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Molecular Cancer Therapeutics May 2020There is a need to develop novel approaches to improve the balance between efficacy and toxicity for transcription factor-targeted therapies. In this study, we exploit...
There is a need to develop novel approaches to improve the balance between efficacy and toxicity for transcription factor-targeted therapies. In this study, we exploit context-dependent differences in RNA polymerase II processivity as an approach to improve the activity and limit the toxicity of the EWS-FLI1-targeted small molecule, mithramycin, for Ewing sarcoma. The clinical activity of mithramycin for Ewing sarcoma is limited by off-target liver toxicity that restricts the serum concentration to levels insufficient to inhibit EWS-FLI1. In this study, we perform an siRNA screen of the druggable genome followed by a matrix drug screen to identify mithramycin potentiators and a synergistic "class" effect with cyclin-dependent kinase 9 (CDK9) inhibitors. These CDK9 inhibitors enhanced the mithramycin-mediated suppression of the EWS-FLI1 transcriptional program leading to a shift in the IC and striking regressions of Ewing sarcoma xenografts. To determine whether these compounds may also be liver protective, we performed a qPCR screen of all known liver toxicity genes in HepG2 cells to identify mithramycin-driven transcriptional changes that contribute to the liver toxicity. Mithramycin induces expression of the gene in HepG2 but not Ewing sarcoma cells, which leads to a liver-specific accumulation of reactive oxygen species (ROS). siRNA silencing of BTG2 rescues the induction of ROS and the cytotoxicity of mithramycin in these cells. Furthermore, CDK9 inhibition blocked the induction of BTG2 to limit cytotoxicity in HepG2, but not Ewing sarcoma cells. These studies provide the basis for a synergistic and less toxic EWS-FLI1-targeted combination therapy for Ewing sarcoma.
Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Bone Neoplasms; Cell Proliferation; Cyclin-Dependent Kinase 9; Drug-Related Side Effects and Adverse Reactions; Female; Humans; Mice; Mice, Nude; Plicamycin; Sarcoma, Ewing; Tumor Cells, Cultured; Xenograft Model Antitumor Assays
PubMed: 32127464
DOI: 10.1158/1535-7163.MCT-19-0775 -
International Journal of Molecular... Apr 2020Cholangiocarcinoma (CCA) is a cancer of biliary epithelium. Late diagnosis and resistance to conventional chemotherapy are the major obstacles in CCA treatment....
Cholangiocarcinoma (CCA) is a cancer of biliary epithelium. Late diagnosis and resistance to conventional chemotherapy are the major obstacles in CCA treatment. Increased expression of anti‑apoptotic proteins are observed in CCA, which might confer chemoresistance. Thus, modulations of anti‑apoptotic proteins leading to apoptotic induction is the focus of this study. Chromomycin A3 (CMA3), an anthraquinone glycoside‑mithramycin A analog, was selected. CMA3 strongly binds to GC‑rich regions in DNA, where specificity protein 1 (Sp1), a common transcription factor of apoptosis‑related proteins, is preferentially bounded. The effects of CMA3 on anti‑proliferation, cell cycle arrest and apoptosis induction in CCA cells were demonstrated by MTT assay, flow cytometry and western blot analysis. The results showed CMA3 suppressed cell proliferation in vitro in the nM range. At low doses, CMA3 inhibited cell cycle progression at S phase, while it promoted caspase‑dependent apoptosis at higher doses. CMA3 induced effects of apoptosis were through the suppression of Sp1‑related anti‑apoptotic proteins, FADD‑like IL‑1β‑converting enzyme‑inhibitory protein, myeloid cell leukemia‑1, X‑linked inhibitor of apoptosis protein, cellular inhibitor of apoptosis and survivin. The anti‑CCA effects of CMA3 were confirmed in the xenograft mouse model. CMA3 retarded xenograft tumor growth. Taken together, CMA3 induced apoptosis in CCA cells by diminishing the Sp1‑related anti‑apoptotic proteins is demonstrated. CMA3 might be useful as a chemosensitizing agent.
Topics: Animals; Bile Duct Neoplasms; Cell Line, Tumor; Cholangiocarcinoma; Chromomycin A3; Humans; Inhibitor of Apoptosis Proteins; Male; Mice; Mice, Inbred BALB C; Mice, Knockout; Neoplasm Proteins; S Phase Cell Cycle Checkpoints; Sp1 Transcription Factor; Xenograft Model Antitumor Assays
PubMed: 32124934
DOI: 10.3892/ijmm.2020.4482 -
Virology Mar 2020Latent HIV reservoir is a major barrier to absolute HIV cure. Studies on latency reversal agents (LRA) have by far focused mainly on CD4 T-lymphocytes, while myeloid...
