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Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds.

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Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds.

Front Immunol. 2017;8:38

Authors: Liu H, Saxena A, Sidhu SS, Wu D

Abstract
Therapeutic monoclonal antibodies have become molecules of choice to treat autoimmune disorders, inflammatory diseases, and cancer. Moreover, bispecific/multispecific antibodies that target more than one antigen or epitope on a target cell or recruit effector cells (T cell, natural killer cell, or macrophage cell) toward target cells have shown great potential to maximize the benefits of antibody therapy. In the past decade, many novel concepts to generate bispecific and multispecific antibodies have evolved successfully into a range of formats from full bispecific immunoglobulin gammas to antibody fragments. Impressively, antibody fragments such as bispecific T-cell engager, bispecific killer cell engager, trispecific killer cell engager, tandem diabody, and dual-affinity-retargeting are showing exciting results in terms of recruiting and activating self-immune effector cells to target and lyse tumor cells. Promisingly, crystallizable fragment (Fc) antigen-binding fragment and monomeric antibody or half antibody may be particularly advantageous to target solid tumors owing to their small size and thus good tissue penetration potential while, on the other hand, keeping Fc-related effector functions such as antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cell-mediated phagocytosis, and extended serum half-life via interaction with neonatal Fc receptor. This review, therefore, focuses on the progress of Fc engineering in generating bispecific molecules and on the use of small antibody fragment as scaffolds for therapeutic development.

PMID: 28184223 [PubMed - in process]



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MARK4 inhibits Hippo signaling to promote proliferation and migration of breast cancer cells.

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MARK4 inhibits Hippo signaling to promote proliferation and migration of breast cancer cells.

EMBO Rep. 2017 Mar;18(3):420-436

Authors: Heidary Arash E, Shiban A, Song S, Attisano L

Abstract
The Hippo pathway is a critical regulator of tissue size, and aberrations in pathway regulation lead to cancer. MST1/2 and LATS1/2 kinases comprise the core of the pathway that, in association with adaptor proteins SAV and MOB, functions in a sequential manner to phosphorylate and inhibit the transcription factors YAP and TAZ. Here we identify mammalian MARK family members as activators of YAP/TAZ. We show that depletion of MARK4 in MDA-MB-231 breast cancer cells results in the loss of nuclear YAP/TAZ and decreases the expression of YAP/TAZ targets. We demonstrate that MARK4 can bind to MST and SAV, leading to their phosphorylation, and that MARK4 expression attenuates the formation of a complex between MST/SAV and LATS, which depends on the kinase activity of MARK4. Abrogation of MARK4 expression using siRNAs and CRISPR/Cas9 gene editing attenuates the proliferation and migration of MDA-MB-231 cells. Our results show that MARK4 acts as a negative regulator of the Hippo kinase cassette to promote YAP/TAZ activity and that loss of MARK4 restrains the tumorigenic properties of breast cancer cells.

PMID: 28183853 [PubMed - indexed for MEDLINE]



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Integrated analysis of proteome, phosphotyrosine-proteome, tyrosine-kinome and tyrosine-phosphatome in acute myeloid leukemia.

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Integrated analysis of proteome, phosphotyrosine-proteome, tyrosine-kinome and tyrosine-phosphatome in acute myeloid leukemia.

Proteomics. 2017 Feb 07;:

Authors: Tong J, Helmy M, Cavalli FM, Jin L, St-Germain J, Karisch R, Taylor P, Minden MD, Taylor MD, Neel BG, Bader GD, Moran MF

Abstract
Reversible protein-tyrosine phosphorylation is catalyzed by the antagonistic actions of protein-tyrosine kinases (PTKs) and phosphatases (PTPs), and represents a major form of cell regulation. Acute myeloid leukemia (AML) is an aggressive hematological malignancy that results from the acquisition of multiple genetic alterations, which in some instances are associated with deregulated protein-phosphotyrosine (pY)-mediated signaling networks. However, although individual PTKs and PTPs have been linked to AML and other malignancies, analysis of protein-pY networks as a function of activated PTKs and PTPs has not been done. In this study, mass spectrometry was used to characterize AML proteomes, and phospho-proteome-subsets including pY proteins, PTKs, and PTPs. AML proteomes resolved into two groups related to high or low degrees of maturation according to French-American-British (FAB) classification, and reflecting differential expression of cell surface antigens. AML pY proteomes reflect canonical, spatially organized signaling networks, unrelated to maturation, with heterogeneous expression of activated receptor and non-receptor PTKs. We present the first integrated analysis of the pY-proteome, activated PTKs, and PTPs. Every PTP and most PTKs have both positive and negative associations with the pY-proteome. pY proteins resolve into groups with shared PTK and PTP correlations. These findings highlight the importance of pY turnover and the PTP phosphatome in shaping the pY-proteome in AML. This article is protected by copyright. All rights reserved.

