Publications by authors named "Laura E Herring"

39 Publications

SPOP and OTUD7A Control EWS-FLI1 Protein Stability to Govern Ewing Sarcoma Growth.

Adv Sci (Weinh) 2021 Jun 1:e2004846. Epub 2021 Jun 1.

Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.

Chromosomal translocation results in development of an Ewing sarcoma breakpoint region 1-Friend leukemia integration 1 (EWS-FLI1) fusion oncogene in the majority of Ewing sarcoma. The persistent dependence of the tumor for this oncoprotein points to EWS-FLI1 as an ideal drug target. Although EWS-FLI1 transcriptional targets and binding partners are evaluated, the mechanisms regulating EWS-FLI1 protein stability remain elusive. Speckle-type POZ protein (SPOP) and OTU domain-containing protein 7A (OTUD7A) are identified as the bona fide E3 ligase and deubiquitinase, respectively, that control EWS-FLI1 protein turnover in Ewing sarcoma. Casein kinase 1-mediated phosphorylation of the VTSSS degron in the FLI1 domain enhances SPOP activity to degrade EWS-FLI1. Opposing this process, OTUD7A deubiquitinates and stabilizes EWS-FLI1. Depletion of OTUD7A in Ewing sarcoma cell lines reduces EWS-FLI1 protein abundance and impedes Ewing sarcoma growth in vitro and in mice. Performing an artificial-intelligence-based virtual drug screen of a 4-million small molecule library, 7Ai is identified as a potential OTUD7A catalytic inhibitor. 7Ai reduces EWS-FLI1 protein levels and decreases Ewing sarcoma growth in vitro and in a xenograft mouse model. This study supports the therapeutic targeting of OTUD7A as a novel strategy for Ewing sarcoma bearing EWS-FLI1 and related fusions, and may also be applicable to other cancers dependent on aberrant FLI1 expression.
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http://dx.doi.org/10.1002/advs.202004846DOI Listing
June 2021

Tau seeds are subject to aberrant modifications resulting in distinct signatures.

Cell Rep 2021 Apr;35(4):109037

Department of Neurology and the UNC Neuroscience Center, University of North Carolina, Chapel Hill, NC 27599, USA; Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA. Electronic address:

The prion-like spread of tau pathology could underlie a spectrum of clinical syndromes including Alzheimer's disease (AD). Although evidence indicates that tau is transmissible, it is unclear how pathogenic tau seeds are processed in neurons. Here, we analyze fibrillar wild-type and disease-associated P301L tau seeds by using in vitro and neuronal assays. We show that P301L seeds are uniquely modified by post-translational modifications (PTMs) within the microtubule-binding region (MTBR). Although these modifications do not alter tau seed trafficking or localization, acetylated tau variants show accelerated tau aggregation, enhanced tau PTM priming, and prion-like templating. To explain the enhanced tau seed acetylation, we demonstrate that P301L seeds undergo auto-acetylation. Moreover, tau acts generally to inhibit HDAC6 deacetylase activity by preventing HDAC6 phosphorylation, leading to increased substrate acetylation. Our study highlights complex post-translational regulation of transmissible tau seeds and provides insight into the biological properties of tau strains in AD and other tauopathies.
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http://dx.doi.org/10.1016/j.celrep.2021.109037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8135111PMC
April 2021

Evidence for the Mycobacterial Mce4 Transporter Being a Multiprotein Complex.

J Bacteriol 2021 Apr 21;203(10). Epub 2021 Apr 21.

Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Mycobacteria possess Mce transporters that import lipids and are thought to function analogously to ATP-binding cassette (ABC) transporters. However, whereas ABC transporters import substrates using a single solute-binding protein (SBP) to deliver a substrate to permease proteins in the membrane, mycobacterial Mce transporters have a potential for six SBPs (MceA to MceF) working with a pair of permeases (YrbEA and YrbEB), a cytoplasmic ATPase (MceG), and multiple Mce-associated membrane (Mam) and orphaned Mam (Omam) proteins to transport lipids. In this study, we used the model mycobacterium to study the requirement for individual Mce, Mam, and Omam proteins in Mce4 transport of cholesterol. All of the Mce4 and Mam4 proteins we investigated were required for cholesterol uptake. However, not all Omam proteins, which are encoded by genes outside loci, proved to contribute to cholesterol import. OmamA and OmamB were required for cholesterol import, while OmamC, OmamD, OmamE, and OmamF were not. In the absence of any single Mce4, Mam4, or Omam protein that we tested, the abundance of Mce4A and Mce4E declined. This relationship between the levels of Mce4A and Mce4E and these additional proteins suggests a network of interactions that assemble and/or stabilize a multiprotein Mce4 transporter complex. Further support for Mce transporters being multiprotein complexes was obtained by immunoprecipitation-mass spectrometry, in which we identified every single Mce, YrbE, MceG, Mam, and Omam protein with a role in cholesterol transport as associating with Mce4A. This study represents the first time any of these Mce4 transporter proteins has been shown to associate. How lipids travel between membranes of diderm bacteria is a challenging mechanistic question because lipids, which are hydrophobic molecules, must traverse a hydrophilic periplasm. This question is even more complex for mycobacteria, which have a unique cell envelope that is highly impermeable to molecules. A growing body of knowledge identifies Mce transporters as lipid importers for mycobacteria. Here, using protein stability experiments and immunoprecipitation-mass spectrometry, we provide evidence for mycobacterial Mce transporters existing as multiprotein complexes.
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http://dx.doi.org/10.1128/JB.00685-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8088607PMC
April 2021

Author Correction: Antigen-capturing nanoparticles improve the abscopal effect and cancer immunotherapy.

Nat Nanotechnol 2021 Feb 12. Epub 2021 Feb 12.

