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1.
Oncotarget ; 8(55): 94619-94634, 2017 Nov 07.
Article in English | MEDLINE | ID: mdl-29212254

ABSTRACT

Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 play a critical role in mobilization and redistribution of immune cells and hematopoietic stem cells (HSCs). We evaluated effects of two CXCR4-targeting agents, peptide antagonist LY2510924 and monoclonal antibody LY2624587, on mobilizing HSCs and white blood cells (WBCs) in humans, monkeys, and mice. Biochemical analysis showed LY2510924 peptide blocked SDF-1/CXCR4 binding in all three species; LY2624587 antibody blocked binding in human and monkey, with minimal activity in mouse. Cellular analysis showed LY2624587 antibody, but not LY2510924 peptide, down-regulated cell surface CXCR4 and induced hematological tumor cell death; both agents have been shown to inhibit SDF-1/CXCR4 interaction and downstream signaling. In animal models, LY2510924 peptide induced robust, prolonged, dose- and time-dependent WBC and HSC increases in mice and monkeys, whereas LY2624587 antibody induced only moderate, transient increases in monkeys. In clinical trials, similar pharmacodynamic effects were observed in patients with advanced cancer: LY2510924 peptide induced sustained WBC and HSC increases, while LY2624587 antibody induced only minimal, transient WBC changes. These distinct pharmacodynamic effects in two different classes of CXCR4 inhibitors are clinically important and should be carefully considered when designing combination studies with immune checkpoint inhibitors or other agents for cancer therapy.

2.
PLoS One ; 11(3): e0150585, 2016.
Article in English | MEDLINE | ID: mdl-26954567

ABSTRACT

SDF-1 and CXCR4 are a chemokine and chemokine receptor pair playing critical roles in tumorigenesis. Overexpression of CXCR4 is a hallmark of many hematological malignancies including acute myeloid leukemia, chronic lymphocytic leukemia and non-Hodgkin's lymphoma, and generally correlates with a poor prognosis. In this study, we developed a humanized anti-CXCR4 monoclonal antibody, LY2624587 as a potent CXCR4 antagonist that was advanced into clinical study for cancer. LY2624587 blocked SDF-1 binding to CXCR4 with an IC50 of 0.26 nM, and inhibited SDF-1-induced GTP binding with a Kb of 0.66 nM. In human lymphoma U937 and leukemia CCRF-CEM cells expressing endogenous CXCR4, LY2624587 inhibited SDF-1-induced cell migration with IC50 values of 3.7 and 0.26 nM, respectively. This antibody also inhibited CXCR4 and SDF-1 mediated cell signaling including activation of MAPK and AKT in tumor cells expressing CXCR4. Bifocal microscopic and flow cytometry analyses revealed that LY2624587 mediated receptor internalization and caused CXCR4 down-regulation on the cell surface. In human hematologic cancer cells, LY2624587 caused dose dependent apoptosis in vitro and in vivo. In mouse xenograft models developed with human leukemia and lymphoma cells expressing high levels of CXCR4, LY2624587 exhibited dose-dependent tumor growth inhibition and provided significant survival benefit in a disseminated lymphoma model. Collectively, we have demonstrated that CXCR4 inhibition by LY2624587 has the potential for the treatment of human hematological malignancies.


Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Hematologic Neoplasms/metabolism , Receptors, CXCR4/antagonists & inhibitors , Animals , Annexin A5/metabolism , Caspase 3/metabolism , Cell Line, Tumor , Cell Movement , Cell Proliferation , Chemokine CXCL12/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Down-Regulation , Extracellular Signal-Regulated MAP Kinases/metabolism , Hematologic Neoplasms/drug therapy , Hematologic Neoplasms/mortality , Hematologic Neoplasms/pathology , Humans , Mice , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Receptors, CXCR4/metabolism , Tumor Burden/drug effects , Xenograft Model Antitumor Assays
3.
Methods Mol Biol ; 988: 291-302, 2013.
Article in English | MEDLINE | ID: mdl-23475727

ABSTRACT

Epitope mapping of antibodies is the identification and characterization of binding sites of monoclonal antibodies (mAbs) on target antigens. This knowledge can be useful in generating novel antibodies to a particular target as well as elucidating an antibody mechanism of action. Several techniques are available to identify antibody epitopes among which are preliminary and simple ones like sequence homology analysis ELISA and Western blotting. However, the more widely used robust methods typically involve the use of mass spectrometry to fully analyze and interpret the data and accurately identify the binding site. Such methods include epitope extortion/excision, hydrogen deuterium exchange.


