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1.
Am J Transplant ; 21(4): 1365-1375, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33251712

RESUMEN

Islet allotransplantation in the United States (US) is facing an imminent demise. Despite nearly three decades of progress in the field, an archaic regulatory framework has stymied US clinical practice. Current regulations do not reflect the state-of-the-art in clinical or technical practices. In the US, islets are considered biologic drugs and "more than minimally manipulated" human cell and tissue products (HCT/Ps). In contrast, across the world, human islets are appropriately defined as "minimally manipulated tissue" and not regulated as a drug, which has led to islet allotransplantation (allo-ITx) becoming a standard-of-care procedure for selected patients with type 1 diabetes mellitus. This regulatory distinction impedes patient access to islets for transplantation in the US. As a result only 11 patients underwent allo-ITx in the US between 2016 and 2019, and all as investigational procedures in the settings of a clinical trials. Herein, we describe the current regulations pertaining to islet transplantation in the United States. We explore the progress which has been made in the field and demonstrate why the regulatory framework must be updated to both better reflect our current clinical practice and to deal with upcoming challenges. We propose specific updates to current regulations which are required for the renaissance of ethical, safe, effective, and affordable allo-ITx in the United States.


Asunto(s)
Productos Biológicos , Diabetes Mellitus Tipo 1 , Trasplante de Islotes Pancreáticos , Costos y Análisis de Costo , Diabetes Mellitus Tipo 1/cirugía , Humanos , Trasplante Heterólogo , Estados Unidos
2.
Biol Blood Marrow Transplant ; 24(5): 997-1004, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29288821

RESUMEN

Haplo/cord transplantation combines an umbilical cord blood (UCB) graft with CD34-selected haploidentical cells and results in rapid hematopoietic recovery followed by durable UCB engraftment. We compared outcomes of transplants in older patients with acute myeloid leukemia (AML) or high-risk myelodysplastic syndromes (MDS) who received either HLA-matched unrelated donor (MUD) cells or haplo/cord grafts. Between 2007 and 2013, 109 adults ages 50 and older underwent similar reduced-intensity conditioning with fludarabine and melphalan and antibody-mediated T cell depletion for AML (n = 83) or high-risk MDS (n = 26) followed by either a MUD (n = 68) or haplo/cord (n = 41) graft. Patient characteristics were similar for each graft source except for more minority patients receiving a haplo/cord transplant (P = .01). One half of the AML patients were not in remission. Two-year progression-free survival (PFS), overall survival (OS), and graft-versus-host disease-free relapse-free survival were 38%, 48%, and 32.1% for MUD and 33%, 48%, and 33.8% for haplo/cord transplants (P = .62 for PFS; P = .97 for OS; P= .84), respectively. Acute grades II to IV and chronic graft-versus-host-disease rates did not differ at 19.5% and 4.9% in haplo/cord compared with 25% and 7.4% after MUD (P = .53 and P = .62, respectively). Multivariate analysis confirmed no significant differences in transplant outcomes by donor type. Haplo/cord reduced-intensity transplantation achieves similar outcomes relative to MUD in older AML and MDS patients, making this a promising option for those without matched donors.


Asunto(s)
Trasplante de Células Madre Hematopoyéticas/métodos , Leucemia Mieloide Aguda/terapia , Síndromes Mielodisplásicos/terapia , Acondicionamiento Pretrasplante/métodos , Anciano , Antígenos CD34/sangre , Femenino , Sangre Fetal/trasplante , Enfermedad Injerto contra Huésped/etiología , Trasplante de Células Madre Hematopoyéticas/mortalidad , Trasplante de Células Madre Hematopoyéticas/normas , Prueba de Histocompatibilidad , Humanos , Leucemia Mieloide Aguda/mortalidad , Depleción Linfocítica , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/mortalidad , Análisis de Supervivencia , Acondicionamiento Pretrasplante/mortalidad , Trasplante Haploidéntico , Donante no Emparentado
3.
Proc Natl Acad Sci U S A ; 112(46): E6359-68, 2015 Nov 17.
Artículo en Inglés | MEDLINE | ID: mdl-26578796

RESUMEN

Anemia is the predominant clinical manifestation of myelodysplastic syndromes (MDS). Loss or deletion of chromosome 7 is commonly seen in MDS and leads to a poor prognosis. However, the identity of functionally relevant, dysplasia-causing, genes on 7q remains unclear. Dedicator of cytokinesis 4 (DOCK4) is a GTPase exchange factor, and its gene maps to the commonly deleted 7q region. We demonstrate that DOCK4 is underexpressed in MDS bone marrow samples and that the reduced expression is associated with decreased overall survival in patients. We show that depletion of DOCK4 levels leads to erythroid cells with dysplastic morphology both in vivo and in vitro. We established a novel single-cell assay to quantify disrupted F-actin filament network in erythroblasts and demonstrate that reduced expression of DOCK4 leads to disruption of the actin filaments, resulting in erythroid dysplasia that phenocopies the red blood cell (RBC) defects seen in samples from MDS patients. Reexpression of DOCK4 in -7q MDS patient erythroblasts resulted in significant erythropoietic improvements. Mechanisms underlying F-actin disruption revealed that DOCK4 knockdown reduces ras-related C3 botulinum toxin substrate 1 (RAC1) GTPase activation, leading to increased phosphorylation of the actin-stabilizing protein ADDUCIN in MDS samples. These data identify DOCK4 as a putative 7q gene whose reduced expression can lead to erythroid dysplasia.


