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Cellular identity requires the concerted action of multiple transcription factors (TFs) bound together to enhancers of cell-type-specific genes. Despite TFs recognizing specific DNA motifs within accessible chromatin, this information is insufficient to explain how TFs select enhancers1. Here we compared four different TF combinations that induce different cell states, analysing TF genome occupancy, chromatin accessibility, nucleosome positioning and 3D genome organization at the nucleosome resolution. We show that motif recognition on mononucleosomes can decipher only the individual binding of TFs. When bound together, TFs act cooperatively or competitively to target nucleosome arrays with defined 3D organization, displaying motifs in particular patterns. In one combination, motif directionality funnels TF combinatorial binding along chromatin loops, before infiltrating laterally to adjacent enhancers. In other combinations, TFs assemble on motif-dense and highly interconnected loop junctions, and subsequently translocate to nearby lineage-specific sites. We propose a guided-search model in which motif grammar on nucleosome fibres acts as signpost elements, directing TF combinatorial binding to enhancers.

Driver mutations in genes encoding histone H3 proteins resulting in p.Lys27Met substitutions (H3-K27M) are frequent in pediatric midline brain tumors. However, the precise mechanisms by which H3-K27M causes tumor initiation remain unclear. Here, we use human hindbrain neural stem cells to model the consequences of H3.3-K27M on the epigenomic landscape in a relevant developmental context. Genome-wide mapping of epitope-tagged histone H3.3 revealed that both the wild type and the K27M mutant incorporate abundantly at pre-existing active enhancers and promoters, and to a lesser extent at Polycomb repressive complex 2 (PRC2)-bound regions. At active enhancers, H3.3-K27M leads to focal H3K27ac loss, decreased chromatin accessibility and reduced transcriptional expression of nearby neurodevelopmental genes. In addition, H3.3-K27M deposition at a subset of PRC2 target genes leads to increased PRC2 and PRC1 binding and augmented transcriptional repression that can be partially reversed by PRC2 inhibitors. Our work suggests that, rather than imposing de novo transcriptional circuits, H3.3-K27M drives tumorigenesis by locking initiating cells in their pre-existing, immature epigenomic state, via disruption of PRC2 and enhancer functions.

Here, Soares et al. show an important role for differential binding of TFs to mitotic chromosomes, governed by their electrostatic properties, in defining the temporal order of transcriptional reactivation during mitosis-to-G1 transition. They report that Brn2, an important regulator of neural stem cell identity, associates with condensed chromatin throughout cell division, as assessed by live-cell imaging of proliferating neural stem cells, and ChIP-seq analysis reveals that Brn2 mitotic chromosome binding does not result in sequence-specific interactions prior to mitotic exit, relying mostly on electrostatic forces.

In this study from Lawlor et al., the authors sought to understand the cell of origin for glioblastoma, and analyzed the interaction between transformed and wild-type neural stem cells (NSCs) isolated from the adult mouse subventricular zone niche. Their findings suggest that oncogenic mutations are propagated in the stem cell niche not just through cell-intrinsic advantages, but also by outcompeting neighboring stem cells through repression of their proliferation.

Despite decades of research, brain tumours remain among the deadliest of all forms of cancer. The ability of these tumours to resist almost all conventional and novel treatments relates, in part, to the unique cell-intrinsic and microenvironmental properties of neural tissues. In an attempt to encourage progress in our understanding and ability to successfully treat patients with brain tumours, Cancer Research UK convened an international panel of clinicians and laboratory-based scientists to identify challenges that must be overcome if we are to cure all patients with a brain tumour. The seven key challenges summarized in this Position Paper are intended to serve as foci for future research and investment.

