ABSTRACT
BACKGROUND: Deregulated transcription is a major driver of diseases such as cancer. Bromodomain and extra-terminal (BET) proteins (BRD2, BRD3, BRD4 and BRDT) are chromatin readers essential for maintaining proper gene transcription by specifically binding acetylated lysine residues. Targeted displacement of BET proteins from chromatin, using BET inhibitors (I-BETs), is a promising therapy, especially for acute myeloid leukemia (AML), and evaluation of resistance mechanisms is necessary to optimize the clinical efficacy of these drugs. RESULTS: To uncover mechanisms of intrinsic I-BET resistance, we quantified chromatin binding and displacement for BRD2, BRD3 and BRD4 after dose response treatment with I-BET151, in sensitive and resistant in vitro models of leukemia, and mapped BET proteins/I-BET interactions genome wide using antibody- and compound-affinity capture methods followed by deep sequencing. The genome-wide map of BET proteins sensitivity to I-BET revealed a bimodal pattern of binding flanking transcription start sites (TSSs), in which drug-mediated displacement from chromatin primarily affects BRD4 downstream of the TSS and prolongs the pausing of RNA Pol II. Correlation of BRD4 binding and drug-mediated displacement at RNA Pol II pause sites with gene expression revealed a differential behavior of sensitive and resistant tumor cells to I-BET and identified a BRD4 signature at promoters of sensitive coding and non-coding genes. CONCLUSIONS: We provide evidence that I-BET-induced shift of Pol II pausing at promoters via displacement of BRD4 is a determinant of intrinsic I-BET sensitivity. This finding may guide pharmacological treatment to enhance the clinical utility of such targeted therapies in AML and potentially other BET proteins-driven diseases.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Heterocyclic Compounds, 4 or More Rings/pharmacology , Proteins/metabolism , RNA Polymerase II/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Cell Line, Tumor , Chromatin/genetics , Chromatin/metabolism , Gene Expression , Humans , K562 Cells , Nuclear Proteins/metabolism , Promoter Regions, Genetic , Protein Domains , Protein Interaction Mapping , Proteins/antagonists & inhibitors , RNA Polymerase II/genetics , Transcription Initiation SiteABSTRACT
Citrullination of joint proteins by the protein arginine deiminase (PAD) family of enzymes is recognized increasingly as a key process in the pathogenesis of rheumatoid arthritis. This present study was undertaken to explore the efficacy of a novel PAD4-selective inhibitor, GSK199, in the murine collagen-induced arthritis model of rheumatoid arthritis. Mice were dosed daily from the time of collagen immunization with GSK199. Efficacy was assessed against a wide range of end-points, including clinical disease scores, joint histology and immunohistochemistry, serum and joint citrulline levels and quantification of synovial autoantibodies using a proteomic array containing joint peptides. Administration of GSK199 at 30 mg/kg led to significant effects on arthritis, assessed both by global clinical disease activity and by histological analyses of synovial inflammation, pannus formation and damage to cartilage and bone. In addition, significant decreases in complement C3 deposition in both synovium and cartilage were observed robustly with GSK199 at 10 mg/kg. Neither the total levels of citrulline measurable in joint and serum, nor levels of circulating collagen antibodies, were affected significantly by treatment with GSK199 at any dose level. In contrast, a subset of serum antibodies reactive against citrullinated and non-citrullinated joint peptides were reduced with GSK199 treatment. These data extend our previous demonstration of efficacy with the pan-PAD inhibitor Cl-amidine and demonstrate robustly that PAD4 inhibition alone is sufficient to block murine arthritis clinical and histopathological end-points.