Asunto(s)
Eritroblastos/metabolismo , Proteínas Activadoras de GTPasa/biosíntesis , Regulación de la Expresión Génica , Síndromes Mielodisplásicos/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/metabolismo , Eritroblastos/patología , Femenino , Proteínas Activadoras de GTPasa/genética , Humanos , Masculino , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/patología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo
4.
Blood ; 125(20): 3144-52, 2015 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-25810490

RESUMEN

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with disease-initiating stem cells that are not eliminated by conventional therapies. Novel therapeutic targets against preleukemic stem cells need to be identified for potentially curative strategies. We conducted parallel transcriptional analysis of highly fractionated stem and progenitor populations in MDS, AML, and control samples and found interleukin 8 (IL8) to be consistently overexpressed in patient samples. The receptor for IL8, CXCR2, was also significantly increased in MDS CD34(+) cells from a large clinical cohort and was predictive of increased transfusion dependence. High CXCR2 expression was also an adverse prognostic factor in The Cancer Genome Atlas AML cohort, further pointing to the critical role of the IL8-CXCR2 axis in AML/MDS. Functionally, CXCR2 inhibition by knockdown and pharmacologic approaches led to a significant reduction in proliferation in several leukemic cell lines and primary MDS/AML samples via induction of G0/G1 cell cycle arrest. Importantly, inhibition of CXCR2 selectively inhibited immature hematopoietic stem cells from MDS/AML samples without an effect on healthy controls. CXCR2 knockdown also impaired leukemic growth in vivo. Together, these studies demonstrate that the IL8 receptor CXCR2 is an adverse prognostic factor in MDS/AML and is a potential therapeutic target against immature leukemic stem cell-enriched cell fractions in MDS and AML.


Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Interleucina-8/metabolismo , Leucemia Mieloide Aguda/metabolismo , Síndromes Mielodisplásicos/metabolismo , Receptores de Interleucina-8B/metabolismo , Transducción de Señal , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Análisis por Conglomerados , Modelos Animales de Enfermedad , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Interleucina-8/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Ratones , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/mortalidad , Células Madre Neoplásicas/metabolismo , Pronóstico , Receptores de Interleucina-8B/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
5.
Biol Blood Marrow Transplant ; 22(6): 1065-1072, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26912055

RESUMEN

Delayed engraftment and cord graft failure (CGF) are serious complications after unrelated cord blood (UCB) hematopoietic stem cell transplantation (HSCT), particularly when using low-cell-dose UCB units. The haplo-cord HSCT approach allows the use of a lower dose single UCB unit by co-infusion of a CD34(+) selected haploidentical graft, which provides early transient engraftment while awaiting durable UCB engraftment. We describe the frequency, complications, and risk factors of CGF after reduced-intensity conditioning haplo-cord HSCT. Among 107 patients who underwent haplo-cord HSCT, 94 were assessable for CGF, defined as <5% cord blood chimerism at day 60 in the myeloid and CD3 compartments, irrespective of neutrophil and platelet counts. CGF occurred in 14 of 94 assessable patients (15%). Median survival after CGF was 12.7 months with haploidentical or mixed haploidentical-autologous hematopoiesis persisting in the 7 surviving. Median progression-free survival after CGF was 7.7 months and was not statistically different from those without CGF (10.47 months; P = .18). In univariate analyses, no UCB factors were associated with CGF, including cell dose, cell viability, recipient major ABO mismatch against the UCB unit, or degree of HLA match. We also found no association of CGF with recipient cytomegalovirus serostatus, haploidentical donor age, or day 30 haploidentical chimerism. However, higher haploidentical total nucleated and CD34(+) cell doses and day 30 UCB chimerism < 5% in either the myeloid or CD3 compartments were associated with greater risk of CGF. We conclude that assessing chimerism at day 30 may foretell impending CGF, and avoidance of high haploidentical cell doses may reduce risk of CGF after haplo-cord HSCT. However, long-term survival is possible after CGF because of predominant haploidentical or mixed chimerism and hematopoietic function.


