RESUMEN
Chimeric antigen receptor (CAR) T cells represent a revolutionary immunotherapy that allows specific tumor recognition by a unique single-chain fragment variable (scFv) derived from monoclonal antibodies (mAbs). scFv selection is consequently a fundamental step for CAR construction, to ensure accurate and effective CAR signaling toward tumor antigen binding. However, conventional in vitro and in vivo biological approaches to compare different scFv-derived CARs are expensive and labor-intensive. With the aim to predict the finest scFv binding before CAR-T cell engineering, we performed artificial intelligence (AI)-guided molecular docking and steered molecular dynamics analysis of different anti-CD30 mAb clones. Virtual computational scFv screening showed comparable results to surface plasmon resonance (SPR) and functional CAR-T cell in vitro and in vivo assays, respectively, in terms of binding capacity and anti-tumor efficacy. The proposed fast and low-cost in silico analysis has the potential to advance the development of novel CAR constructs, with a substantial impact on reducing time, costs, and the need for laboratory animal use.
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Inteligencia Artificial , Antígeno Ki-1 , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Receptores Quiméricos de Antígenos , Anticuerpos de Cadena Única , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Receptores Quiméricos de Antígenos/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética , Humanos , Antígeno Ki-1/inmunología , Antígeno Ki-1/metabolismo , Animales , Ratones , Unión Proteica , Resonancia por Plasmón de SuperficieRESUMEN
Nucleophosmin (NPM1) mutations in acute myeloid leukemia (AML) affect exon 12, but also sporadically affect exons 9 and 11, causing changes at the protein C-terminal end (tryptophan loss, nuclear export signal [NES] motif creation) that lead to aberrant cytoplasmic NPM1 (NPM1c+), detectable by immunohistochemistry. Combining immunohistochemistry and molecular analyses in 929 patients with AML, we found non-exon 12 NPM1 mutations in 5 (1.3%) of 387 NPM1c+ cases. Besides mutations in exons 9 (n = 1) and 11 (n = 1), novel exon 5 mutations were discovered (n = 3). Another exon 5 mutation was identified in an additional 141 patients with AML selected for wild-type NPM1 exon 12. Three NPM1 rearrangements (NPM1/RPP30, NPM1/SETBP1, NPM1/CCDC28A) were detected and characterized among 13 979 AML samples screened by cytogenetic/fluorescence in situ hybridization and RNA sequencing. Functional studies demonstrated that in AML cases, new NPM1 proteins harbored an efficient extra NES, either newly created or already present in the fusion partner, ensuring its cytoplasmic accumulation. Our findings support NPM1 cytoplasmic relocation as critical for leukemogenesis and reinforce the role of immunohistochemistry in predicting AML-associated NPM1 genetic lesions. This study highlights the need to develop new assays for molecular diagnosis and monitoring of NPM1-mutated AML.
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Leucemia Mieloide Aguda/genética , Mutación , Nucleofosmina/genética , Adulto , Exones , Femenino , Fusión Génica , Reordenamiento Génico , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Dissecting the pathogenesis of classical Hodgkin lymphoma (cHL), a common cancer in young adults, remains challenging because of the rarity of tumor cells in involved tissues (usually <5%). Here, we analyzed the coding genome of cHL by microdissecting tumor and normal cells from 34 patient biopsies for a total of â¼50 000 singly isolated lymphoma cells. We uncovered several recurrently mutated genes, namely, STAT6 (32% of cases), GNA13 (24%), XPO1 (18%), and ITPKB (16%), and document the functional role of mutant STAT6 in sustaining tumor cell viability. Mutations of STAT6 genetically and functionally cooperated with disruption of SOCS1, a JAK-STAT pathway inhibitor, to promote cHL growth. Overall, 87% of cases showed dysregulation of the JAK-STAT pathway by genetic alterations in multiple genes (also including STAT3, STAT5B, JAK1, JAK2, and PTPN1), attesting to the pivotal role of this pathway in cHL pathogenesis and highlighting its potential as a new therapeutic target in this disease.
