Afadin is a multimodal scaffolding protein with essential functions in cell–cell adhesion. Although its loss of expression has been linked to breast cancer invasion and metastasis, the underlying mechanisms driving tumour progression upon mutational Afadin ( AFDN ) loss in breast cancers remains unclear. In the current study we identified a somatic frameshift AFDN mutation ( p .Lys630fs) in an invasive breast cancer sample that coincides with loss of Afadin protein expression. Functional studies in E‐cadherin‐expressing breast cancer cells show that Afadin loss leads to immature and aberrant adherens junction (AJ) formation. The lack of AJ maturation results in a noncohesive cellular phenotype accompanied by Actomyosin‐dependent anoikis resistance, which are classical progression hallmarks of single‐cell breast cancer invasion. Reconstitution experiments using Afadin truncates show that proper F‐actin organisation and epithelial cell–cell adhesion critically depend on the Coiled‐Coil domain of Afadin but not on the designated C‐terminal F‐actin binding domain. Mouse xenograft experiments based on cell lines and primary patient‐derived breast cancer organoids demonstrate that Afadin loss induces single‐cell lobular‐type invasion phenotypes and overt dissemination to the lungs and the peritoneum. In short, Afadin is a metastasis suppressor for breast cancer through stabilisation and maturation of a mechanical E‐cadherin to F‐actin outside‐in link. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Zonula adherens junctions (zAJ) are spatially proximal to tight junctions (TJ), in a superstructure known as the apical junctional complex (AJC). A key component of the AJC is a circumferential ring of filamentous (F)-actin, but how actomyosin contractility drives AJC structure and epithelial barrier function is incompletely understood. Here, we show that a central mechanosensitive component of zAJ, α-catenin (α-cat), undergoes force-dependent phosphorylation in an unstructured linker region. This modification in turn primes the α-cat mechanosensitive Middle-region for effector-binding. We credential Afadin, a multi-domain TJ/AJ scaffold protein, as mechano-chemical binding partner of α-cat, identifying residues in α-cat required for this interaction. α-cat phosphorylation and Afadin-binding are required for their co-enrichment at zAJ and epithelial barrier function. A mouse model that prevents α-cat phosphorylation is particularly detrimental to post-natal brain development. These data support a stepwise model where α-cat integrates mechanical and chemical signals to progressively promote zAJ enrichment, effector recruitment and epithelial barrier function.

Supplemental Materials from Computational and Functional Analyses of HER2 Mutations Reveal Allosteric Activation Mechanisms and Altered Pharmacologic Effects

Epithelial cells can become polyploid upon tissue injury, but mechanosensitive cues that trigger this state are poorly understood. Using an Madin Darby Canine Kidney (MDCK) cell knock-out/reconstitution system, we show that α-catenin mutants that alter force-sensitive binding to F-actin or middle (M)-domain promote cytokinesis failure and binucleation, particularly near epithelial wound-fronts. We identified Leucine Zipper Tumor Suppressor 2 (LZTS2), a factor previously implicated in abscission, as a conformation sensitive proximity partner of α-catenin. We show that LZTS2 enriches not only at midbody/intercellular bridges but also at apical adhering junctions. α-Catenin mutants with persistent M-domain opening show elevated junctional enrichment of LZTS2 compared with wild-type cells. LZTS2 knock-down leads to elevated rates of binucleation. These data implicate LZTS2 as a mechanosensitive effector of α-catenin that is critical for cytokinetic fidelity. This model rationalizes how persistent mechanoactivation of α-catenin may drive tension-induced polyploidization of epithelia after injury and suggests an underlying mechanism for how pathogenic α-catenin M-domain mutations drive macular dystrophy.

