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1.
PLoS One ; 12(2): e0171221, 2017.
Article in English | MEDLINE | ID: mdl-28178345

ABSTRACT

Activated B-cell-like diffuse large B-cell lymphoma relies on B-cell receptor signaling to drive proliferation and survival. Downstream of the B-cell receptor, the key signaling kinases Bruton's tyrosine kinase and phosphoinositide 3-kinase δ offer opportunities for therapeutic intervention by agents such as ibrutinib, ONO/GS-4059, and idelalisib. Combination therapy with such targeted agents could provide enhanced efficacy due to complimentary mechanisms of action. In this study, we describe both the additive interaction of and resistance mechanisms to idelalisib and ONO/GS-4059 in a model of activated B-cell-like diffuse large B-cell lymphoma. Significant tumor regression was observed with a combination of PI3Kδ and Bruton's tyrosine kinase inhibitors in the mouse TMD8 xenograft. Acquired resistance to idelalisib in the TMD8 cell line occurred by loss of phosphatase and tensin homolog and phosphoinositide 3-kinase pathway upregulation, but not by mutation of PIK3CD. Sensitivity to idelalisib could be restored by combining idelalisib and ONO/GS-4059. Further evaluation of targeted inhibitors revealed that the combination of idelalisib and the phosphoinositide-dependent kinase-1 inhibitor GSK2334470 or the AKT inhibitor MK-2206 could partially overcome resistance. Characterization of acquired Bruton's tyrosine kinase inhibitor resistance revealed a novel tumor necrosis factor alpha induced protein 3 mutation (TNFAIP3 Q143*), which led to a loss of A20 protein, and increased p-IκBα. The combination of idelalisib and ONO/GS-4059 partially restored sensitivity in this resistant line. Additionally, a mutation in Bruton's tyrosine kinase at C481F was identified as a mechanism of resistance. The combination activity observed with idelalisib and ONO/GS-4059, taken together with the ability to overcome resistance, could lead to a new therapeutic option in activated B-cell-like diffuse large B-cell lymphoma. A clinical trial is currently underway to evaluate the combination of idelalisib and ONO/GS-4059 (NCT02457598).


Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm , Imidazoles/pharmacology , Lymphoma, Large B-Cell, Diffuse/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Purines/pharmacology , Pyrimidines/pharmacology , Quinazolinones/pharmacology , 3-Phosphoinositide-Dependent Protein Kinases/metabolism , Agammaglobulinaemia Tyrosine Kinase , Apoptosis/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Humans , Lymphoma, Large B-Cell, Diffuse/drug therapy , Mutation , PTEN Phosphohydrolase/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects
2.
J Pharmacol Exp Ther ; 360(2): 324-340, 2017 Feb.
Article in English | MEDLINE | ID: mdl-27927912

ABSTRACT

(R)-6-[(3-{[4-(5-{[2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl]amino}pent-1-yn-1-yl)phenyl] carbamoyl}phenyl)sulphonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide trifluoroacetic acid (GS-5759) is a bifunctional ligand composed of a quinolinone-containing pharmacophore [ß2-adrenoceptor agonist orthostere (ß2A)] found in several ß2-adrenoceptor agonists, including indacaterol, linked covalently to a phosphodiesterase 4 (PDE4) inhibitor related to 6-[3-(dimethylcarbamoyl)benzenesulphonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GSK 256066) by a pent-1-yn-1-ylbenzene spacer. GS-5759 had a similar affinity for PDE4B1 and the native ß2-adrenoceptor expressed on BEAS-2B human airway epithelial cells. However, compared with the monofunctional parent compound, ß2A, the KA of GS-5759 for the ß2-adrenoceptor was 35-fold lower. Schild analysis determined that the affinities of the ß-adrenoceptor antagonists, (2R,3R)-1-[(2,3-dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl) amino]-2-butanol (ICI 118551) and propranolol, were agonist-dependent, being significantly lower for GS-5759 than ß2A. Collectively, these data can be explained by "forced proximity," bivalent binding where the pharmacophore in GS-5759 responsible for PDE4 inhibition also interacts with a nonallosteric domain within the ß2-adrenoceptor that enhances the affinity of ß2A for the orthosteric site. Microarray analyses revealed that, after 2-hour exposure, GS-5759 increased the expression of >3500 genes in BEAS-2B cells that were highly rank-order correlated with gene expression changes produced by indacaterol and GSK 256066 in combination (Ind/GSK). Moreover, the line of regression began close to the origin with a slope of 0.88, indicating that the magnitude of most gene expression changes produced by Ind/GSK was quantitatively replicated by GS-5759. Thus, GS-5759 is a novel compound exhibiting dual ß2-adrenoceptor agonism and PDE4 inhibition with potential to interact on target tissues in a synergistic manner. Such polypharmacological behavior may be particularly effective in chronic obstructive pulmonary disease and other complex disorders where multiple processes interact to promote disease pathogenesis and progression.


Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Epithelial Cells/drug effects , Gene Expression Regulation/drug effects , Pulmonary Disease, Chronic Obstructive/genetics , Quinolones/pharmacology , Receptors, Adrenergic, beta-2/metabolism , Respiratory System/pathology , Sulfones/pharmacology , Adrenergic beta-2 Receptor Agonists/pharmacology , Adrenergic beta-2 Receptor Agonists/therapeutic use , Aminoquinolines/pharmacology , Cell Line , Drug Interactions , Epithelial Cells/metabolism , Humans , Indans/pharmacology , Phosphodiesterase 4 Inhibitors/pharmacology , Phosphodiesterase 4 Inhibitors/therapeutic use , Pulmonary Disease, Chronic Obstructive/drug therapy , Pulmonary Disease, Chronic Obstructive/pathology , Quinolones/therapeutic use , Sulfones/therapeutic use
3.
Nat Genet ; 48(1): 59-66, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26618343

ABSTRACT

Gliomas represent approximately 30% of all central nervous system tumors and 80% of malignant brain tumors. To understand the molecular mechanisms underlying the malignant progression of low-grade gliomas with mutations in IDH1 (encoding isocitrate dehydrogenase 1), we studied paired tumor samples from 41 patients, comparing higher-grade, progressed samples to their lower-grade counterparts. Integrated genomic analyses, including whole-exome sequencing and copy number, gene expression and DNA methylation profiling, demonstrated nonlinear clonal expansion of the original tumors and identified oncogenic pathways driving progression. These include activation of the MYC and RTK-RAS-PI3K pathways and upregulation of the FOXM1- and E2F2-mediated cell cycle transitions, as well as epigenetic silencing of developmental transcription factor genes bound by Polycomb repressive complex 2 in human embryonic stem cells. Our results not only provide mechanistic insight into the genetic and epigenetic mechanisms driving glioma progression but also identify inhibition of the bromodomain and extraterminal (BET) family as a potential therapeutic approach.


Subject(s)
Central Nervous System Neoplasms/genetics , Glioma/genetics , Isocitrate Dehydrogenase/genetics , Mutation , Central Nervous System Neoplasms/pathology , DNA Methylation , Embryonic Stem Cells/metabolism , Forkhead Box Protein M1 , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Dosage , Gene Expression Regulation, Neoplastic , Genes, myc , Glioma/pathology , Humans , Isocitrate Dehydrogenase/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Receptor, Notch1/genetics , Receptor, Notch1/metabolism
4.
Pharmacol Res Perspect ; 2(4): e00046, 2014 Aug.
Article in English | MEDLINE | ID: mdl-25505595

ABSTRACT

Bronchodilators are a central therapy for symptom relief in respiratory diseases such as chronic obstructive pulmonary disease (COPD) and asthma, with inhaled ß 2-adrenoceptor agonists and anticholinergics being the primary treatments available. The present studies evaluated the in vivo pharmacology of (R)-6-[[3-[[4-[5-[[2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl]amino]pent-1-ynyl]phenyl]carbamoyl]phenyl]sulfonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GS-5759), a novel bifunctional compound with both phosphodiesterase 4 (PDE4) inhibitor and long-acting ß 2-adrenoceptor agonist (LABA) activity, which has been optimized for inhalation delivery. GS-5759 dose-dependently inhibited pulmonary neutrophilia in a lipopolysaccharide (LPS) aerosol challenge model of inflammation in rats with an ED50 ≤ 10 µg/kg. GS-5759 was also a potent bronchodilator with an ED50 of 0.09 µg/kg in guinea pigs and 3.4 µg/kg in dogs after methylcholine (MCh) and ragweed challenges respectively. In cynomolgus monkeys, GS-5759 was dosed as a fine-particle dry powder and was efficacious in the same dose range in both MCh and LPS challenge models, with an ED50 = 70 µg/kg for bronchodilation and ED50 = 4.9 µg/kg for inhibition of LPS-induced pulmonary neutrophilia. In models to determine therapeutic index (T.I.), efficacy for bronchodilation was evaluated against increased heart rate and GS-5759 had a T.I. of 700 in guinea pigs and >31 in dogs. In a ferret model of emesis, no emesis was seen at doses several orders of magnitude greater than the ED50 observed in the rat LPS inflammation model. GS-5759 is a bifunctional molecule developed for the treatment of COPD, which has both bronchodilator and anti-inflammatory activity and has the potential for combination as a triple therapy with a second compound, within a single inhalation device.