Glioblastoma multiforme (GBM) (grade IV astrocytoma) is the most common and aggressive primary brain tumor. GBM consists of heterogeneous cell types including a subset of stem cell‐like cells thought to sustain tumor growth. These tumor‐initiating glioblastoma multiforme‐derived neural stem (GNS) cells as well as their genetically normal neural stem (NS) counterparts can be propagated in culture as relatively pure populations. Here, we perform quantitative proteomics to globally characterize and compare total proteome plus the secreted proteome (secretome) between GNS cells and NS cells. Proteins and pathways that distinguish malignant cancer (GNS) stem cells from their genetically normal counterparts (NS cells) might have value as new biomarkers or therapeutic targets. Our analysis identified and quantified ∼7,500 proteins in the proteome and ∼2,000 in the secretome, 447 and 138 of which were differentially expressed, respectively. Notable tumor‐associated processes identified using gene set enrichment analysis included: extracellular matrix interactions, focal adhesion, cell motility, and cell signaling. We focused on differentially expressed surface proteins, and identified 26 that participate in ligand‐receptor pairs that play a prominent role in tumorigenesis. Immunocytochemistry and immunoblotting confirmed that CD9, a recently identified marker of adult subventricular zone NS cells, was consistently enriched across a larger set of primary GNS cell lines. CD9 may, therefore, have value as a GNS‐specific surface marker and a candidate therapeutic target. Altogether, these findings support the notion that increased cell‐matrix and cell‐cell adhesion molecules play a crucial role in promoting the tumor initiating and infiltrative properties of GNS cells. StemCells2017;35:967–980

Glioblastoma (GBM) stem cells (GSCs) display phenotypic and molecular features reminiscent of normal neural stem cells and exhibit a spectrum of cell cycle states (dormant, quiescent, proliferative). However, mechanisms controlling the transition from quiescence to proliferation in both neural stem cells (NSCs) and GSCs are poorly understood. Elevated expression of the forebrain transcription factor FOXG1 is often observed in GBMs. Here, using small-molecule modulators and genetic perturbations, we identify a synergistic interaction between FOXG1 and Wnt/β-catenin signaling. Increased FOXG1 enhances Wnt-driven transcriptional targets, enabling highly efficient cell cycle re-entry from quiescence; however, neither FOXG1 nor Wnt is essential in rapidly proliferating cells. We demonstrate that FOXG1 overexpression supports gliomagenesis in vivoand that additional β-catenin induction drives accelerated tumor growth. These data indicate that elevated FOXG1 cooperates with Wnt signaling to support the transition from quiescence to proliferation in GSCs.

ABSTRACTBackground: SOX2 and SOX9 are commonly overexpressed in glioblastoma, and regulate the activity of glioma stem cells (GSCs). Their specific and overlapping roles in GSCs and glioma treatment remain unclear.Methods: SOX2and SOX9levels were examined in human biopsies. Gain and loss of function determined the impact of altering SOX2 and SOX9 on cell proliferation, senescence, stem cell activity, tumorigenesis and chemoresistance.Results: SOX2 and SOX9 expression correlates positively in glioma cells and glioblastoma biopsies. High levels of SOX2 bypass cellular senescence and promote resistance to temozolomide. Mechanistic investigations revealed that SOX2 acts upstream of SOX9. mTOR genetic and pharmacologic (rapamycin) inhibition decreased SOX2 and SOX9 expression, and reversed chemoresistance.Conclusions: Our findings reveal SOX2-SOX9 as an oncogenic axis that regulates stem cell properties and chemoresistance. We identify that rapamycin abrogate SOX protein expression and provide evidence that a combination of rapamycin and temozolomide inhibits tumor growth in cells with high SOX2/SOX9.

To identify therapeutic targets for glioblastoma (GBM), we performed genome-wide CRISPR-Cas9 knockout (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem/progenitors (NSCs), non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers). In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, most likely as a result of oncogenic signaling, causing GBM-specific lethality.

The epigenetic marks displayed by a cancer cell originate from two separate processes: The most prominent epigenetic signatures are associated with the cell of origin, i.e., the lineage and cell type identity imposed during development. The second set comprises those aberrant cancer-specific epigenetic marks that appear during tumor initiation or subsequent malignant progression. These are generally thought to associate with tumor-promoting pathways. As biochemical pathways regulating epigenetic mechanisms are potentially “druggable” and reversible, there is considerable interest in defining their roles in tumor genesis and growth, as they may represent therapeutic targets for treatment of human neoplasias.1However, despite the potential importance of epigenetic modifications in human cancer, it has been difficult to determine when, where and how epigenetic disruptions occur, and if they have important functional roles in sustaining the malignant state.

