RESUMEN
During neural development, stem and precursor cells can divide either symmetrically or asymmetrically. The transition between symmetric and asymmetric cell divisions is a major determinant of precursor cell expansion and neural differentiation, but the underlying mechanisms that regulate this transition are not well understood. Here, we identify the Sonic hedgehog (Shh) pathway as a critical determinant regulating the mode of division of cerebellar granule cell precursors (GCPs). Using partial gain and loss of function mutations within the Shh pathway, we show that pathway activation determines spindle orientation of GCPs, and that mitotic spindle orientation correlates with the mode of division. Mechanistically, we show that the phosphatase Eya1 is essential for implementing Shh-dependent GCP spindle orientation. We identify atypical protein kinase C (aPKC) as a direct target of Eya1 activity and show that Eya1 dephosphorylates a critical threonine (T410) in the activation loop. Thus, Eya1 inactivates aPKC, resulting in reduced phosphorylation of Numb and other components that regulate the mode of division. This Eya1-dependent cascade is critical in linking spindle orientation, cell cycle exit and terminal differentiation. Together these findings demonstrate that a Shh-Eya1 regulatory axis selectively promotes symmetric cell divisions during cerebellar development by coordinating spindle orientation and cell fate determinants.
Asunto(s)
División Celular/fisiología , Cerebelo/metabolismo , Proteínas Hedgehog/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis/fisiología , Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Animales , Cerebelo/embriología , Cerebelo/crecimiento & desarrollo , Ratones , Ratones Mutantes , Células-Madre Neurales/citología , Transducción de Señal/fisiologíaRESUMEN
The TCF4 gene encodes for the basic helix-loop-helix transcription factor 4 (TCF4), which plays an important role in the development of the central nervous system (CNS). Haploinsufficiency of TCF4 was found to cause Pitt-Hopkins syndrome (PTHS), a severe neurodevelopmental disorder. Recently, the screening of a large cohort of medulloblastoma (MB), a highly aggressive embryonal brain tumor, revealed almost 20% of adult patients with MB of the Sonic hedgehog (SHH) subtype carrying somatic TCF4 mutations. Interestingly, many of these mutations have previously been detected as germline mutations in patients with PTHS. We show here that overexpression of wild-type TCF4 in vitro significantly suppresses cell proliferation in MB cells, whereas mutant TCF4 proteins do not to the same extent. Furthermore, RNA sequencing revealed significant upregulation of multiple well-known tumor suppressors upon expression of wild-type TCF4. In vivo, a prenatal knockout of Tcf4 in mice caused a significant increase in apoptosis accompanied by a decreased proliferation and failed migration of cerebellar granule neuron precursor cells (CGNP), which are thought to be the cells of origin for SHH MB. In contrast, postnatal in vitro and in vivo knockouts of Tcf4 with and without an additional constitutive activation of the SHH pathway led to significantly increased proliferation of CGNP or MB cells. Finally, publicly available data from human MB show that relatively low expression levels of TCF4 significantly correlate with a worse clinical outcome. These results not only point to time-specific roles of Tcf4 during cerebellar development but also suggest a functional linkage between TCF4 mutations and the formation of SHH MB, proposing that TCF4 acts as a tumor suppressor during postnatal stages of cerebellar development.
Asunto(s)
Proteínas Hedgehog/genética , Meduloblastoma/genética , Mutación , Factor de Transcripción 4/genética , Animales , Apoptosis/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Facies , Proteínas Hedgehog/metabolismo , Humanos , Hiperventilación/genética , Hiperventilación/metabolismo , Hiperventilación/patología , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Meduloblastoma/metabolismo , Meduloblastoma/patología , Ratones , Ratones Noqueados , Factor de Transcripción 4/metabolismoRESUMEN
BACKGROUND: Genome-wide functional screening using the CRISPR-Cas9 system is a powerful tool to uncover tumor-specific and common genetic dependencies across cancer cell lines. Current CRISPR-Cas9 knockout libraries, however, primarily target protein-coding genes. This limits functional genomics-based investigations of miRNA function. METHODS: We designed a novel CRISPR-Cas9 knockout library (lentiG-miR) of 8107 distinct sgRNAs targeting a total of 1769 human miRNAs and benchmarked its single guide RNA (sgRNA) composition, predicted on- and off-target activity, and screening performance against previous libraries. Using a total of 45 human cancer cell lines, representing 16 different tumor entities, we performed negative selection screens to identify miRNA fitness genes. Fitness miRNAs in each cell line were scored using a combination of supervised and unsupervised essentiality classifiers. Common essential miRNAs across distinct cancer cell lines were determined using the 90th percentile method. For subsequent validation, we performed knockout experiments for selected common essential miRNAs in distinct cancer cell lines and gene expression profiling. RESULTS: We found significantly lower off-target activity for protein-coding genes and a higher miRNA gene coverage for lentiG-miR as compared to previously described miRNA-targeting libraries, while preserving high on-target activity. A minor fraction of miRNAs displayed robust depletion of targeting sgRNAs, and we observed a high level of consistency between redundant sgRNAs targeting the same miRNA gene. Across 45 human cancer cell lines, only 217 (12%) of all targeted human miRNAs scored as a fitness gene in at least one model, and fitness effects for most miRNAs were confined to small subsets of cell lines. In contrast, we identified 49 common essential miRNAs with a homogenous fitness profile across the vast majority of all cell lines. Transcriptional profiling verified highly consistent gene expression changes in response to knockout of individual common essential miRNAs across a diverse set of cancer cell lines. CONCLUSIONS: Our study presents a miRNA-targeting CRISPR-Cas9 knockout library with high gene coverage and optimized on- and off-target activities. Taking advantage of the lentiG-miR library, we define a catalogue of miRNA fitness genes in human cancer cell lines, providing the foundation for further investigation of miRNAs in human cancer.
