RESUMEN
Microenvironment contributes to follicular lymphoma (FL) pathogenesis and impacts survival with macrophages playing a controversial role. In the present study, using FL primary samples and HK follicular dendritic cells (FDC) to mimic the germinal center, together with mouse models, we have analyzed the three-way crosstalk of FL-FDC-macrophages and derived therapeutic opportunities. Ex vivo primary FL-FDC co-cultures (n = 19) and in vivo mouse co-xenografts demonstrated that FL-FDC crosstalk favors tumor growth and, via the secretion of CCL2 and CSF-1, promotes monocyte recruitment, differentiation, and polarization towards an M2-like protumoral phenotype. Moreover, FL-M2 co-cultures displayed enhanced angiogenesis, dissemination, and immunosuppression. Analysis of the CSF-1/CSF-1R pathway uncovered that CSF-1 was significantly higher in serum from grade 3A FL patients, and that high CSF-1R expression in FL biopsies correlated with grade 3A, reduced overall survival and risk of transformation. Furthermore, CSF-1R inhibition with pexidartinib (PLX3397) preferentially affected M2-macrophage viability and polarization program disrupting FL-M2 positive crosstalk. In vivo CSF1-R inhibition caused M2 reduction and repolarization towards M1 macrophages and antitumor effect cooperating with anti-CD20 rituximab. In summary, these results support the role of macrophages in FL pathogenesis and indicate that CSF-1R may be a relevant prognostic factor and a novel therapeutic target cooperating with anti-CD20 immunotherapy.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Regulación Neoplásica de la Expresión Génica , Linfoma Folicular/patología , Macrófagos/patología , Monocitos/patología , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Microambiente Tumoral , Aminopiridinas/farmacología , Animales , Apoptosis , Biomarcadores de Tumor/genética , Diferenciación Celular , Proliferación Celular , Humanos , Linfoma Folicular/tratamiento farmacológico , Linfoma Folicular/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Fosforilación , Pirroles/farmacología , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Survival of infants with KMT2A-rearranged (R) acute lymphoblastic leukemia (ALL) remains dismal despite intensive chemotherapy. We observed constitutive phosphorylation of spleen tyrosine kinase (SYK) and associated signaling proteins in infant ALL patient-derived xenograft (PDX) model specimens and hypothesized that the SYK inhibitor entospletinib would inhibit signaling and cell growth in vitro and leukemia proliferation in vivo. We further predicted that combined entospletinib and chemotherapy could augment anti-leukemia effects. Basal kinase signaling activation and HOXA9/MEIS1 expression differed among KMT2A-R (KMT2A-AFF1 [n=4], KMT2A-MLLT3 [n=1], KMT2A-MLLT1 [n=4]) and non-KMT2A-R [n=3] ALL specimens and stratified by genetic subgroup. Incubation of KMT2A-R ALL cells in vitro with entospletinib inhibited methylcellulose colony formation and SYK pathway signaling in a dose-dependent manner. In vivo inhibition of leukemia proliferation with entospletinib monotherapy was observed in RAS-wild-type KMT2A-AFF1, KMT2A-MLLT3, and KMT2A-MLLT1 ALL PDX models with enhanced activity in combination with vincristine chemotherapy in several models. Surprisingly, entospletinib did not decrease leukemia burden in two KMT2A-AFF1 PDX models with NRAS/ or KRAS mutations, suggesting potential RAS-mediated resistance to SYK inhibition. As hypothesized, superior inhibition of ALL proliferation was observed in KMT2A-AFF1 PDX models treated with entospletinib and the MEK inhibitor selumetinib versus vehicle or inhibitor monotherapies (p.