ABSTRACTLoss of E-cadherin (CDH1) and the adherens junction (AJ) drive development and progression of invasive lobular breast cancer (ILC). However, approximately 40% retain wild typeCDH1alleles, indicating that modulation of other genes attenuates the AJ during ILC etiology. To identify alternative drivers, we performed targeted sequencing inCDH1wild type samples, based on a defined set of 100 AJ, tight junction, and desmosome genes we designated as the ‘Adhesome’. In 146 ILC samples, we identified 62 cases (43%) with wild typeCDH1alleles in which we detected a total of 284 mutations in 36 Adhesome genes. After selection based on occurrence and potential loss of function, we identified an inactivating frameshift mutation in Afadin (AFDN; p.Lys630fs).Functional studies in E-cadherin-expressing breast cancer cells showed that Afadin knockout leads to immature AJs, and a non-cohesive phenotype accompanied by actomyosin dependent anoikis resistance, which are classical ILC hallmarks. Afadin reconstitutions show that F-actin organization critically depends on the ⍰E-catenin binding CC domain. Afadin loss in intraductal xenograft mouse breast cancer models leads to ILC-type morphologies and overt lung metastases.AFDNtruncate reconstitutions revealed that deletion of the C-terminal ⍰E-catenin binding CC domain is sufficient to drive metastatic ILC. In conclusion, we identified and functionally coupled a somatic frameshiftAFDNmutation in breast cancer to destabilization the epithelial AJ and the development of ILC hallmarks such as actomyosin-dependent anoikis resistance and single cell invasion. As such, Afadin represents a candidate tumor suppressor for E-cadherin-positive ILC development and progression.

Mutations in BRAF and RAS are often oncogenic and lead to a constitutively active MAPK pathway that promotes aberrant cell proliferation and tumor growth. Currently approved BRAF inhibitors are selective against monomeric BRAF V600 mutants. These drugs are largely inactive against non-V600 dimeric BRAF mutants and have poor brain penetration. Although there is an FDA-approved KRAS G12C mutant-selective inhibitor, there are no approved inhibitors for cancer patients who harbor other (non-G12C) KRAS and NRAS mutations which promote tumor growth likely through constitutively active RAF dimers. There remains a high unmet clinical need for a highly CNS penetrant oral RAF inhibitor that targets a broad spectrum of BRAF mutations and constitutively active RAF dimers without paradoxical activation of the MAPK signaling pathway. BDTX-4933 is a potent, reversible, CNS penetrant RAF MasterKey inhibitor designed to target a large family of oncogenic BRAF mutations including BRAF monomers and RAF dimers. The compound inhibits not only all classes (I, II, and III) of BRAF mutations but also targets constitutively active RAF dimers promoted by upstream oncogenic MAPK pathway alterations, such as RAS mutations. In a panel of cancer cell lines that endogenously express BRAF or RAS mutations, BDTX-4933 demonstrates inhibition of the MAPK pathway signaling without paradoxical activation, resulting in potent inhibition of cellular proliferation. BDTX-4933 shows target engagement, inhibiting ERK phosphorylation, in tumor models in vivo, achieving strong anti-tumor efficacy and tumor regression across tumor models driven by either BRAF or RAS mutations. Furthermore, BDTX-4933 exhibits high CNS exposure leading to dose-dependent tumor growth inhibition, and survival benefit in mice implanted intracranially with xenograft BRAF mutant tumors. BDTX-4933 has a best-in-class profile to treat cancer patients harboring BRAF mutations or RAF dimer-promoting upstream genetic alterations. IND-enabling studies for BDTX-4933 are on-going. Citation Format: Yoon-Chi Han, Pui-Yee Ng, Luisa Shin Ogawa, Shao Ning Yang, Miao Chen, Noboru Ishiyama, Tai-An Lin, Elizabeth Buck. Preclinical characterization of a brain penetrant RAF inhibitor, BDTX-4933, targeting oncogenic BRAF Class I/II/III and RAS mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3415.

Achondroplasia is a rare disease affecting bone growth and is caused by a missense mutation in the fibroblast growth factor receptor 3 (FGFR3) gene. In the past few years, there were multiple experimental drugs entering into clinical trials for treating achondroplasia including vosoritide, the first precision medicine approved for this indication. This perspective presents the mechanism of action, benefit, and potential mechanistic limitation of the drugs currently being evaluated in clinical trials for achondroplasia. This article also discusses the potential impact of those drugs not only in increasing the growth of individuals living with achondroplasia but also in improving their quality of life.

