From bench to bedside: Advancing towards therapeutic treatment of vestibular schwannomas.

Vestibular schwannomas are rare intracranial tumors originating from Schwann cells of the vestibular nerve. Despite their benign nature, these tumors can exert significant mass effects and debilitating symptoms, including gradual hearing loss, vertigo, facial nerve dysfunction, and headaches. Current clinical management options encompass wait-and-scan, surgery, radiation therapy, and off-label medication. However, each approach exhibits its own challenges and harbors limitations that underscore the urgent need for therapeutic treatments. Over the past 2 decades, extensive elucidation of the molecular underpinnings of vestibular schwannomas has unraveled genetic anomalies, dysregulated signaling pathways, downstream of receptor tyrosine kinases, disrupted extracellular matrix, inflammatory tumor microenvironment, and altered cerebrospinal fluid composition as integral factors in driving the development and progression of the disease. Armed with this knowledge, novel therapeutic interventions tailored to the unique molecular characteristics of those conditions are actively being pursued. This review underscores the urgency of addressing the dearth of Food and Drug Administration-approved drugs for vestibular schwannoma, highlighting the key molecular discoveries and their potential translation into therapeutics. It provides an in-depth exploration of the evolving landscape of therapeutic development, which is currently advancing from bench to bedside. These ongoing efforts hold the promise of significantly transforming the lives of vestibular schwannoma patients in the future.

Mechanisms of acquired resistance to HER2-Positive breast cancer therapies induced by HER3: A comprehensive review.

Receptor tyrosine kinases (RTKs) are cell surface receptors with kinase activity that play a crucial role in diverse cellular processes. Among the RTK family members, Human epidermal growth factor receptor 2 (HER2) and HER3 are particularly relevant to breast cancer. The review delves into the complexities of receptor tyrosine kinase interactions, resistance mechanisms, and the potential of anti-HER3 drugs, offering valuable insights into the clinical implications and future directions in this field of study. It assesses the potential of anti-HER3 drugs, such as pertuzumab, in overcoming resistance observed in HER2-positive breast cancer therapies. The review also explores the resistance mechanisms associated with various drugs, including trastuzumab, lapatinib, and PI3K inhibitors, providing insights into the intricate molecular processes underlying resistance development. The review concludes by emphasizing the necessity for further clinical trials to assess the efficacy of HER3 inhibitors and the potential of developing safe and effective anti-HER3 treatments to improve treatment outcomes for patients with HER2-positive breast cancer.

Decoding how receptor tyrosine kinases (RTKs) mediate nuclear calcium signaling.

Calcium (Ca2+) is a highly versatile intracellular messenger that regulates several cellular processes. Although it is unclear how a single-second messenger coordinates various effects within a cell, there is growing evidence that spatial patterns of Ca2+ signals play an essential role in determining their specificity. Ca2+ signaling patterns can differ in various cell regions, and Ca2+ signals in the nuclear and cytoplasmic compartments have been observed to occur independently. The initiation and function of Ca2+ signaling within the nucleus are not yet fully understood. Receptor tyrosine kinases (RTKs) induce Ca2+ signaling resulting from phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and inositol 1,4,5-trisphosphate (InsP3) formation within the nucleus. This signaling mechanism may be responsible for the effects of specific growth factors on cell proliferation and gene transcription. This review highlights the recent advances in RTK trafficking to the nucleus and explains how these receptors initiate nuclear calcium signaling.

Reconstructing Signaling Networks Using Biosensor Barcoding.

Understanding how signaling networks are regulated offers valuable insights into how cells and organisms react to internal and external stimuli and is crucial for developing novel strategies to treat diseases. To achieve this, it is necessary to delineate the intricate interactions between the nodes in the network, which can be accomplished by measuring the activities of individual nodes under perturbation conditions. To facilitate this, we have recently developed a biosensor barcoding technique that enables massively multiplexed tracking of numerous signaling activities in live cells using genetically encoded fluorescent biosensors. In this chapter, we detail how we employed this method to reconstruct the EGFR signaling network by systematically monitoring the activities of individual nodes under perturbations.

