In vivo functional investigation of oncogenes using somatic gene transfer has been successfully exploited to validate their role in tumorigenesis. For tumour suppressor genes this has proven more challenging due to technical aspects. To provide a flexible and effective method for investigating somatic loss-of-function alterations and their influence on tumorigenesis, we have established CRISPR/Cas9-mediated somatic gene disruption, allowing for in vivo targeting of TSGs. Here we demonstrate the utility of this approach by deleting single (Ptch1) or multiple genes (Trp53, Pten, Nf1) in the mouse brain, resulting in the development of medulloblastoma and glioblastoma, respectively. Using whole-genome sequencing (WGS) we characterized the medulloblastoma-driving Ptch1 deletions in detail and show that no off-targets were detected in these tumours. This method provides a fast and convenient system for validating the emerging wealth of novel candidate tumour suppressor genes and the generation of faithful animal models of human cancer.

Diffuse intrinsic pontine glioma is a uniformly lethal malignant tumor of infancy with no effective therapies. A new study reveals that inhibition of JMJD3 has robust antitumor activity in diffuse intrinsic pontine glioma xenografts.

The majority of metabolic principles are evolutionarily conserved from nematodes to humans. Caenorhabditis elegans has widely accelerated the discovery of new genes important to maintain organismic metabolic homeostasis. Various methods exist to assess the metabolic state in worms, yet they often require large animal numbers and tend to be performed as bulk analyses of whole worm homogenates, thereby largely precluding a detailed studies of metabolic changes in specific worm tissues. Here, we have adapted well-established histochemical methods for the use on C. elegans fresh frozen sections and demonstrate their validity for analyses of morphological and metabolic changes on tissue level in wild type and various mutant strains. We show how the worm presents on hematoxylin and eosin (H&E) stained sections and demonstrate their usefulness in monitoring and the identification of morphological abnormalities. In addition, we demonstrate how Oil-Red-O staining on frozen worm cross-sections permits quantification of lipid storage, avoiding the artifact-prone fixation and permeabilization procedures of traditional whole-mount protocols. We also adjusted standard enzymatic stains for respiratory chain subunits (NADH, SDH, and COX) to monitor metabolic states of various C. elegans tissues. In summary, the protocols presented here provide technical guidance to obtain robust, reproducible and quantifiable tissue-specific data on worm morphology as well as carbohydrate, lipid and mitochondrial energy metabolism that cannot be obtained through traditional biochemical bulk analyses of worm homogenates. Furthermore, analysis of worm cross-sections overcomes the common problem with quantification in three-dimensional whole-mount specimens.

Medulloblastoma is a pediatric brain tumor with a variable prognosis due to clinical and genomic heterogeneity. Among the 4 major genomic sub-groups, patients with MYC amplified tumors have a particularly poor prognosis despite therapy with surgery, radiation and chemotherapy. Targeting the MYC oncogene has traditionally been problematic. Here we report that MYC driven medulloblastoma can be targeted by inhibition of the bromodomain protein BRD4. We show that bromodomain inhibition with JQ1 restricts c-MYC driven transcriptional programs in medulloblastoma, suppresses medulloblastoma cell growth and induces a cell cycle arrest. Importantly JQ1 suppresses stem cell associated signaling in medulloblastoma cells and inhibits medulloblastoma tumor cell self-renewal. Additionally JQ1 also promotes senescence in medulloblastoma cells by activating cell cycle kinase inhibitors and inhibiting activity of E2F1. Furthermore BRD4 inhibition displayed an anti-proliferative, pro-senescence effect in a medulloblastoma model in vivo. In clinical samples we found that transcriptional programs suppressed by JQ1 are associated with adverse risk in medulloblastoma patients. Our work indicates that BRD4 inhibition attenuates stem cell signaling in MYC driven medulloblastoma and demonstrates the feasibility BET domain inhibition as a therapeutic approach in vivo.

