Biology subjects

Eck, M. J.

Publications and source records attributed to Eck, M. J..

12 recordsLinked to original sources

bioRxiv2026

Pathway-wide base editing charts chemical-genetic interactions in MAPK signaling

CRISPR base editor (BE) scanning enables sequence-level interrogation of proteins at scale in their native cellular and genomic contexts. This approach opens new opportunities to dissect cellular pathways, where signaling depends on coordinated interactions among multiple pathway proteins. We apply BE scanning to the RAS-RAF-MEK-ERK (MAPK) cascade, a central oncogenic pathway and major therapeutic target. Compounds targeting the MAPK pathway have mechanisms of action and resistance that remain incompletely characterized. By base editing 22 MAPK genes under either hyperactivation or inhibition at various nodes, we present a chemical-genetic map of the signaling pathway. Pathway-wide analysis shows that drug resistance mutations frequently occur in proteins other than the direct drug target itself, and we exploit these chemical-genetic interactions to recapitulate key pathway connections. Our BE scanning also identified underappreciated functional hotspots, including an allosteric site in the KRAS N-terminus. We also identify catalytically impaired CRAF mutants that confer bidirectional resistance and sensitization to structurally similar MEK inhibitors, distinguishing functional differences between structurally related compounds. These findings establish BE scanning as a scalable approach for network-level chemical-genetic interaction mapping, revealing new mechanistic insights into one of the most studied oncogenic pathways.

cancer biology↗

bioRxiv2026

Cryo-EM structure and biochemical characterization of a BRAF/CRAF heterodimer: Negative charge in the NtA motif is not required for RAF activation

Upon RAS-driven membrane recruitment, RAF kinases ARAF, BRAF and CRAF are activated via formation of homo- or hetero-dimers to initiate signaling through the MAP kinase cascade. Although RAF heterodimers are important for both physiologic and oncogenic signaling, they have been little studied at a structural and biochemical level. Here we report the preparation, biochemical characterization, and the cryo-EM structure of a 14-3-3-bound BRAF/CRAF heterodimer complex. The heterodimer exhibited kinetic parameters and sensitivity to a panel of twelve structurally diverse RAF inhibitors that were closely similar to, or intermediate between, those of BRAF and CRAF homodimers. Cryo-EM structures of the heterodimer with and without MEK1 revealed an overall organization essentially identical to that of RAF homodimers, but with an asymmetric interaction in the MEK1-bound structure in which the BRAF N-terminal acidic (NtA) motif extends across the dimer interface to engage the CRAF RKTR motif. Mutagenesis of this interface unexpectedly revealed that replacing the acidic NtA sequence with a basic RARA sequence yields highly active RAF homodimers and heterodimers, demonstrating that negative charge in the NtA motif is not required for activity. Collectively, our findings suggest that the charge state of the NtA motif influences RAF activity through effects on local backbone dynamics and the stability of the inactive kinase conformation, rather than via stereospecific recognition across the dimer interface.

biophysics↗

bioRxiv2025

RAF isoform selectivity of MEK inhibitors and rational design of a covalent ARAF-MEK inhibitor

Aberrant activation of the RAS/RAF/MEK/ERK pathway is a frequent cause of cancer. Allosteric MEK inhibitors block this pathway by binding RAF-MEK complexes to prevent activation of MEK by RAF. However, how MEK inhibitor potency varies across the three RAF isoforms remains poorly understood. We profiled seven allosteric MEK inhibitors and observed a striking hierarchy of sensitivity: all most potently inhibited CRAF-driven MEK activation while relatively sparing ARAF-driven activation. We identified point mutations in ARAF and CRAF proximate to the MEK inhibitor binding site that markedly altered inhibitor sensitivity. Using a rational design approach, we developed a more potent inhibitor of ARAF-driven MEK signaling, TWG-07-148. Our cryo-EM structure shows how this acrylamide-containing analog of MEK inhibitor trametinib covalently targets Cys514, a residue unique to ARAF. Our studies highlight the importance of the activating RAF isoform as a determinant of MEK inhibitor sensitivity and provide proof-of-concept for development of MEK inhibitors that more effectively block ARAF-driven MEK signaling via covalent targeting of Cys514.

biochemistry↗

bioRxiv2025

In vitro reconstitutions suggest a general model for paradoxical activation of ARAF, BRAF, and CRAF by diverse RAF inhibitor types that does not rely on negative allostery.