Laboratory of Nano- and Translational Medicine, Carolina Center for Cancer Nanotechnology Excellence, Carolina Institute of Nanomedicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, USA.

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http://dx.doi.org/10.1038/s41565-021-00864-wDOI Listing
February 2021

Listeria monocytogenes upregulates mitochondrial calcium signalling to inhibit LC3-associated phagocytosis as a survival strategy.

Nat Microbiol 2021 03 18;6(3):366-379. Epub 2021 Jan 18.

Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, USA.

Mitochondria are believed to have originated ~2.5 billion years ago. As well as energy generation in cells, mitochondria have a role in defence against bacterial pathogens. Despite profound changes in mitochondrial morphology and functions following bacterial challenge, whether intracellular bacteria can hijack mitochondria to promote their survival remains elusive. We report that Listeria monocytogenes-an intracellular bacterial pathogen-suppresses LC3-associated phagocytosis (LAP) by modulation of mitochondrial Ca (mtCa) signalling in order to survive inside cells. Invasion of macrophages by L. monocytogenes induced mtCa uptake through the mtCa uniporter (MCU), which in turn increased acetyl-coenzyme A (acetyl-CoA) production by pyruvate dehydrogenase. Acetylation of the LAP effector Rubicon with acetyl-CoA decreased LAP formation. Genetic ablation of MCU attenuated intracellular bacterial growth due to increased LAP formation. Our data show that modulation of mtCa signalling can increase bacterial survival inside cells, and highlight the importance of mitochondrial metabolism in host-microbial interactions.
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http://dx.doi.org/10.1038/s41564-020-00843-2DOI Listing
March 2021

Identification of BBOX1 as a Therapeutic Target in Triple-Negative Breast Cancer.

Cancer Discov 2020 11 20;10(11):1706-1721. Epub 2020 Jul 20.

Department of Pathology, The University of Texas Southwestern Medical Center, Dallas, Texas.

Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease. Because of its heterogeneity and lack of hormone receptors or HER2 expression, targeted therapy is limited. Here, by performing a functional siRNA screening for 2-OG-dependent enzymes, we identified gamma-butyrobetaine hydroxylase 1 () as an essential gene for TNBC tumorigenesis. BBOX1 depletion inhibits TNBC cell growth while not affecting normal breast cells. Mechanistically, BBOX1 binds with the calcium channel inositol-1,4,5-trisphosphate receptor type 3 (IP3R3) in an enzymatic-dependent manner and prevents its ubiquitination and proteasomal degradation. BBOX1 depletion suppresses IP3R3-mediated endoplasmic reticulum calcium release, therefore impairing calcium-dependent energy-generating processes including mitochondrial respiration and mTORC1-mediated glycolysis, which leads to apoptosis and impaired cell-cycle progression in TNBC cells. Therapeutically, genetic depletion or pharmacologic inhibition of BBOX1 inhibits TNBC tumor growth and . Our study highlights the importance of targeting the previously uncharacterized BBOX1-IP3R3-calcium oncogenic signaling axis in TNBC. SIGNIFICANCE: We provide evidence from unbiased screens that BBOX1 is a potential therapeutic target in TNBC and that genetic knockdown or pharmacologic inhibition of BBOX1 leads to decreased TNBC cell fitness. This study lays the foundation for developing effective BBOX1 inhibitors for treatment of this lethal disease..
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http://dx.doi.org/10.1158/2159-8290.CD-20-0288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642036PMC
November 2020

Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers.

Cell Rep 2020 06;31(11):107764

Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Charité Universitätsmedizin Berlin, Institute of Pathology, Laboratory of Molecular Tumor Pathology and Systems Biology, 10117 Berlin, Germany. Electronic address:

We address whether combinations with a pan-RAF inhibitor (RAFi) would be effective in KRAS mutant pancreatic ductal adenocarcinoma (PDAC). Chemical library and CRISPR genetic screens identify combinations causing apoptotic anti-tumor activity. The most potent combination, concurrent inhibition of RAF (RAFi) and ERK (ERKi), is highly synergistic at low doses in cell line, organoid, and rat models of PDAC, whereas each inhibitor alone is only cytostatic. Comprehensive mechanistic signaling studies using reverse phase protein array (RPPA) pathway mapping and RNA sequencing (RNA-seq) show that RAFi/ERKi induced insensitivity to loss of negative feedback and system failures including loss of ERK signaling, FOSL1, and MYC; shutdown of the MYC transcriptome; and induction of mesenchymal-to-epithelial transition. We conclude that low-dose vertical inhibition of the RAF-MEK-ERK cascade is an effective therapeutic strategy for KRAS mutant PDAC.
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http://dx.doi.org/10.1016/j.celrep.2020.107764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7393480PMC
June 2020

Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B'δ holoenzyme.

J Biol Chem 2020 04 10;295(17):5654-5668. Epub 2020 Mar 10.

Department of Neuroscience and Pharmacology and Iowa Neuroscience Institute, University of Iowa, Iowa City, Iowa 52242. Electronic address:

