Publications by authors named "Stephen C Blacklow"

118 Publications

Time-resolved phosphoproteomics reveals scaffolding and catalysis-responsive patterns of SHP2-dependent signaling.

Elife 2021 03 23;10. Epub 2021 Mar 23.

Department of Cancer Biology, Dana-Farber Cancer Institute Boston, Boston, United States.

SHP2 is a protein tyrosine phosphatase that normally potentiates intracellular signaling by growth factors, antigen receptors, and some cytokines, yet is frequently mutated in human cancer. Here, we examine the role of SHP2 in the responses of breast cancer cells to EGF by monitoring phosphoproteome dynamics when SHP2 is allosterically inhibited by SHP099. The dynamics of phosphotyrosine abundance at more than 400 tyrosine residues reveal six distinct response signatures following SHP099 treatment and washout. Remarkably, in addition to newly identified substrate sites on proteins such as occludin, ARHGAP35, and PLCγ2, another class of sites shows reduced phosphotyrosine abundance upon SHP2 inhibition. Sites of decreased phospho-abundance are enriched on proteins with two nearby phosphotyrosine residues, which can be directly protected from dephosphorylation by the paired SH2 domains of SHP2 itself. These findings highlight the distinct roles of the scaffolding and catalytic activities of SHP2 in effecting a transmembrane signaling response.
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http://dx.doi.org/10.7554/eLife.64251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024022PMC
March 2021

Biophysics of Notch Signaling.

Annu Rev Biophys 2021 May 3;50:157-189. Epub 2021 Feb 3.

Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts 02115, USA; email:

Notch signaling is a conserved system of communication between adjacent cells, influencing numerous cell fate decisions in the development of multicellular organisms. Aberrant signaling is also implicated in many human pathologies. At its core, Notch has a mechanotransduction module that decodes receptor-ligand engagement at the cell surface under force to permit proteolytic cleavage of the receptor, leading to the release of the Notch intracellular domain (NICD). NICD enters the nucleus and acts as a transcriptional effector to regulate expression of Notch-responsive genes. In this article, we review and integrate current understanding of the detailed molecular basis for Notch signal transduction, highlighting quantitative, structural, and dynamic features of this developmentally central signaling mechanism. We discuss the implications of this mechanistic understanding for the functionality of the signaling pathway in different molecular and cellular contexts.
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http://dx.doi.org/10.1146/annurev-biophys-101920-082204DOI Listing
May 2021

Cryo-EM structure of the B cell co-receptor CD19 bound to the tetraspanin CD81.

Science 2021 01;371(6526):300-305

Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA.

Signaling through the CD19-CD81 co-receptor complex, in combination with the B cell receptor, is a critical determinant of B cell development and activation. It is unknown how CD81 engages CD19 to enable co-receptor function. Here, we report a 3.8-angstrom structure of the CD19-CD81 complex bound to a therapeutic antigen-binding fragment, determined by cryo-electron microscopy (cryo-EM). The structure includes both the extracellular domains and the transmembrane helices of the complex, revealing a contact interface between the ectodomains that drives complex formation. Upon binding to CD19, CD81 opens its ectodomain to expose a hydrophobic CD19-binding surface and reorganizes its transmembrane helices to occlude a cholesterol binding pocket present in the apoprotein. Our data reveal the structural basis for CD19-CD81 complex assembly, providing a foundation for rational design of therapies for B cell dysfunction.
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http://dx.doi.org/10.1126/science.abd9836DOI Listing
January 2021

High-efficacy subcellular micropatterning of proteins using fibrinogen anchors.

J Cell Biol 2021 Feb;220(2)

Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.

Protein micropatterning allows proteins to be precisely deposited onto a substrate of choice and is now routinely used in cell biology and in vitro reconstitution. However, drawbacks of current technology are that micropatterning efficiency can be variable between proteins and that proteins may lose activity on the micropatterns. Here, we describe a general method to enable micropatterning of virtually any protein at high specificity and homogeneity while maintaining its activity. Our method is based on an anchor that micropatterns well, fibrinogen, which we functionalized to bind to common purification tags. This enhances micropatterning on various substrates, facilitates multiplexed micropatterning, and dramatically improves the on-pattern activity of fragile proteins like molecular motors. Furthermore, it enhances the micropatterning of hard-to-micropattern cells. Last, this method enables subcellular micropatterning, whereby complex micropatterns simultaneously control cell shape and the distribution of transmembrane receptors within that cell. Altogether, these results open new avenues for cell biology.
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http://dx.doi.org/10.1083/jcb.202009063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7802367PMC
February 2021

Design of biologically active binary protein 2D materials.

Nature 2021 01 6;589(7842):468-473. Epub 2021 Jan 6.

Department of Biochemistry, University of Washington, Seattle, WA, USA.

