ABSTRACT
Photodynamic therapy (PDT) utilizes reactive oxygen species (ROS) for eradication of cancer cells. Its effectiveness is governed by the oxygen content, which is scarce in the hypoxic tumor microenvironment. We report herein two zinc(II) phthalocyanines substituted with two or four nitric oxide (NO)-releasing moieties, namely ZnPc-2NO and ZnPc-4NO, which can suppress the mitochondrial respiration, thereby sparing more intracellular oxygen for PDT. Using HT29 human colorectal adenocarcinoma cells and A549 human lung carcinoma cells, we have demonstrated that both conjugates release NO upon interaction with the intracellular glutathione, which can reduce the cellular oxygen consumption rate and adenosine triphosphate generation and alter the mitochondrial membrane potential. They can also relieve the hypoxic status of cancer cells and decrease the expression of hypoxia-inducible factor protein HIF-1α. Upon light irradiation, both conjugates can generate ROS and induce cytotoxicity even under a hypoxic condition, overcoming the oxygen-dependent nature of PDT. Interestingly, the photodynamic action of ZnPc-2NO elicits the release of damage-associated molecular patterns, inducing the maturation of dendritic cells and triggering an antitumor immune response. The immunogenic cell death caused by this oxygen-economized PDT has been demonstrated through a series of in vitro and in vivo experiments.
ABSTRACT
Manipulation of cell-cell interactions via cell surface modification is crucial in tissue engineering and cell-based therapy. To be able to monitor intercellular interactions, it can also provide useful information for understanding how the cells interact and communicate. We report herein a facile bioorthogonal strategy to promote and monitor cell-cell interactions. It involves the use of a maleimide-appended tetrazine-caged boron dipyrromethene (BODIPY)-based fluorescent probe and a maleimide-substituted bicyclo[6.1.0]non-4-yne (BCN) to modify the membrane of macrophage (RAW 264.7) and cancer (HT29, HeLa, and A431) cells, respectively, via maleimide-thiol conjugation. After modification, the two kinds of cells interact strongly through inverse electron-demand Diels-Alder reaction of the surface tetrazine and BCN moieties. The coupling also disrupts the tetrazine quenching unit, restoring the fluorescence emission of the BODIPY core on the cell-cell interface, and promotes phagocytosis. Hence, this approach can promote and facilitate the detection of intercellular interactions, rendering it potentially useful for macrophage-based immunotherapy.
Subject(s)
Boron Compounds , Cell Communication , Fluorescent Dyes , Humans , Boron Compounds/chemistry , Mice , Animals , Fluorescent Dyes/chemistry , RAW 264.7 Cells , Maleimides/chemistry , HeLa CellsABSTRACT
Plasmacytoid dendritic cells are a rare subset of dendritic cells that exhibit antiviral functions in response to toll-like receptor 7/8 stimulations. Alternative toll-like receptors such as TLR4 have been known to be active in plasmacytoid dendritic cells for immune regulatory functions. However, it is unclear whether these toll-like receptors differentially activate plasmacytoid dendritic cells as compared with canonical toll-like receptor 7/8 stimulation. Here, we assessed alternative plasmacytoid dendritic cell activation states mediated by toll-like receptors other than endosomal toll-like receptors via the RNA sequencing approach. We found that toll-like receptor 4 stimulation induced a high degree of similarity in gene expression pattern to toll-like receptor 7/8 stimulation in plasmacytoid dendritic cells. Despite high resemblance to toll-like receptor 7/8, we discovered unique genes that were activated under toll-like receptor 4 activation only, as well as genes that were induced at a higher magnitude in comparison to toll-like receptor 7/8 activation. In comparison between toll-like receptor 4-activated plasmacytoid dendritic cells and conventional dendritic cells, we revealed that plasmacytoid dendritic cells and conventional dendritic cells expressed distinct gene sets, whereby conventional dendritic cells mostly favored antigen presentation functions for adaptive immune response regulation while plasmacytoid dendritic cells leaned toward immune response against infectious diseases. Last, we determined that toll-like receptor 4 activation sensitized plasmacytoid dendritic cells against SARS-CoV-2 (COVID-19) single-stranded RNA by enhancing antiviral-related responses and type I interferon production. These findings provided greater insights into the toll-like receptor 4 activation state in plasmacytoid dendritic cells, which can be beneficial for alternative therapeutic interventions involving plasmacytoid dendritic cells for various diseases.