Asunto(s)
Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Sangre Fetal/citología , Rechazo de Injerto/prevención & control , Haploidia , Acondicionamiento Pretrasplante/métodos , Adolescente , Adulto , Anciano , Antígenos CD34 , Quimerismo , Trasplante de Células Madre de Sangre del Cordón Umbilical/efectos adversos , Trasplante de Células Madre de Sangre del Cordón Umbilical/mortalidad , Femenino , Rechazo de Injerto/etiología , Histocompatibilidad , Humanos , Masculino , Persona de Mediana Edad , Factores de Riesgo , Análisis de Supervivencia , Trasplante Haploidéntico/métodos , Adulto Joven
6.
Biol Blood Marrow Transplant ; 22(11): 2100-2103, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27497531

RESUMEN

Analysis of the clinical characteristics of hematopoietic stem cell transplant (HSCT) donors has proven beneficial for identifying cases of heritable hematopoietic disorders. This study examines poor peripheral blood hematopoietic stem cell mobilization after granulocyte colony-stimulating factor administration among 328 donors as a potential marker for suspected familial predisposition to myeloid malignancies. Here, we present data comparing the clinical characteristics of poor-mobilizing versus nonpoor-mobilizing donors and the results of panel-based sequencing of hematopoietic genes in poor-mobilizing donors. From this analysis, we identified a novel case of a donor-derived myelodysplastic syndrome in an HSCT recipient that is consistent with clonal evolution of TET2-mutated clonal hematopoiesis of indeterminate potential (CHIP) within the donor. This study demonstrates the potential risk of using hematopoietic stem cells from a donor with CHIP and raises the question of whether there should be increased screening measures to identify such donors.


Asunto(s)
Donantes de Sangre , Movilización de Célula Madre Hematopoyética/normas , Neoplasias/genética , Adolescente , Adulto , Anciano , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Dioxigenasas , Femenino , Predisposición Genética a la Enfermedad , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Hematopoyesis/genética , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/genética , Neoplasias/etiología , Linaje , Proteínas Proto-Oncogénicas/genética , Adulto Joven
7.
Blood ; 123(5): 758-67, 2014 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-24159174

RESUMEN

Tropomodulin (Tmod) is a protein that binds and caps the pointed ends of actin filaments in erythroid and nonerythoid cell types. Targeted deletion of mouse tropomodulin3 (Tmod3) leads to embryonic lethality at E14.5-E18.5, with anemia due to defects in definitive erythropoiesis in the fetal liver. Erythroid burst-forming unit and colony-forming unit numbers are greatly reduced, indicating defects in progenitor populations. Flow cytometry of fetal liver erythroblasts shows that late-stage populations are also decreased, including reduced percentages of enucleated cells. Annexin V staining indicates increased apoptosis of Tmod3(-/-) erythroblasts, and cell-cycle analysis reveals that there are more Ter119(hi) cells in S-phase in Tmod3(-/-) embryos. Notably, enucleating Tmod3(-/-) erythroblasts are still in the process of proliferation, suggesting impaired cell-cycle exit during terminal differentiation. Tmod3(-/-) late erythroblasts often exhibit multilobular nuclear morphologies and aberrant F-actin assembly during enucleation. Furthermore, native erythroblastic island formation was impaired in Tmod3(-/-) fetal livers, with Tmod3 required in both erythroblasts and macrophages. In conclusion, disruption of Tmod3 leads to impaired definitive erythropoiesis due to reduced progenitors, impaired erythroblastic island formation, and defective erythroblast cell-cycle progression and enucleation. Tmod3-mediated actin remodeling may be required for erythroblast-macrophage adhesion, coordination of cell cycle with differentiation, and F-actin assembly and remodeling during erythroblast enucleation.


Asunto(s)
Células Precursoras Eritroides/metabolismo , Eliminación de Gen , Hígado/embriología , Tropomodulina/genética , Animales , Apoptosis , Ciclo Celular , Eritroblastos/citología , Eritroblastos/metabolismo , Células Precursoras Eritroides/citología , Eritropoyesis , Femenino , Regulación del Desarrollo de la Expresión Génica , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados
9.
Haematologica ; 101(5): 634-43, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-26869630

RESUMEN

Umbilical cord blood stem cell transplants are commonly used in adults lacking HLA-identical donors. Delays in hematopoietic recovery contribute to mortality and morbidity. To hasten recovery, we used co-infusion of progenitor cells from a partially matched related donor and from an umbilical cord blood graft (haplo-cord transplant). Here we compared the outcomes of haplo-cord and double-cord transplants. A total of 97 adults underwent reduced intensity conditioning followed by haplo-cord transplant and 193 patients received reduced intensity conditioning followed by double umbilical cord blood transplantation. Patients in the haplo-cord group were more often from minority groups and had more advanced malignancy. Haplo-cord recipients received fludarabine-melphalan-anti-thymocyte globulin. Double umbilical cord blood recipients received fludarabine-cyclophosphamide and low-dose total body irradiation. In a multivariate analysis, haplo-cord had faster neutrophil (HR=1.42, P=0.007) and platelet (HR=2.54, P<0.0001) recovery, lower risk of grade II-IV acute graft-versus-host disease (HR=0.26, P<0.0001) and chronic graft-versus-host disease (HR=0.06, P<0.0001). Haplo-cord was associated with decreased risk of relapse (HR 0.48, P=0.001). Graft-versus-host disease-free, relapse-free survival was superior with haplo-cord (HR 0.63, P=0.002) but not overall survival (HR=0.97, P=0.85). Haplo-cord transplantation using fludarabine-melphalan-thymoglobulin conditioning hastens hematopoietic recovery with a lower risk of relapse relative to double umbilical cord blood transplantation using the commonly used fludarabine-cyclophosphamide-low-dose total body irradiation conditioning. Graft-versus-host disease-free and relapse-free survival is significantly improved. Haplo-cord is a readily available graft source that improves outcomes and access to transplant for those lacking HLA-matched donors. Trials registered at clinicaltrials.gov identifiers 00943800 and 01810588.


