RESUMEN
The development of CRISPR-derived genome editing technologies has enabled the precise manipulation of DNA sequences within the human genome. In this review, we discuss the initial development and cellular mechanism of action of CRISPR nucleases and DNA base editors. We then describe factors that must be taken into consideration when developing these tools into therapeutic agents, including the potential for unintended and off-target edits when using these genome editing tools, and methods to characterize these types of edits. We finish by considering specific challenges associated with bringing a CRISPR-based therapy to the clinic, including manufacturing, regulatory oversight, and considerations for clinical trials that involve genome editing agents.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Terapia Genética , Animales , Proteína 9 Asociada a CRISPR , Ensayos Clínicos como Asunto , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica/métodos , Técnicas de Transferencia de Gen , Ingeniería Genética , Terapia Genética/métodos , Terapia Genética/tendencias , Humanos , Modelos Animales , ARN Guía de Kinetoplastida , Reparación del ADN por Recombinación , Investigación Biomédica Traslacional/métodos , Investigación Biomédica Traslacional/tendenciasRESUMEN
Fbw7 is a tumor suppressor that regulates the degradation of oncogenic substrates such as c-Jun, c-Myc, Notch1 intracellular domain (ICD), and cyclin E by functioning as the substrate recognition protein in the Skp1-Cullin-F-box (SCF) ubiquitin ligase complex. Consequently, low expression or loss of FBXW7 in breast cancer has been hypothesized to result in the accumulation of oncogenic transcription factors that are master regulators of proliferation, apoptosis, and ultimately transformation. Despite this, the direct effect of Fbw7 loss on mammary gland morphology and tumorigenesis has not been examined. Here, we demonstrate that conditional deletion of Fbxw7 in murine mammary tissue initiates breast tumor development and also results in lactation and involution defects. Further, while Fbxw7 loss results in the overexpression of Notch1-ICD, c-Jun, cyclin E, and c-Myc, the downstream transcription factor pathways associated with c-Myc and cyclin E are the most dysregulated, including at the single-cell level. These pathways are dysregulated early after Fbxw7 loss, and their sustained loss results in tumorigenesis and reinforced c-Myc and cyclin E-E2F pathway disruption. We also find that loss of Fbxw7 is linked to the acquisition of Trp53 mutations, similar to the mutational spectrum observed in patients. Our results demonstrate that the loss of Fbxw7 promotes the acquisition of Trp53 mutations and that the two cooperate in breast tumor development. Targeting c-Myc, E2F, or p53 may therefore be a beneficial treatment strategy for FBXW7-altered breast cancer patients.
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Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Factores de Transcripción E2F/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD/deficiencia , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteína p53 Supresora de Tumor/genética , Secuencia de Aminoácidos , Animales , Neoplasias de la Mama , Línea Celular Tumoral , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Proteína 7 que Contiene Repeticiones F-Box-WD/química , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Inmunohistoquímica , Ratones , Ratones Noqueados , Ratones Transgénicos , Mutación , Transcripción GenéticaRESUMEN
PURPOSE: The anti-B-cell maturation antigen BiTE molecule AMG 420 was assessed in patients with relapsed/refractory multiple myeloma. PATIENTS AND METHODS: In this first-in-human study, up to 10 cycles of AMG 420 were given (4-week infusions/6-week cycles). Patients had progression after ≥ 2 lines of prior therapy and no extramedullary disease. Minimal residual disease (MRD) response was defined as < 1 tumor cell/104 bone marrow cells by flow cytometry. RESULTS: Forty-two patients received AMG 420 at 0.2-800 µg/d. Median age was 65 years, and median disease duration was 5.2 years. Median exposure was 1 cycle (range, 1-10 cycles) and 7 cycles (range, 1-10 cycles) for responders. Patients discontinued for disease progression (n = 25), adverse events (AEs; n = 7), death (n = 4), completion of 10 cycles (n = 3), and consent withdrawal (n = 1). Two patients remain on treatment. There were 2 nontreatment-related deaths from AEs, influenza/aspergillosis and adenovirus-related hepatitis. Serious AEs (n = 20; 48%) included infections (n = 14) and polyneuropathy (n = 2); treatment-related serious AEs included 2 grade 3 polyneuropathies and 1 grade 3 edema. There were no grade ≥ 3 CNS toxicities or anti-AMG 420 antibodies. In this study, 800 µg/d was considered to not be tolerable because of 1 instance each of grade 3 cytokine release syndrome and grade 3 polyneuropathy, both of which resolved. The overall response rate was 31% (n = 13 of 42). At the maximum tolerated dose (MTD) of 400 µg/d, the response rate was 70% (n = 7 of 10). Of these, five patients experienced MRD-negative complete responses, and 1 had a partial response, and 1 had a very good partial response; all 7 patients responded during the first cycle, and some responses lasted > 1 year. CONCLUSION: In this study of AMG 420 in patients with relapsed/refractory multiple myeloma, the response rate was 70%, including 50% MRD-negative complete responses, at 400 µg/d, the MTD for this study.
