RESUMEN
Growth and patterning of the cerebellum is compromised if granule cell precursors do not properly expand and migrate. During embryonic and postnatal cerebellar development, the Hedgehog pathway tightly regulates granule cell progenitors to coordinate appropriate foliation and lobule formation. Indeed, granule cells impairment or defects in the Hedgehog signaling are associated with developmental, neurodegenerative and neoplastic disorders. So far, scant and inefficient cellular models have been available to study granule cell progenitors, in vitro. Here, we validated a new culture method to grow postnatal granule cell progenitors as hedgehog-dependent neurospheres with prolonged self-renewal and ability to differentiate into granule cells, under appropriate conditions. Taking advantage of this cellular model, we provide evidence that Ptch1-KO, but not the SMO-M2 mutation, supports constitutive and cell-autonomous activity of the hedgehog pathway.
Asunto(s)
Diferenciación Celular , Cerebelo/metabolismo , Proteínas Hedgehog , Células-Madre Neurales/metabolismo , Transducción de Señal , Receptor Smoothened , Animales , Cerebelo/citología , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Ratones , Ratones Noqueados , Células-Madre Neurales/citología , Receptor Smoothened/genética , Receptor Smoothened/metabolismoRESUMEN
MRE11 is a component of the MRE11/RAD50/NBS1 (MRN) complex, whose activity is essential to control faithful DNA replication and to prevent accumulation of deleterious DNA double-strand breaks. In humans, hypomorphic mutations in these genes lead to DNA damage response (DDR)-defective and cancer-prone syndromes. Moreover, MRN complex dysfunction dramatically affects the nervous system, where MRE11 is required to restrain MYCN-dependent replication stress, during the rapid expansion of progenitor cells. MYCN activation, often due to genetic amplification, represents the driving oncogenic event for a number of human tumors, conferring bad prognosis and predicting very poor responses even to the most aggressive therapeutic protocols. This is prototypically exemplified by neuroblastoma, where MYCN amplification occurs in about 25% of the cases. Intriguingly, MRE11 is highly expressed and predicts bad prognosis in MYCN-amplified neuroblastoma. Due to the lack of direct means to target MYCN, we explored the possibility to trigger intolerable levels of replication stress-dependent DNA damage, by inhibiting MRE11 in MYCN-amplified preclinical models. Indeed, either MRE11 knockdown or its pharmacological inhibitor mirin induce accumulation of replication stress and DNA damage biomarkers in MYCN-amplified cells. The consequent DDR recruits p53 and promotes a p53-dependent cell death, as indicated by p53 loss- and gain-of-function experiments. Encapsulation of mirin in nanoparticles allowed its use on MYCN-amplified neuroblastoma xenografts in vivo, which resulted in a sharp impairment of tumor growth, associated with DDR activation, p53 accumulation, and cell death. Therefore, we propose that MRE11 inhibition might be an effective strategy to treat MYCN-amplified and p53 wild-type neuroblastoma, and suggest that targeting replication stress with appropriate tools should be further exploited to tackle MYCN-driven tumors.
Asunto(s)
Proteína Homóloga de MRE11/antagonistas & inhibidores , Proteína Homóloga de MRE11/genética , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/tratamiento farmacológico , Pirimidinonas/farmacología , Tionas/farmacología , Células 3T3 , Células A549 , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Daño del ADN/genética , Femenino , Células HEK293 , Células Hep G2 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neuroblastoma/patología , Pronóstico , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Dual binding modes: Combined empirical and computational studies of a series of compounds showed adenine and 1-benzyl-4-(dimethylamino)pyridinium fragments to function most efficiently in binding CHOKα1, and also determined how the latter fragment interacts with the choline binding site through two different binding modes. These data provide a basis for the future design of better and more selective inhibitors.
Asunto(s)
Colina Quinasa/metabolismo , Espectrometría de Fluorescencia , Adenina/química , Adenina/metabolismo , Sitios de Unión , Dominio Catalítico , Colina Quinasa/química , Humanos , Simulación del Acoplamiento Molecular , Unión ProteicaRESUMEN
Applying a CHOK hold: Combined experimental and computational studies of the binding mode of a rationally designed inhibitor of the dimeric choline kinaseâ α1 (CHOKα1) explain the molecular mechanism of negative cooperativity (see scheme) and how the monomers are connected. The results give insight into how the symmetry of the dimer can be partially conserved despite a lack of conservation in the static crystal structures.