PMID: 28176486 [PubMed - as supplied by publisher]



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Discovery and structure activity relationship of the first potent cryptosporidium FIKK kinase inhibitor.

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Discovery and structure activity relationship of the first potent cryptosporidium FIKK kinase inhibitor.

Bioorg Med Chem. 2017 Mar 01;25(5):1672-1680

Authors: Osman KT, Ye J, Shi Z, Toker C, Lovato D, Jumani RS, Zuercher W, Huston CD, Edwards AM, Lautens M, Santhakumar V, Hui R

Abstract
FIKKs are parasite-specific protein kinases with distinctive sequence motifs and their biological roles have not been completely elucidated. Here, we report the first potent Cryptosporidium FIKK (CpFIKK) inhibitor. We identified 4b as a potent (IC50=0.2nM) inhibitor of CpFIKK catalytic activity. In addition, we identified both CpCDPK1 selective as well as dually acting CpFIKK-CDPK1 inhibitors from the same structural class of compounds. We evaluated these CpFIKK inhibitors for inhibition of parasite growth in vitro. The observed effects on parasite growth did not correlate with CpFIKK inhibition, suggesting that CpFIKK may not be involved in parasite growth.

PMID: 28162900 [PubMed - indexed for MEDLINE]



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Preclinical evaluation of taxane-binding peptide-modified polymeric micelles loaded with docetaxel in an orthotopic breast cancer mouse model.

Preclinical evaluation of taxane-binding peptide-modified polymeric micelles loaded with docetaxel in an orthotopic breast cancer mouse model.

Biomaterials. 2017 Jan 26;123:39-47

Authors: Logie J, Ganesh AN, Aman AM, Al-Awar RS, Shoichet MS

Abstract
We developed a novel taxane-binding peptide (TBP) modified, biodegradable polymeric micelle that overcomes limitations of drug loading and poor serum stability typically seen with particle delivery, leading to enhanced pharmacokinetics and tumor distribution of docetaxel (DTX). The use of the taxane-binding peptide to increase docetaxel loading is particularly compelling as it takes advantage of a known intracellular binding mechanism in a new way. Docetaxel is a potent chemotherapeutic with a therapeutic index often limited by the toxicity of the excipients that are necessary to enhance its solubility for intravenous delivery. Our polymeric micelle has terminal furan groups that enable facile antibody Fab conjugation by Diels-Alder chemistry for targeted delivery. Compared to the conventional ethanolic polysorbate 80 formulation (Free DTX), our nanoparticle (NP DTX) formulation exhibited a two-fold increase in exposure and tumor accumulation. Notably, the reduced toxicity of the NP DTX formulation increased the therapeutic index and allowed for higher dosing regimens, with a maximum tolerated dose (MTD) 1.6-fold higher than that of the Free DTX formulation, which is significant and similar to enhancements observed in clinical products for docetaxel and other drugs. These improved properties led to enhanced mouse survival in an orthotopic model of breast cancer; however, the targeted formulation of Fab-NP DTX did not further improve efficacy. Together, these results clearly demonstrate the benefits of the TBP-modified polymeric micelles as promising carriers for docetaxel.

PMID: 28161682 [PubMed - as supplied by publisher]



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Multilayered Control of Alternative Splicing Regulatory Networks by Transcription Factors.

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Multilayered Control of Alternative Splicing Regulatory Networks by Transcription Factors.