Subject(s)
Antibodies, Monoclonal/chemistry , Epitopes/chemistry , Animals , Biotinylation/methods , CHO Cells , Chromatography, High Pressure Liquid , Cricetinae , Deuterium Exchange Measurement , Epitope Mapping/methods , Epitopes/isolation & purification , Fas Ligand Protein/chemistry , Fas Ligand Protein/isolation & purification , Humans , Immobilized Proteins/chemistry , Immobilized Proteins/isolation & purification , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Proteolysis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Solutions , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Trypsin/chemistry
4.
Biochemistry ; 48(30): 7251-60, 2009 Aug 04.
Article in English | MEDLINE | ID: mdl-19588926

ABSTRACT

Fas ligand (FasL) is a 40-kDa type II transmembrane protein belonging to the tumor necrosis factor (TNF) family of proteins and binds to its specific receptor, Fas, a member of the TNF receptor family. Membrane-bound FasL can be processed into a soluble form by a metalloprotease similar to that which cleaves TNFalpha. Elevated levels of FasL have been implicated in a wide variety of diseases ranging from cancer to inflammatory abnormalities, which could be targeted by antibody therapy. We generated a fully human high-affinity antibody against FasL that binds to and neutralizes the activity of both soluble and membrane-associated human FasL. In order to elucidate the mechanism of function of this antibody, we have mapped the region and critical residues involved in the recognition of FasL using a combination of homology modeling, immunoprecipitation, hydrogen-deuterium exchange mass spectrometry (H/DXMS), and alanine scanning site-directed mutagenesis. These studies have revealed the antibody binding site on human FasL. Furthermore, through molecular homology modeling, we have proposed a mechanism for the neutralizing activity of this antibody that involves interference with the docking of the ligand to its receptor by the antibody.


Subject(s)
Antibodies , Epitopes/chemistry , Fas Ligand Protein/chemistry , Fas Ligand Protein/immunology , Amino Acid Sequence , Animals , Antibodies/chemistry , Antibodies/immunology , Apoptosis/immunology , Binding Sites , Epitope Mapping , Epitopes/immunology , Fas Ligand Protein/genetics , Humans , Jurkat Cells , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Trypsin/metabolism
5.
Ann N Y Acad Sci ; 1014: 189-98, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15153434

ABSTRACT

Multiple endocrine neoplasia type 1 (MEN1), among all syndromes, causes tumors in the highest number of tissue types. Most of the tumors are hormone producing (e.g., parathyroid, enteropancreatic endocrine, anterior pituitary) but some are not (e.g., angiofibroma). MEN1 tumors are multiple for organ type, for regions of a discontinuous organ, and for subregions of a continuous organ. Cancer contributes to late mortality; there is no effective prevention or cure for MEN1 cancers. Morbidities are more frequent from benign than malignant tumor, and both are indicators for screening. Onset age is usually earlier in a tumor type of MEN1 than of nonhereditary cases. Broad trends contrast with those in nonneoplastic excess of hormones (e.g., persistent hyperinsulinemic hypoglycemia of infancy). Most germline or somatic mutations in the MEN1 gene predict truncation or absence of encoded menin. Similarly, 11q13 loss of heterozygosity in tumors predicts inactivation of the other MEN1 copy. MEN1 somatic mutation is prevalent in nonhereditary, MEN1-like tumor types. Compiled germline and somatic mutations show almost no genotype/phenotype relation. Normal menin is 67 kDa, widespread, and mainly nuclear. It may partner with junD, NF-kB, PEM, SMAD3, RPA2, FANCD2, NM23beta, nonmuscle myosin heavy chain II-A, GFAP, and/or vimentin. These partners have not clarified menin's pathways in normal or tumor tissues. Animal models have opened approaches to menin pathways. Local overexpression of menin in Drosophila reveals its interaction with the jun-kinase pathway. The Men1+/- mouse has robust MEN1; its most important difference from human MEN1 is marked hyperplasia of pancreatic islets, a tumor precursor stage.