Evidence for neurogenesis within the adult brain has challenged traditional views that this tissue is devoid of stem cell activity. This raises the possibility of introducing new cells through cell transplantation or stimulating endogenous neurogenesis as routes to treat disease and injury. Fetal and adult neural stem/progenitor cells can be isolated and expanded in vitroand might provide a cell source for such transplantations. Embryonic stem (ES) cells, which can generate any adult tissues, offer an alternative unlimited supply of neural tissue. We recently showed that both mouse and human ES cells can be converted to adherent neural stem (NS) cell lines [1]. Here we discuss the benefits of working with NS cell lines and how they might be exploited for studies of fundamental cellular processes, such as neuronal specification and differentiation. NS cells also serve as versatile models of disease processes, either through genetic manipulations or direct isolation from disease carriers and can be exploited in pharmaceutical drug screening. Longer term, NS cells offer an opportunity to rigorously test the efficacy of cell-based therapies and develop strategies for tissue engineering.

Basement membranes are thought to be essential for organ formation, providing the scaffold on which individual cells organize to form complex tissues. Laminins are integral components of basement membranes. To understand the development of a simple vertebrate organ, we have used positional cloning to characterize grumpy and sleepy, two zebrafish loci known to control notochord formation, and find that they encode laminin beta1 and laminin gamma1, respectively. Removal of either chain results in the dramatic loss of laminin 1 staining throughout the embryo and prevents formation of the basement membrane surrounding the notochord. Notochord cells fail to differentiate and many die by apoptosis. By transplantation, we demonstrate that, for both grumpy and sleepy, notochord differentiation can be rescued by exogenous sources of the missing laminin chain, although notochordal sources are also sufficient for rescue. These results demonstrate a clear in vivo requirement for laminin beta1 and laminin gamma1 in the formation of a specific vertebrate organ and show that laminin or the laminin-dependent basement membrane is essential for the differentiation of chordamesoderm to notochord.

Basement membranes are thought to be essential for organ formation, providing the scaffold on which individual cells organize to form complex tissues. Laminins are integral components of basement membranes. To understand the development of a simple vertebrate organ, we have used positional cloning to characterize grumpy and sleepy, two zebrafish loci known to control notochord formation, and find that they encode laminin β1 and laminin γ1, respectively. Removal of either chain results in the dramatic loss of laminin 1 staining throughout the embryo and prevents formation of the basement membrane surrounding the notochord. Notochord cells fail to differentiate and many die by apoptosis. By transplantation, we demonstrate that, for both grumpy and sleepy, notochord differentiation can be rescued by exogenous sources of the missing laminin chain, although notochordal sources are also sufficient for rescue. These results demonstrate a clear in vivo requirement for laminin β1 and laminin γ1 in the formation of a specific vertebrate organ and show that laminin or the laminin-dependent basement membrane is essential for the differentiation of chordamesoderm to notochord.

Point mutations within the histone H3.3 are frequent in aggressive childhood brain tumors known as pediatric high-grade gliomas (pHGGs). Intriguingly, distinct mutations arise in discrete anatomical regions: H3.3-G34R within the forebrain and H3.3-K27M preferentially within the hindbrain. The reasons for this contrasting etiology are unknown. By engineering human fetal neural stem cell cultures from distinct brain regions, we demonstrate here that cell-intrinsic regional identity provides differential responsiveness to each mutant that mirrors the origins of pHGGs. Focusing on H3.3-G34R, we find that the oncohistone supports proliferation of forebrain cells while inducing a cytostatic response in the hindbrain. Mechanistically, H3.3-G34R does not impose widespread transcriptional or epigenetic changes but instead impairs recruitment of ZMYND11, a transcriptional repressor of highly expressed genes. We therefore propose that H3.3-G34R promotes tumorigenesis by focally stabilizing the expression of key progenitor genes, thereby locking initiating forebrain cells into their pre-existing immature state.

