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Extensive rewiring of the EGFR network in colorectal cancer cells expressing transforming levels of KRASG13D.

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Extensive rewiring of the EGFR network in colorectal cancer cells expressing transforming levels of KRASG13D.

Nat Commun. 2020 01 24;11(1):499

Authors: Kennedy SA, Jarboui MA, Srihari S, Raso C, Bryan K, Dernayka L, Charitou T, Bernal-Llinares M, Herrera-Montavez C, Krstic A, Matallanas D, Kotlyar M, Jurisica I, Curak J, Wong V, Stagljar I, LeBihan T, Imrie L, Pillai P, Lynn MA, Fasterius E, Al-Khalili Szigyarto C, Breen J, Kiel C, Serrano L, Rauch N, Rukhlenko O, Kholodenko BN, Iglesias-Martinez LF, Ryan CJ, Pilkington R, Cammareri P, Sansom O, Shave S, Auer M, Horn N, Klose F, Ueffing M, Boldt K, Lynn DJ, Kolch W

Abstract
Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes.

PMID: 31980649 [PubMed - indexed for MEDLINE]



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In situ antibody phage display yields optimal inhibitors of integrin α11/β1.

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In situ antibody phage display yields optimal inhibitors of integrin α11/β1.

MAbs. 2020 Jan-Dec;12(1):1717265

Authors: Gallo E, Kelil A, Bayliss PE, Jeganathan A, Egorova O, Ploder L, Adams JA, Giblin P, Sidhu SS

Abstract
Integrins are transmembrane multi-conformation receptors that mediate interactions with the extracellular matrix. In cancer, integrins influence metastasis, proliferation, and survival. Collagen-binding integrin-α11/β1, a marker of aggressive tumors that is involved in stroma-tumor crosstalk, may be an attractive target for anti-cancer therapeutic antibodies. We performed selections with phage-displayed synthetic antibody libraries for binding to either purified integrin-α11/β1 or in situ on live cells. The in-situ strategy yielded many diverse antibodies, and strikingly, most of these antibodies did not recognize purified integrin-α11/β1. Conversely, none of the antibodies selected for binding to purified integrin-α11/β1 were able to efficiently recognize native cell-surface antigen. Most importantly, only the in-situ selection yielded functional antibodies that were able to compete with collagen-I for binding to cell-surface integrin-α11/β1, and thus inhibited cell adhesion. In-depth characterization of a subset of in situ-derived clones as full-length immunoglobulins revealed high affinity cellular binding and inhibitory activities in the single-digit nanomolar range. Moreover, the antibodies showed high selectivity for integrin-α11/β1 with minimal cross-reactivity for close homologs. Taken together, our findings highlight the advantages of in-situ selections for generation of anti-integrin antibodies optimized for recognition and inhibition of native cell-surface proteins, and our work establishes general methods that could be extended to many other membrane proteins.

PMID: 31980006 [PubMed - in process]



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A crucial RNA-binding lysine residue in the Nab3 RRM domain undergoes SET1 and SET3-responsive methylation.

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A crucial RNA-binding lysine residue in the Nab3 RRM domain undergoes SET1 and SET3-responsive methylation.

Nucleic Acids Res. 2020 04 06;48(6):2897-2911

Authors: Lee KY, Chopra A, Burke GL, Chen Z, Greenblatt JF, Biggar KK, Meneghini MD

Abstract
The Nrd1-Nab3-Sen1 (NNS) complex integrates molecular cues to direct termination of noncoding transcription in budding yeast. NNS is positively regulated by histone methylation as well as through Nrd1 binding to the initiating form of RNA PolII. These cues collaborate with Nrd1 and Nab3 binding to target RNA sequences in nascent transcripts through their RRM RNA recognition motifs. In this study, we identify nine lysine residues distributed amongst Nrd1, Nab3 and Sen1 that are methylated, suggesting novel molecular inputs for NNS regulation. We identify mono-methylation of one these residues (Nab3-K363me1) as being partly dependent on the H3K4 methyltransferase, Set1, a known regulator of NNS function. Moreover, the accumulation of Nab3-K363me1 is essentially abolished in strains lacking SET3, a SET domain containing protein that is positively regulated by H3K4 methylation. Nab3-K363 resides within its RRM and physically contacts target RNA. Mutation of Nab3-K363 to arginine (Nab3-K363R) decreases RNA binding of the Nab3 RRM in vitro and causes transcription termination defects and slow growth. These findings identify SET3 as a potential contextual regulator of Nab3 function through its role in methylation of Nab3-K363. Consistent with this hypothesis, we report that SET3 exhibits genetic activation of NAB3 that is observed in a sensitized context.

