Abstract Glioblastoma (GBM) profoundly alters neural circuit dynamics, yet most studies rely on static endpoint measurements that overlook how network dysfunction evolves over time. We address this gap through a longitudinal, multimodal framework that combines large-scale imaging, electrophysiology, behavior, computational modeling, and circuit perturbation to define dynamic biomarkers of GBM progression and restoration. We hypothesize that glioblastoma induces progressive and sex-dependent disruption of inhibitory network balance that weakens long-range cortical communication, and that restoring inhibitory tone can recover both neural synchrony and behavior. Using mesoscale cortical calcium imaging, depth-resolved Neuropixel recordings, and single- and multiphoton microscopy with multiple calcium indicators, we captured cortical and subcortical activity in syngeneic (SB28, KR158) and genetically engineered (GEMM) GBM models. Progressive circuit remodeling emerged as accelerated and directionally biased traveling waves, disrupted inter- and intrahemispheric coupling, and layer-persistent hyperexcitability characterized by delta-band elevation near the tumor core and high-frequency suppression in surrounding regions. Recurrent spatiotemporal motifs revealed evolving network patterns in both hemispheres, paralleling tumor infiltration observed in histology and highlighting widespread reorganization of cortical communication networks. To link these neural changes with functional outcomes, we performed simultaneous pupil and orofacial tracking during spontaneous activity, virtual-reality navigation, and social-interaction paradigms. Our deep learning models (DeepVision and DeepFace) extracted high-resolution behavioral features, while generalized linear models (GLMs) predicted cortical activity from these signals. GBM progression reduced the behavioral predictability of brain dynamics, reflecting degraded sensorimotor coupling and impaired state-dependent coordination across cortical regions. These findings establish behavioral and physiological signatures that mirror neural instability and can serve as scalable, noninvasive biomarkers for longitudinal disease monitoring. Finally, optogenetic activation of inhibitory neurons restored cortical synchrony, normalized oscillatory patterns, and improved behavioral performance. Remarkably, this intervention doubled the survival rate of female mice, revealing a sex-dependent therapeutic benefit likely mediated by hormonal modulation of inhibitory tone and circuit resilience. Together, this integrative platform—spanning cellular to systems scales, spontaneous to social behaviors, and physiological to optogenetic domains—provides a comprehensive view of how glioblastoma disrupts, and how targeted circuit modulation can restore, brain function. By bridging optical, electrophysiological, behavioral, and computational modalities, this work identifies quantitative and translatable network biomarkers for early detection, mechanism-guided intervention, and personalized therapy development, establishing a foundation for precision cancer neuroscience. Citation Format: Murat Yildirim, Tenesha Connor, Maryam Faisal, Omer Dinc, Kemal Ozdemirli, Frederick Bell, Berfin Dinc, Miguel Maldonado, Daniel Silver, Anthony Sloan, Justin Lathia. Dynamic circuit remodeling during glioblastoma progression: Depth-resolved electrophysiology and cortical imaging define network biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB001.

Gaseous hydrogen sulfide (H2S), a by-product of cysteine metabolism, inhibits cancer cell behavior and growth in glioblastoma (GBM) models. H2S production capacity (HPC) is decreased in human GBM specimens compared to non-tumor controls. Thus, boosting HPC is a novel strategy for GBM treatment. Suppression of thyroid hormone signaling increases production of H2S. Hypothesis: methimazole-induced hypothyroidism will increase tumor HPC to enhance chemotherapy efficacy in GBM. Proof of concept that methimazole, by reducing thyroid hormone signaling, can increase HPC in patients with recurrent/progressive GBM (rGBM). 10% increase in HPC and a 1.5-fold increase in peripheral blood sulfhydration signaling. This window of opportunity trial evaluates safety, HPC in plasma and tumor, and efficacy of methimazole + chemotherapy in patients with rGBM. rGBM patients planned for a resection begin methimazole 15 mg/d 5-7 days pre-op with investigator’s choice chemotherapy added 10-28 days post-op. Patients receive combination until progression. Pre- and post-op plasma and resected tumor tissue are assayed for HPC and proteins relevant to H2S production. Key eligibility: rGBM for whom a clinically-indicated resection is planned; normal thyroid function with no history of thyroid dysfunction. Patients may have received unlimited prior regimens including bevacizumab. To date 15 patients (8 male), median age 56 years, enrolled. The trial was amended to increase methimazole to 25 mg/d; 3 patients have received this dose. Median TSH rose from 1.3 mU/ml to 20.5 at cycle 6. Free T3 and T4 did not fall significantly. Median plasma HPC rose 3-fold while tumor HPC did not rise significantly. No treatment-related grade 3-5 toxicities. Efficacy: PFS6 – 15% and OS12 – 21%. Induction of subclinical hypothyroidism is feasible and safe in rGBM with significant increases in plasma but not tumor HPC. Further tumor correlative data for the dose-escalated methimazole will be presented.