Ordered two-dimensional arrays such as S-layers and designed analogues have intrigued bioengineers, but with the exception of a single lattice formed with flexible linkers, they are constituted from just one protein component. Materials composed of two components have considerable potential advantages for modulating assembly dynamics and incorporating more complex functionality. Here we describe a computational method to generate co-assembling binary layers by designing rigid interfaces between pairs of dihedral protein building blocks, and use it to design a p6m lattice. The designed array components are soluble at millimolar concentrations, but when combined at nanomolar concentrations, they rapidly assemble into nearly crystalline micrometre-scale arrays nearly identical to the computational design model in vitro and in cells without the need for a two-dimensional support. Because the material is designed from the ground up, the components can be readily functionalized and their symmetry reconfigured, enabling formation of ligand arrays with distinguishable surfaces, which we demonstrate can drive extensive receptor clustering, downstream protein recruitment and signalling. Using atomic force microscopy on supported bilayers and quantitative microscopy on living cells, we show that arrays assembled on membranes have component stoichiometry and structure similar to arrays formed in vitro, and that our material can therefore impose order onto fundamentally disordered substrates such as cell membranes. In contrast to previously characterized cell surface receptor binding assemblies such as antibodies and nanocages, which are rapidly endocytosed, we find that large arrays assembled at the cell surface suppress endocytosis in a tunable manner, with potential therapeutic relevance for extending receptor engagement and immune evasion. Our work provides a foundation for a synthetic cell biology in which multi-protein macroscale materials are designed to modulate cell responses and reshape synthetic and living systems.
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http://dx.doi.org/10.1038/s41586-020-03120-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855610PMC
January 2021

Characterization of novel neutralizing mouse monoclonal antibody JM1-24-3 developed against MUC18 in metastatic melanoma.

J Exp Clin Cancer Res 2020 Dec 5;39(1):273. Epub 2020 Dec 5.

Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA.

Background: MUC18 is a glycoprotein highly expressed on the surface of melanoma and other cancers which promotes tumor progression and metastasis. However, its mechanism of action and suitability as a therapeutic target are unknown.

Methods: A monoclonal antibody (mAb) (JM1-24-3) was generated from metastatic melanoma tumor live cell immunization, and high-throughput screening identified MUC18 as the target.

Results: Analysis of molecular interactions between MUC18 and JM1-24-3 revealed that the downstream signaling events depended on binding of the mAb to a conformational epitope on the extracellular domain of MUC18. JM1-24-3 inhibited melanoma cell proliferation, migration and invasion in vitro and reduced tumor growth and metastasis in vivo.

Conclusion: These results confirm that MUC18 is mechanistically important in melanoma growth and metastasis, suggest that the MUC18 epitope identified is a promising therapeutic target, and that the JM1-24-3 mAb may serve as the basis for a potential therapeutic agent.
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http://dx.doi.org/10.1186/s13046-020-01722-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7718695PMC
December 2020

, a DNA damage response gene, is required for Notch-mediated induction of squamous cell differentiation.

Elife 2020 09 16;9. Epub 2020 Sep 16.

Department of Pathology, Brigham and Women's Hospital, and Harvard Medical School, Boston, United States.

Notch signaling regulates squamous cell proliferation and differentiation and is frequently disrupted in squamous cell carcinomas, in which Notch is tumor suppressive. Here, we show that conditional activation of Notch in squamous cells activates a context-specific gene expression program through lineage-specific regulatory elements. Among direct Notch target genes are multiple DNA damage response genes, including , which we show is required for Notch-induced differentiation of squamous carcinoma cells and TERT-immortalized keratinocytes. is epistatic to , a gene that encodes the PP2A B55α subunit, which we show interacts with IER5 in cells and in purified systems. Thus, Notch and DNA-damage response pathways converge in squamous cells on common genes that promote differentiation, which may serve to eliminate damaged cells from the proliferative pool. We further propose that crosstalk involving Notch and PP2A enables tuning and integration of Notch signaling with other pathways that regulate squamous differentiation.
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http://dx.doi.org/10.7554/eLife.58081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529455PMC
September 2020

Pharmacological disruption of the Notch transcription factor complex.

Proc Natl Acad Sci U S A 2020 07 29;117(28):16292-16301. Epub 2020 Jun 29.

