PRECLINICAL EXPERIMENTAL THERAPEUTICS AND PHARMACOLOGY

doi:10.1093/neuonc/nor158

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-01. EXPLORING NEW DRUGS THAT CAN AFFECT THE VIABILITY AND TRANSFORMATION OF CHILDHOOD MEDULLOBLASTOMAS

Helena Pavel ¹, Norbert Ajeawung ¹, Robert Faure ², Donald Poirier ², Deepak Kamnasaran ²

Abstract

BACKGROUND: Medulloblastoma is one of the most common malignant tumors of the central nervous system in newborn infants and children and accounts for about 15% to 20% of pediatric brain tumors. Despite current diagnostic and therapeutic advances, the morbidity and mortality rates still remain high. Furthermore, children who survive medulloblastoma are at risk for long-term sequelae related to the neurological effects of the tumor and surgery, radiotherapy, and chemotherapy. Therefore, it is of great importance to identify new anticancer drugs that can significantly assist in improving the survival of children with minimal or no side effects. In this study, our primary objective was to identify and study the efficacy of new structural classes of drugs belonging to a family of steroid biogenesis inhibitors and in the peroxovanadium superfamily. METHODS: We chose to undertake our preclinical testing on malignant pediatric medulloblastoma cell lines. After determining the IC50 values of our experimental drugs, we subjected our preclinical cell line models to a wide variety of cancer assays, including viability/proliferation, cell death, cell cycle regulation and transformation assays. RESULTS AND CONCLUSIONS: Our data so far demonstrate that the panel of new structural classes of drugs examined can significantly affect the viability and transformation of malignant medulloblastoma preclinical cell line models. Most importantly, the toxicity of our drugs was almost devoid in our non-transformed control cell lines. Our current work continues to explore the efficacy of these drugs in additional malignant medulloblastoma preclinical models. This body of work is novel and highly significant in our effort to discover improved treatments for childhood brain tumors.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-02. DISCOVERY OF A NEW CLASS OF MICROTUBULE-BINDING DRUGS THAT SIGNIFICANTLY AFFECT THE VIABILITY AND TRANSFORMATION OF HUMAN GLIOMA STEM CELLS

Norbert Ajeawung ¹, Harish Joshi ², Deepak Kamnasaran ³

Abstract

BACKGROUND: Gliomas are the most common primary brain tumors. Despite current treatment with surgery, irradiation, and temozolomide (TMZ), the median survival for patients diagnosed with malignant gliomas is still less than 2 years. Within the tumor mass reside glioma stem cells that exhibit seminal properties of therapeutic resistance, which leads to tumor re-growth, and hence, patient mortality. The ineffectiveness of current therapies prompted us to identify and characterize new classes of microtubule-binding drugs in an effort to improve the survival of patients with this deadly disease. METHODS: We subjected a panel of human glioma stem cell line preclinical models to a wide variety of cancer assays, including viability/proliferation, cell death, cell-cycle regulation, invasion, and transformation assays. RESULTS: Using a chemical genetic screen of microtubule-binding drugs, which are derivatives of opium alkaloids, we identified new classes of compounds that have profound inhibitory effects on the growth of human glioma stem cells while conferring little or no toxicity to non-transformed cells. Glioma stem cells treated with our drugs succumbed to significant cell cycle arrest and apoptosis, which subsequently caused profound decreases in viability, proliferation, migration, and even transformation. Further testing revealed that these drugs also enhanced the radiosensitivity of glioma stem cells. CONCLUSION: We have discovered novel drugs that can significantly inhibit the viability and growth of human glioma stem cells. These findings are expected to either replace or complement existing therapies within a clinical setting in due course.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-03. A CHEMICAL GENETIC SCREEN IDENTIFIES A NOVEL DRUG THAT TARGETS STEROID BIOGENESIS AND RECEPTOR SIGNALING LEADING TO GROWTH INHIBITION OF PEDIATRIC MALIGNANT ASTROCYTOMA CELL LINES