Subject(s)
Arthritis, Experimental/drug therapy , Arthritis, Experimental/enzymology , Arthritis, Rheumatoid/drug therapy , Arthritis, Rheumatoid/enzymology , Benzimidazoles/administration & dosage , Hydrolases/antagonists & inhibitors , Animals , Arthritis, Experimental/physiopathology , Arthritis, Rheumatoid/chemically induced , Arthritis, Rheumatoid/physiopathology , Autoantibodies/blood , Benzimidazoles/pharmacokinetics , Bone and Bones/pathology , Cartilage/immunology , Cartilage/pathology , Citrulline/analysis , Citrulline/blood , Citrulline/immunology , Collagen/administration & dosage , Complement C3 , Mice , Protein-Arginine Deiminase Type 4 , Proteomics , Synovial Membrane/immunology , Synovial Membrane/physiopathologyABSTRACT
To address the poor prognosis of mixed lineage leukemia (MLL)-rearranged infant acute lymphoblastic leukemia (iALL), we generated a panel of cell lines from primary patient samples and investigated cytotoxic responses to contemporary and novel Food and Drug Administration-approved chemotherapeutics. To characterize representation of primary disease within cell lines, molecular features were compared using RNA-sequencing and cytogenetics. High-throughput screening revealed variable efficacy of currently used drugs, however identified consistent efficacy of three novel drug classes: proteasome inhibitors, histone deacetylase inhibitors and cyclin-dependent kinase inhibitors. Gene expression of drug targets was highly reproducible comparing iALL cell lines to matched primary specimens. Histone deacetylase inhibitors, including romidepsin (ROM), enhanced the activity of a key component of iALL therapy, cytarabine (ARAC) in vitro and combined administration of ROM and ARAC to xenografted mice further reduced leukemia burden. Molecular studies showed that ROM reduces expression of cytidine deaminase, an enzyme involved in ARAC deactivation, and enhances the DNA damage-response to ARAC. In conclusion, we present a valuable resource for drug discovery, including the first systematic analysis of transcriptome reproducibility in vitro, and have identified ROM as a promising therapeutic for MLL-rearranged iALL.
Subject(s)
Depsipeptides/pharmacology , Myeloid-Lymphoid Leukemia Protein/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Animals , Antibiotics, Antineoplastic/pharmacology , Cell Line, Tumor/drug effects , Cyclin-Dependent Kinases/antagonists & inhibitors , Gene Rearrangement , Heterografts , Histone Deacetylase Inhibitors/pharmacology , Humans , Infant, Newborn , Mice , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Proteasome Inhibitors/pharmacologyABSTRACT
Small molecule inhibition of the BET family of proteins, which bind acetylated lysines within histones, has been shown to have a marked therapeutic benefit in pre-clinical models of mixed lineage leukemia (MLL) fusion protein-driven leukemias. Here, we report that I-BET151, a highly specific BET family bromodomain inhibitor, leads to growth inhibition in a human erythroleukemic (HEL) cell line as well as in erythroid precursors isolated from polycythemia vera patients. One of the genes most highly downregulated by I-BET151 was LMO2, an important oncogenic regulator of hematopoietic stem cell development and erythropoiesis. We previously reported that LMO2 transcription is dependent upon Janus kinase 2 (JAK2) kinase activity in HEL cells. Here, we show that the transcriptional changes induced by a JAK2 inhibitor (TG101209) and I-BET151 in HEL cells are significantly over-lapping, suggesting a common pathway of action. We generated JAK2 inhibitor resistant HEL cells and showed that these retain sensitivity to I-BET151. These data highlight I-BET151 as a potential alternative treatment against myeloproliferative neoplasms driven by constitutively active JAK2 kinase.
Subject(s)
Hematologic Neoplasms/pathology , Janus Kinase 2/metabolism , Myeloproliferative Disorders/pathology , Oncogene Proteins/antagonists & inhibitors , Cell Line, Tumor , Chromatin Immunoprecipitation , Hematologic Neoplasms/enzymology , Hematologic Neoplasms/metabolism , Humans , Myeloproliferative Disorders/enzymology , Myeloproliferative Disorders/metabolism , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Recent evidence suggests that inhibition of bromodomain and extra-terminal (BET) epigenetic readers may have clinical utility against acute myeloid leukemia (AML). Here we validate this hypothesis, demonstrating the efficacy of the BET inhibitor I-BET151 across a variety of AML subtypes driven by disparate mutations. We demonstrate that a common 'core' transcriptional program, which is HOX gene independent, is downregulated in AML and underlies sensitivity to I-BET treatment. This program is enriched for genes that contain 'super-enhancers', recently described regulatory elements postulated to control key oncogenic driver genes. Moreover, our program can independently classify AML patients into distinct cytogenetic and molecular subgroups, suggesting that it contains biomarkers of sensitivity and response. We focus AML with mutations of the Nucleophosmin gene (NPM1) and show evidence to suggest that wild-type NPM1 has an inhibitory influence on BRD4 that is relieved upon NPM1c mutation and cytosplasmic dislocation. This leads to the upregulation of the core transcriptional program facilitating leukemia development. This program is abrogated by I-BET therapy and by nuclear restoration of NPM1. Finally, we demonstrate the efficacy of I-BET151 in a unique murine model and in primary patient samples of NPM1c AML. Taken together, our data support the use of BET inhibitors in clinical trials in AML.
