4+ Th1 antitumor immune responses. Using an overlapping peptide screening methodology, we identified nine MHC class II–binding HER3 epitopes that elicited specific Th1 immune response in both healthy donors and breast cancer patients. Most of these peptides were not identified by current binding algorithms. Homology assessment of amino acid sequence BLAST showed >90% sequence similarity between human and murine HER3/ERBB3 peptide sequences. HER3 peptide–pulsed dendritic cell vaccination resulted in anti-HER3 CD4+ Th1 responses that prevented tumor development, significantly delayed tumor growth in prevention models, and caused regression in multiple therapeutic models of HER3-expressing murine tumors, including mammary carcinoma and melanoma. Tumors were robustly infiltrated with CD4+ T cells, suggesting their key role in tumor rejection. Our data demonstrate that class II HER3 promiscuous peptides are effective at inducing HER3-specific CD4+ Th1 responses and suggest their applicability in immunotherapies for human HER3-overexpressing tumors.

Phosphatidylinositol-3’-kinases (PI3Ks) are a family of lipid kinases that phosphorylate the 3’ hydroxyl (OH) of the inositol ring of phosphatidylinositides (PI). Through their downstream effectors, PI3K generated lipids (PI3K-lipids hereafter) such as PI(3,4,5) P(3) and PI(3,4)P(2) regulate myriad biochemical and biological processes in both normal and cancer cells including responses to growth hormones and cytokines; the cell division cycle; cell death; cellular growth; angiogenesis; membrane dynamics; and autophagy and many aspects of cellular metabolism. Engagement of receptor tyrosine kinase by their cognate ligands leads to activation of members of the Class I family of PI3’-kinases (PI3Kα, β, δ & γ) leading to accumulation of PI3K-lipids. Importantly, PI3K-lipid accumulation is antagonized by the hydrolytic action of a number of PI3K-lipid phosphatases, most notably the melanoma suppressor PTEN (lipid phosphatase and tensin homologue). Downstream of PI3K-lipid production, the protein kinases AKT1-3 are believed to be key effectors of PI3’-kinase signalling in cells. Indeed, in preclinical models, activation of the PI3K→AKT signalling axis cooperates with alterations such as expression of the BRAF(V600E) oncoprotein kinase to promote melanoma progression and metastasis. In this review, we describe the different classes of PI3K-lipid effectors, and how they may promote melanomagenesis, influence the tumour microenvironment, melanoma maintenance and progression to metastatic disease. We also provide an update on both FDA-approved or experimental inhibitors of the PI3K→AKT pathway that are currently being evaluated for the treatment of melanoma either in preclinical models or in clinical trials.

Tumor associated macrophages (TAMs) play a major role in regulating mammary tumor growth and in directing the responses of tumor infiltrating leukocytes in the microenvironment. However, macrophage-specific mechanisms regulating the interactions of macrophages with tumor cells and other leukocytes that support tumor progression have not been extensively studied. In this study, we show that the activation of the RON receptor tyrosine kinase signaling pathway specifically in macrophages supports breast cancer growth and metastasis. Using clinically relevant murine models of breast cancer, we demonstrate that loss of macrophage RON expression results in decreases in mammary tumor cell proliferation, survival, cancer stem cell self-renewal, and metastasis. Macrophage RON signaling modulates these phenotypes via direct effects on the tumor proper and indirectly by regulating leukocyte recruitment including macrophages, T-cells, and B-cells in the mammary tumor microenvironment. We further show that macrophage RON expression regulates the macrophage secretome including IL-35 and other immunosuppressive factors. Overall, our studies implicate activation of RON signaling in macrophages as a key player in supporting a thriving mammary pro-tumor microenvironment through novel mechanisms including the augmentation of tumor cell properties through IL-35.