Asunto(s)
Trasplante de Células Madre de Sangre del Cordón Umbilical , Antígenos HLA , Haplotipos , Neoplasias Hematológicas/terapia , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas , Donante no Emparentado , Adulto , Anciano , Anciano de 80 o más Años , Comorbilidad , Trasplante de Células Madre de Sangre del Cordón Umbilical/efectos adversos , Progresión de la Enfermedad , Femenino , Supervivencia de Injerto , Enfermedad Injerto contra Huésped/diagnóstico , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/etiología , Antígenos HLA/genética , Antígenos HLA/inmunología , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/mortalidad , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Prueba de Histocompatibilidad , Humanos , Estimación de Kaplan-Meier , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Mortalidad , Neutrófilos , Recurrencia , Acondicionamiento Pretrasplante/métodos , Trasplante Homólogo , Resultado del Tratamiento , Adulto Joven
10.
Haematologica ; 101(11): 1426-1433, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27662010

RESUMEN

We sought to confirm the prognostic importance of simple clinically available biomarkers of C-reactive protein, serum albumin, and ferritin prior to allogeneic hematopoietic cell transplantation. The study population consisted of 784 adults with acute myeloid leukemia in remission or myelodysplastic syndromes undergoing unrelated donor transplant reported to the Center for International Blood and Marrow Transplant Research. C-reactive protein and ferritin were centrally quantified by ELISA from cryopreserved plasma whereas each center provided pre-transplant albumin. In multivariate analysis, transplant-related mortality was associated with the pre-specified thresholds of C-reactive protein more than 10 mg/L (P=0.008) and albumin less than 3.5 g/dL (P=0.01) but not ferritin more than 2500 ng/mL. Only low albumin independently influenced overall mortality. Optimal thresholds affecting transplant-related mortality were defined as: C-reactive protein more than 3.67 mg/L, log(ferritin), and albumin less than 3.4 g/dL. A 3-level biomarker risk group based on these values separated risks of transplant-related mortality: low risk (reference), intermediate (HR=1.66, P=0.015), and high risk (HR=2.7, P<0.001). One-year survival was 74%, 67% and 56% for low-, intermediate- and high-risk groups. Routinely available pre-transplant biomarkers independently risk-stratify for transplant-related mortality and survival.


Asunto(s)
Proteína C-Reactiva/análisis , Ferritinas/sangre , Trasplante de Células Madre Hematopoyéticas/métodos , Leucemia Mieloide Aguda/terapia , Síndromes Mielodisplásicos/terapia , Albúmina Sérica/análisis , Adolescente , Adulto , Anciano , Biomarcadores de Tumor/sangre , Femenino , Trasplante de Células Madre Hematopoyéticas/mortalidad , Humanos , Leucemia Mieloide Aguda/sangre , Leucemia Mieloide Aguda/mortalidad , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/sangre , Síndromes Mielodisplásicos/mortalidad , Pronóstico , Estudios Retrospectivos , Análisis de Supervivencia , Trasplante Homólogo , Adulto Joven
11.
J Biol Chem ; 288(33): 23814-22, 2013 Aug 16.
Artículo en Inglés | MEDLINE | ID: mdl-23814052

RESUMEN

The mechanisms of generation of the antineoplastic effects of interferons (IFNs) in malignant hematopoietic cells remain to be precisely defined. We examined the activation of type I IFN-dependent signaling pathways in malignant cells transformed by Jak2V617F, a critical pathogenic mutation in myeloproliferative neoplasms (MPNs). Our studies demonstrate that during engagement of the type I IFN receptor (IFNAR), there is activation of Jak-Stat pathways and also engagement of Mnk kinases. Activation of Mnk kinases is regulated by the Mek/Erk pathway and is required for the generation of IFN-induced growth inhibitory responses, but Mnk kinase activation does not modulate IFN-regulated Jak-Stat signals. We demonstrate that for type I IFNs to exert suppressive effects in malignant hematopoietic progenitors from patients with polycythemia vera, induction of Mnk kinase activity is required, as evidenced by studies involving pharmacological inhibition of Mnk or siRNA-mediated Mnk knockdown. Altogether, these findings provide evidence for key and essential roles of the Mnk kinase pathway in the generation of the antineoplastic effects of type I IFNs in Jak2V617F-dependent MPNs.