5.
J Pharmacol Exp Ther ; 349(1): 85-93, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24513870

ABSTRACT

Inhaled long-acting ß(2)-adrenoceptor agonists (LABA) that act as bronchodilators and the oral anti-inflammatory phosphodiesterase 4 (PDE4) inhibitor roflumilast are both approved therapies for chronic obstructive pulmonary disease (COPD). Here we describe the activity of a novel, inhaled, bifunctional, small molecule (R)-6-[(3-{[4-(5-{[2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl]amino}pent-1-yn-1-yl)phenyl]carbamoyl}phenyl)sulfonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GS-5759), which has specific ß(2) agonist and PDE4 inhibitory activity. GS-5759 demonstrated potent and full agonist activity at ß(2) adrenoceptors (EC(50) = 8 ± 4 nM) and is a potent inhibitor of the PDE4 enzyme (IC(50) = 5 ± 3 nM). In cell assays, GS-5759 inhibited lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) production in human peripheral mononuclear cells (PBMC) with an IC(50) = 0.3 nM [confidence interval (CI) 0.1-0.6] and in human neutrophils formyl-methionyl-leucyl-phenylalanine (fMLP)-induced super oxide anion production with an IC(50) = 3 nM (CI 0.8-8). The addition of the ß(2) antagonist ICI 118551 shifted the IC(50) in these cell assays to 4 and 38 nM, respectively, demonstrating the contribution of both ß(2) agonist and PDE4 inhibitory activity to GS-5759. GS-5759 was also a potent inhibitor of profibrotic and proinflammatory mediator release from human lung fibroblasts. GS-5759 relaxed guinea pig airway smooth muscle strips precontracted with carbachol in a concentration-dependent manner with an EC(50) = 0.5 µM (CI 0.2-2) and had slow dissociation kinetics with an Off T(1/2) > 720 minutes at an EC(80) concentration of 3 µM. GS-5759 is a novel bifunctional molecule with both potent ß(2) agonist and PDE4 inhibitor activity that could provide inhaled bronchodilator and anti-inflammatory therapy for COPD.


Subject(s)
Adrenergic beta-2 Receptor Agonists/pharmacology , Fibroblasts/drug effects , Leukocytes, Mononuclear/drug effects , Muscle, Smooth/drug effects , Phosphodiesterase 4 Inhibitors/pharmacology , Quinolones/pharmacology , Respiratory System/drug effects , Sulfones/pharmacology , Adrenergic beta-2 Receptor Agonists/chemical synthesis , Adrenergic beta-2 Receptor Agonists/chemistry , Animals , Cell Culture Techniques , Cytokines/antagonists & inhibitors , Cytokines/immunology , Fibroblasts/enzymology , Fibroblasts/immunology , Fibroblasts/metabolism , Guinea Pigs , Humans , Leukocytes, Mononuclear/enzymology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides/pharmacology , Male , Molecular Structure , Muscle, Smooth/enzymology , Muscle, Smooth/immunology , Muscle, Smooth/metabolism , Phosphodiesterase 4 Inhibitors/chemical synthesis , Phosphodiesterase 4 Inhibitors/chemistry , Pulmonary Disease, Chronic Obstructive/drug therapy , Quinolones/chemical synthesis , Quinolones/chemistry , Respiratory System/enzymology , Respiratory System/immunology , Respiratory System/metabolism , Sulfones/chemical synthesis , Sulfones/chemistry , Time Factors
6.
Respir Res ; 13: 28, 2012 Mar 27.
Article in English | MEDLINE | ID: mdl-22452977

