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In mouse embryonic stem cells (mESCs), chemical blockade of Gsk3α/β and Mek1/2 (2i) instructs a self-renewing ground state whose endogenous inducers are unknown. Here we show that the axon guidance cue Netrin-1 promotes naive pluripotency by triggering profound signalling, transcriptomic and epigenetic changes in mESCs. Furthermore, we demonstrate that Netrin-1 can substitute for blockade of Gsk3α/β and Mek1/2 to sustain self-renewal of mESCs in combination with leukaemia inhibitory factor and regulates the formation of the mouse pluripotent blastocyst. Mechanistically, we reveal how Netrin-1 and the balance of its receptors Neo1 and Unc5B co-regulate Wnt and MAPK pathways in both mouse and human ESCs. Netrin-1 induces Fak kinase to inactivate Gsk3α/β and stabilize β-catenin while increasing the phosphatase activity of a Ppp2r2c-containing Pp2a complex to reduce Erk1/2 activity. Collectively, this work identifies Netrin-1 as a regulator of pluripotency and reveals that it mediates different effects in mESCs depending on its receptor dosage, opening perspectives for balancing self-renewal and lineage commitment.Netrin-1, via precise Neo1/Unc5B stoichiometry, promotes naive pluripotency, embryonic stem cell self-renewal in combination with leukaemia inhibitory factor, and the formation of the mouse epiblast in vivo.

Signaling downstream of Mitogen-Activated Protein Kinase Kinase Kinases (MAP3K) is promiscuous. In the vascular and immune systems the MAP3K, MEKK2, activates different substrates, but the mechanisms of substrate targeting have not been delineated. Here, we determine the 2.4 Å crystal structure of the MEKK2 kinase domain in complex with the small molecule inhibitor, ponatinib. We find that MEKK2 dimerizes by a surface centered on the αG helix and the C-terminal region of the activation segment, that this surface is important for MEKK2 autophosphorylation and dimerization, and that this surface is conserved with MEKK3. We then assess the importance of the surface for phosphorylation and recruitment of two MAP2K substrates, MEK5 and MKK6. We find that both MEK5 and MKK6 require the αG helix-mediated interaction for phosphorylation. In contrast, MEKK2 recruitment of MEK5 is dependent on PB1 domain interactions but MKK6 recruitment is associated with the αG helix-mediated interaction. Our study therefore provides a framework to understand diverse substrate targeting by the MAP3Ks, MEKK2 and MEKK3. MEKK2, a member of the MAP3K family, plays a pivotal role in signaling cascades that regulate cellular responses such as proliferation, differentiation, and stress adaptation. Here the authors determine the crystal structure of the kinase domain of MEKK. Using a structure-directed approach they deconvolute the molecular basis of its autophosphorylation and its recruitment and phosphorylation of MAP2Ks, MEK5 and MKK6.

Coronavirus disease 2019 (COVID-19), the illness caused by a novel coronavirus now called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 260 million confirmed infections and 5 million deaths to date. While vaccination is a powerful tool to control pandemic spread, medication to relieve COVID-19-associated symptoms and alleviate disease progression especially in high-risk patients is still lacking. In this study, we explore the suitability of the rapid accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway as a druggable target in the treatment of SARS-CoV-2 infections. We find that SARS-CoV-2 transiently activates Raf/MEK/ERK signaling in the very early infection phase and that ERK1/2 knockdown limits virus replication in cell culture models. We demonstrate that ATR-002, a specific inhibitor of the upstream MEK1/2 kinases which is currently evaluated in clinical trials as an anti-influenza drug, displays strong anti-SARS-CoV-2 activity in cell lines as well as in primary air–liquid-interphase epithelial cell (ALI) cultures, with a safe and selective treatment window. We also observe that ATR-002 treatment impairs the SARS-CoV-2-induced expression of pro-inflammatory cytokines, and thus might prevent COVID-19-associated hyperinflammation, a key player in COVID-19 progression. Thus, our data suggest that the Raf/MEK/ERK signaling cascade may represent a target for therapeutic intervention strategies against SARS-CoV-2 infections and that ATR-002 is a promising candidate for further drug evaluation.