Glioblastoma (GBM) is the most common malignant primary brain tumor. The tumor microenvironment (TME) has been implicated in the aggressiveness and resistance to chemotherapy. Myeloid cells, consisting of resident microglia and infiltrating macrophages, comprise the majority of the TME and engage in crosstalk with GBM cells, with GBM cells promoting a shift of these cells to an anti-inflammatory phenotype. These pro-tumor myeloid cells promote tumor cell invasion and proliferation and have been associated with decreased survival in humans. PLX3397, Pexidartinib, is a CSF1r inhibitor previously used in clinical trials that has shown efficacy in depleting microglia within the brain. Thus, while many studies have established the pro-tumor polarization of microglia in GBM, a timeline for this shift has not yet been established. Syngeneic GBM models (SB28) were implanted into 6–12-week-old C57Bl/6 male and female mice. PLX3397 was used to deplete microglia, and three timepoints of depletion were investigated: -5, +5, and +10 days post-implantation. Mice were kept on PLX3397 or control chow until neurologic endpoint was reached. Microglia depletion 5 days before implantation of GBM cells decreased survival in both males and females (p<0.001) in a sex-independent manner (median survival 13 versus 19 days). Microglia depletion beginning at 5 days post-implantation also resulted in a decreased survival (p = 0.03), however, at a decreased magnitude (median survival 16 versus 17.5 days). Microglia depletion at 10 days after implantation demonstrated increased survival (p=0.047) compared to control mice (median 16 versus 13 days). Microglia depletion with PLX3397 demonstrates a temporal effect on survival in a murine model of GBM. These results demonstrate the potential time-dependent role of microglia in the acute phase of GBM implantation and provide insight into the evolving phenotype across time.

Metabolic reprogramming enables glioblastoma cells to thrive under adverse conditions by utilizing available fuel sources. While previously explored for its tumor suppressive activities in glioblastoma, hydrogen sulfide (H2S), a gaseous signaling molecule can both inhibit the electron transport chain at complex IV and act as an electron donor by reducing coenzyme Q. The contradictory nature of H2S depends upon the ability of cells to increase sulfide oxidation. Mitochondrial sulfide oxidation eliminates H2S through a series of redox reactions, beginning with two key enzymes, sulfide quinone oxidoreductase (SQOR) and persulfide dioxygenase (ETHE1). Through examination of patient tumor transcriptional data, we identified SQOR and ETHE1 to inversely correlate with patient prognosis, representing a potential driver of glioblastoma progression. Using patient derived glioblastoma cells, we observed increased sulfide oxidation capabilities in response to exogenous H2S. As sulfide oxidation increased in response to exogenous H2S, we examined how manipulating the rate limiting step catalyzed by SQOR impacts tumor growth in vivo. Knockdown of SQOR in murine glioblastoma models led to a robust survival extension. Corresponding in vitro experiments performed with genetic or pharmacological perturbation of SQOR in human glioblastoma cells showed increased intracellular H2S accumulation measured by flow cytometry. The increase in H2S accumulation, lead to decreased self-renewal and proliferation. These observations were driven by a decrease in mitochondrial respiration and ATP production measured via Seahorse assay, with an increased reliance upon glycolytic activity, and an inability to mobilize lipid reserves. Conversely, SQOR overexpression produced the opposite result in all experimental systems. These findings demonstrate how amplification of sulfide oxidation enables glioblastoma cells to shunt H2S into mitochondrial respiration. Targeting sulfide oxidation therefore presents as an intriguing means of disrupting oxidative phosphorylation and suppressing lipid metabolism in highly adaptive glioblastoma stem cells which can be combined with glycolysis inhibitors to suppress tumor growth.