Asunto(s)
Neoplasias de la Médula Ósea/metabolismo , Interferón-alfa/farmacología , Interferón beta/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Trastornos Mieloproliferativos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Neoplasias de la Médula Ósea/patología , Diferenciación Celular , Línea Celular Transformada , Células Eritroides/efectos de los fármacos , Células Eritroides/metabolismo , Células Eritroides/patología , Factor 4E Eucariótico de Iniciación/metabolismo , Humanos , Quinasas Janus/genética , Quinasas Janus/metabolismo , Ratones , Mutación/genética , Trastornos Mieloproliferativos/patología
12.
J Biol Chem ; 288(13): 8805-14, 2013 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-23306203

RESUMEN

Differentiation of hematopoietic stem cells to red cells requires coordinated expression of numerous erythroid genes and is characterized by nuclear condensation and extrusion during terminal development. To understand the regulatory mechanisms governing these widespread phenotypic changes, we conducted a high resolution methylomic and transcriptomic analysis of six major stages of human erythroid differentiation. We observed widespread epigenetic differences between early and late stages of erythropoiesis with progressive loss of methylation being the dominant change during differentiation. Gene bodies, intergenic regions, and CpG shores were preferentially demethylated during erythropoiesis. Epigenetic changes at transcription factor binding sites correlated significantly with changes in gene expression and were enriched for binding motifs for SCL, MYB, GATA, and other factors not previously implicated in erythropoiesis. Demethylation at gene promoters was associated with increased expression of genes, whereas epigenetic changes at gene bodies correlated inversely with gene expression. Important gene networks encoding erythrocyte membrane proteins, surface receptors, and heme synthesis proteins were found to be regulated by DNA methylation. Furthermore, integrative analysis enabled us to identify novel, potential regulatory areas of the genome as evident by epigenetic changes in a predicted PU.1 binding site in intron 1 of the GATA1 gene. This intronic site was found to be conserved across species and was validated to be a novel PU.1 binding site by quantitative ChIP in erythroid cells. Altogether, our study provides a comprehensive analysis of methylomic and transcriptomic changes during erythroid differentiation and demonstrates that human terminal erythropoiesis is surprisingly associated with hypomethylation of the genome.


Asunto(s)
Eritropoyesis/fisiología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Antígenos CD34/biosíntesis , Sitios de Unión , Diferenciación Celular , Islas de CpG , Metilación de ADN , Epigénesis Genética , Epigenómica , Eritrocitos/citología , Citometría de Flujo/métodos , Genoma Humano , Genómica , Humanos , Intrones , Metilación , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Madre/química
13.
Blood ; 118(24): 6438-45, 2011 Dec 08.
Artículo en Inglés | MEDLINE | ID: mdl-21976674

RESUMEN

We conducted a 45 patient prospective study of reduced-intensity conditioning (RIC) and transplantation of unrelated umbilical cord blood (UCB) and CD34(+) stem cells from a haploidentical family member. Median age was 50 years; weight was 80 kg. Fifty-eight percent had active disease. Neutrophil engraftment occurred at 11 days (interquartile range [IQR], 9-15) and platelet engraftment at 19 days (IQR, 15-33). In the majority of patients, early haploidentical engraftment was replaced by durable engraftment of UCB by 100 days, with regular persistence of minor host and/or haplo-hematopoiesis. Percentage of haplochimerism at day 100 correlated with the haplo-CD34 dose (P = .003). Cumulative incidence of acute GVHD (aGVHD) was 25% and chronic GVHD (cGVHD) was 5%. Actuarial survival at 1 year was 55%, progression-free survival (PFS) was 42%, nonrelapse mortality (NRM) was 28%, and relapse was 30%. RIC and haplo-cord transplantation results in fast engraftment of neutrophils and platelets, low incidences of aGVHD and cGVHD, low frequency of delayed opportunistic infections, reduced transfusion requirements, shortened length of hospital stay, and promising long-term outcomes. UCB cell dose had no impact on time to hematopoietic recovery. Therefore, UCB selection can prioritize matching, and better matched donors can be identified rapidly for most patients. This study is registered at http://clinicaltrials.gov as NCI clinical trial no. NCT00943800.


Asunto(s)
Trasplante de Células Madre de Sangre del Cordón Umbilical , Supervivencia de Injerto , Enfermedad Injerto contra Huésped/epidemiología , Trasplante de Células Madre de Sangre Periférica , Acondicionamiento Pretrasplante , Adulto , Células Madre Adultas/metabolismo , Células Madre Adultas/trasplante , Anciano , Antígenos CD34/metabolismo , Trasplante de Células Madre de Sangre del Cordón Umbilical/efectos adversos , Femenino , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/prevención & control , Haplotipos , Humanos , Illinois/epidemiología , Incidencia , Masculino , Persona de Mediana Edad , Trasplante de Células Madre de Sangre Periférica/efectos adversos , Proyectos Piloto , Inducción de Remisión , Análisis de Supervivencia , Trasplante Homólogo , Adulto Joven
14.
Cytokine ; 61(1): 329-35, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23148990

