RESUMEN
Medulloblastoma, a malignant childhood cerebellar tumour, segregates molecularly into biologically distinct subgroups, suggesting that a personalized approach to therapy would be beneficial1. Mouse modelling and cross-species genomics have provided increasing evidence of discrete, subgroup-specific developmental origins2. However, the anatomical and cellular complexity of developing human tissues3-particularly within the rhombic lip germinal zone, which produces all glutamatergic neuronal lineages before internalization into the cerebellar nodulus-makes it difficult to validate previous inferences that were derived from studies in mice. Here we use multi-omics to resolve the origins of medulloblastoma subgroups in the developing human cerebellum. Molecular signatures encoded within a human rhombic-lip-derived lineage trajectory aligned with photoreceptor and unipolar brush cell expression profiles that are maintained in group 3 and group 4 medulloblastoma, suggesting a convergent basis. A systematic diagnostic-imaging review of a prospective institutional cohort localized the putative anatomical origins of group 3 and group 4 tumours to the nodulus. Our results connect the molecular and phenotypic features of clinically challenging medulloblastoma subgroups to their unified beginnings in the rhombic lip in the early stages of human development.
Asunto(s)
Linaje de la Célula , Neoplasias Cerebelosas , Meduloblastoma , Metencéfalo , Animales , Neoplasias Cerebelosas/clasificación , Neoplasias Cerebelosas/embriología , Neoplasias Cerebelosas/patología , Cerebelo/embriología , Humanos , Meduloblastoma/clasificación , Meduloblastoma/embriología , Meduloblastoma/patología , Metencéfalo/embriología , Ratones , Neuronas/patología , Estudios ProspectivosRESUMEN
Medulloblastoma is a malignant childhood cerebellar tumour type that comprises distinct molecular subgroups. Whereas genomic characteristics of these subgroups are well defined, the extent to which cellular diversity underlies their divergent biology and clinical behaviour remains largely unexplored. Here we used single-cell transcriptomics to investigate intra- and intertumoral heterogeneity in 25 medulloblastomas spanning all molecular subgroups. WNT, SHH and Group 3 tumours comprised subgroup-specific undifferentiated and differentiated neuronal-like malignant populations, whereas Group 4 tumours consisted exclusively of differentiated neuronal-like neoplastic cells. SHH tumours closely resembled granule neurons of varying differentiation states that correlated with patient age. Group 3 and Group 4 tumours exhibited a developmental trajectory from primitive progenitor-like to more mature neuronal-like cells, the relative proportions of which distinguished these subgroups. Cross-species transcriptomics defined distinct glutamatergic populations as putative cells-of-origin for SHH and Group 4 subtypes. Collectively, these data provide insights into the cellular and developmental states underlying subtype-specific medulloblastoma biology.
Asunto(s)
Genómica , Meduloblastoma/genética , Meduloblastoma/patología , Análisis de la Célula Individual , Transcriptoma , Adolescente , Adulto , Animales , Linaje de la Célula , Cerebelo/metabolismo , Cerebelo/patología , Niño , Preescolar , Variaciones en el Número de Copia de ADN , Regulación Neoplásica de la Expresión Génica , Ácido Glutámico/metabolismo , Humanos , Lactante , Meduloblastoma/clasificación , Ratones , Neuronas/metabolismo , Neuronas/patologíaRESUMEN
The Smoothened (Smo) receptor, a member of class F G protein-coupled receptors, is the main transducer of the Hedgehog (Hh) signaling pathway implicated in a wide range of developmental and adult processes. Smo is the target of anticancer drugs that bind to a long and narrow cavity in the 7-transmembrane (7TM) domain. X-ray structures of human Smo (hSmo) bound to several ligands have revealed 2 types of 7TM-directed antagonists: those binding mostly to extracellular loops (site 1, e.g., LY2940680) and those penetrating deeply in the 7TM cavity (site 2, e.g., SANT-1). Here we report the development of the acylguanidine MRT-92, which displays subnanomolar antagonist activity against Smo in various Hh cell-based assays. MRT-92 inhibits rodent cerebellar granule cell proliferation induced by Hh pathway activation through pharmacologic (half maximal inhibitory concentration [IC50] = 0.4 nM) or genetic manipulation. Using [(3)H]MRT-92 (Kd = 0.3 nM for hSmo), we created a comprehensive framework for the interaction of small molecule modulators with hSmo and for understanding chemoresistance linked to hSmo mutations. Guided by molecular docking and site-directed mutagenesis data, our work convincingly confirms that MRT-92 simultaneously recognized and occupied both sites 1 and 2. Our data demonstrate the existence of a third type of Smo antagonists, those entirely filling the Smo binding cavity from the upper extracellular part to the lower cytoplasmic-proximal subpocket. Our studies should help design novel potent Smo antagonists and more effective therapeutic strategies for treating Hh-linked cancers and associated chemoresistance.