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Proteína de la Leucemia Mieloide-Linfoide , Leucemia-Linfoma Linfoblástico de Células Precursoras , Animales , Xenoinjertos , Humanos , Indazoles , Lactante , Proteína de la Leucemia Mieloide-Linfoide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , PirazinasRESUMEN
Despite idelalisib approval in relapsed follicular lymphoma (FL), a complete characterization of the immunomodulatory consequences of phosphatidylinositol 3-kinase δ (PI3Kδ) inhibition, biomarkers of response, and potential combinatorial therapies in FL remain to be established. Using ex vivo cocultures of FL patient biopsies and follicular dendritic cells (FDCs) to mimic the germinal center (n = 42), we uncovered that PI3Kδ inhibition interferes with FDC-induced genes related to angiogenesis, extracellular matrix formation, and transendothelial migration in a subset of FL samples, defining an 18-gene signature fingerprint of idelalisib sensitivity. A common hallmark of idelalisib found in all FL cases was its interference with the CD40/CD40L pathway and induced proliferation, together with the downregulation of proteins crucial for B-T-cell synapses, leading to an inefficient cross talk between FL cells and the supportive T-follicular helper cells (TFH). Moreover, idelalisib downmodulates the chemokine CCL22, hampering the recruitment of TFH and immunosupressive T-regulatory cells to the FL niche, leading to a less supportive and tolerogenic immune microenvironment. Finally, using BH3 profiling, we uncovered that FL-FDC and FL-macrophage cocultures augment tumor addiction to BCL-XL and MCL-1 or BFL-1, respectively, limiting the cytotoxic activity of the BCL-2 inhibitor venetoclax. Idelalisib restored FL dependence on BCL-2 and venetoclax activity. In summary, idelalisib exhibits a patient-dependent activity toward angiogenesis and lymphoma dissemination. In all FL cases, idelalisib exerts a general reshaping of the FL immune microenvironment and restores dependence on BCL-2, predisposing FL to cell death, providing a mechanistic rationale for investigating the combination of PI3Kδ inhibitors and venetoclax in clinical trials.
Asunto(s)
Antineoplásicos , Linfoma Folicular , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Humanos , Linfoma Folicular/tratamiento farmacológico , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Microambiente TumoralRESUMEN
Despite the emergence of small molecule inhibitors, current treatment strategies for chronic lymphocytic leukemia (CLL) are not curative, and the search for new therapeutic modalities continues. Prosurvival signaling derived from the microenvironment is often mediated via JAK signaling. However, whether JAK inhibitors are useful in CLL therapy has not been studied extensively. JAK inhibitors are valuable therapeutic agents in myelofibrosis and show promising results in graft-versus-host-disease. However, JAK inhibition is associated with an increased infection risk, presumably because of the effect on other immune cells, a feature shared with other kinase inhibitors used for CLL treatment, such as the BTK inhibitor ibrutinib and the PI3Kδ inhibitor idelalisib. We compared functional effects of the JAK1/2 inhibitors momelotinib and ruxolitinib, the BTK inhibitors ibrutinib and tirabrutinib, and PI3Kδ inhibitor idelalisib on malignant CLL cells but also on healthy human T, B, and NK lymphocytes. We found several interesting differences among the inhibitors, apart from expected and well-known effects. Momelotinib but not ruxolitinib blocked cytokine-induced proliferation of CLL cells. Momelotinib also reduced BCR signaling, in contrast to ruxolitinib, indicating that these JAK inhibitors in fact have a distinct target spectrum. In contrast to tirabrutinib, ibrutinib had inhibitory effects on T cell activation, probably because of ITK inhibition. Remarkably, both BTK inhibitors stimulated IFN-γ production in a mixed lymphocyte reaction. Collectively, our results demonstrate that kinase inhibitors directed at identical targets may have differential effects on lymphocyte function. Their unique profile could be strategically employed to balance desired versus unwanted lymphocyte inhibition.
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Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Antineoplásicos/farmacología , Quinasas Janus/antagonistas & inhibidores , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Linfocitos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Adenina/análogos & derivados , Agammaglobulinemia Tirosina Quinasa/metabolismo , Animales , Benzamidas/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Imidazoles/farmacología , Quinasas Janus/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Linfocitos/metabolismo , Linfocitos/patología , Ratones , Células 3T3 NIH , Nitrilos , Piperidinas , Purinas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Quinazolinonas/farmacologíaRESUMEN
Targeted therapy is revolutionizing the treatment of cancers, but resistance evolves against these therapies and derogates their success. The phosphatidylinositol 3-kinase delta (PI3K-δ) inhibitor idelalisib has been approved for treatment of chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma, but the mechanisms conferring resistance in a subset of patients are unknown. Here, we modeled resistance to PI3K-δ inhibitor in vivo using a serial tumor transfer and treatment scheme in mice. Whole-exome sequencing did not identify any recurrent mutation explaining resistance to PI3K-δ inhibitor. In the murine model, resistance to PI3K-δ inhibitor occurred as a result of a signaling switch mediated by consistent and functionally relevant activation of insulin-like growth factor 1 receptor (IGF1R), resulting in enhanced MAPK signaling in the resistant tumors. Overexpression of IGF1R in vitro demonstrated its prominent role in PI3K-δ inhibitor resistance. IGF1R upregulation in PI3K-δ inhibitor-resistant tumors was mediated by functional activation and enhanced nuclear localization of forkhead box protein O1 transcription factors and glycogen synthase kinase 3ß. In human CLL, high IGF1R expression was associated with trisomy 12. CLL cells from an idelalisib-treated patient showed decreased sensitivity to idelalisib in vitro concomitant with enhanced MAPK signaling and strong upregulation of IGF1R upon idelalisib exposure. Thus, our results highlight that alternative signaling cascades play a predominant role in the resistance and survival of cancer cells under PI3K-δ inhibition. We also demonstrate that these pathway alterations can serve as therapeutic targets, because inhibition of IGF1R offered efficacious salvage treatment of PI3K-δ inhibitor-resistant tumors in vitro and in vivo.