De Novo Multi-Omics Pathway Analysis Designed for Prior Data Independent Inference of Cell Signaling Pathways.

New tools for cell signaling pathway inference from multi-omics data that are independent of previous knowledge are needed. Here, we propose a new de novo method, the de novo multi-omics pathway analysis (DMPA), to model and combine omics data into network modules and pathways. DMPA was validated with published omics data and was found accurate in discovering reported molecular associations in transcriptome, interactome, phosphoproteome, methylome, and metabolomics data, and signaling pathways in multi-omics data. DMPA was benchmarked against module discovery and multi-omics integration methods and outperformed previous methods in module and pathway discovery especially when applied to datasets of relatively low sample sizes. Transcription factor, kinase, subcellular location, and function prediction algorithms were devised for transcriptome, phosphoproteome, and interactome modules and pathways, respectively. To apply DMPA in a biologically relevant context, interactome, phosphoproteome, transcriptome, and proteome data were collected from analyses carried out using melanoma cells to address gamma-secretase cleavage-dependent signaling characteristics of the receptor tyrosine kinase TYRO3. The pathways modeled with DMPA reflected the predicted function and its direction in validation experiments.

Kaempferide triggers apoptosis and paraptosis in pancreatic tumor cells by modulating the ROS production, SHP-1 expression, and the STAT3 pathway.

Pancreatic cancer is one of the deadliest diseases with a poor prognosis and a five-survival rate. The STAT3 pathway is hyperactivated which contributes to the sustained proliferative signals in pancreatic cancer cells. We have isolated kaempferide (KF), an O-methylated flavonol, from the green propolis of Mimosa tenuiflora and examined its effect on two forms of cell death namely, apoptosis and paraptosis. KF significantly increased the cleavage of caspase-3 and PARP. It also downmodulated the expression of Alix (an intracellular inhibitor of paraptosis) and increased the expression of CHOP and ATF4 (transcription factors that promote paraptosis) indicating that KF promotes apoptosis as well as paraptosis. KF also increased intracellular reactive oxygen species (ROS) suggesting the perturbance of the redox state. N-acetylcysteine reverted the apoptosis- and paraptosis-inducing effects of KF. Some ROS inducers are known to suppress the STAT3 pathway and investigation revealed that KF downmodulates STAT3 and its upstream kinases (JAK1, JAK2, and Src). Additionally, KF also elevated the expression of SHP-1, a tyrosine phosphatase which is involved in the negative modulation of the STAT3 pathway. Knockdown of SHP-1 prevented KF-driven STAT3 inhibition. Altogether, KF has been identified as a promoter of apoptosis and paraptosis in pancreatic cancer cells through the elevation of ROS generation and SHP-1 expression.

Molecular dissection of PI3Kß synergistic activation by receptor tyrosine kinases, GßGγ, and Rho-family GTPases.

Phosphoinositide 3-kinase (PI3K) beta (PI3Kß) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kß prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kß localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kß when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kß membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kß prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GßGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kß to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GßGγ or pY/Rac1(GTP), PI3Kß activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kß is synergistically activated by pY/GßGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.

Autophagy cooperates with PDGFRA to support oncogenic growth signaling.

Macroautophagy (referred to as autophagy hereafter) is a highly conserved catabolic process which sequesters intracellular substrates for lysosomal degradation. Autophagy-related proteins have been shown to be involved in various aspects of tumor development by engaging with multiple cellular substrates. We recently uncovered a novel role for autophagy in regulating the signaling and levels of PDGFRA, a receptor tyrosine kinase amplified in several cancers. We discovered that PDGFRA can be targeted to autophagic degradation by binding the autophagy cargo receptor SQSTM1. Surprisingly, PDGFRA-mediated signaling is perturbed in the absence of autophagy despite enhanced receptor levels. We show that this is due to disrupted trafficking of the receptor to late endosomes where signaling activity persists. Conversely, prolonged autophagy inhibition results in a transcriptional downregulation of Pdgfra as a result of inhibited signaling activity demonstrating that short- and long-term autophagy inhibition have opposing effects on receptor levels. We further investigated the consequence of PDGFRA regulation by autophagy using a mouse model for gliomagenesis where we observed a disruption in PDGFA-driven tumor formation when autophagy is inhibited. Activation of downstream signaling through Pten mutation overrides the need for autophagy during tumor development suggesting a genotype-specific role for autophagy during tumorigenesis. Altogether, our findings provide a novel mechanism through which autophagy can support tumor growth.

