Search

Temozolomide (TMZ) is the standard of care chemotherapy drug for treating glioblastomas (GBMs), the most aggressive cancer that affects people of all ages. However, its therapeutic efficacy is limited by the drug resistance mediated by a DNA repair protein, O(6)-methylguanine-DNA methyltransferase (MGMT), which eliminates the TMZ-induced DNA lesions. Here we report the development of an iron oxide nanoparticle (NP) system for targeted delivery of siRNAs to suppress the TMZ-resistance gene (MGMT). We show that our NP is able to overcome biological barriers, bind specifically to tumor cells, and reduce MGMT expression in tumors of mice bearing orthotopic GBM serially-passaged patient-derived xenografts. The treatment with sequential administration of this NP and TMZ resulted in increased apoptosis of GBM stem-like cells, reduced tumor growth, and significantly-prolonged survival as compared to mice treated with TMZ alone. This study introduces an approach that holds great promise to improve the outcomes of GBM patients.

Independent scientific achievements have led to the discovery of aberrant splicing patterns in oncogenesis, while more recent advances have uncovered novel gene fusions involving neurotrophic tyrosine receptor kinases (NTRKs) in gliomas. The exploration of NTRK splice variants in normal and neoplastic brain provides an intersection of these two rapidly evolving fields. Tropomyosin receptor kinase B (TrkB), encoded NTRK2, is known for critical roles in neuronal survival, differentiation, molecular properties associated with memory, and exhibits intricate splicing patterns and post-translational modifications. Here, we show a role for a truncated NTRK2 splice variant, TrkB.T1, in human glioma. TrkB.T1 enhances PDGF-driven gliomas in vivo, augments PDGF-induced Akt and STAT3 signaling in vitro, while next generation sequencing broadly implicates TrkB.T1 in the PI3K signaling cascades in a ligand-independent fashion. These TrkB.T1 findings highlight the importance of expanding upon whole gene and gene fusion analyses to include splice variants in basic and translational neuro-oncology research., Tropomyosin receptor kinase B (TrkB), encoded by the neurotrophic tyrosine receptor kinase 2 (NTRK2) gene, exhibits intricate splicing patterns and post-translational modifications. Here, the authors perform whole gene and transcript-level analyses and report the TrkB.T1 splice variant enhances PDGF-driven gliomas in vivo and augments PI3K signaling cascades in vitro.

The lack of in vitro models that support the growth of glioblastoma (GBM) cancer stem cells (GSCs) that underlie clinical aggressiveness hinders developing new, effective therapies for GBM. While orthotopic patient-derived xenograft models of GBM best reflect in vivo tumor behavior, establishing xenografts is a time consuming, costly and frequently unsuccessful endeavor. To address these limitations, we synthesize a three-dimensional porous scaffold composed of chitosan and hyaluronic acid (CHA), and compared growth and expression of the cancer stem cell (CSC) phenotype of the GSC GBM6 taken directly from fresh xenogratfs grown on scaffolds or as adherent monolayers. While 2D adherent cultures grow as monolayers of flat epitheliod cells, GBM6 cells proliferate within pores of CHA scaffolds as clusters of self-adherent ovoid cells. Growth on scaffolds is accompanied by greater expression of genes that mediate epithelial-mesenchymal transition and maintain a primitive, undifferentiated phenotype, hallmarks of CSCs. Scaffold-grown cells also display higher expression of genes that promote resistance to hypoxia-induced oxidative stress. In accord, scaffold-grown cells show markedly greater resistance to clinically utilized alkylating agents compared to adherent cells. These findings suggest that our CHA scaffolds better mimic in vivo biological and clinical behavior and provide insights for developing novel individualized treatments.

Efforts to reduce immunosuppression in the solid tumor microenvironment by blocking the recruitment or polarization of tumor associated macrophages (TAM), or myeloid derived suppressor cells (MDSCs), have gained momentum in recent years. Expanding our knowledge of the immune cell types, cytokines, or recruitment factors that are associated with high-grade disease, both within the tumor and in circulation, is critical to identifying novel targets for immunotherapy. Furthermore, a better understanding of how therapeutic regimens, such as Dexamethasone (Dex), chemotherapy, and radiation, impact these factors will facilitate the design of therapies that can be targeted to the appropriate populations and retain efficacy when administered in combination with standard of care regimens. Here we perform quantitative analysis of tissue microarrays made of samples taken from grades I-III astrocytoma and glioblastoma (GBM, grade IV astrocytoma) to evaluate infiltration of myeloid markers CD163, CD68, CD33, and S100A9. Serum, flow cytometric, and Nanostring analysis allowed us to further elucidate the impact of Dex treatment on systemic biomarkers, circulating cells, and functional markers within tumor tissue. We found that common myeloid markers were elevated in Dex-treated grade I astrocytoma and GBM compared to non-neoplastic brain tissue and grade II-III astrocytomas. Cell frequencies in these samples differed significantly from those in Dex-naïve patients in a pattern that depended on tumor grade. In contrast, observed changes in serum chemokines or circulating monocytes were independent of disease state and were due to Dex treatment alone. Furthermore, these changes seen in blood were often not reflected within the tumor tissue. Conclusions: Our findings highlight the importance of considering perioperative treatment as well as disease grade when assessing novel therapeutic targets or biomarkers of disease.

Human tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is an attractive cancer therapeutic because of its ability to induce apoptosis in tumor cells while having a negligible effect on normal cells. However, the short serum half-life of TRAIL and lack of efficient in vivo administration approaches have largely hindered its clinical use. Using nanoparticles (NPs) as carriers in gene therapy is considered as an alternative approach to increase TRAIL delivery to tumors as transfected cells would be induced to secrete TRAIL into the tumor microenvironment. To enable effective delivery of plasmid DNA encoding TRAIL into glioblastoma (GBM), we developed a targeted iron oxide NP coated with chitosan-polyethylene glycol-polyethyleneimine copolymer and chlorotoxin (CTX) and evaluated its effect in delivering TRAIL in vitro and in vivo. NP-TRAIL successfully delivers TRAIL into human T98G GBM cells and induces secretion of 40 pg mL(-1) of TRAIL in vitro. Transfected cells show threefold increased apoptosis as compared to the control DNA bound NPs. Systemic administration of NP-TRAIL-CTX to mice bearing T98G-derived flank xenografts results in near-zero tumor growth and induces apoptosis in tumor tissue. Our results suggest that NP-TRAIL-CTX can potentially serve as a targeted anticancer therapeutic for more efficient TRAIL delivery to GBM.

BackgroundCpG methylation in the O6-methylguanine-DNA methyltransferase (MGMT) promoter is associated with better outcome following alkylating agent chemotherapy in glioblastoma (GBM) and anaplastic glioma (AG). To what extent improved response reflects low or absent MGMT activity in glioma tissue has not been unequivocally assessed. This information is central to developing anti-resistance therapies.MethodsWe examined the relationship of MGMT activity in 91 GBMs and 84 AGs with progression-free survival (PFS) following alkylator therapy and with promoter methylation status determined by methylation-specific PCR (MSP).ResultsCox regression analysis revealed that GBMs with high activity had a significantly greater risk for progression in dichotomous (P≤0.001) and continuous (P≤0.003) models, an association observed for different alkylator regimens, including concurrent chemo-radiation with temozolomide. Analysis of MGMT promoter methylation status in 47 of the GBMs revealed that methylated tumors had significantly lower activity (P≤0.005) and longer PFS (P≤0.036) compared to unmethylated tumors, despite overlapping activities. PFS was also significantly greater in methylated vs. unmethylated GBMs with comparable activity (P≤0.005), and among unmethylated tumors with less than median activity (P≤0.026), suggesting that mechanisms in addition to MGMT promote alkylator resistance. Similar associations of MGMT activity with PFS and promoter methylation status were observed for AGs.ConclusionsOur results provide strong support for the hypotheses that MGMT activity promotes alkylator resistance and reflects promoter methylation status in malignant gliomas.General significanceMGMT activity is an attractive target for anti-resistance therapy regardless of methylation status.