Swiss Institute for Experimental Cancer Research, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland;

Notch pathway signaling is implicated in several human cancers. Aberrant activation and mutations of Notch signaling components are linked to tumor initiation, maintenance, and resistance to cancer therapy. Several strategies, such as monoclonal antibodies against Notch ligands and receptors, as well as small-molecule γ-secretase inhibitors (GSIs), have been developed to interfere with Notch receptor activation at proximal points in the pathway. However, the use of drug-like small molecules to target the downstream mediators of Notch signaling, the Notch transcription activation complex, remains largely unexplored. Here, we report the discovery of an orally active small-molecule inhibitor (termed CB-103) of the Notch transcription activation complex. We show that CB-103 inhibits Notch signaling in primary human T cell acute lymphoblastic leukemia and other Notch-dependent human tumor cell lines, and concomitantly induces cell cycle arrest and apoptosis, thereby impairing proliferation, including in GSI-resistant human tumor cell lines with chromosomal translocations and rearrangements in Notch genes. CB-103 produces Notch loss-of-function phenotypes in flies and mice and inhibits the growth of human breast cancer and leukemia xenografts, notably without causing the dose-limiting intestinal toxicity associated with other Notch inhibitors. Thus, we describe a pharmacological strategy that interferes with Notch signaling by disrupting the Notch transcription complex and shows therapeutic potential for treating Notch-driven cancers.
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http://dx.doi.org/10.1073/pnas.1922606117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368267PMC
July 2020

A dynamic interaction between CD19 and the tetraspanin CD81 controls B cell co-receptor trafficking.

Elife 2020 04 27;9. Epub 2020 Apr 27.

Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, United States.

CD81 and its binding partner CD19 are core subunits of the B cell co-receptor complex. While CD19 belongs to the extensively studied Ig superfamily, CD81 belongs to a poorly understood family of four-pass transmembrane proteins called tetraspanins. Tetraspanins play important physiological roles by controlling protein trafficking and other processes. Here, we show that CD81 relies on its ectodomain to traffic CD19 to the cell surface. Moreover, the anti-CD81 antibody 5A6, which binds selectively to activated B cells, recognizes a conformational epitope on CD81 that is masked when CD81 is bound to CD19. Mutations of CD81 in this interface suppress its CD19 export activity. These data indicate that the CD81 - CD19 interaction is dynamically regulated upon B cell activation and this dynamism can be exploited to regulate B cell function. These results are not only valuable for understanding B cell biology, but also have important implications for understanding tetraspanin function generally.
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http://dx.doi.org/10.7554/eLife.52337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228769PMC
April 2020

MAML1-Dependent Notch-Responsive Genes Exhibit Differing Cofactor Requirements for Transcriptional Activation.

Mol Cell Biol 2020 05 14;40(11). Epub 2020 May 14.

Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA

Mastermind proteins are required for transcription of Notch target genes, yet the molecular basis for mastermind function remains incompletely understood. Previous work has shown that Notch can induce transcriptional responses by binding to promoters but more often by binding to enhancers, with and as representative mammalian examples of promoter and enhancer responsiveness, respectively. Here, we show that mastermind dependence of the Notch response at these loci is differentially encoded in Jurkat T-cell acute lymphoblastic leukemia (T-ALL) cells. Knockout of Mastermind-like 1 (MAML1) eliminates Notch-responsive activation of both these genes, and reduced target gene expression is accompanied by a decrease in H3K27 acetylation, consistent with the importance of MAML1 for p300 activity. Add-back of MAML1 variants in knockout cells identifies residues 151 to 350 of MAML1 as essential for expression of either Notch-responsive gene. Fusion of the Notch-binding region of MAML1 to the histone acetyltransferase (HAT) domain of p300 rescues expression of but not , suggesting that an additional activity of MAML1 is needed for gene induction at a distance. Together, these studies establish the functional importance of the MAML1 region from residues 151 to 350 for Notch-dependent transcriptional induction and reveal differential requirements for MAML1-dependent recruitment activities at different Notch-responsive loci, highlighting the molecular complexity of Notch-stimulated transcription.
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http://dx.doi.org/10.1128/MCB.00014-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225564PMC
May 2020

Trib1 regulates T cell differentiation during chronic infection by restraining the effector program.

J Exp Med 2020 05;217(5)

Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.

In chronic infections, the immune response fails to control virus, leading to persistent antigen stimulation and the progressive development of T cell exhaustion. T cell effector differentiation is poorly understood in the context of exhaustion, but targeting effector programs may provide new strategies for reinvigorating T cell function. We identified Tribbles pseudokinase 1 (Trib1) as a central regulator of antiviral T cell immunity, where loss of Trib1 led to a sustained enrichment of effector-like KLRG1+ T cells, enhanced function, and improved viral control. Single-cell profiling revealed that Trib1 restrains a population of KLRG1+ effector CD8 T cells that is transcriptionally distinct from exhausted cells. Mechanistically, we identified an interaction between Trib1 and the T cell receptor (TCR) signaling activator, MALT1, which disrupted MALT1 signaling complexes. These data identify Trib1 as a negative regulator of TCR signaling and downstream function, and reveal a link between Trib1 and effector versus exhausted T cell differentiation that can be targeted to improve antiviral immunity.
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http://dx.doi.org/10.1084/jem.20190888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7201917PMC
May 2020

Development of a covalent inhibitor of gut bacterial bile salt hydrolases.

Nat Chem Biol 2020 03 10;16(3):318-326. Epub 2020 Feb 10.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.