Sex differences in cancer outcomes, including glioblastoma (GBM), are shaped by biological, hormonal, and immune factors, influencing disease progression, treatment responses, survival, and the tumor microenvironment (TME). Platelets, as key regulators of immune responses and tumor progression, may contribute to these sex-based differences by influencing the dynamics of the TME, however, the precise molecular mechanisms remain unclear. Here, we show that GBM patients exhibit heightened platelet reactivity driven by protease-activated receptor 4 (PAR4) signaling. In murine GBM models, targeting PAR4 with BMS986120 prolongs survival in females but not males. This survival advantage is estrogen-dependent and TME-specific, driven by enhanced CD8+ T cell infiltration within the tumor. Inhibiting platelet PAR4 signaling decreases platelet alpha-granule secretion in female tumor-bearing mice while enriching alternative exocytosis pathways, thereby influencing CD8+ T cell activity. PAR4-activated platelets within the TME suppress CD8+ T cell function and CD8+ T cell depletion eliminates the tumor induced platelet reactivity and survival benefit when PAR4 is inhibited. These findings establish platelet-mediated PAR4 signaling as a critical driver of tumor progression and identify sex-specific immune responses as key to therapeutic efficacy. ### Competing Interest Statement The authors have declared no competing interest. NIH

Glioblastoma (GBM) is the most common primary malignant brain tumor, and current therapies provide only palliation, not a cure. GBM is difficult to treat due to its complex, multi-faceted development involving various cell types in the tumor microenvironment (TME). Recent observations highlight sex differences in these interactions, influenced by biological, hormonal, and immune factors affecting disease progression, treatment responses, survival, and the TME. Platelets, key regulators of immune responses and tumor progression, may contribute to these sex-based differences by altering TME dynamics, though precise molecular mechanisms remain unclear. Our previous work showed that thrombin, a major platelet activator via protease-activated receptor 1 (PAR1) and protease-activated receptor 4 (PAR4), is secreted by cancer stem cells into the tumor microenvironment. We now demonstrate that GBM patients exhibit heightened platelet reactivity driven by PAR4 signaling. In murine GBM models, targeting PAR4 with BMS986120 and genetic inhibition of PAR4 prolong survival in females but not males. This survival advantage is estrogen-dependent and TME-specific, driven by enhanced CD8+ T cell infiltration regulated by an estrogen receptor response element on the PAR4 promoter. Inhibiting platelet PAR4 signaling decreases alpha-granule secretion in female tumor-bearing mice while enriching alternative exocytosis pathways, thereby influencing CD8+ T cell activity. PAR4-activated platelets within the TME suppress CD8+ T cell function, and CD8+ T cell depletion eliminates tumor-induced platelet reactivity and the survival benefit of PAR4 inhibition. These findings demonstrate how activated platelets interact and regulate immune cell populations in GBM. These results suggest that the hyper-thrombotic state seen in many GBM patients, which serves as a major risk of death, simultaneously contributes to the immunosuppressive TME. In addition, these results identify therapeutic strategies to leverage the platelet hyperactivity seen in GBM by hyperactive thrombin-PAR4 for sex-dependent therapeutic purposes.

