Divergent transcriptomic signatures from putative mesenchymal stimuli in glioblastoma cells.

In glioblastoma, a mesenchymal phenotype is associated with especially poor patient outcomes. Various glioblastoma microenvironmental factors and therapeutic interventions are purported drivers of the mesenchymal transition, but the degree to which these cues promote the same mesenchymal transitions and the uniformity of those transitions, as defined by molecular subtyping systems, is unknown. Here, we investigate this question by analyzing publicly available patient data, surveying commonly measured transcripts for mesenchymal transitions in glioma-initiating cells (GIC), and performing next-generation RNA sequencing of GICs. Analysis of patient tumor data reveals that TGFß, TNFα, and hypoxia signaling correlate with the mesenchymal subtype more than the proneural subtype. In cultured GICs, the microenvironment-relevant growth factors TGFß and TNFα and the chemotherapeutic temozolomide promote expression of commonly measured mesenchymal transcripts. However, next-generation RNA sequencing reveals that growth factors and temozolomide broadly promote expression of both mesenchymal and proneural transcripts, in some cases with equal frequency. These results suggest that glioblastoma mesenchymal transitions do not occur as distinctly as in epithelial-derived cancers, at least as determined using common subtyping ontologies and measuring response to growth factors or chemotherapeutics. Further understanding of these issues may identify improved methods for pharmacologically targeting the mesenchymal phenotype in glioblastoma.

Understanding current experimental models of glioblastoma-brain microenvironment interactions.

Glioblastoma (GBM) is a common and devastating primary brain tumor, with median survival of 16-18 months after diagnosis in the setting of substantial resistance to standard-of-care and inevitable tumor recurrence. Recent work has implicated the brain microenvironment as being critical for GBM proliferation, invasion, and resistance to treatment. GBM does not operate in isolation, with neurons, astrocytes, and multiple immune populations being implicated in GBM tumor progression and invasiveness. The goal of this review article is to provide an overview of the available in vitro, ex vivo, and in vivo experimental models for assessing GBM-brain interactions, as well as discuss each model's relative strengths and limitations. Current in vitro models discussed will include 2D and 3D co-culture platforms with various cells of the brain microenvironment, as well as spheroids, whole organoids, and models of fluid dynamics, such as interstitial flow. An overview of in vitro and ex vivo organotypic GBM brain slices is also provided. Finally, we conclude with a discussion of the various in vivo rodent models of GBM, including xenografts, syngeneic grafts, and genetically-engineered models of GBM.

The semaphorin 3A/neuropilin-1 pathway promotes clonogenic growth of glioblastoma via activation of TGF-ß signaling.

Glioblastoma (GBM) is the most lethal brain cancer with a dismal prognosis. Stem-like GBM cells (GSCs) are a major driver of GBM propagation and recurrence; thus, understanding the molecular mechanisms that promote GSCs may lead to effective therapeutic approaches. Through in vitro clonogenic growth-based assays, we determined mitogenic activities of the ligand molecules that are implicated in neural development. We have identified that semaphorin 3A (Sema3A), originally known as an axon guidance molecule in the CNS, promotes clonogenic growth of GBM cells but not normal neural progenitor cells (NPCs). Mechanistically, Sema3A binds to its receptor neuropilin-1 (NRP1) and facilitates an interaction between NRP1 and TGF-ß receptor 1 (TGF-ßR1), which in turn leads to activation of canonical TGF-ß signaling in both GSCs and NPCs. TGF-ß signaling enhances self-renewal and survival of GBM tumors through induction of key stem cell factors, but it evokes cytostatic responses in NPCs. Blockage of the Sema3A/NRP1 axis via shRNA-mediated knockdown of Sema3A or NRP1 impeded clonogenic growth and TGF-ß pathway activity in GSCs and inhibited tumor growth in vivo. Taken together, these findings suggest that the Sema3A/NRP1/TGF-ßR1 signaling axis is a critical regulator of GSC propagation and a potential therapeutic target for GBM.

Tissue factor is a critical regulator of radiation therapy-induced glioblastoma remodeling.