Mol Cell. 2017 Feb 02;65(3):539-553.e7

Authors: Han H, Braunschweig U, Gonatopoulos-Pournatzis T, Weatheritt RJ, Hirsch CL, Ha KC, Radovani E, Nabeel-Shah S, Sterne-Weiler T, Wang J, O'Hanlon D, Pan Q, Ray D, Zheng H, Vizeacoumar F, Datti A, Magomedova L, Cummins CL, Hughes TR, Greenblatt JF, Wrana JL, Moffat J, Blencowe BJ

Abstract
Networks of coordinated alternative splicing (AS) events play critical roles in development and disease. However, a comprehensive knowledge of the factors that regulate these networks is lacking. We describe a high-throughput system for systematically linking trans-acting factors to endogenous RNA regulatory events. Using this system, we identify hundreds of factors associated with diverse regulatory layers that positively or negatively control AS events linked to cell fate. Remarkably, more than one-third of the regulators are transcription factors. Further analyses of the zinc finger protein Zfp871 and BTB/POZ domain transcription factor Nacc1, which regulate neural and stem cell AS programs, respectively, reveal roles in controlling the expression of specific splicing regulators. Surprisingly, these proteins also appear to regulate target AS programs via binding RNA. Our results thus uncover a large "missing cache" of splicing regulators among annotated transcription factors, some of which dually regulate AS through direct and indirect mechanisms.

PMID: 28157508 [PubMed - in process]



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Yeast Populations Evolve to Resist CdSe Quantum Dot Toxicity.

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Yeast Populations Evolve to Resist CdSe Quantum Dot Toxicity.

Bioconjug Chem. 2017 Apr 19;28(4):1205-1213

Authors: Strtak A, Sathiamoorthy S, Tang PS, Tsoi KM, Song F, Anderson JB, Chan WCW, Shin JA

Abstract
Engineered nanomaterials are used globally in biomedical, electronic, and optical devices, and are often discarded into the environment. Cell culture experiments have shown that many inorganic nanoparticles are toxic to eukaryotic cells. Here, we show that populations of eukaryotic cells can evolve to survive chronic exposure to toxic CdSe semiconductor quantum dots (QDs). We grew yeast Saccharomyces cerevisiae for 24 days in liquid medium containing QDs prepared daily at half the minimum inhibitory concentration (MIC50) of the progenitor yeast cells. After 24 days, the cells grew normally under constant exposure to QDs. We concluded that these cells evolved to resist QD toxicity. Surprisingly, when we removed QDs from the growth medium, some of the evolved cells grew poorly, i.e., they grew better in the presence of QDs. Finally, genetic analysis confirmed that the ubiquitin ligase gene bul1 was mutated in the evolved cells, which suggests that this gene may be implicated in increased CdSe QD tolerance. This study shows that chronic exposure to QDs can exert selective pressure causing irreversible genetic changes leading to adaptation.

PMID: 28152308 [PubMed - indexed for MEDLINE]



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Genome-Scale Networks Link Neurodegenerative Disease Genes to α-Synuclein through Specific Molecular Pathways.

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Genome-Scale Networks Link Neurodegenerative Disease Genes to α-Synuclein through Specific Molecular Pathways.

Cell Syst. 2017 Jan 25;:

Authors: Khurana V, Peng J, Chung CY, Auluck PK, Fanning S, Tardiff DF, Bartels T, Koeva M, Eichhorn SW, Benyamini H, Lou Y, Nutter-Upham A, Baru V, Freyzon Y, Tuncbag N, Costanzo M, San Luis BJ, Schöndorf DC, Barrasa MI, Ehsani S, Sanjana N, Zhong Q, Gasser T, Bartel DP, Vidal M, Deleidi M, Boone C, Fraenkel E, Berger B, Lindquist S

Abstract
Numerous genes and molecular pathways are implicated in neurodegenerative proteinopathies, but their inter-relationships are poorly understood. We systematically mapped molecular pathways underlying the toxicity of alpha-synuclein (α-syn), a protein central to Parkinson's disease. Genome-wide screens in yeast identified 332 genes that impact α-syn toxicity. To "humanize" this molecular network, we developed a computational method, TransposeNet. This integrates a Steiner prize-collecting approach with homology assignment through sequence, structure, and interaction topology. TransposeNet linked α-syn to multiple parkinsonism genes and druggable targets through perturbed protein trafficking and ER quality control as well as mRNA metabolism and translation. A calcium signaling hub linked these processes to perturbed mitochondrial quality control and function, metal ion transport, transcriptional regulation, and signal transduction. Parkinsonism gene interaction profiles spatially opposed in the network (ATP13A2/PARK9 and VPS35/PARK17) were highly distinct, and network relationships for specific genes (LRRK2/PARK8, ATXN2, and EIF4G1/PARK18) were confirmed in patient induced pluripotent stem cell (iPSC)-derived neurons. This cross-species platform connected diverse neurodegenerative genes to proteinopathy through specific mechanisms and may facilitate patient stratification for targeted therapy.