Medulloblastoma (MB) and ependymoma (EP) are the most common pediatric brain tumors, afflicting 3000 children annually. Radiotherapy (RT) is an integral component in the treatment of these tumors; however, the improvement in survival is often accompanied by radiation‐induced adverse developmental and psychosocial sequelae. Therefore, there is an urgent need to develop strategies that can increase the sensitivity of brain tumors cells to RT while sparing adjacent healthy brain tissue. Apurinic endonuclease 1 (Ape1), an enzyme in the base excision repair pathway, has been implicated in radiation resistance in cancer. Pharmacological and specificity limitations inherent to small molecule inhibitors of Ape1 have hindered their clinical development. Here we report on a nanoparticle (NP) based siRNA delivery vehicle for knocking down Ape1 expression and sensitizing pediatric brain tumor cells to RT. The NP comprises a superparamagnetic iron oxide core coated with a biocompatible, biodegradable coating of chitosan, polyethylene glycol (PEG), and polyethyleneimine (PEI) that is able to bind and protect siRNA from degradation and to deliver siRNA to the perinuclear region of target cells. NPs loaded with siRNA against Ape1 (NP:siApe1) knocked down Ape1 expression over 75% in MB and EP cells, and reduced Ape1 activity by 80%. This reduction in Ape1 activity correlated with increased DNA damage post‐irradiation, which resulted in decreased cell survival in clonogenic assays. The sensitization was specific to therapies generating abasic lesions as evidenced by NP:siRNA not increasing sensitivity to paclitaxel, a microtubule disrupting agent. Our results indicate NP‐mediated delivery of siApe1 is a promising strategy for circumventing pediatric brain tumor resistance to RT.

Emerging evidence implicates cancer stem cells (CSCs) as primary determinants of the clinical behavior of human cancers, representing an ideal target for next-generation anti-cancer therapies. However CSCs are difficult to propagate in vitro, severely limiting the study of CSC biology and drug development. Here we report that growing cells from glioblastoma (GBM) cell lines on three dimensional (3D) porous chitosan-alginate (CA) scaffolds dramatically promotes the proliferation and enrichment of cells possessing the hallmarks of CSCs. CA scaffold-grown cells were found more tumorigenic in nude mouse xenografts than cells grown from monolayers. Growing in CA scaffolds rapidly promoted expression of genes involved in the epithelial-to-mesenchymal transition that has been implicated in the genesis of CSCs. Our results indicate that CA scaffolds have utility as a simple and inexpensive means to cultivate CSCs in vitro in support of studies to understand CSC biology and develop more effective anti-cancer therapies.

Resistance to temozolomide (TMZ) based chemotherapy in glioblastoma multiforme (GBM) has been attributed to the upregulation of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT). Inhibition of MGMT using O(6)-benzylguanine (BG) has shown promise in these patients, but its clinical use is hindered by poor pharmacokinetics that leads to unacceptable toxicity. To improve BG biodistribution and efficacy, we developed superparamagnetic iron oxide nanoparticles (NP) for targeted convection-enhanced delivery (CED) of BG to GBM. The nanoparticles (NPCP-BG-CTX) consist of a magnetic core coated with a redox-responsive, cross-linked, biocompatible chitosan-PEG copolymer surface coating (NPCP). NPCP was modified through covalent attachment of BG and tumor targeting peptide chlorotoxin (CTX). Controlled, localized BG release was achieved under reductive intracellular conditions and NPCP-BG-CTX demonstrated proper trafficking of BG in human GBM cells in vitro. NPCP-BG-CTX treated cells showed a significant reduction in MGMT activity and the potentiation of TMZ toxicity. In vivo, CED of NPCP-BG-CTX produced an excellent volume of distribution (Vd) within the brain of mice bearing orthotopic human primary GBM xenografts. Significantly, concurrent treatment with NPCP-BG-CTX and TMZ showed a 3-fold increase in median overall survival in comparison to NPCP-CTX/TMZ treated and untreated animals. Furthermore, NPCP-BG-CTX mitigated the myelosuppression observed with free BG in wild-type mice when administered concurrently with TMZ. The combination of favorable physicochemical properties, tumor cell specific BG delivery, controlled BG release, and improved in vivo efficacy demonstrates the great potential of these NPs as a treatment option that could lead to improved clinical outcomes.

Periostin is a matricellular fascilin-like protein that was first described as a component of periodontal ligaments.1 Subsequently, periostin has been shown to have critical functions in development, pathologic fibrosis, and cancer biology (reviewed in2–5). In carcinomas, periostin, secreted by cancer and stromal cells, promotes malignancy by promoting invasion, cell survival, treatment resistance, and metastasis. Secreted periostin functions in part through integrin interactions (most commonly with αvβ3 and αvβ5), which in turn activates proinvasive and survival-signaling pathways including focal adhesion kinase (FAK) and PI3K/Akt.5 Periostin promotes epithelial-mesenchymal transition (EMT)4 and is accordingly implicated in metastatic cell colonization and survival.6,7 Unlike carcinomas, relatively little is known about the prognostic or functional significance of periostin in human gliomas, this despite the early recognition that the periostin gene (POSTN) is among the most upregulated genes in GBM compared with normal brain.8,9 The few studies of periostin in glioma have reported direct correlations between expression levels and increased tumor edema, tumor grade, and survival.10,11 Functionally, periostin is reported to promote glioma cell invasion in vitro.11 In addition to expression in glioma cells, periostin is also reported to be a tumor-specific marker of GBM-associated angiogenesis.12 These associations support the potential importance of periostin in glioma malignancy, but the mechanisms by which periostin may contribute to glioma malignancy require further investigation. This study broadly examined periostin expression and function to extend current understanding of its clinical relevance and therapeutic potential. Expression in clinical samples and public databases have confirmed robust prognostic significance across all glioma grades. Periostin gene signatures also predicted outcome and demonstrated relationships of periostin with biological hallmarks of malignancy. In functional analysis, periostin regulated glioma cell invasion, adhesion, migration, cell morphology, and glioma stem cell survival in vivo. Interactions with αvβ3 and αvβ5 integrins and alteration of FAK localization suggested putative mechanisms of action for secreted periostin. Together, these data strongly support the potential of periostin as a biomarker and therapeutic target in human gliomas and provide direction for future investigations critical to realizing this potential.