PMID: 31960028 [PubMed - indexed for MEDLINE]



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A synthetic human antibody antagonizes IL-18Rβ signaling through an allosteric mechanism.

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A synthetic human antibody antagonizes IL-18Rβ signaling through an allosteric mechanism.

J Mol Biol. 2020 Jan 15;:

Authors: Liu S, Miersch S, Li P, Bai B, Liu C, Qin W, Su J, Huang H, Pan J, Sidhu SS, Wu D

Abstract
The interleukin-18 subfamily belongs to the interleukin-1 family and plays important roles in modulating innate and adaptive immune responses. Dysregulation of IL-18 has been implicated in or correlated with numerous diseases including inflammatory diseases, autoimmune disorders and cancer. Thus, blockade of IL-18 signaling may offer therapeutic benefits in many pathological settings. Here, we report the development of synthetic human antibodies that target human IL-18Rβ and block IL-18-mediated IFN-γ secretion by inhibiting NF-κB and MAPK dependent pathways. The crystal structure of a potent antagonist antibody in complex with IL-18Rβ revealed inhibition through an unexpected allosteric mechanism. Our findings offer a novel means for therapeutic intervention in the IL-18 pathway and may provide a new strategy for targeting cytokine receptors.

PMID: 31954129 [PubMed - as supplied by publisher]



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A network analysis to identify mediators of germline-driven differences in breast cancer prognosis.

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A network analysis to identify mediators of germline-driven differences in breast cancer prognosis.

Nat Commun. 2020 01 16;11(1):312

Authors: Escala-Garcia M, Abraham J, Andrulis IL, Anton-Culver H, Arndt V, Ashworth A, Auer PL, Auvinen P, Beckmann MW, Beesley J, Behrens S, Benitez J, Bermisheva M, Blomqvist C, Blot W, Bogdanova NV, Bojesen SE, Bolla MK, Børresen-Dale AL, Brauch H, Brenner H, Brucker SY, Burwinkel B, Caldas C, Canzian F, Chang-Claude J, Chanock SJ, Chin SF, Clarke CL, Couch FJ, Cox A, Cross SS, Czene K, Daly MB, Dennis J, Devilee P, Dunn JA, Dunning AM, Dwek M, Earl HM, Eccles DM, Eliassen AH, Ellberg C, Evans DG, Fasching PA, Figueroa J, Flyger H, Gago-Dominguez M, Gapstur SM, García-Closas M, García-Sáenz JA, Gaudet MM, George A, Giles GG, Goldgar DE, González-Neira A, Grip M, Guénel P, Guo Q, Haiman CA, Håkansson N, Hamann U, Harrington PA, Hiller L, Hooning MJ, Hopper JL, Howell A, Huang CS, Huang G, Hunter DJ, Jakubowska A, John EM, Kaaks R, Kapoor PM, Keeman R, Kitahara CM, Koppert LB, Kraft P, Kristensen VN, Lambrechts D, Le Marchand L, Lejbkowicz F, Lindblom A, Lubiński J, Mannermaa A, Manoochehri M, Manoukian S, Margolin S, Martinez ME, Maurer T, Mavroudis D, Meindl A, Milne RL, Mulligan AM, Neuhausen SL, Nevanlinna H, Newman WG, Olshan AF, Olson JE, Olsson H, Orr N, Peterlongo P, Petridis C, Prentice RL, Presneau N, Punie K, Ramachandran D, Rennert G, Romero A, Sachchithananthan M, Saloustros E, Sawyer EJ, Schmutzler RK, Schwentner L, Scott C, Simard J, Sohn C, Southey MC, Swerdlow AJ, Tamimi RM, Tapper WJ, Teixeira MR, Terry MB, Thorne H, Tollenaar RAEM, Tomlinson I, Troester MA, Truong T, Turnbull C, Vachon CM, van der Kolk LE, Wang Q, Winqvist R, Wolk A, Yang XR, Ziogas A, Pharoah PDP, Hall P, Wessels LFA, Chenevix-Trench G, Bader GD, Dörk T, Easton DF, Canisius S, Schmidt MK

Abstract
Identifying the underlying genetic drivers of the heritability of breast cancer prognosis remains elusive. We adapt a network-based approach to handle underpowered complex datasets to provide new insights into the potential function of germline variants in breast cancer prognosis. This network-based analysis studies ~7.3 million variants in 84,457 breast cancer patients in relation to breast cancer survival and confirms the results on 12,381 independent patients. Aggregating the prognostic effects of genetic variants across multiple genes, we identify four gene modules associated with survival in estrogen receptor (ER)-negative and one in ER-positive disease. The modules show biological enrichment for cancer-related processes such as G-alpha signaling, circadian clock, angiogenesis, and Rho-GTPases in apoptosis.