Asunto(s)
Antineoplásicos/farmacología , Membrana Celular/metabolismo , Neoplasias Cerebelosas/metabolismo , Guanidinas/farmacología , Proteínas Hedgehog/antagonistas & inhibidores , Meduloblastoma/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Adulto , Animales , Sitios de Unión , Western Blotting , Membrana Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Neoplasias Cerebelosas/tratamiento farmacológico , Neoplasias Cerebelosas/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Proteínas Hedgehog/metabolismo , Humanos , Técnicas para Inmunoenzimas , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/patología , Ratones , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Mutación/genética , Unión Proteica , Conformación Proteica , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor SmoothenedRESUMEN
Development of the nervous system depends on signaling centers - specialized cellular populations that produce secreted molecules to regulate neurogenesis in the neighboring neuroepithelium. In some cases, signaling center cells also differentiate to produce key types of neurons. The formation of a signaling center involves its induction, the maintenance of expression of its secreted molecules, and cell differentiation and migration events. How these distinct processes are coordinated during signaling center development remains unknown. By performing studies in mice, we show that Lmx1a acts as a master regulator to orchestrate the formation and function of the cortical hem (CH), a critical signaling center that controls hippocampus development. Lmx1a co-regulates CH induction, its Wnt signaling, and the differentiation and migration of CH-derived Cajal-Retzius neurons. Combining RNAseq, genetic, and rescue experiments, we identified major downstream genes that mediate distinct Lmx1a-dependent processes. Our work revealed that signaling centers in the mammalian brain employ master regulatory genes and established a framework for analyzing signaling center development.
Asunto(s)
Neurogénesis , Neuronas , Animales , Ratones , Transporte Biológico , Diferenciación Celular , Mamíferos , Neurogénesis/genética , Vía de Señalización WntRESUMEN
Historically, qualitative research has complemented quantitative biologic and epidemiologic studies to provide a more complete understanding of pandemics. The COVID-19 pandemic has generated unique and novel challenges for qualitative researchers, who have embraced creative solutions including virtual focus groups and rapid analyses to continue their work. We present our experience conducting a multilingual global qualitative study of healthcare resilience among teams of pediatric oncology professionals during the COVID-19 pandemic. We provide an in-depth description of our methodology and an analysis of factors we believe contributed to our study's success including our use of technology, engagement of a large multilingual team, global partnerships, and framework-based rapid analysis. We hope these techniques may be useful to qualitative researchers conducting studies during the current pandemic, as well as for all pediatric oncology studies including multiple languages or geographically disparate subjects.
RESUMEN
Nearly one-third of children with medulloblastoma, a malignant embryonal tumor of the cerebellum, succumb to their disease. Conventional response monitoring by imaging and cerebrospinal fluid (CSF) cytology remains challenging, and a marker for measurable residual disease (MRD) is lacking. Here, we show the clinical utility of CSF-derived cell-free DNA (cfDNA) as a biomarker of MRD in serial samples collected from children with medulloblastoma (123 patients, 476 samples) enrolled on a prospective trial. Using low-coverage whole-genome sequencing, tumor-associated copy-number variations in CSF-derived cfDNA are investigated as an MRD surrogate. MRD is detected at baseline in 85% and 54% of patients with metastatic and localized disease, respectively. The number of MRD-positive patients declines with therapy, yet those with persistent MRD have significantly higher risk of progression. Importantly, MRD detection precedes radiographic progression in half who relapse. Our findings advocate for the prospective assessment of CSF-derived liquid biopsies in future trials for medulloblastoma.
Asunto(s)
Ácidos Nucleicos Libres de Células/líquido cefalorraquídeo , Neoplasias Cerebelosas/diagnóstico , Meduloblastoma/diagnóstico , Secuenciación Completa del Genoma/métodos , Biomarcadores de Tumor/líquido cefalorraquídeo , Biomarcadores de Tumor/genética , Neoplasias Cerebelosas/líquido cefalorraquídeo , Neoplasias Cerebelosas/genética , Niño , Inestabilidad Cromosómica , Variaciones en el Número de Copia de ADN , Progresión de la Enfermedad , Femenino , Humanos , Biopsia Líquida , Masculino , Meduloblastoma/líquido cefalorraquídeo , Meduloblastoma/genética , Neoplasia Residual , Estudios ProspectivosRESUMEN
The cerebellum develops from a restricted number of cell types that precisely organize to form the circuitry that controls sensory-motor coordination and some higher-order cognitive processes. To acquire an enhanced understanding of the molecular processes that mediate cerebellar development, we performed single-cell RNA-sequencing of 39,245 murine cerebellar cells at twelve critical developmental time points. Using recognized lineage markers, we confirmed that the single-cell data accurately recapitulate cerebellar development. We then followed distinct populations from emergence through migration and differentiation, and determined the associated transcriptional cascades. After identifying key lineage commitment decisions, focused analyses uncovered waves of transcription factor expression at those branching points. Finally, we created Cell Seek, a flexible online interface that facilitates exploration of the dataset. Our study provides a transcriptional summarization of cerebellar development at single-cell resolution that will serve as a valuable resource for future investigations of cerebellar development, neurobiology, and disease.
