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Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in innate immune signaling by Toll-like receptors (TLRs), and loss of IRAK4 activity in mice and humans increases susceptibility to bacterial infections and causes defects in TLR and IL1 ligand sensing. However, the mechanism by which IRAK4 activity regulates the production of downstream inflammatory cytokines is unclear. Using transcriptomic and biochemical analyses of human monocytes treated with a highly potent and selective inhibitor of IRAK4, we show that IRAK4 kinase activity controls the activation of interferon regulatory factor 5 (IRF5), a transcription factor implicated in the pathogenesis of multiple autoimmune diseases. Following TLR7/8 stimulation by its agonist R848, chemical inhibition of IRAK4 abolished IRF5 translocation to the nucleus and thus prevented IRF5 binding to and activation of the promoters of inflammatory cytokines in human monocytes. We also found that IKKß, an upstream IRF5 activator, is phosphorylated in response to the agonist-induced TLR signaling. Of note, IRAK4 inhibition blocked IKKß phosphorylation but did not block the nuclear translocation of NFκB, which was surprising, given the canonical role of IKKß in phosphorylating IκB to allow NFκB activation. Moreover, pharmacological inhibition of either IKKß or the serine/threonine protein kinase TAK1 in monocytes blocked TLR-induced cytokine production and IRF5 translocation to the nucleus, but not nuclear translocation of NFκB. Taken together, our data suggest a mechanism by which IRAK4 activity regulates TAK1 and IKKß activation, leading to the nuclear translocation of IRF5 and induction of inflammatory cytokines in human monocytes.
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Quinasa I-kappa B/metabolismo , Factores Reguladores del Interferón/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Modelos Inmunológicos , Monocitos/metabolismo , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Células Cultivadas , Biología Computacional , Citocinas/agonistas , Citocinas/genética , Citocinas/metabolismo , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Quinasa I-kappa B/química , Factores Reguladores del Interferón/agonistas , Quinasas Asociadas a Receptores de Interleucina-1/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/química , Quinasas Quinasa Quinasa PAM/metabolismo , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/inmunología , FN-kappa B/metabolismo , Subunidad p50 de NF-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Análisis de la Célula Individual , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/metabolismoRESUMEN
Vosaroxin is a first-in-class anticancer quinolone derivative that intercalates DNA and inhibits topoisomerase II. This study assessed the safety and tolerability of vosaroxin plus cytarabine in patients with relapsed/refractory acute myeloid leukemia. Escalating vosaroxin doses (10-minute infusion; 10-90 mg/m(2); days 1, 4) were given in combination with cytarabine on one of two schedules: schedule A (24-hour continuous intravenous infusion, 400 mg/m(2)/day, days 1-5) or schedule B (2-hour intravenous infusion, 1 g/m(2)/day, days 1-5). Following dose escalation, enrollment was expanded at the maximum tolerated dose. Of 110 patients enrolled, 108 received treatment. The maximum tolerated dose of vosaroxin was 80 mg/m(2) for schedule A (dose-limiting toxicities: grade 3 bowel obstruction and stomatitis) and was not reached for schedule B (recommended phase 2 dose: 90 mg/m(2)). In the efficacy population (all patients in first relapse or with primary refractory disease treated with vosaroxin 80-90 mg/m(2); n=69), the complete remission rate was 25% and the complete remission/complete remission with incomplete blood count recovery rate was 28%. The 30-day all-cause mortality rate was 2.5% among all patients treated at a dose of 80-90 mg/m(2). Based upon these results, a phase 3 trial of vosaroxin plus cytarabine was initiated in patients with relapsed/refractory acute myeloid leukemia. (Clinicaltrials.gov identifier: NCT00541866).
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Terapia Recuperativa , Adolescente , Adulto , Anciano , Estudios de Cohortes , Citarabina/administración & dosificación , Femenino , Estudios de Seguimiento , Humanos , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Naftiridinas/administración & dosificación , Recurrencia Local de Neoplasia/mortalidad , Recurrencia Local de Neoplasia/patología , Estadificación de Neoplasias , Pronóstico , Inducción de Remisión , Tasa de Supervivencia , Tiazoles/administración & dosificación , Adulto JovenRESUMEN
BACKGROUND: Homologous recombination repair (HRR) pathway deficiencies have significant implications for cancer predisposition and treatment strategies. Improved quantitative methods for functionally characterizing these deficiencies are required to accurately identify patients at risk of developing cancer and to identify mechanisms of drug resistance or sensitivity. METHODS: Flow cytometry-based single cell network profiling (SCNP) was used to measure drug-induced activation of DNA damage response (DDR) proteins in cell lines with defined HRR pathway mutations (including ATM-/-, ATM+/-, BRCA1+/-, BRCA2-/-) and in primary acute myeloid leukemia (AML) samples. Both non-homologous end joining (NHEJ) and HRR pathways were examined by measuring changes in intracellular readouts (including p-H2AX, p-ATM, p-DNA-PKcs, p-53BP1, p-RPA2/32, p-BRCA1, p-p53, and p21) in response to exposure to mechanistically distinct genotoxins. The cell cycle S/G2/M phase CyclinA2 marker was used to normalize for proliferation rates. RESULTS: Etoposide induced proliferation-independent DNA damage and activation of multiple DDR proteins in primary AML cells and ATM +/+but not ATM -/- cell lines. Treatment with the PARPi AZD2281 +/- temozolomide induced DNA damage in CyclinA2+ cells in both primary AML cells and cell lines and distngiushed cell lines deficient (BRCA2-/-) or impaired (BRCA1+/-) in HRR activity from BRCA1+/+ cell lines based on p-H2AX induction. Application of this assay to primary AML samples identified heterogeneous patterns of repair activity including muted or proficient activation of NHEJ and HRR pathways and predominant activation of NHEJ in a subset of samples. CONCLUSIONS: SCNP identified functional DDR readouts in both NHEJ and HRR pathways, which can be applied to identify cells with BRCA1+/- haploinsuffiency and characterize differential DDR pathway functionality in primary clinical samples.