2044 Background: Gaseous hydrogen sulfide (H2S), a by-product of cysteine metabolism, inhibits the growth of glioblastoma (GBM) cells and impairs GBM progression in mice. Likewise, H2S generation and sulfhydration are decreased in human GBM specimens as compared to non-tumor controls. Thus, boosting H2S production is a novel strategy for GBM treatment. Suppression of thyroid hormone (TH) signaling increases endogenous production of H2S. We hypothesize that methimazole-induced hypothyroidism will enhance the efficacy of chemotherapy in WHO grade 4 gliomas by boosting H2S production capacity (HPC) within the tumor. The goal of this trial is to provide proof of concept that suppression of TH signaling, via methimazole and subsequent augmentation of H2S synthesis and signaling, is feasible in patients with WHO grade 4 gliomas (G4G). Methods: This modified phase 1/2 study evaluates the safety and efficacy of methimazole + chemotherapy with pharmacodynamic correlates in patients with progressive G4G. The main objective is a 10% increase in HPC and a 1.5-fold increase in peripheral blood sulfhydration signaling (SS). Patients who are planned for a clinically-indicated resection receive pre-op (5-7 days) and post-op methimazole with the addition of investigator’s choice of chemotherapy 1 month after starting post op methimazole. Patients receive methimazole + chemotherapy until progression. Resected tumor will be assayed for HPC and for proteins relevant to H2S production. Peripheral blood lead-acetate assays for HPC and SS are obtained at baseline, pre-op, intra-op, post-op, before the addition of chemotherapy, and before each cycle of methimazole + chemo. Key eligibility criteria: progressive G4G for whom a clinically-indicated resection is planned, and normal thyroid function with no history of thyroid disease. Patients may have had unlimited prior regimens including bevacizumab. Results: To date six patients (4 male) ages 49-59 years have enrolled. At all time points tested post-methimazole treatment relative to baseline age/sex matched no-methimazole control patients, there were significant increases in plasma H2S production capacity (table). Conclusions: In this early cohort, it appears methimazole treatment in recurrent GBM patients enhances HPC on a systemic level. Next steps will be to measure sulfide signaling and sulfhydration alterations in patient tumor samples. The protocol is being modified to allow earlier addition (2 weeks) of post-op chemotherapy. Clinical trial information: NCT05607407 . [Table: see text]

Abstract Initial detection of glioblastoma (GBM) often reveals a large mass in the forebrain; however, the hazard for many patients lies in extensive invasion that can extend far from the primary tumor. New evidence suggests that end-stage GBM coincides with malignant infiltration of the pons and brainstem. Mechanisms that direct tumor cells from initial positions within the forebrain to terminal positions in the brainstem are largely unknown. Our analysis indicates that spatially defined, reactive astrocytes instruct GBM cells to either proliferate or invade. During development, astrocytes help establish directional gradients and boundaries that mediate proper routing of migrating neural cells and extending axons. We posit that onco-reactive astrocytes re-engage these guidance programs to direct tumor cells to the brainstem. To test this hypothesis, we exposed a series of human GBM models to media conditioned by cortical, midbrain, or brainstem reactive astrocytes. When treated with cortical conditioned media, tumor cells were driven into a state of hyper-proliferation, which mirrored early-stage forebrain tumor generation. In contrast, midbrain and brainstem conditioned medias suppressed proliferation and enhanced the rate of tumor cell migration. RNA sequencing corroborated these functional data, identifying cell division programs augmented in GBM cells exposed to cortical astrocyte conditioned media. This contrasted with axon development and guidance programs enriched in GBM cells exposed to midbrain and especially brainstem conditioned medias. Finally, mRNA and protein analysis identified region-specific engagement with the Eph-ephrin guidance system patterned along a forebrain to hindbrain trajectory. These data support a new hypothesis that GBM tumors recapitulate a form of neurodevelopment, invading specific anatomic regions based on cues generated by resident reactive astrocytes.

Abstract Tumors represent a highly dynamic system that relies on coordinated signaling to drive growth and adapt to selective pressures, including those induced by anti-cancer therapies. Given the need for tumors to engage in rapid and coordinated cell-cell communication, mechanisms such as gap junction intracellular communication (GJIC) should be essential. However, the connexin proteins that make up gap junctions have traditionally been considered tumor suppressors based on the frequent loss of GJIC and lower connexin expression in tumor cells compared to non-neoplastic tissue. For this reason, connexin 43 (Cx43) has been proposed to suppress the growth of glioblastoma (GBM), the most common primary malignant brain tumor. However, recent data suggest that this tumor-suppressive effect is context dependent and that connexins can also function in a tumor-promoting role. Using next-generation sequencing techniques, we found Cx43 expressed at high levels in a panel of GBM patient-derived xenograft (PDX) CSCs and that these models rely on Cx43 for their survival and self-renewal. Mechanistically, depletion of Cx43 led to a dramatic loss of c-MYC expression through reduced phosphorylation of its upstream mediator WNK lysine-deficient protein kinase 1 (WNK1). Depletion of WNK1 phenocopied knockdown of Cx43 and reduced MYC protein and mRNA as well as tumor growth in vivo. Together, this work defines a novel signaling axis downstream of gap junction protein expression that promotes tumor growth and cancer stem cell phenotypes in GBM. Due to the difficulty in targeting both Cx43 and MYC, the identification of intermediate targetable signaling nodes may lead to improved therapies for patients with GBM.

Abstract Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors that are driven by populations of cancer stem cells (CSCs). In this study, we perform an epigenetic-focused functional genomics screen in GBM organoids and identify WDR5 as an essential epigenetic regulator in the SOX2-enriched, therapy resistant cancer stem cell niche. Despite their importance for tumor growth, 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. Our niche-specific screens identified WDR5, an H3K4 histone methyltransferase responsible for activating specific gene expression, as indispensable for GBM CSC growth and survival. In GBM CSC models, 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, required for stem cell maintenance and including the POU5F1(OCT4)::SOX2 motif. We incorporated a SOX2/OCT4 motif driven GFP reporter system into our CSC cell models and found that WDR5 inhibitor treatment resulted in dose-dependent silencing of stem cell reporter activity. Further, WDR5 inhibitor treatment altered the stem cell state, disrupting CSC in vitro growth and self-renewal as well as in vivo tumor growth. Our results unveiled the role of WDR5 in maintaining the CSC state in GBM and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers. This conceptual and experimental framework can be applied to many cancers, and can unmask unique microenvironmental biology and rationally designed combination therapies.

Glioblastoma (GBM) is a prevalent, malignant glioma, and patient prognosis is poor. Even with surgery and aggressive treatment, survival is a dismal 12-15 months. GBM therapeutic resistance is common, often resulting in tumor recurrence, due in part to cancer stem cells (CSCs) that are resistant to chemo- and radio-therapies. This highlights the need for novel treatments, which requires a deeper understanding of the GBM tumor environment. While controversial, emerging data implicate human cytomegalovirus (CMV) in GBM progression, including findings that CMV drives GBM cell proliferation. Our work confirms CMV accelerates GBM cell proliferation in vitro using patient-derived xenograft (PDX) GBM models. Intracranial injection of these infected cells into murine models also results in decreased survival relative to mock-infected cells, suggesting CMV infection influences survival in vivo. Further, we found the CMV-encoded G-protein coupled receptor (GPCR), US28, influences this, as infection of PDX-derived GBM cells in vitro with US28-deletion virus impacts cell proliferation. In murine models, we find wild type infected PDX-derived GBM cells result in a significant decrease in survival compared to mice receiving cells infected with CMV lacking US28, suggesting US28 expression confers improved overall survival in vivo. Additionally, we find expression of US28 is associated with significantly increased survival for female mice, whereas there was no statistical difference between male mice that received CMV-infected or uninfected GBM cells. This indicates CMV infection, and more specifically, US28 expression, adversely impacts survival of female mice. Collectively, our data further illuminate means by which CMV influences the GBM tumor microenvironment and survival. We are currently working to understand: 1) the impact of US28-mediated signaling pathways on the tumor microenvironment; and 2) the influence of US28 interacting partners on GBM progression, including EphA2, a receptor tyrosine kinase whose increased expression in GBM confers increased CSC invasiveness.

Abstract Glioblastoma (GBM) interaction with neural cells is critical to its pathobiology. Emerging evidence suggests that GBM cells form an interconnected network with astrocytes, facilitating tumor persistence. Given reports of intercellular transfer of mitochondria in ischemic stroke and other pathologic disease states outside the CNS, we hypothesized that this network facilitates mitochondria transfer from astrocytes to GBM with protumorigenic sequelae. Employing transgenic mice and intracranial viral vector transductions in rats, we found that mitochondria transfer from the TME to GBM occurs in intracranial mouse and patient-derived xenograft models (in nude rats) of GBM. Mitochondria transfer from bone marrow-derived immune cells was minimal in bone marrow chimera mouse models of orthotopic GBM, suggesting that neural cells were the primary mitochondria donors. We confirmed this in vitro, where mouse astrocytes were the major mitochondria donors, followed by microglia and to a much smaller extent bone marrow-derived macrophages. Immortalized human astrocytes transduced with mitochondria-localized mCherry (mito-mCherry) also transferred their mitochondria to numerous patient-derived glioma stem cell (GSC) models at rates of ~5-20%, assessed by flow cytometry and confocal microscopy. Mitochondria were visualized along intercellular actin bridges, structurally resembling tumor microtubes. Blocking actin polymerization or knocking down GAP43 (previously linked to microtube formation) decreased mitochondria transfer from astrocytes to GBM in vitro. Functionally, sorted mito-mCherry+ patient-derived GSCs displayed higher mitochondrial respiration, metabolomic reprogramming and proliferation-promoting phospho-signaling. Mito-mCherry+ GBM cells were more likely to be in the proliferative G2/M phases of the cell cycle, and when sorted from co-cultures had high self-renewal (in vitro) and tumor-initiating capacity (in vivo xenograft mouse model). In ongoing work, we are investigating the role of retrograde GBM to astrocyte transfer of mitochondria by dual-color labeling of the organelle, as well as further delineating the protein machinery involved in this fundamental protumorigenic process, with the goal of identifying novel therapeutic targets.

BackgroundGlioblastoma (GBM) is the most common primary malignant brain tumor and shows poor outcomes, with a median survival of 12–18 months using the current standard-of-care therapy. GBM exhibits sex differences in incidence and overall survival, with males experiencing a higher incidence and worse prognosis compared to females. Emerging evidence suggests that these differences extend to genetic/epigenetic and cellular levels, including immune responses. However, the mechanisms driving immunological sex differences are not fully understood.MethodsTo investigate the underlying mechanisms of sex differences in GBM, we used orthotopic GBM mouse models. Male and female mice received intracranial injections of murine syngeneic GBM cells (SB28 and GL261) and underwent survival analysis or immune cell profiling. To interrogate the immune cell-intrinsic and -extrinsic mechanisms, bone marrow chimeras were generated by reconstituting immune system of male or female recipient mice with male or female donor bone marrow cells. Additionally, adoptive transfer of T cells to tumor-bearing mice was performed in a sex-matched or mismatched manner.ResultsSex differences in survival were recapitulated in immune-competent B6 mice, but not in immune-deficient NSG or RAG1KO mice. By depleting CD8+ T cells, we further confirmed that CD8+ T cells play a critical role in driving sex differences in survival. Flow cytometry analysis revealed that male CD8+ T cells from tumors expressed higher levels of exhaustion markers such as PD-1, CTLA-4, and Tox, with a higher frequency of progenitor exhausted T cell subsets, whereas female tumors contained more effector-like CD8+ T cells expressing elevated cytokine production. Treatment with anti-PD-1 antibodies exclusively extended the survival of male mice by inducing decreased exhausted CD8+ T cell subsets and increased effector function and proliferation. Survival analysis using the bone marrow chimera model and adoptive transfer model indicated that T cell-mediated tumor control was predominantly regulated in a cell-intrinsic manner, as the sex of donor cells was a critical contributing factor. Furthermore, we confirmed these observations in GBM patient tumor samples, with male tumors having a higher frequency of progenitor exhausted T cells with increased TOX expression. Lastly, we found that an X chromosome inactivation escape gene, Kdm6a, is associated with sex-biased T cell exhaustion.ConclusionsTaken together, these findings demonstrate sex-biased pre-determined behavior of T cells is critical in inducing sex differences in GBM progression and immunotherapy responses (figure 1).

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.