Subject(s)
Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Transcriptional Activation , Animals , Benzodiazepines/administration & dosage , Benzodiazepines/pharmacology , Cell Cycle Proteins , Cell Line, Tumor , Disease Models, Animal , Drug Evaluation, Preclinical , Gene Expression Profiling , Gene Expression Regulation, Leukemic/drug effects , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/mortality , Mice , Nucleophosmin , Xenograft Model Antitumor AssaysABSTRACT
Therapeutic targeting of the processes that regulate histone modification is a growing area of scientific exploration. Although most interest has concentrated on the various families of enzymes that contribute to these processes, this review focuses on emerging data demonstrating the chemical tractability and therapeutic potential of a hitherto underexplored family of proteins, namely the bromodomain (BRD) family of reader proteins. These proteins perform a crucial role in translating histone modifications with powerful transcriptional consequences. We review current knowledge of the biology of this emergent target class and highlight recent breakthroughs that now make the BRD family of reader proteins attractive for drug discovery.
Subject(s)
Histones/metabolism , Nuclear Proteins/metabolism , Acetylation , Animals , Drug Discovery/methods , Gene Expression , Histones/genetics , Humans , Nuclear Proteins/geneticsABSTRACT
BACKGROUND: PINK1 loss-of-function causes recessive, early-onset parkinsonism. In Tunisia there is a high rate of consanguineous marriage but PINK1 carrier frequency and disease prevalence have yet to be assessed. OBJECTIVES: The frequency of PINK1 mutations in familial parkinsonism, community-based patients with idiopathic Parkinson disease (PD) (non-familial PD), and control subjects was determined. Demographic and clinical characteristics of individuals with PINK1 homozygous or heterozygous variants, or without PINK1 mutations, were compared. METHODS: A total of 92 kindreds (with 208 affected and 340 unaffected subjects), 240 nonfamilial PD, and 368 control participants were recruited from the Institut National de Neurologie, Tunis. Clinical examinations included Hoehn &Yahr, UPDRS, and Epworth scales. PINK1 sequencing and dosage analysis was performed in familial index patients, the variants identified screened in all subjects. Parkin and LRRK2 genes were also examined. RESULTS: Four PINK1 homozygous mutations, three novel (Q129X, Q129fsX157, G440E, and one previously reported; Q456X), segregate with parkinsonism in 46 individuals in 14 of 92 families (15%). Six of 240 patients with nonfamilial PD were found with either homozygous Q456X or Q129X (2.5%) substitutions. In patients with familial disease, PINK1 homozygotes were younger at disease onset (36 +/- 12 years) than noncarriers (57 +/- 15 years) and more often had an akinetic-rigid presentation at examination and slow progression. CONCLUSIONS: Segregation of PINK1 mutations with parkinsonism within families, and frequency estimates within population controls, suggested only four PINK1 mutations were pathogenic. Several PINK1 sequence variants are potentially benign and there was no evidence that PINK1 heterozygosity increases susceptibility to idiopathic Parkinson disease.
Subject(s)
Mutation , Parkinsonian Disorders/genetics , Protein Kinases/genetics , Adult , Aged , Aged, 80 and over , Female , Genetic Predisposition to Disease , Heterozygote , Homozygote , Humans , Lod Score , Male , Middle Aged , Pedigree , TunisiaABSTRACT
Neuronal plasticity plays an important role in physiological and pathological processes within the gastrointestinal (GI) tract. Nogo A is a major contributor to the negative effect central nervous system (CNS) myelin has on neurite outgrowth after injury and may also play a role in maintaining synaptic connections in the healthy CNS. Nogo A is highly expressed during neuronal development but in the CNS declines postnatally concomitantly with a loss of regenerative potential while ganglia of the Peripheral Nervous System (PNS) retain Nogo A. The enteric nervous system shares a number of features in common with the CNS, thus the peripheral distribution of factors affecting plasticity is of interest. We have investigated the distribution of Nogo in the adult mammalian gastrointestinal tract. Nogo A mRNA and protein are detectable in the adult rat GI tract. Nogo A is expressed heterogeneously in enteric neurons throughout the GI tract though expression levels appear not to be correlated with neuronal sub-type. The pattern of expression is maintained in cultured myenteric plexus from the guinea-pig small intestine. As is seen in developing neurons of the CNS, enteric Nogo A is present in both neuronal cell bodies and axons. Our results point to a hitherto unsuspected role for Nogo A in enteric neuronal physiology.
