IFI35 regulates non-canonical NF-κB signaling to maintain glioblastoma stem cells and recruit tumor-associated macrophages.

Glioblastoma (GBM) is the most aggressive malignant primary brain tumor characterized by a highly heterogeneous and immunosuppressive tumor microenvironment (TME). The symbiotic interactions between glioblastoma stem cells (GSCs) and tumor-associated macrophages (TAM) in the TME are critical for tumor progression. Here, we identified that IFI35, a transcriptional regulatory factor, plays both cell-intrinsic and cell-extrinsic roles in maintaining GSCs and the immunosuppressive TME. IFI35 induced non-canonical NF-kB signaling through proteasomal processing of p105 to the DNA-binding transcription factor p50, which heterodimerizes with RELB (RELB/p50), and activated cell chemotaxis in a cell-autonomous manner. Further, IFI35 induced recruitment and maintenance of M2-like TAMs in TME in a paracrine manner. Targeting IFI35 effectively suppressed in vivo tumor growth and prolonged survival of orthotopic xenograft-bearing mice. Collectively, these findings reveal the tumor-promoting functions of IFI35 and suggest that targeting IFI35 or its downstream effectors may provide effective approaches to improve GBM treatment.

Reactivating PTEN to impair glioma stem cells by inhibiting cytosolic iron-sulfur assembly.

Glioblastoma, the most lethal primary brain tumor, harbors glioma stem cells (GSCs) that not only initiate and maintain malignant phenotypes but also enhance therapeutic resistance. Although frequently mutated in glioblastomas, the function and regulation of PTEN in PTEN-intact GSCs are unknown. Here, we found that PTEN directly interacted with MMS19 and competitively disrupted MMS19-based cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) machinery in differentiated glioma cells. PTEN was specifically succinated at cysteine (C) 211 in GSCs compared with matched differentiated glioma cells. Isotope tracing coupled with mass spectrometry analysis confirmed that fumarate, generated by adenylosuccinate lyase (ADSL) in the de novo purine synthesis pathway that is highly activated in GSCs, promoted PTEN C211 succination. This modification abrogated the interaction between PTEN and MMS19, reactivating the CIA machinery pathway in GSCs. Functionally, inhibiting PTEN C211 succination by reexpressing a PTEN C211S mutant, depleting ADSL by shRNAs, or consuming fumarate by the US Food and Drug Administration-approved prescription drug N-acetylcysteine (NAC) impaired GSC maintenance. Reexpressing PTEN C211S or treating with NAC sensitized GSC-derived brain tumors to temozolomide and irradiation, the standard-of-care treatments for patients with glioblastoma, by slowing CIA machinery-mediated DNA damage repair. These findings reveal an immediately practicable strategy to target GSCs to treat glioblastoma by combination therapy with repurposed NAC.

RBBP6 maintains glioblastoma stem cells through CPSF3-dependent alternative polyadenylation.

Glioblastoma is one of the most lethal malignant cancers, displaying striking intratumor heterogeneity, with glioblastoma stem cells (GSCs) contributing to tumorigenesis and therapeutic resistance. Pharmacologic modulators of ubiquitin ligases and deubiquitinases are under development for cancer and other diseases. Here, we performed parallel in vitro and in vivo CRISPR/Cas9 knockout screens targeting human ubiquitin E3 ligases and deubiquitinases, revealing the E3 ligase RBBP6 as an essential factor for GSC maintenance. Targeting RBBP6 inhibited GSC proliferation and tumor initiation. Mechanistically, RBBP6 mediated K63-linked ubiquitination of Cleavage and Polyadenylation Specific Factor 3 (CPSF3), which stabilized CPSF3 to regulate alternative polyadenylation events. RBBP6 depletion induced shortening of the 3'UTRs of MYC competing-endogenous RNAs to release miR-590-3p from shortened UTRs, thereby decreasing MYC expression. Targeting CPSF3 with a small molecular inhibitor (JTE-607) reduces GSC viability and inhibits in vivo tumor growth. Collectively, RBBP6 maintains high MYC expression in GSCs through regulation of CPSF3-dependent alternative polyadenylation, providing a potential therapeutic paradigm for glioblastoma.

Threonine fuels glioblastoma through YRDC-mediated codon-biased translational reprogramming.