Key Points The RAS-regulated RAF–MEK–ERK signalling pathway transmits signals from growth factor receptors to the nucleus and other organelles to regulate cell proliferation, differentiation, survival and invasion. ERK pathway architecture places MEK1 and MEK2 in a unique position, where they process inputs from multiple upstream activating kinases so that ERK1 and ERK2 may be activated alone (following RAF activation) or together with other kinases such as JNK or p38. The majority of cancers exhibit hyper-activation of the ERK pathway owing to deregulation (mutation, gene fusions, amplification, and so on) of receptor tyrosine kinases, RAS, BRAF, CRAF, MEK1 or MEK2 or owing to the loss of negative pathway regulators, such as dual-specificity phosphatases (DUSPs) and NF1. In many cases, these mutations confer a range of pathway dependencies including classical 'oncogene addiction', providing a rationale for ERK pathway inhibitors as therapeutic agents. Melanomas carrying the mutation encoding BRAF-V600E are almost invariably addicted to BRAF activity, and first-generation BRAF inhibitors (BRAFis) have transformed the treatment of this disease. However, an inability to inhibit signalling by RAF dimers drives acquired resistance, and paradoxical RAF activation in tumours with wild-type BRAF limits the success or wider application of BRAFis. As RAF signalling proceeds via the activation of MEK1 and MEK2, and RAF-addicted tumour cells are also MEK-addicted, MEK1 and MEK2 are also attractive drug targets. Additionally, MEK1 and MEK2 contain a unique hydrophobic pocket adjacent to their ATP-binding site, which allows the binding of potent, non-ATP competitive, allosteric inhibitors. However, MEK1 and MEK2 are rarely mutated in cancer, so MEK inhibitors (MEKis) will not preferentially inhibit MEK1 and MEK2 in tumour cells compared with normal tissue; this may contribute to normal tissue toxicity. The first MEKi to receive US Food and Drug Administration approval, trametinib, is being used to treat BRAF -mutant melanoma in combination with first-generation BRAFis. A range of additional MEKis are in late-stage clinical development and exhibit various modes of action; for example, some MEKis can prevent the phosphorylation of MEK1 and MEK2 by RAF, and others can disrupt RAF–MEK1/MEK2 complexes. The extent to which these properties influence clinical activity is currently unclear. Factors that limit the efficacy of MEKis include loss of feedback inhibition and consequent reactivation of ERK1 and ERK2. Intrinsic resistance can be driven by the activation of parallel pathways, including the PI3K pathway, whereas acquired resistance to MEKis arises through the emergence of mutations in MEK1 or MEK2 or the amplification of BRAF V600E or mutant RAS. Adaptation and resistance to MEKis can be overcome by combinations with other targeted agents, including intrapathway dual inhibition. Clinical trials assessing MEKis in combination with other targeted agents or conventional chemotherapy are ongoing. MEK1 and MEK2 have key roles in tumorigenesis and, therefore, represent promising targets for cancer therapy. This Review discusses the mechanisms of action of different inhibitors of MEK1 and MEK2, the mechanisms of resistance to these inhibitors and their current clinical progress. The role of the ERK signalling pathway in cancer is thought to be most prominent in tumours in which mutations in the receptor tyrosine kinases RAS, BRAF, CRAF, MEK1 or MEK2 drive growth factor-independent ERK1 and ERK2 activation and thence inappropriate cell proliferation and survival. New drugs that inhibit RAF or MEK1 and MEK2 have recently been approved or are currently undergoing late-stage clinical evaluation. In this Review, we consider the ERK pathway, focusing particularly on the role of MEK1 and MEK2, the 'gatekeepers' of ERK1/2 activity. We discuss their validation as drug targets, the merits of targeting MEK1 and MEK2 versus BRAF and the mechanisms of action of different inhibitors of MEK1 and MEK2. We also consider how some of the systems-level properties (intrapathway regulatory loops and wider signalling network connections) of the ERK pathway present a challenge for the success of MEK1 and MEK2 inhibitors, discuss mechanisms of resistance to these inhibitors, and review their clinical progress.

Background Abnormalities of in utero testis development are strongly associated with reproductive health conditions, including male infertility and testis cancer. In mouse testes, SOX9 and FGF9 support Sertoli cell development, while VEGF signalling is essential for the establishment of vasculature. The mitogen-activated protein kinase (MAPK) pathway is a major signalling cascade, essential for cell proliferation, differentiation and activation of Sry during primary sex-determination, but little is known about its function during fetal testis morphogenesis. We explored potential functions of MAPK signalling immediately after the establishment of testis cords in embryonic day (E)12.5 Oct4 -eGFP transgenic mouse testes cultured using a MEK1/2 inhibitor. Results RNA sequencing in isolated gonadal somatic cells identified 116 and 114 differentially expressed genes after 24 and 72 h of MEK1/2 inhibition, respectively. Ingenuity Pathway Analysis revealed an association of MEK1/2 signalling with biological functions such as angiogenesis, vasculogenesis and cell migration. This included a failure to upregulate the master transcriptional regulators of vascular development, Sox7 and Sox17, VEGF receptor genes , the cell adhesion factor gene Cd31 and a range of other endothelial cell markers such as Cdh5 (encoding VE-cadherin) and gap junction genes Gja4 and Gja5 . In contrast, only a small number of Sertoli cell enriched genes were affected. Immunofluorescent analyses of control testes revealed that the MEK1/2 downstream target, ERK1/2 was phosphorylated in endothelial cells and Sertoli cells. Inhibition of MEK1/2 eliminated pERK1/2 in fetal testes, and CD31, VE-cadherin, SOX7 and SOX17 and endothelial cells were lost. Consistent with a role for VEGF in driving endothelial cell development in the testis, inhibition of VEGFR also abrogated pERK1/2 and SOX7 and SOX17 expressing endothelial cells. Moreover, while Sertoli cell proliferation and localisation to the testis cord basement membrane was disrupted by inhibition of MEK1/2, it was unaffected by VEGFR inhibition. Instead, inhibition of FGF signalling compromised Sertoli cell proliferation and localisation to the testis cord basement membrane. Conclusions Together, our data highlight an essential role for VEGF-dependent MEK1/2 signalling in promoting vasculature and indicate that FGF signalling through MEK1/2 regulates Sertoli cell organisation in the developing mouse testis.