Subject(s)
Enteric Nervous System/metabolism , Myelin Proteins/biosynthesis , Animals , COS Cells , Chlorocebus aethiops , Female , Gene Expression Regulation/physiology , Guinea Pigs , Humans , Male , Mice , Myelin Proteins/genetics , Nogo Proteins , RatsABSTRACT
The expression of Nogo-A mRNA and protein in the nervous system of adult rats and cultured neurons was studied by in situ hybridisation and immunohistochemistry. Nogo-A mRNA was expressed by many cells in unoperated animals, including spinal motor, DRG, and sympathetic neurons, retinal ganglion cells, and neocortical, hippocampal, and Purkinje neurons. Nogo-A protein was strongly expressed by presumptive oligodendrocytes, but not by NG2+glia and was abundant in motor, DRG, and sympathetic neurons, retinal ganglion cells, and many Purkinje cells, but was difficult to detect in dentate gyrus neurons and some neocortical neurons. Cultured fetal mouse neocortical neurons and adult rat DRG neurons strongly expressed Nogo-A in their perikarya, growth cones, and axonal varicosities. All axons in the intact sciatic nerve contained Nogo-A and many but not all regenerating axons were strongly Nogo-A immunopositive after sciatic nerve transection. Ectopic muscle fibres that developed among the regenerating axons were also Nogo-A immunopositive. Following injury to the spinal cord, Nogo-A mRNA was upregulated around the lesion and Nogo-A protein was strongly expressed in injured dorsal column fibres and their sprouts which entered the lesion site. Following optic nerve crush, Nogo-A accumulated in the proximal and distal stumps bordering the lesions.
Subject(s)
Myelin Proteins/biosynthesis , Neurons/metabolism , Optic Nerve Injuries/metabolism , Sciatic Neuropathy/metabolism , Spinal Cord Injuries/metabolism , Animals , Cells, Cultured , Female , Gene Expression Regulation/physiology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred ICR , Myelin Proteins/genetics , Nervous System/metabolism , Nogo Proteins , Optic Nerve Injuries/genetics , Pregnancy , Rats , Rats, Inbred Lew , Rats, Sprague-Dawley , Sciatic Neuropathy/genetics , Spinal Cord Injuries/geneticsABSTRACT
Whole-cell voltage-clamp techniques were used to investigate the capsaicin-, voltage- and time-dependent properties of the rat vanilloid receptor (rVR1) stably expressed in human embryonic kidney (HEK) 293 cells. At a holding potential of -70 mV, application of capsaicin (0.03-30 microM) to HEK 293 cells expressing the rVR1 receptor led to the appearance of inward currents (EC50, 497 nM; Hill coefficient, nH, 2.85) which were reversibly antagonized by 10 microM capsazepine. Current-voltage relationships, determined using depolarizing or hyperpolarizing voltage ramps, had reversal potentials close to 0 mV, exhibited substantial outward rectification and possessed a region of negative slope conductance at holding potentials negative to around -70 mV. Further experiments indicated that the outward rectification and the region of negative slope conductance did not result from external block of the channel by either Ba2+, Ca2+ or Mg2+. During our characterization of rVR1, it became apparent that the rectification behaviour of this receptor was not entirely instantaneous as might be expected for a ligand-gated ion channel, but rather displayed clear time-dependent components. We characterized the kinetics of these novel gating properties in a series of additional voltage-step experiments. The time-dependent changes in rVR1-mediated conductance due to membrane depolarization or repolarization occurred with bi-exponential kinetics. On depolarization to +70 mV the time-dependent increase in outward current developed with mean time constants of 6.7 +/- 0.7 and 51.8 +/- 18.4 ms, with the faster time constant playing a dominant role (64.4 +/- 3.8 %). Similar kinetics also described the decay of 'tail currents' observed on repolarization. Furthermore, these time-dependent changes appeared to be unaffected by the removal of extracellular divalent cations and were not significantly voltage dependent. Our data reveal that rVR1 exhibits substantial time- and voltage-dependent gating properties that may have significance for the physiology of sensory transduction of nociceptive signals.