Cancers commonly reprogram translation and metabolism, but little is known about how these two features coordinate in cancer stem cells. Here we show that glioblastoma stem cells (GSCs) display elevated protein translation. To dissect underlying mechanisms, we performed a CRISPR screen and identified YRDC as the top essential transfer RNA (tRNA) modification enzyme in GSCs. YRDC catalyzes the formation of N6-threonylcarbamoyladenosine (t6A) on ANN-decoding tRNA species (A denotes adenosine, and N denotes any nucleotide). Targeting YRDC reduced t6A formation, suppressed global translation and inhibited tumor growth both in vitro and in vivo. Threonine is an essential substrate of YRDC. Threonine accumulated in GSCs, which facilitated t6A formation through YRDC and shifted the proteome to support mitosis-related genes with ANN codon bias. Dietary threonine restriction (TR) reduced tumor t6A formation, slowed xenograft growth and augmented anti-tumor efficacy of chemotherapy and anti-mitotic therapy, providing a molecular basis for a dietary intervention in cancer treatment.

Lymphatic endothelial-like cells promote glioblastoma stem cell growth through cytokine-driven cholesterol metabolism.

Glioblastoma is the most lethal primary brain tumor with glioblastoma stem cells (GSCs) atop a cellular hierarchy. GSCs often reside in a perivascular niche, where they receive maintenance cues from endothelial cells, but the role of heterogeneous endothelial cell populations remains unresolved. Here, we show that lymphatic endothelial-like cells (LECs), while previously unrecognized in brain parenchyma, are present in glioblastomas and promote growth of CCR7-positive GSCs through CCL21 secretion. Disruption of CCL21-CCR7 paracrine communication between LECs and GSCs inhibited GSC proliferation and growth. LEC-derived CCL21 induced KAT5-mediated acetylation of HMGCS1 on K273 in GSCs to enhance HMGCS1 protein stability. HMGCS1 promoted cholesterol synthesis in GSCs, favorable for tumor growth. Expression of the CCL21-CCR7 axis correlated with KAT5 expression and HMGCS1K273 acetylation in glioblastoma specimens, informing patient outcome. Collectively, glioblastomas contain previously unrecognized LECs that promote the molecular crosstalk between endothelial and tumor cells, offering potentially alternative therapeutic strategies.

Transgelin Promotes Glioblastoma Stem Cell Hypoxic Responses and Maintenance Through p53 Acetylation.

Glioblastoma (GBM) is a lethal cancer characterized by hypervascularity and necrosis associated with hypoxia. Here, it is found that hypoxia preferentially induces the actin-binding protein, Transgelin (TAGLN), in GBM stem cells (GSCs). Mechanistically, TAGLN regulates HIF1α transcription and stabilizes HDAC2 to deacetylate p53 and maintain GSC self-renewal. To translate these findings into preclinical therapeutic paradigm, it is found that sodium valproate (VPA) is a specific inhibitor of TAGLN/HDAC2 function, with augmented efficacy when combined with natural borneol (NB) in vivo. Thus, TAGLN promotes cancer stem cell survival in hypoxia and informs a novel therapeutic paradigm.

Gα12 signaling regulates transcriptional and phenotypic responses that promote glioblastoma tumor invasion.

In silico interrogation of glioblastoma (GBM) in The Cancer Genome Atlas (TCGA) revealed upregulation of GNA12 (Gα12), encoding the alpha subunit of the heterotrimeric G-protein G12, concomitant with overexpression of multiple G-protein coupled receptors (GPCRs) that signal through Gα12. Glioma stem cell lines from patient-derived xenografts also showed elevated levels of Gα12. Knockdown (KD) of Gα12 was carried out in two different human GBM stem cell (GSC) lines. Tumors generated in vivo by orthotopic injection of Gα12KD GSC cells showed reduced invasiveness, without apparent changes in tumor size or survival relative to control GSC tumor-bearing mice. Transcriptional profiling of GSC-23 cell tumors revealed significant differences between WT and Gα12KD tumors including reduced expression of genes associated with the extracellular matrix, as well as decreased expression of stem cell genes and increased expression of several proneural genes. Thrombospondin-1 (THBS1), one of the genes most repressed by Gα12 knockdown, was shown to be required for Gα12-mediated cell migration in vitro and for in vivo tumor invasion. Chemogenetic activation of GSC-23 cells harboring a Gα12-coupled DREADD also increased THBS1 expression and in vitro invasion. Collectively, our findings implicate Gα12 signaling in regulation of transcriptional reprogramming that promotes invasiveness, highlighting this as a potential signaling node for therapeutic intervention.