Recent work has uncovered oncogenic TRK fusions in a wide range of cancer types, including adult and pediatric gliomas. With some exceptions, many of these fusions tend to occur at very low frequencies below 1–2%, with unclear clinical significance, yet they highlight a potentially important and rapidly evolving role for NTRK1, NTRK2, NTRK3 in glioma biology. Basic scientific and clinical investigation surrounding TRKs’ role in cancer has often been hindered due to the nonspecific nature of antibodies and kinase inhibitors, combined with a lack of precise exon-specific expression data from patient populations. Tropomyosin receptor B (TrkB), encoded by the NTRK2 gene, is most known for its established roles in neuronal survival, proliferation, differentiation, apoptosis, learning, and memory. TrkB exerts diverse effects on cellular outcomes through interactions with downstream signaling cascades and has been shown to exhibit complex alternative splicing patterns. Here we show a novel role for a TrkB splice variant in human gliomas via NTRK2 transcript analyses in normal human brain and gliomas using The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression Project (GTEx). Using a novel antibody designed against this splice variant, immunostaining shows altered receptor localization within human gliomas compared to normal human brain. This NTRK2 splice variant enhances PDGF-driven gliomas in vivo in an RCAS-TVA mouse model and augments PDGF-induced signaling in vitro. Through the lens of NTRK2, these results highlight the importance of expanding upon whole gene-level and kinase-fusion analyses to explore TRK splicing in basic and translational research.

Due largely to the inability to accurately quantify and characterize de novo deletion events, the mechanisms underpinning the pathogenic expansion of mtDNA deletions in aging and neuromuscular disorders remain poorly understood. Here, we outline and validate a new tool termed ‘Digital Deletion Detection’ (3D) that allows for high-resolution analysis of rare deletions occurring at frequencies as low as 1 × 10−8. 3D is a three-step process that includes targeted enrichment for deletion-bearing molecules, single-molecule partitioning of genomes into thousands of droplets for direct quantification via droplet digital PCR, and breakpoint characterization using massively parallel sequencing. Using 3D, we interrogated over 8 billion mitochondrial genomes to analyze the age-related dynamics of mtDNA deletions in human brain tissue. We demonstrate that the total deletion load increases with age, while the total number and diversity of unique deletions remain constant. Our data provide support for the hypothesis that expansion of pre-existing mutations is the primary factor contributing to age-related accumulation of mtDNA deletions.

Nanoparticle-mediated delivery of chemotherapeutics has demonstrated potential in improving anti-cancer efficacy by increasing serum half-life and providing tissue specificity and controlled drug release to improve biodistribution of hydrophobic chemotherapeutics. However, sub-optimal drug loading, particularly for solid core nanoparticles (NPs), remains a challenge that limits their clinical application. In this study we formulated a NP coated with a pH-sensitive polymer of O6-methylguanine-DNA methyltransferase (MGMT) inhibitor analog, dialdehyde modified O6-benzylguanosine (DABGS) to achieve high drug loading and with polyethylene glycol (PEG) to ameliorate water solubility while maintaining NP stability. The base nanovector consists of an iron oxide core (9 nm) coated with hydrazide functionalized PEG (IOPH). DABGS and PEG-dihydrazide were polymerized on the iron oxide nanoparticle surface (IOPH-pBGS) through acid-labile hydrazone bonds utilizing a rapid, freeze-thaw catalysis approach. DABGS polymerization was confirmed by FTIR and quantitated by UV-Vis spectroscopy. IOPH-pBGS demonstrated excellent drug loading of 33.4 ± 5.1% by weight while maintaining small size (36.5 ± 1.8 nm). Drug release was monitored at biologically relevant pHs and demonstrated pH dependent release with maximum release at pH 5.5 (intracellular conditions), and minimal release at physiological pH (7.4). IOPH-pBGS significantly suppressed activity of MGMT and potentiated temozolomide (TMZ) toxicity in vitro, demonstrating the potential as a new treatment option for glioblastomas (GBMs).

Derivatives of methyl 3-(1-methyl-5-(1-methyl-5-(propylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylamino)-3-oxopropane-1-sulfonate (1), a peptide-based DNA minor groove binding methylating agent, were synthesized and characterized. In all cases, the N-terminus was appended with an O-methyl sulfonate ester, while the C-terminus group was varied with nonpolar and polar side chains. In addition, the number of pyrrole rings was varied from 2 (dipeptide) to 3 (tripeptide). The ability of the different analogues to efficiently generate N3-methyladenine was demonstrated as was their selectivity for minor groove (N3-methyladenine) versus major groove (N7-methylguanine) methylation. Induced circular dichroism studies were used to measure the DNA equilibrium binding properties of the stable sulfone analogues; the tripeptide binds with affinity that is >10-fold higher than that of the dipeptide. The toxicities of the compounds were evaluated in alkA/tag glycosylase mutant E. coli and in human WT glioma cells and in cells overexpressing and under-expressing N-methylpurine-DNA glycosylase, which excises N3-methyladenine from DNA. The results show that equilibrium binding correlates with the levels of N3-methyladenine produced and cellular toxicity. The toxicity of 1 was inversely related to the expression of MPG in both the bacterial and mammalian cell lines. The enhanced toxicity parallels the reduced activation of PARP and the diminished rate of formation of aldehyde reactive sites observed in the MPG knockdown cells. It is proposed that unrepaired N3-methyladenine is toxic due to its ability to directly block DNA polymerization.

Apurinic/apyrimidinic endonuclease (Ap endo) is a key DNA repair activity that confers radiation resistance in human cells. Here we examined the association between Ap endo activity and response to radiotherapy in pediatric ependymomas, tumors for which treatment options are limited and survival rates are only about 50%. We assayed Ap endo activity in 36 ependymomas and expression of Ape1/Ref-1, the predominant Ap endo activity in humans, in 44 tumors by immunostaining. Cox proportional hazards regression models were used to analyze the association of activity or expression with progression-free survival or with overall survival. Activity varied 13-fold and was not associated with tumor or patient characteristics. In univariate models with Ap endo activity entered as a continuous variable, the hazard ratio for progression increased by a factor of 2.18 for every 0.01 unit increase in activity (p ≤ 0.003) in 24 grade II ependymomas. Risk for death increased by a factor of 1.89 (p ≤ 0.02) in the same population. The fraction of Ape1/Ref-1 immunopositive cells varied widely within individual tumors and was not associated with either progression-free or with overall survival. Suppressing Ap endo activity in pediatric ependymoma cells significantly increased radiation sensitivity, suggesting that the association of activity with radiation response reflected, at least in part, repair of radiation-induced DNA lesions. Our data indicate that Ap endo activity is predictive of outcome following radiotherapy, and suggest that Ape1/Ref-1 promotes radiation resistance in pediatric ependymomas. Our findings support the use of inhibitors of Ap endo activity to overcome resistance.

Multidrug resistance (MDR) is characterized by the overexpression of ATP-binding cassette (ABC) transporters that actively pump a broad class of hydrophobic chemotherapeutic drugs out of cancer cells. MDR is a major mechanism of treatment resistance in a variety of human tumors, and clinically applicable strategies to circumvent MDR remain to be characterized. Here we describe the fabrication and characterization of a drug-loaded iron oxide nanoparticle designed to circumvent MDR. Doxorubicin (DOX), an anthracycline antibiotic commonly used in cancer chemotherapy and substrate for ABC-mediated drug efflux, was covalently bound to polyethylenimine via a pH sensitive hydrazone linkage and conjugated to an iron oxide nanoparticle coated with amine terminated polyethylene glycol. Drug loading, physiochemical properties and pH lability of the DOX-hydrazone linkage were evaluated in vitro. Nanoparticle uptake, retention, and dose-dependent effects on viability were compared in wild-type and DOX-resistant ABC transporter over-expressing rat glioma C6 cells. We found that DOX release from nanoparticles was greatest at acidic pH, indicative of cleavage of the hydrazone linkage. DOX-conjugated nanoparticles were readily taken up by wild-type and drug-resistant cells. In contrast to free drug, DOX-conjugated nanoparticles persisted in drug-resistant cells, indicating that they were not subject to drug efflux. Greater retention of DOX-conjugated nanoparticles was accompanied by reduction of viability relative to cells treated with free drug. Our results suggest that DOX-conjugated nanoparticles could improve the efficacy of chemotherapy by circumventing MDR.

