Origins and Proliferative States of Human Oligodendrocyte Precursor Cells.

Human cerebral cortex size and complexity has increased greatly during evolution. While increased progenitor diversity and enhanced proliferative potential play important roles in human neurogenesis and gray matter expansion, the mechanisms of human oligodendrogenesis and white matter expansion remain largely unknown. Here, we identify EGFR-expressing "Pre-OPCs" that originate from outer radial glial cells (oRGs) and undergo mitotic somal translocation (MST) during division. oRG-derived Pre-OPCs provide an additional source of human cortical oligodendrocyte precursor cells (OPCs) and define a lineage trajectory. We further show that human OPCs undergo consecutive symmetric divisions to exponentially increase the progenitor pool size. Additionally, we find that the OPC-enriched gene, PCDH15, mediates daughter cell repulsion and facilitates proliferation. These findings indicate properties of OPC derivation, proliferation, and dispersion important for human white matter expansion and myelination.

Functional Enhancers Shape Extrachromosomal Oncogene Amplifications.

Non-coding regions amplified beyond oncogene borders have largely been ignored. Using a computational approach, we find signatures of significant co-amplification of non-coding DNA beyond the boundaries of amplified oncogenes across five cancer types. In glioblastoma, EGFR is preferentially co-amplified with its two endogenous enhancer elements active in the cell type of origin. These regulatory elements, their contacts, and their contribution to cell fitness are preserved on high-level circular extrachromosomal DNA amplifications. Interrogating the locus with a CRISPR interference screening approach reveals a diversity of additional elements that impact cell fitness. The pattern of fitness dependencies mirrors the rearrangement of regulatory elements and accompanying rewiring of the chromatin topology on the extrachromosomal amplicon. Our studies indicate that oncogene amplifications are shaped by regulatory dependencies in the non-coding genome.

Processing Temporal Growth Factor Patterns by an Epidermal Growth Factor Receptor Network Dynamically Established in Space.

The proto-oncogenic epidermal growth factor (EGF) receptor (EGFR) is a tyrosine kinase whose sensitivity and response to growth factor signals that vary over time and space determine cellular behavior within a developing tissue. The molecular reorganization of the receptors on the plasma membrane and the enzyme-kinetic mechanisms of phosphorylation are key determinants that couple growth factor binding to EGFR signaling. To enable signal initiation and termination while simultaneously accounting for suppression of aberrant signaling, a coordinated coupling of EGFR kinase and protein tyrosine phosphatase activity is established through space by vesicular dynamics. The dynamical operation mode of this network enables not only time-varying growth factor sensing but also adaptation of the response depending on cellular context. By connecting spatially coupled enzymatic kinase/phosphatase processes and the corresponding dynamical systems description of the EGFR network, we elaborate on the general principles necessary for processing complex growth factor signals.

Microbial metabolism of L-tyrosine protects against allergic airway inflammation.

The constituents of the gut microbiome are determined by the local habitat, which itself is shaped by immunological pressures, such as mucosal IgA. Using a mouse model of restricted antibody repertoire, we identified a role for antibody-microbe interactions in shaping a community of bacteria with an enhanced capacity to metabolize L-tyrosine. This model led to increased concentrations of p-cresol sulfate (PCS), which protected the host against allergic airway inflammation. PCS selectively reduced CCL20 production by airway epithelial cells due to an uncoupling of epidermal growth factor receptor (EGFR) and Toll-like receptor 4 (TLR4) signaling. Together, these data reveal a gut microbe-derived metabolite pathway that acts distally on the airway epithelium to reduce allergic airway responses, such as those underpinning asthma.

Amphiregulin from regulatory T cells promotes liver fibrosis and insulin resistance in non-alcoholic steatohepatitis.

Production of amphiregulin (Areg) by regulatory T (Treg) cells promotes repair after acute tissue injury. Here, we examined the function of Treg cells in non-alcoholic steatohepatitis (NASH), a setting of chronic liver injury. Areg-producing Treg cells were enriched in the livers of mice and humans with NASH. Deletion of Areg in Treg cells, but not in myeloid cells, reduced NASH-induced liver fibrosis. Chronic liver damage induced transcriptional changes associated with Treg cell activation. Mechanistically, Treg cell-derived Areg activated pro-fibrotic transcriptional programs in hepatic stellate cells via epidermal growth factor receptor (EGFR) signaling. Deletion of Areg in Treg cells protected mice from NASH-dependent glucose intolerance, which also was dependent on EGFR signaling on hepatic stellate cells. Areg from Treg cells promoted hepatocyte gluconeogenesis through hepatocyte detection of hepatic stellate cell-derived interleukin-6. Our findings reveal a maladaptive role for Treg cell-mediated tissue repair functions in chronic liver disease and link liver damage to NASH-dependent glucose intolerance.