Activation of the RAS-RAF-MEK-ERK signaling pathway is thought to be the key driver of pediatric low-grade astrocytoma (PLGA) growth. Sorafenib is a multikinase inhibitor targeting BRAF, VEGFR, PDGFR, and c-kit. This multicenter phase II study was conducted to determine the response rate to sorafenib in patients with recurrent or progressive PLGA.

Recurrent mutations affecting the histone H3.3 residues Lys27 or indirectly Lys36 are frequent drivers of pediatric high-grade gliomas (over 30% of HGGs). To identify additional driver mutations in HGGs, we investigated a cohort of 60 pediatric HGGs using whole-exome sequencing (WES) and compared them to 543 exomes from non-cancer control samples. We identified mutations in SETD2, a H3K36 trimethyltransferase, in 15% of pediatric HGGs, a result that was genome-wide significant (FDR = 0.029). Most SETD2 alterations were truncating mutations. Sequencing the gene in this cohort and another validation cohort (123 gliomas from all ages and grades) showed SETD2 mutations to be specific to high-grade tumors affecting 15% of pediatric HGGs (11/73) and 8% of adult HGGs (5/65) while no SETD2 mutations were identified in low-grade diffuse gliomas (0/45). Furthermore, SETD2 mutations were mutually exclusive with H3F3A mutations in HGGs (P = 0.0492) while they partly overlapped with IDH1 mutations (4/14), and SETD2-mutant tumors were found exclusively in the cerebral hemispheres (P = 0.0055). SETD2 is the only H3K36 trimethyltransferase in humans, and SETD2-mutant tumors showed a substantial decrease in H3K36me3 levels (P < 0.001), indicating that the mutations are loss-of-function. These data suggest that loss-of-function SETD2 mutations occur in older children and young adults and are specific to HGG of the cerebral cortex, similar to the H3.3 G34R/V and IDH mutations. Taken together, our results suggest that mutations disrupting the histone code at H3K36, including H3.3 G34R/V, IDH1 and/or SETD2 mutations, are central to the genesis of hemispheric HGGs in older children and young adults.

Autophagy is a catabolic process that ensures homeostatic cell clearance and is deregulated in a growing number of myopathological conditions. Although FoxO3 was shown to promote the expression of autophagy-related genes in skeletal muscle, the mechanisms triggering autophagy are unclear. We show that TSC1-deficient mice (TSCmKO), characterized by sustained activation of mTORC1, develop a late-onset myopathy related to impaired autophagy. In young TSCmKO mice, constitutive and starvation-induced autophagy is blocked at the induction steps via mTORC1-mediated inhibition of Ulk1, despite FoxO3 activation. Rapamycin is sufficient to restore autophagy in TSCmKO mice and improves the muscle phenotype of old mutant mice. Inversely, abrogation of mTORC1 signaling by depletion of raptor induces autophagy regardless of FoxO inhibition. Thus, mTORC1 is the dominant regulator of autophagy induction in skeletal muscle and ensures a tight coordination of metabolic pathways. These findings may open interesting avenues for therapeutic strategies directed toward autophagy-related muscle diseases.

Recent exon-sequencing studies of human tumours have revealed that subunits of BAF (mammalian SWI/SNF) complexes are mutated in more than 20% of all human malignancies, but the mechanisms involved in tumour suppression are unclear. BAF chromatin-remodelling complexes are polymorphic assemblies that use energy provided by ATP hydrolysis to regulate transcription through the control of chromatin structure and the placement of Polycomb repressive complex 2 (PRC2) across the genome. Several proteins dedicated to this multisubunit complex, including BRG1 (also known as SMARCA4) and BAF250a (also known as ARID1A), are mutated at frequencies similar to those of recognized tumour suppressors. In particular, the core ATPase BRG1 is mutated in 5-10% of childhood medulloblastomas and more than 15% of Burkitt's lymphomas. Here we show a previously unknown function of BAF complexes in decatenating newly replicated sister chromatids, a requirement for proper chromosome segregation during mitosis. We find that deletion of Brg1 in mouse cells, as well as the expression of BRG1 point mutants identified in human tumours, leads to anaphase bridge formation (in which sister chromatids are linked by catenated strands of DNA) and a G2/M-phase block characteristic of the decatenation checkpoint. Endogenous BAF complexes interact directly with endogenous topoisomerase IIα (TOP2A) through BAF250a and are required for the binding of TOP2A to approximately 12,000 sites across the genome. Our results demonstrate that TOP2A chromatin binding is dependent on the ATPase activity of BRG1, which is compromised in oncogenic BRG1 mutants. These studies indicate that the ability of TOP2A to prevent DNA entanglement at mitosis requires BAF complexes and suggest that this activity contributes to the role of BAF subunits as tumour suppressors.

Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that eukaryotic elongation factor 2 kinase (eEF2K), which is activated by AMP-kinase (AMPK), confers cell survival under acute nutrient depletion by blocking translation elongation. Tumor cells exploit this pathway to adapt to nutrient deprivation by reactivating the AMPK-eEF2K axis. Adaptation of transformed cells to nutrient withdrawal is severely compromised in cells lacking eEF2K. Moreover, eEF2K knockdown restored sensitivity to acute nutrient deprivation in highly resistant human tumor cell lines. In vivo, overexpression of eEF2K rendered murine tumors remarkably resistant to caloric restriction. Expression of eEF2K strongly correlated with overall survival in human medulloblastoma and glioblastoma multiforme. Finally, C. elegans strains deficient in efk-1, the eEF2K ortholog, were severely compromised in their response to nutrient depletion. Our data highlight a conserved role for eEF2K in protecting cells from nutrient deprivation and in conferring tumor cell adaptation to metabolic stress. PAPERCLIP:

The microscopic analysis of telomere features provides a wealth of information on the mechanism by which tumor cells maintain their unlimited proliferative potential. Accordingly, the analysis of telomeres in tissue sections of patient tumor samples can be exploited to obtain diagnostic information and to define tumor subgroups. In many instances, however, analysis of the image data is conducted by manual inspection of 2D images at relatively low resolution for only a small part of the sample. As the telomere feature signal distribution is frequently heterogeneous, this approach is prone to a biased selection of the information present in the image and lacks subcellular details. Here we address these issues by using an automated high-resolution imaging and analysis workflow that quantifies individual telomere features on tissue sections for a large number of cells. The approach is particularly suited to assess telomere heterogeneity and low abundant cellular subpopulations with distinct telomere characteristics in a reproducible manner. It comprises the integration of multi-color fluorescence in situ hybridization, immunofluorescence and DNA staining with targeted automated 3D fluorescence microscopy and image analysis. We apply our method to telomeres in glioblastoma and prostate cancer samples, and describe how the imaging data can be used to derive statistically reliable information on telomere length distribution or colocalization with PML nuclear bodies. We anticipate that relating this approach to clinical outcome data will prove to be valuable for pretherapeutic patient stratification.

SOX9 is a master transcription factor that regulates development and stem cell programs. However, its potential oncogenic activity and regulatory mechanisms that control SOX9 protein stability are poorly understood. Here, we show that SOX9 is a substrate of FBW7, a tumor suppressor, and a SCF (SKP1/CUL1/F-box)-type ubiquitin ligase. FBW7 recognizes a conserved degron surrounding threonine 236 (T236) in SOX9 that is phosphorylated by GSK3 kinase and consequently degraded by SCFFBW7α Failure to degrade SOX9 promotes migration, metastasis, and treatment resistance in medulloblastoma, one of the most common childhood brain tumors. FBW7 is either mutated or downregulated in medulloblastoma, and in cases where FBW7 mRNA levels are low, SOX9 protein is significantly elevated and this phenotype is associated with metastasis at diagnosis and poor patient outcome. Transcriptional profiling of medulloblastoma cells expressing a degradation-resistant SOX9 mutant reveals activation of pro-metastatic genes and genes linked to cisplatin resistance. Finally, we show that pharmacological inhibition of PI3K/AKT/mTOR pathway activity destabilizes SOX9 in a GSK3/FBW7-dependent manner, rendering medulloblastoma cells sensitive to cytostatic treatment.