A systematic computational analysis of the endosomal recycling pathway in glioblastoma.

Glioblastoma (GBM) is the most common and aggressive brain cancer in adults. The standard treatment is brutal and has changed little in 20 years, and more than 85% of patients will die within two years of their diagnosis. There is thus an urgent need to identify new drug targets and develop novel therapeutic strategies to increase survival and improve quality of life. Using publicly available genomics, transcriptomics and proteomics datasets, we compared the expression of endosomal recycling pathway regulators in non-tumour brain tissue with their expression in GBM. We found that key regulators of this pathway are dysregulated in GBM and their expression levels can be linked to survival outcomes. Further analysis of the differentially expressed endosomal recycling regulators allowed us to generate an 8-gene prognostic signature that can distinguish low-risk from high-risk GBM and potentially identify tumours that may benefit from treatment with endosomal recycling inhibitors. This study presents the first systematic analysis of the endosomal recycling pathway in glioblastoma and suggests it could be a promising target for the development of novel therapies and therapeutic strategies to improve outcomes for patients.

Targeted Agents in Esophagogastric Cancer Beyond Human Epidermal Growth Factor Receptor-2.

Gastroesophageal cancers are highly diverse tumors in terms of their anatomic and molecular characteristics, making drug development challenging. Recent advancements in understanding the molecular profiles of these cancers have led to the identification of several new biomarkers. Ongoing clinical trials are investigating new targeted agents with promising results. CLDN18.2 has emerged as a biomarker with established activity of associated targeted therapies. Other targeted agents, such as bemarituzumab and DKN-01, are under active investigation. As new agents are incorporated into the treatment continuum, the questions of biomarker overlap, tumor heterogeneity, and toxicity management will need to be addressed.

Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation.

Human Epidermal growth factor Receptor 4 (HER4 or ERBB4) carries out essential functions in the development and maintenance of the cardiovascular and nervous systems. HER4 activation is regulated by a diverse group of extracellular ligands including the neuregulin (NRG) family and betacellulin (BTC), which promote HER4 homodimerization or heterodimerization with other HER receptors. Important cardiovascular functions of HER4 are exerted via heterodimerization with its close homolog and orphan receptor, HER2. To date structural insights into ligand-mediated HER4 activation have been limited to crystallographic studies of HER4 ectodomain homodimers in complex with NRG1ß. Here, we report cryo-EM structures of near full-length HER2/HER4 heterodimers and full-length HER4 homodimers bound to NRG1ß and BTC. We show that the structures of the heterodimers bound to either ligand are nearly identical and that in both cases the HER2/HER4 heterodimer interface is less dynamic than those observed in structures of HER2/EGFR and HER2/HER3 heterodimers. In contrast, structures of full-length HER4 homodimers bound to NRG1ß and BTC display more large-scale dynamics mirroring states previously reported for EGFR homodimers. Our structures also reveal the presence of multiple glycan modifications within HER4 ectodomains, modeled for the first time in HER receptors, that distinctively contribute to the stabilization of HER4 homodimer interfaces over those of HER2/HER4 heterodimers.

Activin E is a transforming growth factor ß ligand that signals specifically through activin receptor-like kinase 7.

