RESUMEN
ABSTRACT: Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis and limited treatment options. Programmed cell death ligand 1(PD-L1) is recognized to be involved in the pathobiology of ATLL. However, what molecules control PD-L1 expression and whether genetic or pharmacological intervention might modify PD-L1 expression in ATLL cells are still unknown. To comprehend the regulatory mechanisms of PD-L1 expression in ATLL cells, we performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening in this work. In ATLL cells, we discovered that the neddylation-associated genes NEDD8, NAE1, UBA3, and CUL3 negatively regulated PD-L1 expression, whereas STAT3 positively did so. We verified, in line with the genetic results, that treatment with the JAK1/2 inhibitor ruxolitinib or the neddylation pathway inhibitor pevonedistat resulted in a decrease in PD-L1 expression in ATLL cells or an increase in it, respectively. It is significant that these results held true regardless of whether ATLL cells had the PD-L1 3' structural variant, a known genetic anomaly that promotes PD-L1 overexpression in certain patients with primary ATLL. Pevonedistat alone showed cytotoxicity for ATLL cells, but compared with each single modality, pevonedistat improved the cytotoxic effects of the anti-PD-L1 monoclonal antibody avelumab and chimeric antigen receptor (CAR) T cells targeting PD-L1 in vitro. As a result, our work provided insight into a portion of the complex regulatory mechanisms governing PD-L1 expression in ATLL cells and demonstrated the in vitro preliminary preclinical efficacy of PD-L1-directed immunotherapies by using pevonedistat to upregulate PD-L1 in ATLL cells.
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Ciclopentanos , Leucemia-Linfoma de Células T del Adulto , Linfoma , Pirimidinas , Adulto , Humanos , Leucemia-Linfoma de Células T del Adulto/tratamiento farmacológico , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/patología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Antígeno B7-H1/metabolismo , Linfoma/genéticaRESUMEN
Anaplastic large cell lymphoma (ALCL), a subgroup of mature T-cell neoplasms with an aggressive clinical course, is characterized by elevated expression of CD30 and anaplastic cytology. To achieve a comprehensive understanding of the molecular characteristics of ALCL pathology and to identify therapeutic vulnerabilities, we applied genome-wide CRISPR library screenings to both anaplastic lymphoma kinase positive (ALK+) and primary cutaneous (pC) ALK- ALCLs and identified an unexpected role of the interleukin-1R (IL-1R) inflammatory pathway in supporting the viability of pC ALK- ALCL. Importantly, this pathway is activated by IL-1α in an autocrine manner, which is essential for the induction and maintenance of protumorigenic inflammatory responses in pC-ALCL cell lines and primary cases. Hyperactivation of the IL-1R pathway is promoted by the A20 loss-of-function mutation in the pC-ALCL lines we analyze and is regulated by the nonproteolytic protein ubiquitination network. Furthermore, the IL-1R pathway promotes JAK-STAT3 signaling activation in ALCLs lacking STAT3 gain-of-function mutation or ALK translocation and enhances the sensitivity of JAK inhibitors in these tumors in vitro and in vivo. Finally, the JAK2/IRAK1 dual inhibitor, pacritinib, exhibited strong activities against pC ALK- ALCL, where the IL-1R pathway is hyperactivated in the cell line and xenograft mouse model. Thus, our studies revealed critical insights into the essential roles of the IL-1R pathway in pC-ALCL and provided opportunities for developing novel therapeutic strategies.
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Linfoma Anaplásico de Células Grandes , Linfoma Anaplásico Cutáneo Primario de Células Grandes , Neoplasias Cutáneas , Humanos , Animales , Ratones , Linfoma Anaplásico de Células Grandes/tratamiento farmacológico , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/patología , Proteínas Tirosina Quinasas Receptoras/genética , Quinasa de Linfoma Anaplásico/genética , Interleucinas/metabolismoRESUMEN
Adult T-cell leukemia/lymphoma (ATLL) is one of the aggressive peripheral T-cell neoplasms with a poor prognosis. Accumulating evidence demonstrates that escape from adaptive immunity is a hallmark of ATLL pathogenesis. However, the mechanisms by which ATLL cells evade natural killer (NK)-cell-mediated immunity have been poorly understood. Here we show that CD48 expression in ATLL cells determines the sensitivity for NK-cell-mediated cytotoxicity against ATLL cells. We performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening using 2 ATLL-derived cell lines and discovered CD48 as one of the best-enriched genes whose knockout conferred resistance to YT1-NK cell line-mediated cytotoxicity. The ability of CD48-knockout ATLL cells to evade NK-cell effector function was confirmed using human primary NK cells with reduced interferon-γ (IFNγ) induction and degranulation. We found that primary ATLL cells had reduced CD48 expression along with disease progression. Furthermore, other subgroups among aggressive peripheral T-cell lymphomas (PTCLs) also expressed lower concentrations of CD48 than normal T cells, suggesting that CD48 is a key molecule in malignant T-cell evasion of NK-cell surveillance. Thus, this study demonstrates that CD48 expression is likely critical for malignant T-cell lymphoma cell regulation of NK-cell-mediated immunity and provides a rationale for future evaluation of CD48 as a molecular biomarker in NK-cell-associated immunotherapies.