Donald Poirier ¹, Norbert Ajeawung ², Deepak Kamnasaran ¹

Abstract

BACKGROUND: Brain tumors are among the leading cause of cancer-related deaths in children, and at least 60% manifest as astrocytomas. Malignant astrocytomas represent 8% to 12% of all pediatric supratentorial brain tumors, with an overall median survival of 11-14 months, whereas those that arise in the brainstem represent an additional 10% to 20%, with a 10-year overall median survival rate of <10%. Despite current therapies, challenges still exist in the treatment of pediatric malignant astrocytomas, thus leading to the need to explore new therapies. Since a wide range of genes involved in steroid biogenesis and signaling is expressed in pediatric malignant astrocytomas, our objective was to investigate whether novel classes of drugs that target these gene products could be effective in inhibiting growth. METHODS AND RESULTS: We screened using a candidate chemical structure approach, a library of 400 drugs that can potentially inhibit steroid biogenesis and cell signaling. Using a panel of human pediatric malignant glioma cell lines established from surgical specimens, we discovered a potent drug that inhibits androsterone (male sex pheromone) biogenesis and has the ability to significantly reduce the viability of pediatric malignant astrocytomas in a dose-dependent manner. Cells treated with this drug responded by undergoing apoptotic, cell-cycle regulatory, and invasive changes. Furthermore, significant inhibition of transformation was noted. Cells also become increasingly radiosensitive upon drug treatment. Most remarkably, the toxicity of the drug in human astrocytes (control) was minimal. CONCLUSION: We have discovered a novel drug from a chemical genetic screen that can significantly inhibit the growth of pediatric malignant astrocytomas with minimal toxicity in non-transformed human astrocytes.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-04. COMBINED MYXOMA VIROTHERAPY WITH RAPAMYCIN TARGETING BRAIN TUMOR-INITIATING CELLS

Xueqing Lun ¹, Franz Zemp ¹, Beichen Sun ¹, Owen Stechishin ², Artee Luchman ², John J Kelly ², Samuel Weiss ², Mark G Hamilton ³, Gregory Cairncross ¹, Donna L Senger ¹, John Bell ⁴, Grant McFadden ⁵, Peter A Forsyth ¹

Abstract

Glioblastoma multiforme (GBM) still has a poor prognosis and remains largely incurable. Although temozolomide (TMZ) improves the survival of some patients with GBM, TMZ resistance is a significant problem. Therefore, new treatments that could overcome TMZ resistance are desperately needed. Recent studies have suggested that brain tumor-initiating cells (BTICs) may be responsible for the initiation of GBM and recurrence, as well as chemoresistance and radioresistance. Therefore, therapeutic strategies that target BTICs may improve GBM treatment. We previously reported that Myxoma virus (MYXV) cures most intracranial brain tumor models in nude mice, and combination with rapamycin improves efficacy in immunocompent hosts. Here, we further investigated the oncolytic potential of MYXV in animal models established using human BTICs. We found that 1) all tested BTICs were susceptible to MYXV infection and killing, even BTICs that were resistant to TMZ; 2) MYXV replicated within BTICs in vivo, and intratumoral administration of MYXV significantly prolonged survival of mice bearing BTIC tumors; 3) more importantly, MYXV combination therapy with rapamycin further improved the efficacy in half of the four BTICs tested, even in mice bearing advanced invasive BTIC tumors; 4) the mechanism of enhancement of MYXV infection/killing was partly due to rapamycin inhibition of mammalian target of rapamycin and activated pAkt in BTICs we classify as type II cells; and 5) IP-10 and MCP-1/CCL2 (monocyte chemoattractant protein-1) are two key factors that contributed to the synergy mechanism of MYXV in combination with rapamycin in some of the BTIC lines. Our study suggests that MYXV in combination with rapamycin should be considered for further exploration in the treatment of MGs that target both the BTIC and more differentiated compartments of GBMs.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-05. SAFE AND EFFECTIVE NONVIRAL DNA DELIVERY TO TREAT GLIOBLASTOMA-DERIVED BRAIN CANCER STEM CELLS