Bile salt hydrolase (BSH) enzymes are widely expressed by human gut bacteria and catalyze the gateway reaction leading to secondary bile acid formation. Bile acids regulate key metabolic and immune processes by binding to host receptors. There is an unmet need for a potent tool to inhibit BSHs across all gut bacteria to study the effects of bile acids on host physiology. Here, we report the development of a covalent pan-inhibitor of gut bacterial BSHs. From a rationally designed candidate library, we identified a lead compound bearing an alpha-fluoromethyl ketone warhead that modifies BSH at the catalytic cysteine residue. This inhibitor abolished BSH activity in conventional mouse feces. Mice gavaged with a single dose of this compound displayed decreased BSH activity and decreased deconjugated bile acid levels in feces. Our studies demonstrate the potential of a covalent BSH inhibitor to modulate bile acid composition in vivo.
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http://dx.doi.org/10.1038/s41589-020-0467-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036035PMC
March 2020

Extension of the Notch intracellular domain ankyrin repeat stack by NRARP promotes feedback inhibition of Notch signaling.

Sci Signal 2019 11 5;12(606). Epub 2019 Nov 5.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.

Canonical Notch signaling relies on regulated proteolysis of the receptor Notch to generate a nuclear effector that induces the transcription of Notch-responsive genes. In higher organisms, one Notch-responsive gene that is activated in many different cell types encodes the Notch-regulated ankyrin repeat protein (NRARP), which acts as a negative feedback regulator of Notch responses. Here, we showed that NRARP inhibited the growth of Notch-dependent T cell acute lymphoblastic leukemia (T-ALL) cell lines and bound directly to the core Notch transcriptional activation complex (NTC), requiring both the transcription factor RBPJ and the Notch intracellular domain (NICD), but not Mastermind-like proteins or DNA. The crystal structure of an NRARP-NICD1-RBPJ-DNA complex, determined to 3.75 Å resolution, revealed that the assembly of NRARP-NICD1-RBPJ complexes relied on simultaneous engagement of RBPJ and NICD1, with the three ankyrin repeats of NRARP extending the Notch1 ankyrin repeat stack. Mutations at the NRARP-NICD1 interface disrupted entry of the proteins into NTCs and abrogated feedback inhibition in Notch signaling assays in cultured cells. Forced expression of NRARP reduced the abundance of NICD in cells, suggesting that NRARP may promote the degradation of NICD. These studies establish the structural basis for NTC engagement by NRARP and provide insights into a critical negative feedback mechanism that regulates Notch signaling.
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http://dx.doi.org/10.1126/scisignal.aay2369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212804PMC
November 2019

Domain integration of ADAM family proteins: Emerging themes from structural studies.

Exp Biol Med (Maywood) 2019 12 23;244(17):1510-1519. Epub 2019 Jul 23.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.

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http://dx.doi.org/10.1177/1535370219865901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920674PMC
December 2019

A Flow-Extension Tethered Particle Motion Assay for Single-Molecule Proteolysis.

Biochemistry 2019 05 12;58(20):2509-2518. Epub 2019 Apr 12.

Department of Biological Chemistry and Molecular Pharmacology , Harvard Medical School , Boston , Massachusetts 02115 , United States.

Regulated proteolysis of signaling proteins under mechanical tension enables cells to communicate with their environment in a variety of developmental and physiologic contexts. The role of force in inducing proteolytic sensitivity has been explored using magnetic tweezers at the single-molecule level with bead-tethered assays, but such efforts have been limited by challenges in ensuring that beads not be restrained by multiple tethers. Here, we describe a multiplexed assay for single-molecule proteolysis that overcomes the multiple-tether problem using a flow-extension strategy on a microscope equipped with magnetic tweezers. Particle tracking and computational sorting of flow-induced displacements allow assignment of tethered substrates to singly captured and multiply tethered bins, with the fraction of fully mobile, single-tether substrates depending inversely on the concentration of substrate loaded on the coverslip. Computational exclusion of multiple-tether beads enables robust assessment of on-target proteolysis by the highly specific tobacco etch virus protease and the more promiscuous metalloprotease ADAM17. This method should be generally applicable to a wide range of proteases and readily extensible to robust evaluation of proteolytic sensitivity as a function of applied magnetic force.
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http://dx.doi.org/10.1021/acs.biochem.9b00106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6607913PMC
May 2019

Bispecific Forkhead Transcription Factor FoxN3 Recognizes Two Distinct Motifs with Different DNA Shapes.

Mol Cell 2019 04 27;74(2):245-253.e6. Epub 2019 Feb 27.

Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA; Committee on Higher Degrees in Biophysics, Harvard University, Cambridge, MA 02138, USA; Program in Biological and Biomedical Sciences, Harvard University, Cambridge, MA 02138, USA; Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Transcription factors (TFs) control gene expression by binding DNA recognition sites in genomic regulatory regions. Although most forkhead TFs recognize a canonical forkhead (FKH) motif, RYAAAYA, some forkheads recognize a completely different (FHL) motif, GACGC. Bispecific forkhead proteins recognize both motifs, but the molecular basis for bispecific DNA recognition is not understood. We present co-crystal structures of the FoxN3 DNA binding domain bound to the FKH and FHL sites, respectively. FoxN3 adopts a similar conformation to recognize both motifs, making contacts with different DNA bases using the same amino acids. However, the DNA structure is different in the two complexes. These structures reveal how a single TF binds two unrelated DNA sequences and the importance of DNA shape in the mechanism of bispecific recognition.
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http://dx.doi.org/10.1016/j.molcel.2019.01.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6474805PMC
April 2019

Oncogenic Notch Promotes Long-Range Regulatory Interactions within Hyperconnected 3D Cliques.

Mol Cell 2019 03 7;73(6):1174-1190.e12. Epub 2019 Feb 7.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Biomedical Informatics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Chromatin loops enable transcription-factor-bound distal enhancers to interact with their target promoters to regulate transcriptional programs. Although developmental transcription factors such as active forms of Notch can directly stimulate transcription by activating enhancers, the effect of their oncogenic subversion on the 3D organization of cancer genomes is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B cell lymphoma, we show that beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes by instructing enhancer repositioning. Moreover, a large fraction of Notch-instructed regulatory loops form highly interacting enhancer and promoter spatial clusters termed "3D cliques." Loss- and gain-of-function experiments show that Notch preferentially targets hyperconnected 3D cliques that regulate the expression of crucial proto-oncogenes. Our observations suggest that oncogenic hijacking of developmental transcription factors can dysregulate transcription through widespread effects on the spatial organization of cancer genomes.
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http://dx.doi.org/10.1016/j.molcel.2019.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485942PMC
March 2019

Structural reorganization of SHP2 by oncogenic mutations and implications for oncoprotein resistance to allosteric inhibition.

Nat Commun 2018 10 30;9(1):4508. Epub 2018 Oct 30.

Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, MA, 02115, USA.

Activating mutations in PTPN11, encoding the cytosolic protein tyrosine phosphatase SHP2, result in developmental disorders and act as oncogenic drivers in patients with hematologic cancers. The allosteric inhibitor SHP099 stabilizes the wild-type SHP2 enzyme in an autoinhibited conformation that is itself destabilized by oncogenic mutations. Here, we report the impact of the highly activated and most frequently observed mutation, E76K, on the structure of SHP2, and investigate the effect of E76K and other oncogenic mutations on allosteric inhibition by SHP099. SHP2 adopts an open conformation but can be restored to the closed, autoinhibited conformation, near-identical to the unoccupied wild-type enzyme, when complexed with SHP099. SHP099 inhibitory activity against oncogenic SHP2 variants in vitro and in cells scales inversely with the activating strength of the mutation, indicating that either oncoselective or vastly more potent inhibitors will be necessary to suppress oncogenic signaling by the most strongly activating SHP2 mutations in cancer.
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http://dx.doi.org/10.1038/s41467-018-06823-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6207684PMC
October 2018

The ectodomains determine ligand function in vivo and selectivity of DLL1 and DLL4 toward NOTCH1 and NOTCH2 in vitro.

Elife 2018 10 5;7. Epub 2018 Oct 5.

Institute for Molecular Biology, Medizinische Hochschule Hannover, Hannover, Germany.

DLL1 and DLL4 are Notch ligands with high structural similarity but context-dependent functional differences. Here, we analyze their functional divergence using cellular co-culture assays, biochemical studies, and in vivo experiments. DLL1 and DLL4 activate NOTCH1 and NOTCH2 differently in cell-based assays and this discriminating potential lies in the region between the N-terminus and EGF repeat three. Mice expressing chimeric ligands indicate that the ectodomains dictate ligand function during somitogenesis, and that during myogenesis even regions C-terminal to EGF3 are interchangeable. Substitution of NOTCH1-interface residues in the MNNL and DSL domains of DLL1 with the corresponding amino acids of DLL4, however, does not disrupt DLL1 function in vivo. Collectively, our data show that DLL4 preferentially activates NOTCH1 over NOTCH2, whereas DLL1 is equally effective in activating NOTCH1 and NOTCH2, establishing that the ectodomains dictate selective ligand function in vivo, and that features outside the known binding interface contribute to their differences.
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http://dx.doi.org/10.7554/eLife.40045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202052PMC
October 2018

Structural and Atropisomeric Factors Governing the Selectivity of Pyrimido-benzodiazipinones as Inhibitors of Kinases and Bromodomains.

ACS Chem Biol 2018 09 31;13(9):2438-2448. Epub 2018 Aug 31.

Department of Cancer Biology , Dana-Farber Cancer Institute , Boston , Massachusetts 02215 , United States.