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Daño del ADN , Reparación del ADN , Análisis de la Célula Individual/métodos , Adulto , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Niño , Ciclina A2/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Inhibidores Enzimáticos/farmacología , Etopósido/farmacología , Haploinsuficiencia/efectos de los fármacos , Histonas/metabolismo , Recombinación Homóloga/efectos de los fármacos , Humanos , Mutágenos/toxicidad , Fosforilación/efectos de los fármacos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/metabolismo , Reproducibilidad de los Resultados , TemozolomidaRESUMEN
BACKGROUND: Single-cell network profiling (SCNP) is a multiparametric flow cytometry-based approach that simultaneously measures evoked signaling in multiple cell subsets. Previously, using the SCNP approach, age-associated immune signaling responses were identified in a cohort of 60 healthy donors. METHODS: In the current study, a high-dimensional analysis of intracellular signaling was performed by measuring 24 signaling nodes in 7 distinct immune cell subsets within PBMCs in an independent cohort of 174 healthy donors [144 elderly (>65 yrs); 30 young (25-40 yrs)]. RESULTS: Associations between age and 9 immune signaling responses identified in the previously published 60 donor cohort were confirmed in the current study. Furthermore, within the current study cohort, 48 additional immune signaling responses differed significantly between young and elderly donors. These associations spanned all profiled modulators and immune cell subsets. CONCLUSIONS: These results demonstrate that SCNP, a systems-based approach, can capture the complexity of the cellular mechanisms underlying immunological aging. Further, the confirmation of age associations in an independent donor cohort supports the use of SCNP as a tool for identifying reproducible predictive biomarkers in areas such as vaccine response and response to cancer immunotherapies.
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Envejecimiento/inmunología , Voluntarios Sanos , Transducción de Señal , Adulto , Anciano , Estudios de Cohortes , HumanosRESUMEN
A greater understanding of the function of the human immune system at the single-cell level in healthy individuals is critical for discerning aberrant cellular behavior that occurs in settings such as autoimmunity, immunosenescence, and cancer. To achieve this goal, a systems-level approach capable of capturing the response of the interdependent immune cell types to external stimuli is required. In this study, an extensive characterization of signaling responses in multiple immune cell subpopulations within PBMCs from a cohort of 60 healthy donors was performed using single-cell network profiling (SCNP). SCNP is a multiparametric flow cytometry-based approach that enables the simultaneous measurement of basal and evoked signaling in multiple cell subsets within heterogeneous populations. In addition to establishing the interindividual degree of variation within a broad panel of immune signaling responses, the possible association of any observed variation with demographic variables including age and race was investigated. Using half of the donors as a training set, multiple age- and race-associated variations in signaling responses in discrete cell subsets were identified, and several were subsequently confirmed in the remaining samples (test set). Such associations may provide insight into age-related immune alterations associated with high infection rates and diminished protection following vaccination and into the basis for ethnic differences in autoimmune disease incidence and treatment response. SCNP allowed for the generation of a functional map of healthy immune cell signaling responses that can provide clinically relevant information regarding both the mechanisms underlying immune pathological conditions and the selection and effect of therapeutics.