PMID: 31949161 [PubMed - indexed for MEDLINE]



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The entry of nanoparticles into solid tumours.

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The entry of nanoparticles into solid tumours.

Nat Mater. 2020 Jan 13;:

Authors: Sindhwani S, Syed AM, Ngai J, Kingston BR, Maiorino L, Rothschild J, MacMillan P, Zhang Y, Rajesh NU, Hoang T, Wu JLY, Wilhelm S, Zilman A, Gadde S, Sulaiman A, Ouyang B, Lin Z, Wang L, Egeblad M, Chan WCW

Abstract
The concept of nanoparticle transport through gaps between endothelial cells (inter-endothelial gaps) in the tumour blood vessel is a central paradigm in cancer nanomedicine. The size of these gaps was found to be up to 2,000 nm. This justified the development of nanoparticles to treat solid tumours as their size is small enough to extravasate and access the tumour microenvironment. Here we show that these inter-endothelial gaps are not responsible for the transport of nanoparticles into solid tumours. Instead, we found that up to 97% of nanoparticles enter tumours using an active process through endothelial cells. This result is derived from analysis of four different mouse models, three different types of human tumours, mathematical simulation and modelling, and two different types of imaging techniques. These results challenge our current rationale for developing cancer nanomedicine and suggest that understanding these active pathways will unlock strategies to enhance tumour accumulation.

PMID: 31932672 [PubMed - as supplied by publisher]



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On-demand serum-free media formulations for human hematopoietic cell expansion using a high dimensional search algorithm.

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On-demand serum-free media formulations for human hematopoietic cell expansion using a high dimensional search algorithm.

Commun Biol. 2019 Feb 01;2(1):48

Authors: Kim MM, Audet J

Abstract
Substitution of serum and other clinically incompatible reagents is requisite for controlling product quality in a therapeutic cell manufacturing process. However, substitution with chemically defined compounds creates a complex, large-scale optimization problem due to the large number of possible factors and dose levels, making conventional process optimization methods ineffective. We present a framework for high-dimensional optimization of serum-free formulations for the expansion of human hematopoietic cells. Our model-free approach utilizes evolutionary computing principles to drive an experiment-based feedback control platform. We validate this method by optimizing serum-free formulations for first, TF-1 cells and second, primary T-cells. For each cell type, we successfully identify a set of serum-free formulations that support cell expansions similar to the serum-containing conditions commonly used to culture these cells, by experimentally testing less than 1 × 10-5 % of the total search space. We also demonstrate how this iterative search process can provide insights into factor interactions that contribute to supporting cell expansion.

PMID: 31924921 [PubMed - in process]



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A community effort to create standards for evaluating tumor subclonal reconstruction.

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A community effort to create standards for evaluating tumor subclonal reconstruction.

Nat Biotechnol. 2020 01;38(1):97-107

Authors: Salcedo A, Tarabichi M, Espiritu SMG, Deshwar AG, David M, Wilson NM, Dentro S, Wintersinger JA, Liu LY, Ko M, Sivanandan S, Zhang H, Zhu K, Ou Yang TH, Chilton JM, Buchanan A, Lalansingh CM, P'ng C, Anghel CV, Umar I, Lo B, Zou W, DREAM SMC-Het Participants, Simpson JT, Stuart JM, Anastassiou D, Guan Y, Ewing AD, Ellrott K, Wedge DC, Morris Q, Van Loo P, Boutros PC

Abstract
Tumor DNA sequencing data can be interpreted by computational methods that analyze genomic heterogeneity to infer evolutionary dynamics. A growing number of studies have used these approaches to link cancer evolution with clinical progression and response to therapy. Although the inference of tumor phylogenies is rapidly becoming standard practice in cancer genome analyses, standards for evaluating them are lacking. To address this need, we systematically assess methods for reconstructing tumor subclonality. First, we elucidate the main algorithmic problems in subclonal reconstruction and develop quantitative metrics for evaluating them. Then we simulate realistic tumor genomes that harbor all known clonal and subclonal mutation types and processes. Finally, we benchmark 580 tumor reconstructions, varying tumor read depth, tumor type and somatic variant detection. Our analysis provides a baseline for the establishment of gold-standard methods to analyze tumor heterogeneity.