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Regulación Neoplásica de la Expresión Génica , Enfermedad de Hodgkin/genética , Quinasas Janus/genética , Mutación , Factores de Transcripción STAT/genética , Línea Celular Tumoral , Análisis Mutacional de ADN , Enfermedad de Hodgkin/metabolismo , Enfermedad de Hodgkin/patología , Humanos , Quinasas Janus/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de SeñalRESUMEN
Hairy cell leukemia (HCL) shows unique clinicopathological and biological features. HCL responds well to purine analogs but relapses are frequent and novel therapies are required. BRAF-V600E is the key driver mutation in HCL and distinguishes it from other B-cell lymphomas, including HCL-like leukemias/lymphomas (HCL-variant and splenic marginal zone lymphoma). The kinase-activating BRAF-V600E mutation also represents an ideal therapeutic target in HCL. Here, we investigated the biological and therapeutic importance of the activated BRAF-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway in HCL by exposing in vitro primary leukemic cells purified from 26 patients to clinically available BRAF (vemurafenib; dabrafenib) or MEK (trametinib) inhibitors. Results were validated in vivo in samples from vemurafenib-treated HCL patients within a phase 2 clinical trial. BRAF and MEK inhibitors caused, specifically in HCL (but not HCL-like) cells, marked MEK/ERK dephosphorylation, silencing of the BRAF-MEK-ERK pathway transcriptional output, loss of the HCL-specific gene expression signature, downregulation of the HCL markers CD25, tartrate-resistant acid phosphatase, and cyclin D1, smoothening of leukemic cells' hairy surface, and, eventually, apoptosis. Apoptosis was partially blunted by coculture with bone marrow stromal cells antagonizing MEK-ERK dephosphorylation. This protective effect could be counteracted by combined BRAF and MEK inhibition. Our results strongly support and inform the clinical use of BRAF and MEK inhibitors in HCL.
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Antineoplásicos , Imidazoles , Indoles , Leucemia de Células Pilosas/tratamiento farmacológico , Leucemia de Células Pilosas/genética , Oximas , Piridonas , Pirimidinonas , Sulfonamidas , Transcriptoma/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Imidazoles/farmacología , Imidazoles/uso terapéutico , Indoles/farmacología , Indoles/uso terapéutico , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Oximas/farmacología , Oximas/uso terapéutico , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Piridonas/farmacología , Piridonas/uso terapéutico , Pirimidinonas/farmacología , Pirimidinonas/uso terapéutico , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Células Tumorales Cultivadas , VemurafenibAsunto(s)
Transformación Celular Neoplásica/patología , Leucemia Linfocítica Crónica de Células B/patología , Linfoma de Células B Grandes Difuso/patología , Miocardio/patología , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/genética , Procedimientos Quirúrgicos Cardíacos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/inmunología , Diagnóstico Diferencial , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/terapia , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/inmunología , Linfoma de Células B Grandes Difuso/terapia , Miocardio/inmunología , Valor Predictivo de las Pruebas , Resultado del TratamientoAsunto(s)
Leucemia Mieloide Aguda/metabolismo , Mutación , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Femenino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Masculino , Proteínas Nucleares/genética , Nucleofosmina , Proteínas Proto-Oncogénicas/genética , Transactivadores/genéticaRESUMEN
BACKGROUND: Hairy-cell leukemia (HCL) is a well-defined clinicopathological entity whose underlying genetic lesion is still obscure. METHODS: We searched for HCL-associated mutations by performing massively parallel sequencing of the whole exome of leukemic and matched normal cells purified from the peripheral blood of an index patient with HCL. Findings were validated by Sanger sequencing in 47 additional patients with HCL. RESULTS: Whole-exome sequencing identified five missense somatic clonal mutations that were confirmed on Sanger sequencing, including a heterozygous mutation in BRAF that results in the BRAF V600E variant protein. Since BRAF V600E is oncogenic in other tumors, further analyses were focused on this genetic lesion. The same BRAF mutation was noted in all the other 47 patients with HCL who were evaluated by means of Sanger sequencing. None of the 195 patients with other peripheral B-cell lymphomas or leukemias who were evaluated carried the BRAF V600E variant, including 38 patients with splenic marginal-zone lymphomas or unclassifiable splenic lymphomas or leukemias. In immunohistologic and Western blot studies, HCL cells expressed phosphorylated MEK and ERK (the downstream targets of the BRAF kinase), indicating a constitutive activation of the RAF-MEK-ERK mitogen-activated protein kinase pathway in HCL. In vitro incubation of BRAF-mutated primary leukemic hairy cells from 5 patients with PLX-4720, a specific inhibitor of active BRAF, led to a marked decrease in phosphorylated ERK and MEK. CONCLUSIONS; The BRAF V600E mutation was present in all patients with HCL who were evaluated. This finding may have implications for the pathogenesis, diagnosis, and targeted therapy of HCL. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).