Circular extrachromosomal DNA promotes tumor heterogeneity in high-risk medulloblastoma.

Circular extrachromosomal DNA (ecDNA) in patient tumors is an important driver of oncogenic gene expression, evolution of drug resistance and poor patient outcomes. Applying computational methods for the detection and reconstruction of ecDNA across a retrospective cohort of 481 medulloblastoma tumors from 465 patients, we identify circular ecDNA in 82 patients (18%). Patients with ecDNA-positive medulloblastoma were more than twice as likely to relapse and three times as likely to die within 5 years of diagnosis. A subset of tumors harbored multiple ecDNA lineages, each containing distinct amplified oncogenes. Multimodal sequencing, imaging and CRISPR inhibition experiments in medulloblastoma models reveal intratumoral heterogeneity of ecDNA copy number per cell and frequent putative 'enhancer rewiring' events on ecDNA. This study reveals the frequency and diversity of ecDNA in medulloblastoma, stratified into molecular subgroups, and suggests copy number heterogeneity and enhancer rewiring as oncogenic features of ecDNA.

LncRNA INHEG promotes glioma stem cell maintenance and tumorigenicity through regulating rRNA 2'-O-methylation.

Glioblastoma (GBM) ranks among the most lethal of human cancers, containing glioma stem cells (GSCs) that display therapeutic resistance. Here, we report that the lncRNA INHEG is highly expressed in GSCs compared to differentiated glioma cells (DGCs) and promotes GSC self-renewal and tumorigenicity through control of rRNA 2'-O-methylation. INHEG induces the interaction between SUMO2 E3 ligase TAF15 and NOP58, a core component of snoRNP that guides rRNA methylation, to regulate NOP58 sumoylation and accelerate the C/D box snoRNP assembly. INHEG activation enhances rRNA 2'-O-methylation, thereby increasing the expression of oncogenic proteins including EGFR, IGF1R, CDK6 and PDGFRB in glioma cells. Taken together, this study identifies a lncRNA that connects snoRNP-guided rRNA 2'-O-methylation to upregulated protein translation in GSCs, supporting an axis for potential therapeutic targeting of gliomas.

EGFR promotes ALKBH5 nuclear retention to attenuate N6-methyladenosine and protect against ferroptosis in glioblastoma.

Growth factor receptors rank among the most important oncogenic pathways, but pharmacologic inhibitors often demonstrate limited benefit as monotherapy. Here, we show that epidermal growth factor receptor (EGFR) signaling repressed N6-methyladenosine (m6A) levels in glioblastoma stem cells (GSCs), whereas genetic or pharmacologic EGFR targeting elevated m6A levels. Activated EGFR induced non-receptor tyrosine kinase SRC to phosphorylate the m6A demethylase, AlkB homolog 5 (ALKBH5), thereby inhibiting chromosomal maintenance 1 (CRM1)-mediated nuclear export of ALKBH5 to permit sustained mRNA m6A demethylation in the nucleus. ALKBH5 critically regulated ferroptosis through m6A modulation and YTH N6-methyladenosine RNA binding protein (YTHDF2)-mediated decay of the glutamate-cysteine ligase modifier subunit (GCLM). Pharmacologic targeting of ALKBH5 augmented the anti-tumor efficacy of EGFR and GCLM inhibitors, supporting an EGFR-ALKBH5-GCLM oncogenic axis. Collectively, EGFR reprograms the epitranscriptomic landscape through nuclear retention of the ALKBH5 demethylase to protect against ferroptosis, offering therapeutic paradigms for the treatment of lethal cancers.

Human plasma-like medium facilitates metabolic tracing and enables upregulation of immune signaling pathways in glioblastoma explants.

