RESUMEN
BACKGROUND: Genetic aberrations in the NFκB pathway lead to primary immunodeficiencies with various degrees of severity. We previously demonstrated that complete ablation of the RelB transcription factor, a key component of the alternative pathway, results in an early manifested combined immunodeficiency requiring stem cell transplantation. OBJECTIVE: To study the molecular basis of a progressive severe autoimmunity and immunodeficiency in three patients. METHODS: Whole exome sequencing was performed to identify the genetic defect. Molecular and cellular techniques were utilized to assess the variant impact on NFκB signaling, canonical and alternative pathway crosstalk, as well as the resultant effects on immune function. RESULTS: Patients presented with multiple autoimmune progressive severe manifestations encompassing the liver, gut, lung, and skin, becoming debilitating in the second decade of life. This was accompanied by a deterioration of the immune system, demonstrating an age-related decline in naïve T cells and responses to mitogens, accompanied by a gradual loss of all circulating CD19+ cells. Whole exome sequencing identified a novel homozygous c. C1091T (P364L) transition in RELB. The P364L RelB protein was unstable, with extremely low expression, but retained some function and could be transiently and partially upregulated following Toll-like receptor stimulation. Stimulation of P364L patient fibroblasts resulted in a marked rise in a cluster of pro-inflammatory hyper-expressed transcripts consistent with the removal of RelB inhibitory effect on RelA function. This is likely the main driver of autoimmune manifestations in these patients. CONCLUSION: Incomplete loss of RelB provided a unique opportunity to gain insights into NFκB's pathway interactions as well as the pathogenesis of autoimmunity. The P364L RelB mutation leads to gradual decline in immune function with progression of severe debilitating autoimmunity.
Asunto(s)
Enfermedades Autoinmunes , Factor de Transcripción ReIB , Humanos , Factor de Transcripción ReIB/genética , Factor de Transcripción ReIB/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Regulación de la Expresión Génica , Enfermedades Autoinmunes/genéticaRESUMEN
Background: STAT1 gain-of-function (GOF) is a primary immune dysregulatory disorder marked by wide infectious predisposition (most notably chronic mucocutaneous Candidiasis), autoimmunity, vascular disease and malignant predisposition. While atopic features have been described in some STAT1 GOF patients, they are not considered a predominant feature of the disease. Additionally, while eosinophilic gastrointestinal infiltration has been reported in some cases, this has always been described in the context of pre-existing oropharyngeal and/or esophageal Candidiasis. Clinical cases: Herein, we report 3 members of a multi-generational family diagnosed with STAT1 GOF caused by a novel mutation in the N-terminal domain, c.194A>C (p.D65A). The proband presented initially with a long-standing history of treatment-refractory eosinophilic esophagitis (EoE) without preceding gastrointestinal tract fungal infections, and her mother was diagnosed with esophagitis as well. Conclusion: EoE has been previously associated with alterations to STAT6 and STAT3 signaling pathways. The current report expands the possible association between JAK/STAT-related disorders and EoE, suggesting that EoE could be a primary disease manifestation of STAT1 GOF, even in the absence of oropharyngeal and/or esophageal Candidiasis.
Asunto(s)
Esofagitis Eosinofílica/diagnóstico , Esofagitis Eosinofílica/etiología , Mutación con Ganancia de Función , Factor de Transcripción STAT1/genética , Alelos , Biomarcadores , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Factor de Transcripción STAT1/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Genetic faults in several components of the nuclear factor-κB pathway cause immunodeficiency. Most defects lead to combined immunodeficiency with a range of severity. Heterozygous mutations in NFKB1 were associated with common variable immunodeficiency, however, homozygous mutations have not been described. OBJECTIVE: We studied the molecular basis of combined immunodeficiency in a patient who presented with failure to thrive, persistent EBV viremia and hepatitis, pneumocystis jirovecii pneumonitis, and generalized lymphadenopathy. METHODS: Whole genome and exome sequencing followed by Sanger confirmation were performed to identify the genetic defect. Molecular and cellular techniques were used to assess the variant impact on the nuclear factor-κB pathway and lymphocyte function. RESULTS: Genetic analysis revealed a novel homozygous mutation in NFKB1, c.2878G>A, p.Gly960Arg (G960R). This affected p105 phosphorylation and p50 formation on antigen and cytokine stimulation, as well as attenuating nuclear signal transmission. As a result, both T- and B-cell maturation and function were perturbed. The number of memory CD4+ T cells were reduced, while CD8+ T cells consisted predominately of expanded differentiated populations. The function of T cells were diminished as shown by reduced responses to mitogens as well as diminished cytokine secretion. B-cell maturation was also affected, with decreased IgD+CD27+ memory B cells while transitional B cells were increased, likely contributing to the reduced ability to produce specific antibodies. CONCLUSION: Homozygous G960R mutation in NFKB1 leads to a severe clinical presentation of combined immunodeficiency. This was associated with blockade of nuclear factor-κB pathway signaling, resulting in aberrations in T- and B-cell maturation and function.