Asunto(s)
Diferenciación Celular , Movimiento Celular , Neurogénesis , Transcriptoma , Animales , Cerebelo/citología , Cerebelo/embriología , Embrión de Mamíferos , Desarrollo Embrionario , Femenino , Ratones , Ratones Endogámicos ICR , Análisis de la Célula IndividualRESUMEN
Cancer cells often express differentiation programs unrelated to their tissue of origin, although the contribution of these aberrant phenotypes to malignancy is poorly understood. An aggressive subgroup of medulloblastoma, a malignant pediatric brain tumor of the cerebellum, expresses a photoreceptor differentiation program normally expressed in the retina. We establish that two photoreceptor-specific transcription factors, NRL and CRX, are master regulators of this program and are required for tumor maintenance in this subgroup. Beyond photoreceptor lineage genes, we identify BCL-XL as a key transcriptional target of NRL and provide evidence substantiating anti-BCL therapy as a rational treatment opportunity for select MB patients. Our results highlight the utility of studying aberrant differentiation programs in cancer and their potential as selective therapeutic vulnerabilities.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Proteínas del Ojo/genética , Proteínas de Homeodominio/genética , Meduloblastoma/genética , Transactivadores/genética , Animales , Diferenciación Celular/genética , Neoplasias Cerebelosas/genética , Humanos , Ratones Desnudos , Retina/patología , Transcripción Genética/genéticaRESUMEN
Medulloblastoma arising from the cerebellum is the most common pediatric brain malignancy, with leptomeningeal metastases often present at diagnosis and recurrence associated with poor clinical outcome. In this study, we used mouse medulloblastoma models to explore the relationship of tumor pathophysiology and dysregulated expression of the NOTCH pathway transcription factor ATOH1, which is present in aggressive medulloblastoma subtypes driven by aberrant Sonic Hedgehog/Patched (SHH/PTCH) signaling. In experiments with conditional ATOH1 mouse mutants crossed to Ptch1+/- mice, which develop SHH-driven medulloblastoma, animals with Atoh1 transgene expression developed highly penetrant medulloblastoma at a young age with extensive leptomeningeal disease and metastasis to the spinal cord and brain, resembling xenografts of human SHH medulloblastoma. Metastatic tumors retained abnormal SHH signaling like tumor xenografts. Conversely, ATOH1 expression was detected consistently in recurrent and metastatic SHH medulloblastoma. Chromatin immunoprecipitation sequencing and gene expression profiling identified candidate ATOH1 targets in tumor cells involved in development and tumorigenesis. Among these targets specific to metastatic tumors, there was an enrichment in those implicated in extracellular matrix remodeling activity, cytoskeletal network and interaction with microenvironment, indicating a shift in transcriptomic and epigenomic landscapes during metastasis. Treatment with bone morphogenetic protein or SHH pathway inhibitors decreased tumor cell proliferation and suppressed metastatic tumor growth, respectively. Our work reveals a dynamic ATOH1-driven molecular cascade underlying medulloblastoma metastasis that offers possible therapeutic opportunities. Cancer Res; 77(14); 3766-77. ©2017 AACR.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Neoplasias Cerebelosas/metabolismo , Meduloblastoma/metabolismo , Meduloblastoma/patología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proliferación Celular , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Proteínas Hedgehog , Xenoinjertos , Humanos , Meduloblastoma/genética , Ratones , Ratones Transgénicos , Metástasis de la Neoplasia , Transducción de SeñalRESUMEN
Cerebellar development is an extensive process that begins during early embryonic stages and persists more than one year after birth in human. Therefore, the cerebellum is susceptible to acquire various developmental abnormalities leading to numerous diseases such as medulloblastoma, the most common pediatric malignant brain tumor. One third of the patients with medulloblastoma are incurable and survivors have a poor quality of life due to the aggressiveness of the broad-spectrum treatments. Within the past few years, it has been highlighted that medulloblastoma is a heterogeneous disease that is divided in four molecular subgroups. This recent advance in the field, combined with the development of associated preclinical models for each subgroup, should enable, in the future, the discovery and use of targeted therapy in clinical treatments for each subtype of medulloblastoma. In this review, we first aim to show how deregulation of cerebellar development can lead to medulloblastoma formation and then to present the advances in the molecular subgrouping of medulloblastoma and the associated preclinical models.