As protein-protein interactions (PPIs) are involved in many cellular events, development of mammalian cytosolic PPI detection systems is important for drug discovery as well as understanding biological phenomena. We have previously reported a c-kit-based PPI screening (KIPPIS) system, in which proteins of interest were fused with a receptor tyrosine kinase c-kit, leading to intracellular PPI-dependent cell growth. However, it has not been investigated whether PPI can be detected using other receptors. In this study, we employed a thrombopoietin receptor, which belongs to the Type I cytokine receptor family, to develop a thrombopoietin receptor-based PPI screening (THROPPIS) system. To improve the sensitivity of THROPPIS, we examined two strategies of (i) localization of the chimeric receptors on the cell membrane, and (ii) addition of a helper module to the chimeric receptors. Intriguingly, the nonlocalized chimeric receptor showed the best performance of THROPPIS. Furthermore, the addition of the helper module dramatically improved the detection sensitivity. In total, 5 peptide-domain interactions were detected successfully, demonstrating the versatility of THROPPIS. In addition, a peptide-domain interaction was detected even when insulin receptor or epidermal growth factor receptor was used as a signaling domain, demonstrating that this PPI detection system can be extended to other receptors.

Activated Cdc42-associated kinase 1 (ACK1/TNK2) is a nonreceptor tyrosine kinase with a unique structure. It not only can act as an activated transmembrane effector of receptor tyrosine kinases (RTKs) to transmit various RTK signals but also can play a corresponding role in epigenetic regulation. A number of studies have shown that ACK1 is a carcinogenic factor. Blockage of ACK1 has been proven to be able to inhibit cancer cell survival, proliferation, migration, and radiation resistance. Thus, ACK1 is a promising potential antitumor target. To date, despite many efforts to develop ACK1 inhibitors, no specific small molecule inhibitors have entered clinical trials. This Perspective provides an overview of the structural features, biological functions, and association with diseases of ACK1 and in vitro and in vivo activities, selectivity, and therapeutic potential of small molecule ACK1 inhibitors with different chemotypes.

Background Human epidermal growth factor receptor 2 (HER2) is tyrosine kinase receptor that belongs to the ErbB family and is overexpressed on the membrane surface of various cancer cells, including small cell lung cancer (SCLC); however, no HER2 targeted therapy for SCLC have yet been established. Near‐infrared photoimmunotherapy (NIR‐PIT) is a novel cancer therapy based on photo‐absorber, IRDye‐700DX (IR700), ‐antibody conjugates, and near‐infrared (NIR) light. Methods We used HER2‐positive SCLC parental cell lines (SBC‐3) and its chemoresistant cell lines, and examined therapeutic efficacy of HER2 targeting NIR‐PIT using anti HER2 antibody trastuzumab. Results We found that HER2 expression was upregulated on chemoresistant cell lines, especially cisplatin‐resistance (SBC‐3/CDDP). In vitro, the rate of cell death increased with the amount of NIR‐light irradiation, and it was significantly higher in SBC‐3/CDDP than in SBC‐3. In vivo, tumor growth was more suppressed in SBC‐3/CDDP group than in SBC‐3 group, and survival period tended to be prolonged. Conclusion In this study, we demonstrated that HER2 targeting NIR‐PIT using trastuzumab is promising therapy for HER2‐positive SCLC, and is more effective when HER2 expression is upregulated due to CDDP resistance, suggesting that the HER2 expression level positively corelated with the efficacy of NIR‐PIT., We found that human epidermal growth factor receptor 2 (HER2) was expressed in small‐cell lung cancer (SCLC) parental cell lines (SBC‐3) and its chemoresistant cell lines, and HER2 was upregulated in chemoresistant SCLC cells, especially CDDP‐resistant cells (SBC‐3/CDDP). The HER2‐targeting near‐infrared photoimmunotherapy (NIR‐PIT) using an anti‐HER2 antibody, trastuzumab, induced HER2‐expressing SCLC cells death, and efficacy of this therapy was significantly higher in SBC‐3/CDDP than in SBC‐3 in vitro and in vivo. These findings suggest a positive correlation between the cancer antigen expression level and the NIR‐PIT effect. Implications of all the available evidence. Our finding suggests that NIR‐PIT targeting HER2 using trastuzumab is a novel therapeutic approach for HER2‐positive SCLC when SCLC acquired CDDP‐based chemotherapy resistance and HER2 expression was upregulated.