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Anticuerpos Biespecíficos/administración & dosificación , Antígeno de Maduración de Linfocitos B/antagonistas & inhibidores , Mieloma Múltiple/terapia , Adulto , Anciano , Anticuerpos Biespecíficos/inmunología , Antígeno de Maduración de Linfocitos B/inmunología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Cancer-associated mutations of the core splicing factor 3 B1 (SF3B1) result in selection of novel 3' splice sites (3'SS), but precise molecular mechanisms of oncogenesis remain unclear. SF3B1 stabilizes the interaction between U2 snRNP and branch point (BP) on the pre-mRNA. It has hence been speculated that a change in BP selection is the basis for novel 3'SS selection. Direct quantitative determination of BP utilization is however technically challenging. To define BP utilization by SF3B1-mutant spliceosomes, we used an overexpression approach in human cells as well as a complementary strategy using isogenic murine embryonic stem cells with monoallelic K700E mutations constructed via CRISPR/Cas9-based genome editing and a dual vector homology-directed repair methodology. A synthetic minigene library with degenerate regions in 3' intronic regions (3.4 million individual minigenes) was used to compare BP usage of SF3B1K700E and SF3B1WT. Using this model, we show that SF3B1K700E spliceosomes utilize non-canonical sequence variants (at position -1 relative to BP adenosine) more frequently than wild-type spliceosomes. These predictions were confirmed using minigene splicing assays. Our results suggest a model of BP utilization by mutant SF3B1 wherein it is able to utilize non-consensus alternative BP sequences by stabilizing weaker U2-BP interactions.
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Factores de Empalme de ARN/metabolismo , Animales , Emparejamiento Base , Células Cultivadas , Células Madre Embrionarias/metabolismo , Biblioteca de Genes , Células HEK293 , Humanos , Ratones , Mutación , Motivos de Nucleótidos , Fosfoproteínas/genética , Sitios de Empalme de ARN , Factores de Empalme de ARN/genética , ARN Mensajero/metabolismoRESUMEN
Splicing factor 3B1 (SF3B1) is a core splicing protein that stabilizes the interaction between the U2 snRNA and the branch point in the mRNA target during splicing. SF3B1 is heavily phosphorylated at its N terminus and a substrate of cyclin-dependent kinases (CDKs). Although SF3B1 phosphorylation coincides with splicing catalysis, the functional significance of SF3B1 phosphorylation is largely undefined. Here, we show that SF3B1 phosphorylation follows a dynamic pattern during cell cycle progression that depends on CDK activity. SF3B1 is known to interact with chromatin, and we found that SF3B1 maximally interacts with nucleosomes during G1/S and that this interaction requires CDK2 activity. In contrast, SF3B1 disassociates from nucleosomes at G2/M, coinciding with a peak in CDK1-mediated SF3B1 phosphorylation. Thus, CDK1 and CDK2 appear to have opposing roles in regulating SF3B1-nucleosome interactions. Importantly, these interactions were modified by the presence and phosphorylation status of linker histone H1, particularly the H1.4 isoform. Performing genome-wide analysis of SF3B1-chromatin binding in synchronized cells, we observed that SF3B1 preferentially bound exons. Differences in SF3B1 chromatin binding to specific sites, however, did not correlate with changes in RNA splicing, suggesting that the SF3B1-nucleosome interaction does not determine cell cycle-dependent changes to mRNA splicing. Our results define a cell cycle stage-specific interaction between SF3B1 and nucleosomes that is mediated by histone H1 and depends on SF3B1 phosphorylation. Importantly, this interaction does not seem to be related to SF3B1's splicing function and, rather, points toward its potential role as a chromatin modifier.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Cromatina/metabolismo , Fosfoproteínas/metabolismo , Factores de Empalme de ARN/metabolismo , Ciclo Celular , Células HeLa , Histonas/metabolismo , Humanos , Nucleosomas/metabolismo , Fosforilación , Unión Proteica , Empalme del ARNRESUMEN