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Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Receptor IGF Tipo 1/metabolismo , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/mortalidad , Leucemia Linfocítica Crónica de Células B/patología , Ratones , Mutación , Receptor IGF Tipo 1/genética , Resultado del Tratamiento , Secuenciación del Exoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chronic graft-versus-host disease (cGVHD) is a leading cause of morbidity and mortality following allotransplant. Activated donor effector T cells can differentiate into pathogenic T helper (Th)-17 cells and germinal center (GC)-promoting T follicular helper (Tfh) cells, resulting in cGVHD. Phosphoinositide-3-kinase-δ (PI3Kδ), a lipid kinase, is critical for activated T cell survival, proliferation, differentiation, and metabolism. We demonstrate PI3Kδ activity in donor T cells that become Tfh cells is required for cGVHD in a nonsclerodermatous multiorgan system disease model that includes bronchiolitis obliterans (BO), dependent upon GC B cells, Tfhs, and counterbalanced by T follicular regulatory cells, each requiring PI3Kδ signaling for function and survival. Although B cells rely on PI3Kδ pathway signaling and GC formation is disrupted resulting in a substantial decrease in Ig production, PI3Kδ kinase-dead mutant donor bone marrow-derived GC B cells still supported BO cGVHD generation. A PI3Kδ-specific inhibitor, compound GS-649443, that has superior potency to idelalisib while maintaining selectivity, reduced cGVHD in mice with active disease. In a Th1-dependent and Th17-associated scleroderma model, GS-649443 effectively treated mice with active cGVHD. These data provide a foundation for clinical trials of US Food and Drug Administration (FDA)-approved PI3Kδ inhibitors for cGVHD therapy in patients.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/enzimología , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Animales , Linfocitos B/inmunología , Trasplante de Médula Ósea/efectos adversos , Bronquiolitis Obliterante/tratamiento farmacológico , Bronquiolitis Obliterante/enzimología , Bronquiolitis Obliterante/etiología , Enfermedad Crónica , Fosfatidilinositol 3-Quinasa Clase I/deficiencia , Fosfatidilinositol 3-Quinasa Clase I/genética , Modelos Animales de Enfermedad , Enfermedad Injerto contra Huésped/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Esclerodermia Localizada/tratamiento farmacológico , Esclerodermia Localizada/enzimología , Esclerodermia Localizada/etiología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Acute lymphoblastic leukaemia (ALL) has a marked propensity to metastasize to the central nervous system (CNS). In contrast to brain metastases from solid tumours, metastases of ALL seldom involve the parenchyma but are isolated to the leptomeninges, which is an infrequent site for carcinomatous invasion. Although metastasis to the CNS occurs across all subtypes of ALL, a unifying mechanism for invasion has not yet been determined. Here we show that ALL cells in the circulation are unable to breach the blood-brain barrier in mice; instead, they migrate into the CNS along vessels that pass directly between vertebral or calvarial bone marrow and the subarachnoid space. The basement membrane of these bridging vessels is enriched in laminin, which is known to coordinate pathfinding of neuronal progenitor cells in the CNS. The laminin receptor α6 integrin is expressed in most cases of ALL. We found that α6 integrin-laminin interactions mediated the migration of ALL cells towards the cerebrospinal fluid in vitro. Mice with ALL xenografts were treated with either a PI3Kδ inhibitor, which decreased α6 integrin expression on ALL cells, or specific α6 integrin-neutralizing antibodies and showed significant reductions in ALL transit along bridging vessels, blast counts in the cerebrospinal fluid and CNS disease symptoms despite minimally decreased bone marrow disease burden. Our data suggest that α6 integrin expression, which is common in ALL, allows cells to use neural migratory pathways to invade the CNS.