Selective inhibition of MEK isoforms remains a central challenge in MAPK-targeted drug discovery, largely due to the structural similarity between MEK1 and MEK2. While MEK1 has been extensively characterized, the structural basis of MEK2-specific ligand recognition is not fully understood. Here, we present crystal structures of human MEK2 in complex with the noncompetitive inhibitor U0126 and the allosteric inhibitor refametinib at resolutions of 3.15 Å and 3.30 Å, respectively. Despite a conserved kinase fold, MEK2 exhibits isoform-specific features within the N-lobe β-sheet. Additional differences are observed in the relative orientation of the helix C and activation segment, and the helix F-supported regulatory spine. Structural differences are reflected in micromolar binding affinities for U0126 (Kd = 9.8 μM) and refametinib (Kd = 7.4 μM). Notably, a single N-lobe substitution (Thr87 in MEK2 versus Phe83 in MEK1) selectively enhanced U0126 binding. The MEK2 T87F mutant exhibited an approximately twofold increase in affinity, while refametinib binding remained largely unchanged. SEC–MALS analysis demonstrated that MEK2 predominantly exists as a monomer in solution, contrasting with the reported homodimeric behavior of MEK1. Molecular dynamics simulations supported these findings by revealing isoform-specific differences in oligomeric state-dependent flexibility and inhibitor-induced dynamics. Collectively, our findings define the structural basis underlying the differential inhibitor recognition of MEK2 and MEK1, providing mechanistic insight into isoform-selective MEK-targeted drug design.

Selumetinib (KOSELUGO TM ; AZD6244, ARRY-142886) is a mitogen-activated protein kinase 1 and 2 (MEK1/2) inhibitor being developed by AstraZeneca for the treatment of tumours associated with neurofibromatosis and various cancers. Selumetinib has been granted orphan drug status as adjuvant treatment for thyroid cancer (in the USA) and as treatment for neurofibromatosis type 1 (in the USA and the EU) and, based on the results of the phase II SPRINT trial, was recently approved in the USA in paediatric patients with neurofibromatosis type 1 and symptomatic, inoperable plexiform neurofibromas. This article summarizes the milestones in the development of selumetinib leading to this first approval for the treatment of paediatric patients aged ≥ 2 years with neurofibromatosis type 1 who have symptomatic, inoperable plexiform neurofibromas.

KSR–MEK complexes allosterically activate BRAF through the action of N-terminal regulatory region and kinase domain contacts, thus challenging the accepted role of KSR as a scaffold for MEK recruitment to RAF. Non-catalytic role for MEK in BRAF activation The Ras–ERK signalling pathway regulates diverse cellular processes and its aberrant function is a common oncogenic driver in cancer. RAF family kinases transmit signals from activated Ras at the plasma membrane to the downstream kinases MEK and ERK and are targets for drug development. Here, Marc Therrien and colleagues use crystallography and biochemistry to explore the mechanistic details of how of BRAF is activated. They show that the BRAF substrate MEK promotes dimerization between BRAF and the pseudokinase KSR1 in a manner that is dependent on a direct interaction with KSR1 but independent of MEK kinase activity. Therefore, KSR1 functions as a MEK-dependent allosteric activator of BRAF in a feed-forward mechanism. The work also reveals a modular interaction surface between BRAF and KSR1 that may be amenable to targeting by small-molecule inhibitors. RAF family kinases have prominent roles in cancer 1 . Their activation is dependent on dimerization of their kinase domains, which has emerged as a hindrance for drug development 2 , 3 . In mammals, RAF family kinases include three catalytically competent enzymes (ARAF, BRAF and CRAF) and two pseudokinases (KSR1 and KSR2) that have been described as scaffolds owing to their apparent ability to bridge RAF isoforms and their substrate, mitogen-activated protein kinase kinase (MEK) 4 . Kinase suppressor of Ras (KSR) pseudokinases were also shown to dimerize with kinase-competent RAFs to stimulate catalysis allosterically 5 . Although GTP-bound RAS can modulate the dimerization of RAF isoforms by engaging their RAS-binding domains, KSR1 and KSR2 lack an RAS-binding domain and therefore the regulatory principles underlying their dimerization with other RAF family members remain unknown. Here we show that the selective heterodimerization of BRAF with KSR1 is specified by direct contacts between the amino-terminal regulatory regions of each protein, comprising in part a novel domain called BRS in BRAF and the coiled-coil-sterile α motif (CC-SAM) domain in KSR1. We also discovered that MEK binding to the kinase domain of KSR1 asymmetrically drives BRAF–KSR1 heterodimerization, resulting in the concomitant stimulation of BRAF catalytic activity towards free MEK molecules. These findings demonstrate that KSR–MEK complexes allosterically activate BRAF through the action of N-terminal regulatory region and kinase domain contacts and challenge the accepted role of KSR as a scaffold for MEK recruitment to RAF.