EGFR Ligands Differentially Stabilize Receptor Dimers to Specify Signaling Kinetics.

Epidermal growth factor receptor (EGFR) regulates many crucial cellular programs, with seven different activating ligands shaping cell signaling in distinct ways. Using crystallography and other approaches, we show how the EGFR ligands epiregulin (EREG) and epigen (EPGN) stabilize different dimeric conformations of the EGFR extracellular region. As a consequence, EREG or EPGN induce less stable EGFR dimers than EGF-making them partial agonists of EGFR dimerization. Unexpectedly, this weakened dimerization elicits more sustained EGFR signaling than seen with EGF, provoking responses in breast cancer cells associated with differentiation rather than proliferation. Our results reveal how responses to different EGFR ligands are defined by receptor dimerization strength and signaling dynamics. These findings have broad implications for understanding receptor tyrosine kinase (RTK) signaling specificity. Our results also suggest parallels between partial and/or biased agonism in RTKs and G-protein-coupled receptors, as well as new therapeutic opportunities for correcting RTK signaling output.

Spatial and Temporal Regulation of Receptor Tyrosine Kinase Activation and Intracellular Signal Transduction.

Epidermal growth factor (EGF) and insulin receptor tyrosine kinases (RTKs) exemplify how receptor location is coupled to signal transduction. Extracellular binding of ligands to these RTKs triggers their concentration into vesicles that bud off from the cell surface to generate intracellular signaling endosomes. On the exposed cytosolic surface of these endosomes, RTK autophosphorylation selects the downstream signaling proteins and lipids to effect growth factor and polypeptide hormone action. This selection is followed by the recruitment of protein tyrosine phosphatases that inactivate the RTKs and deliver them by membrane fusion and fission to late endosomes. Coincidentally, proteinases inside the endosome cleave the EGF and insulin ligands. Subsequent inward budding of the endosomal membrane generates multivesicular endosomes. Fusion with lysosomes then results in RTK degradation and downregulation. Through the spatial positioning of RTKs in target cells for EGF and insulin action, the temporal extent of signaling, attenuation, and downregulation is regulated.

EGFR Dynamics Change during Activation in Native Membranes as Revealed by NMR.

The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. We propose that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase.

Unlocking the EGFR-mediated epitranscriptome: A pathway to novel therapies.

In this issue, Lv et al.1 explore EGFR-driven epitranscriptomic reprogramming in glioblastoma, revealing the pivotal role of the EGFR-ALKBH5-GCLM axis in ferroptosis protection. Their findings offer mechanistic insight and therapeutic strategies involving novel combination targets to enhance tumor responses.

A structural perspective on the regulation of the epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays a critical role in the pathogenesis of many cancers. The structure of intact forms of this receptor has yet to be determined, but intense investigations of fragments of the receptor have provided a detailed view of its activation mechanism, which we review here. Ligand binding converts the receptor to a dimeric form, in which contacts are restricted to the receptor itself, allowing heterodimerization of the four EGFR family members without direct ligand involvement. Activation of the receptor depends on the formation of an asymmetric dimer of kinase domains, in which one kinase domain allosterically activates the other. Coupling between the extracellular and intracellular domains may involve a switch between alternative crossings of the transmembrane helices, which form dimeric structures. We also discuss how receptor regulation is compromised by oncogenic mutations and the structural basis for negative cooperativity in ligand binding.

Tumor-derived exosomes antagonize innate antiviral immunity.

Malignancies can compromise innate immunity, but the mechanisms of this are largely unknown. Here we found that, via tumor-derived exosomes (TEXs), cancers were able to transfer activated epidermal growth factor receptor (EGFR) to host macrophages and thereby suppress innate antiviral immunity. Screening of the human kinome identified the kinase MEKK2 in macrophages as an effector of TEX-delivered EGFR that negatively regulated the antiviral immune response. In the context of experimental tumor implantation, MEKK2-deficient mice were more resistant to viral infection than were wild-type mice. Injection of TEXs into mice reduced innate immunity, increased viral load and increased morbidity in an EGFR- and MEKK2-dependent manner. MEKK2 phosphorylated IRF3, a transcription factor crucial for the production of type I interferons; this triggered poly-ubiquitination of IRF3 and blocked its dimerization, translocation to the nucleus and transcriptional activity after viral infection. These findings identify a mechanism by which cancer cells can dampen host innate immunity and potentially cause patients with cancer to become immunocompromised.