Subject(s)
COVID-19 , Toll-Like Receptor 7 , Humans , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 4/metabolism , SARS-CoV-2/metabolism , COVID-19/metabolism , Toll-Like Receptors/metabolism , Dendritic Cells , Antiviral Agents/pharmacology , Antiviral Agents/metabolism , Toll-Like Receptor 9/metabolismABSTRACT
Site-selective acetylation of a single lysine residue in a protein that reaches a lysine acetyltransferase's accuracy, precision, and reliability is challenging. Here, we report a peptide-guided, proximity-driven group transfer reaction that acetylates a single lysine residue, Lys 248, of the fragment crystallizable region (Fc region) in the heavy chain of the human Immunoglobulin G (IgG). An Fc-interacting peptide bound with the Fc domain and positioned a phenolic ester close to Lys 248, which induced a nucleophilic reaction and resulted in the transfer of an acetyl group to Lys 248. The acetylation reaction proceeded to a decent yield under the physiological condition without the need for deglycosylation, unnatural amino acids, or catalysts. Along with acetylation, functional moieties such as azide, alkyne, fluorescent molecules, or biotin could also be site-selectively installed on Lys 248, allowing IgG's further derivatization. We then synthesized an antibody-lipid conjugate and constructed antibody-conjugated liposomes (immunoliposomes), targeting HER2-positive (HER2+) cancer cells. We also built a bispecific antibody complex (bsAbC) covalently linking an anti-HER2 antibody and an anti-CD3 antibody. The bsAbC showed in vitro effector-cell-mediated cytotoxicity at nanomolar concentrations. Compared with bispecific antibodies (bsAbs), bsAbCs are constructed based on native IgGs and contain two antigen-binding sites to each antigen, twice that of bsAbs. Altogether, this work reports a method of site-selective acetylation of native antibodies, highlights a facile way of site-selective IgG functionalization, and underscores the potential of bsAbCs in cancer immunotherapy.
Subject(s)
Antibodies, Bispecific , Lysine Acetyltransferases , Acetylation , Alkynes , Antibodies, Bispecific/chemistry , Azides , Biotin , Esters , Humans , Immunoglobulin G/chemistry , Lipids , Liposomes , Lysine , Reproducibility of ResultsABSTRACT
Immune modulation is a hallmark of cancer. Cancer-immune interaction shapes the course of disease progression at every step of tumorigenesis, including metastasis, of which circulating tumor cells (CTCs) are regarded as an indicator. These CTCs are a heterogeneous population of tumor cells that have disseminated from the tumor into circulation. They have been increasingly studied in recent years due to their importance in diagnosis, prognosis, and monitoring of treatment response. Ample evidence demonstrates that CTCs interact with immune cells in circulation, where they must evade immune surveillance or modulate immune response. The interaction between CTCs and the immune system is emerging as a critical point by which CTCs facilitate metastatic progression. Understanding the complex crosstalk between the two may provide a basis for devising new diagnostic and treatment strategies. In this review, we will discuss the current understanding of CTCs and the complex immune-CTC interactions. We also present novel options in clinical interventions, targeting the immune-CTC interfaces, and provide some suggestions on future research directions.