In murine fetal germ cells, retinoic acid (RA) is an extrinsic cue for meiotic initiation that stimulates transcriptional activation of the Stimulated by retinoic acid gene 8 (Stra8), which is required for entry of germ cells into meiotic prophase I. Canonically, the biological activities of RA are mediated by nuclear RA receptors. Recent studies in somatic cells found that RA noncanonically stimulates intracellular signal transduction pathways to regulate multiple cellular processes. In this study, using a germ cell culture system, we investigated (1) whether RA treatment activates any mitogen-activated protein kinase (MAPK) pathways in fetal germ cells at the time of sex differentiation, and (2) if this is the case, whether the corresponding RA-stimulated signaling pathway regulates Stra8 expression in fetal germ cells and their entry into meiosis. When XX germ cells at embryonic day (E) 12.5 were cultured with RA, the extracellular-signal-regulated kinase (ERK) 1/2 pathway was predominantly activated. MEK1/2 inhibitor (U0126) treatment suppressed the mRNA expressions of RA-induced Stra8 and meiotic marker genes (Rec8, Spo11, Dmc1, and Sycp3) in both XX and XY fetal germ cells. Furthermore, U0126 treatment dramatically reduced STRA8 protein levels and numbers of meiotic cells among cultured XX and XY fetal germ cells even in the presence of RA. Taken together, our results suggest the novel concept that the RA functions by stimulating the ERK1/2 pathway and that this activity is critical for Stra8 expression and meiotic progression in fetal germ cells.

Inhibition of MEK stops cell proliferation and induces apoptosis; therefore, this enzyme is a key anticancer target. Trametinib is a selective, orally administered MEK1/MEK2 inhibitor. We aimed to define the maximum tolerated dose and recommended phase 2 dose of trametinib and to assess its safety, pharmacokinetics, pharmacodynamics, and response rate in individuals with advanced solid tumours. We undertook a multicentre phase 1 study in patients with advanced solid tumours and adequate organ function. The study was in three parts: dose escalation to define the maximum tolerated dose; identification of the recommended phase 2 dose; and assessment of pharmacodynamic changes. Intermittent and continuous dosing regimens were analysed. Blood samples and tumour biopsy specimens were taken to assess pharmacokinetic and pharmacodynamic changes. Adverse events were defined with common toxicity criteria, and tumour response was measured by Response Evaluation Criteria In Solid Tumors. This study is registered with ClinicalTrials.gov, number NCT00687622. We enrolled 206 patients (median age 58·5 years, range 19–92). Dose-limiting toxic effects included rash (n=2), diarrhoea (n=1), and central serous retinopathy (n=2). The most common treatment-related adverse events were rash or dermatitis acneiform (n=165; 80%) and diarrhoea (87; 42%), most of which were grade 1 and 2. The maximum tolerated dose was 3 mg once daily and the recommended phase 2 dose was 2 mg a day. The effective half-life of trametinib was about 4 days. At the recommended phase 2 dose, the exposure profile of the drug showed low interpatient variability and a small peak:trough ratio of 1·81. Furthermore, mean concentrations in plasma were greater than the preclinical target concentration throughout the dosing interval. Pathway inhibition and clinical activity were seen, with 21 (10%) objective responses recorded. The recommended phase 2 dose of 2 mg trametinib once a day is tolerable, with manageable side-effects. Trametinib's inhibition of the expected target and clinical activity warrants its further development as a monotherapy and in combination. GlaxoSmithKline.