PMID: 31919445 [PubMed - indexed for MEDLINE]



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Modular mimicry and engagement of the Hippo pathway by Marburg virus VP40: Implications for filovirus biology and budding.

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Modular mimicry and engagement of the Hippo pathway by Marburg virus VP40: Implications for filovirus biology and budding.

PLoS Pathog. 2020 01;16(1):e1008231

Authors: Han Z, Dash S, Sagum CA, Ruthel G, Jaladanki CK, Berry CT, Schwoerer MP, Harty NM, Freedman BD, Bedford MT, Fan H, Sidhu SS, Sudol M, Shtanko O, Harty RN

Abstract
Ebola (EBOV) and Marburg (MARV) are members of the Filoviridae family, which continue to emerge and cause sporadic outbreaks of hemorrhagic fever with high mortality rates. Filoviruses utilize their VP40 matrix protein to drive virion assembly and budding, in part, by recruitment of specific WW-domain-bearing host proteins via its conserved PPxY Late (L) domain motif. Here, we screened an array of 115 mammalian, bacterially expressed and purified WW-domains using a PPxY-containing peptide from MARV VP40 (mVP40) to identify novel host interactors. Using this unbiased approach, we identified Yes Associated Protein (YAP) and Transcriptional co-Activator with PDZ-binding motif (TAZ) as novel mVP40 PPxY interactors. YAP and TAZ function as downstream transcriptional effectors of the Hippo signaling pathway that regulates cell proliferation, migration and apoptosis. We demonstrate that ectopic expression of YAP or TAZ along with mVP40 leads to significant inhibition of budding of mVP40 VLPs in a WW-domain/PPxY dependent manner. Moreover, YAP colocalized with mVP40 in the cytoplasm, and inhibition of mVP40 VLP budding was more pronounced when YAP was localized predominantly in the cytoplasm rather than in the nucleus. A key regulator of YAP nuclear/cytoplasmic localization and function is angiomotin (Amot); a multi-PPxY containing protein that strongly interacts with YAP WW-domains. Interestingly, we found that expression of PPxY-containing Amot rescued mVP40 VLP egress from either YAP- or TAZ-mediated inhibition in a PPxY-dependent manner. Importantly, using a stable Amot-knockdown cell line, we found that expression of Amot was critical for efficient egress of mVP40 VLPs as well as egress and spread of authentic MARV in infected cell cultures. In sum, we identified novel negative (YAP/TAZ) and positive (Amot) regulators of MARV VP40-mediated egress, that likely function in part, via competition between host and viral PPxY motifs binding to modular host WW-domains. These findings not only impact our mechanistic understanding of virus budding and spread, but also may impact the development of new antiviral strategies.

PMID: 31905227 [PubMed - indexed for MEDLINE]



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Acquisition of a Unique Mesenchymal Precursor-like Blastema State Underlies Successful Adult Mammalian Digit Tip Regeneration.

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Acquisition of a Unique Mesenchymal Precursor-like Blastema State Underlies Successful Adult Mammalian Digit Tip Regeneration.

Dev Cell. 2020 02 24;52(4):509-524.e9

Authors: Storer MA, Mahmud N, Karamboulas K, Borrett MJ, Yuzwa SA, Gont A, Androschuk A, Sefton MV, Kaplan DR, Miller FD

Abstract
Here, we investigate the origin and nature of blastema cells that regenerate the adult murine digit tip. We show that Pdgfra-expressing mesenchymal cells in uninjured digits establish the regenerative blastema and are essential for regeneration. Single-cell profiling shows that the mesenchymal blastema cells are distinct from both uninjured digit and embryonic limb or digit Pdgfra-positive cells. This unique blastema state is environmentally determined; dermal fibroblasts transplanted into the regenerative, but not non-regenerative, digit express blastema-state genes and contribute to bone regeneration. Moreover, lineage tracing with single-cell profiling indicates that endogenous osteoblasts or osteocytes acquire a blastema mesenchymal transcriptional state and contribute to both dermis and bone regeneration. Thus, mammalian digit tip regeneration occurs via a distinct adult mechanism where the regenerative environment promotes acquisition of a blastema state that enables cells from tissues such as bone to contribute to the regeneration of other mesenchymal tissues such as the dermis.

PMID: 31902657 [PubMed - indexed for MEDLINE]



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