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The balance of stem cell quiescence versus proliferation must be tightly regulated under normal homeostasis and becomes disrupted in cancers. The brain cancer glioblastoma is driven by cells with neural stem cell characteristics. These glioblastoma stem cells (GSCs) can reside in dormant, quiescent and proliferative states, yet the molecular transitions between these remain poorly understood. Quiescent GSCs are refractory to anti-mitotic cytotoxic therapies, and contribute to regrowth of the tumour; therefore elucidating molecular pathways that control GSC exit from quiescence may uncover new therapeutic strategies. The role of Wnt signaling in GBM has remained enigmatic. Here I show that the transcription factor FOXG1, which is frequently overexpressed in GSCs, cooperates with Wnt signaling to drive efficient exit from quiescence. However, Wnt signaling is dispensable once GSCs are fully proliferative. Using a phenotypic chemical screen (303 small molecule regulators of stem cell related pathways) I identified a potent synergy between glycogen synthase kinase 3 (GSK3) inhibitors and FOXG1 in driving exit from NSC quiescence. Pharmacological and genetic perturbations confirmed that this was due to activation of the Wnt/beta catenin pathway. The FOXG1/Wnt synergy is also relevant in vivo, and in human glioblastoma cell models. Mechanistically, these data suggest that the excessive FOXG1 both leads to an accumulation of beta-catenin and sequesters the Wnt/TCF co-repressor Groucho/TLE, thereby derepressing Wnt/beta catenin target genes and enabling their maximal activation. I conclude that FOXG1 and Wnt cooperate in the critical process of exiting the quiescent state. These findings suggest inhibition of Wnt signaling will have limited impact on the proliferative GSC compartment, but may be critical in preventing reactivation of the quiescent cells that drive regrowth of the tumour after chemo- and radiotherapy.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain cancer in adults. Relapse after conventional surgery and chemo/radiotherapy is thought to be driven by glioblastoma stem cells (GSCs). GSCs have phenotypic sumularities to normal neural stem cells (NSCs) and frequently overexpress many key neurodevelopmental transcription factors, including FOXG1, a key forebrain transcription factor with reporgramming activity. Evidence points to an important funtional role of FOXG1 in driving the NSC-like identity of GSCs. Here we explore the mechanisms by which FOXG1 acts, by characterising its key downstream transcritional targets and protein-protein interactions. Analysis of the transcriptional changes induced by FOXG1 overexpression identified key targets, including cell cycle and epigenetic regulators, such as Fox06 and Chd3. Fox06 was identified as a clear downstream target with a functional role in enabling FOXG1 to drive exit from quiescence. This also revealed the FOXG1, a transcriptional repressor, can lead to gene activation. The H3K4 demethylase JARID1B (KDM5B/PLU-1), a negative regulator of promoter activation, is thought to be a protein partner of FOXG1. We hypothesised that FOXG1 may activate gene expression by sequestration of this repressor. However, our studies suggest JARID1B is not essential for FOXG1's ability to drive a proliferative NS cell-like state, nor transcriptional activiation of Fox06 or Chd3. FOXG1 immunoprecipitation studies highlighted difficulties in obtaining good quality, protein-specific antibodies for characterising protein partners. we therefore exploited CRISPR/Cas9 technology to epitope-tag endogenous FOXG1 in patient-derived glioblastoma NS (GNS) cells. These cellular models can now be used in future studies to define FOXG1's key protein partners and aid the identification of ways to therapeutically target FOXG1 in GBM.

Glioblastoma (GBM) is the most common intrinsic primary brain tumour. It is uniformly fatal, with median survival approximately 14 months. These tumours comprise a mixture of neural stem cell-like cells and more differentiated astrocytic cells. The former are thought to be responsible for tumour development and recurrence, and display self-renewal and differentiation capacity in vitro. Glioma stem cells (GSCs) are defined operationally by their capacity to initiate tumours on orthotopic transplant into immunocompromised mice. The Pollard lab has identified the neural developmental transcription factor Forkhead Box G1 (FOXG1) as the most consistently overexpressed gene in GBM-derived neural stem (GNS) cells compared to their genetically normal neural stem (NS) cell counterparts. Here we explore the function and critical downstream effectors of FOXG1 in NS and GNS cells. We find that, although FOXG1 is not essential for sustaining proliferation of NS or GNS cells (in vitro), high FOXG1 restricts astrocyte differentiation in response to BMP and can drive dedifferentiation of postmitotic astrocytes. We identify a potential cooperation with SOX2. ChIP-Seq and RNA-Seq were used to define transcriptional targets. FOXG1 directly controls critical cell cycle regulators FOXO3 and FOXO6 (two forkhead family proteins), as well as the proto-oncogene MYCN and key regulators of both DNA and chromatin methylation, including TET3 and CHD3. Pharmacological inhibitors of MYC block FOXG1-driven de-differentiation, whereas Vitamin C and 5-azacytidine – agents that disrupt DNA and chromatin methylation – can facilitate de-differentiation. CRISPR/Cas genome editing was used to genetically ablate the cell cycle inhibitor FOXO3, or remove the FOXG1-bound cis-regulatory region. These data suggest direct transcriptional repression of FOXO3 by FOXG1 may drive cells into cycle. We conclude that high levels of FOXG1 in GBM limit astrocyte differentiation commitment by direct transcriptional control of core cell cycle regulators and DNA/histone methylation.

Disease modeling for neurodevelopmental disorders has long been a challenging endeavor, necessitating innovative approaches to understand the underlying mechanisms. Traditional animal models, while informative, often fail to fully capture the intricacies of human brain development and associated diseases. FOXG1 syndrome, caused by mutations in the Forkhead box protein G1 (FOXG1) gene and characterized by forebrain developmental abnormalities, represents one such disorder with a range of neurological symptoms including motor deficits, intellectual retardation, and seizures. In recent years, cortical brain organoid models derived from human pluripotent stem cells have emerged as a revolutionary tool in neuroscience research, providing a three-dimensional representation of the human brain. These organoids exhibit structural and functional resemblance to developing brains, encompassing the formation of diverse cell types and the establishment of functional networks. In this study, we were successfully able to develop a cortical organoid model using both healthy and patient derived cell lines for investigating FOXG1 syndrome, with the patient lines accurately reproducing the microcephaly phenotype observed in FOXG1 syndrome We explored strategies to enhance the cortical identity of the model using Wingless/Integrated pathway (WNT pathway) inhibition, achieving promising results. Furthermore, we successfully recapitulated neural tube development by constructing a single rosette organoid, thereby increasing its biomimetic properties. Our findings highlight the potential of cortical organoids as powerful tools for studying human neurodevelopment and neurodevelopmental disorders, offering new avenues for understanding disease pathogenesis and facilitating the development of targeted therapeutic interventions.