RESUMEN
Glioblastoma (GBM) is one of the most aggressive forms of cancer. It has been proposed that the presence within these tumors of a population of cells with stem-like features termed Glioma Initiating Cells (GICs) is responsible for the relapses that take place in the patients with this disease. Targeting this cell population is therefore an issue of great therapeutic interest in neuro-oncology. We had previously found that the neurotrophic factor MIDKINE (MDK) promotes resistance to glioma cell death. The main objective of this work is therefore investigating the role of MDK in the regulation of GICs. Methods: Assays of gene and protein expression, self-renewal capacity, autophagy and apoptosis in cultures of GICs derived from GBM samples subjected to different treatments. Analysis of the growth of GICs-derived xenografts generated in mice upon blockade of the MDK and its receptor the ALK receptor tyrosine kinase (ALK) upon exposure to different treatments. Results: Genetic or pharmacological inhibition of MDK or ALK decreases the self-renewal and tumorigenic capacity of GICs via the autophagic degradation of the transcription factor SOX9. Blockade of the MDK/ALK axis in combination with temozolomide depletes the population of GICs in vitro and has a potent anticancer activity in xenografts derived from GICs. Conclusions: The MDK/ALK axis regulates the self-renewal capacity of GICs by controlling the autophagic degradation of the transcription factor SOX9. Inhibition of the MDK/ALK axis may be a therapeutic strategy to target GICs in GBM patients.
Asunto(s)
Quinasa de Linfoma Anaplásico/metabolismo , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Midkina/metabolismo , Células Madre Neoplásicas/metabolismo , Temozolomida/farmacología , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Animales , Antineoplásicos Alquilantes/farmacología , Autofagia/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Línea Celular , Femenino , Glioma/tratamiento farmacológico , Glioma/patología , Humanos , Ratones , Ratones Desnudos , Midkina/antagonistas & inhibidores , Transducción de Señal , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Directed gene therapy mediated by nucleases has become a new alternative to lead targeted integration of therapeutic genes in specific regions in the genome. In this work, we have compared the efficiency of two nuclease types, TALEN and meganucleases (MN), to introduce an EGFP reporter gene in a specific site in a safe harbor locus on chromosome 21 in an intergenic region, named here SH6. The efficiency of targeted integration mediated by SH6v5-MN and SH6-TALEN in HEK-293H cells was up to 16.3 and 15.0%. A stable expression was observed both in the pool of transfected cells and in established pseudoclones, with no detection of off-target integrations by Southern blot. In human hematopoietic stem and progenitor CD34+ cells, the nucleofection process preserved the viability and clonogenic capacity of nucleofected cells, reaching up to 3.1% of specific integration of the transgene in colony forming cells when the SH6-TALEN was used, although no expression of the transgene could be found in these cells. Our results show the possibility to specifically integrate genes at the SH6 locus in CD34+ progenitor cells, although further improvements in the efficacy of the procedure are required before this approach could be used for the gene editing of hematopoietic stem cells in patients with hematopoietic diseases.
Asunto(s)
Terapia Genética , Células Madre Hematopoyéticas , Genes Reporteros , Humanos , Nucleasas de los Efectores Tipo Activadores de la Transcripción , TransgenesRESUMEN
We report the case of a Caucasian Spanish boy, who showed profound neonatal hypotonia, feeding difficulties, apnea, severe developmental delay, epilepsy, bilateral convergent strabismus, poor verbal language development and a large brainstem. Whole-exome sequence uncovered a novel de novo mutation in the purine-rich element binding protein A gene (PURA; NM_005859.4:c.72del:p.(-Gly25AlafsTer53)) that encodes the transcriptional activator protein Pur-alpha (PURA). Mutations in this gene have been identified in patients with PURA syndrome, a rare disorder characterized by an early hypotonia, developmental delay, severe intellectual disability with or without epilepsy, and disability in expressive language development. Although, up to 75 cases have been identified worldwide, to the best of our knowledge, this is the first patient described with a brainstem larger than normal. In conclusion, our data expand both geneticand phenotypic spectrum associated with PURA gene mutations.