Gliomas are the most frequent and aggressive malignancies of the central nervous system. Decades of molecular analyses have demonstrated that gliomas accumulate genetic alterations that culminate in enhanced activity of receptor tyrosine kinases and downstream mediators. While the genetic alterations, like gene amplification or loss, have been well characterized, little information exists about changes in the proteome of gliomas of different grades.

High morbidity and mortality are common traits of malignant tumours and identification of the cells responsible is a focus of on-going research. Many studies are now reporting the use of antibodies specific to Clusters of Differentiation (CD) cell surface antigens to identify tumour-initiating cell (TIC) populations in neural tumours. Medulloblastoma is one of the most common malignant brain tumours in children and despite a considerable amount of research investigating this tumour, the identity of the TICs, and the means by which such cells can be targeted remain largely unknown. Current prognostication and stratification of medulloblastoma using clinical factors, histology and genetic profiling have classified this tumour into four main subgroups: WNT, Sonic hedgehog (SHH), Group 3 and Group 4. Of these subgroups, SHH remains one of the most studied tumour groups due to the ability to model medulloblastoma formation through targeted deletion of the Shh pathway inhibitor Patched1 (Ptch1). Here we sought to utilise CD antibody expression to identify and isolate TIC populations in Ptch1 deleted medulloblastoma, and determine if these antibodies can help classify the identity of human medulloblastoma subgroups. Using a fluorescence-activated cell sorted (FACS) CD antibody panel, we identified CD24 as a marker of TICs in Ptch1 deleted medulloblastoma. CD24 expression was not correlated with markers of astrocytes or oligodendrocytes, but co-labelled with markers of neural progenitor cells. In conjunction with CD15, proliferating CD24+/CD15+ granule cell precursors (GCPs) were identified as a TIC population in Ptch1 deleted medulloblastoma. On human medulloblastoma, CD24 was found to be highly expressed on Group 3, Group 4 and SHH subgroups compared with the WNT subgroup, which was predominantly positive for CD15, suggesting CD24 is an important marker of non-WNT medulloblastoma initiating cells and a potential therapeutic target in human medulloblastoma. This study reports the use of CD24 and CD15 to isolate a GCP-like TIC population in Ptch1 deleted medulloblastoma, and suggests CD24 expression as a marker to help stratify human WNT tumours from other medulloblastoma subgroups.

Dysembryoplastic neuroepithelial tumors (DNT) share V600E mutation in the BRAF gene with other low grade neuroepithelial tumors (LGNTs). FGFR1 internal tandem duplication of the tyrosine-kinase domain (FGFR1-ITD), another genetic alteration that also leads to MAP kinase pathway alteration, has been previously reported in LGNTs by whole-genome sequencing. In the present study we searched for FGFR1-ITD by droplet digital PCR (DDPCR™) and for FGFR1 point mutations by HRM-sequencing in a series of formalin-fixed paraffin-embedded (FFPE) LGNTs including 12 DNT, 2 oligodendrogliomas lacking IDH mutation and 1p/19q co- deletion (pediatric-type oligodendrogliomas; PTOs), 3 pediatric diffuse astrocytomas (PDAs), 14 gangliogliomas (GGs) and 5 pilocytic astrocytomas (PAs). We showed by DDPCR™ that 5/12 DNT, but none of the other LGNTs, demonstrated FGFR1-ITD. In addition, these cases also accumulated phosphorylated-FGFR1 protein as shown by immunohistochemistry. FGFR1G539R point mutation was only recorded in one DNT that also showed FGFR1-ITD. Interestingly, these FGFR1 alterations were mutually exclusive from BRAFV600E mutation that was recorded in 13 LGNTs (3 DNTs, 1 PTO, 2 PDAs, 5 GGs and 2 PAs). Therefore, FGFR1 alteration mainly represented by FGFR1-ITD is a frequent event in DNT. DDPCR™ is an easy and alternative method than whole-genome sequencing to detect FGFR1-ITD in FFPE brain tumors, in routine practice.