Subject(s)
Gene Expression Regulation, Neoplastic , Multiple Endocrine Neoplasia/genetics , Multiple Endocrine Neoplasia/physiopathology , Proto-Oncogene Proteins/genetics , Animals , Humans , Multiple Endocrine Neoplasia/pathology
6.
Oncogene ; 22(41): 6347-58, 2003 Sep 25.
Article in English | MEDLINE | ID: mdl-14508515

ABSTRACT

MEN1 is a likely tumor suppressor gene that encodes a novel protein, menin. Menin is a 610 amino-acid residue protein with as yet unknown function(s). We have used tandem affinity purification and mass spectroscopy to isolate and identify proteins associating with menin from cultured HeLa cell extracts. This strategy has resulted in the isolation and identification of nonmuscle myosin type II-A heavy chain (NMHC II-A) as a menin interacting protein. This interaction was confirmed by glutathione-S-transferase pulldown assays, by coimmunoprecipitation, and by actin selection of myosin. We have further identified the amino-terminal region of menin and the head domain of NMHC II-A to be regions required for this interaction. Moreover menin was seen to colocalize with this myosin isoform in the cleavage furrow of dividing cells by indirect immunofluoresence. These data indicate that menin through binding to NMHC II-A could participate in cell division and in other processes that involve NMHC II-A.


Subject(s)
Myosin Heavy Chains/metabolism , Neoplasm Proteins/metabolism , Nonmuscle Myosin Type IIA/metabolism , Proto-Oncogene Proteins , Cell Nucleus/metabolism , Fluorescent Antibody Technique , Humans , Mutation , Myosin Heavy Chains/isolation & purification , Neoplasm Proteins/genetics , Nonmuscle Myosin Type IIA/isolation & purification , Precipitin Tests
7.
Mol Cell Biol ; 23(2): 493-509, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12509449

ABSTRACT

Menin is a 70-kDa protein encoded by MEN1, the tumor suppressor gene disrupted in multiple endocrine neoplasia type 1. In a yeast two-hybrid system based on reconstitution of Ras signaling, menin was found to interact with the 32-kDa subunit (RPA2) of replication protein A (RPA), a heterotrimeric protein required for DNA replication, recombination, and repair. The menin-RPA2 interaction was confirmed in a conventional yeast two-hybrid system and by direct interaction between purified proteins. Menin-RPA2 binding was inhibited by a number of menin missense mutations found in individuals with multiple endocrine neoplasia type 1, and the interacting regions were mapped to the N-terminal portion of menin and amino acids 43 to 171 of RPA2. This region of RPA2 contains a weak single-stranded DNA-binding domain, but menin had no detectable effect on RPA-DNA binding in vitro. Menin bound preferentially in vitro to free RPA2 rather than the RPA heterotrimer or a subcomplex consisting of RPA2 bound to the 14-kDa subunit (RPA3). However, the 70-kDa subunit (RPA1) was coprecipitated from HeLa cell extracts along with RPA2 by menin-specific antibodies, suggesting that menin binds to the RPA heterotrimer or a novel RPA1-RPA2-containing complex in vivo. This finding was consistent with the extensive overlap in the nuclear localization patterns of endogenous menin, RPA2, and RPA1 observed by immunofluorescence.


Subject(s)
DNA-Binding Proteins/metabolism , Neoplasm Proteins/metabolism , Proto-Oncogene Proteins , Animals , Bacterial Proteins/metabolism , Blotting, Western , Cell Line , Cell Nucleus/metabolism , Chromatography, Gel , DNA/metabolism , DNA Damage , DNA, Complementary/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , G1 Phase , Glutathione Transferase/metabolism , HeLa Cells , Humans , Mice , Microscopy, Fluorescence , Multiple Endocrine Neoplasia/genetics , Mutation, Missense , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Plasmids/metabolism , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/metabolism , Replication Protein A , S Phase , Transfection , Two-Hybrid System Techniques
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