Activins are one of the three distinct subclasses within the greater Transforming growth factor ß (TGFß) superfamily. First discovered for their critical roles in reproductive biology, activins have since been shown to alter cellular differentiation and proliferation. At present, members of the activin subclass include activin A (ActA), ActB, ActC, ActE, and the more distant members myostatin and GDF11. While the biological roles and signaling mechanisms of most activins class members have been well-studied, the signaling potential of ActE has remained largely unknown. Here, we characterized the signaling capacity of homodimeric ActE. Molecular modeling of the ligand:receptor complexes showed that ActC and ActE shared high similarity in both the type I and type II receptor binding epitopes. ActE signaled specifically through ALK7, utilized the canonical activin type II receptors, ActRIIA and ActRIIB, and was resistant to the extracellular antagonists follistatin and WFIKKN. In mature murine adipocytes, ActE invoked a SMAD2/3 response via ALK7, like ActC. Collectively, our results establish ActE as a specific signaling ligand which activates the type I receptor, ALK7.

Antibody dependent cellular cytotoxicity-inducing anti-EGFR antibodies as effective therapeutic option for cutaneous melanoma resistant to BRAF inhibitors.

Introduction: About 50% of cutaneous melanoma (CM) patients present activating BRAF mutations that can be effectively targeted by BRAF inhibitors (BRAFi). However, 20% of CM patients exhibit intrinsic drug resistance to BRAFi, while most of the others develop adaptive resistance over time. The mechanisms involved in BRAFi resistance are disparate and globally seem to rewire the cellular signaling profile by up-regulating different receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR). RTKs inhibitors have not clearly demonstrated anti-tumor activity in BRAFi resistant models. To overcome this issue, we wondered whether the shared up-regulated RTK phenotype associated with BRAFi resistance could be exploited by using immune weapons as the antibody-dependent cell cytotoxicity (ADCC)-mediated effect of anti-RTKs antibodies, and kill tumor cells independently from the mechanistic roots. Methods and results: By using an in vitro model of BRAFi resistance, we detected increased membrane expression of EGFR, both at mRNA and protein level in 4 out of 9 BRAFi-resistant (VR) CM cultures as compared to their parental sensitive cells. Increased EGFR phosphorylation and AKT activation were observed in the VR CM cultures. EGFR signaling appeared dispensable for maintaining resistance, since small molecule-, antibody- and CRISPR-targeting of EGFR did not restore sensitivity of VR cells to BRAFi. Importantly, immune-targeting of EGFR by the anti-EGFR antibody cetuximab efficiently and specifically killed EGFR-expressing VR CM cells, both in vitro and in humanized mouse models in vivo, triggering ADCC by healthy donors' and patients' peripheral blood cells. Conclusion: Our data demonstrate the efficacy of immune targeting of RTKs expressed by CM relapsing on BRAFi, providing the proof-of-concept supporting the assessment of anti-RTK antibodies in combination therapies in this setting. This strategy might be expected to concomitantly trigger the crosstalk of adaptive immune response leading to a complementing T cell immune rejection of tumors.

Targeting Receptor Tyrosine Kinases as a Novel Strategy for the Treatment of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) comprises a group of aggressive and heterogeneous breast carcinoma. Chemotherapy is the mainstay for the treatment of triple-negative tumors. Nevertheless, the success of chemotherapeutic treatments is limited by their toxicity and development of acquired resistance leading to therapeutic failure and tumor relapse. Hence, there is an urgent need to explore novel targeted therapies for TNBC. Receptor tyrosine kinases (RTKs) are a family of transmembrane receptors that are key regulators of intracellular signaling pathways controlling cell proliferation, differentiation, survival, and motility. Aberrant activity and/or expression of several types of RTKs have been strongly connected to tumorigenesis. RTKs are frequently overexpressed and/or deregulated in triple-negative breast tumors and are further associated with tumor progression and reduced survival in patients. Therefore, targeting RTKs could be an appealing therapeutic strategy for the treatment of TNBC. This review summarizes the current evidence regarding the antitumor activity of RTK inhibitors in preclinical models of TNBC. The review also provides insights into the clinical trials evaluating the use of RTK inhibitors for the treatment of patients with TNBC.

Nuclear translocation of Axl contributes to the malignancy of oral cancer cells.