ABSTRACT
Pathogen recognition receptor (PRR) signaling is critical for triggering innate immune activation and the expression of immune response genes, including genes that impart restriction against virus replication. RIG-I-like receptors and TLRs are PRRs that signal immune activation and drive the expression of antiviral genes and the production of type I IFN leading to induction of IFN-stimulated genes, in part through the interferon regulatory factor (IRF) family of transcription factors. Previous studies with West Nile virus (WNV) showed that IRF3 and IRF7 regulate IFN expression in fibroblasts and neurons, whereas macrophages and dendritic cells (DCs) retained the ability to induce IFN-ß in the absence of IRF3 and IRF7 in a manner implicating IRF5 in PRR signaling actions. Here we assessed the contribution of IRF5 to immune gene induction in response to WNV infection in DCs. We examined IRF5-dependent gene expression and found that loss of IRF5 in mice resulted in modest and subtle changes in the expression of WNV-regulated genes. Anti-IRF5 chromatin immunoprecipitation with next-generation sequencing of genomic DNA coupled with mRNA analysis revealed unique IRF5 binding motifs within the mouse genome that are distinct from the canonical IRF binding motif and that link with IRF5-target gene expression. Using integrative bioinformatics analyses, we identified new IRF5 primary target genes in DCs in response to virus infection. This study provides novel insights into the distinct and unique innate immune and immune gene regulatory program directed by IRF5.
Subject(s)
Dendritic Cells/metabolism , Dendritic Cells/virology , Gene Expression Regulation , Interferon Regulatory Factors/metabolism , West Nile Fever/genetics , West Nile virus/physiology , Animals , Base Sequence , Binding Sites , DNA/metabolism , Interferon Regulatory Factors/deficiency , Macrophages/metabolism , Macrophages/virology , Mice, Inbred C57BL , Transcription, Genetic , West Nile Fever/pathologyABSTRACT
We examined the signaling pathways and cell type-specific responses of IFN regulatory factor (IRF) 5, an immune-regulatory transcription factor. We show that the protein kinases IKKα, IKKß, IKKε, and TANK-binding kinase 1 each confer IRF5 phosphorylation/dimerization, thus extending the family of IRF5 activator kinases. Among primary human immune cell subsets, we found that IRF5 is most abundant in plasmacytoid dendritic cells (pDCs). Flow cytometric cell imaging revealed that IRF5 is specifically activated by endosomal TLR signaling. Comparative analyses revealed that IRF3 is activated in pDCs uniquely through RIG-I-like receptor (RLR) signaling. Transcriptomic analyses of pDCs show that the partitioning of TLR7/IRF5 and RLR/IRF3 pathways confers differential gene expression and immune cytokine production in pDCs, linking IRF5 with immune regulatory and proinflammatory gene expression. Thus, TLR7/IRF5 and RLR-IRF3 partitioning serves to polarize pDC response outcome. Strategies to differentially engage IRF signaling pathways should be considered in the design of immunotherapeutic approaches to modulate or polarize the immune response for specific outcome.
Subject(s)
Dendritic Cells/immunology , Interferon Regulatory Factor-3/immunology , Interferon Regulatory Factors/immunology , Signal Transduction/immunology , Cells, Cultured , Dendritic Cells/metabolism , Gene Expression Regulation/immunology , Humans , Interferon Regulatory Factor-3/metabolism , Interferon Regulatory Factors/metabolismABSTRACT
Pattern recognition receptors (PRRs) survey intra- and extracellular spaces for pathogen-associated molecular patterns (PAMPs) within microbial products of infection. Recognition and binding to cognate PAMP ligand by specific PRRs initiates signaling cascades that culminate in a coordinated intracellular innate immune response designed to control infection. In particular, our immune system has evolved specialized PRRs to discriminate viral nucleic acid from host. These are critical sensors of viral RNA to trigger innate immunity in the vertebrate host. Different families of PRRs of virus infection have been defined and reveal a diversity of PAMP specificity for wide viral pathogen coverage to recognize and extinguish virus infection. In this review, we discuss recent insights in pathogen recognition by the RIG-I-like receptors, related RNA helicases, Toll-like receptors, and other RNA sensor PRRs, to present emerging themes in innate immune signaling during virus infection.