Asunto(s)
Subunidad p50 de NF-kappa B/genética , Inmunodeficiencia Combinada Grave/genética , Homocigoto , Humanos , Lactante , Masculino , Mutación , LinajeRESUMEN
Signal transducer and activator of transcription 1 (STAT1) regulates multiple biological processes downstream of a variety of cytokine receptors in many cell types. Heterozygous gain-of-function (GOF) mutations in STAT1 have been associated with a diverse phenotype encompassing chronic mucocutaneous candidiasis (CMCC) and declining immunity. There is no clear correlation between STAT1 domain-specific mutations and phenotype, and it remains unclear why GOF mutations in STAT1 result in such a wide spectrum of clinical presentations. To begin exploring this dilemma, we have studied the patterns of gene expression mediated by two different GOF mutations. Analysis of IFN-γ response elements using RNA microarrays in cells transfected with the rare H629Y mutant or the common R274G mutant showed distinct patterns of gene expression. We show here that the impact of GOF mutations in STAT1 is variant-specific. This difference in gene expression may explain the diversity in clinical manifestations experienced by these patients.
RESUMEN
BACKGROUND: Combined immunodeficiency (CID) is a T-cell defect frequently presenting with recurrent infections, as well as associated immune dysregulation manifesting as autoimmunity or allergic inflammation. OBJECTIVE: We sought to identify the genetic aberration in 4 related patients with CID, early-onset asthma, eczema, and food allergies, as well as autoimmunity. METHODS: We performed whole-exome sequencing, followed by Sanger confirmation, assessment of the genetic variant effect on cell signaling, and evaluation of the resultant immune function. RESULTS: A heterozygous novel c.C88T 1-bp substitution resulting in amino acid change R30W in caspase activation and recruitment domain family member 11 (CARD11) was identified by using whole-exome sequencing and segregated perfectly to family members with severe atopy only but was not found in healthy subjects. We demonstrate that the R30W mutation results in loss of function while also exerting a dominant negative effect on wild-type CARD11. The CARD11 defect altered the classical nuclear factor κB pathway, resulting in poor in vitro T-cell responses to mitogens and antigens caused by reduced secretion of IFN-γ and IL-2. CONCLUSION: Unlike patients with biallelic mutations in CARD11 causing severe CID, the R30W defect results in a less profound yet prominent susceptibility to infections, as well as multiorgan atopy and autoimmunity.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/inmunología , Guanilato Ciclasa/genética , Guanilato Ciclasa/inmunología , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Adulto , Proteínas Adaptadoras de Señalización CARD/deficiencia , Preescolar , Femenino , Guanilato Ciclasa/deficiencia , Humanos , Interferón gamma/genética , Interleucina-2/genética , Masculino , Mutación , FN-kappa B/genética , Estudios Prospectivos , Estudios Retrospectivos , Transducción de Señal/genética , Transducción de Señal/inmunología , Linfocitos T/inmunología , Secuenciación del Exoma/métodosRESUMEN
BACKGROUND: We previously reported a novel syndrome characterized by combined immunodeficiency associated with severe developmental defects-subsequently known as Roifman-Chitayat syndrome (RCS; OMIM 613328). Linkage analysis identified 2 disease-associated loci. OBJECTIVES: We sought to identify the genetic defect in these patients and characterize their immunologic cellular abnormalities. METHODS: Genetic, immunologic, protein, and cellular functional analyses were used to identify and characterize patient genetic deficiencies and aberrant patient cell behavior. RESULTS: Deleterious variants were found at both loci identified by linkage analysis: a homozygous stop codon in PI3-kinase p110δ (PIK3CD) and a homozygous frame shift mutation in SKAP (KNSTRN), both ablating protein expression. Patients with RCS display aberrant B-cell development, similar to p110δ-deficient mice, but also aberrant T-cell spreading, cell-cell interaction, and migration. Patients also display significant developmental abnormalities not seen in p110δ knockouts (eg, optic nerve atrophy and skeletal anomalies) that we ascribe to loss of SKAP. Aberrant SKAP expression can prolong anaphase and this may contribute to developmental defects. However, we also identified microtubule-associated protein 4 microtubule-binding protein as a novel SKAP-binding partner and show that it undergoes relocalization in patient T cells, with associated areas of aberrant microtubule hyperstabilization, likely contributing not only to the altered properties of RCS lymphoid cells but also to developmental defects. CONCLUSIONS: The complex RCS presentation, with combined developmental and immunologic defects, is associated with a combined deficiency of 2 genes products, PI3-kinase p110δ and SKAP, both of which appear to play a significant role in the disease.