Asunto(s)
Sistema Nervioso Central/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Animales , Anticuerpos Neutralizantes/inmunología , Membrana Basal/metabolismo , Barrera Hematoencefálica/metabolismo , Médula Ósea , Movimiento Celular , Sistema Nervioso Central/irrigación sanguínea , Sistema Nervioso Central/metabolismo , Líquido Cefalorraquídeo/metabolismo , Circulación Cerebrovascular , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Progresión de la Enfermedad , Femenino , Xenoinjertos/inmunología , Xenoinjertos/patología , Integrina alfa6/inmunología , Integrina alfa6/metabolismo , Laminina/metabolismo , Masculino , Ratones , Ratones SCID , Trasplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores de Laminina/antagonistas & inhibidores , Receptores de Laminina/inmunología , Receptores de Laminina/metabolismo , Cráneo , Espacio SubaracnoideoRESUMEN
PI3Kδ (idelalisib) and BTK (ibrutinib) inhibitors have demonstrated significant clinical activity in chronic lymphocytic leukemia (CLL) interfering with the cross-talk between CLL cells and the lymph node microenviroment, yet their mechanism of action remains to be fully elucidated. Here, we developed an ex vivo model with the aim of reproducing the effects of the microenvironment that would help shed light on the in vivo mechanism of action of idelalisib and ibrutinib and predict their clinical efficacy in individual patients. First we explored the effects of various cell-extrinsic elements on CLL apoptosis and proliferation and found that the combination of CpG+IL2+HS5 stromal cell line + human serum +CLL plasma and erythrocyte fractions represented the best co-culture conditions to test the effects of the novel inhibitors. Then, using this assay, we investigated the impact of idelalisib and ibrutinib on both survival and proliferation in 30 CLL patients. While both drugs had a limited direct pro-apoptotic activity, a potent inhibition of proliferation was achieved at clinically achievable concentrations. Notably, up to 10% of CLL cells still proliferated even at the highest concentrations, likely mirroring the known difficulty to achieve complete responses in vivo. Altogether, this novel assay represents an appropriate ex vivo drug testing system to potentially predict the clinical response to novel inhibitors in particular by quantifying the antiproliferative effect.
RESUMEN
Idelalisib is a highly selective oral inhibitor of PI3Kδ indicated for the treatment of patients with relapsed chronic lymphocytic leukemia in combination with rituximab. Despite additive clinical effects, previous studies have paradoxically demonstrated that targeted therapies potentially negatively affect anti-CD20 mAb effector mechanisms. To address these potential effects, we investigated the impact of PI3Kδ inhibition by idelalisib on the effector mechanisms of rituximab and obinutuzumab. At clinically relevant concentrations, idelalisib minimally influenced rituximab- and obinutuzumab-mediated Ab-dependent cellular cytotoxicity and phagocytosis on human lymphoma cell lines, while maintaining the superiority of obinutuzumab-mediated Ab-dependent cellular cytotoxicity. Consistent with this, idelalisib did not influence obinutuzumab-mediated B cell depletion in whole-blood B cell-depletion assays. Further, idelalisib significantly enhanced obinutuzumab-mediated direct cell death of chronic lymphocytic leukemia cells. In murine systems, in vivo inhibition of PI3Kδ minimally interfered with maximal rituximab- or obinutuzumab-mediated depletion of leukemic targets. In addition, the duration of rituximab- and obinutuzumab-mediated depletion of leukemia cells was extended by combination with PI3Kδ inhibition. Collectively, these data demonstrate that PI3Kδ inhibition does not significantly affect the effector mechanisms induced by rituximab or obinutuzumab and provides an effective in vivo therapeutic combination. Therefore, combinations of obinutuzumab and idelalisib are currently being assessed in clinical studies.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Antineoplásicos/farmacología , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Fagocitosis/efectos de los fármacos , Purinas/farmacología , Quinazolinonas/farmacología , Rituximab/farmacología , Animales , Línea Celular Tumoral , Interacciones Farmacológicas , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Linfoma/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Ratones TransgénicosRESUMEN
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).