E2F-2 is a retinoblastoma (Rb)-regulated transcription factor induced during terminal erythroid maturation. Cyclin E-mediated Rb hyperphosphorylation induces E2F transcriptional activator functions. We previously reported that deregulated cyclin E activity causes defective terminal maturation of nucleated erythroblasts in vivo Here, we found that these defects are normalized by E2F-2 deletion; however, anemia in mice with deregulated cyclin E is not improved by E2F-2-loss, which itself causes reduced peripheral red blood cell (RBC) counts without altering relative abundances of erythroblast subpopulations. To determine how E2F-2 regulates RBC production, we comprehensively studied erythropoiesis using knockout mice and hematopoietic progenitors. We found that efficient stress erythropoiesis in vivo requires E2F-2, and we also identified an unappreciated role for E2F-2 in erythroblast enucleation. In particular, E2F-2 deletion impairs nuclear condensation, a morphological feature of maturing erythroblasts. Transcriptome profiling of E2F-2-null, mature erythroblasts demonstrated widespread changes in gene expression. Notably, we identified citron Rho-interacting kinase (CRIK), which has known functions in mitosis and cytokinesis, as induced in erythroblasts in an E2F-2-dependent manner, and we found that CRIK activity promotes efficient erythroblast enucleation and nuclear condensation. Together, our data reveal novel, lineage-specific functions for E2F-2 and suggest that some mitotic kinases have specialized roles supporting enucleation of maturing erythroblasts.
Asunto(s)
Núcleo Celular/metabolismo , Factor de Transcripción E2F2/genética , Factor de Transcripción E2F2/metabolismo , Eritroblastos/citología , Eritropoyesis , Animales , Ciclo Celular , Diferenciación Celular , Ciclina E/metabolismo , Eritroblastos/metabolismo , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteína de Retinoblastoma/metabolismoRESUMEN
Mutations of HSPB5 (also known as CRYAB or αB-crystallin), a bona fide heat shock protein and molecular chaperone encoded by the HSPB5 (crystallin, alpha B) gene, are linked to multisystem disorders featuring variable combinations of cataracts, cardiomyopathy, and skeletal myopathy. This study aimed to investigate the pathological mechanisms involved in an early-onset myofibrillar myopathy manifesting in a child harboring a homozygous recessive mutation in HSPB5, 343delT. To study HSPB5 343delT protein dynamics, we utilize model cell culture systems including induced pluripotent stem cells derived from the 343delT patient (343delT/343delT) along with isogenic, heterozygous, gene-corrected control cells (WT KI/343delT) and BHK21 cells, a cell line lacking endogenous HSPB5 expression. 343delT/343delT and WT KI/343delT-induced pluripotent stem cell-derived skeletal myotubes and cardiomyocytes did not express detectable levels of 343delT protein, contributable to the extreme insolubility of the mutant protein. Overexpression of HSPB5 343delT resulted in insoluble mutant protein aggregates and induction of a cellular stress response. Co-expression of 343delT with WT prevented visible aggregation of 343delT and improved its solubility. Additionally, in vitro refolding of 343delT in the presence of WT rescued its solubility. We demonstrate an interaction between WT and 343delT both in vitro and within cells. These data support a loss-of-function model for the myopathy observed in the patient because the insoluble mutant would be unavailable to perform normal functions of HSPB5, although additional gain-of-function effects of the mutant protein cannot be excluded. Additionally, our data highlight the solubilization of 343delT by WT, concordant with the recessive inheritance of the disease and absence of symptoms in carrier individuals.