Epithelial cells can become polyploid upon tissue injury, but mechanosensitive cues that trigger this state are poorly understood. Using α-catenin (α-cat) knock-out Madin Darby Canine Kidney (MDCK) cells reconstituted with wild-type and mutant forms of α-cat as a model system, we find that an established α-cat actin-binding domain unfolding mutant designed to reduce force-sensitive binding to F-actin (α-cat-H0-FABD+) can promote cytokinesis failure, particularly along epithelial wound-fronts. Enhanced α-cat coupling to cortical actin is neither sufficient nor mitotic cell-autonomous for cytokinesis failure, but critically requires the mechanosensitive Middle-domain (M1-M2-M3) and neighboring cells. Disease relevant α-cat M-domain missense mutations known to cause a form of retinal pattern dystrophy (α-cat E307K or L436P) are associated with elevated binucleation rates via cytokinesis failure. Similar binucleation rates are seen in cells expressing an α-cat salt-bridge destabilizing mutant (R551A) designed to promote M2-M3 domain unfurling at lower force thresholds. Since binucleation is strongly enhanced by removal of the M1 as opposed to M2-M3 domains, cytokinetic fidelity is most sensitive to α-cat M2-M3 domain opening. To identify α-cat conformation-dependent proximity partners that contribute to cytokinesis, we used a biotin-ligase approach to distinguished proximity partners that show enhanced recruitment upon α-cat M-domain unfurling (R551A). We identified Leucine Zipper Tumor Suppressor 2 (LZTS2), an abscission factor previously implicated in cytokinesis. We confirm that LZTS2 enriches at the midbody, but discover it also localizes to tight and tricellular junctions. LZTS2 knock-down promotes binucleation in both MDCK and Retinal Pigmented Epithelial (RPE) cells. α-cat mutants with persistent M2-M3 domain opening showed elevated junctional enrichment of LZTS2 from the cytosol compared α-cat wild-type cells. These data implicate LZTS2 as a mechanosensitive effector of α-cat that is critical for cytokinetic fidelity. This model rationalizes how persistent mechano-activation of α-cat may drive tension-induced polyploidization of epithelia post-injury and suggests an underlying mechanism for how pathogenic α-cat mutations drive macular dystrophy.

One central question for cell and developmental biologists is defining how epithelial cells can change shape and move during embryonic development without tearing tissues apart. This requires robust yet dynamic connections of cells to one another, via the cell-cell adherens junction, and of junctions to the actin and myosin cytoskeleton, which generates force. The last decade revealed that these connections involve a multivalent network of proteins, rather than a simple linear pathway. We focus on Drosophila Canoe, homolog of mammalian Afadin, as a model for defining the underlying mechanisms. Canoe and Afadin are complex, multidomain proteins that share multiple domains with defined and undefined binding partners. Both also share a long carboxy-terminal intrinsically disordered region (IDR), whose function is less well defined. IDRs are found in many proteins assembled into large multiprotein complexes. We have combined bioinformatic analysis and the use of a series of canoe mutants with early stop codons to explore the evolution and function of the IDR. Our bioinformatic analysis reveals that the IDRs of Canoe and Afadin differ dramatically in sequence and sequence properties. When we looked over shorter evolutionary time scales, we identified multiple conserved motifs. Some of these are predicted by AlphaFold to be alpha-helical, and two correspond to known protein interaction sites for alpha-catenin and F-actin. We next identified the lesions in a series of eighteen canoe mutants, which have early stop codons across the entire protein coding sequence. Analysis of their phenotypes are consistent with the idea that the IDR, including the conserved motifs in the IDR, are critical for protein function. These data provide the foundation for further analysis of IDR function.