Subject(s)
DEAD Box Protein 58/metabolism , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Virus Diseases/etiology , Virus Diseases/metabolism , Viruses/immunology , Animals , DEAD-box RNA Helicases/metabolism , Humans , Protein Processing, Post-Translational , RNA Helicases/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Receptors, Immunologic , Signal Transduction , Toll-Like Receptors/metabolismABSTRACT
Interferons (IFNs) are crucial cytokines of antimicrobial, antitumor, and immunomodulatory activity. The three types of IFN (I, II, and III) are classified by their receptor specificity and sequence homology. IFNs are produced and secreted by cells in response to specific stimuli. Here, we review the subsequent IFN signaling events occurring through unique receptors leading to regulation of gene expression for modulation of innate and adaptive immunity. To view this SnapShot, open or download the PDF.
Subject(s)
Interferons/metabolism , Signal Transduction , Animals , Humans , Interferons/classification , Receptors, InterferonABSTRACT
Growth factor independence genes (Gfi1 and Gfi1b) repress recombination activating genes (Rag) transcription in developing B lymphocytes. Because all blood lineages originate from hematopoietic stem cells (HSCs) and different lineage progenitors have been shown to share transcription factor networks prior to cell fate commitment, we hypothesized that GFI family proteins may also play a role in repressing Rag transcription or a global lymphoid transcriptional program in other blood lineages. We tested the level of Rag transcription in various blood cells when Gfi1 and Gfi1b were deleted, and observed an upregulation of Rag expression in plasmacytoid dendritic cells (pDCs). Using microarray analysis, we observed that Gfi1 and Gfi1b do not regulate a lymphoid or pDC-specific transcriptional program. This study establishes a role for Gfi1 and Gfi1b in Rag regulation in a non-B lineage cell type.
Subject(s)
DNA-Binding Proteins/metabolism , Dendritic Cells/metabolism , Gene Expression Regulation/genetics , Homeodomain Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Repressor Proteins/metabolism , Transcription Factors/metabolism , Animals , Computational Biology , DNA-Binding Proteins/genetics , Flow Cytometry , Humans , Mice , Microarray Analysis , Proto-Oncogene Proteins/genetics , Real-Time Polymerase Chain Reaction , Repressor Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/geneticsABSTRACT
Foxo1 is a critical, direct regulator of Rag (recombination activating gene) transcription during B cell development and is thus essential for the generation of a diverse repertoire of antigen receptors. Although Foxo1 regulation has been widely studied in many cell types, pathways regulating Foxo1 in B cells have not been fully elucidated. By screening a panel of Foxo1 mutants, we identified serine 215 on Foxo1 as a novel phosphorylation site that is essential for the activation of Rag transcription. Mutation of S215 strongly attenuated transactivation of Rag but did not affect most other Foxo1 target genes. We show that MK5, a MAPK-activated protein kinase, is a previously unidentified upstream regulator of Foxo1. MK5 was necessary and sufficient to activate Rag transcription in transformed and primary pro-B cells. Together, our experiments show that MK5 positively regulates Rag transcription via phosphorylation of Foxo1 in developing B cells.