RAF kinases are central regulators of the RAS/MAP kinase pathway and important targets in cancer therapy. Paradoxically, RAF inhibitors can activate wild-type RAF signaling. Negative allostery is a central feature of the prevailing model for this phenomenon, wherein inhibitors induce RAF dimers in which inhibitor binding to one protomer promotes an active but inhibitor-resistant conformation in the other protomer. Here we systematically examined paradoxical activation of ARAF, BRAF, and CRAF using biochemical assays with isolated RAF/MEK kinase domain complexes. We found that type I and type II inhibitors induce paradoxical activation of all three isoforms, and that phosphomimetic mutation of the N-terminal acidic motif of ARAF and CRAF dramatically sensitized these isoforms to activation by type II inhibitors. The inhibition phase of paradoxical activation curves for type II inhibitors was suggestive of positive cooperativity, a finding in conflict with the prevailing model which implies negative cooperativity. In contrast to the kinase domain RAF/MEK preparations, full-length autoinhibited RAF/MEK/14-3-3 complexes were refractory to activation. Mass photometry confirmed that paradoxical activators promote BRAF dimerization. These findings support a revised model that does not rely on negative allostery. Inhibitors act on the RAS-engaged "open monomer" state to induce dimerization and activation. The open monomer and active dimer are structurally distinct species with differing affinities for inhibitor and ATP, creating a concentration window in which paradoxical activation occurs.

biochemistry↗

bioRxiv2025

Overcoming EGFR resistance by monovalent and bident inhibitors targeting Cys775

Covalent targeting of EGFR cysteine 797 by osimertinib is one of the most successful breakthroughs in targeted therapy, fundamentally transforming the treatment landscape for non-small cell lung cancer (NSCLC) patients. However, resistance driven by mutation of C797 remains a major clinical challenge. Developing novel covalent strategies beyond C797 targeting presents a compelling opportunity for next-generation EGFR inhibitors. We first demonstrated that cysteine 775, located deep within the ATP-binding pocket, is accessible by a rationally designed covalent molecule ZNL-3, which as the first-in-class covalent cysteine 775 inhibitor exhibited strong efficacy in osimertinib-resistant mouse models. To further enhance resilience to resistance-causing mutations, we developed a dual-warhead, bident compound--YNW-1--which covalently targets both cysteine 775 and 797 simultaneously. YNW-1 is the first intramolecular lock to exhibit balanced reactive efficiency on both cysteines, rendering single-site mutations ineffective to confer resistance. The discovery of ZNL-3 and YNW-1 represents significant advancements in EGFR-targeted drug development, and further optimization toward clinical translation is a worthwhile strategy. SIGNIFICANCE: This study establishes the therapeutic potential of an EGFR covalent inhibitor through unprecedented targeting of cysteine 775 and provides the first demonstration that dual cysteine engagement offers superior efficacy over conventional covalent inhibitors by delaying resistance.