Abstract Amplification of HER2 can drive the proliferation of cancer cells, and several inhibitors of HER2 have been successfully developed. Recent advances in next-generation sequencing now reveal that HER2 is subject to mutation, with over 2,000 unique variants observed in human cancers. Several examples of oncogenic HER2 mutations have been described, and these primarily occur at allosteric sites outside the ATP-binding site. To identify the full spectrum of oncogenic HER2 driver mutations aside from a few well-studied mutations, we developed mutation-allostery-pharmacology (MAP), an in silico prediction algorithm based on machine learning. By applying this computational approach to 820 single-nucleotide variants, a list of 222 known and potential driver mutations was produced. Of these 222 mutations, 111 were screened by Ba/F3-retrovirus proliferation assays; 37 HER2 mutations were experimentally determined to be driver mutations, comprising 15 previously characterized and 22 newly identified oncogenic mutations. These oncogenic mutations mostly affected allosteric sites in the extracellular domain (ECD), transmembrane domain, and kinase domain of HER2, with only a single mutation in the HER2 orthosteric ATP site. Covalent homodimerization was established as a common mechanism of activation among HER2 ECD allosteric mutations, including the most prevalent HER2 mutation, S310F. Furthermore, HER2 allosteric mutants with enhanced covalent homodimerization were characterized by altered pharmacology that reduces the activity of existing anti-HER2 agents, including the mAb trastuzumab and the tyrosine kinase inhibitor lapatinib. Overall, the MAP-scoring and functional validation analyses provided new insights into the oncogenic activity and therapeutic targeting of HER2 mutations in cancer. Significance: This study identified new oncogenic HER2 allosteric mutations, including ECD mutations that share covalent dimerization as a mechanism of oncogenicity, suggesting the need for novel inhibitors to treat HER2-mutant cancers.

Background: FDA-approved BRAF inhibitors target V600 (Class I) mutant monomers and are largely inactive against mutant BRAF dimers. These dimeric mutants found in many solid tumors including primary CNS tumors and brain metastases can drive RAS-independent (Class II) or RAS-dependent (Class III) oncogenic tumor growth. Furthermore, the approved BRAF inhibitors can induce paradoxical RAF activation that limits their activity. Although currently approved BRAF V600 mutation-selective inhibitors demonstrated efficacy in brain tumors and metastases when combined with MEK inhibitors in clinical trials, duration of response tends to be short partly due to limited BBB permeability. There remains a high unmet clinical need for a broad Class I/II/III BRAF inhibitor with high CNS penetrant activity for patients with RAF-dependent tumors carrying a broad spectrum of BRAF alterations. Materials and Methods: The MAP platform used NGS data and a proprietary machine-learning algorithm to predict and then validate the oncogenicity of previously uncharacterized groups of oncogenic BRAF mutations across Class I/II/III and to identify small molecule MasterKey drug candidates against this spectrum of mutations. Candidates were further optimized for brain penetration properties. Results: BDTX-4933 is a potent, selective, CNS penetrant BRAF Class I/ II/III mutation inhibitor that targets MAP-predicted oncogenic BRAF alterations while sparing wild type BRAF with >10-fold selectivity in cell proliferation assays. BDTX-4933 inhibits the RAF-MEK-ERK signaling pathway and cell proliferation across a panel of cancer cell lines endogenously expressing Class I/II/III mutations without paradoxical RAF activation. BDTX-4933 achieves target engagement, inhibiting the BRAF signaling pathway in in vivo models, and anti-tumor activity across tumor models representing all three classes of BRAF mutations including NSCLC and melanoma. Preclinical data shows that BDTX-4933 achieves high CNS exposure, and results in tumor growth inhibition and survival benefit in BRAF mutant intracranial models. Conclusions: BDTX-4933 has all the attributes of a best-in-class CNS penetrant BRAF inhibitor to address patients with and without CNS disease whose tumors express monomeric (Class I) or dimeric (Class II and III) BRAF mutants. BDTX-4933 achieves on-target inhibition of the RAF-MEK-ERK signaling pathway and anti-tumor activity in multiple preclinical tumor models, including intracranial models. IND-enabling studies for BDTX-4933 are underway. Conflict of interest: Ownership: Black Diamond Therapeutics