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Envejecimiento/inmunología , Negro o Afroamericano , Sistema Inmunológico/metabolismo , Leucocitos Mononucleares/inmunología , Transducción de Señal , Análisis de la Célula Individual/métodos , Población Blanca , Adulto , Anciano , Enfermedades Autoinmunes/inmunología , Células Cultivadas , Estudios de Cohortes , Citocinas/biosíntesis , Femenino , Citometría de Flujo/métodos , Humanos , Sistema Inmunológico/inmunología , Inmunidad Celular , Masculino , Persona de Mediana Edad , Linfocitos T/inmunologíaRESUMEN
INTRODUCTION: High baseline neutrophil-to-lymphocyte ratio (NLR) in rheumatoid arthritis (RA) has been associated with positive responses to biologic tumor necrosis factor inhibition and negative responses to conventional synthetic disease-modifying antirheumatic drug (csDMARD) triple therapy. Datasets from three randomized clinical trials in patients with RA were used to test the hypothesis that baseline NLR is associated with improved clinical response to filgotinib in methotrexate (MTX)-naïve or MTX-experienced RA populations. METHODS: Patients from FINCH 1 (inadequate response to MTX, MTX-IR; NCT02889796), FINCH 2 (inadequate response to biologic DMARDs; NCT02873936), and FINCH 3 (MTX-naïve; NCT02886728) were classified as baseline NLR-High or baseline NLR-Low based on a previously published cut point of 2.7. In total, 3365 patients were included across the three studies. Differences in clinical outcomes and patient-reported outcomes (PROs) were determined using linear-regression models. RESULTS: Control-arm patients (placebo + MTX/placebo + csDMARD) classified as NLR-High exhibited worse continuous clinical and PRO responses at week 12 across clinical trials compared to NLR-Low patients. In contrast, NLR-High patients who received FIL 200 mg + MTX/csDMARD exhibited consistently better responses after 12 weeks compared to NLR-Low patients across clinical trials, clinical endpoints, and PROs. These trends were most prominent among the MTX-IR population. CONCLUSION: The 2.7 baseline NLR cut point could be used to enrich for patients most likely to benefit from the addition of filgotinib to background MTX/csDMARD. Use of baseline NLR as part of therapeutic decision-making would not require additional diagnostics and could contribute to improved outcomes for patients with RA. TRIAL REGISTRATION: Clinicaltrials.gov: NCT02889796; NCT02873936; NCT02886728.
Rheumatoid arthritis is a disease that results in swollen and painful joints. There is currently no method to determine which treatment will work best for an individual patient. However, there may be identifying markers found in the blood that could indicate how a patient will respond to treatment. One of these possible markers is a ratio of two types of white blood cells, neutrophils and lymphocytes, which are part of the body's immune system and help the body detect and fight infection and other diseases. This ratio is referred to as the neutrophil-to-lymphocyte ratio. The current study evaluated whether the neutrophil-to-lymphocyte ratio at the beginning of treatment was associated with rheumatoid arthritis treatment outcomes. Blood test results were used from 3365 patients receiving filgotinib (a medicine used to treat rheumatoid arthritis) or other therapies as part of the FINCH clinical trials. Patients were classified as having a high or low neutrophil-to-lymphocyte ratio at the start of treatment. Patients receiving filgotinib over 24 weeks who had a high neutrophil-to-lymphocyte ratio showed less disease activity than patients whose ratio was low. This study provides support for the use of the neutrophil-to-lymphocyte ratio as a way to help determine whether a patient would benefit from filgotinib as part of their rheumatoid arthritis treatment and may help improve rheumatoid arthritis treatment outcomes.
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OBJECTIVES: To assess cigarette smoking's effects on efficacy of the preferential Janus kinase (JAK) 1 inhibitor filgotinib and drug persistence in patients with rheumatoid arthritis (RA). METHODS: Efficacy in non-smokers, former smokers, and current smokers from phase 3 filgotinib trials was analyzed, including patients with inadequate response (IR) to methotrexate (MTX) or biologic disease-modifying antirheumatic drugs (bDMARDs) or who were MTX-naïve. Proportions achieving Disease Activity Score in 28 joints with C-reactive protein (DAS28[CRP]) ≤ 3.2 were compared using logistic regression. Retrospective claims-based switching data were reviewed. RESULTS: Week 12 (W12) DAS28(CRP) ≤ 3.2 was achieved by 50, 61, and 62% of MTX-IR non-smokers, former smokers, and current smokers taking filgotinib 200 mg (FIL200) + MTX vs. 23, 16, and 32% taking placebo + MTX (p < 0.001, < 0.001, and 0.001) and 50, 34, and 33% taking adalimumab + MTX (p = 0.97, 0.013, and 0.006 vs. FIL200 + MTX). W12 DAS28(CRP) ≤ 3.2 was achieved by 46, 48, and 32% of bDMARD-IR non-smokers, former smokers, and current smokers taking FIL200 + conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) vs. 16, 23, and 5% taking placebo + csDMARD (p < 0.001, 0.077, and 0.051); 57, 58, and 59% of respective MTX-naïve smoking groups achieved W12 DAS28(CRP) ≤ 3.2 with FIL200 + MTX vs. 28, 37, and 18% with MTX (p < 0.001, 0.026, and < 0.001). Claims data showed former/current smokers were likelier than non-smokers to switch from adalimumab to other biologics or JAK inhibitors. CONCLUSIONS: Greater proportions of MTX-IR current/former smokers responded to FIL200 + MTX vs. adalimumab + MTX. In non-smoking MTX-IR, bDMARD-IR, and MTX-naïve patients with RA, FIL200 + MTX demonstrated increased response vs. controls. Current/former smokers were likelier to discontinue adalimumab vs. non-smokers in real-world clinical settings. TRIAL REGISTRATION: NCT02889796, NCT02873936, NCT02886728.