Subject(s)
Ion Channel Gating/physiology , Neurons/chemistry , Neurons/physiology , Receptors, Drug/genetics , Receptors, Drug/metabolism , Action Potentials/drug effects , Action Potentials/physiology , Animals , Barium/pharmacology , Calcium/pharmacology , Capsaicin/analogs & derivatives , Capsaicin/pharmacology , Cells, Cultured , Ganglia, Spinal/cytology , Humans , Ion Channel Gating/drug effects , Kidney/cytology , Kinetics , Magnesium/pharmacology , Neurons/cytology , Nociceptors/physiology , Patch-Clamp Techniques , Rats , Rats, Inbred Strains , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Time Factors , TransfectionABSTRACT
Neuropilins are receptors for class 3 secreted semaphorins, most of which can function as potent repulsive axon guidance cues. We have generated mice with a targeted deletion in the neuropilin-2 (Npn-2) locus. Many Npn-2 mutant mice are viable into adulthood, allowing us to assess the role of Npn-2 in axon guidance events throughout neural development. Npn-2 is required for the organization and fasciculation of several cranial nerves and spinal nerves. In addition, several major fiber tracts in the brains of adult mutant mice are either severely disorganized or missing. Our results show that Npn-2 is a selective receptor for class 3 semaphorins in vivo and that Npn-1 and Npn-2 are required for development of an overlapping but distinct set of CNS and PNS projections.
Subject(s)
Axons/physiology , Carrier Proteins/metabolism , Glycoproteins/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Age Factors , Animals , Axons/chemistry , Brain Chemistry/physiology , COS Cells , Gene Deletion , Gene Expression Regulation, Developmental , Habenula/chemistry , Habenula/embryology , Habenula/pathology , Mice , Mice, Knockout , Mossy Fibers, Hippocampal/chemistry , Mossy Fibers, Hippocampal/embryology , Mossy Fibers, Hippocampal/pathology , Motor Neurons/chemistry , Motor Neurons/physiology , Motor Neurons/ultrastructure , Neuropilin-1 , Peripheral Nervous System/chemistry , Peripheral Nervous System/embryology , Peripheral Nervous System/pathology , Protein Binding/physiology , Rats , Semaphorin-3A , Spinal Nerves/chemistry , Spinal Nerves/pathology , Spinal Nerves/physiology , Superior Cervical Ganglion/chemistry , Superior Cervical Ganglion/embryology , Superior Cervical Ganglion/pathology , Thalamus/chemistry , Thalamus/embryology , Thalamus/pathology , Trochlear Nerve/chemistry , Trochlear Nerve/embryology , Trochlear Nerve/pathologyABSTRACT
We have used degenerate oligonucleotides, derived from the amino acid sequence of transgelin peptides [Shapland et al., 1993: J. Cell Biol. 121:1065-1073], to isolate and sequence overlapping cDNA clones encoding this actin gelling protein. Primers with 5' restriction enzyme sites directed against the N and C terminal amino acids present in these clones were then used to amplify and clone the entire transgelin coding region from reverse transcribed rat small intestine cDNA (RT-PCR). These studies have shown that transgelin is the product of a single gene which is conserved between yeast, Drosophila, molluscs, and humans. Transgelin is expressed as a single message that is regulated at the level of transcription in SV40 transformed 3T3 cells. Our data have shown that transgelin and several other proteins of unknown function, SM22 alpha [Pearlstone et al., 1987: J. Biol. Chem. 262:5985-5991], mouse p27 [Almendral et al., 1989: Exp. Cell Res. 181:518-530], and human WS3-10 [Thweatt et al., 1992: Biochem. Biophys. Res. Commun. 187:1-7], share extensive homology. More limited regions of homology shared between transgelin and other proteins such as rat NP25 (unpublished), chicken calponins alpha and beta [Takahashi and Nadal-Ginard, 1991: J. Biol. Chem. 266:13284-13288], and Drosophila mp20 [Ayme-Southgate et al., 1989: J. Cell Biol. 108:521-531] suggest that all of these proteins may be classified as members of a new transgelin multigene family.