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Glioblastomas harbor diverse cell populations, including rare glioblastoma stem cells (GSCs) that drive tumorigenesis. To characterize functional diversity within this population, we performed single-cell RNA sequencing on >69,000 GSCs cultured from the tumors of 26 patients. We observed a high degree of inter- and intra-GSC transcriptional heterogeneity that could not be fully explained by DNA somatic alterations. Instead, we found that GSCs mapped along a transcriptional gradient spanning two cellular states reminiscent of normal neural development and inflammatory wound response. Genome-wide CRISPR–Cas9 dropout screens independently recapitulated this observation, with each state characterized by unique essential genes. Further single-cell RNA sequencing of >56,000 malignant cells from primary tumors found that the majority organize along an orthogonal astrocyte maturation gradient yet retain expression of founder GSC transcriptional programs. We propose that glioblastomas grow out of a fundamental GSC-based neural wound response transcriptional program, which is a promising target for new therapy development. Pugh and colleagues use single-cell RNA sequencing, CRISPR screens and functional assays to define a gradient of developmental and wound-response cell states in glioblastoma stem cells, revealing insights into glioblastoma origins and potential therapeutic targets.

Glioblastoma multiforme (GBM) contains a subpopulation of cells, GBM stem cells (GSCs), that maintain the bulk tumor and represent a key therapeutic target. Norrin is a Wnt ligand that binds Frizzled class receptor 4 (FZD4) to activate canonical Wnt signaling. Although Norrin, encoded by NDP, has a well-described role in vascular development, its function in human tumorigenesis is largely unexplored. Here, we show that NDP expression is enriched in neurological cancers, including GBM, and its levels positively correlated with survival in a GBM subtype defined by low expression of ASCL1, a proneural factor. We investigated the function of Norrin and FZD4 in GSCs and found that it mediated opposing tumor-suppressive and -promoting effects on ASCL1lo and ASCL1hi GSCs. Consistent with a potential tumor-suppressive effect of Norrin suggested by the tumor outcome data, we found that Norrin signaling through FZD4 inhibited growth in ASCL1lo GSCs. In contrast, in ASCL1hi GSCs Norrin promoted Notch signaling, independently of WNT, to promote tumor progression. Forced ASCL1 expression reversed the tumor-suppressive effects of Norrin in ASCL1lo GSCs. Our results identify Norrin as a modulator of human brain cancer progression and reveal an unanticipated Notch-mediated function of Norrin in regulating cancer stem cell biology. This study identifies an unanticipated role of Norrin in human brain cancer progression. In addition, we provide preclinical evidence suggesting Norrin and canonical Wnt signaling as potential therapeutic targets for GBM subtype-restricted cancer stem cells.

Glioblastoma stem cells (GSCs) exhibit latent neuronal lineage differentiation potential governed by the proneural transcription factor Achaete-scute homolog 1 (ASCL1) and harnessing and promoting terminal neuronal differentiation has been proposed as a novel therapeutic strategy. Here, using a genome-wide CRISPR suppressor screen we identified genes required for ASCL1-mediated neuronal differentiation. This approach revealed a specialized function of the Mediator complex tail module and of its subunits MED24 and MED25 for this process in GSCs, human fetal neural stem cells and pluripotent stem cells. We show that upon induction of neuronal differentiation MED25 is recruited to genomic loci co-occupied by ASCL1 to regulate neurogenic gene expression programs. MED24 and MED25 are sufficient to induce neuronal differentiation in GSC cultures and to mediate neuronal differentiation in multiple contexts. Collectively our data expand our understanding of the mechanisms underlying directed terminal neuronal differentiation in brain tumor stem cells and point to a unique function of the Mediator tail in neuronal reprogramming.

Human glioblastomas harbour a subpopulation of glioblastoma stem cells that drive tumorigenesis. However, the origin of intratumoural functional heterogeneity between glioblastoma cells remains poorly understood. Here we study the clonal evolution of barcoded glioblastoma cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of glioblastoma clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in glioblastoma stem cells. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, which in turn generates non-proliferative cells. We also identify rare 'outlier' clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant glioblastoma stem cells. Finally, we show that functionally distinct glioblastoma stem cells can be separately targeted using epigenetic compounds, suggesting new avenues for glioblastoma-targeted therapy.