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The aim of this study was to assess the feasibility of applying the single cell network profiling (SCNP) assay to the examination of signaling networks in epithelial cancer cells, using bladder washings from 29 bladder cancer (BC) and 15 nonbladder cancer (NC) subjects. This report describes the methods we developed to detect rare epithelial cells (within the cells we collected from bladder washings), distinguish cancer cells from normal epithelial cells, and reproducibly quantify signaling within these low frequency cancer cells. Specifically, antibodies against CD45, cytokeratin, EpCAM, and cleaved-PARP (cPARP) were used to differentiate nonapoptotic epithelial cells from leukocytes, while measurements of DNA content to determine aneuploidy (DAPI stain) allowed for distinction between tumor and normal epithelial cells. Signaling activity in the PI3K and MAPK pathways was assessed by measuring intracellular levels of p-AKT and p-ERK at baseline and in response to pathway modulation; 66% (N = 19) of BC samples and 27% (N = 4) of NC samples met the "evaluable" criteria, i.e., at least 400,000 total cells available upon sample receipt with >2% of cells showing an epithelial phenotype. The majority of epithelial cells detected in BC samples were nonapoptotic and all signaling data were generated from identified cPARP negative cells. In four of 19 BC samples but in none of the NC specimens, SCNP assay identified epithelial cancer cells with a quantifiable increase in epidermal growth factor-induced p-AKT and p-ERK levels. Furthermore, preincubation with the PI3K inhibitor GDC-0941 reduced or completely inhibited basal and epidermal growth factor-induced p-AKT but, as expected, had no effect on p-ERK levels. This study demonstrates the feasibility of applying SCNP assay using multiparametric flow cytometry to the functional characterization of rare, bladder cancer cells collected from bladder washing. Following assay standardization, this method could potentially serve as a tool for disease characterization and drug development in bladder cancer and other solid tumors.
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Biomarcadores de Tumor/genética , Células Epiteliales/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Proto-Oncogénicas c-akt/genética , Neoplasias de la Vejiga Urinaria/genética , Aneuploidia , Biomarcadores de Tumor/metabolismo , Inhibidores Enzimáticos/farmacología , Células Epiteliales/clasificación , Células Epiteliales/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Citometría de Flujo/métodos , Humanos , Indazoles/farmacología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Análisis de la Célula Individual/métodos , Sulfonamidas/farmacología , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
While many prognostic markers in B-cell chronic lymphocytic leukemia provide insight into the biology of the disease, few have been demonstrated to be useful in the daily management of patients. B-cell receptor signaling is a driving event in the progression of B-cell chronic lymphocytic leukemia and markers of B-cell receptor responsiveness have been shown to be of prognostic value. Single cell network profiling, a multiparametric flow cytometry-based assay, allows functional signaling analysis at the level of the single cell. B-cell receptor signaling proteins (i.e. p-SYK, p-NF-κB p65, p-ERK, p-p38, p-JNK) were functionally characterized by single cell network profiling in samples from patients with B-cell chronic lymphocytic leukemia in an exploratory study (n=27) after stimulation with anti-IgM. Significant associations of single cell network profiling data with clinical outcome (i.e. time to first treatment), as assessed by Cox regression models, were then confirmed in patients' samples in two other sequential independent studies, i.e. test study 1 (n=30), and test study 2 (n=37). In the exploratory study, higher responsiveness of the B-cell receptor signaling proteins to anti-IgM was associated with poor clinical outcomes. Patients' clustering based on signaling response was at least as powerful in discriminating different disease courses as traditional prognostic markers. In an unselected subgroup of patients with Binet stage A disease (n=21), increased anti-IgM-modulated p-ERK signaling was shown to be a significant, independent predictor of shorter time to first treatment. This result was independently confirmed in two test cohorts from distinct populations of patients. In conclusion, these findings support the utility of the single cell network profiling assay in elucidating signaling perturbations with the potential for the development of a clinically useful prognostic test in patients with early stage B-cell chronic lymphocytic leukemia. These data support the clinical relevance of B-cell receptor signaling in B-cell chronic lymphocytic leukemia, and suggest a key role of ERK activation in the physiopathology of this leukemia.
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Leucemia Linfocítica Crónica de Células B/metabolismo , Leucocitos Mononucleares/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Análisis de la Célula Individual/métodos , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antiidiotipos/farmacología , Células Cultivadas , Progresión de la Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Citometría de Flujo/métodos , Citometría de Flujo/estadística & datos numéricos , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Estimación de Kaplan-Meier , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/patología , Leucocitos Mononucleares/efectos de los fármacos , Masculino , Persona de Mediana Edad , Análisis Multivariante , FN-kappa B/metabolismo , Pronóstico , Modelos de Riesgos Proporcionales , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Quinasa SykRESUMEN
BACKGROUND: Single-cell network profiling (SCNP) is a multi-parametric flow cytometry-based approach that simultaneously measures basal and modulated intracellular signaling activity in multiple cell subpopulations. Previously, SCNP analysis of a broad panel of immune signaling pathways in cell subsets within PBMCs from 60 healthy donors identified a race-associated difference in B cell anti-IgD-induced PI3K pathway activity. METHODS: The present study extended this analysis to a broader range of signaling pathway components downstream of the B cell receptor (BCR) in European Americans and African Americans using a subset of donors from the previously analyzed cohort of 60 healthy donors. Seven BCR signaling nodes (a node is defined as a paired modulator and intracellular readout) were measured at multiple time points by SCNP in PBMCs from 10 healthy donors [5 African Americans (36-51 yrs), 5 European Americans (36-56 yrs), all males]. RESULTS: Analysis of BCR signaling activity in European American and African American PBMC samples revealed that, compared to the European American donors, B cells from African Americans had lower anti-IgD induced phosphorylation of multiple BCR pathway components, including the membrane proximal proteins Syk and SFK as well as proteins in the PI3K pathway (S6 and Akt), the MAPK pathways (Erk and p38), and the NF-κB pathway (NF-κB). In addition to differences in the magnitude of anti-IgD-induced pathway activation, racial differences in BCR signaling kinetic profiles were observed. Further, the frequency of IgD+ B cells differed by race and strongly correlated with BCR pathway activation. Thus, the race-related difference in BCR pathway activation appears to be attributable at least in part to a race-associated difference in IgD+ B cell frequencies. CONCLUSIONS: SCNP analysis enabled the identification of statistically significant race-associated differences in BCR pathway activation within PBMC samples from healthy donors. Understanding race-associated contrasts in immune cell signaling responses may be one critical component for elucidation of differences in immune-mediated disease prevalence and treatment responses.