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Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Catarata/genética , Catarata/metabolismo , Enfermedades Musculares/genética , Enfermedades Musculares/metabolismo , Cadena B de alfa-Cristalina/genética , Cadena B de alfa-Cristalina/metabolismo , Cardiomiopatías/etiología , Catarata/etiología , Femenino , Homocigoto , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Modelos Biológicos , Fibras Musculares Esqueléticas/metabolismo , Enfermedades Musculares/etiología , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Miocitos Cardíacos/metabolismo , Linaje , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Solubilidad , Cadena B de alfa-Cristalina/químicaRESUMEN
MicroRNAs (miRNAs) comprise a class of small, regulatory noncoding RNAs (ncRNAs) with pivotal roles in posttranscriptional gene regulation. Since their initial discovery in 1993, numerous miRNAs have been identified in mammalian genomes, many of which play important roles in diverse cellular processes in development and disease. These small ncRNAs regulate the expression of many protein-coding genes posttranscriptionally, thus adding a substantial complexity to the molecular networks underlying physiological development and disease. In part, this complexity arises from the distinct gene structures, the extensive genomic redundancy, and the complex regulation of the expression and biogenesis of miRNAs. These characteristics contribute to the functional robustness and versatility of miRNAs and provide important clues to the functional significance of these small ncRNAs. The unique structure and function of miRNAs will continue to inspire many to explore the vast noncoding genome and to elucidate the molecular basis for the functional complexity of mammalian genomes.
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Regulación de la Expresión Génica/fisiología , Genoma Humano/fisiología , MicroARNs/metabolismo , Conformación de Ácido Nucleico , Animales , Humanos , MicroARNs/genéticaRESUMEN
To maintain hematologic homeostasis, hematopoietic stem cells (HSCs) undergo multiple rounds of cell division throughout their lives. Under steady-state conditions, adult HSCs are relatively quiescent and reside primarily in hypoxic bone marrow niches. In response to physiologic stimuli, normal HSCs either reenter the cell division cycle or remain in quiescence. A large body of work has focused on understanding the mechanistic underpinnings balancing differentiation against self-renewal programs in cycling HSCs. Numerous reports from genetically engineered mouse models harboring mutations in key pathways governing proliferation control, DNA damage responses, and metabolic regulation indicate the critical roles these processes play in determining HSC self-renewing versus blood-lineage-reconstituting divisions. In this chapter, we integrate these findings and highlight the cellular networks that control HSC function and fitness by regulating HSC cycling.
Asunto(s)
Puntos de Control del Ciclo Celular/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Biología de Sistemas/métodos , Adulto , Células Madre Adultas/citología , Células Madre Adultas/fisiología , Animales , Proliferación Celular/genética , Humanos , RatonesRESUMEN
The Fbw7 ubiquitin ligase critically regulates hematopoietic stem cell (HSC) function, though the precise contribution of individual substrate ubiquitination pathways to HSC homeostasis is unknown. In the work reported here, we used a mouse model in which we introduced two knock-in mutations (T74A and T393A [changes of T to A at positions 74 and 393]) to disrupt Fbw7-dependent regulation of cyclin E, its prototypic substrate, and to examine the consequences of cyclin E dysregulation for HSC function. Serial transplantation revealed that cyclin E(T74A T393A) HSCs self-renewed normally; however, we identified defects in their multilineage reconstituting capacity. By inducing hematologic stress, we exposed an impaired self-renewal phenotype in cyclin E knock-in HSCs that was associated with defective cell cycle exit and the emergence of chromosome instability (CIN). Importantly, p53 deletion induced both defects in self-renewal and multilineage reconstitution in cyclin E knock-in HSCs with serial transplantation and CIN in hematopoietic stem and progenitor cells. Moreover, CIN was a feature of fatal T-cell malignancies that ultimately developed in recipients of cyclin E(T74A T393A); p53-null HSCs. Together, our findings demonstrate the importance of Fbw7-dependent cyclin E control to the hematopoietic system and highlight CIN as a characteristic feature of HSC dysfunction and malignancy induced by deregulated cyclin E.