Most known for their essential roles in the development and maintenance of the nervous system, the neurotrophins consist of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), NT-3, NT-4 and their respective tropomyosin receptor kinases (TRKs) TrkA, TrkB, and TrkC along with the low affinity nerve growth factor receptor, p75. In addition to known roles in neuronal survival, proliferation, differentiation, and apoptosis, TRKs exert diverse effects on cellular outcomes through their interactions with downstream signaling cascades. Prior to TRKs’ established roles in neurobiology, oncogenic TRK was discovered as tropomysoin 3 (TPM3)-TrkA fusion and as a result, oncological studies of the neurotrophin family have identified additional TRK fusions. Putative oncogenic TRK fusions have been observed in various cancer types, but their clinical significance remains unclear and these fusions tend to occur at very low frequencies below 1–2%. The low incidence of these fusions combined with significant overexpression of various TRKs in a multitude of cancers raises the possibility that another aspect of TRK biology, in addition to kinase-domain fusions, may be relevant. Basic scientific and clinical investigation surrounding TRKs’ role in cancer has often been hindered due to the nonspecific nature of antibodies and kinase inhibitors, combined with a lack of precise exon-specific expression data from patient populations. Tropomyosin receptor B (TrkB), encoded by the NTRK2 gene, exhibits complex alternative splicing patterns. Here we show a novel role for a TrkB splice variant in gliomas via NTRK2 transcript analyses and immunostaining using a novel antibody engineered specifically to this variant. This NTRK2 splice variant enhances PDGF-driven gliomas in vivo and augments PDGF-induced signaling in vitro. Through the lens of NTRK2, these results highlight the importance of expanding upon whole gene-level and kinase-fusion analyses to explore TRK splicing in basic and translational research.

Cerebral vasospasm is a major cause of morbidity and mortality in patients with subarachnoid hemorrhage (SAH), causing delayed neurological deficits in as many as one third of cases. Existing therapy targets induction of cerebral vasodilation through use of various drugs and mechanical means, with a range of observed efficacy. Here, we perform a literature review supporting our hypothesis that transcranially delivered ultrasound may have the ability to induce therapeutic cerebral vasodilation and, thus, may one day be used therapeutically in the context of SAH. Prior studies demonstrate that ultrasound can induce vasodilation in both normal and vasoconstricted blood vessels in peripheral tissues, leading to reduced ischemia and cell damage. Among the proposed mechanisms is alteration of several nitric oxide (NO) pathways, where NO is a known vasodilator. While in vivo studies do not point to a specific physical mechanism, results of in vitro studies favor cavitation induction by ultrasound, where the associated shear stresses likely induce NO production. Two papers discussed the effects of ultrasound on the cerebral vasculature. One study applied clinical transcranial Doppler ultrasound to a rodent complete middle cerebral artery occlusion model and found reduced infarct size. A second involved the application of pulsed ultrasound in vitro to murine brain endothelial cells and showed production of a variety of vasodilatory chemicals, including by-products of arachidonic acid metabolism. In sum, nine reviewed studies demonstrated evidence of either cerebrovascular dilation or elaboration of vasodilatory compounds. Of particular interest, all of the reviewed studies used ultrasound capable of transcranial application: pulsed ultrasound, with carrier frequencies ranging between 0.5 and 2.0 MHz, and intensities not substantially above FDA-approved intensity values. We close by discussing potential specific treatment paradigms of SAH and other cerebral ischemic disorders based on MRI-guided transcranial ultrasound.

Purpose: Defining the cytotoxicity of individual adducts in DNA is necessary for mechanistic understanding of human brain tumor resistance to therapeutic alkylating agents and for design of DNA repair-related antiresistance strategies. Our purpose is to characterize the sensitivity of human glioma cells to methyl-lexitropsin (Me-lex), a sequence-specific alkylator that produces 3-methyladenine (3-meA) as the predominant (>90%) DNA lesion. Experimental Design: We quantitated the Me-lex cytotoxicity of 10 human glioma cell lines that differ in O6-methylguanine (O6-meG)-DNA methyltransferase (MGMT) and mismatch repair activity. We used antisense suppression of alkyladenine DNA glycosylase (AAG) and Ape1 to assess the contribution of 3-meA and abasic sites to lethality and measured abasic sites. Results: (a) The LD10 for Me-lex varied widely among the cell lines. (b) MGMT-proficient lines were more resistant than MGMT-deficient lines, an unexpected finding because Me-lex produces very little O6-meG. (c) Suppression of AAG increased Me-lex killing and reduced abasic site content. (d) Suppression of Ape1 increased Me-lex killing and increased abasic site content. (e) Ablation of MGMT had no effect on Me-lex cytotoxicity. Conclusions: (a) Me-lex is cytotoxic in human glioma cells and AAG promotes resistance, indicating that 3-meA is a lethal lesion in these cells. (b) Abasic sites resulting from 3-meA repair are cytotoxic and Ape1 promotes resistance to these derivative lesions. (c) A factor(s) associated with MGMT expression, other than repair of O6-meG, contributes to Me-lex resistance. (d) Me-lex may have clinical utility in the adjuvant therapy of gliomas. (e) AAG and Ape1 inhibitors may be useful in targeting alkylating agent resistance.

Radiotherapy (RT) is an integral component of the treatment for medulloblastoma (MB) and the only effective adjuvant therapy for ependymoma (EP), two of the most common pediatric brain tumors. However, survival is frequently accompanied by one or more radiation-induced adverse developmental and psychosocial sequelae. Therefore, strategies that enhance the tumoricidal action of RT while sparing adjacent normal brain are expected to reduce the amount of radiation required to generate a therapeutic effect in these tumors. The multifunctional DNA repair protein Ape1/Ref-1 has been implicated in conferring radiation resistance in pediatric brain tumors. However, inhibiting Ape1 activity in the clinic has been hindered by the lack of safe and effective drugs and siRNA delivery vehicles. Here, we used our nanoparticle delivery vehicles to deliver siRNA against Ape1 (siApe1) and improve cell kill after RT. Nanoparticles were loaded with siApe1, or siGFP as a control, and used to treat UW228 (MB) and Res196 (EP) cells. Ape1 expression and activity were measured using PCR, Western blot, and an abasic endonuclease activity assay. DNA repair and clonogenic survival were assessed after exposure to 137Cs-γ-rays. We found that nanoparticle-mediated siApe1 delivery reduced Ape1 expression and activity by greater than 80%, which was accompanied by a greater abundance of DNA damage. This diminished the shoulder of resistance in survival curves decreasing survival to ∼50% at 1 Gy. Sensitization was specific to abasic site generating treatments as response to paclitaxel was not affected. Therefore, nanoparticle-mediated inhibition of Ape1 may help enhance the therapeutic effect of RT in pediatric brain tumor patients and reduce treatment-induced morbidity.