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Grupos Raciales , Receptores de Antígenos de Linfocitos B/inmunología , Transducción de Señal/inmunología , Adulto , Negro o Afroamericano , Linfocitos B/inmunología , Citometría de Flujo , Humanos , Inmunoglobulina D/inmunología , Cinética , Masculino , Persona de Mediana Edad , Donantes de Tejidos , Población BlancaRESUMEN
Inhibitors of cyclin-dependent kinases (Cdks) have been reported to have activities in chronic lymphocytic leukemia cells by inhibiting Cdk7 and Cdk9, which control transcription. Here we studied the novel Cdk inhibitor SNS-032, which exhibits potent and selective inhibitory activity against Cdk2, Cdk7, and Cdk9. We hypothesized that transient inhibition of transcription by SNS-032 would decrease antiapoptotic proteins, resulting in cell death. SNS-032 effectively killed chronic lymphocytic leukemia cells in vitro regardless of prognostic indicators and treatment history. This was associated with inhibition of phosphorylation of RNA polymerase II and inhibition of RNA synthesis. Consistent with the intrinsic turnover rates of their transcripts and proteins, antiapoptotic proteins, such as Mcl-1 and X-linked inhibitor of apoptosis protein (XIAP), were rapidly reduced on exposure to SNS-032, whereas Bcl-2 protein was not affected. The initial decrease of Mcl-1 protein was the result of transcriptional inhibition rather than cleavage by caspase. Compared with flavopiridol and roscovitine, SNS-032 was more potent, both in inhibition of RNA synthesis and at induction of apoptosis. SNS-032 activity was readily reversible; removal of SNS-032 reactivated RNA polymerase II, which led to resynthesis of Mcl-1 and cell survival. Thus, these data support the clinical development of SNS-032 in diseases that require short-lived oncoproteins for survival.
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Apoptosis/efectos de los fármacos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/enzimología , Oxazoles/farmacología , ARN Polimerasa II/antagonistas & inhibidores , ARN Neoplásico/metabolismo , Tiazoles/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Caspasas/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Femenino , Flavonoides/farmacología , Humanos , Immunoblotting , Leucemia Linfocítica Crónica de Células B/patología , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Linfocitos/patología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Persona de Mediana Edad , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Fosforilación/efectos de los fármacos , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Purinas/farmacología , ARN Polimerasa II/metabolismo , ARN Neoplásico/antagonistas & inhibidores , Roscovitina , Transcripción Genética/efectos de los fármacos , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
Rheumatoid arthritis (RA) is a systemic and incurable autoimmune disease characterized by chronic inflammation in synovial lining of joints. To identify the signaling pathways involved in RA, its disease activity, and treatment response, we adapted a systems immunology approach to simultaneously quantify 42 signaling nodes in 21 immune cell subsets (e.g., IFNαâp-STAT5 in B cells) in peripheral blood mononuclear cells (PBMC) from 194 patients with longstanding RA (including 98 patients before and after treatment), and 41 healthy controls (HC). We found multiple differences between patients with RA compared to HC, predominantly in cytokine-induced Jak/STAT signaling in many immune cell subsets, suggesting pathways that may be associated with susceptibility to RA. We also found that high RA disease activity, compared to low disease activity, was associated with decreased (e.g., IFNαâp-STAT5, IL-10âp-STAT1) or increased (e.g., IL-6âSTAT3) response to stimuli in multiple cell subsets. Finally, we compared signaling in patients with established, refractory RA before and six months after initiation of methotrexate (MTX) or TNF inhibitors (TNFi). We noted significant changes from pre-treatment to post-treatment in IFNαâp-STAT5 signaling and IL-10âp-STAT1 signaling in multiple cell subsets; these changes brought the aberrant RA signaling profiles toward those of HC. This large, comprehensive functional signaling pathway study provides novel insights into the pathogenesis of RA and shows the potential of quantification of cytokine-induced signaling as a biomarker of disease activity or treatment response.