A kinase-independent role for EGF receptor in autophagy initiation.

The epidermal growth factor receptor (EGFR) is upregulated in numerous human cancers. Inhibition of EGFR signaling induces autophagy in tumor cells. Here, we report an unanticipated role for the inactive EGFR in autophagy initiation. Inactive EGFR interacts with the oncoprotein LAPTM4B that is required for the endosomal accumulation of EGFR upon serum starvation. Inactive EGFR and LAPTM4B stabilize each other at endosomes and recruit the exocyst subcomplex containing Sec5. We show that inactive EGFR, LAPTM4B, and the Sec5 subcomplex are required for basal and starvation-induced autophagy. LAPTM4B and Sec5 promote EGFR association with the autophagy inhibitor Rubicon, which in turn disassociates Beclin 1 from Rubicon to initiate autophagy. Thus, the oncoprotein LAPTM4B facilitates the role of inactive EGFR in autophagy initiation. This pathway is positioned to control tumor metabolism and promote tumor cell survival upon serum deprivation or metabolic stress.

ARAF protein kinase activates RAS by antagonizing its binding to RASGAP NF1.

RAF protein kinases are effectors of the GTP-bound form of small guanosine triphosphatase RAS and function by phosphorylating MEK. We showed here that the expression of ARAF activated RAS in a kinase-independent manner. Binding of ARAF to RAS displaced the GTPase-activating protein NF1 and antagonized NF1-mediated inhibition of RAS. This reduced ERK-dependent inhibition of RAS and increased RAS-GTP. By this mechanism, ARAF regulated the duration and consequences of RTK-induced RAS activation and supported the RAS output of RTK-dependent tumor cells. In human lung cancers with EGFR mutation, amplification of ARAF was associated with acquired resistance to EGFR inhibitors, which was overcome by combining EGFR inhibitors with an inhibitor of the protein tyrosine phosphatase SHP2 to enhance inhibition of nucleotide exchange and RAS activation.

Injury prevents Ras mutant cell expansion in mosaic skin.

Healthy skin is a mosaic of wild-type and mutant clones1,2. Although injury can cooperate with mutated Ras family proteins to promote tumorigenesis3-12, the consequences in genetically mosaic skin are unknown. Here we show that after injury, wild-type cells suppress aberrant growth induced by oncogenic Ras. HrasG12V/+ and KrasG12D/+ cells outcompete wild-type cells in uninjured, mosaic tissue but their expansion is prevented after injury owing to an increase in the fraction of proliferating wild-type cells. Mechanistically, we show that, unlike HrasG12V/+ cells, wild-type cells respond to autocrine and paracrine secretion of EGFR ligands, and this differential activation of the EGFR pathway explains the competitive switch during injury repair. Inhibition of EGFR signalling via drug or genetic approaches diminishes the proportion of dividing wild-type cells after injury, leading to the expansion of HrasG12V/+ cells. Increased proliferation of wild-type cells via constitutive loss of the cell cycle inhibitor p21 counteracts the expansion of HrasG12V/+ cells even in the absence of injury. Thus, injury has a role in switching the competitive balance between oncogenic and wild-type cells in genetically mosaic skin.

Tissue-Specific Macrophage Responses to Remote Injury Impact the Outcome of Subsequent Local Immune Challenge.

Myocardial infarction, stroke, and sepsis trigger systemic inflammation and organism-wide complications that are difficult to manage. Here, we examined the contribution of macrophages residing in vital organs to the systemic response after these injuries. We generated a comprehensive catalog of changes in macrophage number, origin, and gene expression in the heart, brain, liver, kidney, and lung of mice with myocardial infarction, stroke, or sepsis. Predominantly fueled by heightened local proliferation, tissue macrophage numbers increased systemically. Macrophages in the same organ responded similarly to different injuries by altering expression of tissue-specific gene sets. Preceding myocardial infarction improved survival of subsequent pneumonia due to enhanced bacterial clearance, which was caused by IFNÉ£ priming of alveolar macrophages. Conversely, EGF receptor signaling in macrophages exacerbated inflammatory lung injury. Our data suggest that local injury activates macrophages in remote organs and that targeting macrophages could improve resilience against systemic complications following myocardial infarction, stroke, and sepsis.

Self-eating limits EGFR-dependent tumor growth.

Autophagy is a cell-autonomous, catabolic process that plays context-dependent roles in tumor growth and progression. Wei et al. report that EGFR signaling promotes tumor growth through phosphorylation and functional inactivation of Beclin 1 and the consequent suppression of autophagy.