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Leucemia de Células Pilosas/genética , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Adulto , Anciano , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Leucemia de Células Pilosas/metabolismo , Leucemia de Células Pilosas/patología , Linfoma de Células B/genética , Linfoma de Células B/patología , Quinasas Quinasa Quinasa PAM/metabolismo , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ADNRESUMEN
OBJECTIVES: Growing evidence suggests that IL-17-producing T cells, lacking both CD4 and CD8 molecules and defined as double negative (DN) cells, play a pivotal role in the pathogenesis of a number of systemic autoimmune disorders. We recently demonstrated that this T-cell subset is expanded in the peripheral blood (PB) of patients with primary Sjögren's syndrome (pSS), produces IL-17 and accumulates in minor salivary glands (MSGs). We aimed to investigate glandular and PB DN T cells in early pSS in order to verify a possible correlation with MSGs histological patterns and clinical parameters. METHODS: Paired samples of PB mononuclear cells and MSGs from pSS patients were evaluated at the diagnosis by flow cytometry and immunofluorescence staining respectively. Histological analysis to identify histological scores, B/T cell segregation and the presence of germinal center (GC)-like structures was also performed. RESULTS: In early stages of pSS, circulating DN T cells appear to be not yet expanded and inversely correlated with circulating CD4(+)Th17 cells. The number of infiltrating DN T cells were associated with extent of glandular involvement, presence of GC-like structures and dryness symptoms and were inversely correlated with circulating DN T cells. CONCLUSIONS: Our findings suggest that DN T cells are actively involved in the pathogenic mechanisms leading to glandular dysfunction and damage in pSS and may play a role in ectopic lymphoneogenesis development occurring during the disease.
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Glándulas Salivales Menores/inmunología , Glándulas Salivales Menores/patología , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/patología , Subgrupos de Linfocitos T/inmunología , Adulto , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Femenino , Humanos , Inmunohistoquímica , Inmunofenotipificación , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Síndrome de Sjögren/metabolismo , Subgrupos de Linfocitos T/metabolismoRESUMEN
OBJECTIVES: It has been recently observed that a T-cell subset, lacking of both CD4 and CD8 molecules and defined as double negative (DN), is expanded in the blood of patients with systemic lupus erythematosus, produces IL-17 and accumulates in the kidney during nephritis. Since IL-17 production is enhanced in salivary gland infiltrates of primary Sjögren's syndrome (SS) patients, we investigated whether DN T cells may be involved in the pathogenesis of salivary gland damage. METHODS: Phenotypic characterisation of peripheral blood mononuclear cells from SS patients and controls was performed by flow cytometry in freshly isolated and anti-CD3-stimulated cells. SS minor salivary glands were processed for immunofluorescence staining. RESULTS: CD3(+)CD4(-)CD8(-) DN T cells were major producers of IL-17 in SS and expressed ROR-γt. They were expanded in the peripheral blood, spontaneously produced IL-17 and infiltrated salivary glands. In addition, the expansion of αß-TCR(+) DN T cells was associated with disease activity. Notably, IL-17-producing DN T cells from SS patients, but not from healthy controls, were strongly resistant to the in vitro effect of dexamethasone. CONCLUSIONS: These findings appear to be of great interest since the identification of a peculiar T-cell subset with pro-inflammatory activity, but resistant to corticosteroids, in an autoimmune disorder such as SS may help to design new specific treatments for the disease.