Bromodomains have been pursued intensively over the past several years as emerging targets for the development of anticancer and anti-inflammatory agents. It has recently been shown that some kinase inhibitors are able to potently inhibit the bromodomains of BRD4. The clinical activities of PLK inhibitor BI-2536 and JAK2-FLT3 inhibitor TG101348 have been attributed to this unexpected polypharmacology, indicating that dual-kinase/bromodomain activity may be advantageous in a therapeutic context. However, for target validation and biological investigation, a more selective target profile is desired. Here, we report that benzo[e]pyrimido-[5,4- b]diazepine-6(11H)-ones, versatile ATP-site directed kinase pharmacophores utilized in the development of inhibitors of multiple kinases, including several previously reported kinase chemical probes, are also capable of exhibiting potent BRD4-dependent pharmacology. Using a dual kinase-bromodomain inhibitor of the kinase domains of ERK5 and LRRK2, and the bromodomain of BRD4 as a case study, we define the structure-activity relationships required to achieve dual kinase/BRD4 activity, as well as how to direct selectivity toward inhibition of either ERK5 or BRD4. This effort resulted in identification of one of the first reported kinase-selective chemical probes for ERK5 (JWG-071), a BET selective inhibitor with 1 μM BRD4 IC (JWG-115), and additional inhibitors with rationally designed polypharmacology (JWG-047, JWG-069). Co-crystallography of seven representative inhibitors with the first bromodomain of BRD4 demonstrate that distinct atropisomeric conformers recognize the kinase ATP-site and the BRD4 acetyl lysine binding site, conformational preferences supported by rigid docking studies.
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http://dx.doi.org/10.1021/acschembio.7b00638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333101PMC
September 2018

The Molecular Mechanism of Notch Activation.

Adv Exp Med Biol 2018 ;1066:47-58

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.

Research in the last several years has shown that Notch proteolysis, and thus Notch activation, is conformationally controlled by the extracellular juxtamembrane NRR of Notch, which sterically occludes the S2 protease site until ligand binds. The question of how conformational exposure of the protease site is achieved during physiologic activation, and thus how normal activation is bypassed in disease pathogenesis, has been the subject of intense study in the last several years, and is the subject of this chapter. Here, we summarize the structural features of the NRR domains of Notch receptors that establish the autoinhibited state and then review a number of recent studies aimed at testing the mechanotransduction model for Notch signaling using force spectroscopy and molecular tension sensors.
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http://dx.doi.org/10.1007/978-3-319-89512-3_3DOI Listing
February 2019

Dual Allosteric Inhibition of SHP2 Phosphatase.

ACS Chem Biol 2018 03 18;13(3):647-656. Epub 2018 Jan 18.

Novartis Institutes for Biomedical Research , Cambridge , Massachusetts 02139 , United States.

SHP2 is a cytoplasmic protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell proliferation, differentiation, and survival. Recently, we reported an allosteric mechanism of inhibition that stabilizes the auto-inhibited conformation of SHP2. SHP099 (1) was identified and characterized as a moderately potent, orally bioavailable, allosteric small molecule inhibitor, which binds to a tunnel-like pocket formed by the confluence of three domains of SHP2. In this report, we describe further screening strategies that enabled the identification of a second, distinct small molecule allosteric site. SHP244 (2) was identified as a weak inhibitor of SHP2 with modest thermal stabilization of the enzyme. X-ray crystallography revealed that 2 binds and stabilizes the inactive, closed conformation of SHP2, at a distinct, previously unexplored binding site-a cleft formed at the interface of the N-terminal SH2 and PTP domains. Derivatization of 2 using structure-based design resulted in an increase in SHP2 thermal stabilization, biochemical inhibition, and subsequent MAPK pathway modulation. Downregulation of DUSP6 mRNA, a downstream MAPK pathway marker, was observed in KYSE-520 cancer cells. Remarkably, simultaneous occupation of both allosteric sites by 1 and 2 was possible, as characterized by cooperative biochemical inhibition experiments and X-ray crystallography. Combining an allosteric site 1 inhibitor with an allosteric site 2 inhibitor led to enhanced pharmacological pathway inhibition in cells. This work illustrates a rare example of dual allosteric targeted protein inhibition, demonstrates screening methodology and tactics to identify allosteric inhibitors, and enables further interrogation of SHP2 in cancer and related pathologies.
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http://dx.doi.org/10.1021/acschembio.7b00980DOI Listing
March 2018

Structural Basis for Regulated Proteolysis by the α-Secretase ADAM10.