cancer biology↗

bioRxiv2025

Characterization and inhibitor sensitivity of ARAF, BRAF, and CRAF complexes

The RAS-RAF-MEK-ERK signaling pathway controls cellular growth and proliferation, and mutational activation of this pathway is a frequent cause of cancer. Most prominently, the V600E mutation in BRAF causes malignant melanoma, papillary thyroid cancer and other malignancies. Rare but recurrent activating mutations in the other two RAF isoforms, ARAF and CRAF, have also been identified in diverse cancers. Distinct classes of RAF inhibitors have been developed, particularly for BRAFV600E, but their potencies against the three RAF isoforms have not been systematically compared. Here we biochemically characterize monomeric and dimeric preparations of ARAF, BRAF, and CRAF and measure the potencies of a panel of thirteen type I, type I.5, and type II RAF inhibitors against each active RAF preparation. Type I inhibitor SB590885 is roughly equipotent across RAF isoforms and, as expected, type I.5 inhibitors are typically most potent against BRAFV600E. Despite their reputation as pan-RAF inhibitors, type II inhibitors as a class are potent inhibitors of CRAF but exhibit relative sparing of ARAF and intermediate potencies against BRAF. Type II compounds inhibit BRAF and CRAF with marked positive cooperativity, and their apparent potencies are insensitive to ATP-concentrations. Crystal structures of CRAF in complex with type I.5 inhibitor PLX4720 reveal an asymmetric CRAF dimer with one CRAF subunit bound in the inactive state and the second bound in an C-helix-in, active conformation with an altered inhibitor pose. Our findings have important implications for understanding the pharmacology of current RAF inhibitors and will inform development of new agents with distinct isoform selectivity.

biochemistry↗

bioRxiv2024

Structure of the CABIT2 domain of THEMIS reveals a novel protein fold with an inserted SH3-like domain

Maturation of thymocytes into T cells is critical for proper function of the adaptive immune system. During this developmental process, thymocytes undergo a highly-regulated selection process regulated by the signaling characteristics of the T cell receptor (TCR) pathway. Thymocyte-Expressed Molecule Involved in Selection (THEMIS) is an essential protein for T cell development. THEMIS regulates phosphatases downstream of the T cell receptor to ensure signaling thresholds are met during selection. Important features of THEMIS are its two uncharacterized CABIT (Cysteine-containing All-Beta In THEMIS) domains, which are intriguing because they have been proposed to participate in important protein-protein interactions (PPIs) that modulate immunological signals. Here, we report the 2.9 [A] crystal structure of the THEMIS CABIT2 domain determined via heavy atom phasing. The structure revealed a novel protein domain fold comprised mainly of {beta}-sheets with two distinct subdomains. This domain appears to have a different C-terminal boundary than predicted or found in previously used experimental constructs. Inclusion of the proline rich segment enables GRB2 to bind CABIT2. Isolated CABIT2 domain is unable to bind or modulate the function of SHP1 phosphatase. This structure will provide the foundation for future structure-function studies of CABIT domains and THEMIS.

biochemistry↗

bioRxiv2024

A diverse landscape of FGFR alterations and co-mutations defines novel therapeutic strategies in pediatric low-grade gliomas

Alterations in Fibroblast growth factor receptor (FGFR)-family proteins frequently occur as oncogenes in many cancers, including a subset of pediatric gliomas. Here, we performed a genomic analysis of 11,635 gliomas across ages and found that 4.5% of all gliomas harbor FGFR alterations including structural variants (SV) and single nucleotide variants (SNV), with an incidence of almost 10% in pediatric gliomas. FGFR family members are differentially enriched by age, tumor grade, and histological subtype, with FGFR1-alterations associated with glioneuronal histologies and pediatric low-grade gliomas. Across development, we find FGFR1 expression in both neuronal and glial precursors, while FGFR3 expression is largely restricted to astrocytic lineages. Leveraging novel isogenic model systems, we confirm FGFR1 alterations to be sufficient to activate MAPK and mTOR signaling, drive gliomagenesis, activate neuronal transcriptional programs and exhibit sensitivity to MAPK pathway inhibitors, including pan-FGFR inhibitors. Models driven by FGFR1 SVs exhibited different patterns of sensitivity compared to those driven by SNVs. Finally, we performed a retrospective analysis of clinical responses in children diagnosed with FGFR-driven gliomas and found that targeted MAPK or FGFR-inhibition with currently available inhibitors is largely associated with stability of disease. This study provides key insights into the biology of FGFR1-altered gliomas, therapeutic strategies to target them and associated challenges that still need to be overcome.