Abstract The canonical BRAF V600E (Class I) mutation is a potent oncogene uniquely active as a RAS-independent monomer, successfully targeted by several FDA-approved inhibitors. However, these first-generation BRAF inhibitors are not active against non-canonical BRAF oncogenic mutations, including BRAF-fusions, that drive RAS-independent (Class II) or RAS-dependent (Class III) dimers. As such, developing inhibitors directed against dimeric BRAF oncogenic mutations that avoid paradoxical activation is a major unmet clinical need. We applied proprietary Mutation-Allostery-Pharmacology (MAP) platform technology developed by Black Diamond Therapeutics to identify and validate a group of previously uncharacterized non-canonical oncogenic Class II and Class III BRAF mutation clusters. We further demonstrate that this ensemble of novel and previously validated non-canonical oncogenic BRAF mutants can form the basis of a differentiated drug discovery program aimed at identifying small molecules that potently and selectively target this family of dimeric BRAF mutations. Herein, we describe a small molecule inhibitor, BDTX BRAF-A, with potent anti-proliferative activity directed against tumor cells expressing a wide spectrum of non-canonical Class II/III mutations. This broad activity (“MasterKey” profile) of BDTX BRAF-A is further demonstrated in cell lines that harbor endogenous oncogenic dimer-inducing BRAF mutations and in various solid tumor patient-derived xenograft (PDX) models ex vivo. Importantly, BDTX BRAF-A did not induce paradoxical RAF activation characteristic of Class I BRAF inhibitors. Finally, BDTX BRAF-A achieves robust anti-tumor efficacy and target engagement of dimeric BRAF oncogenes in mouse models. These data support the continued development of rationally designed molecules targeting a broad range of non-canonical BRAF dimer-promoting mutations to extend the prospect of precision medicine in patients. Citation Format: Yoon-Chi Han, Pui Yee Ng, Ryan Schulz, Shao Ning Yang, Alana Lelo, Luisa Shin, Matthew O'Connor, Ivan Jewett, Noboru Ishiyama, Darlene Romashko, Shalabh Thakur, Andrei Salomatov, Sherri Smith, Elizabeth Buck, Christopher Roberts, Matthew Lucas, Tai-An Lin. Pre-clinical evaluation of next-generation inhibitor targeting a wide spectrum of oncogenic BRAF dimers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P229.

Cadherin-mediated cell adhesion requires anchoring via the β-catenin-α-catenin complex to the actin cytoskeleton, yet, α-catenin only binds F-actin weakly. A covalent fusion of VE-cadherin to α-catenin enhances actin anchorage in endothelial cells and strongly stabilizes endothelial junctions in vivo, blocking inflammatory responses. Here, we have analyzed the underlying mechanism. We found that VE-cadherin-α-catenin constitutively recruits the actin adaptor vinculin. However, removal of the vinculin-binding region of α-catenin did not impair the ability of VE-cadherin-α-catenin to enhance junction integrity. Searching for an alternative explanation for the junction-stabilizing mechanism, we found that an antibody-defined epitope, normally buried in a short α1-helix of the actin-binding domain (ABD) of α-catenin, is openly displayed in junctional VE-cadherin-α-catenin chimera. We found that this epitope became exposed in normal α-catenin upon triggering thrombin-induced tension across the VE-cadherin complex. These results suggest that the VE-cadherin-α-catenin chimera stabilizes endothelial junctions due to conformational changes in the ABD of α-catenin that support constitutive strong binding to actin.