ABSTRACT

BACKGROUND: Small airway narrowing is an important pathology which impacts lung function in chronic obstructive pulmonary disease (COPD). The accumulation of fibroblasts and myofibroblasts contribute to inflammation, remodeling and fibrosis by production and release of mediators such as cytokines, profibrotic factors and extracellular matrix proteins. This study investigated the effects of the phosphodiesterase 4 inhibitor roflumilast, combined with the long acting ß2 adrenergic agonist indacaterol, both approved therapeutics for COPD, on fibroblast functions that contribute to inflammation and airway fibrosis. METHODS: The effects of roflumilast and indacaterol treatment were characterized on transforming growth factor ß1 (TGFß1)-treated normal human lung fibroblasts (NHLF). NHLF were evaluated for expression of the profibrotic mediators endothelin-1 (ET-1) and connective tissue growth factor (CTGF), expression of the myofibroblast marker alpha smooth muscle actin, and fibronectin (FN) secretion. Tumor necrosis factor-α (TNF-α) was used to induce secretion of chemokine C-X-C motif ligand 10 (CXCL10), chemokine C-C motif ligand 5 (CCL5) and granulocyte macrophage colony-stimulating factor (GM-CSF) from NHLF and drug inhibition was assessed. RESULTS: Evaluation of roflumilast (1-10 µM) showed no significant inhibition alone on TGFß1-induced ET-1 and CTGF mRNA transcripts, ET-1 and FN protein production, alpha smooth muscle expression, or TNF-α-induced secretion of CXCL10, CCL5 and GM-CSF. A concentration-dependent inhibition of ET-1 and CTGF was shown with indacaterol treatment, and a submaximal concentration was chosen for combination studies. When indacaterol (0.1 nM) was added to roflumilast, significant inhibition was seen on all inflammatory and fibrotic mediators evaluated, which was superior to the inhibition seen with either drug alone. Roflumilast plus indacaterol combination treatment resulted in significantly elevated phosphorylation of the transcription factor cAMP response element-binding protein (CREB), an effect that was protein kinase A-dependent. Inhibition of protein kinase A was also found to reverse the inhibition of indacaterol and roflumilast on CTGF. CONCLUSIONS: These results demonstrate that addition of roflumilast to a LABA inhibits primary fibroblast/myofibroblast function and therapeutically this may impact lung fibroblast proinflammatory and profibrotic mediator release which contributes to small airway remodeling and airway obstruction in COPD.


Subject(s)
Adrenergic beta-2 Receptor Agonists/pharmacology , Aminopyridines/pharmacology , Benzamides/pharmacology , Fibroblasts/metabolism , Indans/pharmacology , Inflammation/prevention & control , Lung/metabolism , Phosphodiesterase 4 Inhibitors/pharmacology , Pulmonary Fibrosis/prevention & control , Quinolones/pharmacology , Actins/metabolism , Cells, Cultured , Chemokine CCL5/metabolism , Chemokine CXCL10/metabolism , Connective Tissue Growth Factor/metabolism , Cyclopropanes/pharmacology , Endothelin-1/metabolism , Fibroblasts/cytology , Fibroblasts/drug effects , Fibronectins/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , Inflammation/metabolism , Lung/cytology , Lung/drug effects , Pulmonary Fibrosis/metabolism , Transforming Growth Factor beta1/pharmacology , Tumor Necrosis Factor-alpha/pharmacology
7.
Pulm Pharmacol Ther ; 25(2): 178-84, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22306235

ABSTRACT

The phosphodiesterase 4 inhibitor (PDE4i) roflumilast has been approved in the US and EU for treatment of GOLD stage 3 and 4 chronic obstructive pulmonary disease (COPD). Inhaled ß2 adrenoceptor agonist bronchodilators and anti-inflammatory glucocorticosteroids are also used as standard of care in COPD. We investigated the anti-inflammatory interaction of roflumilast in combination with long-acting ß2 agonists (LABA), salmeterol or formoterol, or a glucocorticosteroid, dexamethasone, on cytokine production from LPS-stimulated human primary peripheral blood mononuclear cells (PBMC). Salmeterol or formoterol caused a concentration-dependent inhibition of tumor necrosis factor-α (TNFα) secretion with an IC50 of 0.33 pM (C.I. 0.006-19) and 34 pM (C.I. 13-87), respectively. When roflumilast was evaluated, the addition of salmeterol (1 nM) to roflumilast caused the IC50 for roflumilast to shift from 1.8 nM (C.I. 0.8-4) to 4.1 pM (C.I.0.3-69) (p < 0.01), and maximal inhibition increased from 72.5 ± 3.2% to 90.9 ± 3.1%. Addition of formoterol to roflumilast also produced an increased TNFα inhibition more than either drug alone (p < 0.05). The inhibition of TNFα production with salmeterol was both ß2 adrenoceptor- and protein kinase A-dependent. Addition of roflumilast (10 nM) in the presence of dexamethasone increased the inhibition of LPS-induced TNFα and CCL3. Roflumilast in combination with salmeterol, formoterol, or dexamethasone increased the inhibition of LPS-induced TNFα from human PBMC, in an additive manner. Addition of roflumilast to either a ß2 adrenoceptor agonist or a glucocorticosteroid may provide superior anti-inflammatory activity and greater efficacy in COPD patients and be dose sparing.