Background/purpose: Dysregulation of receptor tyrosine kinases is implicated in cancer development. This study aimed to investigate the nuclear translocation of Axl, a membrane protein and receptor tyrosine kinase in cancer malignancy. Materials and methods: We examined Axl's entry into the cell nucleus and validated it with the nuclear export inhibitor leptomycin. Transfection experiments with mutated nuclear localization signals were conducted to assess the impact of reduced nuclear Axl levels on cancer cell malignancy. Additionally, we evaluated the effects of decreased nuclear Axl on sensitivity to radiation and cisplatin, a chemotherapeutic drug. Results: In the present study, we observed nuclear translocation of Axl in cancer cells. Reducing nuclear Axl levels led to a decrease in cancer cell malignancy. This nuclear translocation was further validated using a nuclear export inhibitor, leptomycin. Additionally, transfection experiments with mutated nuclear localization signals confirmed the functional significance of Axl's nuclear localization. Notably, decreased nuclear Axl levels also increased the sensitivity of cancer cells to radiation and cisplatin treatment. Conclusion: This study suggests that Axl's nuclear translocation plays a significant role in cancer malignancy. Targeting Axl's nuclear localization could offer a potential strategy to inhibit cancer progression and improve the efficacy of radiation and chemotherapy treatments.

Corrigendum: Targeting HER3 to overcome RGFR TKI resistance in NSCLC.

[This corrects the article DOI: 10.3389/fimmu.2023.1332057.].

Identification of natural product-based effective inhibitors of spleen tyrosine kinase (SYK) through virtual screening and molecular dynamics simulation approaches.

Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that plays an essential role in signal transduction across different cell types. In the context of allergy and autoimmune disorders, it is a crucial regulator of immune receptor signaling in inflammatory cells such as B cells, mast cells, macrophages, and neutrophils. Developing SYK kinase inhibitors has gained significant interest for potential therapeutic applications in neurological and cancer-related conditions. The clinical use of the most advanced SYK inhibitor, Fostamatinib, has been limited due to its unwanted side effects. Thus, a more targeted approach to SYK inhibition would provide a more comprehensive treatment window. In this study, we used a virtual screening approach to identify potential SYK inhibitors from natural compounds from the IMPPAT database. We identified two compounds, Isolysergic acid and Michelanugine, which showed strong affinity and specificity for the SYK binding pocket. All-atom molecular dynamics (MD) simulations were also performed to explore the stability, conformational changes, and interaction mechanism of SYK in complexes with the identified compounds. The identified compounds might have the potential to be developed into promising SYK inhibitors for the treatment of various diseases, including autoimmune disorders, cancer, and inflammatory diseases. This work aims to identify potential phytochemicals to develop a new protein kinase inhibitor for treating advanced malignancies by providing an updated understanding of the role of SYK.Communicated by Ramaswamy H. Sarma.

From nature to cancer therapy: Evaluating the Streptomyces clavuligerus secondary metabolites for potential protein kinase inhibitors.

The study aimed to evaluate the antioxidant, protein kinase inhibitory (PKIs) potential, cytotoxicity activity of Streptomyces clavuligerus extract. DPPH assay revealed a robust free radical scavenging capacity (IC50 28.90 ± 0.24 µg/mL) of organic extract with a maximum inhibition percentage of 61 ± 1.04%. PKIs assay revealed the formation of a whitish bald zone by S. clavuligerus extracts which indicates the presence of PKIs. The cytotoxicity activity of organic fraction of extract through Sulforhodamine B assay on MCF-7, Hop-62, SiHa, and PC-3 cell lines demonstrated the lowest GI50 value against the MCF-7 cell line followed by the PC-3 cell line, showing potent growth inhibitory potential against human breast cancer and human prostate cancer cell line. HR-LCMS analysis identified multiple secondary metabolites from the organic and aqueous extracts of S. clavuligerus when incubated at 30°C under 200 rpm for 3 days. All the secondary metabolites were elucidated for their potential to inhibit RTKs by molecular docking, molecular dynamic simulation, MM/GBSA calculations, and free energy approach. It revealed the superior inhibitory potential of epirubicin (Epi) and dodecaprenyl phosphate-galacturonic acid (DPGA) against fibroblast growth factors receptor (FGFR). Epi also exhibited excellent inhibitory activity against the platelet-derived growth factor receptor (PDGFR), while DPGA effectively inhibited the vascular endothelial growth factor receptor. Additionally, the presence Epi in S. clavuligerus extract was validated through the HPLC technique. Thus, our findings highlight a superior inhibitory potential of Epi against FGFR and PDGFR RTKs than the FDA-approved drug.