Asunto(s)
Inestabilidad Cromosómica/fisiología , Ciclina E/genética , Ciclina E/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Células Madre Hematopoyéticas/fisiología , Linfoma de Células T/etiología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Sustitución de Aminoácidos , Animales , Linaje de la Célula , Inestabilidad Cromosómica/genética , Proteína 7 que Contiene Repeticiones F-Box-WD , Genes p53 , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/patología , Linfoma de Células T/genética , Linfoma de Células T/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Estabilidad Proteica , Proteolisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The inherited bone marrow failure syndromes, like severe congenital neutropenia (SCN) and Shwachman-Diamond syndrome (SDS), provide unique insights into normal and impaired myelopoiesis. The inherited neutropenias are heterogeneous in both clinical presentation and genetic associations, and their causative mechanisms are not well established. SCN, for example, is a genetically heterogeneous syndrome associated with mutations of ELANE, HAX1, GFI1, WAS, G6PC3, or CSF3R. The genetic diversity in SCN, along with congenital neutropenias associated with other genetically defined bone marrow failure syndromes (e.g., SDS), suggests that various pathways may be involved in their pathogenesis. Alternatively, all may lead to a final common pathway of enhanced apoptosis. The pursuit for a more complete understanding of the molecular mechanisms that drive inherited neutropenias remains at the forefront of pediatric translational and basic science investigation. Advances in our understanding of these disorders have greatly increased over the last 10 years concomitant with identification of their genetic lesions. Emerging themes include induction of the unfolded protein response (UPR), defective ribosome assembly, and p53-dependent apoptosis. Additionally, defects in metabolism, disruption of mitochondrial membrane potential, and mislocalization have been found. When perturbed, each of these lead to an intracellular stress that triggers apoptosis in the vulnerable granulocytic precursor.
Asunto(s)
Hemoglobinuria Paroxística/metabolismo , Neutropenia/metabolismo , Neutrófilos/metabolismo , Transducción de Señal , Estrés Fisiológico , Anemia Aplásica , Animales , Apoptosis , Enfermedades de la Médula Ósea , Trastornos de Fallo de la Médula Ósea , Predisposición Genética a la Enfermedad , Hemoglobinuria Paroxística/congénito , Hemoglobinuria Paroxística/inmunología , Hemoglobinuria Paroxística/patología , Humanos , Mutación , Neutropenia/congénito , Neutropenia/inmunología , Neutropenia/patología , Neutrófilos/inmunología , Neutrófilos/patología , Fenotipo , Factores de Riesgo , Transducción de Señal/genética , Estrés Fisiológico/genéticaRESUMEN
mir-17-92, a potent polycistronic oncomir, encodes six mature miRNAs with complex modes of interactions. In the Eµ-myc Burkitt's lymphoma model, mir-17-92 exhibits potent oncogenic activity by repressing c-Myc-induced apoptosis, primarily through its miR-19 components. Surprisingly, mir-17-92 also encodes the miR-92 component that negatively regulates its oncogenic cooperation with c-Myc. This miR-92 effect is, at least in part, mediated by its direct repression of Fbw7, which promotes the proteosomal degradation of c-Myc. Thus, overexpressing miR-92 leads to aberrant c-Myc increase, imposing a strong coupling between excessive proliferation and p53-dependent apoptosis. Interestingly, miR-92 antagonizes the oncogenic miR-19 miRNAs; and such functional interaction coordinates proliferation and apoptosis during c-Myc-induced oncogenesis. This miR-19:miR-92 antagonism is disrupted in B-lymphoma cells that favor a greater increase of miR-19 over miR-92. Altogether, we suggest a new paradigm whereby the unique gene structure of a polycistronic oncomir confers an intricate balance between oncogene and tumor suppressor crosstalk. DOI:http://dx.doi.org/10.7554/eLife.00822.001.