The canonical BRAF V600E (class I) mutation is a potent oncogene which is uniquely active as a RAS-independent monomer, and which has been successfully targeted by several FDA-approved inhibitors. While active against monomeric BRAF V600E, these first generation BRAF inhibitors induce paradoxical activation of RAS-driven BRAF dimers in cells expressing wild-type RAF, and this can lead to secondary malignancies. More recently, numerous non-canonical BRAF oncogenic mutations including BRAF-fusions have been described as oncogenes that drive RAS-independent (class II) or RAS-dependent (class III) dimers. These non-canonical dimeric BRAF oncogenes are resistant to the first-generation drugs, effective only against the monomeric BRAF V600E mutation. Discovery of an inhibitor directed against the family of dimeric BRAF oncogenic mutations which avoids paradoxical activation is a major unmet need. We applied our proprietary Mutation-Allostery-Pharmacology (MAP) platform technology developed by Black Diamond Therapeutics to identify and validate a group of previously uncharacterized non-canonical oncogenic class II and class III BRAF mutation clusters. We further demonstrate that this ensemble of both novel and previously validated non-canonical oncogenic BRAF mutants can form the basis of a differentiated drug discovery program aimed at identifying small molecules that potently and selectively target this family of dimeric BRAF mutations. Herein, we describe a series of small molecule inhibitors with potent anti-proliferative activity directed against tumor cells harboring dimer-inducing BRAF oncogenic mutations and which are devoid of paradoxical RAF activation. Leading exemplars of BDTX compounds are orally available inhibitors that achieve target engagement of BRAF dimer oncogenes in vivo and robust anti-tumor efficacy in xenograft models in mice. These data support continued development of rationally designed molecules targeting a broad range of non-canonical BRAF dimer-promoting mutations to extend the prospect of precision medicine in patients with BRAF mutant tumors.

KRAS4b is a small guanosine triphosphatase (GTPase) protein that regulates several signal transduction pathways that underlie cell proliferation, differentiation, and survival. KRAS4b function requires prenylation of its C terminus and recruitment to the plasma membrane, where KRAS4b activates effector proteins including the RAF family of kinases. The Ca2+-sensing protein calmodulin (CaM) has been suggested to regulate the localization of KRAS4b through direct, Ca2+-dependent interaction, but how CaM and KRAS4b functionally interact is controversial. Here, we determined a crystal structure, which was supported by solution nuclear magnetic resonance (NMR), that revealed the sequestration of the prenyl moiety of KRAS4b in the hydrophobic pocket of the C-terminal lobe of Ca2+-bound CaM. Our engineered fluorescence resonance energy transfer (FRET)-based biosensor probes (CaMeRAS) showed that, upon stimulation of Ca2+ influx by extracellular ligands, KRAS4b reversibly translocated in a Ca2+-CaM-dependent manner from the plasma membrane to the cytoplasm in live HeLa and HEK293 cells. These results reveal a mechanism underlying the inhibition of KRAS4b activity by Ca2+ signaling pathways.

Store operated calcium (Ca2+) entry (SOCE) is the process whereby endoplasmic reticulum (ER) Ca2+ store depletion causes Orai1-composed Ca2+ channels on the plasma membrane (PM) to open, mediating a rise in cytosolic Ca2+ levels. Stromal interaction molecules (STIMs) are the proteins that directly sense ER Ca2+ content and gate Orai1 channels due to store depletion. The trigger for STIM activation is Ca2+ unbinding from the ER lumen-oriented domains, which consist of a nonconserved amino (N) terminal region and EF-hand and sterile alpha motif (SAM) domains (EF-SAM), highly conserved from humans to Caenorhabditis elegans. Solution NMR structures of the human EF-SAM domains have been determined at high Ca2+ concentrations; however, no direct structural view of the Ca2+ binding mode has been elucidated. Further, no atomic resolution data currently exists on EF-SAM at low Ca2+ levels. Here, we determined the X-ray crystal structure of the C. elegans STIM lumina] domain, revealing that EF-SAM binds a single Ca2+ ion with pentagonal bipyramidal geometry and an ancillary alpha-helix formed by the N-terminal region acts as a brace to stabilize EF-SAM. Using solution NMR, we observed EF-hand domain unfolding and a conformational exchange between folded and unfolded states involving the ancillary alpha-helix and the canonical EF-hand in low Ca2+. Remarkably, we also detected an alpha-helix (+Ca2+) to beta-strand (-Ca2+) transition at the terminal SAM domain alpha-helix. Collectively, our analyses indicate that one canonically bound Ca2+ ion is sufficient to stabilize the quiescent luminal domain structure, precluding unfolding, conformational exchange, and secondary structure transformation. (C) 2019 Elsevier Ltd. All rights reserved.