Receptor tyrosine kinase gene expression profiling of orbital rhabdomyosarcoma unveils MET as a potential biomarker and therapeutic target.

Receptor tyrosine kinases (RTKs) serve as molecular targets for the development of novel personalized therapies in many malignancies. In the present study, expression pattern of receptor tyrosine kinases and its clinical significance in orbital RMS has been explored. Eighteen patients with histopathologically confirmed orbital RMS formed part of this study. Comprehensive q-PCR gene expression profiles of 19 RTKs were generated in the cases and controls. The patients were followed up for 59.53 ± 20.93 years. Clustering and statistical analysis tools were applied to identify the significant combination of RTKs associated with orbital rhabdomyosarcoma patients. mRNA overexpression of RTKs which included MET, AXL, EGFR was seen in 60-80% of cases; EGFR3, IGFR2, FGFR1, RET, PDGFR1, VEGFR2, PDGFR2 in 30-60% of cases; and EGFR4, FGFR3,VEGFR3 and ROS,IGFR1, EGFR1, FGFR2, VEGFR1 in 10-30% of cases. Immunoexpression of MET was seen in 89% of cases. A significant association was seen between MET mRNA and its protein expression. In all the cases MET gene expression was associated with worst overall survival (P = 0.03).There was a significant correlation of MET mRNA expression with RET, ROS, AXL, FGFR1, FGFR3, PDGFR1, IGFR1, VEGFR2, and EGFR3 genes. Association between MET gene and collective expression of RTKs was further evaluated by semi-supervised gene cluster analysis and Principal component analysis, which showed well-separated tumor clusters. MET gene overexpression could be a useful biomarker for identifying high risk orbital rhabdomyosarcoma patients. Well-separated tumor clusters confirmed the association between MET gene and collective expression of RTK genes. Therefore, the therapeutic potential of multi-kinase inhibitors targeting MET and the 9 other significant RTKs needs to be explored.

Quantitative live imaging reveals a direct interaction between CD44v6 and MET in membrane domains upon activation with both MET ligands, HGF and internalin B.

Deregulation of the receptor tyrosine kinase MET/hepatocyte growth factor (HGF) pathway results in several pathological processes involved in tumor progression and metastasis. In a different context, MET can serve as an entry point for the bacterium Listeria monocytogenes, when activated by the internalin B (InlB) protein during infection of non-phagocytic cells. We have previously demonstrated that MET requires CD44v6 for its ligand-induced activation. However, the stoichiometry and the steps required for the formation of this complex, are still unknown. In this work, we studied the dynamics of the ligand-induced interaction of CD44v6 with MET at the plasma membrane. Using Förster resonance energy transfer-based fluorescence lifetime imaging microscopy in T-47D cells, we evidenced a direct interaction between MET and CD44v6 promoted by HGF and InlB in live cells. In the absence of MET, fluorescence correlation spectroscopy experiments further showed the dimerization of CD44v6 and the increase of its diffusion induced by HGF and InlB. In the presence of MET, stimulation of the cells by HGF or InlB significantly decreased the diffusion of CD44v6, in line with the formation of a ternary complex of MET with CD44v6 and HGF/InlB. Finally, similarly to HGF/InlB, disruption of liquid-ordered domains (Lo) by methyl-ß-cyclodextrin increased CD44v6 mobility suggesting that these factors induce the exit of CD44v6 from the Lo domains. Our data led us to propose a model for MET activation, where CD44v6 dimerizes and diffuses rapidly out of Lo domains to form an oligomeric MET/ligand/CD44v6 complex that is instrumental for MET activation.