Latent HIV reservoir is a major barrier to absolute HIV cure. Studies on latency reversal agents (LRA) have by far focused mainly on CD4 T-lymphocytes, while myeloid reservoirs remain under-represented despite their persistence and key contribution to HIV pathogenesis. cAMP has been shown to increase HIV-1 transcription in latently-infected monocytes/macrophages. In this communication, we explored the potential of commercially available pharmacological drugs and phosphodiesterase inhibitors to reactivate HIV in latently-infected monocytic cell-line, U1. We showed that increased levels of intracellular cAMP reverse HIV latency in vitro, which is specific to cells of the myeloid lineage. High throughput RNA-seq analysis revealed that cAMP modulates transcriptional profile of latently HIV-infected cells and provides favourable cellular environment for HIV to produce viral proteins. This reactivation of latent HIV was inhibited by Mithramycin A, a selective Sp1 inhibitor, indicating that the reversal of HIV latency in monocytes is driven by transcription factor Sp1.
Topics: 1-Methyl-3-isobutylxanthine; Colforsin; Cyclic AMP; Gene Expression Profiling; Gene Regulatory Networks; Genome, Viral; HIV-1; Host Microbial Interactions; Humans; Jurkat Cells; Models, Biological; Monocytes; Phosphodiesterase Inhibitors; Sp1 Transcription Factor; U937 Cells; Virus Activation; Virus Latency
PubMed: 32056667
DOI: 10.1016/j.virol.2020.01.006 -
Frontiers in Aging Neuroscience 2019Alzheimer's disease (AD) is a growing global threat to healthcare in the aging population. In the USA alone, it is estimated that one in nine persons over the age of 65...
Inhibition of Amyloid-Beta Production, Associated Neuroinflammation, and Histone Deacetylase 2-Mediated Epigenetic Modifications Prevent Neuropathology in Alzheimer's Disease Model.
Alzheimer's disease (AD) is a growing global threat to healthcare in the aging population. In the USA alone, it is estimated that one in nine persons over the age of 65 years is living with AD. The pathology is marked by the accumulation of amyloid-beta (Aβ) deposition in the brain, which is further enhanced by the neuroinflammatory process. Nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 3 (NLRP3) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) are the major neuroinflammatory pathways that intensify AD pathogenesis. Histone deacetylase 2 (HDAC2)-mediated epigenetic mechanisms play a major role in the genesis and neuropathology of AD. Therefore, therapeutic drugs, which can target Aβ production, NLRP3 activation, and HDAC2 levels, may play a major role in reducing Aβ levels and the prevention of associated neuropathology of AD. In this study, we demonstrate that withaferin A (WA), an extract from plant, significantly inhibits the Aβ production and NF-κB associated neuroinflammatory molecules' gene expression. Furthermore, we demonstrate that cytokine release inhibitory drug 3 (CRID3), an inhibitor of NLRP3, significantly prevents inflammasome-mediated gene expression in our AD model system. We have also observed that mithramycin A (MTM), an HDAC2 inhibitor, significantly upregulated the synaptic plasticity gene expression and downregulated HDAC2 in SH-SY5Y cells overexpressing amyloid precursor protein (SH-APP cells). Therefore, the introduction of these agents targeting Aβ production, NLRP3-mediated neuroinflammation, and HDAC2 levels will have a translational significance in the prevention of neuroinflammation and associated neurodegeneration in AD patients.
PubMed: 32009938
DOI: 10.3389/fnagi.2019.00342 -
Journal of the American Heart... Jan 2020Background Activation of the YAP (Yes-associated protein) pathway has been demonstrated to be related to smooth muscle cells (SMCs) phenotypic modulation and vessel...
Background Activation of the YAP (Yes-associated protein) pathway has been demonstrated to be related to smooth muscle cells (SMCs) phenotypic modulation and vessel restenosis. The aim of this study was to illustrate the molecular mechanisms that regulate the expression of YAP during the process of SMCs phenotypic switch. Whether the molecular basis identified in the study could be a potential therapeutic target for drug-eluting stents is further tested. Methods and Results In cell culture and in rat carotid arterial injury models, Sp-1 (specificity protein 1) expression was significantly induced, and correlated with SMCs proliferative phenotype. Overexpression of Sp-1 promoted SMCs proliferation and migration. Conversely, siSp-1 transfection or Sp-1 inhibitor Mithramycin A treatment attenuates SMC proliferation and migration. Through gain- and loss-function assays, we demonstrated that YAP was involved in Sp-1-mediated SMC phenotypic switch. Mechanistically, activated Sp-1 regulated YAP transcriptional expression through binding to its promoter. Moreover, we fabricated a Sp-1 inhibitor Mithramycin A-eluting stent and further tested it. In the rabbit carotid model, Mithramycin A-eluting stent inhibited YAP transcription and attenuated in-stent restenosis through regulating YAP-mediated SMC phenotypic switch. Conclusions Sp-1 controls phenotypic modulation of SMC by regulating transcription factor YAP. Drug-eluting stent targeting Sp-1 might represent a novel therapeutic strategy to prevent in-stent restenosis.