Asunto(s)
Neoplasias Cerebelosas , Cerebelo/embriología , Cerebelo/crecimiento & desarrollo , Meduloblastoma , Animales , Neoplasias Cerebelosas/clasificación , Neoplasias Cerebelosas/diagnóstico , Neoplasias Cerebelosas/etiología , Neoplasias Cerebelosas/terapia , Cerebelo/anatomía & histología , Niño , Modelos Animales de Enfermedad , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/fisiología , Humanos , Ilustración Médica , Meduloblastoma/clasificación , Meduloblastoma/diagnóstico , Meduloblastoma/etiología , Meduloblastoma/terapia , Ratones , Mutación , Pronóstico , Calidad de VidaRESUMEN
Signaling networks controlled by Sonic hedgehog (SHH) and the transcription factor Atoh1 regulate the proliferation and differentiation of cerebellar granule neuron progenitors (GNPs). Deregulations in those developmental processes lead to medulloblastoma formation, the most common malignant brain tumor in childhood. Although the protein Atoh1 is a key factor during both cerebellar development and medulloblastoma formation, up-to-date detailed mechanisms underlying its function and regulation have remained poorly understood. Here, we report that SHH regulates Atoh1 stability by preventing its phosphodependent degradation by the E3 ubiquitin ligase Huwe1. Our results reveal that SHH and Atoh1 contribute to a positive autoregulatory loop promoting neuronal precursor expansion. Consequently, Huwe1 loss in mouse SHH medulloblastoma illustrates the disruption of this developmental mechanism in cancer. Hence, the crosstalk between SHH signaling and Atoh1 during cerebellar development highlights a collaborative network that could be further targeted in medulloblastoma.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Proteínas Hedgehog/metabolismo , Neuronas/metabolismo , Receptores de Superficie Celular/fisiología , Células Madre/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Animales , Diferenciación Celular , Células Cultivadas , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/metabolismo , Neoplasias Cerebelosas/mortalidad , Cromatografía de Afinidad , Femenino , Proteínas Hedgehog/genética , Humanos , Masculino , Meduloblastoma/genética , Meduloblastoma/metabolismo , Meduloblastoma/mortalidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/citología , Receptores Patched , Fosforilación , Proteolisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Células Madre/citología , Tasa de Supervivencia , Proteínas Supresoras de TumorRESUMEN
We devised a high-throughput, cell-based assay to identify compounds to treat Group3 medulloblastoma (G3 MB). Mouse G3 MBs neurospheres were screened against a library of approximately 7,000 compounds including US Food and Drug Administration-approved drugs. We found that pemetrexed and gemcitabine preferentially inhibited G3 MB proliferation in vitro compared to control neurospheres and substantially inhibited G3 MB proliferation in vivo. When combined, these two drugs significantly increased survival of mice bearing cortical implants of mouse and human G3 MBs that overexpress MYC compared to each agent alone, while having little effect on mouse MBs of the sonic hedgehog subgroup. Our findings strongly suggest that combination therapy with pemetrexed and gemcitabine is a promising treatment for G3 MBs.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Cerebelosas/tratamiento farmacológico , Meduloblastoma/tratamiento farmacológico , Animales , Línea Celular Tumoral , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Glutamatos/administración & dosificación , Guanina/administración & dosificación , Guanina/análogos & derivados , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Ratones Transgénicos , Pemetrexed , Pronóstico , GemcitabinaRESUMEN
Medulloblastoma, originating in the cerebellum, is the most common malignant brain tumor in children. Medulloblastoma consists of four major groups where constitutive activation of the Sonic Hedgehog (SHH) signaling pathway is a hallmark of one group. Mouse and human SHH medulloblastomas exhibit increased expression of microRNAs encoded by the miR-17~92 and miR-106b~25 clusters compared with granule progenitors and postmitotic granule neurons. Here, we assessed the therapeutic potential of 8-mer seed-targeting locked nucleic acid (LNA)-modified anti-miR oligonucleotides, termed tiny LNAs, that inhibit microRNA seed families expressed by miR-17~92 and miR-106b~25 in two mouse models of SHH medulloblastomas. We found that tumor cells (medulloblastoma cells) passively took up 8-mer LNA-anti-miRs and specifically inhibited targeted microRNA seed-sharing family members. Inhibition of miR-17 and miR-19a seed families by anti-miR-17 and anti-miR-19, respectively, resulted in diminished tumor cell proliferation in vitro. Treatment of mice with systemic delivery of anti-miR-17 and anti-miR-19 reduced tumor growth in flank and brain allografts in vivo and prolonged the survival of mice with intracranial transplants, suggesting that inhibition of the miR-17~92 cluster family by 8-mer LNA-anti-miRs might be considered for the treatment of SHH medulloblastomas.