Purpose: Metabolism within the tumor microenvironment (TME) represents an increasing area of interest to understand glioma initiation and progression. Stable isotope tracing is a technique critical to the study of tumor metabolism. Cell culture models of this disease are not routinely cultured under physiologically relevant nutrient conditions and do not retain cellular heterogeneity present in the parental TME. Moreover, in vivo, stable isotope tracing in intracranial glioma xenografts, the gold standard for metabolic investigation, is time consuming and technically challenging. To provide insights into glioma metabolism in the presence of an intact TME, we performed stable isotope tracing analysis of patient-derived, heterocellular Surgically eXplanted Organoid (SXO) glioma models in human plasma-like medium (HPLM). Methods: Glioma SXOs were established and cultured in conventional media or transitioned to HPLM. We evaluated SXO cytoarchitecture and histology, then performed spatial transcriptomic profiling to identify cellular populations and differential gene expression patterns. We performed stable isotope tracing with 15N2-glutamine to evaluate intracellular metabolite labeling patterns. Results: Glioma SXOs cultured in HPLM retain cytoarchitecture and cellular constituents. Immune cells in HPLM-cultured SXOs demonstrated increased transcription of immune-related signatures, including innate immune, adaptive immune, and cytokine signaling programs. 15N isotope enrichment from glutamine was observed in metabolites from diverse pathways, and labeling patterns were stable over time. Conclusion: To enable ex vivo, tractable investigations of whole tumor metabolism, we developed an approach to conduct stable isotope tracing in glioma SXOs cultured under physiologically relevant nutrient conditions. Under these conditions, SXOs maintained viability, composition, and metabolic activity while exhibiting increased immune-related transcriptional programs.

Dual Role of CXCL8 in Maintaining the Mesenchymal State of Glioblastoma Stem Cells and M2-Like Tumor-Associated Macrophages.

PURPOSE: The dynamic interplay between glioblastoma stem cells (GSC) and tumor-associated macrophages (TAM) sculpts the tumor immune microenvironment (TIME) and promotes malignant progression of glioblastoma (GBM). However, the mechanisms underlying this interaction are still incompletely understood. Here, we investigate the role of CXCL8 in the maintenance of the mesenchymal state of GSC populations and reprogramming the TIME to an immunosuppressive state. EXPERIMENTAL DESIGN: We performed an integrative multi-omics analyses of RNA sequencing, GBM mRNA expression datasets, immune signatures, and epigenetic profiling to define the specific genes expressed in the mesenchymal GSC subsets. We then used patient-derived GSCs and a xenograft murine model to investigate the mechanisms of tumor-intrinsic and extrinsic factor to maintain the mesenchymal state of GSCs and induce TAM polarization. RESULTS: We identified that CXCL8 was preferentially expressed and secreted by mesenchymal GSCs and activated PI3K/AKT and NF-κB signaling to maintain GSC proliferation, survival, and self-renewal through a cell-intrinsic mechanism. CXCL8 induced signaling through a CXCR2-JAK2/STAT3 axis in TAMs, which supported an M2-like TAM phenotype through a paracrine, cell-extrinsic pathway. Genetic- and small molecule-based inhibition of these dual complementary signaling cascades in GSCs and TAMs suppressed GBM tumor growth and prolonged survival of orthotopic xenograft-bearing mice. CONCLUSIONS: CXCL8 plays critical roles in maintaining the mesenchymal state of GSCs and M2-like TAM polarization in GBM, highlighting an interplay between cell-autonomous and cell-extrinsic mechanisms. Targeting CXCL8 and its downstream effectors may effectively improve GBM treatment.

Glioblastoma Stem Cell Targeting Peptide Isolated Through Phage Display Binds Cadherin 2.

Glioblastoma stem cells (GSCs) have unique properties of self-renewal and tumor initiation that make them potential therapeutic targets. Development of effective therapeutic strategies against GSCs requires both specificity of targeting and intracranial penetration through the blood-brain barrier. We have previously demonstrated the use of in vitro and in vivo phage display biopanning strategies to isolate glioblastoma targeting peptides. Here we selected a 7-amino acid peptide, AWEFYFP, which was independently isolated in both the in vitro and in vivo screens and demonstrated that it was able to target GSCs over differentiated glioma cells and non-neoplastic brain cells. When conjugated to Cyanine 5.5 and intravenously injected into mice with intracranially xenografted glioblastoma, the peptide localized to the site of the tumor, demonstrating intracranial tumor targeting specificity. Immunoprecipitation of the peptide with GSC proteins revealed Cadherin 2 as the glioblastoma cell surface receptor targeted by the peptides. Peptide targeting of Cadherin 2 on GSCs was confirmed through ELISA and in vitro binding analysis. Interrogation of glioblastoma databases demonstrated that Cadherin 2 expression correlated with tumor grade and survival. These results confirm that phage display can be used to isolate unique tumor-targeting peptides specific for glioblastoma. Furthermore, analysis of these cell specific peptides can lead to the discovery of cell specific receptor targets that may serve as the focus of future theragnostic tumor-homing modalities for the development of precision strategies for the treatment and diagnosis of glioblastomas.