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Artritis Reumatoide/patología , Interferón-alfa/farmacología , Interleucina-10/farmacología , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Abatacept/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/metabolismo , Biomarcadores/metabolismo , Estudios de Casos y Controles , Femenino , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Masculino , Metotrexato/uso terapéutico , Persona de Mediana Edad , Fosforilación , Índice de Severidad de la EnfermedadRESUMEN
INTRODUCTION: The Janus kinase (JAK) inhibitor therapeutic class has shown significant clinical benefit in the treatment of rheumatoid arthritis (RA). We sought to gain insight into the mode of action and immunological effects of filgotinib, a JAK1 selective inhibitor, in active RA by analyzing secreted and cell-based biomarkers key to RA pathophysiology in two phase 2b trials of filgotinib in active RA. METHODS: Immune cell subsets and 34 serum biomarkers were analyzed longitudinally over 12 weeks using blood samples collected from patients with active RA receiving filgotinib (100 or 200 mg once daily) or placebo (PBO) in the two phase 2b trials (DARWIN 1, on a background of methotrexate, and DARWIN 2, as monotherapy). RESULTS: Consistently across both studies, filgotinib treatment decreased multiple immune response biomarkers that have key roles in RA for immune response, and decreased markers that promote matrix degradation, angiogenesis, leukocyte adhesion, and recruitment. Filgotinib did not significantly modulate T and natural killer (NK) lymphoid subsets, but slightly increased B cell numbers after 12 weeks. Multiple correlations were observed for changes in biomarkers with disease activity score 28-CRP. MIP1ß showed modest predictivity at baseline for ACR50 response at 12 weeks in the 100 mg filgotinib dose across both studies (AUROC, 0.65 and 0.67, p < 0.05). CONCLUSIONS: Filgotinib regulates biomarkers from multiple pathways, indicative of direct and indirect network effects on the immune system and the stromal response. These effects were not associated with reductions of major circulating lymphoid populations. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01888874, NCT01894516.
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BACKGROUND: We previously reported the results of a multicentric prospective randomized trial of chemo-refractory metastatic breast cancer patients testing the efficacy of two doses of TGFß blockade during radiotherapy. Despite a lack of objective responses to the combination, patients who received a higher dose of TGFß blocking antibody fresolimumab had a better overall survival when compared to those assigned to lower dose (hazard ratio of 2.73, p = 0.039). They also demonstrated an improved peripheral blood mononuclear cell (PBMC) counts and increase in the CD8 central memory pool. We performed additional analysis on residual PBMC using single cell network profiling (SCNP). METHODS: The original trial randomized metastatic breast cancer patients to either 1 or 10 mg/kg of fresolimumab, every 3 weeks for 5 cycles, combined with radiotherapy to a metastatic site at week 1 and 7 (22.5 Gy given in 3 doses of 7.5 Gy). Trial immune monitoring results were previously reported. In 15 patients with available residual blood samples, additional functional studies were performed, and compared with data obtained in parallel from seven healthy female donors (HD): SCNP was applied to analyze T cell receptor (TCR) modulated signaling via CD3 and CD28 crosslinking and measurement of evoked phosphorylation of AKT and ERK in CD4 and CD8 T cell subsets defined by PD-1 expression. RESULTS: At baseline, a significantly higher level of expression (p < 0.05) of PD-L1 was identified in patient monocytes compared to HD. TCR modulation revealed dysfunction of circulating T-cells in patient baseline samples as compared to HD, and this was more pronounced in PD-1+ cells. Treatment with radiotherapy and fresolimumab did not resolve this dyfunctional signaling. However, in vitro PD-1 blockade enhanced TCR signaling in patient PD-1+ T cells and not in PD-1- T cells or in PD-1+ T cells from HD. CONCLUSIONS: Functional T cell analysis suggests that baseline T cell functionality is hampered in metastatic breast cancer patients, at least in part mediated by the PD-1 signaling pathway. These preliminary data support the rationale for investigating the possible beneficial effects of adding PD-1 blockade to improve responses to TGFß blockade and radiotherapy. TRIAL REGISTRATION: NCT01401062 .
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos/uso terapéutico , Antígeno B7-H1/inmunología , Neoplasias de la Mama , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Adulto , Anciano , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/radioterapia , Terapia Combinada , Femenino , Humanos , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T/inmunología , Adulto JovenRESUMEN
There is growing recognition that immunotherapy is likely to significantly improve health outcomes for cancer patients in the coming years. Currently, while a subset of patients experience substantial clinical benefit in response to different immunotherapeutic approaches, the majority of patients do not but are still exposed to the significant drug toxicities. Therefore, a growing need for the development and clinical use of predictive biomarkers exists in the field of cancer immunotherapy. Predictive cancer biomarkers can be used to identify the patients who are or who are not likely to derive benefit from specific therapeutic approaches. In order to be applicable in a clinical setting, predictive biomarkers must be carefully shepherded through a step-wise, highly regulated developmental process. Volume I of this two-volume document focused on the pre-analytical and analytical phases of the biomarker development process, by providing background, examples and "good practice" recommendations. In the current Volume II, the focus is on the clinical validation, validation of clinical utility and regulatory considerations for biomarker development. Together, this two volume series is meant to provide guidance on the entire biomarker development process, with a particular focus on the unique aspects of developing immune-based biomarkers. Specifically, knowledge about the challenges to clinical validation of predictive biomarkers, which has been gained from numerous successes and failures in other contexts, will be reviewed together with statistical methodological issues related to bias and overfitting. The different trial designs used for the clinical validation of biomarkers will also be discussed, as the selection of clinical metrics and endpoints becomes critical to establish the clinical utility of the biomarker during the clinical validation phase of the biomarker development. Finally, the regulatory aspects of submission of biomarker assays to the U.S. Food and Drug Administration as well as regulatory considerations in the European Union will be covered.
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Bioensayo , Biomarcadores de Tumor , Inmunoterapia , Neoplasias/diagnóstico , Neoplasias/terapia , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Bioensayo/métodos , Bioensayo/normas , Unión Europea , Humanos , Inmunoterapia/métodos , Terapia Molecular Dirigida , Neoplasias/mortalidad , Pronóstico , Curva ROC , Reproducibilidad de los Resultados , Resultado del Tratamiento , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Immunotherapies have emerged as one of the most promising approaches to treat patients with cancer. Recently, there have been many clinical successes using checkpoint receptor blockade, including T cell inhibitory receptors such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death-1 (PD-1). Despite demonstrated successes in a variety of malignancies, responses only typically occur in a minority of patients in any given histology. Additionally, treatment is associated with inflammatory toxicity and high cost. Therefore, determining which patients would derive clinical benefit from immunotherapy is a compelling clinical question. Although numerous candidate biomarkers have been described, there are currently three FDA-approved assays based on PD-1 ligand expression (PD-L1) that have been clinically validated to identify patients who are more likely to benefit from a single-agent anti-PD-1/PD-L1 therapy. Because of the complexity of the immune response and tumor biology, it is unlikely that a single biomarker will be sufficient to predict clinical outcomes in response to immune-targeted therapy. Rather, the integration of multiple tumor and immune response parameters, such as protein expression, genomics, and transcriptomics, may be necessary for accurate prediction of clinical benefit. Before a candidate biomarker and/or new technology can be used in a clinical setting, several steps are necessary to demonstrate its clinical validity. Although regulatory guidelines provide general roadmaps for the validation process, their applicability to biomarkers in the cancer immunotherapy field is somewhat limited. Thus, Working Group 1 (WG1) of the Society for Immunotherapy of Cancer (SITC) Immune Biomarkers Task Force convened to address this need. In this two volume series, we discuss pre-analytical and analytical (Volume I) as well as clinical and regulatory (Volume II) aspects of the validation process as applied to predictive biomarkers for cancer immunotherapy. To illustrate the requirements for validation, we discuss examples of biomarker assays that have shown preliminary evidence of an association with clinical benefit from immunotherapeutic interventions. The scope includes only those assays and technologies that have established a certain level of validation for clinical use (fit-for-purpose). Recommendations to meet challenges and strategies to guide the choice of analytical and clinical validation design for specific assays are also provided.
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Bioensayo , Biomarcadores de Tumor , Inmunoterapia , Neoplasias/diagnóstico , Neoplasias/terapia , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Bioensayo/métodos , Bioensayo/normas , Antígeno CTLA-4/antagonistas & inhibidores , Ensayos Clínicos como Asunto , Humanos , Inmunoterapia/métodos , Terapia Molecular Dirigida , Neoplasias/mortalidad , Pronóstico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Reproducibilidad de los Resultados , Resultado del TratamientoRESUMEN
Chronic lymphocytic leukemia (CLL) is characterized by a progressive accumulation of B lymphocytes. T cell abnormalities are a common feature of CLL and contribute to impaired immune function in these patients. T cells are ineffective in eliminating the leukemic clone and may actually promote tumor growth and survival. Previous work from our laboratory documented elevated circulating levels of IL-17A-producing Th17 cells in CLL patients as compared to healthy age-matched control subjects. These high circulating Th17 levels associated with better prognostic markers and significantly longer overall survival, even among patients whose clones used unmutated IGHVs (U-CLL). Recent studies suggest that Th17 cells are heterogeneous, expressing different profiles of cytokines, and that different subsets of Th17s mediate different biological functions. In the present study, we found significantly higher levels of IL-17F-expressing CD4(+) T cells in CLL versus healthy peripheral blood mononuclear cells following in vitro stimulation in the presence of Th17-promoting cytokines. Furthermore, the differentiation of IL-17F-expressing Th17 cells was significantly enhanced when purified CD4(+) T cells from CLL patients were cultured in the presence of autologous CLL B cells. Lastly, single-cell network profiling revealed that IL-17F triggers NFκB phosphorylation in T and B cells from patients with CLL, but not age-matched healthy controls. Taken together, our data suggest that the phenotype of Th17 cells in CLL patients is distinct from healthy individuals, expressing higher levels of IL-17F, and that B and T cells from CLL patients are particularly responsive to IL-17F, as compared to healthy age-matched control individuals.