Asunto(s)
Apoptosis/fisiología , MicroARNs/fisiología , Oncogenes , Animales , Apoptosis/genética , Células Cultivadas , RatonesRESUMEN
Cancers of diverse cell lineages express high levels of cyclin E, and in various studies, cyclin E overexpression correlates with increased tumor aggression. One way that normal control of cyclin E expression is disabled in cancer cells is via loss-of-function mutations sustained by FBXW7. This gene encodes the Fbw7 tumor suppressor protein that provides substrate specificity for a ubiquitin ligase complex that targets multiple oncoproteins for degradation. Numerous other mechanisms besides Fbw7 mutations can deregulate cyclin E expression and activity in cancer cells. Recent reports demonstrate that inappropriate cyclin E expression may have far-reaching biological consequences for cell physiology, including altering gene expression programs governing proliferation, differentiation, survival and senescence. In this review, we discuss the function of mammalian cyclin E in the context of these new data as well as the complex network that connects cyclin E functions to the cellular controls regulating its expression and activity.
Asunto(s)
Ciclina E/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ciclina E/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Factores de Transcripción E2F/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD , Técnicas de Sustitución del Gen , Humanos , Interfase , Ratones , Fosforilación , Especificidad por Sustrato , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The classical myeloproliferative neoplasms (MPNs) feature an overproduction of mature blood elements. Phenotypic conversion, including transformation to myelofibrosis (MF) in those with antecedent ET and PV is a feared complication. Hypereosinophilic syndromes (HESs), especially those with myeloproliferative variants, can display similar features, including organomegaly, marrow fibrosis, clonality, thrombotic tendencies, and acute myeloid leukemia (AML) transformation. However, this group of illnesses is typically clinically and molecularly distinct from the classical MPNs. We report a case of a 59-yr-old woman with complex hypereosinophilia in the setting of post-polycythemic myelofibrosis (post-PVMF), with multi-system end-organ damage characteristic of HES.
RESUMEN
Increased cyclin E expression has been identified in human tumors of diverse histologies, and in studies of primary breast cancers, high cyclin E is associated with poor prognosis. We have studied dysregulated cyclin E in epithelial tissues using organotypic cultures of human mammary epithelial cells and a murine model. We unexpectedly discovered that dysregulated cyclin E impairs normal acinar morphogenesis in vitro, and this is associated with the induction of p21(Cip1), p27(Kip1), and cellular senescence. Cyclin E-induced morphogenesis arrest is dependent upon hypoxia-inducible factor 1α (HIF-1α), which itself is induced by high cyclin E both in cultured mammary acini and in mammary epithelial tissues in a mouse model of deregulated cyclin E expression. We next determined that E2F activity directly regulates and is required for induction of HIF1A by cyclin E. Additionally, we found that cyclin E deregulation in mammary acini decreases, in an E2F-independent manner, expression of the EGLN1 prolyl hydroxylase that regulates HIF-1α degradation within the VHL ubiquitin ligase pathway. Together, our findings reveal a direct link between cyclin E and HIF-1 activities in mammary epithelial cells and implicate HIF-1 as a mediator of proliferation-independent phenotypes associated with high cyclin E expression in some human breast cancers.
Asunto(s)
Ciclina E , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Glándulas Mamarias Animales/citología , Glándulas Mamarias Humanas/citología , Animales , Neoplasias de la Mama/metabolismo , Ciclo Celular , Línea Celular , Senescencia Celular , Inmunoprecipitación de Cromatina , Ciclina E/biosíntesis , Ciclina E/genética , Ciclina E/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Factores de Transcripción E2F/metabolismo , Femenino , Células HEK293 , Humanos , Ratones , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
F-box and WD-40 domain protein 7 (Fbw7) provides substrate specificity for the Skp1-Cullin1-F-box protein (SCF) ubiquitin ligase complex that targets multiple oncoproteins for degradation, including cyclin E, c-Myc, c-Jun, Notch, and mammalian target of rapamycin (mTOR). Fbw7 is a bona fide tumor suppressor, and loss-of-function mutations in FBXW7 have been identified in diverse human tumors. Although much is known about targets of the Fbw7 ubiquitin ligase pathway, relatively little is known about the regulation of Fbw7 expression. We identified a panel of candidate microRNA regulators of Fbw7 expression within a study of gene expression alterations in primary erythroblasts obtained from cyclin E(T74A T393A) knock-in mice, which have markedly dysregulated cyclin E expression. We found that overexpression of miR-223, in particular, significantly reduces FBXW7 mRNA levels, increases endogenous cyclin E protein and activity levels, and increases genomic instability. We next confirmed that miR-223 targets the FBXW7 3'-untranslated region. We then found that reduced miR-223 expression in primary mouse embryonic fibroblasts leads to increased Fbw7 expression and decreased cyclin E activity. Finally, we found that miR-223 expression is responsive to acute alterations in cyclin E regulation by the Fbw7 pathway. Together, our data indicate that miR-223 regulates Fbw7 expression and provide the first evidence that activity of the SCF(Fbw7) ubiquitin ligase can be modulated directly by the microRNA pathway.