Platelet-derived growth factor-beta (PDGFRB) gene maps for the receptor tyrosine kinase PDGRFβ. PDGFRB gene fusions have been implicated in multiple myeloid and lymphoid neoplasms and have shown exquisite sensitivity to tyrosine kinase inhibitors. We report a case of a 29-year-old male who presented with acute myeloid leukemia who was eventually found to harbor a unique three-way translocation t(5;7;7)(q33.2;q32;q11.2) involving the PDGFRB gene. The patient initially achieved a complete response after induction with daunorubicin and cytarabine, but when he returned for consolidation, his white cell count had increased, and he was found to have an underlying myeloproliferative neoplasm. He was given consolidation with high-dose cytarabine and imatinib with excellent response, and ultimately received a matched unrelated donor transplant. The patient remains in remission to this day more than eight years later.

Mitochondria are ubiquitous membrane-bound organelles that not only play a key role in maintaining cellular energy homeostasis and metabolism but also in signaling and apoptosis. Aryl hydrocarbons receptors (AhRs) are ligand-activated transcription factors that recognize a wide variety of xenobiotics, including polyaromatic hydrocarbons and dioxins, and activate diverse detoxification pathways. These receptors are also activated by natural dietary compounds and endogenous metabolites. In addition, AhRs can modulate the expression of a diverse array of genes related to mitochondrial biogenesis and function. The aim of the present review is to analyze scientific data available on the AhR signaling pathway and its interaction with the intracellular signaling pathways involved in mitochondrial functions, especially those related to cell cycle progression and apoptosis. Various evidence have reported the crosstalk between the AhR signaling pathway and the nuclear factor κB (NF-κB), tyrosine kinase receptor signaling and mitogen-activated protein kinases (MAPKs). The AhR signaling pathway seems to promote cell cycle progression in the absence of exogenous ligands, whereas the presence of exogenous ligands induces cell cycle arrest. However, its effects on apoptosis are controversial since activation or overexpression of AhR has been observed to induce or inhibit apoptosis depending on the cell type. Regarding the mitochondria, although activation by endogenous ligands is related to mitochondrial dysfunction, the effects of endogenous ligands are not well understood but point towards antiapoptotic effects and inducers of mitochondrial biogenesis.

The human EGF receptor family plays pivotal roles in physiology and cancer, which contains four closely-related members: HER1/EGFR, HER2, HER3 and HER4. Previously, it was found that the mitogen-inducible gene 6 (Mig6) protein is a negative regulator of EGFR and HER2 by using its S1 segment to bind at the kinase dimerization interface. However, it is still unclear whether the S1 segment can also effectively target HER3 and HER4? Here, we performed a systematic investigation to address this issue. The segment can bind to all the four HER kinases with a varying affinity and moderate selectivity; breaking of the segment into shorter hotspot peptides would largely impair the affinity and selectivity, indicating that the full-length sequence is required for the effective binding of S1 to these kinases. The hs2 peptide, which corresponds to the middle hotspot region of S1 segment, can partially retain the affinity to HER kinases, can moderately compete with S1 segment at the dimerization interfaces, and can mimic the biological function of Mig6 protein to suppress HER4+ esophageal cancer at cellular level. In addition, we also analyzed the binding potency of S1 segment and hs2 peptide to the kinase domains of other five widely documented growth factor receptors (GFRs). It was showed that both the S1 and hs2 cannot effectively interact with these receptors. Overall, the Mig6 is suggested as a specific pan-HER inhibitor, which can target and suppress HER family members with a broad selectivity, but exhibits weak or no activity towards other GFRs.