Cell 2017 Dec 7;171(7):1638-1648.e7. Epub 2017 Dec 7.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Cleavage of membrane-anchored proteins by ADAM (a disintegrin and metalloproteinase) endopeptidases plays a key role in a wide variety of biological signal transduction and protein turnover processes. Among ADAM family members, ADAM10 stands out as particularly important because it is both responsible for regulated proteolysis of Notch receptors and catalyzes the non-amyloidogenic α-secretase cleavage of the Alzheimer's precursor protein (APP). We present here the X-ray crystal structure of the ADAM10 ectodomain, which, together with biochemical and cellular studies, reveals how access to the enzyme active site is regulated. The enzyme adopts an unanticipated architecture in which the C-terminal cysteine-rich domain partially occludes the enzyme active site, preventing unfettered substrate access. Binding of a modulatory antibody to the cysteine-rich domain liberates the catalytic domain from autoinhibition, enhancing enzymatic activity toward a peptide substrate. Together, these studies reveal a mechanism for regulation of ADAM activity and offer a roadmap for its modulation.
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http://dx.doi.org/10.1016/j.cell.2017.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773094PMC
December 2017

MAFB enhances oncogenic Notch signaling in T cell acute lymphoblastic leukemia.

Sci Signal 2017 11 14;10(505). Epub 2017 Nov 14.

Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104, USA.

Activating mutations in the gene encoding the cell-cell contact signaling protein Notch1 are common in human T cell acute lymphoblastic leukemias (T-ALLs). However, expressing mutant alleles in mice fails to efficiently induce the development of leukemia. We performed a gain-of-function screen to identify proteins that enhanced signaling by leukemia-associated Notch1 mutants. The transcription factors MAFB and ETS2 emerged as candidates that individually enhanced Notch1 signaling, and when coexpressed, they synergistically increased signaling to an extent similar to that induced by core components of the Notch transcriptional complex. In mouse models of T-ALL, MAFB enhanced leukemogenesis by the naturally occurring Notch1 mutants, decreased disease latency, and increased disease penetrance. Decreasing MAFB abundance in mouse and human T-ALL cells reduced the expression of Notch1 target genes, including and , and sustained MAFB knockdown impaired T-ALL growth in a competitive setting. MAFB bound to ETS2 and interacted with the acetyltransferases PCAF and P300, highlighting its importance in recruiting coactivators that enhance Notch1 signaling. Together, these data identify a mechanism for enhancing the oncogenic potential of weak Notch1 mutants in leukemia models, and they reveal the MAFB-ETS2 transcriptional axis as a potential therapeutic target in T-ALL.
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http://dx.doi.org/10.1126/scisignal.aam6846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5885022PMC
November 2017

Identification of an allosteric benzothiazolopyrimidone inhibitor of the oncogenic protein tyrosine phosphatase SHP2.

Bioorg Med Chem 2017 12 20;25(24):6479-6485. Epub 2017 Oct 20.

Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, MA, USA; Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA. Electronic address:

The PTPN11 oncogene encodes the cytoplasmic protein tyrosine phosphatase SHP2, which, through its role in multiple signaling pathways, promotes the progression of hematological malignancies and other cancers. Here, we employ high-throughput screening to discover a lead chemical scaffold, the benzothiazolopyrimidones, that allosterically inhibits this oncogenic phosphatase by simultaneously engaging the C-SH2 and PTP domains. We improved our lead to generate an analogue that better suppresses SHP2 activity in vitro. Suppression of Erk phopsphorylation by the lead compound is also consistent with SHP2 inhibition in AML cells. Our findings provide an alternative starting point for therapeutic intervention and will catalyze investigations into the relationship between SHP2 conformational regulation, activity, and disease progression.
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http://dx.doi.org/10.1016/j.bmc.2017.10.025DOI Listing
December 2017

A B Cell Regulome Links Notch to Downstream Oncogenic Pathways in Small B Cell Lymphomas.

Cell Rep 2017 Oct;21(3):784-797

Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Gain-of-function Notch mutations are recurrent in mature small B cell lymphomas such as mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), but the Notch target genes that contribute to B cell oncogenesis are largely unknown. We performed integrative analysis of Notch-regulated transcripts, genomic binding of Notch transcription complexes, and genome conformation data to identify direct Notch target genes in MCL cell lines. This B cell Notch regulome is largely controlled through Notch-bound distal enhancers and includes genes involved in B cell receptor and cytokine signaling and the oncogene MYC, which sustains proliferation of Notch-dependent MCL cell lines via a Notch-regulated lineage-restricted enhancer complex. Expression of direct Notch target genes is associated with Notch activity in an MCL xenograft model and in CLL lymph node biopsies. Our findings provide key insights into the role of Notch in MCL and other B cell malignancies and have important implications for therapeutic targeting of Notch-dependent oncogenic pathways.
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http://dx.doi.org/10.1016/j.celrep.2017.09.066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5687286PMC
October 2017

The common oncogenomic program of NOTCH1 and NOTCH3 signaling in T-cell acute lymphoblastic leukemia.

PLoS One 2017 12;12(10):e0185762. Epub 2017 Oct 12.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, United States of America.