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Leucemia-Linfoma de Células T del Adulto , Linfoma de Células T Periférico , Adulto , Humanos , Antígeno CD48/genética , Antígeno CD48/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Leucemia-Linfoma de Células T del Adulto/genética , Linfoma de Células T Periférico/genética , Células Asesinas NaturalesRESUMEN
Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis with current therapy. Here we report genome-wide CRISPR-Cas9 screening of ATLL models, which identified CDK6, CCND2, BATF3, JUNB, STAT3, and IL10RB as genes that are essential for the proliferation and/or survival of ATLL cells. As a single agent, the CDK6 inhibitor palbociclib induced cell cycle arrest and apoptosis in ATLL models with wild-type TP53. ATLL models that had inactivated TP53 genetically were relatively resistant to palbociclib owing to compensatory CDK2 activity, and this resistance could be reversed by APR-246, a small molecule activator of mutant TP53. The CRISPR-Cas9 screen further highlighted the dependence of ATLL cells on mTORC1 signaling. Treatment of ATLL cells with palbociclib in combination with mTORC1 inhibitors was synergistically toxic irrespective of the TP53 status. This work defines CDK6 as a novel therapeutic target for ATLL and supports the clinical evaluation of palbociclib in combination with mTORC1 inhibitors in this recalcitrant malignancy.
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Leucemia-Linfoma de Células T del Adulto , Linfoma , Adulto , Apoptosis/genética , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , Humanos , Leucemia-Linfoma de Células T del Adulto/tratamiento farmacológico , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/patología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Transducción de SeñalRESUMEN
B cell receptor (BCR) signalling has emerged as a therapeutic target in B cell lymphomas, but inhibiting this pathway in diffuse large B cell lymphoma (DLBCL) has benefited only a subset of patients1. Gene expression profiling identified two major subtypes of DLBCL, known as germinal centre B cell-like and activated B cell-like (ABC)2,3, that show poor outcomes after immunochemotherapy in ABC. Autoantigens drive BCR-dependent activation of NF-κB in ABC DLBCL through a kinase signalling cascade of SYK, BTK and PKCß to promote the assembly of the CARD11-BCL10-MALT1 adaptor complex, which recruits and activates IκB kinase4-6. Genome sequencing revealed gain-of-function mutations that target the CD79A and CD79B BCR subunits and the Toll-like receptor signalling adaptor MYD885,7, with MYD88(L265P) being the most prevalent isoform. In a clinical trial, the BTK inhibitor ibrutinib produced responses in 37% of cases of ABC1. The most striking response rate (80%) was observed in tumours with both CD79B and MYD88(L265P) mutations, but how these mutations cooperate to promote dependence on BCR signalling remains unclear. Here we used genome-wide CRISPR-Cas9 screening and functional proteomics to determine the molecular basis of exceptional clinical responses to ibrutinib. We discovered a new mode of oncogenic BCR signalling in ibrutinib-responsive cell lines and biopsies, coordinated by a multiprotein supercomplex formed by MYD88, TLR9 and the BCR (hereafter termed the My-T-BCR supercomplex). The My-T-BCR supercomplex co-localizes with mTOR on endolysosomes, where it drives pro-survival NF-κB and mTOR signalling. Inhibitors of BCR and mTOR signalling cooperatively decreased the formation and function of the My-T-BCR supercomplex, providing mechanistic insight into their synergistic toxicity for My-T-BCR+ DLBCL cells. My-T-BCR supercomplexes characterized ibrutinib-responsive malignancies and distinguished ibrutinib responders from non-responders. Our data provide a framework for the rational design of oncogenic signalling inhibitors in molecularly defined subsets of DLBCL.
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Carcinogénesis , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Complejos Multiproteicos/metabolismo , Transducción de Señal , Adenina/análogos & derivados , Animales , Biopsia , Sistemas CRISPR-Cas/genética , Carcinogénesis/genética , Diseño de Fármacos , Femenino , Humanos , Linfoma de Células B Grandes Difuso/genética , Ratones , Complejos Multiproteicos/química , Mutación , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/metabolismo , Piperidinas , Proteómica , Pirazoles/farmacología , Pirazoles/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Receptores de Antígenos de Linfocitos B/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The success of programmed cell death protein 1 (PD-1)/PD-L1-based immunotherapy highlights the critical role played by PD-L1 in cancer progression and reveals an urgent need to develop new approaches to attenuate PD-L1 function by gaining insight into how its expression is controlled. Anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma (ALK+ ALCL) expresses a high level of PD-L1 as a result of the constitutive activation of multiple oncogenic signaling pathways downstream of ALK activity, making it an excellent model in which to define the signaling processes responsible for PD-L1 upregulation in tumor cells. Here, using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 library screening, we sought a comprehensive understanding of the molecular effectors required for PD-L1 regulation in ALK+ ALCL. Indeed, we determined that PD-L1 induction is dependent on the nucleophosmin-ALK oncoprotein activation of STAT3, as well as a signalosome containing GRB2/SOS1, which activates the MEK-ERK and PI3K-AKT signaling pathways. These signaling networks, through STAT3 and the GRB2/SOS1, ultimately induce PD-L1 expression through the action of transcription factors IRF4 and BATF3 on the enhancer region of the PD-L1 gene. IRF4 and BATF3 are essential for PD-L1 upregulation, and IRF4 expression is correlated with PD-L1 levels in primary ALK+ ALCL tissues. Targeting this oncogenic signaling pathway in ALK+ ALCL largely inhibited the ability of PD-L1-mediated tumor immune escape when cocultured with PD-1-positive T cells and natural killer cells. Thus, our identification of this previously unrecognized regulatory hub not only accelerates our understanding of the molecular circuitry that drives tumor immune escape but also provides novel opportunities to improve immunotherapeutic intervention strategies.
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Antígeno B7-H1/biosíntesis , Regulación Neoplásica de la Expresión Génica/fisiología , Linfoma Anaplásico de Células Grandes/metabolismo , Transducción de Señal/fisiología , Quinasa de Linfoma Anaplásico/genética , Quinasa de Linfoma Anaplásico/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Humanos , Linfoma Anaplásico de Células Grandes/genética , Regulación hacia ArribaRESUMEN
Antithymocyte globulin (ATG) reduces severe acute and chronic graft-versus-host disease (GVHD) in allogeneic peripheral blood stem cell transplantation (PBSCT). However, risk factors for severe acute GVHD in PBSCT using ATG remain to be determined. We conducted a single-center, retrospective study to analyze the association of acute GVHD requiring systemic corticosteroid (SC-aGVHD) with absolute lymphocyte counts (ALC) before the administration of ATG or conditioning in 53 patients with HLA-matched PBSCT using low-dose thymoglobulin (2 mg/kg) after myeloablative conditioning. The cumulative incidence of SC-aGVHD was 17.0% and ALC before ATG were significantly higher in patients with SC-aGVHD compared to that in patients without it (median, 0.15 × 109/L vs 0.06 × 109/L, P = 0.047). The cumulative incidence of SC-aGVHD was significantly higher in patients with high ALC before ATG (≥ 0.15 × 109/L) than in those with low ALC (38.5% vs 10.0%, P = 0.016). Non-relapse mortality (NRM) was also significantly higher in the high ALC before ATG group than the low ALC before ATG group (2-year NRM: 23.9% vs 6.0%, P = 0.048), leading to worse survival (2-year overall survival: 69.2% vs 83.5%, P = 0.039). Our study suggested that high ALC before ATG is a risk factor for SC-aGVHD.
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Suero Antilinfocítico/uso terapéutico , Enfermedad Injerto contra Huésped/diagnóstico , Inmunosupresores/uso terapéutico , Recuento de Linfocitos , Trasplante de Células Madre de Sangre Periférica , Corticoesteroides/administración & dosificación , Corticoesteroides/efectos adversos , Corticoesteroides/uso terapéutico , Adulto , Anciano , Suero Antilinfocítico/administración & dosificación , Femenino , Enfermedad Injerto contra Huésped/sangre , Enfermedad Injerto contra Huésped/etiología , Humanos , Inmunosupresores/administración & dosificación , Masculino , Persona de Mediana Edad , Trasplante de Células Madre de Sangre Periférica/efectos adversos , Trasplante de Células Madre de Sangre Periférica/métodos , Pronóstico , Estudios Retrospectivos , Acondicionamiento Pretrasplante/efectos adversos , Acondicionamiento Pretrasplante/métodos , Adulto JovenRESUMEN
Although a combination of calcineurin inhibitor and methotrexate (MTX) is used for graft-versus-host disease (GVHD) prophylaxis in umbilical cord blood transplantation (CBT), optimal dose of MTX for CBT remains to be determined.We conducted a retrospective study to evaluate the safety and efficacy of standard-dose MTX (St-MTX, 15 mg/m2 on day 1 and 10 mg/m2 on days 3 and 6) and mini-dose MTX (Mini-MTX, 5 mg/m2 on days 1, 3 and 6) for GVHD prophylaxis in patients who underwent single unit CBT against hematological malignancies.Thirty-two and 26 patients received St-MTX and Mini-MTX, respectively. Cumulative incidence of neutrophil engraftment was significantly higher in the Mini-MTX group than in the St-MTX group (88.5% vs 65.6%, P = 0.00448). Cumulative incidences of grade II to IV and grade III to IV of acute graft-versus-host disease (GVHD) were 34.4% and 6.2% in the St-MTX group, and 34.6% and 7.7% in the Mini-MTX group with no statistical significance. One-year non-relapse mortality (NRM) was significantly lower in the Mini-MTX group compared to the St-MTX group (31.2% vs 3.8%, P = 0.00938), whereas relapse rate was not different between the groups. Multivariate analysis also indicated that Mini-MTX significantly improved engraftment (HR, 0.5359; 95% CI, 0.3082 to 0.9318; P = 0.0270) and reduced NRM (HR, 0.117; 95% CI, 0.0151 to 0.9067; P = 0.040).Our study suggests that GVHD prophylaxis using Mini-MTX in CBT is feasible and associated with improvement of engraftment and reduction in NRM.
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Trasplante de Células Madre de Sangre del Cordón Umbilical , Supervivencia de Injerto/efectos de los fármacos , Enfermedad Injerto contra Huésped , Neoplasias Hematológicas , Metotrexato/administración & dosificación , Adolescente , Adulto , Anciano , Aloinjertos , Supervivencia sin Enfermedad , Femenino , Enfermedad Injerto contra Huésped/mortalidad , Enfermedad Injerto contra Huésped/prevención & control , Neoplasias Hematológicas/mortalidad , Neoplasias Hematológicas/terapia , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
Activating Janus kinase (JAK) and signal transducer and activator of transcription (STAT) mutations have been discovered in many T-cell malignancies, including anaplastic lymphoma kinase (ALK)- anaplastic large cell lymphomas (ALCLs). However, such mutations occur in a minority of patients. To investigate the clinical application of targeting JAK for ALK- ALCL, we treated ALK- cell lines of various histological origins with JAK inhibitors. Interestingly, most exogenous cytokine-independent cell lines responded to JAK inhibition regardless of JAK mutation status. JAK inhibitor sensitivity correlated with the STAT3 phosphorylation status of tumor cells. Using retroviral shRNA knockdown, we have demonstrated that these JAK inhibitor-sensitive cells are dependent on both JAK1 and STAT3 for survival. JAK1 and STAT3 gain-of-function mutations were found in some, but not all, JAK inhibitor-sensitive cells. Moreover, the mutations alone cannot explain the JAK1/STAT3 dependency, given that wild-type JAK1 or STAT3 was sufficient to promote cell survival in the cells that had either JAK1or STAT3 mutations. To investigate whether other mechanisms were involved, we knocked down upstream receptors GP130 or IL-2Rγ. Knockdown of GP130 or IL-2Rγ induced cell death in selected JAK inhibitor-sensitive cells. High expression levels of cytokines, including IL-6, were demonstrated in cell lines as well as in primary ALK- ALCL tumors. Finally, ruxolitinib, a JAK1/2 inhibitor, was effective in vivo in a xenograft ALK- ALCL model. Our data suggest that cytokine receptor signaling is required for tumor cell survival in diverse forms of ALK- ALCL, even in the presence of JAK1/STAT3 mutations. Therefore, JAK inhibitor therapy might benefit patients with ALK- ALCL who are phosphorylated STAT3.
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Janus Quinasa 1/genética , Linfoma Anaplásico de Células Grandes/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Citocinas/metabolismo , Factor de Transcripción STAT3/genética , Quinasa de Linfoma Anaplásico , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Receptor gp130 de Citocinas/genética , Receptor gp130 de Citocinas/metabolismo , Citocinas/genética , Citocinas/metabolismo , Humanos , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 1/metabolismo , Linfoma Anaplásico de Células Grandes/tratamiento farmacológico , Linfoma Anaplásico de Células Grandes/genética , Ratones , Nitrilos , Fosforilación , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Citocinas/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The requirement for the B-cell transcription factor OCT2 (octamer-binding protein 2, encoded by Pou2f2) in germinal center B cells has proved controversial. Here, we report that germinal center B cells are formed normally after depletion of OCT2 in a conditional knockout mouse, but their proliferation is reduced and in vivo differentiation to antibody-secreting plasma cells is blocked. This finding led us to examine the role of OCT2 in germinal center-derived lymphomas. shRNA knockdown showed that almost all diffuse large B-cell lymphoma (DLBCL) cell lines are addicted to the expression of OCT2 and its coactivator OCA-B. Genome-wide chromatin immunoprecipitation (ChIP) analysis and gene-expression profiling revealed the broad transcriptional program regulated by OCT2 that includes the expression of STAT3, IL-10, ELL2, XBP1, MYC, TERT, and ADA. Importantly, genetic alteration of OCT2 is not a requirement for cellular addiction in DLBCL. However, we detected amplifications of the POU2F2 locus in DLBCL tumor biopsies and a recurrent mutation of threonine 223 in the DNA-binding domain of OCT2. This neomorphic mutation subtly alters the DNA-binding preference of OCT2, leading to the transactivation of noncanonical target genes including HIF1a and FCRL3 Finally, by introducing mutations designed to disrupt the OCT2-OCA-B interface, we reveal a requirement for this protein-protein interface that ultimately might be exploited therapeutically. Our findings, combined with the predominantly B-cell-restricted expression of OCT2 and the absence of a systemic phenotype in our knockout mice, suggest that an OCT2-targeted therapeutic strategy would be efficacious in both major subtypes of DLBCL while avoiding systemic toxicity.
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Linfocitos B/citología , Diferenciación Celular , Supervivencia Celular , Linfoma de Células B Grandes Difuso/patología , Proteínas de Transporte de Catión Orgánico/fisiología , Animales , Línea Celular Tumoral , Ratones , Ratones Noqueados , Proteínas de Transporte de Catión Orgánico/genética , Transportador 2 de Cátion OrgánicoRESUMEN
Benign precursors of B lymphocytes, termed hematogones, are observed in the regenerative state of hematopoiesis following chemotherapy or allogeneic hematopoietic stem cell transplantation (allo-HSCT). Previous studies have demonstrated that expansion of hematogones correlates with better clinical outcomes after allo-HSCT. We retrospectively analyzed the association between hematogones and clinical outcomes in 309 consecutive patients who underwent allo-HSCT, which is the largest population-based cohort reported so far. The incidence of hematogones was significantly higher in complete remission (CR) patients at the time of transplantation than in non-CR patients, after myeloablative conditioning than after reduced-intensity conditioning, with tacrolimus-based graft-versus-host disease (GVHD) prophylaxis than with cyclosporine-based prophylaxis, and with disease other than malignant lymphoma (all P < .05). Patients with hematogones developed less acute GVHD and infections than did those without them (P < .05). Emergence of hematogones was associated with superior GVHD-free relapse-free survival and lower nonrelapse mortality, and was an independent prognostic factor for overall survival, irrespective of donor sources.
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Neoplasias Hematológicas , Trasplante de Células Madre Hematopoyéticas , Acondicionamiento Pretrasplante , Adolescente , Adulto , Anciano , Aloinjertos , Ciclosporina/administración & dosificación , Supervivencia sin Enfermedad , Femenino , Enfermedad Injerto contra Huésped/mortalidad , Enfermedad Injerto contra Huésped/prevención & control , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/mortalidad , Neoplasias Hematológicas/patología , Neoplasias Hematológicas/terapia , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Inducción de Remisión , Estudios Retrospectivos , Tasa de Supervivencia , Tacrolimus/administración & dosificaciónRESUMEN
We identified a novel heterozygous ITGB3 p.T720del mutation in a pedigree with macrothrombocytopenia exhibiting aggregation dysfunction. Platelet aggregation induced by ADP and collagen was significantly reduced, while ristocetin aggregation was normal. Integrin αIIbß3 was partially activated in a resting status, but platelet expression of αIIbß3 was downregulated. Functional analysis using a cell line showed spontaneous phosphorylation of FAK in αIIb/ß3 (p.T720del)-transfected 293T cells in suspension conditions. Abnormal cytoplasmic protrusions, membrane ruffling, and cytoplasmic localization of αIIbß3 were observed in αIIb/ß3 (p.T720del)-transfected CHO cells. Such morphological changes were reversed by treatment with an FAK inhibitor. These findings imply spontaneous, but partial, activation of αIIbß3 followed by phosphorylation of FAK as the initial mechanism of abnormal thrombopoiesis. Internalization and decreased surface expression of αIIbß3 would contribute to aggregation dysfunction. We reviewed the literature of congenital macrothrombocytopenia associated with heterozygous ITGA2B or ITGB3 mutations. Reported mutations were highly clustered at the membrane proximal region of αIIbß3, which affected the critical interaction between αIIb R995 and ß3 D723, resulting in a constitutionally active form of the αIIbß3 complex. Macrothrombocytopenia caused by a heterozygous activating mutation of ITGA2B or ITGB3 at the membrane proximal region forms a distinct entity of rare congenital thrombocytopenia.
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Eliminación de Gen , Genes Dominantes , Heterocigoto , Integrina beta3/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/agonistas , Trombocitopenia/genética , Adulto , Animales , Células CHO , Cricetulus , Salud de la Familia , Femenino , Células HEK293 , Humanos , Integrina beta3/metabolismo , Japón , Masculino , Persona de Mediana Edad , Mutagénesis Sitio-Dirigida , Linaje , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Proteínas Recombinantes/metabolismo , Trombocitopenia/sangre , Trombocitopenia/metabolismo , Trombocitopenia/fisiopatología , Adulto JovenRESUMEN
BackgroundIn the clinical setting, verapamil is contraindicated in neonates and infants, because of the perceived risk of hypotension or bradyarrhythmia. However, it remains unclear whether there is an age-dependent difference in the sensitivity of cardiac L-type Ca2+ channel current (ICa,L) to inhibition by verapamil.MethodsVentricular myocytes were enzymatically dissociated from the hearts of six different age groups (0, 7, 14, 21, 28 days, and 10-15 weeks) of mice, using a similar Langendorff-perfusion method. Whole-cell patch-clamp technique was applied to examine the sensitivity of ICa,L to inhibition, by three classes of structurally different L-type Ca2+ channel antagonists.ResultsVerapamil, nifedipine, and diltiazem concentration-dependently blocked the ventricular ICa,L in all six age groups. However, although nifedipine and diltiazem blocked ventricular ICa,L with a similar potency in all age groups, verapamil more potently blocked ventricular ICa,L in day 0, day 7, day 14, and day 21 mice, than in day 28, and 10-15-week mice.ConclusionIn a mouse heart model, ventricular ICa,L before the weaning age (~21 days of age) exhibited a higher sensitivity to inhibition by verapamil than that after the weaning age, which may explain one possible mechanism associated with the development of verapamil-induced hypotension in human neonates and infants.
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Bradicardia/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/metabolismo , Ventrículos Cardíacos/efectos de los fármacos , Miocardio/metabolismo , Verapamilo/farmacología , Animales , Diltiazem/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Corazón/efectos de los fármacos , Corazón/crecimiento & desarrollo , Ventrículos Cardíacos/crecimiento & desarrollo , Hipotensión , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/citología , Nifedipino/farmacología , Técnicas de Placa-Clamp , Perfusión , Riesgo , Factores de TiempoRESUMEN
Intestinal microbiota plays an important role in the regulation of allogeneic immune reaction after allogeneic hematopoietic stem cell transplantation (allo-SCT). Intestinal graft-vs-host disease (GVHD) is one of the major causes of mortality after allo-SCT and often complicated with intestinal dysbiosis. Recent studies suggest that antibiotic-induced dysbiosis is a risk factor for intestinal GVHD. We retrospectively evaluated the impacts of antibiotic use on the incidence of intestinal GVHD occurring before day 100 after allo-SCT. Among 213 patients who underwent allo-SCT, 200 patients achieving engraftment were analyzed. Antibiotics were classified into carbapenem, quinolone, penicillin, cephem, and glycopeptide. Among 128 patients who developed acute GVHD, intestinal GVHD developed in 36 patients. Patients with intestinal GVHD received significantly longer administration of carbapenem and glycopeptide compared to those without it in periengraftment period. In multivariate analysis, use of carbapenem for greater than 7 days was associated with an increased risk of intestinal GVHD. However, use of antibiotics for greater than 7 days was not associated with poor overall survival and high nonrelapse mortality. Long use of carbapenem in periengraftment period may be a risk for intestinal GVHD. Prospective studies are required to validate our findings.
Asunto(s)
Antibacterianos/efectos adversos , Microbioma Gastrointestinal/efectos de los fármacos , Enfermedad Injerto contra Huésped/epidemiología , Neoplasias Hematológicas/terapia , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Enfermedades Intestinales/tratamiento farmacológico , Adolescente , Adulto , Anciano , Femenino , Estudios de Seguimiento , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/patología , Humanos , Enfermedades Intestinales/microbiología , Japón/epidemiología , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Retrospectivos , Factores de Riesgo , Adulto JovenRESUMEN
In order to assess the development, approval and early introduction into clinical practice of biologics in the pediatric field, we herein describe the current status of the development to approval of biologics as anti-rheumatic agents for children in Japan, discuss the present problems and provide a proposal for the future. It has become apparent that the duration of the review period required for the preparation of clinical trials and Pharmaceuticals and Medical Devices Agency approval is clearly reduced compared with the past. Thus, it was speculated that a rate-limiting step in the process from development to approval was the duration of clinical trials from start to end. Hence, we focused on the following key words with regard to promotion of the development of biologics and their early practical use: "registry", "centralization", and "global cooperation", all of which are related to the reduction of duration of a clinical trial. In conclusion, to reduce the duration of a clinical trial, it is essential to complete a world-scale registry system by developing the registry system established by the Pediatric Rheumatology Association of Japan. The next step is then to carefully plan to participate in the international network using the world-scale registry system, and develop global cooperative trials in which we can ensure a sufficient number of entries from Japan.
Asunto(s)
Antirreumáticos/uso terapéutico , Productos Biológicos/uso terapéutico , Ensayos Clínicos como Asunto/métodos , Aprobación de Drogas/métodos , Niño , Humanos , Japón , Pediatría , Sistema de Registros , Reumatología/métodos , Factores de TiempoRESUMEN
With the emerging success of treating CD19 expressing B cell malignancies with ex vivo modified, autologous T cells that express CD19-directed chimeric antigen receptors (CAR), there is intense interest in expanding this evolving technology to develop effective modalities to treat other malignancies including solid tumors. Exploiting this approach to develop a therapeutic modality for T cell malignancies for which the available regimens are neither curative, nor confer long term survival we generated a lentivirus-based CAR gene transfer system to target the chemokine receptor CCR4 that is over-expressed in a spectrum of T cell malignancies as well as in CD4+ CD25+ Foxp3+ T regulatory cells that accumulate in the tumor microenvironment constituting a barrier against anti-tumor immunity. Ex vivo modified, donor-derived T cells that expressed CCR4 directed CAR displayed antigen-dependent potent cytotoxicity against patient-derived cell lines representing ATL, CTCL, ALCL and a subset of HDL. Furthermore, these CAR T cells also eradicated leukemia in a mouse xenograft model of ATL illustrating the potential utility of this modality in the treatment of a wide spectrum of T cell malignancies.
Asunto(s)
Neoplasias Hematológicas , Receptores de Antígenos de Linfocitos T , Receptores CCR4/antagonistas & inhibidores , Linfocitos T Reguladores/inmunología , Animales , Línea Celular Tumoral , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/patología , Neoplasias Hematológicas/terapia , Humanos , Leucemia de Células T/genética , Leucemia de Células T/inmunología , Leucemia de Células T/patología , Leucemia de Células T/terapia , Ratones , Ratones Endogámicos NOD , Ratones SCID , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores CCR4/genética , Receptores CCR4/inmunología , Linfocitos T Reguladores/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We herein report a patient who had disseminated toxoplasmosis after hematopoietic stem cell transplantation showing atypical clinical presentation and neuroimaging. Parkinsonism symptoms such as muscle rigidity, bradykinesia, tremor, and postural instability were initial manifestations. Magnetic resonance imaging showed diffuse symmetrical lesions of bilateral basal ganglia lacking ringed enhancement. Post-mortem analysis revealed multiple tachyzoites of Toxoplasma gondii in the basal ganglia, mid brain, cerebellum, and cardiac muscle.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas/efectos adversos , Leucemia Mieloide/cirugía , Trastornos Parkinsonianos/diagnóstico por imagen , Toxoplasma/aislamiento & purificación , Toxoplasmosis Cerebral/diagnóstico por imagen , Encéfalo/diagnóstico por imagen , Encéfalo/parasitología , Encéfalo/patología , Resultado Fatal , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Trastornos Parkinsonianos/etiología , Toxoplasmosis Cerebral/complicaciones , Toxoplasmosis Cerebral/parasitología , Toxoplasmosis Cerebral/patologíaRESUMEN
Noonan syndrome is characterized by short stature, facial dysmorphia and a wide spectrum of congenital heart defects. Mutations of PTPN11, KRAS and SOS1 in the RAS-MAPK pathway cause approximately 60% of cases of Noonan syndrome. However, the gene(s) responsible for the remainder are unknown. We have identified five different mutations in RAF1 in ten individuals with Noonan syndrome; those with any of four mutations causing changes in the CR2 domain of RAF1 had hypertrophic cardiomyopathy (HCM), whereas affected individuals with mutations leading to changes in the CR3 domain did not. Cells transfected with constructs containing Noonan syndrome-associated RAF1 mutations showed increased in vitro kinase and ERK activation, and zebrafish embryos with morpholino knockdown of raf1 demonstrated the need for raf1 for the development of normal myocardial structure and function. Thus, our findings implicate RAF1 gain-of-function mutations as a causative agent of a human developmental disorder, representing a new genetic mechanism for the activation of the MAPK pathway.