Subject(s)
B-Lymphocytes/cytology , B-Lymphocytes/metabolism , Forkhead Transcription Factors/metabolism , Genes, RAG-1 , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Transcription, Genetic , Amino Acid Sequence , Animals , Benzamides/pharmacology , Cell Line, Transformed , Forkhead Box Protein O1 , Forkhead Transcription Factors/chemistry , Forkhead Transcription Factors/genetics , Gene Expression , Gene Expression Regulation/drug effects , Imatinib Mesylate , Mice , Mice, Knockout , Molecular Sequence Data , Piperazines/pharmacology , Protein Binding , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Pyrimidines/pharmacology , Sequence Alignment , Serine/metabolism , Trans-Activators/genetics , Trans-Activators/metabolismABSTRACT
Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Long-term survival of patients with AML has changed little over the past decade, necessitating the identification and validation of new AML targets. Integration of genomic approaches with small-molecule and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. Here, we identified a role for glycogen synthase kinase 3α (GSK-3α) in AML by performing 2 independent small-molecule library screens and an shRNA screen for perturbations that induced a differentiation expression signature in AML cells. GSK-3 is a serine-threonine kinase involved in diverse cellular processes, including differentiation, signal transduction, cell cycle regulation, and proliferation. We demonstrated that specific loss of GSK-3α induced differentiation in AML by multiple measurements, including induction of gene expression signatures, morphological changes, and cell surface markers consistent with myeloid maturation. GSK-3α-specific suppression also led to impaired growth and proliferation in vitro, induction of apoptosis, loss of colony formation in methylcellulose, and anti-AML activity in vivo. Although the role of GSK-3ß has been well studied in cancer development, these studies support a role for GSK-3α in AML.
Subject(s)
Gene Expression Regulation, Neoplastic , Glycogen Synthase Kinase 3/metabolism , Leukemia, Myeloid, Acute/enzymology , Leukemia, Myeloid, Acute/metabolism , Neoplasms/metabolism , Cell Differentiation , Cell Proliferation , Cell Survival , Gene Expression Profiling , Genomics , HL-60 Cells , Humans , RNA Interference , Technology, Pharmaceutical , U937 CellsABSTRACT
Precise regulation of Rag (recombination-activating gene) expression is crucial to prevent genomic instability caused by the generation of Rag-mediated DNA breaks. Although mechanisms of Rag activation have been well characterized, the mechanism by which Rag expression is down-regulated in early B cell development has not been fully elucidated. Using a complementary DNA library screen, we identified the transcriptional repressor Gfi1b as negative regulator of the Rag locus. Expression of Gfi1b causes repression of Rag1 and Rag2 in cell lines and primary mouse cells. Conversely, Gfi1b-deficient cell lines exhibit increased Rag expression, double-strand breaks and recombination, and cell cycle defects. In primary cells, transcription of Gfi1b inversely correlates with Rag transcription, and simultaneous inactivation of Gfi1 and Gfi1b leads to an increase in Rag transcription early in B cell development. In addition, deletion of Gfi1 and Gfi1b in vivo results in a severe block in B cell development. Gfi1b orchestrates Rag repression via a dual mechanism. Direct binding of Gfi1b to a site 5' of the B cell-specific Erag enhancer results in epigenetic changes in the Rag locus, whereas indirect inhibition is achieved through repression of the trans-activator Foxo1. Together, our experiments show that Gfi family members are essential for normal B cell development and play an important role in modulating expression of the V(D)J recombinase.
Subject(s)
Forkhead Transcription Factors/metabolism , Homeodomain Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Repressor Proteins/metabolism , Animals , B-Lymphocytes/cytology , B-Lymphocytes/metabolism , Cell Differentiation/genetics , Chromatin Assembly and Disassembly , DNA Replication , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Forkhead Box Protein O1 , Gene Deletion , Gene Expression Regulation , Gene Library , Gene Targeting , HEK293 Cells , Homeodomain Proteins/genetics , Humans , Mice , Mice, Inbred BALB C , Proto-Oncogene Proteins/deficiency , Proto-Oncogene Proteins/genetics , Recombination, Genetic , Repressor Proteins/deficiency , Repressor Proteins/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Somatic rearrangements of transcription factors are common abnormalities in the acute leukemias. With rare exception, however, the resultant protein products have remained largely intractable as pharmacologic targets. One example is AML1-ETO, the most common translocation reported in acute myeloid leukemia (AML). To identify AML1-ETO modulators, we screened a small molecule library using a chemical genomic approach. Gene expression signatures were used as surrogates for the expression versus loss of the translocation in AML1-ETO-expressing cells. The top classes of compounds that scored in this screen were corticosteroids and dihydrofolate reductase (DHFR) inhibitors. In addition to modulating the AML1-ETO signature, both classes induced evidence of differentiation, dramatically inhibited cell viability, and ultimately induced apoptosis via on-target activity. Furthermore, AML1-ETO-expressing cell lines were exquisitely sensitive to the effects of corticosteroids on cellular viability compared with nonexpressers. The corticosteroids diminished AML1-ETO protein in AML cells in a proteasome- and glucocorticoid receptor-dependent manner. Moreover, these molecule classes demonstrated synergy in combination with standard AML chemotherapy agents and activity in an orthotopic model of AML1-ETO-positive AML. This work suggests a role for DHFR inhibitors and corticosteroids in treating patients with AML1-ETO-positive disease.