Protein phosphatase 2A (PP2A) is a large enzyme family responsible for most cellular Ser/Thr dephosphorylation events. PP2A substrate specificity, localization, and regulation by second messengers rely on more than a dozen regulatory subunits (including B/R2, B'/R5, and B″/R3), which form the PP2A heterotrimeric holoenzyme by associating with a dimer comprising scaffolding (A) and catalytic (C) subunits. Because of partial redundancy and high endogenous expression of PP2A holoenzymes, traditional approaches of overexpressing, knocking down, or knocking out PP2A regulatory subunits have yielded only limited insights into their biological roles and substrates. To this end, here we sought to reduce the complexity of cellular PP2A holoenzymes. We used tetracycline-inducible expression of pairs of scaffolding and regulatory subunits with complementary charge-reversal substitutions in their interaction interfaces. For each of the three regulatory subunit families, we engineered A/B charge-swap variants that could bind to one another, but not to endogenous A and B subunits. Because endogenous Aα was targeted by a co-induced shRNA, endogenous B subunits were rapidly degraded, resulting in expression of predominantly a single PP2A heterotrimer composed of the A/B charge-swap pair and the endogenous catalytic subunit. Using B'δ/PPP2R5D, we show that PP2A complexity reduction, but not PP2A overexpression, reveals a role of this holoenzyme in suppression of extracellular signal-regulated kinase signaling and protein kinase A substrate dephosphorylation. When combined with global phosphoproteomics, the PP2A/B'δ reduction approach identified consensus dephosphorylation motifs in its substrates and suggested that residues surrounding the phosphorylation site play roles in PP2A substrate specificity.
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http://dx.doi.org/10.1074/jbc.RA119.011270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7186168PMC
April 2020

THEMIS-SHP1 Recruitment by 4-1BB Tunes LCK-Mediated Priming of Chimeric Antigen Receptor-Redirected T Cells.

Cancer Cell 2020 02 30;37(2):216-225.e6. Epub 2020 Jan 30.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Electronic address:

Chimeric antigen receptor (CAR) T cell costimulation mediated by CD28 and 4-1BB is essential for CAR-T cell-induced tumor regression. However, CD28 and 4-1BB differentially modulate kinetics, metabolism and persistence of CAR-T cells, and the mechanisms governing these differences are not fully understood. We found that LCK recruited into the synapse of CD28-encoding CAR by co-receptors causes antigen-independent CAR-CD3ζ phosphorylation and increased antigen-dependent T cell activation. In contrast, the synapse formed by 4-1BB-encoding CAR recruits the THEMIS-SHP1 phosphatase complex that attenuates CAR-CD3ζ phosphorylation. We further demonstrated that the CAR synapse can be engineered to recruit either LCK to enhance the kinetics of tumor killing of 4-1BB CAR-T cells or SHP1 to tune down cytokine release of CD28 CAR-T cells.
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http://dx.doi.org/10.1016/j.ccell.2019.12.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397569PMC
February 2020

Mass spectrometry-based selectivity profiling identifies a highly selective inhibitor of the kinase MELK that delays mitotic entry in cancer cells.

J Biol Chem 2020 02 2;295(8):2359-2374. Epub 2020 Jan 2.

Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599; UNC Michael Hooker Proteomics Core Facility, University of North Carolina, Chapel Hill, North Carolina 27599. Electronic address:

The maternal embryonic leucine zipper kinase (MELK) has been implicated in the regulation of cancer cell proliferation. RNAi-mediated MELK depletion impairs growth and causes G/M arrest in numerous cancers, but the mechanisms underlying these effects are poorly understood. Furthermore, the MELK inhibitor OTSSP167 has recently been shown to have poor selectivity for MELK, complicating the use of this inhibitor as a tool compound to investigate MELK function. Here, using a cell-based proteomics technique called multiplexed kinase inhibitor beads/mass spectrometry (MIB/MS), we profiled the selectivity of two additional MELK inhibitors, NVS-MELK8a (8a) and HTH-01-091. Our results revealed that 8a is a highly selective MELK inhibitor, which we further used for functional studies. Resazurin and crystal violet assays indicated that 8a decreases triple-negative breast cancer cell viability, and immunoblotting revealed that impaired growth is due to perturbation of cell cycle progression rather than induction of apoptosis. Using double-thymidine synchronization and immunoblotting, we observed that MELK inhibition delays mitotic entry, which was associated with delayed activation of Aurora A, Aurora B, and cyclin-dependent kinase 1 (CDK1). Following this delay, cells entered and completed mitosis. Using live-cell microscopy of cells harboring fluorescent proliferating cell nuclear antigen, we confirmed that 8a significantly and dose-dependently lengthens G phase. Collectively, our results provide a rationale for using 8a as a tool compound for functional studies of MELK and indicate that MELK inhibition delays mitotic entry, likely via transient G/M checkpoint activation.
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http://dx.doi.org/10.1074/jbc.RA119.011083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039562PMC
February 2020

Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader.

Cell Chem Biol 2020 01 9;27(1):47-56.e15. Epub 2019 Dec 9.

Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address:

Protein degradation via the use of bivalent chemical degraders provides an alternative strategy to block protein function and assess the biological roles of putative drug targets. This approach capitalizes on the advantages of small-molecule inhibitors while moving beyond the restrictions of traditional pharmacology. Here, we report a chemical degrader (UNC6852) that targets polycomb repressive complex 2 (PRC2). UNC6852 contains an EED226-derived ligand and a ligand for VHL which bind to the WD40 aromatic cage of EED and CRL2, respectively, to induce proteasomal degradation of PRC2 components, EED, EZH2, and SUZ12. Degradation of PRC2 with UNC6852 blocks the histone methyltransferase activity of EZH2, decreasing H3K27me3 levels in HeLa cells and diffuse large B cell lymphoma (DLBCL) cells containing EZH2 gain-of-function mutations. UNC6852 degrades both wild-type and mutant EZH2, and additionally displays anti-proliferative effects in this cancer model system.
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http://dx.doi.org/10.1016/j.chembiol.2019.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7004250PMC
January 2020

TBK1 Is a Synthetic Lethal Target in Cancer with Loss.

Cancer Discov 2020 03 6;10(3):460-475. Epub 2019 Dec 6.

Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina.