Subject(s)
Adrenergic beta-2 Receptor Agonists/pharmacology , Aminopyridines/pharmacology , Benzamides/pharmacology , Dexamethasone/pharmacology , Inflammation/drug therapy , Adrenergic beta-2 Receptor Agonists/administration & dosage , Albuterol/administration & dosage , Albuterol/analogs & derivatives , Albuterol/pharmacology , Aminopyridines/administration & dosage , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/pharmacology , Benzamides/administration & dosage , Cyclopropanes/administration & dosage , Cyclopropanes/pharmacology , Dexamethasone/administration & dosage , Dose-Response Relationship, Drug , Drug Therapy, Combination , Ethanolamines/administration & dosage , Ethanolamines/pharmacology , Formoterol Fumarate , Glucocorticoids/administration & dosage , Glucocorticoids/pharmacology , Humans , Inflammation/physiopathology , Inhibitory Concentration 50 , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides , Male , Phosphodiesterase 4 Inhibitors/administration & dosage , Phosphodiesterase 4 Inhibitors/pharmacology , Salmeterol Xinafoate , Tumor Necrosis Factor-alpha/metabolism
8.
Mol Cancer Res ; 2(11): 643-52, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15561780

ABSTRACT

A breast cancer cell line developed in our laboratory (SUM-52PE) has a 12-fold amplification and high-level overexpression of the oncogene fibroblast growth factor receptor 2 (FGFR2). Previously, nine different alternatively spliced FGFR2 variants were isolated from this cell line. Overexpression of two variants that differ only in their carboxyl termini (C1 and C3) has been successfully accomplished in the immortalized human mammary epithelial cell line H16N2. FGFR2 expression led to the activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling cascades. Phosphorylation of the adapter protein FGF receptor substrate 2 is much more robust in the cells expressing the C3 variant of FGFR2 compared with the C1 variant. H16N2 cells expressing the full-length FGFR2 with the C1 or C3 carboxyl terminus were tested for their ability to grow under epidermal growth factor (EGF)-independent conditions, in soft agar, and for their ability to invade naturally occurring basement membranes and compared with the parental SUM-52PE cell line. All three cell lines grew under EGF-independent conditions and all were inhibited by the FGFR family specific inhibitor PD173074. The full-length FGFR2-C1 and FGFR2-C3 variants grew robustly in soft agar similar to the parental cell line SUM-52PE. However, cells expressing the C3 variant formed large colonies in agar in both insulin-free and EGF-free medium, whereas the cells expressing the C1 variant required insulin for growth. Soft agar growth was also inhibited by PD173074. Because SUM-52PE was developed from a metastatic breast carcinoma, the FGFR2-overexpressing cell lines were assessed for their ability to invade sea urchin embryo cell membranes. H16N2 cells expressing the C1 carboxyl terminus failed to invade sea urchin embryo cell membranes. By contrast, FGFR2-C3-expressing cells were as invasive as the SUM-52 breast cancer cells and erbB-2-overexpressing H16N2 cells. These results indicate that FGFR2 is a transforming oncogene in human mammary epithelial cells when expressed to levels similar to that found in breast cancer cells with FGFR2 gene amplification. Furthermore, the results suggest that different splice variants have differing transforming activities and that signaling from variants expressing the C3 carboxyl terminus results in more autonomous signaling, cell growth, and invasion.


Subject(s)
Alternative Splicing , Breast Neoplasms/genetics , Breast Neoplasms/physiopathology , Cell Transformation, Neoplastic/genetics , Epithelial Cells/physiology , Mammary Glands, Human/cytology , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Fibroblast Growth Factor/genetics , Breast Neoplasms/pathology , Cell Adhesion/physiology , Cell Division/physiology , Cell Line, Transformed , Cells, Cultured , Epithelial Cells/cytology , Gene Expression Regulation, Neoplastic , Humans , Receptor, Fibroblast Growth Factor, Type 2 , Signal Transduction/physiology
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