Conformational coupling across the plasma membrane in activation of the EGF receptor.

How the epidermal growth factor receptor (EGFR) activates is incompletely understood. The intracellular portion of the receptor is intrinsically active in solution, and to study its regulation, we measured autophosphorylation as a function of EGFR surface density in cells. Without EGF, intact EGFR escapes inhibition only at high surface densities. Although the transmembrane helix and the intracellular module together suffice for constitutive activity even at low densities, the intracellular module is inactivated when tethered on its own to the plasma membrane, and fluorescence cross-correlation shows that it fails to dimerize. NMR and functional data indicate that activation requires an N-terminal interaction between the transmembrane helices, which promotes an antiparallel interaction between juxtamembrane segments and release of inhibition by the membrane. We conclude that EGF binding removes steric constraints in the extracellular module, promoting activation through N-terminal association of the transmembrane helices.

Architecture and membrane interactions of the EGF receptor.

Dimerization-driven activation of the intracellular kinase domains of the epidermal growth factor receptor (EGFR) upon extracellular ligand binding is crucial to cellular pathways regulating proliferation, migration, and differentiation. Inactive EGFR can exist as both monomers and dimers, suggesting that the mechanism regulating EGFR activity may be subtle. The membrane itself may play a role but creates substantial difficulties for structural studies. Our molecular dynamics simulations of membrane-embedded EGFR suggest that, in ligand-bound dimers, the extracellular domains assume conformations favoring dimerization of the transmembrane helices near their N termini, dimerization of the juxtamembrane segments, and formation of asymmetric (active) kinase dimers. In ligand-free dimers, by holding apart the N termini of the transmembrane helices, the extracellular domains instead favor C-terminal dimerization of the transmembrane helices, juxtamembrane segment dissociation and membrane burial, and formation of symmetric (inactive) kinase dimers. Electrostatic interactions of EGFR's intracellular module with the membrane are critical in maintaining this coupling.

EGFR-mediated Beclin 1 phosphorylation in autophagy suppression, tumor progression, and tumor chemoresistance.

Cell surface growth factor receptors couple environmental cues to the regulation of cytoplasmic homeostatic processes, including autophagy, and aberrant activation of such receptors is a common feature of human malignancies. Here, we defined the molecular basis by which the epidermal growth factor receptor (EGFR) tyrosine kinase regulates autophagy. Active EGFR binds the autophagy protein Beclin 1, leading to its multisite tyrosine phosphorylation, enhanced binding to inhibitors, and decreased Beclin 1-associated VPS34 kinase activity. EGFR tyrosine kinase inhibitor (TKI) therapy disrupts Beclin 1 tyrosine phosphorylation and binding to its inhibitors and restores autophagy in non-small-cell lung carcinoma (NSCLC) cells with a TKI-sensitive EGFR mutation. In NSCLC tumor xenografts, the expression of a tyrosine phosphomimetic Beclin 1 mutant leads to reduced autophagy, enhanced tumor growth, tumor dedifferentiation, and resistance to TKI therapy. Thus, oncogenic receptor tyrosine kinases directly regulate the core autophagy machinery, which may contribute to tumor progression and chemoresistance.

Glioblastoma mutations alter EGFR dimer structure to prevent ligand bias.

The epidermal growth factor receptor (EGFR) is frequently mutated in human cancer1,2, and is an important therapeutic target. EGFR inhibitors have been successful in lung cancer, where mutations in the intracellular tyrosine kinase domain activate the receptor1, but not in glioblastoma multiforme (GBM)3, where mutations occur exclusively in the extracellular region. Here we show that common extracellular GBM mutations prevent EGFR from discriminating between its activating ligands4. Different growth factor ligands stabilize distinct EGFR dimer structures5 that signal with different kinetics to specify or bias outcome5,6. EGF itself induces strong symmetric dimers that signal transiently to promote proliferation. Epiregulin (EREG) induces much weaker asymmetric dimers that drive sustained signalling and differentiation5. GBM mutations reduce the ability of EGFR to distinguish EREG from EGF in cellular assays, and allow EGFR to form strong (EGF-like) dimers in response to EREG and other low-affinity ligands. Using X-ray crystallography, we further show that the R84K GBM mutation symmetrizes EREG-driven extracellular dimers so that they resemble dimers normally seen with EGF. By contrast, a second GBM mutation, A265V, remodels key dimerization contacts to strengthen asymmetric EREG-driven dimers. Our results argue for an important role of altered ligand discrimination by EGFR in GBM, with potential implications for therapeutic targeting.