TANK binding kinase 1 (TBK1) is an important kinase involved in the innate immune response. Here we discover that TBK1 is hyperactivated by von Hippel-Lindau () loss or hypoxia in cancer cells. Tumors from patients with kidney cancer with loss display elevated TBK1 phosphorylation. Loss of TBK1 via genetic ablation, pharmacologic inhibition, or a new cereblon-based proteolysis targeting chimera specifically inhibits VHL-deficient kidney cancer cell growth, while leaving VHL wild-type cells intact. TBK1 depletion also significantly blunts kidney tumorigenesis in an orthotopic xenograft model . Mechanistically, TBK1 hydroxylation on Proline 48 triggers VHL as well as the phosphatase PPM1B binding that leads to decreased TBK1 phosphorylation. We identify that TBK1 phosphorylates p62/SQSTM1 on Ser366, which is essential for p62 stability and kidney cancer cell proliferation. Our results establish that TBK1, distinct from its role in innate immune signaling, is a synthetic lethal target in cancer with VHL loss. SIGNIFICANCE: The mechanisms that lead to TBK1 activation in cancer and whether this activation is connected to its role in innate immunity remain unclear. Here, we discover that TBK1, distinct from its role in innate immunity, is activated by VHL loss or hypoxia in cancer...
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http://dx.doi.org/10.1158/2159-8290.CD-19-0837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058506PMC
March 2020

Site-specific phosphorylation and caspase cleavage of GFAP are new markers of Alexander disease severity.

Elife 2019 11 4;8. Epub 2019 Nov 4.

Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, United States.

Alexander disease (AxD) is a fatal neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP), which supports the structural integrity of astrocytes. Over 70 GFAP missense mutations cause AxD, but the mechanism linking different mutations to disease-relevant phenotypes remains unknown. We used AxD patient brain tissue and induced pluripotent stem cell (iPSC)-derived astrocytes to investigate the hypothesis that AxD-causing mutations perturb key post-translational modifications (PTMs) on GFAP. Our findings reveal selective phosphorylation of GFAP-Ser13 in patients who died young, independently of the mutation they carried. AxD iPSC-astrocytes accumulated pSer13-GFAP in cytoplasmic aggregates within deep nuclear invaginations, resembling the hallmark Rosenthal fibers observed in vivo. Ser13 phosphorylation facilitated GFAP aggregation and was associated with increased GFAP proteolysis by caspase-6. Furthermore, caspase-6 was selectively expressed in young AxD patients, and correlated with the presence of cleaved GFAP. We reveal a novel PTM signature linking different GFAP mutations in infantile AxD.
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http://dx.doi.org/10.7554/eLife.47789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927689PMC
November 2019

TBK1 Limits mTORC1 by Promoting Phosphorylation of Raptor Ser877.

Sci Rep 2019 09 17;9(1):13470. Epub 2019 Sep 17.

Division of Pulmonary Diseases and Critical Care Medicine, Department of Medicine, University of North Carolina, Chapel Hill, NC, 27599, USA.

While best known for its role in the innate immune system, the TANK-binding kinase 1 (TBK1) is now known to play a role in modulating cellular growth and autophagy. One of the major ways that TBK1 accomplishes this task is by modulating the mechanistic Target of Rapamycin (mTOR), a master regulator that when activated promotes cell growth and inhibits autophagy. However, whether TBK1 promotes or inhibits mTOR activity is highly cell type and context dependent. To further understand the mechanism whereby TBK1 regulates mTOR, we tested the hypothesis that TBK1 phosphorylates a key component of the mTOR complex 1 (mTORC1), Raptor. Using kinase assays coupled with mass spectrometry, we mapped the position of the TBK1 dependent phosphorylation sites on Raptor in vitro. Among the sites identified in vitro, we found that TBK1 promotes Raptor Ser877 phosphorylation in cells both basally and in response to pathogen-associated molecules known to induce TBK1 activity. The levels of Raptor Ser877 phosphorylation were inversely correlated with the levels of mTOR activity. Expression of a mutant Raptor that could not be phosphorylated at Ser877 led to an increase in mTORC1 activity. We conclude that TBK1 limits mTORC1 activity by promoting Raptor Ser877 phosphorylation.
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http://dx.doi.org/10.1038/s41598-019-49707-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748941PMC
September 2019

Data-Driven Construction of Antitumor Agents with Controlled Polypharmacology.

J Am Chem Soc 2019 10 20;141(39):15700-15709. Epub 2019 Sep 20.

Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy , University of North Carolina at Chapel Hill , Chapel Hill , North Carolina 27599-7363 , United States.

Controlling which particular members of a large protein family are targeted by a drug is key to achieving a desired therapeutic response. In this study, we report a rational data-driven strategy for achieving restricted polypharmacology in the design of antitumor agents selectively targeting the TYRO3, AXL, and MERTK (TAM) family tyrosine kinases. Our computational approach, based on the concept of fragments in structural environments (FRASE), distills relevant chemical information from structural and chemogenomic databases to assemble a three-dimensional inhibitor structure directly in the protein pocket. Target engagement by the inhibitors designed led to disruption of oncogenic phenotypes as demonstrated in enzymatic assays and in a panel of cancer cell lines, including acute lymphoblastic and myeloid leukemia (ALL/AML) and nonsmall cell lung cancer (NSCLC). Structural rationale underlying the approach was corroborated by X-ray crystallography. The lead compound demonstrated potent target inhibition in a pharmacodynamic study in leukemic mice.
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http://dx.doi.org/10.1021/jacs.9b08660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894422PMC
October 2019

Conservation and divergence of protein pathways in the vertebrate heart.

PLoS Biol 2019 09 6;17(9):e3000437. Epub 2019 Sep 6.