Stephany Y Tzeng ¹, Hugo Guerrero-Cazares ¹, Elliott E Martinez ², Noah P Young ², Joel C Sunshine ¹, Alfredo Quinones-Hinojosa ², Jordan J Green ¹

Abstract

INTRODUCTION: Glioblastoma (GB) is the most common tumor of the central nervous system in the United States and has a 14-month median survival time even with the gold standard of treatment. Brain cancer stem cells (BCSCs) are thought to be tumor-initiating cells and a major cause of recurrence. Because BCSCs are refractory to many conventional treatments, experimental approaches like gene delivery are attractive for targeting this crucial cell type. Nonviral gene therapy is preferable to viral methods for preventing potential health risks; however, its efficiency in previously studied methods must be improved while maintaining a good safety profile. METHODS AND RESULTS: We optimized delivery of eGFP DNA to malignant GB astrocytes derived from human patients with GB using a combinatorial library of poly(beta-amino esters) (PBAEs). Some PBAEs were significantly more effective than leading commercial (liposomal) agents like Lipofectamine 2000 or FuGENE HD (p < 0.0001 compared to each) in 10% serum. GB astrocytes were transfected with up to 61 ± 5% efficiency with less than 20% toxicity. Testing was repeated in human BCSCs by focusing on polymers that were highly effective for GB astrocytes. Nonadherent BCSC neurospheres were transfected with up to 67 ± 1.4% efficiency with less than 10% toxicity. Importantly, both GB astrocytes and GB BCSCs were transfected with significantly greater efficacy than healthy (human fetal) control neural stem cells (p = 0.0149) and astrocytes (p = 0.0388). We studied DNA delivery mechanisms by observing nanoparticle uptake and trafficking through cells and by tracking the duration of transgene expression. We also developed a formulation that could be stored for up to 3 months with no loss in efficacy, which could facilitate the practical, clinical use of DNA nanoparticles. CONCLUSION: Synthetic PBAEs are effective for DNA delivery to GB cells with specificity compared to healthy cells.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-06. PRECLINICAL STUDIES REVEAL MECHANISMS OF RESPONSE AND ADAPTATION OF PRONEURAL GLIOBLASTOMA TO SUNITINIB

Liang Lei ¹, Randy D'Amico ¹, Julia Sisti ¹, Richard Leung ¹, Adam M Sonabend ¹, Paolo Guarnieri ¹, Steven S Rosenfeld ¹, Jeffrey N Bruce ¹, Peter Canoll ¹

Abstract

Glioblastoma (GBM) is remarkably heterogeneous and may actually represent several distinct entities with different cells of origin, different genetic alterations, and different clinical behaviors. This heterogeneity and the ability of tumor cells to evolve and adapt are major obstacles to finding effective treatments of GBM, particularly therapies that target specific signaling pathways. By delivering a combination of transforming genetic alterations to glial progenitors in adult mouse brain tissue, we generated remarkably robust models resembling a specific subtype of high-grade glioma (proneural GBM). The most aggressive of these models is induced by platelet-derived growth factor (PDGF) signaling in combination with deletion of Pten and p53. Treating this model with sunitinib, which inhibits multiple receptor tyrosine kinases, including PDGFRα, led to slower tumor growth and significant survival advantage. However, all animals developed tumor recurrence. Histological analysis revealed that the recurrent tumors had dramatically changed morphology, which was characterized by diffuse infiltration of the brain and the absence of necrosis. Global expression analysis revealed consistent changes in the treated tumor that pointed to alterations in specific signaling pathways in response to sunitinib. Comparative genomic hybridization showed that the treated tumors accumulated copy number alterations not seen in the untreated tumors, thereby suggesting that the tumors genetically adapted to therapy. These studies identified new candidates that could be targeted in combination therapy to overcome the tumor relapse. Taken together, our results illustrate how a new preclinical model could be used to advance treatment discovery for a common subtype of human GBM.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-07. COMPARISON OF VERUBULIN ACTIVITY USING DIFFERENT DOSING SCHEDULES IN XENOGRAFT MODELS