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Interleucina-17/biosíntesis , Glándulas Salivales/inmunología , Síndrome de Sjögren/inmunología , Subgrupos de Linfocitos T/inmunología , Células Th17/inmunología , Corticoesteroides/farmacología , Dexametasona/farmacología , Femenino , Citometría de Flujo , Humanos , Interleucina-17/inmunología , Persona de Mediana Edad , Glándulas Salivales/patología , Síndrome de Sjögren/patología , Subgrupos de Linfocitos T/efectos de los fármacos , Células Th17/efectos de los fármacos , Células Th17/metabolismoRESUMEN
AIMS: The aim of this study was to search for a molecule selectively expressed by marginal zone (MZ) lymphomas (MZLs), whose diagnosis is currently based on morphological criteria and negativity for markers detectable in other B-cell lymphomas. METHODS AND RESULTS: Two thousand one hundred and four peripheral lymphomas of various types were immunostained with a monoclonal antibody against immunoglobulin superfamily receptor translocation-associated 1 (IRTA1), which recognizes the equivalents of MZ in human lymphoid tissues other than spleen. IRTA1 expression was restricted to extranodal (93%) and nodal MZLs (73%) and to lymphomas with MZ differentiation. Extranodal MZL cells with the strongest IRTA1 expression were usually located adjacent to epithelia, mimicking the IRTA1 expression pattern of normal and acquired mucosa-associated lymphoid tissue (MALT). The cytological features, growth pattern and IRTA1 positivity in nodal MZLs suggest they may derive from IRTA1(+) perifollicular B cells or monocytoid B cells detectable in reactive lymph nodes. Double immunostaining for IRTA1/bcl-6 tracked the colonization of B-cell follicles by MZL cells, and showed modulation of their phenotype (e.g. acquisition of bcl-6) during recirculation through germinal centres. MZL cells differentiating into plasma cells usually lost IRTA1. CONCLUSIONS: These results further expand our knowledge of the biology of MZLs, and highlight IRTA1 as the first positive marker for MZLs, enabling more accurate diagnosis of these neoplasms.
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Linfoma de Células B de la Zona Marginal/inmunología , Linfoma de Células B de la Zona Marginal/patología , Receptores Fc/metabolismo , Linfocitos B/inmunología , Linfocitos B/patología , Biomarcadores de Tumor/inmunología , Biomarcadores de Tumor/metabolismo , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Linfoma de Células B de la Zona Marginal/genética , Bazo/inmunología , Bazo/patología , Translocación GenéticaRESUMEN
NPM1 is the most frequently mutated gene in adults with acute myeloid leukemia (AML). The interaction between mutant NPM1 (NPM1c) and exportin-1 (XPO1) causes aberrant cytoplasmic dislocation of NPM1c and promotes the high expression of homeobox (HOX) genes, which is critical for maintaining the leukemic state of NPM1-mutated cells. Although there is a rationale for using XPO1 inhibitors in NPM1-mutated AML, selinexor administered once or twice per week did not translate into clinical benefit in patients with NPM1 mutations. Here, we show that this dosing strategy results in only a temporary disruption of the XPO1-NPM1c interaction, limiting the efficacy of selinexor. Because the second-generation XPO1 inhibitor eltanexor can be administered more frequently, we tested the antileukemic activity of prolonged XPO1 inhibition in NPM1-mutated AML models. Eltanexor caused irreversible HOX downregulation, induced terminal AML differentiation, and prolonged the survival of leukemic mice. This study provides essential information for the appropriate design of clinical trials with XPO1 inhibitors in NPM1-mutated AML.
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Antineoplásicos , Leucemia Mieloide Aguda , Ratones , Animales , Regulación Leucémica de la Expresión Génica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Carioferinas/genética , Carioferinas/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéuticoAsunto(s)
Leucemia de Células Pilosas/metabolismo , Leucemia de Células Pilosas/patología , Mutación Missense , Proteínas Proto-Oncogénicas B-raf/biosíntesis , Sustitución de Aminoácidos , Línea Celular Tumoral , Humanos , Leucemia de Células Pilosas/genética , Proteínas Proto-Oncogénicas B-raf/genéticaRESUMEN
Creation of a nuclear export signal (NES) motif and loss of tryptophans (W) 288 and 290 (or 290 only) at the COOH terminus of nucleophosmin (NPM) are both crucial for NPM aberrant cytoplasmic accumulation in acute myelogenous leukemia (AML) carrying NPM1 mutations. Hereby, we clarify how these COOH-terminal alterations functionally cooperate to delocalize NPM to the cytoplasm. Using a Rev(1.4)-based shuttling assay, we measured the nuclear export efficiency of six different COOH-terminal NES motifs identified in NPM mutants and found significant strength variability, the strongest NES motifs being associated with NPM mutants retaining W288. When artificially coupled with a weak NES, W288-retaining NPM mutants are not exported efficiently into cytoplasm because the force (W288) driving the mutants toward the nucleolus overwhelms the force (NES) exporting the mutants into cytoplasm. We then used this functional assay to study the physiologic NH(2)-terminal NES motifs of wild-type NPM and found that they are weak, which explains the prominent nucleolar localization of wild-type NPM. Thus, the opposing balance of forces (tryptophans and NES) seems to determine the subcellular localization of NPM. The fact that W288-retaining mutants always combine with the strongest NES reveals mutational selective pressure toward efficient export into cytoplasm, pointing to this event as critical for leukemogenesis.