Asunto(s)
Regiones no Traducidas 3'/fisiología , Proteínas de Ciclo Celular/biosíntesis , Ciclina E/metabolismo , Proteínas F-Box/biosíntesis , Regulación de la Expresión Génica/fisiología , MicroARNs/metabolismo , Ubiquitina-Proteína Ligasas/biosíntesis , Animales , Proteínas de Ciclo Celular/genética , Ciclina E/genética , Proteínas F-Box/genética , Proteína 7 que Contiene Repeticiones F-Box-WD , Humanos , Células K562 , Ratones , Ratones Transgénicos , MicroARNs/genética , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas Ligasas SKP Cullina F-box/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Phosphorylations within N- and C-terminal degrons independently control the binding of cyclin E to the SCF(Fbw7) and thus its ubiquitination and proteasomal degradation. We have now determined the physiologic significance of cyclin E degradation by this pathway. We describe the construction of a knockin mouse in which both degrons were mutated by threonine to alanine substitutions (cyclin E(T74A T393A)) and report that ablation of both degrons abolished regulation of cyclin E by Fbw7. The cyclin E(T74A T393A) mutation disrupted cyclin E periodicity and caused cyclin E to continuously accumulate as cells reentered the cell cycle from quiescence. In vivo, the cyclin E(T74A T393A) mutation greatly increased cyclin E activity and caused proliferative anomalies. Cyclin E(T74A T393A) mice exhibited abnormal erythropoiesis characterized by a large expansion of abnormally proliferating progenitors, impaired differentiation, dysplasia, and anemia. This syndrome recapitulates many features of early stage human refractory anemia/myelodysplastic syndrome, including ineffective erythropoiesis. Epithelial cells also proliferated abnormally in cyclin E knockin mice, and the cyclin E(T74A T393A) mutation delayed mammary gland involution, implicating cyclin E degradation in this anti-mitogenic response. Hyperproliferative mammary epithelia contained increased apoptotic cells, suggesting that apoptosis contributes to tissue homeostasis in the setting of cyclin E deregulation. Overall these data show the critical role of both degrons in regulating cyclin E activity and reveal that complete loss of Fbw7-mediated cyclin E degradation causes spontaneous and cell type-specific proliferative anomalies.
Asunto(s)
Proliferación Celular , Ciclina E/metabolismo , Ciclina E/fisiología , Células Epiteliales/fisiología , Células Madre Hematopoyéticas/fisiología , Animales , Linaje de la Célula/fisiología , Células Cultivadas , Ciclina E/genética , Células Epiteliales/metabolismo , Células Eritroides/patología , Eritropoyesis/genética , Femenino , Marcación de Gen , Enfermedades Hematológicas/genética , Células Madre Hematopoyéticas/metabolismo , Masculino , Glándulas Mamarias Animales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosforilación , Embarazo , Proteínas Quinasas/metabolismo , Proteínas Quinasas/fisiología , Procesamiento Proteico-Postraduccional/fisiologíaRESUMEN
SCF ubiquitin ligases regulate the degradation of many proteins involved in the control of cell division and growth. F-box proteins are the SCF components that bind to substrates, and this binding is usually signaled by substrate phosphorylation. The Fbw7/hCdc4 F-box protein was first recognized by its ability to bind cyclin E, and the SCF (Fbw7) is now known to target c-Myc, c-Jun and Notch for degradation in addition to its role in cyclin E proteolysis. Fbw7 thus negatively regulates several key oncoproteins. Accordingly, Fbw7 is a tumor suppressor that is mutated in a wide spectrum of human cancers, and Fbw7 functions as a haploin sufficient tumor suppressor in mice. Because there are three Fbw7 isoforms that reside in different subcellular compartments, as well as multiple Fbw7 substrates that are the products of proto-oncogenes, the mechanisms of tumor suppression by Fbw7 are complex and incompletely understood. In this review we discuss the activities of the SCF(Fbw7) in the context of its role as a tumor suppressor and highlight recent findings demonstrating that dominant oncogenes disable Fbw7 function.