University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

Heart disease is the leading cause of death in the western world. Attaining a mechanistic understanding of human heart development and homeostasis and the molecular basis of associated disease states relies on the use of animal models. Here, we present the cardiac proteomes of 4 model vertebrates with dual circulatory systems: the pig (Sus scrofa), the mouse (Mus musculus), and 2 frogs (Xenopus laevis and Xenopus tropicalis). Determination of which proteins and protein pathways are conserved and which have diverged within these species will aid in our ability to choose the appropriate models for determining protein function and to model human disease. We uncover mammalian- and amphibian-specific, as well as species-specific, enriched proteins and protein pathways. Among these, we find and validate an enrichment in cell-cycle-associated proteins within Xenopus laevis. To further investigate functional units within cardiac proteomes, we develop a computational approach to profile the abundance of protein complexes across species. Finally, we demonstrate the utility of these data sets for predicting appropriate model systems for studying given cardiac conditions by testing the role of Kielin/chordin-like protein (Kcp), a protein found as enriched in frog hearts compared to mammals. We establish that germ-line mutations in Kcp in Xenopus lead to valve defects and, ultimately, cardiac failure and death. Thus, integrating these findings with data on proteins responsible for cardiac disease should lead to the development of refined, species-specific models for protein function and disease states.
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http://dx.doi.org/10.1371/journal.pbio.3000437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6750614PMC
September 2019

Application of a MYC degradation screen identifies sensitivity to CDK9 inhibitors in KRAS-mutant pancreatic cancer.

Sci Signal 2019 07 16;12(590). Epub 2019 Jul 16.

Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Stabilization of the MYC oncoprotein by KRAS signaling critically promotes the growth of pancreatic ductal adenocarcinoma (PDAC). Thus, understanding how MYC protein stability is regulated may lead to effective therapies. Here, we used a previously developed, flow cytometry-based assay that screened a library of >800 protein kinase inhibitors and identified compounds that promoted either the stability or degradation of MYC in a KRAS-mutant PDAC cell line. We validated compounds that stabilized or destabilized MYC and then focused on one compound, UNC10112785, that induced the substantial loss of MYC protein in both two-dimensional (2D) and 3D cell cultures. We determined that this compound is a potent CDK9 inhibitor with a previously uncharacterized scaffold, caused MYC loss through both transcriptional and posttranslational mechanisms, and suppresses PDAC anchorage-dependent and anchorage-independent growth. We discovered that CDK9 enhanced MYC protein stability through a previously unknown, KRAS-independent mechanism involving direct phosphorylation of MYC at Ser Our study thus not only identifies a potential therapeutic target for patients with KRAS-mutant PDAC but also presents the application of a screening strategy that can be more broadly adapted to identify regulators of protein stability.
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http://dx.doi.org/10.1126/scisignal.aav7259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728149PMC
July 2019

Therapeutic Effect of Y-27632 on Tumorigenesis and Cisplatin-Induced Peripheral Sensory Loss through RhoA-NF-κB.

Mol Cancer Res 2019 09 12;17(9):1910-1919. Epub 2019 Jun 12.

Department of Anatomy and Cell Biology, The Brody School of Medicine at East Carolina University, Greenville, North Carolina.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major side effect of cancer therapy that frequently requires a reduction or cessation of treatments and negatively impacts the patient's quality of life. There is currently no effective means to prevent or treat CIPN. In this study, we developed and applied CIPN in an immunocompetent, syngeneic murine Lewis Lung Carcinoma (LLCab) model that enabled the elucidation of both tumor and host responses to cisplatin and treatments of Y-27632, a selective inhibitor of Rho kinase/p160. Y-27632 not only preserved cisplatin's efficacy toward tumor suppression but also the combination treatment inhibited tumor cell proliferation and increased cellular apoptosis. By alleviating the cisplatin-induced loss of epidermal nerve fibers (ENFs), Y-27632 protected tumor-bearing mice from cisplatin-induced reduction of touch sensation. Furthermore, quantitative proteomic analysis revealed the striking cisplatin-induced dysregulation in cellular stress (inflammation, mitochondrial deficiency, DNA repair, etc.)-associated proteins. Y-27632 was able to reverse the changes of these proteins that are associated with Rho GTPase and NF-κB signaling network, and also decreased cisplatin-induced NF-κB hyperactivation in both footpad tissues and tumor. Therefore, Y-27632 is an effective adjuvant in tumor suppression and peripheral neuroprotection. These studies highlight the potential of targeting the RhoA-NF-κB axis as a combination therapy to treat CIPN. IMPLICATIONS: This study, for the first time, demonstrated the dual antineoplastic and neuroprotective effects of Rho kinase/p160 inhibition in a syngeneic immunocompetent tumor-bearing mouse model, opening the door for further clinical adjuvant development of RhoA-NF-κB axis to improve chemotherapeutic outcomes.
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http://dx.doi.org/10.1158/1541-7786.MCR-19-0024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774348PMC
September 2019

Pathway-Based High-Throughput Chemical Screen Identifies Compounds That Decouple Heterochromatin Transformations.

SLAS Discov 2019 09 30;24(8):802-816. Epub 2019 May 30.

1 The Eshelman School of Pharmacy, Division of Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Heterochromatin protein 1 (HP1) facilitates the formation of repressive heterochromatin domains by recruiting histone lysine methyltransferase enzymes to chromatin, resulting in increased levels of histone H3K9me3. To identify chemical inhibitors of the HP1-heterochromatin gene repression pathway, we combined a cell-based assay that utilized chemical-mediated recruitment of HP1 to an endogenous active gene with high-throughput flow cytometry. Here we characterized small molecule inhibitors that block HP1-mediated heterochromatin formation. Our lead compounds demonstrated dose-dependent inhibition of HP1-stimulated gene repression and were validated in an orthogonal cell-based system. One lead inhibitor was improved by a change in stereochemistry, resulting in compound 2, which was further used to decouple the inverse relationship between H3K9 and H3K4 methylation states. We identified molecular components that bound compound 2, either directly or indirectly, by chemical affinity purification with a biotin-tagged derivative, followed by quantitative proteomic techniques. In summary, our pathway-based chemical screening approach resulted in the discovery of new inhibitors of HP1-mediated heterochromatin formation while identifying exciting new molecular interactions in the pathway to explore in the future. This modular platform can be expanded to test a wide range of chromatin modification pathways yielding inhibitors that are cell permeable and function in a physiologically relevant setting.
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http://dx.doi.org/10.1177/2472555219849838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893849PMC
September 2019

Mitochondrial Protease ClpP is a Target for the Anticancer Compounds ONC201 and Related Analogues.