Vijay R Baichwal ¹, Leslie Reeves ¹, Bradford L Chad ¹, Kenton H Zavitz ¹, Andrew P Beelen ¹, Gary G Mather ¹, Robert O Carlson ¹

Abstract

BACKGROUND: Verubulin is a microtubule-disrupting agent that crosses the blood-brain barrier and achieves brain-to-plasma concentration ratios of up to 30 in animal models. Verubulin (intravenous [IV] administration once weekly) is being evaluated in a phase IIb study in combination with radiation and temozolomide, for the treatment of newly diagnosed GBM. Verubulin is active in multiple xenograft models when administered weekly. Here, we explore its activity when administered daily or continuously for 24 hours. METHODS: B16 mouse melanoma or OVCAR3 human ovarian cancer cells were implanted subcutaneously into athymic mice. Verubulin was administered IV at its maximum tolerated dose of 7.5 mg/kg once or daily at 1 mg/kg for 5 days (B16) or at 5 mg/kg once IV or at 2.5-, 5-, or 10-mg/kg/day intraperitoneally (IP) with an Alzet mini-pump for 24 hours (OVCAR3). RESULTS: In the B16 model, verubulin at 7.5 mg/kg resulted in 72% tumor growth inhibition (TGI) on day 7 relative to vehicle (p = 0.01), whereas 1 mg/kg resulted in 22% TGI (p = 0.68). In OVCAR3, verubulin at 2.5 mg/kg over 24 hours resulted in no TGI; the single 5-mg/kg IV dose resulted in 50% tumor regression. Verubulin plasma concentration was 35 ng/mL at 24 hours (2.5 mg/kg/day IP), whereas C_max was 1291 and 105 ng/mL with a 5- and 1-mg/kg IV dose, respectively. The area under the curve for the 5- and 1-mg/kg doses were 657- and 79 hr*ng/mL, respectively. CONCLUSIONS: Verubulin showed significant activity in animal models when administered intermittently at 5 mg/kg or higher. Anti-xenograft activity was not observed when the single dose was divided into proportionately smaller daily doses or was administered continuously. Thus, the optimal dosing regimen for verubulin in xenograft models is intermittent high doses, which result in transiently elevated plasma concentrations. These data provide further rationale for the once-weekly dosing schedule being studied in the clinic.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-08. MECHANISMS THAT DETERMINE THE EFFICACY OF TWO PROTEASOME INHIBITORS, BORTEZOMIB AND MARIZOMIB, IN GLIOBLASTOMA

Christa Manton ¹, Joya Chandra ¹

Abstract

The proteasome degrades the bulk of cellular proteins, thus putting it in a position to influence protein homeostasis and important regulatory pathways. Catalytic activities in three of the beta subunits of the barrel-shaped 20S core complex carry out protein degradation. Inhibitors targeting these catalytic subunits have been shown to have a selective anticancer effect. This study explored determinants of the efficacy of bortezomib, a reversible proteasome inhibitor, and marizomib, an irreversible inhibitor, in glioblastoma cells. We previously found that bortezomib treatment caused greater DNA fragmentation and caspase 3 activation than equimolar marizomib in glioblastoma cells. This finding counters the assertion that marizomib, as an irreversible inhibitor, should inhibit the proteasome longer and cause more apoptosis. To better understand why bortezomib was more cytotoxic in these cells, we performed a timecourse to measure chymotrypsin-like proteasome activity. Both drugs caused inhibition of this activity by 2 hours, but bortezomib treatment led to more sustained inhibition. Another factor causing the differential efficacy of the drugs could be the efflux of marizomib. The channel blocker verapamil did not lead to large increases in DNA fragmentation when combined with either proteasome inhibitor, thereby suggesting that efflux through P-glycoprotein does not decrease the effectiveness of marizomib. Finally, we assessed whether drug treatment has an effect on proteasome protein levels. At 2 hours after treatment with bortezomib, there was a significant decrease in the protein level of the Β5 catalytic subunit of the proteasome. This decrease was not seen after marizomib treatment. Further studies revealed changes in other catalytic subunits, including the immunoproteasome subunit LMP7, after drug treatment. Further studies of drug-induced changes to proteasome composition are currently under way. Based on our studies, we conclude that the superior cytotoxicity of bortezomib is attributed to a number of factors, including increased duration of proteasome inhibition and changes to protein levels of proteasome subunits.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-09. MIBEFRADIL, A NOVEL THERAPY FOR GLIOBLATOMA MULTIFORME: INTERLACED THERAPY IN A MURINE MODEL

