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Epidermal Growth Factor Receptor (EGFR) signalling is supposed to be triggered by a ligand-induced conformational change of the extracellular domain from a closed, self-inhibited tethered monomer, to an open untethered state with a free dimerization arm. The ectodomain also untethers spontaneously, but the molecular mechanism is still unknown. Expe...
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Clinical data acquired over the last decade on non-small cell lung cancer (NSCLC) treatment with small molecular weight Epidermal Growth Factor Receptor (EGFR) inhibitors have shown significant influence of EGFR point mutations and in-frame deletions on clinical efficacy. Identification of small molecules capable of inhibiting the clinically releva...
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... Further MD simulations revealed that GBM mutations favor spontaneous ECD opening following the lowest frequency modes, to acquire a transient conformation known to exist but never trapped experimentally. This ENM/MD intermediate was validated through structural, in vitro, and in vivo experiments [75,124,125], is shared by missense mutants from different ECD hotspots, and mimics the configuration of the most frequent change in GBM, the deletion EGFRvIII (Figure 1d). Specifically, the first tandem repeat of EGFR is deleted in EGFRvIII but rotates in missense mutations. ...
At the very deepest molecular level, the mechanisms of life depend on the operation of proteins, the so-called “workhorses” of the cell. Proteins are nanoscale machines that transform energy into useful cellular work, such as ion or nutrient transport, information processing, or energy transformation. Behind every biological task, there is a nanometer-sized molecule whose shape and intrinsic motions, binding, and sensing properties have been evolutionarily polished for billions of years. With the emergence of structural biology, the most crucial property of biomolecules was thought to be their 3D shape, but how this relates to function was unclear. During the past years, Elastic Network Models have revealed that protein shape, motion and function are deeply intertwined, so that each structure displays robustly shape-encoded functional movements that can be extraordinarily conserved across the tree of life. Here, we briefly review the growing literature exploring the interplay between sequence evolution, protein shape, intrinsic motions and function, and highlight examples from our research in which fundamental movements are conserved from bacteria to mammals or selected by cancer cells to modulate function.
... Shorter µs-simulations by Kaszuba et al. (2015) also led to predictions about the impact of glycans on EGFR conformation, which were tested by monitoring the accessibility of glycosylation-sensitive surface-epitopes. Recently, we combined first a mutational screening partly based on ENMs, followed by MD simulations of "dynamically" hot EGFR ectodomain mutations ( Orellana et al., 2014Orellana et al., , 2019b) in a multiscale CG-MD scheme similar to that proposed by Saunders and Voth (2013). This approach highlighted how, as happens often experimentally, mutagenesis can help to trap intermediate states. ...
Large-scale conformational changes are essential to link protein structures with their function at the cell and organism scale, but have been elusive both experimentally and computationally. Over the past few years developments in cryo-electron microscopy and crystallography techniques have started to reveal multiple snapshots of increasingly large and flexible systems, deemed impossible only short time ago. As structural information accumulates, theoretical methods become central to understand how different conformers interconvert to mediate biological function. Here we briefly survey current in silico methods to tackle large conformational changes, reviewing recent examples of cross-validation of experiments and computational predictions, which show how the integration of different scale simulations with biological information is already starting to break the barriers between the in silico, in vitro, and in vivo worlds, shedding new light onto complex biological problems inaccessible so far.
... Our work aimed to understand how the most frequent GBM missense mutations (I-II, see below) activate EGFR, leading to important mechanistic and therapeutically relevant insights. 4,5 The ECD consists of four subdomains (I-IV), which are held in a compact and inactive conformation by an inter-domain tether (II-IV). Upon ligand binding, the tether breaks and the ECD opens, releasing a dimerization arm that forms interreceptor interactions in the active dimer. ...
... While II-IV tether mutations clearly favour untethering, the mechanism of I-II mutations, located at an interface away from both the tether and the ligand-binding site, was unclear. 6 Our I-II mutant simulations revealed that these mutations also promote untethering towards a not fully open but intermediate state, 4 which unexpectedly, exposes a cryptic epitope recognized by the cancer-specific antibody mAb806, raised against the main GBM variant, the large deletion EGFRvIII. Although it was known that this peculiar antibody recognized a transitional conformer as EGFR activates, 7 different from both the closed and open crystallographic structures, it had eluded structural determination. ...