PMID: 28131822 [PubMed - as supplied by publisher]



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Functional Analysis of Kinases and Transcription Factors in Saccharomyces cerevisiae Using an Integrated Overexpression Library.

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Functional Analysis of Kinases and Transcription Factors in Saccharomyces cerevisiae Using an Integrated Overexpression Library.

G3 (Bethesda). 2017 Jan 25;:

Authors: Youn JY, Friesen H, Nguyen Ba AN, Liang W, Messier V, Cox MJ, Moses AM, Andrews B

Abstract
Kinases and transcription factors (TFs) are key modulators of important signaling pathways and their activities underlie the proper function of many basic cellular processes such as cell division, differentiation and development. Changes in kinase and TF dosage are often associated with disease, yet a systematic assessment of the cellular phenotypes caused by the combined perturbation of kinases and TFs has not been undertaken. We used a reverse-genetics approach to study the phenotypic consequences of kinase and TF overexpression in the budding yeast, Saccharomyces cerevisiae We constructed a collection of strains expressing stably integrated inducible alleles of kinases and TFs and used a variety of assays to characterize the phenotypes caused by TF and kinase overexpression. We used the Synthetic Genetic Array (SGA) method to examine dosage-dependent genetic interactions (GIs) between 239 gain-of-function (overexpression) alleles of TFs and six loss-of-function and seven over-expression kinase alleles, the former identifying Synthetic Dosage Lethal (SDL) interactions and the latter testing a genetic interaction we call Double Dosage Lethality (DDL). We identified and confirmed 94 genetic interactions between 65 overexpression alleles of TFs and nine kinase alleles. Follow-up experiments validated regulatory relationships between genetically interacting pairs (Cdc28 - Stb1 and Pho85 - Pdr1), suggesting that genetic interaction studies involving overexpression alleles of regulatory proteins will be a rich source of new functional information.

PMID: 28122947 [PubMed - as supplied by publisher]



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Drosophila larvae synthesize the putative oncometabolite L-2-hydroxyglutarate during normal developmental growth.

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Drosophila larvae synthesize the putative oncometabolite L-2-hydroxyglutarate during normal developmental growth.

Proc Natl Acad Sci U S A. 2017 Jan 23;:

Authors: Li H, Chawla G, Hurlburt AJ, Sterrett MC, Zaslaver O, Cox J, Karty JA, Rosebrock AP, Caudy AA, Tennessen JM

Abstract
L-2-hydroxyglutarate (L-2HG) has emerged as a putative oncometabolite that is capable of inhibiting enzymes involved in metabolism, chromatin modification, and cell differentiation. However, despite the ability of L-2HG to interfere with a broad range of cellular processes, this molecule is often characterized as a metabolic waste product. Here, we demonstrate that Drosophila larvae use the metabolic conditions established by aerobic glycolysis to both synthesize and accumulate high concentrations of L-2HG during normal developmental growth. A majority of the larval L-2HG pool is derived from glucose and dependent on the Drosophila estrogen-related receptor (dERR), which promotes L-2HG synthesis by up-regulating expression of the Drosophila homolog of lactate dehydrogenase (dLdh). We also show that dLDH is both necessary and sufficient for directly synthesizing L-2HG and the Drosophila homolog of L-2-hydroxyglutarate dehydrogenase (dL2HGDH), which encodes the enzyme that breaks down L-2HG, is required for stage-specific degradation of the L-2HG pool. In addition, dLDH also indirectly promotes L-2HG accumulation via synthesis of lactate, which activates a metabolic feed-forward mechanism that inhibits dL2HGDH activity and stabilizes L-2HG levels. Finally, we use a genetic approach to demonstrate that dLDH and L-2HG influence position effect variegation and DNA methylation, suggesting that this compound serves to coordinate glycolytic flux with epigenetic modifications. Overall, our studies demonstrate that growing animal tissues synthesize L-2HG in a controlled manner, reveal a mechanism that coordinates glucose catabolism with L-2HG synthesis, and establish the fly as a unique model system for studying the endogenous functions of L-2HG during cell growth and proliferation.

PMID: 28115720 [PubMed - as supplied by publisher]



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