Purpose: Apurinic/apyrimidinic endonuclease (Ap endo) is a key DNA repair activity that confers resistance to radiation- and alkylator-induced cytotoxic abasic sites in human cells. We assayed apurinic/apyrimidinic endonuclease activity in medulloblastomas and primitive neuroectodermal tumors (PNET) to establish correlates with tumor and patient characteristics and with response to adjuvant radiation plus multiagent chemotherapy. Experimental Design: Ap endo activity was assayed in 52 medulloblastomas and 10 PNETs from patients 0.4 to 21 years old. Ape1/Ref-1, the predominant human Ap endo activity, was measured in 42 medulloblastomas by immunostaining. Cox proportional hazards regression models were used to analyze the association of activity with time to tumor progression (TTP). Results: Tumor Ap endo activity varied 180-fold and was significantly associated with age and gender. Tumor Ape1/Ref-1 was detected almost exclusively in nuclei. In a multivariate model, with Ap endo activity entered as a continuous variable, the hazard ratio for progression after adjuvant treatment in 46 medulloblastomas and four PNETs increased by a factor of 1.073 for every 0.01 unit increase in activity (P ≤ 0.001) and was independent of age and gender. Suppressing Ap endo activity in a human medulloblastoma cell line significantly increased sensitivity to 1,3-bis(2-chlororethyl)-1-nitrosourea and temozolomide, suggesting that the association of tumor activity with TTP reflected, at least in part, abasic site repair. Conclusions: Our data (a) suggest that Ap endo activity promotes resistance to radiation plus chemotherapy in medulloblastomas/PNETs, (b) provide a potential marker of treatment outcome, and (c) suggest clinical use of Ap endo inhibitors to overcome resistance.

The Werner syndrome (WS) protein (WRN), a DNA helicase/exonuclease, is required for genomic stability and avoidance of cancer. Current evidence suggests that WRN is involved in the resolution of stalled and/or collapsed replication forks. This function is indicated, in part, by replication defects in WS cells and by hypersensitivity to agents causing major structural aberrations in DNA that block replication. We show here that antisense suppression of WRN in two human glioma cell lines reproduces hallmarks of the drug cytotoxicity profile of WS cells, namely, hypersensitivity to 4-nitroquinoline 1-oxide, camptothecin and hydroxyurea. We also show that antisense-treated cells are hypersensitive to methyl-lexitropsin, a site-specific alkylating agent that produces mainly N3-methyladenine, a cytotoxic and replication-blocking lesion. Antisense-treated cells are hypersensitive to O(6)-methylguanine adducts as well, but only when repair by O(6)-methylguanine-DNA methyltransferase is lacking. Our results illustrate the drug sensitivity caused by deficiency of WRN in a uniform genetic background. They extend the WRN DNA damage sensitivity spectrum to methyl base adducts that can result in blocked replication, and suggest that WRN may be required for resumption of processive replication when incomplete repair of DNA damage leaves blocking lesions at forks. The evidence that highly disparate lesions fall within the purview of WRN, and that abrogating DNA repair can reveal dependence on WRN, suggests that WRN may protect the genome from the lethal, mutagenic and carcinogenic effects of widely diverse DNA damage arising from endogenous processes and environmental agents.

Background CpG methylation in the O6-methylguanine-DNA methyltransferase (MGMT) promoter is associated with better outcome following alkylating agent chemotherapy in glioblastoma (GBM) and anaplastic glioma (AG). To what extent improved response reflects low or absent MGMT activity in glioma tissue has not been unequivocally assessed. This information is central to developing anti-resistance therapies. Methods We examined the relationship of MGMT activity in 91 GBMs and 84 AGs with progression-free survival (PFS) following alkylator therapy and with promoter methylation status determined by methylation-specific PCR (MSP). Results Cox regression analysis revealed that GBMs with high activity had a significantly greater risk for progression in dichotomous (P ≤ 0.001) and continuous (P ≤ 0.003) models, an association observed for different alkylator regimens, including concurrent chemo-radiation with temozolomide. Analysis of MGMT promoter methylation status in 47 of the GBMs revealed that methylated tumors had significantly lower activity (P ≤ 0.005) and longer PFS (P ≤ 0.036) compared to unmethylated tumors, despite overlapping activities. PFS was also significantly greater in methylated vs. unmethylated GBMs with comparable activity (P ≤ 0.005), and among unmethylated tumors with less than median activity (P ≤ 0.026), suggesting that mechanisms in addition to MGMT promote alkylator resistance. Similar associations of MGMT activity with PFS and promoter methylation status were observed for AGs. Conclusions Our results provide strong support for the hypotheses that MGMT activity promotes alkylator resistance and reflects promoter methylation status in malignant gliomas. General significance MGMT activity is an attractive target for anti-resistance therapy regardless of methylation status., Highlights • Largest study to date of association of MGMT activity with treatment response. • MGMT activity is inversely associated with alkylator response in malignant gliomas. • Mean activity is significantly lower in MGMT promoter-methylated tumors. • Better response in methylated tumors is unlikely due to lower MGMT activity alone. • Supports the use of MGMT inhibitors to improve responsiveness to alkylator therapy.

Background Drug sensitivity is commonly determined by assays that utilize colony formation to discriminate between surviving and lethally treated cells. These assays require cells with high plating efficiency that form discernible colonies, are time-consuming and laborious, and require manual counting of large numbers of colonies. To overcome these drawbacks, we developed a flow cytometric technique that assays survival of proliferative capacity in cultured cells. Methods Labeling with bromodeoxyuridine for 72 h followed by bivariate Hoechst 33258/ethidium bromide flow cytometry allows discrimination of nonproliferating cells from those that have undergone one to three divisions. Addition of an internal standard, chicken erythrocyte nuclei, permits determination of total cell number. To validate our assay, we used flow and colony-forming assays to determine the sensitivity of cell lines derived from Werner syndrome patients and unaffected individuals to 4-nitroquinoline-1-oxide (4NQO) and camptothecin. Results The flow and colony-forming assays yielded comparable sensitivity for each drug and essentially identical increases in drug sensitivity exhibited by Werner syndrome cells. Conclusion Our results indicate that the flow assay is a less laborious surrogate for colony-forming assays. The flow technique will also facilitate the analysis of drug sensitivity in cells that are not amenable to colony-forming assays. Cytometry 48:1–5, 2002. © 2002 Wiley-Liss, Inc.

Background The extent of intratumoral mutational heterogeneity remains unclear in gliomas, the most common primary brain tumors, especially with respect to point mutation. To address this, we applied single molecule molecular inversion probes targeting 33 cancer genes to assay both point mutations and gene amplifications within spatially distinct regions of 14 glial tumors. Results We find evidence of regional mutational heterogeneity in multiple tumors, including mutations in TP53 and RB1 in an anaplastic oligodendroglioma and amplifications in PDGFRA and KIT in two glioblastomas (GBMs). Immunohistochemistry confirms heterogeneity of TP53 mutation and PDGFRA amplification. In all, 3 out of 14 glial tumors surveyed have evidence for heterogeneity for clinically relevant mutations. Conclusions Our results underscore the need to sample multiple regions in GBM and other glial tumors when devising personalized treatments based on genomic information, and furthermore demonstrate the importance of measuring both point mutation and copy number alteration while investigating genetic heterogeneity within cancer samples. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0530-z) contains supplementary material, which is available to authorized users.