Human neural stem cell cultures provide progenitor cells that are potential cells of origin for brain cancers. However, the extent to which genetic predisposition to tumor formation can be faithfully captured in stem cell lines is uncertain. Here, we evaluated neuroepithelial stem (NES) cells, representative of cerebellar progenitors. We transduced NES cells with MYCN, observing medulloblastoma upon orthotopic implantation in mice. Significantly, transcriptomes and patterns of DNA methylation from xenograft tumors were globally more representative of human medulloblastoma compared to a MYCN-driven genetically engineered mouse model. Orthotopic transplantation of NES cells generated from Gorlin syndrome patients, who are predisposed to medulloblastoma due to germline-mutated PTCH1, also generated medulloblastoma. We engineered candidate cooperating mutations in Gorlin NES cells, with mutation of DDX3Xor loss of GSE1both accelerating tumorigenesis. These findings demonstrate that human NES cells provide a potent experimental resource for dissecting genetic causation in medulloblastoma.

Glioblastoma multiforme (GBM) is one of the deadliest human cancers. Despite increasing knowledge of the genetic and epigenetic changes that underlie tumour initiation and growth, the prognosis for GBM patients remains dismal. Genome analysis has failed to lead to success in the clinic. Fresh approaches are needed that can stimulate new discoveries across all levels: cell-intrinsic mechanisms (transcriptional/epigenetic and metabolic), cell-cell signalling, niche and microenvironment, systemic signals, immune regulation, and tissue-level physical forces. GBMs are inherently extremely challenging: tumour detection occurs too late, and cells infiltrate widely, hiding in quiescent states behind the blood-brain barrier. The complexity of the brain tissue also provides varied and complex microenvironments that direct cancer cell fates. Phenotypic heterogeneity is therefore superimposed onto pervasive genetic heterogeneity. Despite this bleak outlook, there are reasons for optimism. A myriad of complementary, and increasingly sophisticated, experimental approaches can now be used across the research pipeline, from simple reductionist models devised to delineate molecular and cellular mechanisms, to complex animal models required for preclinical testing of new therapeutic approaches. No single model can cover the breadth of unresolved questions. This Review therefore aims to guide investigators in choosing the right model for their question. We also discuss the recent convergence of two key technologies: human stem cell and cancer stem cell culture, as well as CRISPR/Cas tools for precise genome manipulations. New functional genetic approaches in tailored models will likely fuel new discoveries, new target identification and new therapeutic strategies to tackle GBM.

Glioblastoma multiforme (GBM) is an aggressive incurable brain cancer. The cells that fuel the growth of tumours resemble neural stem cells found in the developing and adult mammalian forebrain. These are referred to as glioma stem cells (GSCs). Similar to neural stem cells, GSCs exhibit a variety of phenotypic states: dormant, quiescent, proliferative and differentiating. How environmental cues within the brain influence these distinct states is not well understood. Laboratory models of GBM can be generated using either genetically engineered mouse models, or via intracranial transplantation of cultured tumour initiating cells (mouse or human). Unfortunately, these approaches are expensive, time-consuming, low-throughput and ill-suited for monitoring live cell behaviours. Here, we explored whole adult brain coronal organotypic slices as an alternative model. Mouse adult brain slices remain viable in a serum-free basal medium for several weeks. GSCs can be easily microinjected into specific anatomical sites ex vivo, and we demonstrate distinct responses of engrafted GSCs to diverse microenvironments in the brain tissue. Within the subependymal zone – one of the adult neural stem cell niches – injected tumour cells could effectively engraft and respond to endothelial niche signals. Tumour-transplanted slices were treated with the antimitotic drug temozolomide as proof of principle of the utility in modelling responses to existing treatments. Engraftment of mouse or human GSCs onto whole brain coronal organotypic brain slices therefore provides a simplified, yet flexible, experimental model. This will help to increase the precision and throughput of modelling GSC-host brain interactions and complements ongoing in vivo studies. This article has an associated First Person interview with the first author of the paper.