EGFR mutations display striking organ-site asymmetry and heterogeneity. We have shown that structurally diverse extracellular mutations, typical of glioblastomas, converge to a similar intermediate conformation, which can be synergistically targeted extra- and intracelullarly by antibody mAb806 and type-II kinase inhibitors. Our findings reveal convergence behind heterogeneity, paving the way for allostery-based co-targeting.
... The antibody-drug conjugate ABT-414, which specifically recognizes EGFRvIII and WT EGFR when expressed at amplified levels, has shown promise in phase I/II clinical trials for GBM patients (Gan et al., 2017;Phillips et al., 2016;Reardon et al., 2017). Previously, we have proposed that certain mutations such as EGFR A289V could bind and respond to mAb806 based on simulations (Orellana et al., 2014). To examine the potential efficacy of this therapy for patients with GBM expressing EGFR A289V , we used flow cytometry to confirm the ability of mAb806 to bind EGFR A289V . ...
... Thus, the overall impact of these mutations could be anticipated to be over-activation of EGFR-driven pathways, leading to increased cell proliferation and invasion (Talasila et al., 2013;Xing et al., 2013). In addition, we have shown that certain mutations, such as EGFR A289V , can expose the mAb806 binding epitope, which is accessible in EGFRvIII (Orellana et al., 2014). ...
We explored the clinical and pathological impact of epidermal growth factor receptor (EGFR) extracellular domain missense mutations. Retrospective assessment of 260 de novo glioblastoma patients revealed a significant reduction in overall survival of patients having tumors with EGFR mutations at alanine 289 (EGFRA289D/T/V). Quantitative multi-parametric magnetic resonance imaging analyses indicated increased tumor invasion for EGFRA289D/T/V mutants, corroborated in mice bearing intracranial tumors expressing EGFRA289V and dependent on ERK-mediated expression of matrix metalloproteinase-1. EGFRA289V tumor growth was attenuated with an antibody against a cryptic epitope, based on in silico simulation. The findings of this study indicate a highly invasive phenotype associated with the EGFRA289V mutation in glioblastoma, postulating EGFRA289V as a molecular marker for responsiveness to therapy with EGFR-targeting antibodies.
... In this respect, aptamer-based strategies may address the possibility to target the different EGFR alterations existing in a tumor, which determine differential biological activities and response to therapy. Indeed, apart from the most common deletion in the ECD leading to EGFRvIII mutant, point mutations which reside in the ECD of EGFR have been identified in GBM [150] most of which are oncogenic because favor conformational changes inducing constitutive receptor activation [151,152]. Aptamers can be selected to identify GBM subpopulations harboring specific EGFR ECD mutations. Still, cell-SELEX to a tumor cell type without prior knowledge of the target molecules reveals among the leading techniques to identify new biomarkers for targeted orphan tumors, including markers of cancer stem cells [153]. ...
Transmembrane receptor tyrosine kinases (RTKs) play crucial roles in cancer cell proliferation, survival, migration and differentiation. Area of intense research is searching for effective anticancer therapies targeting these receptors and, to date, several monoclonal antibodies and small-molecule tyrosine kinase inhibitors have entered the clinic. However, some of these drugs show limited efficacy and give rise to acquired resistance. Emerging highly selective compounds for anticancer therapy are oligonucleotide aptamers that interact with their targets by recognizing a specific three-dimensional structure. Because of their nucleic acid nature, the rational design of advanced strategies to manipulate aptamers for both diagnostic and therapeutic applications is greatly simplified over antibodies.
In this manuscript, we will provide a comprehensive overview of oligonucleotide aptamers as next generation strategies to efficiently target RTKs in human cancers.
... Several reports have described the role of the expression level and mutational status of EGFR in predicting the efficacy of anti-EGFR therapies [67][68][69][70][71], but the association between the expression of EGFR mutations and response to anti-EGFR treatments in patients is still controversial. This responds to variations in the assessed expressions, caused by the use of different cut-off values for EGFR immunostaining [72], the use of antibodies that do not discriminate between the wild-type and mutated forms of the EGFR [73] and discordances between the expression of EGFR in the primary tumor and the metastatic sites [74,75]. Moreover, the interactions between the EGFR´s ligand(s) and the receptor (wild type or mutated), the molecular mechanism of EGFR activation and its impact on patient´s clinical outcome, are not fully elucidated [73]. ...