Notch is a major oncogenic driver in T cell acute lymphoblastic leukemia (T-ALL), in part because it binds to an enhancer that increases expression of MYC. Here, we exploit the capacity of activated NOTCH1 and NOTCH3 to induce T-ALL, despite substantial divergence in their intracellular regions, as a means to elucidate a broad, common Notch-dependent oncogenomic program through systematic comparison of the transcriptomes and Notch-bound genomic regulatory elements of NOTCH1- and NOTCH3-dependent T-ALL cells. ChIP-seq studies show a high concordance of functional NOTCH1 and NOTCH3 genomic binding sites that are enriched in binding motifs for RBPJ, the transcription factor that recruits activated Notch to DNA. The interchangeability of NOTCH1 and NOTCH3 was confirmed by rescue of NOTCH1-dependent T-ALL cells with activated NOTCH3 and vice versa. Despite remarkable overall similarity, there are nuanced differences in chromatin landscapes near critical common Notch target genes, most notably at a Notch-dependent enhancer that regulates MYC, which correlates with responsiveness to Notch pathway inhibitors. Overall, a common oncogenomic program driven by binding of either Notch is sufficient to maintain T-ALL cell growth, whereas cell-context specific differences appear to influence the response of T-ALL cells to Notch inhibition.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185762PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5638296PMC
October 2017

Diffuse Staining for Activated NOTCH1 Correlates With NOTCH1 Mutation Status and Is Associated With Worse Outcome in Adenoid Cystic Carcinoma.

Am J Surg Pathol 2017 Nov;41(11):1473-1482

*Department of Pathology, Massachusetts General Hospital ¶Department of Surgery, Massachusetts General Hospital ‡Department of Pathology, Brigham and Women's Hospital §Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School ∥Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA #Department of Laboratory Medicine & Pathology, Mayo Clinic, Rochester, MN †Northern Institute for Cancer Research, University of Newcastle, Newcastle on Tyne, UK.

NOTCH1 is frequently mutated in adenoid cystic carcinoma (ACC). To test the idea that immunohistochemical (IHC) staining can identify ACCs with NOTCH1 mutations, we performed IHC for activated NOTCH1 (NICD1) in 197 cases diagnosed as ACC from 173 patients. NICD1 staining was positive in 194 cases (98%) in 2 major patterns: subset positivity, which correlated with tubular/cribriform histology; and diffuse positivity, which correlated with a solid histology. To determine the relationship between NICD1 staining and NOTCH1 mutational status, targeted exome sequencing data were obtained on 14 diffusely NICD1-positive ACC specimens from 11 patients and 15 subset NICD1-positive ACC specimens from 15 patients. This revealed NOTCH1 gain-of-function mutations in 11 of 14 diffusely NICD1-positive ACC specimens, whereas all subset-positive tumors had wild-type NOTCH1 alleles. Notably, tumors with diffuse NICD1 positivity were associated with significantly worse outcomes (P=0.003). To determine whether NOTCH1 activation is unique among tumors included in the differential diagnosis with ACC, we performed NICD1 IHC on a cohort of diverse salivary gland and head and neck tumors. High fractions of each of these tumor types were positive for NICD1 in a subset of cells, particularly in basaloid squamous cell carcinomas; however, sequencing of basaloid squamous cell carcinomas failed to identify NOTCH1 mutations. These findings indicate that diffuse NICD1 positivity in ACC correlates with solid growth pattern, the presence of NOTCH1 gain-of-function mutations, and unfavorable outcome, and suggest that staining for NICD1 can be helpful in distinguishing ACC with solid growth patterns from other salivary gland and head and neck tumors.
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http://dx.doi.org/10.1097/PAS.0000000000000945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5657508PMC
November 2017

Structural Basis for Regulation of ESCRT-III Complexes by Lgd.

Cell Rep 2017 05;19(9):1750-1757

Institute of Genetics, Heinrich-Heine-University, Dusseldorf 40225, Germany. Electronic address:

The ESCRT-III complex induces outward membrane budding and fission through homotypic polymerization of its core component Shrub/CHMP4B. Shrub activity is regulated by its direct interaction with a protein called Lgd in flies, or CC2D1A or B in humans. Here, we report the structural basis for this interaction and propose a mechanism for regulation of polymer assembly. The isolated third DM14 repeat of Lgd binds Shrub, and an Lgd fragment containing only this DM14 repeat and its C-terminal C2 domain is sufficient for in vivo function. The DM14 domain forms a helical hairpin with a conserved, positively charged tip, that, in the structure of a DM14 domain-Shrub complex, occupies a negatively charged surface of Shrub that is otherwise used for homopolymerization. Lgd mutations at this interface disrupt its function in flies, confirming functional importance. Together, these data argue that Lgd regulates ESCRT activity by controlling access to the Shrub self-assembly surface.
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http://dx.doi.org/10.1016/j.celrep.2017.05.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5528166PMC
May 2017