ACS Chem Biol 2019 05 1;14(5):1020-1029. Epub 2019 May 1.

Department of Pharmacology and the Lineberger Comprehensive Cancer Center , University of North Carolina at Chapel Hill , Chapel Hill , North Carolina 27599 , United States.

ONC201 is a first-in-class imipridone molecule currently in clinical trials for the treatment of multiple cancers. Despite enormous clinical potential, the mechanism of action is controversial. To investigate the mechanism of ONC201 and identify compounds with improved potency, we tested a series of novel ONC201 analogues (TR compounds) for effects on cell viability and stress responses in breast and other cancer models. The TR compounds were found to be ∼50-100 times more potent at inhibiting cell proliferation and inducing the integrated stress response protein ATF4 than ONC201. Using immobilized TR compounds, we identified the human mitochondrial caseinolytic protease P (ClpP) as a specific binding protein by mass spectrometry. Affinity chromatography/drug competition assays showed that the TR compounds bound ClpP with ∼10-fold higher affinity compared to ONC201. Importantly, we found that the peptidase activity of recombinant ClpP was strongly activated by ONC201 and the TR compounds in a dose- and time-dependent manner with the TR compounds displaying a ∼10-100 fold increase in potency over ONC201. Finally, siRNA knockdown of ClpP in SUM159 cells reduced the response to ONC201 and the TR compounds, including induction of CHOP, loss of the mitochondrial proteins (TFAM, TUFM), and the cytostatic effects of these compounds. Thus, we report that ClpP directly binds ONC201 and the related TR compounds and is an important biological target for this class of molecules. Moreover, these studies provide, for the first time, a biochemical basis for the difference in efficacy between ONC201 and the TR compounds.
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http://dx.doi.org/10.1021/acschembio.9b00222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6528275PMC
May 2019

Organ-specific metastases obtained by culturing colorectal cancer cells on tissue-specific decellularized scaffolds.

Nat Biomed Eng 2018 06 23;2(6):443-452. Epub 2018 Apr 23.

Laboratory of Nano- and Translational Medicine, Department of Radiation Oncology, Lineberger Comprehensive Cancer Center, Carolina Center for Cancer Nanotechnology Excellence, Carolina Institute of Nanomedicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Metastatic disease remains the primary cause of mortality in cancer patients. Yet the number of available in vitro models to study metastasis is limited by challenges in the recapitulation of the metastatic microenvironment in vitro, and by difficulties in maintaining colonized-tissue specificity in the expansion and maintenance of metastatic cells. Here, we show that decellularized scaffolds that retain tissue-specific extracellular-matrix components and bound signalling molecules enable, when seeded with colorectal cancer cells, the spontaneous formation of three-dimensional cell colonies that histologically, molecularly and phenotypically resemble in vivo metastases. Lung and liver metastases obtained by culturing colorectal cancer cells on, respectively, lung and liver decellularized scaffolds retained their tissue-specific tropism when injected in mice. We also found that the engineered metastases contained signet ring cells, which has not previously been observed ex vivo. A culture system with tissue-specific decellularized scaffolds represents a simple and powerful approach for the study of organ-specific cancer metastases.
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http://dx.doi.org/10.1038/s41551-018-0231-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166886PMC
June 2018

O-GlcNAc Transferase Suppresses Inflammation and Necroptosis by Targeting Receptor-Interacting Serine/Threonine-Protein Kinase 3.

Immunity 2019 03 12;50(3):576-590.e6. Epub 2019 Feb 12.

Department of Microbial Infection and Immunity, Infectious Disease Institute, The Ohio State University Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA. Electronic address:

Elevated glucose metabolism in immune cells represents a hallmark feature of many inflammatory diseases, such as sepsis. However, the role of individual glucose metabolic pathways during immune cell activation and inflammation remains incompletely understood. Here, we demonstrate a previously unrecognized anti-inflammatory function of the O-linked β-N-acetylglucosamine (O-GlcNAc) signaling associated with the hexosamine biosynthesis pathway (HBP). Despite elevated activities of glycolysis and the pentose phosphate pathway, activation of macrophages with lipopolysaccharide (LPS) resulted in attenuated HBP activity and protein O-GlcNAcylation. Deletion of O-GlcNAc transferase (OGT), a key enzyme for protein O-GlcNAcylation, led to enhanced innate immune activation and exacerbated septic inflammation. Mechanistically, OGT-mediated O-GlcNAcylation of the serine-threonine kinase RIPK3 on threonine 467 (T467) prevented RIPK3-RIPK1 hetero- and RIPK3-RIPK3 homo-interaction and inhibited downstream innate immunity and necroptosis signaling. Thus, our study identifies an immuno-metabolic crosstalk essential for fine-tuning innate immune cell activation and highlights the importance of glucose metabolism in septic inflammation.
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http://dx.doi.org/10.1016/j.immuni.2019.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426684PMC
March 2019

Lack of GAS2L2 Causes PCD by Impairing Cilia Orientation and Mucociliary Clearance.

Am J Hum Genet 2019 02 18;104(2):229-245. Epub 2019 Jan 18.

Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, NC 27599, USA. Electronic address:

Primary ciliary dyskinesia (PCD) is a genetic disorder in which impaired ciliary function leads to chronic airway disease. Exome sequencing of a PCD subject identified an apparent homozygous frameshift variant, c.887_890delTAAG (p.Val296Glyfs13), in exon 5; this frameshift introduces a stop codon in amino acid 308 of the growth arrest-specific protein 2-like 2 (GAS2L2). Further genetic screening of unrelated PCD subjects identified a second proband with a compound heterozygous variant carrying the identical frameshift variant and a large deletion (c.867_343+1207del; p.?) starting in exon 5. Both individuals had clinical features of PCD but normal ciliary axoneme structure. In this research, using human nasal cells, mouse models, and X.laevis embryos, we show that GAS2L2 is abundant at the apical surface of ciliated cells, where it localizes with basal bodies, basal feet, rootlets, and actin filaments. Cultured GAS2L2-deficient nasal epithelial cells from one of the affected individuals showed defects in ciliary orientation and had an asynchronous and hyperkinetic (GAS2L2-deficient = 19.8 Hz versus control = 15.8 Hz) ciliary-beat pattern. These results were recapitulated in Gas2l2 mouse tracheal epithelial cell (mTEC) cultures and in X. laevis embryos treated with Gas2l2 morpholinos. In mice, the absence of Gas2l2 caused neonatal death, and the conditional deletion of Gas2l2 impaired mucociliary clearance (MCC) and led to mucus accumulation. These results show that a pathogenic variant in GAS2L2 causes a genetic defect in ciliary orientation and impairs MCC and results in PCD.
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http://dx.doi.org/10.1016/j.ajhg.2018.12.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372263PMC
February 2019

O-GlcNAc Transferase Links Glucose Metabolism to MAVS-Mediated Antiviral Innate Immunity.

Cell Host Microbe 2018 12;24(6):791-803.e6

Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA; Holland Regenerative Medicine Program, University of Nebraska Medical Center, Omaha, NE 68198, USA. Electronic address:

Increased glucose metabolism in immune cells not only serves as a hallmark feature of acute inflammation but also profoundly affects disease outcome following bacterial infection and tissue damage. However, the role of individual glucose metabolic pathways during viral infection remains largely unknown. Here we demonstrate an essential function of the hexosamine biosynthesis pathway (HBP)-associated O-linked β-N-acetylglucosamine (O-GlcNAc) signaling in promoting antiviral innate immunity. Challenge of macrophages with vesicular stomatitis viruses (VSVs) enhances HBP activity and downstream protein O-GlcNAcylation. Human and murine cells deficient of O-GlcNAc transferase, a key enzyme for protein O-GlcNAcylation, show defective antiviral immune responses upon VSV challenge. Mechanistically, O-GlcNAc transferase-mediated O-GlcNAcylation of the signaling adaptor MAVS on serine 366 is required for K63-linked ubiquitination of MAVS and subsequent downstream retinoic-acid inducible gene-like receptor -antiviral signaling activation. Thus, our study identifies a molecular mechanism by which HBP-mediated O-GlcNAcylation regulates MAVS function and highlights the importance of glucose metabolism in antiviral innate immunity.
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http://dx.doi.org/10.1016/j.chom.2018.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6296827PMC
December 2018

Dasatinib Is Preferentially Active in the Activated B-Cell Subtype of Diffuse Large B-Cell Lymphoma.

J Proteome Res 2019 01 21;18(1):522-534. Epub 2018 Dec 21.

Department of Biomedical Sciences , College of Veterinary Medicine, Cornell University , T8 004b Veterinary Research Tower, Box 17 , Ithaca , New York 14853 , United States.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, and at least one-third of its patients relapse after treatment with the current chemotherapy regimen, R-CHOP. By gene-expression profiling, patients with DLBCL can be categorized into two clinically relevant subtypes: activated B-cell (ABC) and germinal center B-cell (GCB) DLBCL. Patients with the ABC subtype have a worse prognosis than those with GCB, and the subtype is defined by chronic, over-active signaling through the B-cell receptor and NF-κB pathways. We examined the effects of the Src family kinase (SFK) inhibitor dasatinib in a panel of ABC and GCB DLBCL cell lines and found that the former are much more sensitive to dasatinib than the latter. However, using multiplexed inhibitor bead coupled to mass spectrometry (MIB/MS) kinome profiling and Western blot analysis, we found that both subtypes display inhibition of the SFKs in response to dasatinib after both short- and long-term treatment. The MIB/MS analyses revealed that several cell-cycle kinases, including CDK4, CDK6, and the Aurora kinases, are down-regulated by dasatinib treatment in the ABC, but not in the GCB, subtype. The present findings have potential implications for the clinical use of dasatinib for the treatment of ABC DLBCL, either alone or in combination with other agents.
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http://dx.doi.org/10.1021/acs.jproteome.8b00841DOI Listing
January 2019

KRAS Suppression-Induced Degradation of MYC Is Antagonized by a MEK5-ERK5 Compensatory Mechanism.

Cancer Cell 2018 11;34(5):807-822.e7

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address:

Our recent ERK1/2 inhibitor analyses in pancreatic ductal adenocarcinoma (PDAC) indicated ERK1/2-independent mechanisms maintaining MYC protein stability. To identify these mechanisms, we determined the signaling networks by which mutant KRAS regulates MYC. Acute KRAS suppression caused rapid proteasome-dependent loss of MYC protein, through both ERK1/2-dependent and -independent mechanisms. Surprisingly, MYC degradation was independent of PI3K-AKT-GSK3β signaling and the E3 ligase FBWX7. We then established and applied a high-throughput screen for MYC protein degradation and performed a kinome-wide proteomics screen. We identified an ERK1/2-inhibition-induced feedforward mechanism dependent on EGFR and SRC, leading to ERK5 activation and phosphorylation of MYC at S62, preventing degradation. Concurrent inhibition of ERK1/2 and ERK5 disrupted this mechanism, synergistically causing loss of MYC and suppressing PDAC growth.
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http://dx.doi.org/10.1016/j.ccell.2018.10.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321749PMC
November 2018

Approximating Isotope Distributions of Biomolecule Fragments.