Stephen T Keir ¹, David A Reardon ¹, Julia R Saling ², Lloyd S Gray ³, Darell D Bigner ¹, Henry S Friedman ¹

Abstract

INTRODUCTION: Finding new ways to improve the efficacy of and/or overcome resistance to conventional chemotherapy for patients with brain tumor is an ongoing research paradigm. Interlaced Therapy is a synergistic cancer therapy that involves sequential administration of a T-type calcium channel blocker and a cytotoxic agent. For the purpose of this study, we interlaced mibefradil dihydrochloride (MBFD), a T-type calcium channel blocker, with the standard of care treatment, temozolomide (TMZ), for GBM. METHODS: Intracranial (ic) adult brain tumor xenografts were grown in athymic BALB/c mice. Three days after ic implantation, groups of 10 mice were randomly treated with drug vehicle/control, MBFD alone PO, TMZ alone IP, or the Interlaced Therapy of agents. Tumor responses for ic xenografts were assessed by difference in median survival. Studies were conducted in the following xenograft lines: D-54 MG, D-245 (PR) MG, D-270 MG, and D-317 MG. RESULTS: The Interlaced Therapy of MBFD and TMZ demonstrated statistically significant (p ≤ 0.026) increases in median survival of 719%, 648%, 564%, and 30% vs. control animals in D-270 MG, D-317 MG, D-54 MG, and D-245 (PR) MG, respectively. More importantly, Interlaced Therapy produced significant (p ≤ 0.004) increases in survival of 43%, 23%, and 18% vs. best single agent in D-317 MG, D-270 MG, and D-54 MG lines, respectively. MBFD alone provided significant (p ≤ 0.007) benefit in survival of 50%, 27%, and 13% in D-317 MG, D-54 MG, and D-245 (PR) MG lines, respectively. CONCLUSION: Our results demonstrate the therapeutic efficacy of the delivery of MBFD and TMZ via Interlaced Therapy against a panel of GBM xenografts grown intracranially. The blockade of T-type calcium channels may well be a target that produces clinically effective, safe cell cycle synchronization that can be used in conjunction with conventional chemotherapy to render it more effective. These results warrant further exploration of Interlaced Therapy for brain tumors.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-10. ONCOLYTIC THERAPEUTIC POTENCY OF FARMINGTON VIRUS AND MODIFIED MARABA VIRUS IN IMMUNOCOMPETENT INTRACRANIAL GLIOMA MODELS AND IN MICE BEARING HUMAN BRAIN TUMOR INITIATING CELLS MODELS

Jiqing Zhang ¹, Jan Brun ², Henry Ogbomo ¹, Franz Zemp ¹, Zhigang Wang ³, David J Stojdl ², Xueqing Lun ¹, Peter A Forsyth ¹