Asunto(s)
Linfocitos B/inmunología , Enfermedades del Desarrollo Óseo/genética , Linfocitos T CD4-Positivos/inmunología , Proteínas de Ciclo Celular/genética , Fosfatidilinositol 3-Quinasa Clase I/genética , Síndromes de Inmunodeficiencia/genética , Proteínas Asociadas a Microtúbulos/genética , Enfermedades del Nervio Óptico/genética , Eliminación de Secuencia/genética , Adolescente , Ciclo Celular/genética , Diferenciación Celular/genética , Línea Celular , Preescolar , Discapacidades del Desarrollo/genética , Facies , Femenino , Humanos , Inmunofenotipificación , Lactante , Linaje , Enfermedades de Inmunodeficiencia Primaria , Hermanos , Secuenciación Completa del Genoma , Adulto JovenRESUMEN
Multiple receptors that control cell growth and inflammation activate the NFκB pathway that comprises of two pathways. Dysfunction of the classical pathway leads to impaired adaptive and innate immunity in humans. In contrast the exact role of the alternative NFκB pathway mediated by RelB in humans remains largely elusive. We have recently identified deleterious mutations in RelB in patients with combined immunodeficiency and autoimmunity. We studied here the biological effects of RelB deficiency on the immune system. We show that the thymus in this patient is dysplastic and consequently new thymus emigrants are rare and there is an accumulation of CD45 RO(+) T cells with an increase in CD62L(+) central memory cells. The TCR repertoire of these cells appears skewed with selective clonal expansion. In vitro responses to T cell mitogens were markedly depressed and so were PHA induced IL2 and IFNγ production. In addition, the TH1 promoting T bet and STAT1 were reduced. In contrast, hyper-activation was seen in response to anti-CD3 and CD28. T cell dependent antibody responses were low to absent in all patients. We found that BAFF-R was reduced and CD40 signaling aberrant. Critically, CD27(+) memory cells were absent. We have shown here for the first time the role of RelB on lymphocyte development in humans. In the absence of RelB, B cells development is arrested, resulting in poor production of immunoglobulins and specific antibodies. T cell maturation in the thymus appears altered with reduced output and production of a skewed T cell repertoire with expansion of clones which are likely the cause of the autoimmune features observed in these patients.
Asunto(s)
Linfocitos B/fisiología , Subgrupos de Linfocitos T/fisiología , Timo/anomalías , Factor de Transcripción ReIB/inmunología , Autoinmunidad , Diferenciación Celular/genética , Proliferación Celular , Preescolar , Humanos , Lactante , Recién Nacido , Interleucina-2/inmunología , Masculino , Subunidad p50 de NF-kappa B/inmunología , Transducción de Señal/inmunología , Timo/inmunología , Timo/patología , Factor de Transcripción ReIB/genéticaRESUMEN
BACKGROUND: Mutations in the gene for the signal transducer and activator of transcription 1, STAT1, have been shown to be associated with death at an early age due to overwhelming viral infection (complete STAT1 deficiency) or, more commonly, selective deficiencies to mycobacterial or fungal infection (typically heterozygous STAT1 mutations). OBJECTIVES: To define the molecular basis of progressive combined immunodeficiency in a group of patients with fatal infections. METHODS: We studied a group of unrelated patients who displayed an unusual progressive form of combined immunodeficiency. Whole exome sequencing assisted in confirming a common genetic defect in this group, which consisted of a heterozygous mutation of the STAT1 gene. STAT1 protein level as well as function was assessed, and a detailed evaluation of the immune system, including analysis of thymus tissue, was performed. RESULTS: Patients were found to carry de novo heterozygous mutations in STAT1 encoding T385A, I294T, or C284R amino acid substitutions. STAT1 expression appeared significantly decreased as a result of these changes but not completely absent, with diminished signaling responses. This group display progressive loss in lymphocyte number and function accompanied by increasing autoimmune features as well as severe, fatal infections. CONCLUSIONS: These findings show that some heterozygous aberrations of STAT1 can be associated with progressive combined immunodeficiency, quite distinct from the limited susceptibilities to infection previously reported for heterozygous STAT1 mutations. These mutations were not inherited, rather, arose de novo in each case. Accompanied by significant patient mortality, this finding suggests that this class of STAT1 mutation is ultimately fatal due to overwhelming infection.