Paclitaxel (Taxol), a cytotoxic natural product that disrupts microtubule integrity, is being clinically evaluated for use against gliomas. We examined paclitaxel-induced killing in seven cell lines derived from human malignant astrocytic gliomas and medulloblastomas with the goal of characterizing range of sensitivity, contribution of P-glycoprotein 170-mediated drug efflux to resistance, and cross-resistance with alkylating agents. Exposure to paclitaxel for 8 h or less produced biphasic survival curves for all lines, with 40-75% of cells comprising a subpopulation that was 9-26 times more resistant to paclitaxel than the more sensitive fraction. Increasing exposure to 24 h eliminated the resistant subpopulation, increasing sensitivity 50- to 400-fold. The dose producing one log of kill (LD10) after a 24-h exposure ranged from 4 to 18 nM, comparable to concentrations in the cerebrospinal fluid of brain tumor patients given a 3-h infusion of paclitaxel. Concurrent exposure to paclitaxel and either nimodipine or verapamil, inhibitors of P-glycoprotein activity, did not increase sensitivity, demonstrating that the fivefold range in sensitivity was not due to P-glycoprotein-mediated drug efflux. Importantly, there was no correlation between LD10 for paclitaxel and LD10 for 1,3-bis(2-chloroethyl)-1-nitrosourea, streptozotocin, and temozolomide, indicating no expression of cross-resistance to these different classes of tumoricidal agents. Our results suggest that greater clinical efficacy of paclitaxel against malignant brain tumors may be obtained by infusion for 24 h or longer and support the use of paclitaxel in combination with alkylating agents.

Purpose. To reduce the systemic toxicity and prolong the systemic presence of l-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU), a lipid-based drug carrier was designed and characterized.

Nucleotide excision repair (NER) is an ordered process in nonmalignant cells, in both human and nonhuman systems. We previously reported that in human brain there is discordant mRNA expression of excision repair cross-complementing (ERCC) 1 and ERCC2 in malignant tissues, concurrent with excellent concordance of these genes in nonmalignant tissues from the same patients. Here we have extended these studies to compare low-grade tumors to high-grade tumors and to include ERCC3 (which links DNA repair with DNA transcription) and ERCC6 (which is essential for gene-specific repair). Glial tumor and adjacent normal brain specimens from 19 individuals were studied. Paired malignant and nonmalignant tissues were obtained from 12 of these patients. For ERCC3, there was excellent concordance of mRNA expression between malignant and nonmalignant tissues from the same individuals (P = 0.003). For ERCC6, no concordance was observed (P = 0.314). Tumor tissue from patients with high-grade gliomas exhibited marked discordance of mRNA expression patterns in situations in which good concordance was observed in tumor tissue from low-grade gliomas. We previously established that malignant brain tumors show increased disorder of genes in the NER process, as compared with nonmalignant tissues. These data suggest that increasing disorder in the NER process may occur as cells move from low-grade to high-grade malignancy.

p53 is a tumor suppressor gene found on the short arm of chromosome 17. Loss of one p53 allele and alteration of the other is found in a variety of tumors, including highgrade glial tumors. Point mutations in the remaining allele occur in a highly conserved region of the gene encompassing exons 5-8. Although 40-50% of medulloblastomas lose sequences on the short arm of chromosome 17, alteration of p53 in these tumors is infrequent. To further characterize genetic alteration of p53 in medulloblastoma, we performed a mutational analysis of four medulloblastoma-derived cell lines established by our laboratory. Using two variable-number tandem repeat markers which map distally to p53, we found evidence indicating loss of sequences on the distal end of chromosome 17p in all four lines. However, no gross alterations of the p53 gene were detected. Northern analysis revealed expression of equivalent amounts of full-length p53 messenger RNA in each cell line. Using the polymerase chain reaction to amplify exons 5-8 of the p53 gene, we directly sequenced the amplified fragments and detected no mutations in any of the cell lines. Our results demonstrate that loss of p53 function through gene deletion and/or recessive mutation is not required for growth in our cell lines.

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) has been implicated in resistance of human brain tumors to alkylating agents. We observed that 14 human medulloblastoma- and glioma-derived cell lines differ in sensitivity to the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as shown by their 28-fold range in 10% survival dose (LD10). By using the substrate analogue inhibitor O6-benzylguanine (O6-BG), we showed that the contribution of MGMT to resistance varies widely, as evidenced by 3- to 30-fold reductions in LD10 among the lines, and varies up to 20-fold among subpopulations of individual lines. Importantly, variability in resistance, manifested as a 20-fold range in LD10, persists after measurable MGMT is eliminated, disclosing differential contributions of other resistance mechanisms to survival. Cells exposed to MNNG while suspended in growth medium are more resistant than cells alkylated as subconfluent monolayers, and MGMT accounts for a smaller proportion of their resistance. Notably, the MGMT content of the lines is not statistically correlated with MNNG resistance or with potentiation of killing by O6-BG, even though MGMT is a biochemically demonstrated determinant of resistance. In contrast, the same lines vary less in resistance to the ethylating agent N-ethylnitrosourea (ENU), and MGMT makes only a small contribution to resistance. Our results strongly indicate that resistance to both MNNG and ENU is multifactorial.

Alkylating agents have long played a central role in the adjuvant therapy of glioblastoma multiforme (GBM). More recently, inclusion of temozolomide (TMZ), an orally administered methylating agent with low systemic toxicity, during radiotherapy and afterward has markedly improved survival. Extensive in vitro and in vivo evidence has shown that TMZ-induced O6-methylguanine (O6-meG) mediates GBM cell killing. Moreover, low or absent expression of O6-methylguanine-DNA methyltransferase (MGMT), the sole human repair protein that removes O6-meG from DNA, is frequently associated with longer survival in GBMs treated with TMZ, promoting interest in developing inhibitors of MGMT to counter resistance. However, the clinical efficacy of TMZ is unlikely to be due solely to O6-meG, as the agent produces approximately a dozen additional DNA adducts, including cytotoxic N3-methyladenine (3-meA) and abasic sites. Repair of 3-meA and abasic sites, both of which are produced in greater abundance than O6-meG, is mediated by the base excision repair (BER) pathway, and occurs independently of removal of O6-meG. These observations indicate that BER activities are also potential targets for strategies to potentiate TMZ cytotoxicity. Here we review the evidence that 3-meA and abasic sites mediate killing of GBM cells. We also present in vitro and in vivo evidence that alkyladenine-DNA–glycosylase, the sole repair activity that excises 3-meA from DNA, and Ape1, the major human abasic site endonuclease, mediate TMZ resistance in GBMs and represent potential anti-resistance targets.

Summary Increasing age is the most robust predictor of greater malignancy and treatment resistance in human gliomas. However, the adverse association of clinical course with aging is rarely considered in animal glioma models, impeding delineation of the relative importance of organismal versus progenitor cell aging in the genesis of glioma malignancy. To address this limitation, we implanted transformed neural stem/progenitor cells (NSPCs), the presumed cells of glioma origin, from 3- and 18-month-old mice into 3- and 20-month host animals. Transplantation with progenitors from older animals resulted in significantly shorter (P ≤ 0.0001) median survival in both 3-month (37.5 vs. 83 days) and 20-month (38 vs. 67 days) hosts, indicating that age-dependent changes intrinsic to NSPCs rather than host animal age accounted for greater malignancy. Subsequent analyses revealed that increased invasiveness, genomic instability, resistance to therapeutic agents, and tolerance to hypoxic stress accompanied aging in transformed NSPCs. Greater tolerance to hypoxia in older progenitor cells, as evidenced by elevated HIF-1 promoter reporter activity and hypoxia response gene (HRG) expression, mirrors the upregulation of HRGs in cohorts of older vs. younger glioma patients revealed by analysis of gene expression databases, suggesting that differential response to hypoxic stress may underlie age-dependent differences in invasion, genomic instability, and treatment resistance. Our study provides strong evidence that progenitor cell aging is responsible for promoting the hallmarks of age-dependent glioma malignancy and that consideration of progenitor aging will facilitate development of physiologically and clinically relevant animal models of human gliomas.