Subject(s)
Antineoplastic Agents/pharmacology , Combinatorial Chemistry Techniques , Core Binding Factor Alpha 2 Subunit/antagonists & inhibitors , Core Binding Factor Alpha 2 Subunit/metabolism , Genomics , Neoplasms/drug therapy , Oncogene Proteins, Fusion/antagonists & inhibitors , Oncogene Proteins, Fusion/metabolism , Pharmaceutical Preparations/metabolism , Acetylation , Animals , Apoptosis/drug effects , Cell Differentiation , Cell Proliferation/drug effects , Core Binding Factor Alpha 2 Subunit/genetics , Flow Cytometry , Gene Expression Profiling , Histone Deacetylase Inhibitors , Histone Deacetylases/metabolism , Histones/metabolism , Humans , Immunoblotting , Male , Mice , Mice, Inbred NOD , Myeloid Cells/drug effects , Myeloid Cells/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Oligonucleotide Array Sequence Analysis , Oncogene Proteins, Fusion/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , RUNX1 Translocation Partner 1 Protein , Reverse Transcriptase Polymerase Chain Reaction , Translocation, GeneticABSTRACT
BACKGROUND: The presence of tumor-specific mutations in the cancer genome represents a potential opportunity for pharmacologic intervention to therapeutic benefit. Unfortunately, many classes of oncoproteins (e.g., transcription factors) are not amenable to conventional small-molecule screening. Despite the identification of tumor-specific somatic mutations, most cancer therapy still utilizes nonspecific, cytotoxic drugs. One illustrative example is the treatment of Ewing sarcoma. Although the EWS/FLI oncoprotein, present in the vast majority of Ewing tumors, was characterized over ten years ago, it has never been exploited as a target of therapy. Previously, this target has been intractable to modulation with traditional small-molecule library screening approaches. Here we describe a gene expression-based approach to identify compounds that induce a signature of EWS/FLI attenuation. We hypothesize that screening small-molecule libraries highly enriched for FDA-approved drugs will provide a more rapid path to clinical application. METHODS AND FINDINGS: A gene expression signature for the EWS/FLI off state was determined with microarray expression profiling of Ewing sarcoma cell lines with EWS/FLI-directed RNA interference. A small-molecule library enriched for FDA-approved drugs was screened with a high-throughput, ligation-mediated amplification assay with a fluorescent, bead-based detection. Screening identified cytosine arabinoside (ARA-C) as a modulator of EWS/FLI. ARA-C reduced EWS/FLI protein abundance and accordingly diminished cell viability and transformation and abrogated tumor growth in a xenograft model. Given the poor outcomes of many patients with Ewing sarcoma and the well-established ARA-C safety profile, clinical trials testing ARA-C are warranted. CONCLUSIONS: We demonstrate that a gene expression-based approach to small-molecule library screening can identify, for rapid clinical testing, candidate drugs that modulate previously intractable targets. Furthermore, this is a generic approach that can, in principle, be applied to the identification of modulators of any tumor-associated oncoprotein in the rare pediatric malignancies, but also in the more common adult cancers.