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Linfocitos B/inmunología , Interleucina-17/metabolismo , Leucemia Linfocítica Crónica de Células B/diagnóstico , FN-kappa B/metabolismo , Células Th17/inmunología , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Femenino , Humanos , Interleucina-17/genética , Leucemia Linfocítica Crónica de Células B/inmunología , Masculino , Fosforilación , Pronóstico , Transducción de Señal , TranscriptomaRESUMEN
FMS-like tyrosine kinase 3 receptor (FLT3) internal tandem duplication (ITD) mutations result in constitutive activation of this receptor and have been shown to increase the risk of relapse in patients with acute myeloid leukemia (AML); however, substantial heterogeneity in clinical outcomes still exists within both the ITD mutated and unmutated AML subgroups, suggesting alternative mechanisms of disease relapse not accounted by FLT3 mutational status. Single cell network profiling (SCNP) is a multiparametric flow cytometry based assay that simultaneously measures, in a quantitative fashion and at the single cell level, both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators. We previously reported an initial characterization of FLT3 ITD-mediated signaling using SCNP. Herein SCNP was applied sequentially to two separate cohorts of samples collected from elderly AML patients at diagnosis. In the first (training) study, AML samples carrying unmutated, wild-type FLT3 (FLT3 WT) displayed a wide range of induced signaling, with a fraction having signaling profiles comparable to FLT3 ITD AML samples. Conversely, the FLT3 ITD AML samples displayed more homogeneous induced signaling, with the exception of patients with low (<40%) mutational load, which had profiles comparable to FLT3 WT AML samples. This observation was then confirmed in an independent (verification) cohort. Data from the second cohort were also used to assess the association between SCNP data and disease-free survival (DFS) in the context of FLT3 and nucleophosmin (NPM1) mutational status among patients who achieved complete remission (CR) to induction chemotherapy. The combination of SCNP read outs together with FLT3 and NPM1 molecular status improved the DFS prediction accuracy of the latter. Taken together, these results emphasize the value of comprehensive functional assessment of biologically relevant signaling pathways in AML as a basis for the development of highly predictive tests for guidance of post-remission therapy.
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Leucemia Mieloide Aguda/genética , Mutación , Transducción de Señal , Tirosina Quinasa 3 Similar a fms/genética , Anciano , Anciano de 80 o más Años , Apoptosis , Células Cultivadas , Supervivencia sin Enfermedad , Femenino , Humanos , Quimioterapia de Inducción , Estimación de Kaplan-Meier , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Masculino , Persona de Mediana Edad , Análisis Multivariante , Mutagénesis , Nucleofosmina , Análisis de Componente Principal , Pronóstico , Análisis de la Célula Individual , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
Gemtuzumab ozogamicin (GO), an immunoconjugate between an anti-CD33 antibody and a calicheamicin-γ(1) derivative, induces remissions and improves survival in a subset of patients with acute myeloid leukemia (AML). As the mechanisms underlying GO and calicheamicin-γ(1) resistance are incompletely understood, we herein used flow cytometry-based single cell network profiling (SCNP) assays to study cellular responses of primary human AML cells to GO. Our data indicate that the extent of DNA damage is quantitatively impacted by CD33 expression and drug efflux activity. However, although DNA damage is required for GO-induced cytotoxicity, it is not sufficient for effective cell kill, suggesting that downstream anti-apoptotic pathways may function as relevant resistance mechanisms. Supporting this notion, we found activated PI3K/AKT signaling to be associated with GO resistance in vitro in primary AML cells. Consistently, the investigational AKT inhibitor MK-2206 significantly sensitized various human AML cells to GO or free calicheamicin-γ(1) with particularly pronounced effects in otherwise GO or free calicheamicin-γ(1)-resistant cells. Likewise, MK-2206 also sensitized primary AML cells to calicheamicin-γ(1). Together, our findings illustrate the capacity of SCNP assays to discover chemotherapy-related biological pathways and signaling networks relevant to GO-induced genotoxic stress. The identification of AKT signaling as being associated with GO resistance in vitro may provide a novel approach to improve the in vivo efficacy of GO/calicheamicin-γ(1) and, by extrapolation, other DNA damage-based therapeutics.
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Aminoglicósidos/farmacología , Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/farmacología , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/terapia , Proteínas Proto-Oncogénicas c-akt/fisiología , Aminoglicósidos/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Línea Celular Tumoral , Daño del ADN , Resistencia a Antineoplásicos , Enediinos/farmacología , Gemtuzumab , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Lectina 3 Similar a Ig de Unión al Ácido Siálico/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Análisis de la Célula Individual , Células Tumorales CultivadasRESUMEN
This study uses single cell network profiling (SCNP) to characterize biological pathways associated with in vitro resistance or sensitivity to chemotherapeutics commonly used in acute myeloid leukemia (AML) (i.e. cytarabine/daunorubicin, gemtuzumab ozogamicin (GO), decitabine, azacitidine, clofarabine). Simultaneous measurements at the single cell level of changes in DNA damage, apoptosis and signaling pathway responses in AML blasts incubated in vitro with the above drugs showed distinct profiles for each sample and mechanistically different profiles between distinct classes of agents. Studies are ongoing to assess the clinical predictive value of these findings.