Objective: The methylating agent temozolomide (TMZ) has markedly improved clinical outcome for patients with glioblastoma and other gliomas. While TMZ has comparatively low systemic toxicity, a minority of patients experience severe myelotoxicity that compromises TMZ treatment, necessitating dose reductions and treatment delays. These limitations emphasize the need to develop markers to identify individuals susceptible to TMZ-induced myelosuppression. The purpose of this small pilot study is to examine the association between treatment-limiting myelosuppression in primary brain tumor patients receiving TMZ and expression of O6-methylguanine-DNA methyltransferase (MGMT) in peripheral blood leukocytes (PBL). MGMT is the sole human activity that removes TMZ-induced, cytotoxic O6-methylguanine adducts from DNA. Methods: MGMT biochemical activity and MGMT promoter methylation status, a surrogate measure of MGMT expression, were assayed in PBL from 10 patients who experienced treatment-limiting myelotoxicity during TMZ therapy, 8 patients who experienced no myelotoxicity during TMZ treatment, and 10 disease-free, untreated controls. Results: MGMT activity was detectable in all 28 PBL samples, and all displayed an unmethylated promoter indicative of MGMT expression. Mean PBL MGMT activity was 2-fold lower in patients who experienced myelotoxicity compared to patients without myelotoxicity (8.9 ± 3.9 vs. 18 ± 8.1 fmol/106 cells; P 0.015) and to untreated controls (8.9 ± 3.9 vs. 16 ± 6.8 fmol/106 cells; P 0.015). Conclusions: These preliminary data indicate that low MGMT activity in PBL is associated with myelotoxicity in primary brain tumor patients receiving TMZ, and may have value if confirmed in a larger study as a marker to identify patients at greater risk of treatment-limiting myelosuppression.

Gliomas are the most frequent adult primary brain tumor, and are invariably fatal. The most common diagnosis glioblastoma multiforme (GBM) afflicts 12,500 new patients in the U.S. annually, and has a median survival of approximately one year when treated with the current standard of care. Alkylating agents have long been central in the chemotherapy of GBM and other gliomas. The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT), the principal human activity that removes cytotoxic O(6)-alkylguanine adducts from DNA, promotes resistance to anti-glioma alkylators, including temozolomide and BCNU, in GBM cell lines and xenografts. Moreover, MGMT expression assessed by immunohistochemistry, biochemical activity or promoter CpG methylation status is associated with the response of GBM to alkylator-based therapies, providing evidence that MGMT promotes clinical resistance to alkylating agents. These observations suggest a role for MGMT in directing adjuvant therapy of GBM and other gliomas. Promoter methylation status is the most clinically tractable measure of MGMT, and there is considerable enthusiasm for exploring its utility as a marker to assign therapy to individual patients. Here, we provide an overview of the biochemical, genetic and biological characteristics of MGMT as they relate to glioma therapy. We consider current methods to assess MGMT expression and discuss their utility as predictors of treatment response. Particular emphasis is given to promoter methylation status and the methodological and conceptual impediments that limit its use to direct treatment. We conclude by considering approaches that may improve the utility of MGMT methylation status in planning optimal therapies tailored to individual patients.

18 páginas, 9 figuras, 1 tabla.-- et al., [Background]: Tumor cell invasion into adjacent normal brain is a mesenchymal feature of GBM and a major factor contributing to their dismal outcomes. Therefore, better understandings of mechanisms that promote mesenchymal change in GBM are of great clinical importance to address invasion. We previously showed that the bHLH transcription factor TWIST1 which orchestrates carcinoma metastasis through an epithelial mesenchymal transition (EMT) is upregulated in GBM and promotes invasion of the SF767 GBM cell line in vitro. [Results]: To further define TWIST1 functions in GBM we tested the impact of TWIST1 over-expression on invasion in vivo and its impact on gene expression. We found that TWIST1 significantly increased SNB19 and T98G cell line invasion in orthotopic xenotransplants and increased expression of genes in functional categories associated with adhesion, extracellular matrix proteins, cell motility and locomotion, cell migration and actin cytoskeleton organization. Consistent with this TWIST1 reduced cell aggregation, promoted actin cytoskeletal re-organization and enhanced migration and adhesion to fibronectin substrates. Individual genes upregulated by TWIST1 known to promote EMT and/or GBM invasion included SNAI2, MMP2, HGF, FAP and FN1. Distinct from carcinoma EMT, TWIST1 did not generate an E- to N-cadherin "switch" in GBM cell lines. The clinical relevance of putative TWIST target genes SNAI2 and fibroblast activation protein alpha (FAP) identified in vitro was confirmed by their highly correlated expression with TWIST1 in 39 human tumors. The potential therapeutic importance of inhibiting TWIST1 was also shown through a decrease in cell invasion in vitro and growth of GBM stem cells. [Conclusions]: Together these studies demonstrated that TWIST1 enhances GBM invasion in concert with mesenchymal change not involving the canonical cadherin switch of carcinoma EMT. Given the recent recognition that mesenchymal change in GBMs is associated with increased malignancy, these findings support the potential therapeutic importance of strategies to subvert TWIST1-mediated mesenchymal change., This research was funded in part through an NIH/NINDS T32-NS-0007144 Clinical Neuroscience Training Grant (RO, LK, JM) and a University of Washington Institutional Bridge Funding Grant (RCR, AKM). Research in ISG group is supported partially by FEDER and by MICINN (SAF2009-08803), Junta de Castilla y León (CSI13A08 and proyecto Biomedicina 2009-2010), MEC OncoBIO Consolider-Ingenio 2010 (Ref. CSD2007-0017), Sandra Ibarra Foundation, NIH grant (R01 CA109335-04A1) and by Group of Excellence Grant (GR15) from Junta de Castilla y Leon.

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is a cardinal defense against the mutagenic and carcinogenic effects of alkylating agents. We have reported evidence that absence of detectable MGMT activity (MGMT(-) phenotype) in human brain is a predisposing factor for primary brain tumors that affects ca. 12% of individuals [J.R. Silber, A. Blank, M.S. Bobola, B.A. Mueller, D.D. Kolstoe, G.A. Ojemann, M.S. Berger, Lack of the DNA repair protein O(6)-methylguanine-DNA methyltransferase in histologically normal brain adjacent to primary brain tumors, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 6941-6946]. We report here that MGMT(-) phenotype in the brain of children and adults, and the apparent increase in risk of neurocarcinogenesis, may arise during gestation. We found that MGMT activity in 71 brain specimens at 6-19 weeks post-conception was positively correlated with gestational age (P

This is a report of a 53 year-old man with a glioblastoma multiforme (GBM) treated with an excessive dose of temozolomide (TMZ).This is a single case review of all clinically relevant records. O6-methylguanine-DNA methyltransferase activity was determined by a biochemical assay.Following conventional radiotherapy (RT) without concurrent chemotherapy, the patient received 5,500 mg of TMZ over 2 days. At the standard dose of 200 mg/m2/day his total 5-day dose should have been 1,940 mg. Acutely he had nausea, vomiting and diarrhea for 2 days which cleared. The dominant severe toxicity was pancytopenia between one and four weeks after TMZ which was complicated by secondary infections that were successfully managed. Transient transaminitis occurred but there were no significant pulmonary, renal or other systemic toxicities. His progression free survival was 22 months and overall survival 24 months.His outcome suggests that TMZ may prove to be a good agent for dose-escalation trials with hematopoietic stem cell rescue.

Purpose: Primary brain tumors are the leading cause of cancer death in children. Our purpose is (a) to assess the contribution of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) to the resistance of pediatric brain tumor cell lines to clinical alkylating agents and (b) to evaluate variables for maximal potentiation of cell killing by the MGMT inhibitor O6-benzylguanine, currently in clinical trials. Few such data for pediatric glioma lines, particularly those from low-grade tumors, are currently available. Experimental design: We used clonogenic assays of proliferative survival to quantitate cytoxicity of the chloroethylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and the methylating agent temozolomide in 11 glioma and five medulloblastoma lines. Twelve lines are newly established and characterized here, nine of them from low-grade gliomas including pilocytic astrocytomas. Results: (a) MGMT is a major determinant of BCNU resistance and the predominant determinant of temozolomide resistance in both our glioma and medulloblastoma lines. On average, O6-benzylguanine reduced LD10 for BCNU and temozolomide, 2.6- and 26-fold, respectively, in 15 MGMT-expressing lines. (b) O6-Benzylguanine reduced DT (the threshold dose for killing) for BCNU and temozolomide, 3.3- and 138-fold, respectively. DT was decreased from levels higher than, to levels below, clinically achievable plasma doses for both alkylators. (c) Maximal potentiation by O6-benzylguanine required complete and prolonged suppression of MGMT. Conclusions: Our results support the use of O6-benzylguanine to achieve full benefit of alkylating agents, particularly temozolomide, in the chemotherapy of pediatric brain tumors.

Purpose: Apurinic/apyrimidinic endonuclease (Ap endo) is a key DNA repair enzyme that cleaves DNA at cytotoxic abasic sites caused by alkylating agents and radiation. We have observed that human glioma cells deficient in Ap endo activity are hypersensitive to clinically used alkylators (Silber et al., Clin Cancer Res 2002;8:3008.). Here we examine the association of glioma Ap endo activity with clinical response after alkylating agent-based chemotherapy or after radiotherapy. Experimental Design: Cox proportional hazards regression models were used to analyze the relationship of Ap endo activity with time to tumor progression (TTP). Results: In a univariate model with Ap endo activity entered as a continuous variable, the hazard ratio (HR) for progression after alkylator therapy in 30 grade III gliomas increased by a factor of 1.061 for every 0.01 increase in activity (P = 0.013). Adjusting for age, gender, extent of resection, and prior treatment strengthened slightly the association (HR = 1.094; P = 0.003). Similarly, the HR for progression after radiotherapy in 44 grade II and III tumors increased by a factor of 1.069 (P = 0.008). Adjusting for the aforementioned variables had little effect on the association. In contrast, we observed no association between activity and TTP in grade IV gliomas after either alkylator therapy in 34 tumors or radiotherapy in 26 tumors. Conclusions: Our data suggest that Ap endo activity mediates resistance to alkylating agents and radiation and may be a useful predictor of progression after adjuvant therapy in a subset of gliomas.

Alkylating agents are standard components of adjuvant chemotherapy for gliomas. We provide evidence here that Ape1/Ref-1, the major mammalian apurinic/apyrimidinic endonuclease (Ap endo), contributes to alkylating agent resistance in human glioma cells by incising DNA at abasic sites. We show that antisense oligonucleotides directed against Ape1/Ref-1 in SNB19, a human glioma cell line lacking O(6)-methylguanine-DNA-methyltransferase, mediate both reduction in Ape1/Ref-1 protein and Ap endo activity and concurrent reduction in resistance to methyl methanesulfonate and the clinical alkylators temozolomide and 1,3-(2-chloroethyl)-1-nitrosourea. An accompanying increase in the level of abasic sites indicates that the DNA repair activity of Ape1/Ref-1 contributes to resistance. Conversely, we also show that exposure of SNB19 cells to HOCl, a generator of reactive oxygen species (ROS), results in elevated Ape1/Ref-1 protein and Ap endo activity, enhanced alkylator resistance, and reduced levels of abasic sites. Given current evidence that heightened oxidative stress prevails within brain tumors, the finding that ROS increase resistance to clinical alkylators in glioma cells may have significance for the response of gliomas to alkylating agent-based chemotherapy. Our results may also be relevant to the design of therapeutic regimens using concurrent ionizing radiation (a generator of ROS) and alkylating agent-based chemotherapy.

The clinical phenotype of Werner Syndrome (WRN) includes features reminiscent of accelerated aging and an increased incidence of sarcomas and other tumors of mesenchymal origin. This syndrome results from mutations in the WRN DNA helicase/exonuclease gene. We found that WRN deficient primary fibroblasts, as well as lymphoblastoid cell lines (LCLs), show reduced proliferative survival in response to 4-nitroquinoline-N-oxide (4NQO) and 8-methoxypsoralen (8MOP), compared with WRN-proficient cells. This is the first demonstration of drug hypersensitivity in primary cells of mesenchymal origin from WRN patients. Notably, 8MOP-induced DNA interstrand crosslinks, but not 8MOP mono-adducts, produced S-phase apoptosis in WRN-deficient LCLs. In contrast, 8MOP did not induce S-phase apoptosis in WRN-deficient diploid fibroblasts, in which drug hypersensitivity was entirely due to reduced cell proliferation. Such reduced proliferation of damaged mesenchymal cells in WRN patients may lead to earlier proliferative senescence. In addition, failure of WRN-deficient mesenchymal cells to undergo apoptosis in response to DNA damage in S-phase may promote genomic instability and could help clarify the increased risk of sarcoma in WRN patients. Because interstrand crosslinks are believed to be repaired through homologous recombination, these results suggest an important role for WRN in recombinational resolution of stalled replication forks.

Pediatric brain tumors are the leading cause of death in children, and survival is frequently accompanied by one or more radiation-induced adverse developmental and psychosocial sequelae. Radiotherapy (RT) is an integral component of the treatment for medulloblastoma (MB) and the only effective adjuvant therapy for ependymoma (EP). Therefore, there is an urgent need to develop strategies to enhance the tumoricidal action of RT while sparing adjacent normal tissue. The multifunctional DNA repair protein Ape1/Ref-1 has been implicated in conferring radiation resistance in pediatric brain tumors. However, inhibiting Ape1 activity in the clinic has been hindered by the lack of safe and effective drugs and siRNA delivery vehicles. We have previously developed a nanoparticle that can deliver siRNA specifically to brain tumors for efficient knockdown of GFP. Here, we aimed to deliver siRNA against Ape1 to improve tumor cell kill after RT. Nanoparticles were loaded with siApe1, or siGFP as a control, and used to treat UW228 (MB) and Res196 (EP) cells. Ape1 expression levels were measured using PCR and Western blot, and the abasic endonuclease activity of Ape1 was determined using an Ape activity assay. Cells were then treated with 137Cs-γ-rays and cell survival monitored using clonogenic assays. DNA repair in cells was assessed though quantification of abasic sites, and resulting double-strand breaks were imaged by γH2AX foci immunostaining. We found that nanoparticle-mediated siApe1 delivery reduced Ape1 expression and activity by greater than 80%. This diminished the shoulder of resistance in survival curves decreasing survival to ∼50% at 1 Gy, and was accompanied by inhibition of DNA repair. Sensitization was specific to abasic site generating treatments as response to paclitaxel was not affected. Therefore, siApe1 loaded nanoparticles may help enhance the therapeutic effect of RT in pediatric brain tumor patients by inhibiting DNA repair.