cancer biology↗

bioRxiv2022

Structural analysis of the macrocyclic inhibitor BI-4020 binding to EGFR kinase

A novel macrocyclic inhibitor of mutant EGFR (BI-4020) has shown promise in pre-clinical studies of T790M and C797S drug-resistant non-small cell lung cancer. To better understand the molecular basis for BI-4020 selectivity and potency, we have carried out biochemical activity assays and structural analysis with X-ray crystallography. Biochemical potencies agree with previous studies indicating that BI-4020 is uniquely potent against drug-resistant L858R/T790M and L858R/T790M/C797S variants. Structures show that BI-4020 is likely rendered selective due to interactions with the kinase domain hinge region as well as T790M, akin to Osimertinib. Additionally, BI-4020 is also rendered more potent due to its constrained macrocycle geometry as well as additional H-bonds to conserved K745 and T845 residues in both active and inactive conformations. These findings taken together show how this novel macrocyclic inhibitor is both highly potent and selective for mutant EGFR in a reversible mechanism and motivate structure-inspired approaches to developing targeted therapies in medicinal oncology.

pharmacology and toxicology↗

bioRxiv2022

Cryo-EM structure of a RAS/RAF recruitment complex

Cryo-EM structures of a KRAS/BRAF/MEK1/14-3-3 complex reveal KRAS bound to the flexible Ras-binding domain of BRAF, captured in two orientations. Autoinhibitory interactions are unperturbed by binding of KRAS and in vitro activation studies confirm that KRAS binding is insufficient to activate BRAF, absent membrane recruitment. These structures illustrate the separability of binding and activation of BRAF by Ras and suggest stabilization of this pre-activation intermediate as an alternative to blocking binding of KRAS.

biochemistry↗

bioRxiv2022

PH Domain-Mediated Autoinhibition and Oncogenic Activation of Akt

Previously, we reported that the conformation of autoinhibited Akt, a Ser/Thr protein kinase that plays a central role in metabolism and cancer, may be shifted by small molecule allosteric inhibitors limiting the mechanistic insights from existing X-ray structures that have relied on such compounds (Chu, Viennet, et al, 2020). Here we discover unexpectedly that a single mutation, R86A, in Akts PH domain exhibits intensified autoinhibitory features with enhanced PH domain-kinase domain affinity. Structural and biochemical analysis uncovers the importance of a key interaction network involving Arg86, Glu17, and Tyr18 that controls Akt conformation and activity. Our studies also shed light on the molecular basis for E17K Akt activation as an oncogenic driver.

biochemistry↗

bioRxiv2019

The Eya1 phosphatase mediates Shh-driven symmetric cell division of cerebellar granule cell precursors

During neural development, stem and precursor cells can divide either symmetrically or asymmetrically. The transition between symmetric and asymmetric cell divisions is a major determinant of precursor cell expansion and neural differentiation, but the underlying mechanisms that regulate this transition are not well understood. Here, we identify the Sonic hedgehog (Shh) pathway as a critical determinant regulating the mode of division of cerebellar granule cell precursors (GCPs). Using partial gain and loss of function mutations within the Shh pathway, we show that pathway activation determines spindle orientation of GCPs, and that mitotic spindle orientation directly correlates with the mode of division. Mechanistically, we show that the phosphatase Eya1 is essential for implementing Shh-dependent GCP spindle orientation. We identify atypical protein kinase C (aPKC) as a direct target of Eya1 activity and show that Eya1 dephosphorylates Threonine (T410) in the activation loop of this polarity complex component. Thus, Eya1 inactivates the cell polarity complex, resulting in reduced phosphorylation of Numb and other components that regulate the mode of division. This Eya1-dependent cascade is critical in linking spindle orientation, cell cycle exit and terminal differentiation. Together these findings demonstrate that a Shh-Eya1 regulatory axis selectively promotes symmetric cell divisions during cerebellar development by coordinating spindle orientation and cell fate determinants.\n\nSummary statementBiological responses to Shh signaling are specified by the magnitude of pathway activation and the cellular context. This study shows that potent Shh signaling regulates mitotic orientation and symmetric division of cerebellar granule cell precursors in a process that requires the phosphatase Eya1 and unequal distribution of cell fate determinants to daughter cells.

neuroscience↗