Asunto(s)
Sistemas CRISPR-Cas , MicroARNs , Neoplasias , Humanos , MicroARNs/genética , Línea Celular Tumoral , Neoplasias/genética , Técnicas de Inactivación de Genes , ARN Guía de Sistemas CRISPR-Cas/genética , Regulación Neoplásica de la Expresión Génica , Perfilación de la Expresión Génica , Genes EsencialesRESUMEN
Sonic hedgehog subgroup of medulloblastoma (SHH-MB) is characterized by aberrant activation of the SHH signaling pathway. An inhibition of the positive SHH regulator Smoothened (SMO) has demonstrated promising clinical efficacy. Yet, primary and acquired resistance to SMO inhibitors limit their efficacy. An understanding of underlying molecular mechanisms of resistance to therapy is warranted to bridge this unmet need. Here, we make use of genome-wide CRISPR-Cas9 knockout screens in murine SMB21 and human DAOY cells, in order to unravel genetic dependencies and drug-related genetic interactors that could serve as alternative therapeutic targets for SHH-MB. Our screens reinforce SMB21 cells as a faithful model system for SHH-MB, as opposed to DAOY cells, and identify members of the epigenetic machinery including DNA methyltransferase 1 (DNMT1) as druggable targets in SHH-dependent tumors. We show that Dnmt1 plays a crucial role in normal murine cerebellar development and is required for SHH-MB growth in vivo. Additionally, DNMT1 pharmacological inhibition alone and in combination with SMO inhibition effectively inhibits tumor growth in murine and human SHH-MB cell models and prolongs survival of SHH-MB mouse models by inhibiting SHH signaling output downstream of SMO. In conclusion, our data highlight the potential of inhibiting epigenetic regulators as a novel therapeutic avenue in SMO-inhibitor sensitive as well as resistant SHH-MBs.
Asunto(s)
Sistemas CRISPR-Cas , Neoplasias Cerebelosas , ADN (Citosina-5-)-Metiltransferasa 1 , Proteínas Hedgehog , Meduloblastoma , Meduloblastoma/genética , Meduloblastoma/metabolismo , Meduloblastoma/patología , Animales , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/metabolismo , Neoplasias Cerebelosas/patología , Humanos , Ratones , Línea Celular Tumoral , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Técnicas de Inactivación de Genes/métodosRESUMEN
BACKGROUND: The DNA damage response (DDR) is a physiological network preventing malignant transformation, e.g. by halting cell cycle progression upon DNA damage detection and promoting DNA repair. Glioblastoma are incurable primary tumors of the nervous system and DDR dysregulation contributes to acquired treatment resistance. Therefore, DDR targeting is a promising therapeutic anti-glioma strategy. Here, we investigated Ataxia telangiectasia and Rad3 related (ATR) inhibition (ATRi) and functionally-instructed combination therapies involving ATRi in experimental glioma. METHODS: We used acute cytotoxicity to identify treatment efficacy as well as RNAseq and DigiWest protein profiling to characterize ATRi-induced modulations within the molecular network in glioma cells. Genome-wide CRISPR/Cas9 functional genomic screens and subsequent validation with functionally-instructed compounds and selected shRNA-based silencing were employed to discover and investigate molecular targets modifying response to ATRi in glioma cell lines in vitro, in primary cultures ex vivo and in zebrafish and murine models in vivo. RESULTS: ATRi monotherapy displays anti-glioma efficacy in vitro and ex vivo and modulates the molecular network. We discovered molecular targets by genome-wide CRISPR/Cas9 loss-of-function and activation screens that enhance therapeutic ATRi effects. We validated selected druggable targets by a customized drug library and functional assays in vitro, ex vivo and in vivo. CONCLUSION: In conclusion, our study leads to the identification of novel combination therapies involving ATRi that could inform future preclinical studies and early phase clinical trials.