Abstract

Oncolytic virus therapy is a novel and promising treatment option for malignant gilomas (MGs). We investigated the oncolytic potency of Farmington virus (FMT) and modified Maraba virus (MBGG) in vitro and in experimental models of immunocompetent animals and in animals bearing established brain tumor-initiating cells (BTICs). Our results showed that the majority of glioma cell lines tested (12 of 14, 85.7%) were susceptible to FMT, and 92.9% (13 of 14) of them were susceptible to MBGG. Around 3 of 7 (43%) BTIC lines were permissive to FMT, but more than 85.7% (6 of 7) of them were killed by MBGG. First, in vivo, intracerebral (i.c.) FMT or MBGG inoculation into nontumor-bearing rats was well tolerated and produced only minimal focal inflammatory changes at the site of viral inoculation. Second, a single intratumoral (i.t.) injection of MBGG prolonged survival in rats bearing RG₂-Fluc i.c. tumors but FMT did not; multiple i.t. administration of FMT or MBGG prolonged survival in the same tumor model. Third, we also found that both FMT and MBGG (whether administered via i.v. or i.t. injection) could prolong survival in a syngeneic glioma cell line (1492) model. Fourth, multiple i.t. or i.v. administration of both viruses significantly prolonged the survival of BTIC-bearing (BT025, BT048) animal models. Our findings demonstrate that both FMT and MBGG viruses are non-neurotoxic when delivered via i.c. administration and effectively kill glioma cell lines and BTIC cells in vitro. Multiple i.t. or i.v. administration of FMT or MBGG significantly prolongs survival of immunocompetent mice or rats bearing a glioma model and BTICs bearing i.c. mice models. These very promising results suggest that both FMT and MBGG may be novel oncolytic therapeutic agents against MGs in the future.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-11. THE INFLUENCE OF THE TUMOR MICROENVIRONMENT EXPRESSION OF P-STAT3 ON THERAPEUTIC EFFICACY: CNS TUMORS PREFERENTIALLY IMPACTED

Ling-Yuan Kong ¹, Mustafa A Hatiboglu ¹, Jun Wei ¹, Yongtao Wang ¹, Kayla A McEnery ¹, Gregory N Fuller ¹, Wei Qiao ¹, Michael A Davies ¹, Waldemar Priebe ¹, Amy B Heimberger ¹

Abstract

Melanoma is a common and deadly tumor that upon metastasis to the central nervous system (CNS) has a median survival duration of less than 5 months. Activation of the signal transducer and activator of transcription 3 (STAT3) has been identified as a key mediator that drives the fundamental components of melanoma malignancy. We hypothesized that WP1066, a novel inhibitor of STAT3 signaling, would enhance the antitumor activity of cyclophosphamide (CTX) against melanoma, including disease within the CNS. We investigated the mechanisms of efficacy by conducting tumor- and immune-mediated cytotoxic assays, by evaluating the reduction of regulatory T cells (Tregs) in vivo, and by determining intratumoral p-STAT3 expression using immunohistochemistry. Combinational therapy of WP1066, with both metronomic and cytotoxic dosing of CTX, was investigated in a model system of systemic and intracerebral melanoma in syngeneic mice. Inhibition of p-STAT3 by WP1066 was enhanced with CTX in a dose-dependent manner. The enhancement of the therapeutic effect of CTX with WP1066 was minimal in the pulmonary model of melanoma that had low levels of p-STAT3 expression. However, in mice with intracerebral melanoma, the greatest therapeutic benefit was seen in animals treated with cytotoxic CTX dosing and WP1066, whose median survival time was 120 days, an increase of 375%, with 57% long-term survivors. This treatment efficacy correlated with the higher p-STAT3 expression levels within the CNS microenvironment, which provides an effective target for WP1066. The efficacy of the combination of cytotoxic dosing of CTX with WP1066 is attributed to the direct tumor cytotoxic effects of the agents as well as their combined inhibition on p-STAT3 and has the greatest therapeutic potential for the treatment of CNS melanoma.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-12. A CLINICALLY AVAILABLE INHIBITOR OF RHO KINASE BLOCKS GLIOMA DISPERSION IN VITRO AND IN VIVO

Benjamin Amendolara ¹, Orlando Gil ¹, Liang Lei ¹, Sanja Ivkovic ¹, Jeffrey Bruce ¹, Peter Canoll ¹, Steven Rosenfeld ¹