Background Circular RNAs (circRNAs) are increasingly recognized as important regulators in cancer including ovarian cancer (OC). This work focuses on the effects of circ_0000745 on the OC development of and molecules involved. Methods Expression of circ_0000745 in collected OC tissues and the acquired OC cell lines was examined by RT-qPCR. The stability of circ_0000745 in cells was examined by RNase R treatment. The target transcripts interacted with circ_0000745 were predicted using bioinformatic systems. Gain- and loss-of-function studies of circ_0000745, microRNA (miR)-3187-3p and erb-b2 receptor tyrosine kinase 4 (ERBB4) were conducted to determine their functions on proliferation, migration, invasion and stem cell property of OC cells. Results Circ_0000745 and ERBB4 were abundantly expressed while miR-3187-3p was poorly expressed in OC tissues and cells. Circ_0000745 sequestered miR-3187-3p and blocked its repressive effect on ERBB4. Downregulation of circ_0000745 reduced proliferation, aggressiveness, epithelial-mesenchymal transition, and stemness of SK-OV-3 cells, but this reduction was blocked upon miR-3187-3p inhibition or ERBB4 upregulation. By contrast, artificial induction of circ_0000745 upregulation, miR-3187-3p upregulation and ERBB4 downregulation led to inverse trends in ES-2 cells. ERBB4 promoted the phosphorylation of the PI3K/AKT signaling pathway. An RNA binding protein IGF2BP2 was found to circ_0000745 bind to and promote its expression and stability. Conclusion This study demonstrated that circ_0000745 upregulated by IGF2BP2 promotes aggressiveness and stemness of OC cells through a miR-3187-3p/ERBB4/PI3K/AKT axis. Circ_0000745 may serve as a promising target for OC treatment.

The NTRK gene family is composed of NTRK1, NTRK2, and NTRK3, which encode three tropomyosin-receptor kinases, belonging to a class of tyrosine kinase receptors. These proteins are known to play roles in cell proliferation, differentiation, apoptosis, and survival. Fusions involving the NTRK genes are long known as drivers in many tumors. Although they occur in less than 5% of all malignancies, their occurrence in a great diversity of tumors has been documented. Several rare tumors including infantile fibrosarcoma, secretory breast carcinoma, and mammary analogue secretory carcinoma are accompanied by NTRK fusions in more than 90% of cases, demonstrating a diagnostic value for the NTRK fusion testing in these tumors. More recently, the development of effective targeted therapies has created a demand for their detection in all malignancies. A variety of approaches are available for testing including immunohistochemistry, fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR), and DNA- and RNA-based next-generation sequencing (NGS). This article reviews the molecular biology and tumorigenesis of NTRK fusions, their prevalence and clinical significance with a focus on available methods for fusion detection. The advantages and limitations of different technologies, the best practice algorithms for NTRK fusion detection, and the future direction of NTRK testing are also discussed.

PURPOSE The tyrosine kinase receptor anaplastic lymphoma kinase (ALK) can be abnormally activated in neuroblastoma, and somatic ALK mutations occur in 6%-10% of patients. The differential clinical impact of these mutations has not been clearly elucidated. METHODS Data on patients with neuroblastoma harboring ALK mutations were retrospectively analyzed. ALK sequencing was performed by whole-genome sequencing, hybrid-based capture of targeted exomes, or hotspot ALK mutation profiling. The differential impact of ALK mutation site on clinical characteristics, response to treatment, and survival was analyzed. In a subgroup of patients with locoregional neuroblastoma diagnosed after 2014, the impact of all ALK mutations was compared with wild-type ALK. RESULTS Of 641 patients with neuroblastoma with ALK status analyzed on at least one tumor sample, 103 (16%) had tumors harboring ALK mutations. Mutations existed across all ages (birth to 67.8 years), stages (30% locoregional and 70% metastatic), and risk groups (20%, 11%, and 69% with low-, intermediate-, and high-risk disease, respectively). Mutation sites included F1174 (51%), R1275 (29%), R1245 (10%), and others (10%). Mutation site was not prognostic for progression-free survival or overall survival in the entire cohort, high-risk subgroup, or locoregional subgroup. Locoregional tumors with any ALK mutation were generally invasive: L2 by International Neuroblastoma Research Group staging in 30/31 patients with a 2-year progression-free survival (59%, 95% CI, 37.4 to 80.5) that was inferior to historical controls. This observation was corroborated in the post-2014 subgroup in which gross total resection was less likely for ALK-mutated tumors. CONCLUSION Somatic ALK mutations are present across all stages and risk groups of neuroblastoma. No specific mutation carries differential prognostic significance. Locoregional neuroblastoma has an invasive phenotype when harboring somatic ALK mutations in this population.