Topics: Angioplasty, Balloon; Animals; Apoptosis Regulatory Proteins; Cardiovascular Agents; Carotid Arteries; Carotid Artery Injuries; Carotid Stenosis; Cell Movement; Cell Plasticity; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Drug-Eluting Stents; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phenotype; Plicamycin; Prosthesis Design; Rabbits; Rats; Signal Transduction; Sp1 Transcription Factor; YAP-Signaling Proteins
PubMed: 31880978
DOI: 10.1161/JAHA.119.014103 -
Genes Nov 2019Prenyl diphosphate synthase subunit 2 (PDSS2) is the first key enzyme in the CoQ biosynthesis pathway, and contributes to various metabolic and nephritic diseases. It...
Prenyl diphosphate synthase subunit 2 (PDSS2) is the first key enzyme in the CoQ biosynthesis pathway, and contributes to various metabolic and nephritic diseases. It has been reported that is downregulated in several types of tumors and acts as a potential tumor suppressor gene to inhibit the proliferation and migration of cancer cells. However, the regulatory mechanism of expression remains elusive. In the present study, we first identified and characterized the promoter region. We established four different luciferase reporter constructs which mainly cover the 2 kb region upstream of the gene transcription initiation site. Series luciferase reporter assay demonstrated that all four constructs have prominent promoter activity, and the core promoter of is mainly located within the 202 bp region near its transcription initiation site. Transcription factor binding site analysis revealed that the promoter contains binding sites for canonical transcription factors such as Sp1 and GATA-1. Overexpression of significantly inhibited promoter activity, as well as its endogenous expression, at both mRNA and protein levels in lung cancer cells. Site-directed mutagenesis assay further confirmed that the Sp1 binding sites are essential for proximal prompter activity of . Consistently, a selective Sp1 inhibitor, mithramycin A, treatment repressed the promoter activity, as well as its endogenous expression. Chromatin immunoprecipitation (ChIP) assay revealed that Sp1 binds to the promoter in vivo. Of note, the expression of and are negatively correlated, and higher expression with low expression is significantly associated with poor prognosis in lung cancer. Taken together, our results strongly suggest the essential role of Sp1 in maintaining the basic constitutive expression of , and the pathogenic implication of Sp1-mediated transcriptional repression in lung cancer cells.
Topics: A549 Cells; Alkyl and Aryl Transferases; Binding Sites; Cell Line, Tumor; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Plicamycin; Prognosis; Promoter Regions, Genetic; Sp1 Transcription Factor; Survival Analysis; Transcription Initiation Site
PubMed: 31783675
DOI: 10.3390/genes10120977 -
International Journal For Parasitology.... Dec 2019The free-living amebae Naegleria, Acanthamoeba, and Balamuthia cause rare but life-threatening infections. All three parasites can cause meningoencephalitis....
Identification of plicamycin, TG02, panobinostat, lestaurtinib, and GDC-0084 as promising compounds for the treatment of central nervous system infections caused by the free-living amebae Naegleria, Acanthamoeba and Balamuthia.
The free-living amebae Naegleria, Acanthamoeba, and Balamuthia cause rare but life-threatening infections. All three parasites can cause meningoencephalitis. Acanthamoeba can also cause chronic keratitis and both Balamuthia and Acanthamoeba can cause skin and systemic infections. There are minimal drug development pipelines for these pathogens despite a lack of available treatment regimens and high fatality rates. To identify anti-amebic drugs, we screened 159 compounds from a high-value repurposed library against trophozoites of the three amebae. Our efforts identified 38 compounds with activity against at least one ameba. Multiple drugs that bind the ATP-binding pocket of mTOR and PI3K are active, highlighting these compounds as important inhibitors of these parasites. Importantly, 24 active compounds have progressed at least to phase II clinical studies and overall 15 compounds were active against all three amebae. Based on central nervous system (CNS) penetration or exceptional potency against one amebic species, we identified sixteen priority compounds for the treatment of meningoencephalitis caused by these pathogens. The top five compounds are (i) plicamycin, active against all three free-living amebae and previously U.S. Food and Drug Administration (FDA) approved, (ii) TG02, active against all three amebae, (iii and iv) FDA-approved panobinostat and FDA orphan drug lestaurtinib, both highly potent against Naegleria, and (v) GDC-0084, a CNS penetrant mTOR inhibitor, active against at least two of the three amebae. These results set the stage for further investigation of these clinically advanced compounds for treatment of infections caused by the free-living amebae, including treatment of the highly fatal meningoencephalitis.