Radiation therapy (RT) provides therapeutic benefits for patients with glioblastoma (GBM), but inevitably induces poorly understood global changes in GBM and its microenvironment (TME) that promote radio-resistance and recurrence. Through a cell surface marker screen, we identified that CD142 (tissue factor or F3) is robustly induced in the senescence-associated ß-galactosidase (SA-ßGal)-positive GBM cells after irradiation. F3 promotes clonal expansion of irradiated SA-ßGal+ GBM cells and orchestrates oncogenic TME remodeling by activating both tumor-autonomous signaling and extrinsic coagulation pathways. Intratumoral F3 signaling induces a mesenchymal-like cell state transition and elevated chemokine secretion. Simultaneously, F3-mediated focal hypercoagulation states lead to activation of tumor-associated macrophages (TAMs) and extracellular matrix (ECM) remodeling. A newly developed F3-targeting agent potently inhibits the aforementioned oncogenic events and impedes tumor relapse in vivo. These findings support F3 as a critical regulator for therapeutic resistance and oncogenic senescence in GBM, opening potential therapeutic avenues.

Identification of ritanserin analogs that display DGK isoform specificity.

The diacylglycerol kinase (DGK) family of lipid enzymes catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). Both DAG and PA are lipid signaling molecules that are of notable importance in regulating cell processes such as proliferation, apoptosis, and migration. There are ten mammalian DGK enzymes that appear to have distinct biological functions. DGKα has emerged as a promising therapeutic target in numerous cancers including glioblastoma (GBM) and melanoma as treatment with small molecule DGKα inhibitors results in reduced tumor sizes and prolonged survival. Importantly, DGKα has also been identified as an immune checkpoint due to its promotion of T cell anergy, and its inhibition has been shown to improve T cell activation. There are few small molecule DGKα inhibitors currently available, and the application of existing compounds to clinical settings is hindered by species-dependent variability in potency, as well as concerns regarding isotype specificity particularly amongst other type I DGKs. In order to resolve these issues, we have screened a library of compounds structurally analogous to the DGKα inhibitor, ritanserin, in an effort to identify more potent and specific alternatives. We identified two compounds that more potently and selectively inhibit DGKα, one of which (JNJ-3790339) demonstrates similar cytotoxicity in GBM and melanoma cells as ritanserin. Consistent with its inhibitor profile towards DGKα, JNJ-3790339 also demonstrated improved activation of T cells compared with ritanserin. Together our data support efforts to identify DGK isoform-selective inhibitors as a mechanism to produce pharmacologically relevant cancer therapies.

Loss of Diacylglycerol Kinase α Enhances Macrophage Responsiveness.

The diacylglycerol kinases (DGKs) are a family of enzymes responsible for the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). In addition to their primary function in lipid metabolism, DGKs have recently been identified as potential therapeutic targets in multiple cancers, including glioblastoma (GBM) and melanoma. Aside from its tumorigenic properties, DGKα is also a known promoter of T-cell anergy, supporting a role as a recently-recognized T cell checkpoint. In fact, the only significant phenotype previously observed in Dgka knockout (KO) mice is the enhancement of T-cell activity. Herein we reveal a novel, macrophage-specific, immune-regulatory function of DGKα. In bone marrow-derived macrophages (BMDMs) cultured from wild-type (WT) and KO mice, we observed increased responsiveness of KO macrophages to diverse stimuli that yield different phenotypes, including LPS, IL-4, and the chemoattractant MCP-1. Knockdown (KD) of Dgka in a murine macrophage cell line resulted in similar increased responsiveness. Demonstrating in vivo relevance, we observed significantly smaller wounds in Dgka-/- mice with full-thickness cutaneous burns, a complex wound healing process in which macrophages play a key role. The burned area also demonstrated increased numbers of macrophages. In a cortical stab wound model, Dgka-/- brains show increased Iba1+ cell numbers at the needle track versus that in WT brains. Taken together, these findings identify a novel immune-regulatory checkpoint function of DGKα in macrophages with potential implications for wound healing, cancer therapy, and other settings.

Focused Ultrasound Preconditioning for Augmented Nanoparticle Penetration and Efficacy in the Central Nervous System.

Microbubble activation with focused ultrasound (FUS) facilitates the noninvasive and spatially-targeted delivery of systemically administered therapeutics across the blood-brain barrier (BBB). FUS also augments the penetration of nanoscale therapeutics through brain tissue; however, this secondary effect has not been leveraged. Here, 1 MHz FUS sequences that increase the volume of transfected brain tissue after convection-enhanced delivery of gene-vector "brain-penetrating" nanoparticles were first identified. Next, FUS preconditioning is applied prior to trans-BBB nanoparticle delivery, yielding up to a fivefold increase in subsequent transgene expression. Magnetic resonance imaging (MRI) analyses of tissue temperature and Ktrans confirm that augmented transfection occurs through modulation of parenchymal tissue with FUS. FUS preconditioning represents a simple and effective strategy for markedly improving the efficacy of gene vector nanoparticles in the central nervous system.

Tumor pharmacokinetics and pharmacodynamics of the CDK4/6 inhibitor ribociclib in patients with recurrent glioblastoma.

INTRODUCTION: We conducted a phase Ib study (NCT02345824) to determine whether ribociclib, an inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6), penetrates tumor tissue and modulates downstream signaling pathways including retinoblastoma protein (Rb) in patients with recurrent glioblastoma (GBM). METHODS: Study participants received ribociclib (600 mg QD) for 8-21 days before surgical resection of their recurrent GBM. Total and unbound concentrations of ribociclib were measured in samples of tumor tissue, plasma, and cerebrospinal fluid (CSF). We analyzed tumor specimens obtained from the first (initial/pre-study) and second (recurrent/on-study) surgery by immunohistochemistry for Rb status and downstream signaling of CDK4/6 inhibition. Participants with Rb-positive recurrent tumors continued ribociclib treatment on a 21-day-on, 7-day-off schedule after surgery, and were monitored for toxicity and disease progression. RESULTS: Three participants with recurrent Rb-positive GBM participated in this study. Mean unbound (pharmacologically active) ribociclib concentrations in plasma, CSF, MRI-enhancing, MRI-non-enhancing, and tumor core regions were 0.337 µM, 0.632 µM, 1.242 nmol/g, 0.484 nmol/g, and 1.526 nmol/g, respectively, which exceeded the in vitro IC50 (0.04 µM) for inhibition of CDK4/6 in cell-free assay. Modulation of pharmacodynamic markers of ribociclib CDK 4/6 inhibition in tumor tissues were inconsistent between study participants. No participants experienced serious adverse events, but all experienced early disease progression. CONCLUSIONS: This study suggests that ribociclib penetrated recurrent GBM tissue at concentrations predicted to be therapeutically beneficial. Our study was unable to demonstrate tumor pharmacodynamic correlates of drug activity. Although well tolerated, ribociclib monotherapy seemed ineffective for the treatment of recurrent GBM.

Deconstructing Lipid Kinase Inhibitors by Chemical Proteomics.

Diacylglycerol kinases (DGKs) regulate lipid metabolism and cell signaling through ATP-dependent phosphorylation of diacylglycerol to biosynthesize phosphatidic acid. Selective chemical probes for studying DGKs are currently lacking and are needed to annotate isoform-specific functions of these elusive lipid kinases. Previously, we explored fragment-based approaches to discover a core fragment of DGK-α (DGKα) inhibitors responsible for selective binding to the DGKα active site. Here, we utilize quantitative chemical proteomics to deconstruct widely used DGKα inhibitors to identify structural regions mediating off-target activity. We tested the activity of a fragment (RLM001) derived from a nucleotide-like region found in the DGKα inhibitors R59022 and ritanserin and discovered that RLM001 mimics ATP in its ability to broadly compete at ATP-binding sites of DGKα as well as >60 native ATP-binding proteins (kinases and ATPases) detected in cell proteomes. Equipotent inhibition of activity-based probe labeling by RLM001 supports a contiguous ligand-binding site composed of C1, DAGKc, and DAGKa domains in the DGKα active site. Given the lack of available crystal structures of DGKs, our studies highlight the utility of chemical proteomics in revealing active-site features of lipid kinases to enable development of inhibitors with enhanced selectivity against the human proteome.

The Ligand Binding Landscape of Diacylglycerol Kinases.

Diacylglycerol kinases (DGKs) are integral components of signal transduction cascades that regulate cell biology through ATP-dependent phosphorylation of the lipid messenger diacylglycerol. Methods for direct evaluation of DGK activity in native biological systems are lacking and needed to study isoform-specific functions of these multidomain lipid kinases. Here, we utilize ATP acyl phosphate activity-based probes and quantitative mass spectrometry to define, for the first time, ATP and small-molecule binding motifs of representative members from all five DGK subtypes. We use chemical proteomics to discover an unusual binding mode for the DGKα inhibitor, ritanserin, including interactions at the atypical C1 domain distinct from the ATP binding region. Unexpectedly, deconstruction of ritanserin yielded a fragment compound that blocks DGKα activity through a conserved binding mode and enhanced selectivity against the kinome. Collectively, our studies illustrate the power of chemical proteomics to profile protein-small molecule interactions of lipid kinases for fragment-based lead discovery.

Dual activities of ritanserin and R59022 as DGKα inhibitors and serotonin receptor antagonists.

Diacylglycerol kinase alpha (DGKα) catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). Recently, DGKα was identified as a therapeutic target in various cancers, as well as in immunotherapy. Application of small-molecule DGK inhibitors, R59022 and R59949, induces cancer cell death in vitro and in vivo. The pharmacokinetics of these compounds in mice, however, are poor. Thus, there is a need to discover additional DGK inhibitors not only to validate these enzymes as targets in oncology, but also to achieve a better understanding of their biology. In the present study, we investigate the activity of ritanserin, a compound structurally similar to R59022, against DGKα. Ritanserin, originally characterized as a serotonin (5-HT) receptor (5-HTR) antagonist, underwent clinical trials as a potential medicine for the treatment of schizophrenia and substance dependence. We document herein that ritanserin attenuates DGKα kinase activity while increasing the enzyme's affinity for ATP in vitro. In addition, R59022 and ritanserin function as DGKα inhibitors in cultured cells and activate protein kinase C (PKC). While recognizing that ritanserin attenuates DGK activity, we also find that R59022 and R59949 are 5-HTR antagonists. In conclusion, ritanserin, R59022 and R59949 are combined pharmacological inhibitors of DGKα and 5-HTRs in vitro.

Interstitial flow differentially increases patient-derived glioblastoma stem cell invasion via CXCR4, CXCL12, and CD44-mediated mechanisms.

Glioblastoma (GBM) prognosis remains dismal due in part to the invasiveness of GBM cells. Interstitial fluid flow (IFF) has been shown to increase invasion of glioma cells in vitro through the CXCR4 receptor interacting with autologous, pericellular gradients of CXCL12 (autologous chemotaxis) or through the CD44 receptor interactions with the extracellular matrix (hyaluronan-mediated mechanotransduction). These mechanisms have not been examined together and thus we hypothesized that both mechanisms contribute to invasion in populations of cancer cells. Therefore, we examined IFF-stimulated CXCR4-, CXCL12-, and CD44-dependent invasion in patient-derived glioblastoma stem cells (GSCs). Using our 3D in vitro assay and correlative in vivo studies we demonstrated GSC lines show increased invasion with flow. This flow-stimulated invasion was reduced by blockade of CXCR4, CXCL12, and/or CD44, revealing that GSC invasion may be mediated simultaneously by both mechanisms. Characterization of CXCR4+, CXCL12+, and CD44+ populations in four GSC lines revealed different percentages of protein positive subpopulations for each line. We developed an agent-based model to identify the contributions of each subpopulation to flow-stimulated invasion and validated the model through comparisons with experimental blocking studies. Clinically relevant radiation therapy increased flow-stimulated invasion in one GSC line. Our agent-based model predicted that IFF-stimulated invasion is driven primarily by CXCR4+CXCL12+ populations, and, indeed our irradiated cells had an increase in this subpopulation. Together, these data indicate that different mechanisms govern the flow response across GSCs, but that within a single patient, there are subpopulations of GSCs that respond to flow via either CD44- or CXCR4-CXCL12 mechanisms.

A Multicenter, Phase II, Randomized, Noncomparative Clinical Trial of Radiation and Temozolomide with or without Vandetanib in Newly Diagnosed Glioblastoma Patients.

PURPOSE: Vandetanib, a tyrosine kinase inhibitor of KDR (VEGFR2), EGFR, and RET, may enhance sensitivity to chemotherapy and radiation. We conducted a randomized, noncomparative, phase II study of radiation (RT) and temozolomide with or without vandetanib in patients with newly diagnosed glioblastoma (GBM). EXPERIMENTAL DESIGN: We planned to randomize a total of 114 newly diagnosed GBM patients in a ratio of 2:1 to standard RT and temozolomide with (76 patients) or without (38 patients) vandetanib 100 mg daily. Patients with age ≥ 18 years, Karnofsky performance status (KPS) ≥ 60, and not on enzyme-inducing antiepileptics were eligible. Primary endpoint was median overall survival (OS) from the date of randomization. Secondary endpoints included median progression-free survival (PFS), 12-month PFS, and safety. Correlative studies included pharmacokinetics as well as tissue and serum biomarker analysis. RESULTS: The study was terminated early for futility based on the results of an interim analysis. We enrolled 106 patients (36 in the RT/temozolomide arm and 70 in the vandetanib/RT/temozolomide arm). Median OS was 15.9 months [95% confidence interval (CI), 11.0-22.5 months] in the RT/temozolomide arm and 16.6 months (95% CI, 14.9-20.1 months) in the vandetanib/RT/temozolomide (log-rank P = 0.75). CONCLUSIONS: The addition of vandetanib at a dose of 100 mg daily to standard chemoradiation in patients with newly diagnosed GBM or gliosarcoma was associated with potential pharmacodynamic biomarker changes and was reasonably well tolerated. However, the regimen did not significantly prolong OS compared with the parallel control arm, leading to early termination of the study.

Sustained radiosensitization of hypoxic glioma cells after oxygen pretreatment in an animal model of glioblastoma and in vitro models of tumor hypoxia.

Glioblastoma multiforme (GBM) is the most common and lethal form of brain cancer and these tumors are highly resistant to chemo- and radiotherapy. Radioresistance is thought to result from a paucity of molecular oxygen in hypoxic tumor regions, resulting in reduced DNA damage and enhanced cellular defense mechanisms. Efforts to counteract tumor hypoxia during radiotherapy are limited by an attendant increase in the sensitivity of healthy brain tissue to radiation. However, the presence of heightened levels of molecular oxygen during radiotherapy, while conventionally deemed critical for adjuvant oxygen therapy to sensitize hypoxic tumor tissue, might not actually be necessary. We evaluated the concept that pre-treating tumor tissue by transiently elevating tissue oxygenation prior to radiation exposure could increase the efficacy of radiotherapy, even when radiotherapy is administered after the return of tumor tissue oxygen to hypoxic baseline levels. Using nude mice bearing intracranial U87-luciferase xenografts, and in vitro models of tumor hypoxia, the efficacy of oxygen pretreatment for producing radiosensitization was tested. Oxygen-induced radiosensitization of tumor tissue was observed in GBM xenografts, as seen by suppression of tumor growth and increased survival. Additionally, rodent and human glioma cells, and human glioma stem cells, exhibited prolonged enhanced vulnerability to radiation after oxygen pretreatment in vitro, even when radiation was delivered under hypoxic conditions. Over-expression of HIF-1α reduced this radiosensitization, indicating that this effect is mediated, in part, via a change in HIF-1-dependent mechanisms. Importantly, an identical duration of transient hyperoxic exposure does not sensitize normal human astrocytes to radiation in vitro. Taken together, these results indicate that briefly pre-treating tumors with elevated levels of oxygen prior to radiotherapy may represent a means for selectively targeting radiation-resistant hypoxic cancer cells, and could serve as a safe and effective adjuvant to radiation therapy for patients with GBM.

Novel anti-apoptotic microRNAs 582-5p and 363 promote human glioblastoma stem cell survival via direct inhibition of caspase 3, caspase 9, and Bim.

Glioblastoma is the most common and lethal primary brain tumor. Tumor initiation and recurrence are likely caused by a sub-population of glioblastoma stem cells, which may derive from mutated neural stem and precursor cells. Since CD133 is a stem cell marker for both normal brain and glioblastoma, and to better understand glioblastoma formation and recurrence, we looked for dys-regulated microRNAs in human CD133+ glioblastoma stem cells as opposed to CD133+ neural stem cells isolated from normal human brain. Using FACS sorting of low-passage cell samples followed by microRNA microarray analysis, we found 43 microRNAs that were dys-regulated in common in three separate CD133+ human glioblastomas compared to CD133+ normal neural stem cells. Among these were several microRNAs not previously associated with cancer. We then verified the microRNAs dys-regulated in glioblastoma using quantitative real time PCR and Taqman analysis of the original samples, as well as human GBM stem cell and established cell lines and many human specimens. We show that two candidate oncogenic microRNAs, miR-363 and miR-582-5p, can positively influence glioblastoma survival, as shown by forced expression of the microRNAs and their inhibitors followed by cell number assay, Caspase 3/7 assay, Annexin V apoptosis/fluorescence activated cell sorting, siRNA rescue of microRNA inhibitor treatment, as well as 3'UTR mutagenesis to show luciferase reporter rescue of the most successful targets. miR-582-5p and miR-363 are shown to directly target Caspase 3, Caspase 9, and Bim.

Diacylglycerol kinase α is a critical signaling node and novel therapeutic target in glioblastoma and other cancers.

Although diacylglycerol kinase α (DGKα) has been linked to several signaling pathways related to cancer cell biology, it has been neglected as a target for cancer therapy. The attenuation of DGKα activity via DGKα-targeting siRNA and small-molecule inhibitors R59022 and R59949 induced caspase-mediated apoptosis in glioblastoma cells and in other cancers, but lacked toxicity in noncancerous cells. We determined that mTOR and hypoxia-inducible factor-1α (HIF-1α) are key targets of DGKα inhibition, in addition to its regulation of other oncogenes. DGKα regulates mTOR transcription via a unique pathway involving cyclic AMP. Finally, we showed the efficacy of DGKα inhibition with short hairpin RNA or a small-molecule agent in glioblastoma and melanoma xenograft treatment models, with growth delay and decreased vascularity. This study establishes DGKα as a central signaling hub and a promising therapeutic target in the treatment of cancer.

A novel fixative for immunofluorescence staining of CD133-positive glioblastoma stem cells.

Isolation of glioblastoma stem cells requires incubation of tumor cells in a neural stem cell media. Neurospheres containing these glioblastoma stem cells are formed after approximately a five-day period. These cells can then be analyzed for the presence of stem cell markers. Immunofluorescence staining for these markers can serve as a valuable tool for analyzing the intact neurosphere directly in stem cell media. Here we present the use of a novel fixative (1,4-benzoquinone) for immunoflourescence staining of neurospheres.

Glioblastoma genetics: in rapid flux.

Glioblastoma is the most common and most lethal primary brain tumor. While small progress has been made in treating this cancer in recent years, glioblastoma remains largely resistant to all existing therapies. It has been hoped that dissection of the genetics of this cancer would lead to more targeted and effective treatments, and new advances may finally be bringing this closer to fruition. Within the last few years, high-throughput efforts such as The Cancer Genome Atlas and a massive sequencing project have yielded novel insights and classifications of this dreaded cancer. The likely impact on care delivery in the clinic may only be a few years away. The rapid and exciting pace of advances in glioblastoma genetics has prompted this up-to-date review.

Neuro-oncology: Isocitrate dehydrogenase mutations in low-grade gliomas.

Most grade II and grade III gliomas, as well as the secondary glioblastomas that arise from these tumors, possess point mutations that affect the substrate binding site of isocitrate dehydrogenase. These mutations are essentially unique to gliomas, seem to represent an early step in gliomagenesis, and confer a favorable prognosis.

Notch-1 regulates transcription of the epidermal growth factor receptor through p53.

The Notch pathway plays a key role in the development and is increasingly recognized for its importance in cancer. We demonstrated previously the overexpression of Notch-1 and its ligands in gliomas and showed that their knockdown inhibits glioma cell proliferation and survival. To elucidate the mechanisms downstream of Notch-1 in glioma cells, we performed microarray profiling of glioma cells transfected with Notch-1 small interfering RNA. Notable among downregulated transcripts was the epidermal growth factor receptor (EGFR), known to be overexpressed or amplified in gliomas and prominent in other cancers as well. Further studies confirmed that Notch-1 inhibition decreased EGFR messenger RNA (mRNA) and EGFR protein in glioma and other cell lines. Transfection with Notch-1 increased EGFR expression. Additionally, we found a significant correlation in levels of EGFR and Notch-1 mRNA in primary high-grade human gliomas. Subsequent experiments showed that p53, an activator of the EGFR promoter, is regulated by Notch-1. Experiments with p53-positive and -null cell lines confirmed that p53 partially mediates the effects of Notch-1 on EGFR expression. These results show for the first time that Notch-1 upregulates EGFR expression and also demonstrate Notch-1 regulation of p53 in gliomas. These observations have significant implications for understanding the mechanisms of Notch in cancer and development.