There have been great efforts on the nanoscale 3D probing of brain tissues to image subcellular morphologies. However, limitations in terms of tissue coverage, anisotropic resolution, stain dependence, and complex sample preparation all hinder achieving a better understanding of the human brain functioning in the subcellular context. Herein, X-ray nanoholotomography is introduced as an emerging synchrotron radiation-based technology for large-scale, label-free, direct imaging with isotropic voxel sizes down to 25 nm, exhibiting a spatial resolution down to 88 nm. The procedure is nondestructive as it does not require physical slicing. Hence, it allows subsequent imaging by complementary techniques, including histology. The feasibility of this 3D imaging approach is demonstrated on human cerebellum and neocortex specimens derived from paraffin-embedded tissue blocks. The obtained results are compared to hematoxylin and eosin stained histological sections and showcase the ability for rapid hierarchical neuroimaging and automatic rebuilding of the neuronal architecture at the level of a single cell nucleolus. The findings indicate that nanoholotomography can complement microscopy not only by large isotropic volumetric data but also by morphological details on the sub-100 nm level, addressing many of the present challenges in brain tissue characterization and probably becoming an important tool in nanoanatomy.

Corpora amylacea are cell-derived structures that appear physiologically in the aged human brain. While their histological identification is straightforward, their ultrastructural composition and microenvironment at the nanoscale have remained unclear so far, as has their relevance to aging and certain disease states that involve the sequestration of toxic cellular metabolites. Here, we apply correlative serial block-face scanning electron microscopy and transmission electron tomography to gain three-dimensional insight into the ultrastructure and surrounding microenvironment of cerebral Corpora amylacea in the human brainstem and hippocampal region. We find that cerebral Corpora amylacea are composed of dense labyrinth-like sheets of lipid membranes, contain vesicles as well as morphologically preserved mitochondria, and are in close proximity to blood vessels and the glymphatic system, primarily within the cytoplasm of perivascular glial cells. Our results clarify the nature of cerebral Corpora amylacea and provide first hints on how they may arise and develop in the aging brain.

Pilocytic astrocytoma is the most common childhood brain tumor, characterized by constitutive MAPK activation. MAPK signaling induces oncogene-induced senescence (OIS), which may cause unpredictable growth behavior of pilocytic astrocytomas. The senescence-associated secretory phenotype (SASP) has been shown to regulate OIS, but its role in pilocytic astrocytoma remains unknown.Experimental Design: The patient-derived pilocytic astrocytoma cell culture model, DKFZ-BT66, was used to demonstrate presence of the SASP and analyze its impact on OIS in pilocytic astrocytoma. The model allows for doxycycline-inducible switching between proliferation and OIS. Both states were studied using gene expression profiling (GEP), Western blot, ELISA, and cell viability testing. Primary pilocytic astrocytoma tumors were analyzed by GEP and multiplex assay.