Abnormal vascular smooth muscle cell (VSMC) proliferation has an important role in the pathogenesis of both atherosclerosis restenosis and hypertension. Vascular endothelial growth factor (VEGF) has been shown to stimulate VSMC proliferation. In addition, angiogenesis is one of the hallmarks of cancerous growth. VEGF is the key modulator for the initial stages of angiogenesis that acts through the endothelial-specific receptor tyrosine kinases (VEGFRs). VEGFR-2 blockage is a good approach for suppression of angiogenesis. In order to discover novel VEGFR-2 TK inhibitors, we have designed and synthesized three new series of pyridine-containing compounds. The new compounds were all screened against a panel of three cell lines (HepG-2, HCT-116, and MCF-7). Promising results encouraged us to additionally evaluate the most active members for their in vitro VEGFR-2 inhibitory effect. Compound 7a, which is the most potent candidate, revealed a significant increase in caspase-3 level by 7.80-fold when compared to the control. In addition, Bax and Bcl-2 concentration levels showed an increase in the proapoptotic protein Bax (261.4 Pg/ml) and a decrease of the antiapoptotic protein Bcl-2 (1.25 Pg/ml) compared to the untreated cells. Furthermore, compound 7a arrested the cell cycle in the G2/M phase with induction of apoptosis. The immunomodulatory effect of compound 7a, the most active member, showed a reduction in TNF-α by 87%. Also, compound 7a caused a potent inhibitory effect on smooth muscle proliferation. Docking studies were also performed to get better insights into the possible binding mode of the target compounds with VEGFR-2 active sites.

Cabozantinib, mainly targeting cMet and vascular endothelial growth factor receptor 2, is the second-line treatment for patients with advanced hepatocellular carcinoma (HCC). However, the lower response rate and resistance limit its enduring clinical benefit. In this study, we found that cMet-low HCC cells showed primary resistance to cMet inhibitors, and the combination of cabozantinib and mammalian target of rapamycin (mTOR) inhibitor, rapamycin, exhibited a synergistic inhibitory effect on the in vitro cell proliferation and in vivo tumor growth of these cells. Mechanically, the combination of rapamycin with cabozantinib resulted in the remarkable inhibition of AKT, extracellular signal-regulated protein kinases, mTOR, and common downstream signal molecules of receptor tyrosine kinases; decreased cyclin D1 expression; and induced cell cycle arrest. Meanwhile, rapamycin enhanced the inhibitory effects of cabozantinib on the migration and tubule formation of human umbilical vascular endothelial cells and human growth factor-induced invasion of cMet inhibitor-resistant HCC cells under hypoxia condition. These effects were further validated in xenograft models. In conclusion, our findings uncover a potential combination therapy of cabozantinib and rapamycin to combat cabozantinib-resistant HCC.

Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2) plays a role in receptor tyrosine kinase (RTK), neurofibromin-1 (NF-1), and Kirsten rat sarcoma virus (KRAS) mutant-driven cancers, as well as in RTK-mediated resistance, making the identification of small-molecule therapeutics that interfere with its function of high interest. Our quest to identify potent, orally bioavailable, and safe SHP2 inhibitors led to the discovery of a promising series of pyrazolopyrimidinones that displayed excellent potency but had a suboptimal in vivo pharmacokinetic (PK) profile. Hypothesis-driven scaffold optimization led us to a series of pyrazolopyrazines with excellent PK properties across species but a narrow human Ether-a-go-go-Related Gene (hERG) window. Subsequent optimization of properties led to the discovery of the pyrimidinone series, in which multiple members possessed excellent potency, optimal in vivo PK across species, and no off-target activities including no hERG liability up to 100 μM. Importantly, compound 30 (IACS-15414) potently suppressed the mitogen-activated protein kinase (MAPK) pathway signaling and tumor growth in RTK-activated and KRASmut xenograft models in vivo.