Asunto(s)
Síndromes de Inmunodeficiencia/genética , Mutación , Factor de Transcripción STAT1/genética , Adolescente , Niño , Preescolar , Femenino , Heterocigoto , Humanos , Síndromes de Inmunodeficiencia/mortalidad , Masculino , Factor de Transcripción STAT1/análisis , Factor de Transcripción STAT1/fisiología , Receptor fas/análisisRESUMEN
BACKGROUND: ζ-associated protein, 70 kd (ZAP70), deficiency in human subjects results in a combined immunodeficiency characterized by normal numbers of circulating CD4 T cells and CD8 lymphocytopenia. Patients who live beyond infancy can also experience autoimmune manifestations. OBJECTIVES: We sought to further characterize the nature of the T-cell populations found in ZAP70-deficient patients and explored the mechanisms that might predispose them to autoimmunity. METHODS: T-cell development was assessed by examining T-cell receptor (TCR) gene rearrangements and thymopoiesis by measuring TCR exclusion circle levels. TCR repertoire on CD4 and CD8 T-cell populations was assessed by means of flow cytometry. T-cell gene expression patterns were examined by means of exonic microarray analysis and apoptotic responses by means of Annexin V binding and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. RESULTS: Cells displaying recombination events from all stages of TCR gene rearrangement were present in the peripheral blood of ZAP70-deficient patients; however, the late TCRD-deleting rearrangement was significantly reduced. TCR exclusion circle levels were also found to be low. Surprisingly, all Vß families were detected in both CD4(+) and CD8(+) circulating T cells. Several Vß families were significantly overrepresented, which is reminiscent of autoimmune disorders. Levels of mRNA for cytotoxic T lymphocyte-associated antigen 4, TGF-ß, and IL-10 were found to be low, a signature of autoimmunity. Finally, Fas-mediated CD4 T-cell apoptosis was found to be reduced in vitro, and staining of thymus biopsy specimens revealed reduced thymocyte apoptosis. CONCLUSION: We show that in the absence of ZAP70, thymopoiesis is altered and differentiation to double-positive cells is hampered. Circulating T cells appear poorly regulated, do not differentiate into T(H)2 T cells, lack a number of inhibitory growth controls, and display reduced apoptosis, all predisposing patients to exaggerated inflammation and autoimmunity.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Timo/metabolismo , Proteína Tirosina Quinasa ZAP-70/metabolismo , Apoptosis/genética , Autoinmunidad , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Procesos de Crecimiento Celular/genética , Procesos de Crecimiento Celular/inmunología , Genes Codificadores de los Receptores de Linfocitos T/inmunología , Humanos , Inflamación , Linfopenia , Análisis por Micromatrices , Inmunodeficiencia Combinada Grave/inmunología , Inmunodeficiencia Combinada Grave/patología , Inmunodeficiencia Combinada Grave/fisiopatología , Timo/inmunología , Timo/patología , Proteína Tirosina Quinasa ZAP-70/deficiencia , Proteína Tirosina Quinasa ZAP-70/genética , Proteína Tirosina Quinasa ZAP-70/inmunologíaRESUMEN