Developmental signal transduction pathways act diversely, with context-dependent roles across systems and disease types. Glioblastomas (GBMs), which are the poorest prognosis primary brain cancers, strongly resemble developmental systems, but these growth processes have not been exploited therapeutically, likely in part due to the extreme cellular and genetic heterogeneity observed in these tumors. The role of Wnt/βcatenin signaling in GBM stem cell (GSC) renewal and fate decisions remains controversial. Here, we report context-specific actions of Wnt/βcatenin signaling in directing cellular fate specification and renewal. A subset of primary GBM-derived stem cells requires Wnt proteins for self-renewal, and this subset specifically relies on Wnt/βcatenin signaling for enhanced tumor burden in xenograft models. In an orthotopic Wnt reporter model, Wnthi GBM cells (which exhibit high levels of βcatenin signaling) are a faster-cycling, highly self-renewing stem cell pool. In contrast, Wntlo cells (with low levels of signaling) are slower cycling and have decreased self-renewing potential. Dual inhibition of Wnt/βcatenin and Notch signaling in GSCs that express high levels of the proneural transcription factor ASCL1 leads to robust neuronal differentiation and inhibits clonogenic potential. Our work identifies new contexts for Wnt modulation for targeting stem cell differentiation and self-renewal in GBM heterogeneity, which deserve further exploration therapeutically.

Glioblastomas (GBM) grow in a rich neurochemical milieu, but the impact of neurochemicals on GBM growth is largely unexplored. We interrogated 680 neurochemical compounds in patient-derived GBM neural stem cells (GNS) to determine the effects on proliferation and survival. Compounds that modulate dopaminergic, serotonergic, and cholinergic signaling pathways selectively affected GNS growth. In particular, dopamine receptor D4 (DRD4) antagonists selectively inhibited GNS growth and promoted differentiation of normal neural stem cells. DRD4 antagonists inhibited the downstream effectors PDGFRβ, ERK1/2, and mTOR and disrupted the autophagy-lysosomal pathway, leading to accumulation of autophagic vacuoles followed by G0/G1 arrest and apoptosis. These results demonstrate a role for neurochemical pathways in governing GBM stem cell proliferation and suggest therapeutic approaches for GBM.

Mutations in the histone 3 variant H3.3 have been identified in one-third of pediatric glioblastomas (GBMs), but not in adult tumors. Here we show that H3.3 is a dynamic determinant of functional properties in adult GBM. H3.3 is repressed by mixed lineage leukemia 5 (MLL5) in self-renewing GBM cells. MLL5 is a global epigenetic repressor that orchestrates reorganization of chromatin structure by punctuating chromosomes with foci of compacted chromatin, favoring tumorigenic and self-renewing properties. Conversely, H3.3 antagonizes self-renewal and promotes differentiation. We exploited these epigenetic states to rationally identify two small molecules that effectively curb cancer stem cell properties in a preclinical model. Our work uncovers a role for MLL5 and H3.3 in maintaining self-renewal hierarchies in adult GBM.

The tumor microenvironment is an important contributor to malignancy in glioblastoma (GBM) by influencing stemness properties in glioma stem cells (GSCs) and their capacity for differentiation into various cell types. Tumor hypoxia is a common feature of the GBM microenvironment and can influence cell state through a variety of processes including the inhibition of oxygen-dependent lysine histone demethylases (KDMs) and subsequent epigenetic changes required for differentiation or maintenance of the stem cell state. However, the precise mechanisms underlying hypoxic restriction of differentiation to specific neural lineages is unclear. Here, we show that hypoxia suppresses astrocytic differentiation induced by bone morphogenetic protein 4 (BMP4) in patient-derived GSCs. In 8/10 patient derived GSC lines, BMP4 induced expression of astrocytic markers, such as glial fibrillary acidic protein, and this induction was repressed by hypoxia in 7/10 lines. Interestingly, significant anti-proliferative effects of BMP4 induced differentiation were only observed in 3/10 lines and was rescued in only one GSC line by hypoxia, suggesting that these processes are uncoupled. Although hypoxia may directly limit the activity of oxygen-dependent KDMs important in this process, levels of S-2-hydroxyglutarate (S-2HG), an endogenous KDM inhibitor, were also increased in 5/8 GSC lines under hypoxia. Exogenous treatment with S-2HG or a small molecule KDM6 family inhibitor, phenocopied the repressive effects of hypoxia on BMP4 induced differentiation. In patient tumors, hypoxic areas surrounding pseudopalisading necrosis showed high expression of hypoxia markers, and these hypoxia markers were anti-correlated with markers of astrocytic differentiation. Together, these results demonstrate that the hypoxic tumor microenvironment in GBM, through multiple mechanisms limiting oxygen-dependent KDM activity, promote maintenance of the malignant stemness state by inhibiting astrocytic differentiation. This study implicates tumour hypoxia as a key regulator of stemness and a therapeutic target to promote differentiation in GBM. Treatment strategies that target hypoxic cells are urgently needed to overcome these challenges. Citation Format: Ronald S. Wu, Elisabeth Liedtke, Sheila Mansouri, Samantha Brazier, Paul Guilhamon, Nicole I. Park, Eric Chen, Mathieu Lupien, Daniel D. De Carvalho, Peter G. Dirks, Gelareh Zadeh, Bradley G. Wouters. Hypoxia inhibits BMP4-induced astrocytic differentiation in glioma stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3438.