Abstract

The propensity of gliomas to diffusely invade the brain and the enhancement of this dispersion produced by antiangiogenics highlight the need to develop effective anti-invasive therapies for this tumor type. We previously showed that glioma invasion critically depends on the function of the molecular motor myosin II and its immediate upstream activator, rho kinase (ROCK). In this study, we investigated the efficacy of fasudil, a clinically available ROCK inhibitor, in glioma dispersion, using both in vitro and in vivo invasion assays. We found that fasudil reduced the migration of C6 glioma cells through 3 µm transwell pores over 14-fold compared to control and at non-toxic doses. We also examined the effects of fasudil on the migration of green fluorescent protein-expressing glioma cells in a retroviral model of glioblastoma. We generated 300-μm thick brain slices from these tumors and monitored glioma migration with time-lapse microscopy. While tumor cells were highly motile under control conditions, fasudil treatment abolished their motility. We also found that Y27632, another ROCK inhibitor, produced identical results, thus implying that our observations with fasudil were not due to off-target effects. Finally, we treated mice bearing luciferase-expressing glioma cells with either fasudil or vehicle to examine how this blood-brain barrier-permeable drug affects tumor invasion in vivo. All mice were euthanized when the first control mouse developed tumor morbidity. Fasudil reduced tumor growth by over 10-fold, as measured by bioluminescence imaging. It also significantly reduced tumor invasion of white matter tracts, with markedly fewer cells crossing the corpus callosum to the contralateral hemisphere. We conclude that a blood-brain barrier-permeable ROCK inhibitor shows promise as an effective anti-invasive treatment of glioblastoma.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-13. TRAIL CONJUGATED TO NANOPARTICLES EXHIBITS INCREASED STABILITY AND ANTITUMOR ACTIVITY IN GLIOMA CELLS AND GLIOMA STEM CELLS IN VITRO AND IN VIVO

Susan Finniss ¹, Benny Perlstein ², Cathie Miller ¹, Hana Okhrimenko ², Gila Kazimirsky ², Simona Cazacu ¹, Nancy Lemke ¹, Shlomit Brodie ¹, Sandra A Rempel ¹, Mark Rosenblum ¹, Tom Mikkelsen ¹, Shlomo Margel ², Chaya Brodie ¹

Abstract

Glioblastomas are characterized by resistance to chemotherapy and radiotherapy; therefore, alternative therapeutic approaches are needed. TRAIL induces apoptosis in cancer but not in normal cells and is considered a promising antitumor agent. However, its poor solubility and short in vivo half-life are serious impediments to its therapeutic efficacy. Nanoparticles (NPs) have been used as effective delivery tools for various anticancer drugs. In this study, we conjugated TRAIL to magnetic ferric oxide NP by binding the TRAIL primary amino groups to activated double bonds on the surface of the NP. We found that the conjugation of TRAIL to the NP increased the stabilization of TRAIL. In addition, NP-TRAIL exhibited a 2-10-fold increase in apoptotic activity against different human glioma cells and glioma stem cells (GSCs) compared to free recombinant TRAIL, without altering its lack of effect on normal cells. Using rhodamine-labeled NPs, we demonstrated that NP-TRAIL localized to the plasma membrane of glioma cells and GSCs within 10 minutes of treatment followed by internalization of NP-TRAIL to the perinuclear region 30 minutes thereafter. Using U251 cell-derived human xenografts, we found that NP-TRAIL migrated to the tumor site, whereas a significantly lower degree of migration was observed with control NPs. Administration of NP-TRAIL resulted in a significant increase in cell apoptosis, a decrease in tumor volume, and an increase in animal survival. Combined administration of NP-TRAIL and gamma-radiation or proteasome inhibitors sensitized TRAIL-resistant glioma cells and GSCs to NP-TRAIL. In summary, we found that conjugation of TRAIL to NP results in the stabilization of TRAIL and in a significant increase in its apoptotic activity both in vitro and in vivo. We suggest that conjugation of TRAIL to NP represents a targeted anticancer agent with prolonged action for the treatment of glioblastomas.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-14. TARGETING THERAPY-RESISTANT GLIOMA CELLS WITH NEW COMPOUNDS THAT INHIBIT ACTION OF SURVIVIN