RESUMEN

Erythropoietin (EPO) and Stem Cell Factor (SCF) have partially distinct functions in erythroid cell development. The primary functions of EPO are to prevent apoptosis and promote differentiation, with a minor role as a mitogen. On the other hand SCF acts primarily as a mitogenic factor promoting erythroid cell proliferation with a minor role in inhibition of apoptosis. The concerted effects of these two growth factors are responsible for guiding initial commitment, expansion and differentiation of progenitors. The aim of the study was to identify signaling elements pertinent to translational control and elucidate whether both cytokines can contribute to protein translation providing some functional redundancy as seen with respect to apoptosis. The current study focused on non-apoptotic functions of SCF mediated through mTOR/p70S6 leading to protein translation and cell proliferation. We utilized a human primary erythroid progenitors and erythroblasts that are responsive to EPO and SCF to investigate the activation of mTOR/p70S6 kinases and their downstream effectors, the pathway primarily responsible for protein translation. We showed that mTOR, p70S6 kinases and their downstream signaling elements 4EBP1 and S6 ribosomal protein are all activated by SCF but not by EPO in primary erythroid progenitors. We also found that SCF is the sole contributor to activation of the protein translational machinery and activation of mTOR/p70S6 pathway is confined to the proliferative phase of erythroid differentiation program. Altogether these results demonstrate that unlike the survival function which is supported by both EPO and SCF protein translation essential for proliferation is governed by only SCF.


Asunto(s)
Células Precursoras Eritroides/metabolismo , Eritropoyetina/farmacología , Factor de Células Madre/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Apoptosis , Diferenciación Celular , Proliferación Celular , Eritropoyetina/metabolismo , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal , Factor de Células Madre/metabolismo , Serina-Treonina Quinasas TOR/efectos de los fármacos
15.
J Biol Chem ; 286(28): 25211-23, 2011 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-21532034

RESUMEN

Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors, very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel, aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally, array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes, thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis, potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4, a GTPase regulator located in the commonly deleted 7q31 region, was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets, providing further validation of our findings. Finally, DOCK4 knockdown in primary marrow CD34(+) stem cells led to decreased erythroid colony formation and increased apoptosis, thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region.


Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 7/metabolismo , Epigénesis Genética , Proteínas Activadoras de GTPasa/biosíntesis , Leucocitos/metabolismo , Síndromes Mielodisplásicos/metabolismo , Apoptosis/genética , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Cromosomas Humanos Par 7/genética , Islas de CpG/genética , Metilación de ADN/genética , Femenino , Proteínas Activadoras de GTPasa/genética , Marcadores Genéticos , Humanos , Leucocitos/patología , Masculino , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/patología , Células Madre/metabolismo , Células Madre/patología
16.
Blood ; 116(17): 3331-40, 2010 Oct 28.
Artículo en Inglés | MEDLINE | ID: mdl-20644112

RESUMEN

Enucleation of mammalian erythroblasts is a process whose mechanism is largely undefined. The prevailing model suggests that nuclear extrusion occurs via asymmetric cytokinesis. To test this hypothesis, we treated primary erythroblasts with inhibitors of cytokinesis, including blebbistatin, hesperadin, and nocodazole, and then assayed for enucleation. Although these agents inhibited cell-cycle progression and subsequent enucleation when added early in culture, they failed to block enucleation proper when added to postmitotic cells. These results suggest that contraction of the actomyosin ring is not essential for nuclear expulsion. Next, by ultrastructural examination of primary erythroblasts, we observed an accumulation of vacuoles in the cytoplasm proximal to the extruding nucleus. This finding led us to hypothesize that vesicle trafficking contributes to erythroblast enucleation. Here, we show that chemical inhibitors of vesicle trafficking block enucleation of primary erythroblasts without affecting differentiation, cell division, or apoptosis. Moreover, knock-down of clathrin inhibited the enucleation of late erythroblasts. In contrast, vacuolin-1, a small molecule that induces vacuole formation, increased the percentage of enucleated cells. Together, these results illustrate that vesicle trafficking, specifically the formation, movement, and subsequent coalescence of vacuoles at the junction of the nucleus and the cytoplasm, is a critical component of mammalian erythroblast enucleation.


Asunto(s)
Eritroblastos/citología , Eritropoyesis , Animales , Células Cultivadas , Clatrina/genética , Clatrina/metabolismo , Citocinesis/efectos de los fármacos , Endocitosis , Eritroblastos/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Compuestos Heterocíclicos de 4 o más Anillos/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Hígado/citología , Ratones , Monensina/farmacología , Nocodazol/farmacología , Bazo/citología , Vacuolas/efectos de los fármacos , Vacuolas/metabolismo
17.
Haematologica ; 97(10): 1471-9, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22491741

RESUMEN

BACKGROUND: Nucleus free red blood cells are unique to mammals. During their terminal stage of differentiation, mammalian erythroblasts exit the cell cycle and enucleate. We previously found that survivin, a member of the chromosomal passenger complex that is required for cytokinesis, is highly expressed in late non-dividing cells. The role of survivin in enucleating erythroblasts is not known. DESIGN AND METHODS: In order to identify the role of survivin in these late erythroblasts, we performed proteomic analysis on survivin-bound protein complexes purified from murine erythroleukemia cells. Various molecular and cell biological techniques were used to confirm the presence and function of this novel complex. Furthermore, we used survivin(fl/fl) mice to study the effect of loss of survivin in enucleating erythroblasts. RESULTS: We found that survivin failed to co-localize with its known partners' inner centromere protein or Aurora-B in enucleating erythroblasts but rather exists in a multi-protein complex with epidermal growth factor receptor substrate15 and clathrin, two proteins that mediate endocytic vesicle trafficking. As evidence for a direct role of this latter complex in enucleation, we found that knockdown of the genes reduced the efficiency of enucleation of primary human erythroblasts. We also observed that loss of survivin in murine erythroblasts inhibited enucleation and that survivin-deficient cells harbored smaller cytoplasmic vacuoles. Interestingly, vacuolin-1, a small molecule that induces vacuole fusion, rescued the defective enucleation caused by survivin deficiency. CONCLUSIONS: This study identified a novel role for survivin in erythroblast enucleation through previously unknown protein partners.


Asunto(s)
Diferenciación Celular , Núcleo Celular/metabolismo , Eritroblastos/citología , Eritroblastos/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Proteínas Represoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Expresión Génica , Técnicas de Inactivación de Genes , Compuestos Heterocíclicos de 4 o más Anillos/metabolismo , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Ratones , Unión Proteica , Transporte de Proteínas , Proteínas Represoras/genética , Survivin , Vacuolas/metabolismo
18.
Cytotherapy ; 14(6): 707-15, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22548695

RESUMEN

BACKGROUND AIMS: The rising use of allogeneic transplantation in older recipients necessitates considering older related donors. The effect of related donor age for peripheral blood stem cell allografts (PBSC) on graft maintenance and outcomes, independent of CD34(+)cell dose, has not been well-characterized. METHODS: HLA-related donors (98% siblings) underwent a uniform filgrastim-based mobilization regimen aiming to collect and infuse 5 × 10(6) CD34(+) cells/recipient kg. Donor and recipient age were modeled in multiple ways to account for the correlation, and outcomes reported by decade of donor age. RESULTS: The median donor and recipient ages were 52 years and 54 years, respectively. The mean CD34(+) cell dose infused was 5.6 × 10(6) CD34(+)/kg and 75% of patients received a narrow range between 4.4 and 6.6 × 10(6) CD34(+) cells/kg. Neither better PBSC mobilization nor higher CD34(+) content of allografts was significantly associated with engraftment or transplant outcomes. After adjusting for recipient age and other prognostic factors, older donor age by decade conferred a lower risk of non-relapse mortality (NRM) [hazard ratio (HR) = 0.64, 95% confidence interval (CI) 0.45-0.91, P = 0.013] and borderline improvement in overall survival (OS) (HR = 0.76, 95% CI 0.58-0.99, P = 0.045) without altering progression-free survival (PFS) (HR = 0.85, 95% CI 0.66-1.07, P = 0.18). CONCLUSIONS: Older donor age does not worsen outcome after matched related donor PBSC transplantation in patients receiving a narrow range CD34(+) cells. The relatively small sample size mandates that the finding of similar to improved outcomes for older related donor age must be confirmed in larger studies.


Asunto(s)
Trasplante de Células Madre de Sangre Periférica , Donantes de Tejidos , Adolescente , Adulto , Factores de Edad , Anciano , Antígenos CD34 , Supervivencia sin Enfermedad , Familia , Femenino , Movilización de Célula Madre Hematopoyética , Humanos , Illinois/epidemiología , Masculino , Persona de Mediana Edad , Trasplante de Células Madre de Sangre Periférica/mortalidad , Pronóstico , Acondicionamiento Pretrasplante , Trasplante Homólogo , Resultado del Tratamiento , Adulto Joven
19.
Blood Cancer Discov ; 3(5): 444-467, 2022 09 06.
Artículo en Inglés | MEDLINE | ID: mdl-35820129

RESUMEN

Cytosine hypermethylation in and around DNA-binding sites of master transcription factors, including PU.1, occurs in aging hematopoietic stem cells following acquired loss-of-function mutations of DNA methyl-cytosine dioxygenase ten-eleven translocation-2 (TET2), albeit functional relevance has been unclear. We show that Tet2-deficient mouse hematopoietic stem and progenitor cells undergo malignant transformation upon compromised gene regulation through heterozygous deletion of an upstream regulatory region (UREΔ/WT) of the PU.1 gene. Although compatible with multilineage blood formation at young age, Tet2-deficient PU.1 UREΔ/WT mice develop highly penetrant, transplantable acute myeloid leukemia (AML) during aging. Leukemic stem and progenitor cells show hypermethylation at putative PU.1-binding sites, fail to activate myeloid enhancers, and are hallmarked by a signature of genes with impaired expression shared with human AML. Our study demonstrates that Tet2 and PU.1 jointly suppress leukemogenesis and uncovers a methylation-sensitive PU.1-dependent gene network as a unifying molecular vulnerability associated with AML. SIGNIFICANCE: We identify moderately impaired PU.1 mRNA expression as a biological modality predisposing Tet2-deficient hematopoietic stem and progenitor cells to malignant transformation. Our study furthermore uncovers a methylation-sensitive PU.1 gene network as a common feature of myeloid leukemia potentially allowing for the identification of patients at risk for malignant transformation. See related commentary by Schleicher and Pietras, p. 378. This article is highlighted in the In This Issue feature, p. 369.