Watching the clock in glioblastoma.

Glioblastoma (GBM) is the most prevalent malignant primary brain tumor, accounting for 14.2% of all diagnosed tumors and 50.1% of all malignant tumors, and the median survival time is approximately 8 months irrespective of whether a patient receives treatment without significant improvement despite expansive research (Ostrom QT, Price M, Neff C, et al. CBTRUS statistical report: primary brain and other central nervous system tumors diagnosed in the United States in 2015-2019. Neurooncology. 2022; 24(suppl 5):v1-v95.). Recently, important roles for the circadian clock in GBM tumorigenesis have been reported. Positive regulators of circadian-controlled transcription, brain and muscle ARNT-like 1 (BMAL1), and circadian locomotor output cycles kaput (CLOCK), are highly expressed also in GBM and correlated with poor patient prognosis. BMAL1 and CLOCK promote the maintenance of GBM stem cells (GSCs) and the establishment of a pro-tumorigenic tumor microenvironment (TME), suggesting that targeting the core clock proteins may augment GBM treatment. Here, we review findings that highlight the critical role the circadian clock plays in GBM biology and the strategies by which the circadian clock can be leveraged for GBM treatment in the clinic moving forward.

Lysine catabolism reprograms tumour immunity through histone crotonylation.

Cancer cells rewire metabolism to favour the generation of specialized metabolites that support tumour growth and reshape the tumour microenvironment1,2. Lysine functions as a biosynthetic molecule, energy source and antioxidant3-5, but little is known about its pathological role in cancer. Here we show that glioblastoma stem cells (GSCs) reprogram lysine catabolism through the upregulation of lysine transporter SLC7A2 and crotonyl-coenzyme A (crotonyl-CoA)-producing enzyme glutaryl-CoA dehydrogenase (GCDH) with downregulation of the crotonyl-CoA hydratase enoyl-CoA hydratase short chain 1 (ECHS1), leading to accumulation of intracellular crotonyl-CoA and histone H4 lysine crotonylation. A reduction in histone lysine crotonylation by either genetic manipulation or lysine restriction impaired tumour growth. In the nucleus, GCDH interacts with the crotonyltransferase CBP to promote histone lysine crotonylation. Loss of histone lysine crotonylation promotes immunogenic cytosolic double-stranded RNA (dsRNA) and dsDNA generation through enhanced H3K27ac, which stimulates the RNA sensor MDA5 and DNA sensor cyclic GMP-AMP synthase (cGAS) to boost type I interferon signalling, leading to compromised GSC tumorigenic potential and elevated CD8+ T cell infiltration. A lysine-restricted diet synergized with MYC inhibition or anti-PD-1 therapy to slow tumour growth. Collectively, GSCs co-opt lysine uptake and degradation to shunt the production of crotonyl-CoA, remodelling the chromatin landscape to evade interferon-induced intrinsic effects on GSC maintenance and extrinsic effects on immune response.

Sterol regulatory element-binding protein 2 maintains glioblastoma stem cells by keeping the balance between cholesterol biosynthesis and uptake.