Glioblastomas exhibit a hierarchical cellular organization, suggesting that they are driven by neoplastic stem cells that retain partial yet abnormal differentiation potential. Here, we show that a large subset of patient-derived glioblastoma stem cells (GSCs) express high levels of Achaete-scute homolog 1 (ASCL1), a proneural transcription factor involved in normal neurogenesis. ASCL1hi GSCs exhibit a latent capacity for terminal neuronal differentiation in response to inhibition of Notch signaling, whereas ASCL1lo GSCs do not. Increasing ASCL1 levels in ASCL1lo GSCs restores neuronal lineage potential, promotes terminal differentiation, and attenuates tumorigenicity. ASCL1 mediates these effects by functioning as a pioneer factor at closed chromatin, opening new sites to activate a neurogenic gene expression program. Directing GSCs toward terminal differentiation may provide therapeutic applications for a subset of GBM patients and strongly supports efforts to restore differentiation potential in GBM and other cancers.

Cancer genomes accumulate frequent and diverse chromosomal abnormalities as well as gene mutations but must maintain the ability to survive in vivo. We hypothesize that genetic selection acts to maintain tumour survival by preserving copy number of specific genes and genomic regions. Genomic regions and genes that remain unaltered in copy number and expression, respectively, may be essential for maintaining tumour survival.We analyzed copy number data of 243 previously reported breast tumours and computationally derived stable copy number regions. To identify genes in stable copy number regions with nominal changes in expression, datasets for tumour and normal samples were compared. Results were replicated by analysis of a series of independent copy number, expression and genomic sequencing studies. A subset of stable regions, including stable paralogous regions, were confirmed by quantitative PCR and fluorescence in situ hybridization (FISH) in 5 breast cancer cell lines. We deduced a comprehensive set of dually stable genes (i.e. maintaining nominal copy number and expression) which were categorized according to pathway and ontology assignments. The stability of genes encoding therapeutic drug targets was also assessed.Tumour genome analysis revealed 766 unstable (amplified and/or deleted) and 812 stable contiguous genomic regions. Replication analysis of an independent set of 171 breast tumours confirmed copy number stability of 1.3 Gb of the genome. We found that 5804 of these genes were dually stable. The composition of this gene set remained essentially unchanged (2% reduction) after accounting for commonly mutated breast cancer genes found by sequencing and differential expression. The stable breast cancer genome is enriched for cellular metabolism, regulation of gene expression, DNA packaging (chromatin and nucleosome assembly), and regulation of apoptosis functions. Stable genes participating in multiple essential pathways were consistently found to be targets of chemotherapies. Preservation of stable, essential genes may be related to the effectiveness of certain chemotherapeutic agents that act on multiple gene products in this set.

Glioblastoma multiforme (GBM) is the most common malignant tumour in the central nervous system with a prevalence of 2-3 cases per 100 000 people. Although the standard treatment of surgery, chemotherapy and radiotherapy improve survival, the median survival continues to remain at only 15 months with a 5-year survival rate of under 10%. Glioma neural stem-like (GNS) cells have been identified in GBM and have the capability of regenerating the tumour. Treatment strategies that target the majority of the tumour may be incapable of also targeting GNS cells and thus characterization of GNS cells may provide insight into additional treatment options. The Wnt signalling pathway has been linked to several cancers including GBM. Wnt signalling involves the secretion of Wnt ligand proteins that bind to specific Frizzled (FZD) receptor complexes on the cell surface of Wnt-responding cells to activate intracellular signalling cascades. The transcriptional and epigenetic regulation in GNS cells is the focus of several recent studies. The transcription factor ASCL1 was identified to be overexpressed and to lead to Wnt signalling activation by repressing Dickkopf (DKK1, Wnt inhibitor). Using microarray data we analyzed the expression of FZD receptors and Wnt target genes in over 50 primary GNS cell lines cultured in serum free conditions in order to maintain the GIC population and identified a subgroup of glioma lines with activated Wnt signalling. To determine the requirement of autocrine Wnt signalling for GNS cell renewal we inhibited Wnt secretion, using the porcupine inhibitor LGK974, and measured self-renewal using a limited dilution assay. There was a significant reduction in GNS cell frequency with LGK974 (1uM) treatment in four out of eight lines tested (G432NS, G472NS, G511NS and G523NS). Furthermore, a secondary sphere assay with G523NS cells also showed a significant reduction in GNS cell frequency. When G511NS and G523NS cells were treated with LGK974 (1uM) over a two week period, there was a significant increase in the percentage of GFAP (astrocytic marker) expressing cells whereas a two week treatment with Bio (1uM, Wnt activator), significantly increased the percentage of Tuj1 (neuronal marker) expressing cells. This finding suggest that these cells require a specific amount of Wnt signalling for self-renewal and Wnt inhibition or activation may lead to differentiation. RNAseq analysis comparing the four LGK974 responsive lines with the four LGK974 unresponsive lines identified ASCL1 to be highly expressed in the responsive lines. Gene set enrichment analysis identified four gene sets significantly enriched in the responsive group including the Glioblastoma Proneural gene set whereas genes from the Glioblastoma Mesenchymal gene set were significantly enriched in the unresponsive group. We have identified a subset of GNS cells that are dependent on Wnt secretion for self-renewal and RNAseq analysis suggests that GBMs that fall under the Proneural subtype may be sensitive to Wnt inhibition. Citation Format: Nishani Rajakulendran, Hayden Selvadurai, Katherine Rowland, Nicole Park, Nizar Batada, Peter Dirks, Stephane Angers. Wnt signaling circuits in glioblastoma multiforme. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B14.

Glioblastoma (GBM), the most common and lethal adult primary brain tumour, exhibits cellular heterogeneity and a subpopulation of tumour cells exhibits the neural precursor phenotype and drives tumourigenesis. These findings suggest that tumours may represent aberrant organogenesis with growth caused by unlimited proliferation and failure of differentiation of malignant precursor cells. As GBM-neural stem (GNS) cells have precursor cell properties, promotion of differentiation represents a potential strategy for treatment. A key regulator of neuronal differentiation is ASCL1 during normal development and adult neurogenesis. Microarray analysis of ASCL1 expression in primary GNS cultures (n = 33) revealed two subgroups of GNS cultures as having either high or low expression. Evidence suggests that GNS cells with high expression of ASCL1 exhibit neuronal lineage commitment whereas GNS cells with low expression of ASCL1 do not undergo lineage commitment. This was confirmed using immunocytochemistry, quantitative real-time PCR and Western blot analysis. Furthermore, a dependent relationship was observed between ASCL1 subgroups and GNS subgroups responsive to inhibitors of Notch signaling. Treatment of the responder subgroup with a gamma-secretase inhibitor resulted in an increase in neuronal lineage markers and concomitant decrease in stem cell frequencies, as measured by immunocytochemistry and in vitro limiting dilution assays, respectively. Dominant-negative studies of ASCL1 suggest that ASCL1 is necessary for these effects in vitro and have been validated in vivo. This study aims to determine whether neuronal differentiation by blocking Notch signaling is mediated by ASCL1

Glioblastoma (GBM), the most common and lethal adult primary brain tumour, exhibits cellular heterogeneity and a subpopulation of tumour cells exhibits the neural precursor phenotype and drives tumourigenesis. These findings suggest that tumours may represent aberrant organogenesis with growth caused by unlimited proliferation and failure of differentiation of malignant precursor cells. It has yet to be determined the relative importance of aberrant self-renewal versus blocked differentiation in the brain tumour phenotype. As GBM-initiating cells have precursor cell properties, promotion of differentiation represents a potential strategy for treatment. Forcing glioblastoma precursor cells (GPCs) to differentiate terminally would limit clonal expansion and attenuate tumour growth. Data generated in our lab suggests that differentiation potential varies between different patient-derived GPC cultures. This study aims to determine what defines a blocked differentiation phenotype in human glioblastoma and to determine whether differentiation therapy is a feasible strategy for glioblastoma treatment. A key regulator of neuronal differentiation is ASCL1 during normal development and adult neurogenesis. Microarray analysis of ASCL1 expression in GPCs (n = 33) revealed two subgroups of GPCs as having either high or low expression. Preliminary evidence suggests that GPCs with high expression of ASCL1 exhibit neuronal lineage commitment whereas GPCs with low expression of ASCL1 do not undergo lineage commitment. This was confirmed using immunocytochemistry, quantitative real-time PCR and Western blot analysis. Furthermore, an inverse correlation was observed between transcript levels of ASCL1 and DKK1, an antagonist of the Wnt signalling pathway, while a direct correlation was observed between ASCL1 and activated β-catenin (ABC). Treatment of GPCs with a GSK3β inhibitor resulted in the activation of Wnt signalling and concomitant increase in transcript levels of ASCL1 and neuronal lineage markers (i.e. TUJ1, MAP2). This study aims to further investigate the role of ASCL1 in glioblastoma and to determine whether neuronal lineage commitment in the context of ASCL1 activity is mediated in a Wnt-dependent manner. Gain- and loss-of-function studies are in progress to determine whether ASCL1 is sufficient and/or necessary for neuronal lineage commitment and consequent effects on tumour-initiating properties (i.e. proliferation and self-renewal) will be examined in vitro and in vivo. Citation Format: Nicole I. Park, Peter B. Dirks. Investigating the role of ASCL1 in regulating differentiation of gliobastoma precursor cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A45.