Asunto(s)
Mutación Missense , Síndrome de Noonan/genética , Proteínas Proto-Oncogénicas c-raf/genética , Animales , Línea Celular , Línea Celular Transformada , Femenino , Corazón/embriología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Miocardio/metabolismo , Estructura Terciaria de Proteína , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas c-raf/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
BACKGROUND: The aim of this study was to determine whether electrocardiographic signs correlate with hemodynamics and the magnitude of the intracardiac shunt in children with ostium secundum atrial septal defects (ASD). METHODS: A total of 100 ASD patients (median age, 6 years 4 months; 54 girls) underwent cardiac catheterization between August 1980 and April 2010. We retrospectively investigated the relationship between electrocardiographic signs and the pulmonary/systemic blood flow ratio (Qp/Qs) in these patients. We also compared 63 postoperative electrocardiograms with those recorded before surgery. RESULTS: The mean Qp/Qs ratio of the 100 patients was 2.46 ± 0.81 (range, 1.1-5.0). The Qp/Qs ratio in patients with and without right bundle branch block (RBBB) was 2.57 ± 0.82 (n = 73) and 2.15 ± 0.72 (n = 27), respectively (P = 0.016). The Qp/Qs ratio in patients with and without isolated negative T-wave was 2.85 ± 0.87 (n = 38) and 2.22 ± 0.68 (n = 62), respectively (P = 0.0003). None of the patients with low Qp/Qs ratio (Qp/Qs ratio ≤ 1.5) had both RBBB and isolated negative T-wave. The prevalence of these two signs decreased from 73.0% (n = 46) and 36.5% (n = 23) to 15.9% (n = 10) and 15.9% (n = 10) after surgical repair, respectively. CONCLUSIONS: RBBB and isolated negative T-wave in the precordial leads are well correlated with high Qp/Qs ratio in ASD patients.
Asunto(s)
Procedimientos Quirúrgicos Cardíacos/métodos , Electrocardiografía , Defectos del Tabique Interatrial/diagnóstico , Adolescente , Cateterismo Cardíaco , Niño , Preescolar , Ecocardiografía/métodos , Femenino , Defectos del Tabique Interatrial/fisiopatología , Defectos del Tabique Interatrial/cirugía , Humanos , Lactante , Masculino , Periodo Preoperatorio , Estudios RetrospectivosRESUMEN
Clonal heterogeneity in lymphoid malignancies has been recently reported in adult T-cell lymphoma/leukemia, peripheral T-cell lymphoma, not otherwise specified, and mantle cell lymphoma. Our analysis was extended to other types of lymphoma including marginal zone lymphoma, follicular lymphoma and diffuse large B-cell lymphoma. To determine the presence of clonal heterogeneity, 332 cases were examined using array comparative genomic hybridization analysis. Results showed that incidence of clonal heterogeneity varied from 25% to 69% among different types of lymphoma. Survival analysis revealed that mantle cell lymphoma and diffuse large B-cell lymphoma with clonal heterogeneity showed significantly poorer prognosis, and that clonal heterogeneity was confirmed as an independent predictor of poor prognosis for both types of lymphoma. Interestingly, 8q24.1 (MYC) gain, 9p21.3 (CDKN2A/2B) loss and 17p13 (TP53, ATP1B2, SAT2, SHBG) loss were recurrent genomic lesions among various types of lymphoma with clonal heterogeneity, suggesting at least in part that alterations of these genes may play a role in clonal heterogeneity.