BACKGROUND: Glioblastomas (GBMs) display striking dysregulation of metabolism to promote tumor growth. Glioblastoma stem cells (GSCs) adapt to regions of heterogeneous nutrient availability, yet display dependency on de novo cholesterol biosynthesis. The transcription factor Sterol Regulatory Element-Binding Protein 2 (SREBP2) regulates cholesterol biosynthesis enzymes and uptake receptors. Here, we investigate adaptive behavior of GSCs under different cholesterol supplies. METHODS: In silico analysis of patient tumors demonstrated enrichment of cholesterol synthesis associated with decreased angiogenesis. Comparative gene expression of cholesterol biosynthesis enzymes in paired GBM specimens and GSCs were performed. In vitro and in vivo loss-of-function genetic and pharmacologic assays were conducted to evaluate the effect of SREBP2 on GBM cholesterol biosynthesis, proliferation, and self-renewal. Chromatin immunoprecipitation quantitative real-time PCR was leveraged to map the regulation of SREBP2 to cholesterol biosynthesis enzymes and uptake receptors in GSCs. RESULTS: Cholesterol biosynthetic enzymes were expressed at higher levels in GBM tumor cores than in invasive margins. SREBP2 promoted cholesterol biosynthesis in GSCs, especially under starvation, as well as proliferation, self-renewal, and tumor growth. SREBP2 governed the balance between cholesterol biosynthesis and uptake in different nutrient conditions. CONCLUSIONS: SREBP2 displays context-specific regulation of cholesterol biology based on its availability in the microenvironment with induction of cholesterol biosynthesis in the tumor core and uptake in the margin, informing a novel treatment strategy for GBM.

A neurodevelopmental epigenetic programme mediated by SMARCD3-DAB1-Reelin signalling is hijacked to promote medulloblastoma metastasis.

How abnormal neurodevelopment relates to the tumour aggressiveness of medulloblastoma (MB), the most common type of embryonal tumour, remains elusive. Here we uncover a neurodevelopmental epigenomic programme that is hijacked to induce MB metastatic dissemination. Unsupervised analyses of integrated publicly available datasets with our newly generated data reveal that SMARCD3 (also known as BAF60C) regulates Disabled 1 (DAB1)-mediated Reelin signalling in Purkinje cell migration and MB metastasis by orchestrating cis-regulatory elements at the DAB1 locus. We further identify that a core set of transcription factors, enhancer of zeste homologue 2 (EZH2) and nuclear factor I X (NFIX), coordinates with the cis-regulatory elements at the SMARCD3 locus to form a chromatin hub to control SMARCD3 expression in the developing cerebellum and in metastatic MB. Increased SMARCD3 expression activates Reelin-DAB1-mediated Src kinase signalling, which results in a MB response to Src inhibition. These data deepen our understanding of how neurodevelopmental programming influences disease progression and provide a potential therapeutic option for patients with MB.

Sorting nexin 10 sustains PDGF receptor signaling in glioblastoma stem cells via endosomal protein sorting.

Glioblastoma is the most malignant primary brain tumor, the prognosis of which remains dismal even with aggressive surgical, medical, and radiation therapies. Glioblastoma stem cells (GSCs) promote therapeutic resistance and cellular heterogeneity due to their self-renewal properties and capacity for plasticity. To understand the molecular processes essential for maintaining GSCs, we performed an integrative analysis comparing active enhancer landscapes, transcriptional profiles, and functional genomics profiles of GSCs and non-neoplastic neural stem cells (NSCs). We identified sorting nexin 10 (SNX10), an endosomal protein sorting factor, as selectively expressed in GSCs compared with NSCs and essential for GSC survival. Targeting SNX10 impaired GSC viability and proliferation, induced apoptosis, and reduced self-renewal capacity. Mechanistically, GSCs utilized endosomal protein sorting to promote platelet-derived growth factor receptor ß (PDGFRß) proliferative and stem cell signaling pathways through posttranscriptional regulation of the PDGFR tyrosine kinase. Targeting SNX10 expression extended survival of orthotopic xenograft-bearing mice, and high SNX10 expression correlated with poor glioblastoma patient prognosis, suggesting its potential clinical importance. Thus, our study reveals an essential connection between endosomal protein sorting and oncogenic receptor tyrosine kinase signaling and suggests that targeting endosomal sorting may represent a promising therapeutic approach for glioblastoma treatment.

WDR5 represents a therapeutically exploitable target for cancer stem cells in glioblastoma.

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors driven by populations of cancer stem cells (CSCs). However, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy-resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1 family-mediated Lys4 methylation of histone H3 (H3K4me), associated with positive regulation of transcription. In GBM CSCs, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. Use of a SOX2/OCT4 reporter demonstrated that WDR5 inhibitor treatment diminished cells with high reporter activity. Furthermore, WDR5 inhibitor treatment and WDR5 knockdown altered the stem cell state, disrupting CSC in vitro growth and self-renewal, as well as in vivo tumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers.