RESUMEN
BACKGROUND: Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. METHODS: We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry. RESULTS: Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients. CONCLUSIONS: Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay Kendall Leukaemia Fund and others.).
Asunto(s)
Calreticulina/genética , Mutación , Síndromes Mielodisplásicos/genética , Mielofibrosis Primaria/genética , Trombocitemia Esencial/genética , Secuencia de Aminoácidos , Enfermedades de la Médula Ósea/genética , Calreticulina/análisis , Exones , Humanos , Janus Quinasa 2/genética , Leucemia Mieloide/genética , Datos de Secuencia Molecular , Neoplasias/genética , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
Most myeloproliferative neoplasm (MPN) patients lacking JAK2 mutations harbour somatic CALR mutations that are thought to activate cytokine signalling although the mechanism is unclear. To identify kinases important for survival of CALR-mutant cells, we developed a novel strategy (KISMET) that utilizes the full range of kinase selectivity data available from each inhibitor and thus takes advantage of off-target noise that limits conventional small-interfering RNA or inhibitor screens. KISMET successfully identified known essential kinases in haematopoietic and non-haematopoietic cell lines and identified the mitogen activated protein kinase (MAPK) pathway as required for growth of the CALR-mutated MARIMO cells. Expression of mutant CALR in murine or human haematopoietic cell lines was accompanied by myeloproliferative leukemia protein (MPL)-dependent activation of MAPK signalling, and MPN patients with CALR mutations showed increased MAPK activity in CD34 cells, platelets and megakaryocytes. Although CALR mutations resulted in protein instability and proteosomal degradation, mutant CALR was able to enhance megakaryopoiesis and pro-platelet production from human CD34+ progenitors. These data link aberrant MAPK activation to the MPN phenotype and identify it as a potential therapeutic target in CALR-mutant positive MPNs.
Asunto(s)
Calreticulina/genética , Diferenciación Celular , Megacariocitos/citología , Megacariocitos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Transducción de Señal , Antígenos CD34/metabolismo , Calreticulina/antagonistas & inhibidores , Línea Celular , Descubrimiento de Drogas , Expresión Génica Ectópica/efectos de los fármacos , Sangre Fetal/citología , Humanos , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/genética , Megacariocitos/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Estabilidad Proteica , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Transducción de Señal/efectos de los fármacos , Trombopoyesis/genética , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
With an eye toward the design of new nitroxide-based magnetic resonance imaging (MRI) contrast agents, the effect of ring size and the presence of proximate polar groups on the rate of reduction of cyclic nitroxides by the biologically relevant reducing agent ascorbate ion have been determined. To the extent that reduction by ascorbate ion is indicative of the behavior of nitroxides toward other in vivo reducing agents, our results indicate that saturated five-membered cyclic nitroxides are to be preferred over smaller or larger ring sizes or acyclic nitroxides. Proximate polar groups tend to enhance the susceptibility toward reduction by ascorbate.