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.
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•CREBBP mutations in human medulloblastoma deplete acetyltransferase activity•Crebbp mutations exert opposing effects during cerebellar development•Embryonal loss of Crebbp impairs normal cerebellar development•Postnatal loss of Crebbp synergizes with Shh to drive growth of medulloblastoma
Merk et al. show that the developmental time frame of CREBBP mutation acquisition in cerebellar granule neurons determines the pathogenic effect of these alterations in the cerebellum. These time-sensitive consequences explain phenotypic differences seen in patients with germline (Rubinstein-Taybi syndrome) or somatic mutations (adult SHH medulloblastoma) of CREBBP.
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.
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. Keywords: CRISPR-Cas9 knockout, Non-coding RNA, miRNA, Common essentiality, Negative selection screens, Functional annotation
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.
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.
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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The purpose of this study was to determine the long-term impact of surgical repair and subsequent 6-week immobilization of an Achilles tendon rupture on muscle strength, muscle strength endurance and ...muscle activity. 63 patients participated in this study on average 10.8 ± 3.4 years after surgically repaired Achilles tendon rupture and short-term immobilization. Clinical function was assessed and muscle strength, strength endurance and muscle activity were measured using a dynamometer and electromyography. Ankle ROM, heel height during heel-raise tests and calf circumference were smaller on the injured than on the contralateral side. Ankle torques during the concentric dorsiflexion tasks at 60 °/sec and 180 °/sec and ankle torques during the eccentric plantarflexion task and during the concentric plantarflexion task at 60 °/sec for the injured leg were significantly lower than those for the contralateral leg. The total work during a plantarflexion exercise at 180 °/sec was 14.9% lower in the injured compared to the contralateral leg (p < 0.001). Muscle activity for the gastrocnemius muscle during dorsiflexion tasks was significantly higher in the injured than in the contralateral limb. Limited ankle joint ROM and increased muscle activity in the injured leg suggest compensatory mechanisms to account for differences in muscle morphology and physiology caused by the injury.
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.
Background
The differential outcomes of clinical studies of the targeted therapies for non-small cell lung cancer (NSCLC) indicate that better stratification of patients is required. This could be ...achieved with the help of patient-derived xenografts (PDX) of epidermal growth factor receptor (EGFR) wild-type patients resistant to erlotinib treatment.
Objective
To explore the potential of patient-derived NSCLC xenografts to optimize therapy using 24 well-characterized early-stage NSCLC PDX.
Method
Patient tumor tissue was transplanted subcutaneously into nude mice. After engraftment, tumors were expanded and the sensitivity was tested. Gene expression analysis was used to identify differentially expressed genes between erlotinib responder (
n
= 3) and non-responder (
n
= 21). Tumor tissue was analyzed with TaqMan PCR, immunohistochemistry and ELISA to examine the response of the models.
Results
Gene expression analysis revealed vascular endothelial growth factor A (VEGFA) to be up-regulated in erlotinib non-responder. Because of that, the combination of erlotinib with bevacizumab was evaluated in one erlotinib-sensitive and four erlotinib-resistant PDX. Combination treatment was superior to monotherapy, leading to the highest and significant inhibition of tumor growth in all models investigated. A decline of VEGFA protein and an increase of
VEGFA
-mRNA were observed after bevacizumab treatment. Bevacizumab treatment resulted in a distinct decrease of blood vessel number.
Conclusion
This study showed that with the help of preclinical PDX models, drug combinations for therapy improvement can be identified on a rational basis. It was observed that a dual blockage of EGFR and VEGFA was more effective than a monotherapy for the treatment of NSCLC in selected PDX models. PDX could be employed to optimize the treatment of cancer patients.
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::CBP
, 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.
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