Cancer invasiveness plays a major role in the mortality of patients with solid tumors, and deregulated cell adhesion and migration are suspected to drive invasive behavior. Since Eph receptor tyrosine kinases control both cell attachment and migration, they may act to define the level of cancer invasiveness. EphB6 is an unusual Eph receptor, lacking catalytic capacity due to alterations in its kinase domain. Interestingly, increased metastatic activity is associated with reduced EphB6 receptor expression in several tumor types, including breast cancer. This emphasizes the potential of EphB6 to act as a suppressor of cancer aggressiveness; however, the mechanism of its action is not well understood. We show that restoration of EphB6 expression in invasive breast cancer cells supports actin-dependent spreading and attachment and blocks invasiveness. EphB6 stimulation induces its tyrosine phosphorylation, which is crucial for its function and is mediated by the EphB4 receptor. This is accompanied by EphB6-c-Cbl interaction and phosphorylation of c-Cbl partner, the Abl kinase. Cbl silencing suppresses Abl phosphorylation, cell adhesion, and morphologic changes and blocks the ability of EphB6 to inhibit invasiveness, confirming its importance for EphB6 activity. Despite its crucial role in EphB6 responses, EphB4 also acts in an EphB6-independent manner to enhance invasive activity, suggesting that cancer invasiveness may be defined by the balance in the EphB6-EphB4 system. Overall, our observations suggest a new role for EphB6 in suppressing cancer invasiveness through c-Cbl-dependent signaling, morphologic changes, and cell attachment and indicate that EphB6 may represent a useful prognostic marker and a promising target for therapeutic approaches.
Asunto(s)
Movimiento Celular , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Actinas/metabolismo , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Adhesión Celular , Línea Celular , Línea Celular Tumoral , Citoesqueleto/metabolismo , Fibronectinas/metabolismo , Humanos , Invasividad Neoplásica , Unión Proteica , Proteínas Proto-Oncogénicas c-cbl/genética , Interferencia de ARN , Proteínas Tirosina Quinasas Receptoras/genética , Receptor EphB4/genética , Receptor EphB4/metabolismo , Receptores de la Familia Eph , TransfecciónAsunto(s)
Sustitución de Aminoácidos , Candidiasis Mucocutánea Crónica/genética , Mutación Missense , Proteína Tirosina Fosfatasa no Receptora Tipo 22/genética , Adolescente , Adulto , Candidiasis Mucocutánea Crónica/enzimología , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Proteína Tirosina Fosfatasa no Receptora Tipo 22/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína AIRERESUMEN
Progressive interaction with other cells is critical to all aspects of T-cell biology - from migration through tissues, to recognition of antigen-presenting cells. We demonstrate a novel mechanism for regulating T lymphocyte adhesion and thus cell-cell interactions, showing that T-cell-expressed EphA and ephrin-A proteins not only regulate adhesive properties, but do so in a diametrically opposing fashion. Integrin-mediated adhesion is stimulated by ephrin-A activation, while EphA signalling is inhibitory. Increasing ephrin-A expression enhances T-cell interactions not only with purified integrin ligands but also endothelial cells, while EphA activation down-regulates these interactions. In accordance with an in vivo role for these proteins in regulating cell-cell interactions, activation of ephrin-A signalling was found to alter the trafficking of injected T lymphocytes to peripheral lymph nodes in vivo. Given the ubiquitous nature of EphA/ephrin-A expression in tissues, these proteins likely play a significant role in regulating T-cell interactions.