... This responds to variations in the assessed expressions, caused by the use of different cut-off values for EGFR immunostaining [72], the use of antibodies that do not discriminate between the wild-type and mutated forms of the EGFR [73] and discordances between the expression of EGFR in the primary tumor and the metastatic sites [74,75]. Moreover, the interactions between the EGFR´s ligand(s) and the receptor (wild type or mutated), the molecular mechanism of EGFR activation and its impact on patient´s clinical outcome, are not fully elucidated [73]. The existence of multiple routes for EGFR activation, including a ligandindependent untethering in wild type EGFR (wEGFR), sensitive to differential ligand concentrations, has also been suggested [73,76]. ...
... Moreover, the interactions between the EGFR´s ligand(s) and the receptor (wild type or mutated), the molecular mechanism of EGFR activation and its impact on patient´s clinical outcome, are not fully elucidated [73]. The existence of multiple routes for EGFR activation, including a ligandindependent untethering in wild type EGFR (wEGFR), sensitive to differential ligand concentrations, has also been suggested [73,76]. This alternative activation might explain the response to small molecules erlotinib and gefitinib [77], achieved in NSCLC patients with wEGFR (not harboring EGFRvIII). ...
Background: Literature reports contradictory findings regarding the capacity of serum EGF concentrations ([EGF]) to differentiate non-small cell lung cancer (NSCLC) patients from healthy individuals. Therefore, the diagnostic capacity of [EGF], suggestive of dependency on this growth factor in NSCLC patients (tumors) and hence indicative of possible response to therapies directed to EGF/EGFR, is still an open question. Inconsistencies likely derive from the lack of harmonization and standardization in methodologies for blood and sera processing.Methods: A cohort of NSCLC patients was evaluated at diagnosis (25) and after first-line-therapy (18/25). Sera were collected 1h and 4h after phlebotomy, controlling the variables influencing [EGF]. EGF was quantified by ELISA. Platelets count was also estimated. The values obtained for several combined and/or normalized by platelets count, variables, were compared to those in selected cohorts of healthy controls.Results: We found differences between healthy individuals and NSCLC patients in the accessibility of EGF to circulation, but not in the total available EGF stock. Indeed, we observed a higher fraction of free EGF in the circulation of patients and consequently a lower amount of EGF stored in platelets. Interestingly, an aberrant relationship between EGF and platelets counts was also observed, especially in patients with thrombocytosis. Moreover, several EGF-related variables, with enough accuracy for discrimination, were identified. Particularly, those variables normalized by platelets count made more evident the differences between patients and healthy controls. Therefore, they might be potential biomarkers in NSCLC.Conclusion: Our results suggest the increase in free/accessible EGF in blood circulation as relevant to the biology of NSCLC, most likely because it reflects a higher accessibility of this growth factor for the tumor. They also suggest some of the study variables to be further evaluated on its predictive value, to select good responders to CIMAvax-EGF® or other therapies targeting the EGF/EGFR system.
The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. The binding of EGF ligand to EGFR extracellular domain (EGFR-ECD) promotes its inactive-to-active conformational transition (activation) but the relevant detailed mechanism has remained elusive yet. Here, the structural characterization and energetics of EGFR-ECD conformational transition with and without the binding of EGF are quantitatively explored using an innovative enhanced sampling MD simulation method. Intriguingly, the EGF offers the hydrophobic interaction (e.g., EGF residues of Tyr44, Leu47) and particularly the electrostatic interaction (e.g., EGF residues of Glu5, Asp11, Asp17, Arg41) to play a dominant role in dragging domain III to close the ligand binding domain gap. Subsequently, the correlation between domains III and II is enhanced through the salt-bridges among Glu376, Arg403, Arg405 from domain III and Glu293, Glu295, Arg300 from domain II. Finally, the structure bending of domain II is regulated to facilitate the disengagement of domain II from domain IV. In this regard, the functional conformational transition of EGFR-ECD is a consequence of the cooperative motion of protein domains driven by the EGF ligand binding. The present study displays a detailed scenario of the EGF induced activation of EGFR-ECD and provides valuable information for drug discovery targeting EGFR.