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Regulación Neoplásica de la Expresión Génica , Proteínas Nucleares/química , Proteínas Nucleares/genética , Secuencias de Aminoácidos , Animales , Transformación Celular Neoplásica , Citoplasma/metabolismo , Análisis Mutacional de ADN , Fibroblastos/metabolismo , Leucemia Mieloide Aguda/metabolismo , Ratones , Microscopía Fluorescente , Mutación , Células 3T3 NIH , Nucleofosmina , Plásmidos/metabolismo , Estructura Terciaria de ProteínaRESUMEN
OBJECTIVE: A standardization of minor salivary gland (MSG) histopathology in primary Sjögren's syndrome (pSS) has been recently proposed. Although there is strong agreement that germinal center (GC)-like structures should be routinely identified, due to their prognostic value, a consensus regarding the best protocol is still lacking. Aim of this study was to compare the performance of different histological techniques and operators to identify GC-like structures in pSS MSGs. MSG biopsies from 50 pSS patients were studied. METHODS: Three blinded operators (one pathologist and two rheumatologists with different years of experience in pSS MSG assessment) assessed 50 MSGs of which one slide was stained with haematoxylin and eosin (H&E) and consecutive slides were processed to investigate CD3/CD20, CD21 and Bcl-6 expression. RESULTS: By assessing 225 foci, the best agreement was between H&E-stained sections evaluated by the rheumatologist with more years of experience in pSS MSG assessment and CD3/CD20 segregation. In the foci with CD21 positivity, the agreement further increased. Bcl-6- foci could display a GC, detected with other staining, but not vice versa. CONCLUSION: GC assessment on H&E-stained sections should be performed with caution, being operator-dependent. The combination of H&E with CD3/CD20 and CD21 staining should be recommended as it is reliable, feasible, able to overcome the bias of operator experience and easily transferrable into routine practice.
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Antígenos CD/biosíntesis , Síndrome de Sjögren/metabolismo , Síndrome de Sjögren/patología , Biopsia , Femenino , Humanos , Masculino , Persona de Mediana Edad , Glándulas Salivales Menores/metabolismo , Glándulas Salivales Menores/patologíaRESUMEN
BACKGROUND: Nucleophosmin (NPM), a nucleocytoplasmic shuttling protein with prominent nucleolar localization, regulates the ARF-p53 tumor-suppressor pathway. Translocations involving the NPM gene cause cytoplasmic dislocation of the NPM protein. METHODS: We used immunohistochemical methods to study the subcellular localization of NPM in bone marrow-biopsy specimens from 591 patients with primary acute myelogenous leukemia (AML). We then correlated the presence of cytoplasmic NPM with clinical and biologic features of the disease. RESULTS: Cytoplasmic NPM was detected in 208 (35.2 percent) of the 591 specimens from patients with primary AML but not in 135 secondary AML specimens or in 980 hematopoietic or extrahematopoietic neoplasms other than AML. It was associated with a wide spectrum of morphologic subtypes of the disease, a normal karyotype, and responsiveness to induction chemotherapy, but not with recurrent genetic abnormalities. There was a high frequency of FLT3 internal tandem duplications and absence of CD34 and CD133 in AML specimens with a normal karyotype and cytoplasmic dislocation of NPM, but not in those in which the protein was restricted to the nucleus. AML specimens with cytoplasmic NPM carried mutations of the NPM gene that were predicted to alter the protein at its C-terminal; this mutant gene caused cytoplasmic localization of NPM in transfected cells. CONCLUSIONS: Cytoplasmic NPM is a characteristic feature of a large subgroup of patients with AML who have a normal karyotype, NPM gene mutations, and responsiveness to induction chemotherapy.