Glioblastoma multiform (GBM) is the most common brain tumor characterized by a dismal prognosis. GBM cancer stem cells (gCSC) or tumor-initiating cells are the cell population within the tumor-driving therapy resistance and recurrence. While temozolomide (TMZ), an alkylating agent, constitutes the first-line chemotherapeutic significantly improving survival in GBM patients, resistance against this compound commonly leads to GBM recurrence and treatment failure. Although the roles of protein-coding transcripts, proteins and microRNA in gCSC, and therapy resistance have been comprehensively investigated, very little is known about the role of long noncoding RNAs (lncRNAs) in this context. Using nonoverlapping, independent RNA sequencing and gene expression profiling datasets, we reveal that TP73-AS1 constitutes a clinically relevant lncRNA in GBM. Specifically, we demonstrate significant overexpression of TP73-AS1 in primary GBM samples, which is particularly increased in the gCSC. More importantly, we demonstrate that TP73-AS1 comprises a prognostic biomarker in glioma and in GBM with high expression identifying patients with particularly poor prognosis. Using CRISPRi to downregulate our candidate lncRNA in gCSC, we demonstrate that TP73-AS1 promotes TMZ resistance in gCSC and is linked to regulation of the expression of metabolism- related genes and ALDH1A1, a protein known to be expressed in cancer stem cell markers and protects gCSC from TMZ treatment. Taken together, our results reveal that high TP73-AS1 predicts poor prognosis in primary GBM cohorts and that this lncRNA promotes tumor aggressiveness and TMZ resistance in gCSC.

N-Myc is a transcription factor that is aberrantly expressed in many tumor types and is often correlated with poor patient prognosis. Recently, several lines of evidence pointed to the fact that oncogenic activation of Myc family proteins is concomitant with reprogramming of tumor cells to cope with an enhanced need for metabolites during cell growth. These adaptions are driven by the ability of Myc proteins to act as transcriptional amplifiers in a tissue-of-origin specific manner. Here, we describe the effects of N-Myc overexpression on metabolic reprogramming in neuroblastoma cells. Ectopic expression of N-Myc induced a glycolytic switch that was concomitant with enhanced sensitivity towards 2-deoxyglucose, an inhibitor of glycolysis. Moreover, global metabolic profiling revealed extensive alterations in the cellular metabolome resulting from overexpression of N-Myc. Limited supply with either of the two main carbon sources, glucose or glutamine, resulted in distinct shifts in steady-state metabolite levels and significant changes in glutathione metabolism. Interestingly, interference with glutamine-glutamate conversion preferentially blocked proliferation of N-Myc overexpressing cells, when glutamine levels were reduced. Thus, our study uncovered N-Myc induction and nutrient levels as important metabolic master switches in neuroblastoma cells and identified critical nodes that restrict tumor cell proliferation.

Ependymomas in children can arise throughout all compartments of the central nervous system (CNS). Highly malignant paediatric ependymoma subtypes are Group A tumours of the posterior fossa (PF-EPN-A) and RELA-fusion positive (ST-EPN-RELA) tumours in the supratentorial compartment. It was repeatedly reported in smaller series that accumulation of p53 is frequently observed in ependymomas and that immunohistochemical staining correlates with poor clinical outcome, while TP53 mutations are rare. Our TP53 mutation analysis of 130 primary ependymomas identified a mutation rate of only 3%. Immunohistochemical analysis of 398 ependymomas confirmed previous results correlating the accumulation of p53 with inferior outcome. Among the p53-positive ependymomas, the vast majority exhibited a RELA fusion leading to the hypothesis that p53 inactivation might be linked to RELA positivity.In order to assess the potential of p53 reactivation through MDM2 inhibition in ependymoma, we evaluated the effects of Actinomycin-D and Nutlin-3 treatment in two preclinical ependymoma models representing the high-risk subtypes PF-EPN-A and ST-EPN-RELA. The IC-50 of the agent as determined by metabolic activity assays was in the lower nano-molar range (0.2-0.7 nM). Transcriptome analyses of high-dose (100 nM), low-dose (5 nM) and non-treated cells revealed re-expression of p53 dependent genes including p53 upregulated modulator of apoptosis (PUMA) after low-dose treatment. At the protein level, we validated the Actinomycin-D induced upregulation of PUMA, and of p53 interaction partners MDM2 and p21. Proapoptotic effects of low-dose application of the agent were confirmed by flow cytometry. Thus, Actinomycin-D could constitute a promising therapeutic option for ST-EPN-RELA ependymoma patients, whose tumours frequently exhibit p53 inactivation.