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Imidazoles/farmacología , Linfoma de Células B Grandes Difuso/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Purinas/farmacología , Pirimidinas/farmacología , Quinazolinonas/farmacología , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/metabolismo , Agammaglobulinemia Tirosina Quinasa , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Mutación , Fosfohidrolasa PTEN/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
(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.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Enfermedad Pulmonar Obstructiva Crónica/genética , Quinolonas/farmacología , Receptores Adrenérgicos beta 2/metabolismo , Sistema Respiratorio/patología , Sulfonas/farmacología , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Agonistas de Receptores Adrenérgicos beta 2/uso terapéutico , Aminoquinolinas/farmacología , Línea Celular , Interacciones Farmacológicas , Células Epiteliales/metabolismo , Humanos , Indanos/farmacología , Inhibidores de Fosfodiesterasa 4/farmacología , Inhibidores de Fosfodiesterasa 4/uso terapéutico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/patología , Quinolonas/uso terapéutico , Sulfonas/uso terapéuticoRESUMEN
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.
Asunto(s)
Neoplasias del Sistema Nervioso Central/genética , Glioma/genética , Isocitrato Deshidrogenasa/genética , Mutación , Neoplasias del Sistema Nervioso Central/patología , Metilación de ADN , Células Madre Embrionarias/metabolismo , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Dosificación de Gen , Regulación Neoplásica de la Expresión Génica , Genes myc , Glioma/patología , Humanos , Isocitrato Deshidrogenasa/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/farmacología , Fibroblastos/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Inhibidores de Fosfodiesterasa 4/farmacología , Quinolonas/farmacología , Sistema Respiratorio/efectos de los fármacos , Sulfonas/farmacología , Agonistas de Receptores Adrenérgicos beta 2/síntesis química , Agonistas de Receptores Adrenérgicos beta 2/química , Animales , Técnicas de Cultivo de Célula , Citocinas/antagonistas & inhibidores , Citocinas/inmunología , Fibroblastos/enzimología , Fibroblastos/inmunología , Fibroblastos/metabolismo , Cobayas , Humanos , Leucocitos Mononucleares/enzimología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/farmacología , Masculino , Estructura Molecular , Músculo Liso/enzimología , Músculo Liso/inmunología , Músculo Liso/metabolismo , Inhibidores de Fosfodiesterasa 4/síntesis química , Inhibidores de Fosfodiesterasa 4/química , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Quinolonas/síntesis química , Quinolonas/química , Sistema Respiratorio/enzimología , Sistema Respiratorio/inmunología , Sistema Respiratorio/metabolismo , Sulfonas/síntesis química , Sulfonas/química , Factores de TiempoRESUMEN
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.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/farmacología , Aminopiridinas/farmacología , Benzamidas/farmacología , Fibroblastos/metabolismo , Indanos/farmacología , Inflamación/prevención & control , Pulmón/metabolismo , Inhibidores de Fosfodiesterasa 4/farmacología , Fibrosis Pulmonar/prevención & control , Quinolonas/farmacología , Actinas/metabolismo , Células Cultivadas , Quimiocina CCL5/metabolismo , Quimiocina CXCL10/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Ciclopropanos/farmacología , Endotelina-1/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibronectinas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Inflamación/metabolismo , Pulmón/citología , Pulmón/efectos de los fármacos , Fibrosis Pulmonar/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
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.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/farmacología , Aminopiridinas/farmacología , Benzamidas/farmacología , Dexametasona/farmacología , Inflamación/tratamiento farmacológico , Agonistas de Receptores Adrenérgicos beta 2/administración & dosificación , Albuterol/administración & dosificación , Albuterol/análogos & derivados , Albuterol/farmacología , Aminopiridinas/administración & dosificación , Antiinflamatorios/administración & dosificación , Antiinflamatorios/farmacología , Benzamidas/administración & dosificación , Ciclopropanos/administración & dosificación , Ciclopropanos/farmacología , Dexametasona/administración & dosificación , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Etanolaminas/administración & dosificación , Etanolaminas/farmacología , Fumarato de Formoterol , Glucocorticoides/administración & dosificación , Glucocorticoides/farmacología , Humanos , Inflamación/fisiopatología , Concentración 50 Inhibidora , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos , Masculino , Inhibidores de Fosfodiesterasa 4/administración & dosificación , Inhibidores de Fosfodiesterasa 4/farmacología , Xinafoato de Salmeterol , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.