Asunto(s)
Glioma , Pez Cebra , Ratones , Animales , Línea Celular Tumoral , Reparación del ADN , Daño del ADN , Proteínas de la Ataxia Telangiectasia Mutada/metabolismoRESUMEN
Background: The clinical utility of molecular profiling and targeted therapies for neuro-oncology patients outside of clinical trials is not established. We aimed at investigating feasibility and clinical utility of molecular profiling and targeted therapy in adult patients with advanced tumors in the nervous system within a prospective observational study. Methods: molecular tumor board (MTB)@ZPM (NCT03503149) is a prospective observational precision medicine study for patients with advanced tumors. After inclusion of patients, we performed comprehensive molecular profiling, formulated ranked biomarker-guided therapy recommendations based on consensus by the MTB, and collected prospective clinical outcome data. Results: Here, we present initial data of 661 adult patients with tumors of the nervous system enrolled by December 31, 2021. Of these, 408 patients were presented at the MTB. Molecular-instructed therapy recommendations could be made in 380/408 (93.1%) cases and were prioritized by evidence levels. Therapies were initiated in 86/380 (22.6%) cases until data cutoff. We observed a progression-free survival ratio >1.3 in 31.3% of patients. Conclusions: Our study supports the clinical utility of biomarker-guided therapies for neuro-oncology patients and indicates clinical benefit in a subset of patients. Our data might inform future clinical trials, translational studies, and even clinical care.
RESUMEN
Mutations in the gene encoding for the histone acetyltransferase (HAT) CREBBP are common driver events in multiple types of human cancer, such as small cell lung cancer (SCLC) or Sonic Hedgehog medulloblastoma (SHH MB). Therefore, therapeutic options targeting such alterations are highly desired. We used human cell lines from SCLC as well as primary mouse tumor cells and genetically engineered mouse models for SHH MB to test treatment options with histone deacetylase inhibitors (HDACi) in CREBBP wild-type and mutated tumors. In contrast to CREBBP wild-type SCLC cells, CREBBP-mutated SCLC cells showed significantly lower IC50 values after treatment with HDACi. In addition, both in vitro and in vivo, HDACi had significant effects on cell proliferation of SHH-driven tumor MB cells harboring a CREBBP-mutation as compared to CREBBP wild-type controls. These data suggest that HDACi may serve as an additional therapeutic option for patients suffering from tumors driven by CREBBP mutations.
Asunto(s)
Proteína de Unión a CREB/genética , Neoplasias Cerebelosas/tratamiento farmacológico , Inhibidores de Histona Desacetilasas/administración & dosificación , Meduloblastoma/tratamiento farmacológico , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Modelos Animales de Enfermedad , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ácidos Hidroxámicos/administración & dosificación , Meduloblastoma/genética , Meduloblastoma/patología , Ratones , Ratones Transgénicos , Panobinostat/administración & dosificación , Cultivo Primario de Células , Receptor Smoothened/genéticaRESUMEN
CREB (cyclic AMP response element binding protein) binding protein (CBP, CREBBP) is a ubiquitously expressed transcription coactivator with intrinsic histone acetyltransferase (KAT) activity. Germline mutations within the CBP gene are known to cause Rubinstein-Taybi syndrome (RSTS), a developmental disorder characterized by intellectual disability, specific facial features and physical anomalies. Here, we investigate mechanisms of CBP function during brain development in order to elucidate morphological and functional mechanisms underlying the development of RSTS. Due to the embryonic lethality of conventional CBP knockout mice, we employed a tissue specific knockout mouse model (hGFAP-cre::CBPFl/Fl, mutant mouse) to achieve a homozygous deletion of CBP in neural precursor cells of the central nervous system.Our findings suggest that CBP plays a central role in brain size regulation, correct neural cell differentiation and neural precursor cell migration. We provide evidence that CBP is both important for stem cell viability within the ventricular germinal zone during embryonic development and for unhindered establishment of adult neurogenesis. Prominent histological findings in adult animals include a significantly smaller hippocampus with fewer neural stem cells. In the subventricular zone, we observe large cell aggregations at the beginning of the rostral migratory stream due to a migration deficit caused by impaired attraction from the CBP-deficient olfactory bulb. The cerebral cortex of mutant mice is characterized by a shorter dendrite length, a diminished spine number, and a relatively decreased number of mature spines as well as a reduced number of synapses.In conclusion, we provide evidence that CBP is important for neurogenesis, shaping neuronal morphology, neural connectivity and that it is involved in neuronal cell migration. These findings may help to understand the molecular basis of intellectual disability in RSTS patients and may be employed to establish treatment options to improve patients' quality of life.