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Médula Ósea/patología , Citoplasma/química , Leucemia Mieloide Aguda/genética , Mutación , Proteínas Nucleares/genética , Adolescente , Adulto , Anticuerpos Monoclonales , Antineoplásicos/uso terapéutico , Secuencia de Bases , Nucléolo Celular , Análisis Mutacional de ADN , Humanos , Cariotipificación , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Persona de Mediana Edad , Proteínas Nucleares/análisis , Proteínas Nucleares/inmunología , Nucleofosmina , Inducción de Remisión , Transfección , Translocación GenéticaRESUMEN
We investigated the NPM1 mutation status or subcellular expression of NPM protein (nuclear vs. aberrant cytoplasmic) at diagnosis and relapse in 125 patients with acute myeloid leukemia from Italy and Germany. All 52 patients with acute myeloidleukemia carrying at diagnosis mutated or cytoplasmic NPM (NPMc(+) acute myeloid leukemia) retained this feature at relapse. Notably, cytoplasmic mutated NPM has now been retained for eight years in a xenotransplant model of NPMc(+) acute myeloid leukemia in immunodeficient mice. None of 73 acute myeloid leukemia patients carrying at diagnosis wild-type NPM1 gene or showing at immunohistochemistry nucleus-restricted expression of nucleophosmin (NPMc(-) acute myeloid leukemia), which is predictive of NPM1 gene in germline configuration, acquired cytoplasmic mutated NPM at relapse. This finding further confirms that NPMc(+) acute myeloid leukemia represents a primary event rather than a transformation stage of NPMc(-) acute myeloid leukemia. The stability of cytoplasmic mutated NPM in patients with acute myeloid leukemia, even at relapse in extramedullary sites, and in a xenotransplant model, suggest this event is crucial for leukemogenesis and represents the rationale for monitoring minimal residual disease and molecular targeted therapy in NPMc(+) acute myeloid leukemia.
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Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutación , Proteínas Nucleares/genética , Adulto , Animales , Aberraciones Cromosómicas , Citoplasma/metabolismo , Femenino , Humanos , Inmunohistoquímica/métodos , Cariotipificación , Ratones , Ratones SCID , Trasplante de Neoplasias , Proteínas Nucleares/metabolismo , Nucleofosmina , RecurrenciaRESUMEN
Fever plays a role in activating innate immunity while its relevance in activating adaptive immunity is less clear. Even brief exposure to elevated temperatures significantly impacts on the immunostimulatory capacity of dendritic cells (DCs), but the consequences on immune response remain unclear. To address this issue, we analyzed the gene expression profiles of normal human monocyte-derived DCs from nine healthy adults subjected either to fever-like thermal conditions (39°C) or to normal temperature (37°C) for 180 minutes. Exposure of DCs to 39°C caused upregulation of 43 genes and downregulation of 24 genes. Functionally, the up/downregulated genes are involved in post-translational modification, protein folding, cell death and survival, and cellular movement. Notably, when compared to monocytes, DCs differentially upregulated transcription of the secreted protein IGFBP-6, not previously known to be specifically linked to hyperthermia. Exposure of DCs to 39°C induced apoptosis/necrosis and resulted in accumulation of IGFBP-6 in the conditioned medium at 48 h. IGFBP-6 may have a functional role in the hyperthermic response as it induced chemotaxis of monocytes and T lymphocytes, but not of B lymphocytes. Thus, temperature regulates complex biological DC functions that most likely contribute to their ability to induce an efficient adaptive immune response.
RESUMEN
We recently identified aberrant cytoplasmic expression of nucleophosmin (NPM) as the immunohistochemical marker of a large subgroup of acute myeloid leukemia (AML) (about one-third of adult AML) that is characterized by normal karyotype and mutations occurring at the exon-12 of the NPM gene. In this paper, we have elucidated the molecular mechanism underlying the abnormal cytoplasmic localization of NPM. All 29 AML-associated mutated NPM alleles so far identified encode abnormal proteins which have acquired at the C-terminus a nuclear export signal (NES) motif and lost both tryptophan residues 288 and 290 (or only the residue 290) which determine nucleolar localization. We show for the first time that both alterations are crucial for NPM mutant export from nucleus to cytoplasm. In fact, the cytoplasmic accumulation of NPM is blocked by leptomycin-B and ratjadones, specific exportin-1/Crm1-inhibitors, and by reinsertion of tryptophan residues 288 and 290, which respectively relocate NPM mutants in the nucleoplasm and nucleoli. NPM leukemic mutants in turn recruit the wild-type NPM from nucleoli to nucleoplasm and cytoplasm. These findings indicate that potential therapeutic strategies aimed to retarget NPM to its physiological sites will have to overcome 2 obstacles, the new NES motif and the mutated tryptophan(s) at the NPM mutant C-terminus.