The ERBB3 is a member of human receptor tyrosine kinase ErbB family involved in various cellular processes, which has been recognized as a new and promising druggable target of breast cancer and some other gynecological tumors. The ERRFI1 protein is a natural negative regulator of ErbB signaling and has been found to inactivate ERBB3 kinase activity by directly disrupting the kinase dimerization with its ErbB-binding region (EBR). The ERRFI1 EBR domain contains two functional segments 1 and 2 as well as a flexible loop (FL) linker between the two segments, which can only weakly interact with ERBB3 kinase domain to form a reversible, transient encounter complex. Here, we demonstrated that the binding affinity and recognition specificity of ERBB3 interaction with ERRFI1 FL peptides can be improved essentially by just adding a designed nonbonded interaction system across the kinase–peptide complex interface. The nonbonded system consists of one or two hydrogen bonds as well as a double aromatic stacking. High-level theoretical calculations indicated that these nonbonded forces can work together to confer considerable stability and specificity to the complex, thus making ERRFI1-derived peptides ‘bindable’ to ERBB3 kinase domain. The theoretical analysis was further substantiated by calorimetric study, which revealed that the designed nonbonded system can improve the kinase–peptide binding free energy by − 5 to – 20 kJ/mol with different arrangements of its nonbonded forces.

The ErbB family of receptor tyrosine kinases (RTKs) contains four members: EGFR, ErbB2, ErbB3 and ErbB4; they are involved in the tumorigenesis of diverse cancers and can be inhibited natively by receptor-associated late transducer (RALT), a negative feedback regulator of ErbB signaling in human hepatocytes and hepatocellular carcinoma. Although the biological effects of RALT on EGFR kinase have been widely documented previously, the binding behavior of RALT to other ErbB/RTK kinases still remains largely unexplored. Here, the intermolecular interactions of RALT ErbB-binding region (EBR) as well as its functional sections and peptide segments with ErbBs and other human RTKs were systematically investigated at molecular and structural levels, from which we were able to identify those potential kinase targets of RALT protein, and to profile the affinity, specificity and cross-reactivity of RALT EBR domain and its sub-regions against various RTKs. It is revealed that RALT can target all the four ErbB kinases with high affinity for EGFR/ErbB2/ErbB4 and moderate affinity for ErbB3, but generally exhibits modest affinity to other RTKs, albeit few kinases such as LTK, EPHB6, MET and MUSK were also top-ranked as the unexpected targets of RALT. Peptide segments covering the key binding regions of RALT EBR domain were identified with computational alanine scanning, which were then optimized to obtain a number of designed peptide mutants with improved selectivity between different top-ranked RTKs.

Background Urothelial carcinoma (UC) accounts for > 90% of canine tumors occurring in the urinary bladder. Toceranib phosphate (TOC) is a multi-target receptor tyrosine kinase (RTK) inhibitor that exhibits activity against members of the split kinase family of RTKs. The purpose of this study was to evaluate primary UC tumors and UC cell lines for the expression and activation of VEGFR2, PDGFRα, PDGFRβ, and KIT to assess whether dysregulation of these RTKs may contribute to the observed biological activity of TOC. Results Transcript for VEGFR2, PDGFRα, PDGFRβ, and KIT was detected in all UC tissue samples and UC cell lines. The Proteome Profiler™ Human Phospho-RTK Array Kit (R & D Systems) provided a platform to assess phosphorylation of 42 different RTKs in primary UC tumors and UC cell lines. Evidence of PDGFRα and PDGFRβ phosphorylation was present in only 11% or 33% of UC tumors, respectively, and 25% of UC cell lines. Treatment of UC cell lines with TOC had no significant impact on cell proliferation, including UC cell lines with evidence of PDGFRβ phosphorylation. Conclusions Phosphorylation of several key RTKs targeted by TOC is present in a small subset of primary UC tumors and UC cell lines, suggesting that these RTKs do not exist in a state of continuous activation. These data suggest that activation of RTKs targeted by TOC is present in a small subset of UC tumors and UC cell lines and that treatment with TOC at physiologically relevant concentrations has no direct anti-proliferative effect on UC cells.