ACS Omega 2018 Sep 19;3(9):11383-11391. Epub 2018 Sep 19.

Department of Computer Science, Lineberger Comprehensive Cancer Center, Bioinformatics and Computational Biology, UNC Michael Hooker Proteomics Core Facility, Department of Pharmacology, and Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.

In mass spectrometry (MS)-based proteomics, protein and peptide sequences are determined by the isolation and subsequent fragmentation of precursor ions. When an isolation window captures only part of a precursor's isotopic distribution, the isotope distributions of the fragments depend on the subset of isolated precursor isotopes. Approximation of the expected isotope distributions of these fragments prior to sequence determination enables MS2 deisotoping, monoisotopic mass calculation, charge assignment of fragment peaks, and deconvolution of chimeric spectra. However, currently such methods do not exist, and precursor isotope distributions are often used as a proxy. Here, we present methods that approximate the isotope distribution of a biomolecule's fragment given its monoisotopic mass, the monoisotopic mass of its precursor, the set of isolated precursor isotopes, and optionally sulfur atom content. Our methods use either the Averagine model or splines, the latter of which have similar accuracy to the Averagine approach, but are 20 times faster to compute. Theoretical and approximated isotope distributions are consistent for fragments of in silico digested peptides. Furthermore, mass spectrometry experiments with the angiotensin I peptide and HeLa cell lysate demonstrate that the fragment methods match isotope peaks in MS2 spectra more accurately than the precursor Averagine approach. The algorithms for the approximation of fragment isotope distributions have been added to the OpenMS software library. By providing the means for analyzing fragment isotopic distributions, these methods will improve MS2 spectra interpretation.
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http://dx.doi.org/10.1021/acsomega.8b01649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166224PMC
September 2018

Application of Integrated Drug Screening/Kinome Analysis to Identify Inhibitors of Gemcitabine-Resistant Pancreatic Cancer Cell Growth.

SLAS Discov 2018 09 9;23(8):850-861. Epub 2018 May 9.

1 Department of Pharmacology, University of North Carolina at Chapel Hill, NC, USA.

Continuous exposure of a pancreatic cancer cell line MIA PaCa-2 (Mia) to gemcitabine resulted in the formation of a gemcitabine-resistant subline (Mia). In an effort to discover kinase inhibitors that inhibited Mia growth, Mia cells were exposed to kinase inhibitors (PKIS-1 library) in a 384-well screening format. Three compounds (UNC10112721A, UNC10112652A, and UNC10112793A) were identified that inhibited the growth of Mia cells by more than 50% (at 50 nM). Two compounds (UNC10112721A and UNC10112652A) were classified as cyclin-dependent kinase (CDK) inhibitors, whereas UNC10112793A was reported to be a PLK inhibitor. Dose-response experiments supported the efficacy of these compounds to inhibit growth and increase apoptosis in 2D cultures of these cells. However, only UNC10112721A significantly inhibited the growth of 3D spheroids composed of Mia cells and GFP-tagged cancer-associated fibroblasts. Multiplexed inhibitor bead (MIB)-mass spectrometry (MS) kinome competition experiments identified CDK9, CLK1-4, DYRK1A, and CSNK1 as major kinase targets for UNC10112721A in Mia cells. Another CDK9 inhibitor (CDK-IN-2) replicated the growth inhibitory effects of UNC10112721A, whereas inhibitors against the CLK, DYRK, or CSNK1 kinases had no effect. In summary, these studies describe a coordinated approach to discover novel kinase inhibitors, evaluate their efficacy in 3D models, and define their specificity against the kinome.
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http://dx.doi.org/10.1177/2472555218773045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102050PMC
September 2018

Chronic E-Cigarette Exposure Alters the Human Bronchial Epithelial Proteome.

Am J Respir Crit Care Med 2018 07;198(1):67-76

1 Marsico Lung Institute.

Rationale: E-cigarettes vaporize propylene glycol/vegetable glycerin (PG/VG), nicotine, and flavorings. However, the long-term health effects of exposing lungs to vaped e-liquids are unknown.

Objectives: To determine the effects of chronic vaping on pulmonary epithelia.

Methods: We performed research bronchoscopies on healthy nonsmokers, cigarette smokers, and e-cigarette users (vapers) and obtained bronchial brush biopsies and lavage samples from these subjects for proteomic investigation. We further employed in vitro and murine exposure models to support our human findings.

Measurements And Main Results: Visual inspection by bronchoscopy revealed that vaper airways appeared friable and erythematous. Epithelial cells from biopsy samples revealed approximately 300 proteins that were differentially expressed in smoker and vaper airways, with only 78 proteins being commonly altered in both groups and 113 uniquely altered in vapers. For example, CYP1B1 (cytochrome P450 family 1 subfamily B member 1), MUC5AC (mucin 5 AC), and MUC4 levels were increased in vapers. Aerosolized PG/VG alone significantly increased MUC5AC protein in human airway epithelial cultures and in murine nasal epithelia in vivo. We also found that e-liquids rapidly entered cells and that PG/VG reduced membrane fluidity and impaired protein diffusion.

Conclusions: We conclude that chronic vaping exerts marked biological effects on the lung and that these effects may in part be mediated by the PG/VG base. These changes are likely not harmless and may have clinical implications for the development of chronic lung disease. Further studies will be required to determine the full extent of vaping on the lung.
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http://dx.doi.org/10.1164/rccm.201710-2033OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034122PMC
July 2018