Asunto(s)
Efrina-A1/fisiología , Integrinas/metabolismo , Receptor EphA1/fisiología , Linfocitos T/fisiología , Animales , Adhesión Celular , Comunicación Celular , Células Cultivadas , Células Endoteliales/citología , Endotelio Vascular/citología , Efrina-A3 , Humanos , Ratones , Ratones Endogámicos BALB C , Receptor EphA2RESUMEN
Severe combined immunodeficiency (SCID) is a heterogeneous group of diseases that are invariably fatal in infancy unless treated by hematopoietic stem cell replacement. For many years we have worked to better manage patients affected by SCID through rapid and accurate diagnosis followed by treatment aimed at achieving long-lasting immune reconstitution. By extensive immunological, biochemical, and genetic studies of patient samples, and with the realization of differences between human and murine T cell development, we have successfully been able to identify some of the molecular defects causing SCID. Among these discoveries, we described the first mutated signal transduction protein in T cells (ZAP-70); the first genetic defect leading to SCID and autoimmune phenomena (IL2R alpha); and, recently, the critical importance of CD3delta in the development of T cells. Our efforts have significantly advanced the understanding of the role of some of the signal-transducing proteins in T cell maturation and function. This review summarizes several of these discoveries and some of their impact on our understanding of T cells development, function, and homeostasis in humans.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Síndromes de Inmunodeficiencia/genética , Inmunodeficiencia Combinada Grave/genética , Transducción de Señal/genética , Proteína Tirosina Quinasa ZAP-70/fisiología , Animales , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/enzimología , Humanos , Síndromes de Inmunodeficiencia/inmunología , Interleucinas/genética , Interleucinas/fisiología , Janus Quinasa 3 , Ratones , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/fisiología , Inmunodeficiencia Combinada Grave/enzimología , Inmunodeficiencia Combinada Grave/inmunología , Proteína Tirosina Quinasa ZAP-70/genéticaRESUMEN
The EphA receptor tyrosine kinases interact with membrane-bound ligands of the ephrin-A subfamily. Interaction induces EphA receptor oligomerization, tyrosine phosphorylation, and, as a result, EphA receptor signaling. EphA receptors have been shown to regulate cell survival, migration, and cell-cell and cell-matrix interactions. However, their functions in lymphoid cells are only beginning to be described. We show in this study that functional EphA receptors are expressed by murine thymocytes, including CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) subpopulations. We demonstrate that activation of EphA receptors by the ephrin-A1 ligand inhibits the anti-CD3-induced apoptosis of CD4(+)CD8(+) double-positive thymocytes. Furthermore, ephrin-A1 costimulation suppresses up-regulation of both the IL-2R alpha-chain (CD25) and early activation Ag CD69 and can block IL-2 production by CD4(+) single-positive cells. In agreement, EphA receptor activation in thymocytes also inhibits TCR-induced activation of the Ras-MAPK pathway. Our findings suggest that EphA receptor activation is antithetical to TCR signaling in thymocytes, and that the level of engagement by ephrin-A proteins on thymic APCs regulates thymocyte selection.
Asunto(s)
Anticuerpos/inmunología , Apoptosis/inmunología , Complejo CD3/inmunología , Receptores de la Familia Eph/inmunología , Receptores de la Familia Eph/metabolismo , Timo/citología , Timo/inmunología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Apoptosis/efectos de los fármacos , Células Cultivadas , Efrina-A1/farmacología , Femenino , Regulación de la Expresión Génica , Interleucina-2/metabolismo , Lectinas Tipo C , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos BALB C , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Interleucina-2/metabolismo , Timo/efectos de los fármacos , Timo/metabolismo , Proteínas ras/metabolismoRESUMEN
A series of substituted styryl-acrylonitriles was designed and synthesized. The new compounds, called tyrenes, were tested for the ability to inhibit acute lymphocytic leukemia (ALL) cancer cell growth, as well as on their toxicity to normal bone marrow (NBM) cells. The results showed that 3,4-dihydroxystyryl-acrylonitriles, in particular CR-4, revealed great potency as antitumor agents, and also exhibited low toxicity to normal cells. The effectiveness of these compounds with extended conjugation may be due to their possible functioning as reactive Michael acceptors.