Mutation of both the intracellular catalytic domain and the extracellular domain of the receptor for epidermal growth factor (EGFR) can drive oncogenicity. Despite clinical success with targeting EGFR catalytic site mutations, no drugs have proven effective in patients expressing allosteric extracellular domain EGFR mutations, including glioblastomas (GBM) where these mutations are highly expressed. We define the molecular mechanism for oncogenic activation of families of extracellular EGFR mutations and reveal how this mechanism renders current generation small molecule ATP-site inhibitors ineffective. We demonstrate that a group of commonly expressed extracellular domain EGFR mutants expressed in GBM is activated by disulfide-bond mediated covalent dimerization, collectively referred to as locked dimerization (LoDi) EGFR oncogenes. Strikingly, small molecules binding to the active kinase conformation (Type I), but not those binding to the inactive kinase conformation (Type II), potently inhibit catalytic site mutants, but induce covalent dimerization and activate LoDi-EGFR oncogenes, manifesting in paradoxical acceleration of proliferation. Significance Our data demonstrate how the locked-dimer mechanism of EGFR oncogenesis has a profound impact on the activity of small molecule inhibitors. This provides a mechanistic understanding for the failure of current generation EGFR inhibitors to effectively treat LoDi-EGFR mutants in GBM, and sets guidelines for discovery of selective LoDi-EGFR inhibitors.

α-catenin is a key mechanosensor that forms force-dependent interactions with F-actin, thereby coupling the cadherin-catenin complex to the actin cytoskeleton at adherens junctions (AJs). However, the molecular mechanisms by which α-catenin engages F-actin under tension remained elusive. Here we show that the α1-helix of the α-catenin actin-binding domain (αcat-ABD) is a mechanosensing motif that regulates tension-dependent F-actin binding and bundling. αcat-ABD containing an α1-helix-unfolding mutation (H1) shows enhanced binding to F-actin in vitro. Although full-length α-catenin-H1 can generate epithelial monolayers that resist mechanical disruption, it fails to support normal AJ regulation in vivo. Structural and simulation analyses suggest that α1-helix allosterically controls the actin-binding residue V796 dynamics. Crystal structures of αcat-ABD-H1 homodimer suggest that α-catenin can facilitate actin bundling while it remains bound to E-cadherin. We propose that force-dependent allosteric regulation of αcat-ABD promotes dynamic interactions with F-actin involved in actin bundling, cadherin clustering, and AJ remodeling during tissue morphogenesis.

The PAR-1-MARK pathway controls cell polarity through the phosphorylation of microtubule-associated proteins. Rho-Rac guanine nucleotide exchange factor 2 (ARHGEF2), which activates Ras homolog family member A (RHOA), is anchored to the microtubule network and sequestered in an inhibited state through binding to dynein light chain Tctex-1 type 1 (DYNLT1). We showed in mammalian cells that liver kinase B1 (LKB1) activated the microtubule affinity-regulating kinase 3 (MARK3), which in turn phosphorylated ARHGEF2 at Ser(151). This modification disrupted the interaction between ARHGEF2 and DYNLT1 by generating a 14-3-3 binding site in ARHGEF2, thus causing ARHGEF2 to dissociate from microtubules. Phosphorylation of ARHGEF2 by MARK3 stimulated RHOA activation and the formation of stress fibers and focal adhesions, and was required for organized cellular architecture in three-dimensional culture. Protein phosphatase 2A (PP2A) dephosphorylated Ser(151) in ARHGEF2 to restore the inhibited state. Thus, we have identified a regulatory switch controlled by MARK3 that couples microtubules to the actin cytoskeleton to establish epithelial cell polarity through ARHGEF2.