Asunto(s)
Proteína de Unión a CREB/deficiencia , Movimiento Celular/fisiología , Células-Madre Neurales/metabolismo , Síndrome de Rubinstein-Taybi/metabolismo , Activación Transcripcional/fisiología , Animales , Proteína de Unión a CREB/genética , Preescolar , Femenino , Humanos , Lactante , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Estudios Retrospectivos , Síndrome de Rubinstein-Taybi/diagnóstico por imagen , Síndrome de Rubinstein-Taybi/genéticaRESUMEN
BACKGROUND: Medulloblastoma (MB) is one of the most frequent malignant brain tumors of children, and a large set of these tumors is characterized by aberrant activation of the sonic hedgehog (SHH) pathway. While some tumors initially respond to inhibition of the SHH pathway component Smoothened (SMO), tumors ultimately recur due to downstream resistance mechanisms, indicating a need for novel therapeutic options. METHODS: Here we performed a targeted small-molecule screen on a stable, SHH-dependent murine MB cell line (SMB21). Comprehensive isotype profiling of histone deacetylase (HDAC) inhibitors was performed, and effects of HDAC inhibition were evaluated in cell lines both sensitive and resistant to SMO inhibition. Lastly, distinct mouse models of SHH MB were used to demonstrate pharmacologic efficacy in vivo. RESULTS: A subset of the HDAC inhibitors tested significantly inhibit tumor growth of SMB21 cells by preventing SHH pathway activation. Isotype profiling of HDAC inhibitors, together with genetic approaches suggested that concerted inhibition of multiple class I HDACs is necessary to achieve pathway inhibition. Of note, class I HDAC inhibitors were also efficacious in suppressing growth of diverse SMO inhibitorâresistant clones of SMB21 cells. Finally, we show that the novel HDAC inhibitor quisinostat targets multiple class I HDACs, is well tolerated in mouse models, and robustly inhibits growth of SHH MB cells in vivo as well as in vitro. CONCLUSIONS: Our data provide strong evidence that quisinostat or other class I HDAC inhibitors might be therapeutically useful for patients with SHH MB, including those resistant to SMO inhibition.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Neoplasias Cerebelosas/tratamiento farmacológico , Proteínas Hedgehog/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Meduloblastoma/tratamiento farmacológico , Anilidas , Animales , Compuestos de Bifenilo , Línea Celular Tumoral , Neoplasias Cerebelosas/metabolismo , Resistencia a Antineoplásicos/genética , Proteínas Hedgehog/metabolismo , Ensayos Analíticos de Alto Rendimiento , Concentración 50 Inhibidora , Meduloblastoma/metabolismo , Ratones , Proteínas/genética , Piridinas , Proteínas Represoras/genética , Transducción de Señal , Receptor Smoothened/antagonistas & inhibidores , Receptor Smoothened/metabolismoRESUMEN
The Hedgehog (Hh) pathway is a highly conserved signaling system regulating a range of developmental processes. A 1997 paper by Goodrich and colleagues provided major contributions to understanding the Hh pathway by mutating the gene encoding the Hh receptor, Patched, and thereby developing a mouse model for a human cancer predisposition syndrome, known as Gorlin syndrome. These studies provided one of the first genetically engineered mouse models for brain tumors.
Asunto(s)
Proteínas Hedgehog/fisiología , Mutación , Neurogénesis/fisiología , Receptor Patched-1/genética , Animales , Ratones , Neurogénesis/genéticaRESUMEN
Recurrent mutations in chromatin modifiers are specifically prevalent in adolescent or adult patients with Sonic hedgehog-associated medulloblastoma (SHH MB). Here, we report that mutations in the acetyltransferase CREBBP have opposing effects during the development of the cerebellum, the primary site of origin of SHH MB. Our data reveal that loss of Crebbp in cerebellar granule neuron progenitors (GNPs) during embryonic development of mice compromises GNP development, in part by downregulation of brain-derived neurotrophic factor (Bdnf). Interestingly, concomitant cerebellar hypoplasia was also observed in patients with Rubinstein-Taybi syndrome, a congenital disorder caused by germline mutations of CREBBP. By contrast, loss of Crebbp in GNPs during postnatal development synergizes with oncogenic activation of SHH signaling to drive MB growth, thereby explaining the enrichment of somatic CREBBP mutations in SHH MB of adult patients. Together, our data provide insights into time-sensitive consequences of CREBBP mutations and corresponding associations with human diseases.