Asunto(s)
Proteínas F-Box/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Humanos , Unión Proteica , Especificidad por SustratoRESUMEN
The Skp1-Cullin1 F-box protein-Fbw7 ubiquitin ligase regulates phosphorylation-dependent cyclin E degradation, and disruption of this pathway is associated with genetic instability and tumorigenesis. Fbw7 is a human tumor suppressor that is targeted for mutation in primary cancers. However, mechanisms other than mutation of Fbw7 may also disrupt cyclin E proteolysis in cancers. We show that oncogenic Ha-Ras activity regulates cyclin E degradation by the Fbw7 pathway. Activated Ras impairs Fbw7-driven cyclin E degradation, and, conversely, inhibition of normal Ras activity decreases cyclin E abundance. Moreover, activation of the mitogen-activated protein kinase pathway is the essential Ras function that inhibits cyclin E turnover, and activated Ha-Ras expression inhibits both the binding of cyclin E to Fbw7 and cyclin E ubiquitination. Last, we found that oncogenic Ras activity potentiates cyclin E-induced genetic instability but only when cyclin E is susceptible to degradation by Fbw7. Thus, we conclude that Ras activity regulates Fbw7-mediated cyclin E proteolysis and suggest that impaired cyclin E proteolysis is a mechanism through which Ras mutations promote tumorigenesis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclina E/metabolismo , Proteínas F-Box/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de Señal/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Proteína 7 que Contiene Repeticiones F-Box-WD , Células HeLa , Humanos , Immunoblotting , Inmunoprecipitación , Ratones , Pruebas de Micronúcleos , Microscopía Fluorescente , Células 3T3 NIH , Fosforilación , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Cyclin E, in conjunction with its catalytic partner cdk2, is rate limiting for entry into the S phase of the cell cycle. Cancer cells frequently contain mutations within the cyclin D-Retinoblastoma protein pathway that lead to inappropriate cyclin E-cdk2 activation. Although deregulated cyclin E-cdk2 activity is believed to directly contribute to the neoplastic progression of these cancers, the mechanism of cyclin E-induced neoplasia is unknown. RESULTS: We studied the consequences of deregulated cyclin E expression in primary cells and found that cyclin E initiated a p53-dependent response that prevented excess cdk2 activity by inducing expression of the p21Cip1 cdk inhibitor. The increased p53 activity was not associated with increased expression of the p14ARF tumor suppressor. Instead, cyclin E led to increased p53 serine15 phosphorylation that was sensitive to inhibitors of the ATM/ATR family. When either p53 or p21cip1 was rendered nonfunctional, then the excess cyclin E became catalytically active and caused defects in S phase progression, increased ploidy, and genetic instability. CONCLUSIONS: We conclude that p53 and p21 form an inducible barrier that protects cells against the deleterious consequences of cyclin E-cdk2 deregulation. A response that restrains cyclin E deregulation is likely to be a general protective mechanism against neoplastic transformation. Loss of this response may thus be required before deregulated cyclin E can become fully oncogenic in cancer cells. Furthermore, the combination of excess cyclin E and p53 loss may be particularly genotoxic, because cells cannot appropriately respond to the cell cycle anomalies caused by excess cyclin E-cdk2 activity.