Topics: Acanthamoeba; Amebiasis; Amoebozoa; Antiprotozoal Agents; Carbazoles; Cell Culture Techniques; Central Nervous System Protozoal Infections; Culture Media; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Enzyme Inhibitors; Furans; Heterocyclic Compounds, 4 or More Rings; Inhibitory Concentration 50; Naegleria; Oxazines; Panobinostat; Plicamycin; Pyrimidines
PubMed: 31707263
DOI: 10.1016/j.ijpddr.2019.10.003 -
Angewandte Chemie (International Ed. in... Jan 2020MtmOIV and MtmW catalyze the final two reactions in the mithramycin (MTM) biosynthetic pathway, the Baeyer-Villiger opening of the fourth ring of premithramycin B...
MtmOIV and MtmW catalyze the final two reactions in the mithramycin (MTM) biosynthetic pathway, the Baeyer-Villiger opening of the fourth ring of premithramycin B (PMB), creating the C3 pentyl side chain, strictly followed by reduction of the distal keto group on the new side chain. Unexpectedly this results in a C2 stereoisomer of mithramycin, iso-mithramycin (iso-MTM). Iso-MTM undergoes a non-enzymatic isomerization to MTM catalyzed by Mg ions. Crystal structures of MtmW and its complexes with co-substrate NADPH and PEG, suggest a catalytic mechanism of MtmW. The structures also show that a tetrameric assembly of this enzyme strikingly resembles the ring-shaped β subunit of a vertebrate ion channel. We show that MtmW and MtmOIV form a complex in the presence of PMB and NADPH, presumably to hand over the unstable MtmOIV product to MtmW, yielding iso-MTM, as a potential self-resistance mechanism against MTM toxicity.
Topics: Biological Products; Catalysis; Plicamycin
PubMed: 31702856
DOI: 10.1002/anie.201910241 -
Frontiers in Immunology 2019B-cell receptor (BCR)-dependent signaling is central for leukemia B-cell homeostasis, as underscored by the promising clinical results obtained in patients with chronic...
B-cell receptor (BCR)-dependent signaling is central for leukemia B-cell homeostasis, as underscored by the promising clinical results obtained in patients with chronic lymphocytic leukemia (CLL) treated with novel agents targeting components of this pathway. Herein, we demonstrate that the mithralog EC-7072 displays high cytotoxic activity against leukemia cells from CLL patients independently from high-risk prognostic markers and IGHV mutational status. EC-7072 was significantly less toxic against T cells and NK cells and did not alter the production of the immune effector molecules IFN-γ and perforin. EC-7072 directly triggered caspase-3-dependent CLL cell apoptosis, which was not abrogated by microenvironment-derived factors that sustain leukemia cell survival. RNA-sequencing analyses revealed a dramatic EC-7072-driven reprograming of the transcriptome of CLL cells, including a wide downregulation of multiple components and targets of the BCR signaling pathway. Accordingly, we found decreased levels of phosphorylated signaling nodes downstream of the BCR. Crosslinking-mediated BCR activation antagonized CLL cell death triggered by EC-7072, increased the phosphorylation levels of the abovementioned signaling nodes and upregulated expression, suggesting that the mithralog disrupts CLL cell viability by targeting the BCR signaling axis at multiple levels. EC-7072 exerted similar or higher antileukemic activity than that of several available CLL therapies and displayed additive or synergistic interaction with these drugs in killing CLL cells. Overall, our findings provide rationale for future investigation to test whether EC-7072 may be a potential therapeutic option for patients with CLL and other B-cell malignancies.
Topics: Antibiotics, Antineoplastic; Apoptosis; Caspase 3; Cell Survival; Gene Expression Profiling; Gene Expression Regulation, Leukemic; Humans; Interferon-gamma; Killer Cells, Natural; Leukemia, Lymphocytic, Chronic, B-Cell; Phosphorylation; Plicamycin; Receptors, Antigen, B-Cell; Signal Transduction; T-Lymphocytes; Tumor Cells, Cultured; Tumor Microenvironment
PubMed: 31681329
DOI: 10.3389/fimmu.2019.02455