Asunto(s)
Proteínas de Unión al ADN/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Mutación , Fenotipo , Factores de Transcripción/genética , Adolescente , Tronco Encefálico/anomalías , Discapacidades del Desarrollo/diagnóstico , Discapacidades del Desarrollo/genética , Epilepsia/diagnóstico , Epilepsia/genética , Humanos , Imagen por Resonancia Magnética , Masculino , Linaje , Eliminación de SecuenciaRESUMEN
Glioblastoma multiforme (GBM) is the most frequent and aggressive type of brain tumor due, at least in part, to its poor response to current anticancer treatments. These features could be explained, at least partially, by the presence within the tumor mass of a small population of cells termed Glioma Initiating Cells (GICs) that has been proposed to be responsible for the relapses occurring in this disease. Thus, the development of novel therapeutic approaches (and specifically those targeting the population of GICs) is urgently needed to improve the survival of the patients suffering this devastating disease. Previous observations by our group and others have shown that Δ9-Tetrahydrocannabinol (THC, the main active ingredient of marijuana) and other cannabinoids including cannabidiol (CBD) exert antitumoral actions in several animal models of cancer, including gliomas. We also found that the administration of THC (or of THCâ¯+â¯CBD at a 1:1 ratio) in combination with temozolomide (TMZ), the benchmark agent for the treatment of GBM, synergistically reduces the growth of glioma xenografts. In this work we investigated the effect of the combination of TMZ and THC:CBD mixtures containing different ratios of the two cannabinoids in preclinical glioma models, including those derived from GICs. Our findings show that TMZâ¯+â¯THC:CBD combinations containing a higher proportion of CDB (but not TMZâ¯+â¯CBD alone) produce a similar antitumoral effect as the administration of TMZ together with THC and CBD at a 1:1 ratio in xenografts generated with glioma cell lines. In addition, we also found that the administration of TMZâ¯+â¯THC:CBD at a 1:1 ratio reduced the growth of orthotopic xenografts generated with GICs derived from GBM patients and enhanced the survival of the animals bearing these intracranial xenografts. Remarkably, the antitumoral effect observed in GICs-derived xenografts was stronger when TMZ was administered together with cannabinoid combinations containing a higher proportion of CBD. These findings support the notion that the administration of TMZ together with THC:CBD combinations - and specifically those containing a higher proportion of CBD - may be therapeutically explored to target the population of GICs in GBM.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Cannabidiol/uso terapéutico , Dronabinol/uso terapéutico , Glioblastoma/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Temozolomida/uso terapéutico , Animales , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Femenino , Glioblastoma/patología , Humanos , Masculino , Ratones Desnudos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs) from peripheral blood mononuclear cells (PB-MNCs) of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR). Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses.
Asunto(s)
Anemia Hemolítica Congénita no Esferocítica/genética , Anemia Hemolítica Congénita no Esferocítica/terapia , Células Eritroides/citología , Células Madre Pluripotentes Inducidas/metabolismo , Piruvato Quinasa/deficiencia , Piruvato Quinasa/genética , Errores Innatos del Metabolismo del Piruvato/genética , Errores Innatos del Metabolismo del Piruvato/terapia , Alelos , Secuencia de Bases , Recuento de Células , ADN Complementario/genética , Células Eritroides/metabolismo , Marcación de Gen , Terapia Genética , Humanos , Leucocitos Mononucleares/metabolismo , Recombinación GenéticaRESUMEN
Δ9-tetrahydrocannabinol (THC), the main active component of marijuana, promotes cancer cell death via autophagy stimulation. We find that activation of the tyrosine kinase receptor ALK by its ligand midkine interferes with the signaling mechanism by which THC promotes autophagy-mediated glioma cell death.
Asunto(s)
Autofagia/efectos de los fármacos , Citocinas/farmacología , Glioma/enzimología , Glioma/patología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Quinasa de Linfoma Anaplásico , Dronabinol/farmacología , Humanos , Midkina , Modelos BiológicosRESUMEN
Glioblastoma multiforme (GBM) is highly resistant to current anticancer treatments, which makes it crucial to find new therapeutic strategies aimed at improving the poor prognosis of patients suffering from this disease. Δ(9)-Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoid receptor agonists inhibit tumor growth in animal models of cancer, including glioma, an effect that relies, at least in part, on the stimulation of autophagy-mediated apoptosis in tumor cells. Here, we show that the combined administration of THC and temozolomide (TMZ; the benchmark agent for the management of GBM) exerts a strong antitumoral action in glioma xenografts, an effect that is also observed in tumors that are resistant to TMZ treatment. Combined administration of THC and TMZ enhanced autophagy, whereas pharmacologic or genetic inhibition of this process prevented TMZ + THC-induced cell death, supporting that activation of autophagy plays a crucial role on the mechanism of action of this drug combination. Administration of submaximal doses of THC and cannabidiol (CBD; another plant-derived cannabinoid that also induces glioma cell death through a mechanism of action different from that of THC) remarkably reduces the growth of glioma xenografts. Moreover, treatment with TMZ and submaximal doses of THC and CBD produced a strong antitumoral action in both TMZ-sensitive and TMZ-resistant tumors. Altogether, our findings support that the combined administration of TMZ and cannabinoids could be therapeutically exploited for the management of GBM.