Asunto(s)
Acrilonitrilo/análogos & derivados , Acrilonitrilo/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Estirenos/química , Estirenos/farmacología , Acrilonitrilo/metabolismo , Antineoplásicos/química , Línea Celular Tumoral , Humanos , Estirenos/síntesis químicaRESUMEN
In recent years, synthetic tyrosine kinase inhibitors have made a rapid transition from basic research to therapeutic application. These compounds represent a major clinical advance in the approach to cancer in their relative specificity of action and decreased toxicity. We report here the effects of a novel tyrosine kinase inhibitor CR4 that interferes with growth-promoting pathways to markedly inhibit the growth and survival of both Philadelphia-positive and -negative acute lymphoblastic leukemia (ALL) as well as acute myeloid leukemia (AML). While efficiently ablating leukemic cell growth, normal cell growth and differentiation remain unaffected by CR4. CR4 demonstrates an ability to inhibit the function of multiple growth-critical kinases and yet exhibits a low level of cytotoxicity. These findings suggest that CR4 may prove to be highly effective as a therapeutic agent.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Leucemia Mieloide/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Enfermedad Aguda , Animales , Antineoplásicos/farmacología , Antineoplásicos/toxicidad , División Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/toxicidad , Humanos , Leucemia Mieloide/patología , Leucemia Mieloide/prevención & control , Infiltración Leucémica/tratamiento farmacológico , Infiltración Leucémica/prevención & control , Ratones , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/prevención & control , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Eph receptor tyrosine kinases are expressed by T lineage cells, and stimulation with their ligands, the ephrins, has recently been shown to modulate T cell behavior. We show that ephrin-A1 stimulation of Jurkat T cells induces tyrosine phosphorylation of EphA3 receptors and cytoplasmic proteins, including the c-cbl proto-oncogene. Cbl phosphorylation was also observed in peripheral blood T cells. In contrast, stimulation of Jurkat cells with the EphB receptor ligand ephrin-B1 does not cause Cbl phosphorylation. EphA activation also induced Cbl association with Crk-L and Crk-II adapters, but not the related Grb2 protein. Induction of Cbl phosphorylation upon EphA activation appeared to be dependent upon Src family kinase activity, as Cbl phosphorylation was selectively abrogated by the Src family inhibitor 4-amino-5(4-chlorophenyl-7-(tert-butyl)pyrazolo[3,4-d]pyrimidine, while EphA phosphorylation was unimpaired. Ephrin-A1 stimulation of Jurkat cells was also found to cause down-regulation of endogenous EphA3 receptors from the cell surface and their degradation. In accordance with the role of Cbl as a negative regulator of receptor tyrosine kinases, overexpression of wild-type Cbl, but not its 70-Z mutant, was found to down-regulate EphA receptor expression. Receptor down-regulation could also be inhibited by blockage of Src family kinase activity. Our findings show that EphA receptors can actively signal in T cells, and that Cbl performs multiple roles in this signaling pathway, functioning to transduce signals from the receptors as well as regulating activated EphA receptor expression.
Asunto(s)
Regulación hacia Abajo , Efrina-A1/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Receptores de la Familia Eph/antagonistas & inhibidores , Receptores de la Familia Eph/metabolismo , Subgrupos de Linfocitos T/metabolismo , Ubiquitina-Proteína Ligasas , Línea Celular , Membrana Celular/inmunología , Membrana Celular/metabolismo , Membrana Celular/fisiología , Regulación hacia Abajo/inmunología , Activación Enzimática/efectos de los fármacos , Efrina-A1/antagonistas & inhibidores , Efrina-A1/metabolismo , Humanos , Células Jurkat , Fosforilación/efectos de los fármacos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-cbl , Pirimidinas/farmacología , Receptor EphA3/antagonistas & inhibidores , Receptor EphA3/metabolismo , Receptor EphA4/antagonistas & inhibidores , Receptor EphA4/genética , Receptores de la Familia Eph/biosíntesis , Transducción de Señal/fisiología , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/enzimología , Transfección , Tirosina/metabolismo , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
EphB6 is the most recently identified member of the Eph receptor tyrosine kinase family. EphB6 is primarily expressed in thymocytes and a subpopulation of T cells, suggesting that it may be involved in regulation of T lymphocyte differentiation and functions. We show here that overexpression of EphB6 in Jurkat T cells and stimulation with the EphB6 ligand, ephrin-B1, results in the selective inhibition of TCR-mediated activation of JNK but not the MAPK pathway. EphB6 appears to suppress the JNK pathway by preventing T cell receptor (TCR)-induced activation of the small GTPase Rac1, a critical event in initiating the JNK cascade. Furthermore, EphB6 blocked anti-CD3-induced secretion of IL-2 and CD25 expression in a ligand-dependent manner. Dominant negative EphB6 suppressed the inhibitory activity of the endogenous receptor and enhanced anti-CD3-induced JNK activation, CD25 expression, and IL-2 secretion, confirming the requirement for EphB6-specific signaling. Activation of the JNK pathway and the establishment of an IL-2/IL-2R autocrine loop have been shown to play a role in the negative selection of CD4(+)CD8(+) self-reacting thymocytes. In agreement, stimulation of murine thymocytes with ephrin-B1 not only blocked anti-CD3-induced CD25 up-regulation and IL-2 production, but also inhibited TCR-mediated apoptosis. Thus, EphB6 may play an important role in regulating thymocyte differentiation and modulating responses of mature T cells.