Asunto(s)
Acetiltransferasas/metabolismo , Proteína de Unión a CREB/metabolismo , Proteína de Unión a CREB/fisiología , Proteínas Hedgehog/metabolismo , Meduloblastoma/patología , Mutación , Síndrome de Rubinstein-Taybi/patología , Adulto , Animales , Proteína de Unión a CREB/genética , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/metabolismo , Neoplasias Cerebelosas/patología , Femenino , Proteínas Hedgehog/genética , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Ratones , Ratones Noqueados , Neuronas , Fenotipo , Síndrome de Rubinstein-Taybi/genética , Síndrome de Rubinstein-Taybi/metabolismo , Transducción de SeñalRESUMEN
We identified a rare undifferentiated cell population that is intermingled with the Bergmann glia of the adult murine cerebellar cortex, expresses the stem cell markers Sox2 and Nestin, and lacks markers of glial or neuronal differentiation. Interestingly, such Sox2+ S100- cells of the adult cerebellum expanded after adequate physiological stimuli in mice (exercise), and Sox2+ precursors acquired positivity for the neuronal marker NeuN over time and integrated into cellular networks. In human patients, SOX2+ S100- cells similarly increased in number after relevant pathological insults (infarcts), suggesting a similar expansion of cells that lack terminal glial differentiation.
Asunto(s)
Células Madre Adultas/citología , Corteza Cerebelosa/citología , Factores de Transcripción SOXB1/metabolismo , Células Madre Adultas/metabolismo , Animales , Diferenciación Celular , Corteza Cerebelosa/metabolismo , Ejercicio Físico , Humanos , Ratones , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis , Factores de Transcripción SOXB1/genética , Nicho de Células MadreRESUMEN
Embryonal tumors with multilayered rosettes (ETMRs) have recently been described as a new entity of rare pediatric brain tumors with a fatal outcome. We show here that ETMRs are characterized by a parallel activation of Shh and Wnt signaling. Co-activation of these pathways in mouse neural precursors is sufficient to induce ETMR-like tumors in vivo that resemble their human counterparts on the basis of histology and global gene-expression analyses, and that point to apical radial glia cells as the possible tumor cell of origin. Overexpression of LIN28A, which is a hallmark of human ETMRs, augments Sonic-hedgehog (Shh) and Wnt signaling in these precursor cells through the downregulation of let7-miRNA, and LIN28A/let7a interaction with the Shh pathway was detected at the level of Gli mRNA. Finally, human ETMR cells that were transplanted into immunocompromised host mice were responsive to the SHH inhibitor arsenic trioxide (ATO). Our work provides a novel mouse model in which to study this tumor type, demonstrates the driving role of Wnt and Shh activation in the growth of ETMRs and proposes downstream inhibition of Shh signaling as a therapeutic option for patients with ETMRs.
Asunto(s)
Antineoplásicos/farmacología , Arsenicales/farmacología , Neoplasias Encefálicas/genética , Proteínas Hedgehog/genética , Neoplasias de Células Germinales y Embrionarias/genética , Óxidos/farmacología , Vía de Señalización Wnt/genética , Animales , Trióxido de Arsénico , Western Blotting , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Regulación hacia Abajo , Perfilación de la Expresión Génica , Proteínas Hedgehog/antagonistas & inhibidores , Humanos , Inmunohistoquímica , Ratones , Ratones Transgénicos , MicroARNs/genética , Neoplasias de Células Germinales y Embrionarias/metabolismo , Proteínas de Unión al ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína con Dedos de Zinc GLI1/genéticaRESUMEN
Canonical Wnt signaling is known to promote proliferation of olfactory stem cells. In order to investigate the effects of a constitutive activation of Wnt signaling in Sox2-positive precursor cells of the olfactory epithelium, we used transgenic mice that allowed an inducible deletion of exon 3 of the Ctnnb1 gene, which is responsible for the phosphorylation and degradation of Ctnnb1 protein. After induction of aberrant Wnt activation by Ctnnb1 deletion at embryonic day 14, such mice developed tumor-like lesions in upper parts of the nasal cavity. We still observed areas of epithelial hyperplasia within the olfactory epithelium following early postnatal Wnt activation, but the olfactory epithelial architecture remained unaffected in most parts when Wnt was activated at postnatal day 21 or later. In summary, our results suggest an age-dependent tumorigenic potential of aberrant Wnt signaling in the olfactory epithelium of mice.