Asunto(s)
Proteínas de Unión al ADN , Dioxigenasas , Leucemia Mieloide Aguda , Proteínas Proto-Oncogénicas , Transactivadores , Animales , Transformación Celular Neoplásica/genética , Citosina , Proteínas de Unión al ADN/genética , Dioxigenasas/genética , Elementos de Facilitación Genéticos , Hematopoyesis/genética , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética
20.
Elife ; 112022 08 30.
Artículo en Inglés | MEDLINE | ID: mdl-36040792

RESUMEN

Background: Mutations in the SF3B1 splicing factor are commonly seen in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), yet the specific oncogenic pathways activated by mis-splicing have not been fully elucidated. Inflammatory immune pathways have been shown to play roles in the pathogenesis of MDS, though the exact mechanisms of their activation in splicing mutant cases are not well understood. Methods: RNA-seq data from SF3B1 mutant samples was analyzed and functional roles of interleukin-1 receptor-associated kinase 4 (IRAK4) isoforms were determined. Efficacy of IRAK4 inhibition was evaluated in preclinical models of MDS/AML. Results: RNA-seq splicing analysis of SF3B1 mutant MDS samples revealed retention of full-length exon 6 of IRAK4, a critical downstream mediator that links the Myddosome to inflammatory NF-kB activation. Exon 6 retention leads to a longer isoform, encoding a protein (IRAK4-long) that contains the entire death domain and kinase domain, leading to maximal activation of NF-kB. Cells with wild-type SF3B1 contain smaller IRAK4 isoforms that are targeted for proteasomal degradation. Expression of IRAK4-long in SF3B1 mutant cells induces TRAF6 activation leading to K63-linked ubiquitination of CDK2, associated with a block in hematopoietic differentiation. Inhibition of IRAK4 with CA-4948, leads to reduction in NF-kB activation, inflammatory cytokine production, enhanced myeloid differentiation in vitro and reduced leukemic growth in xenograft models. Conclusions: SF3B1 mutation leads to expression of a therapeutically targetable, longer, oncogenic IRAK4 isoform in AML/MDS models. Funding: This work was supported by Cincinnati Children's Hospital Research Foundation, Leukemia Lymphoma Society, and National Institute of Health (R35HL135787, RO1HL111103, RO1DK102759, RO1HL114582), Gabrielle's Angel Foundation for Cancer Research, and Edward P. Evans Foundation grants to DTS. AV is supported by Edward P. Evans Foundation, National Institute of Health (R01HL150832, R01HL139487, R01CA275007), Leukemia and Lymphoma Society, Curis and a gift from the Jane and Myles P. Dempsey family. AP and JB are supported by Blood Cancer UK (grants 13042 and 19004). GC is supported by a training grant from NYSTEM. We acknowledge support of this research from The Einstein Training Program in Stem Cell Research from the Empire State Stem Cell Fund through New York State Department of Health Contract C34874GG. MS is supported by a National Institute of Health Research Training and Career Development Grant (F31HL132420).


Genes contain blocks of code that tell cells how to make each part of a protein. Between these blocks are sections of linking DNA, which cells remove when they are preparing to use their genes. Scientists call this process 'splicing'. Cells can splice some genes in more than one way, allowing them to make different proteins from the same genetic code. Mutations that affect the splicing process can change the way cells make their proteins, leading to disease. For example, the myelodysplastic syndromes are a group of blood cancers often caused by mutations in splicing proteins, such as SF3B1. The disorder stops blood cells from maturing and causes abnormal inflammation. So far, the link between splicing, blood cell immaturity, inflammation and cancer is not clear. To find out more, Choudhary, Pellagatti et al. looked at the spliced genetic code from people with myelodysplastic syndromes. Mutations in the splicing protein SF3B1 changed the way cells spliced an important signalling molecule known as IRAK4. Affected cells cut out less genetic code and made a longer version of this signalling protein, named IRAK4-Long. This altered protein activated inflammation and stopped blood cells from maturing. Blocking IRAK4-Long reversed the effects. It also reduced tumour formation in mice carrying affected human cells. The molecule used to block IRAK4, CA-4948 ­ also known as Emavusertib ­ is currently being evaluated in clinical trials for myelodysplastic syndromes and other types of blood cancer. The work of Choudhary, Pellagatti et al. could help scientists to design genetic tests to predict which patients might benefit from this treatment.


Asunto(s)
Leucemia Mieloide Aguda , Síndromes Mielodisplásicos , Fosfoproteínas/metabolismo , Factores de Empalme de ARN/metabolismo , Niño , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/genética , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Mutación , Síndromes Mielodisplásicos/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Isoformas de Proteínas/metabolismo , Empalme del ARN
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