Hacer Guvenc ¹, Habibe Demir ¹, Snehalata Gupta ¹, Sarmistha Mazumder ¹, Abhik Ray-Chaundhury ¹, Tom Li ¹, Chenglong Li ¹, Ichiro Nakano ¹

Abstract

Glioblastoma multiforme (GBM) is one of the most lethal diseases in adults. Despite intensive treatment with surgery, chemotherapy, and radiation therapy, recurrence of tumors is inevitable in the majority of cases. It is therefore essential to characterize the mechanisms underlying therapy resistance in GBM cells. Survivin is a member of the family of inhibitor-of-apoptosis proteins, which functions as a key regulator of mitosis and cell survival. Here, we sought to determine whether survivin is a therapeutic target of therapy-resistant GBM and to identify a novel anti-glioma agent that could inhibit the survivin-mediated pathway. Survivin expression was analyzed via immunohistochemistry with primary and recurrent GBM samples derived from 10 matched and 75 unmatched patients. Survivin expression in GBM neurospheres and differentiated cells was analyzed using immunocytochemistry and qRT-PCR. In silico drug design was used to synthesize novel compounds that inhibit survivin dimerization. The effects of these compounds were examined using patient-derived tumor spheres. In 8 of 10 patients, survivin expression was increased in recurrent GBM samples compared to primary GBM samples. The same result was obtained when we compared the unmatched cases. Survivin expression was higher in GSC compared to non-stem tumor cells. In silico drug design suggested 13 novel compounds, LLP_{1-9, 13-16}. Among these compounds, LLP₃ was found to be the most potent agent. Treatment of four patient-derived GBM samples with 50 µM of LLP₃ completely abolished their sphere-forming ability, and IC₅₀ of LLP₃ ranged between 25 and 50 µM. LLP₃ Treatment of mouse intracranial tumors derived from GBM patient cells will also be described. Upregulated survivin expression in recurrent tumors after chemoradiotherapy suggests that survivin is preferentially expressed in therapy-resistant GBM cells. Our novel compound, LLP_3, strongly arrests tumor cell growth and induces apoptosis in sphere cultures. Our results indicate a novel therapeutic strategy for GBM and, more specifically, for therapy-resistant recurrent tumors.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-15. EVALUATING NOVEL POLYMERIC MICROPARTICLE-BASED INJECTABLE MATRICES FOR LOCAL CHEMOTHERAPEUTIC DELIVERY

Ruman Rahman ¹, Cheryl Rahman ¹, Stuart Smith ¹, Donald Macarthur ¹, Felicity Rose ¹, Kevin Shakesheff ¹, Richard G Grundy ¹

Abstract

INTRODUCTION: Administering chemotherapeutic agents into the surgical resection cavity circumvents the blood-brain barrier, thereby allowing for the delivery of effective local doses to the brain surface but low toxic doses systemically. We investigated a novel polymer formulation based on poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) blended microparticles for local chemotherapeutic drug release. The biodegradable microparticles are injected at room temperature and solidify into a matrix upon delivery into the body. This mechanism potentially allows persistent tissue contact in the resection cavity lining with sustained multiple drug release, thereby targeting micro-deposits of residual neoplastic cells. METHODS: The rate of viability/growth of pediatric brain tumor cells cultured on matrices made from PLGA/PEG microparticles was assessed (live/dead staining; SEM; alamar blue proliferation assay). UV absorbance and HPLC were used to determine single/multiple release of etoposide, methotrexate, and the histone deacetylase inhibitor trichostatin A (TSA) from matrices. Cytotoxic response from cells cultured on drug-loaded matrices was evaluated using alamar blue assay. RESULTS: PLGA/PEG matrices were non-toxic to brain tumor cells and amenable to drug penetration. Sustained TSA, etoposide, and methotrexate release over 3 weeks from PLGA/PEG matrices was achieved by diffusion kinetics and followed near zero-order kinetics after an initial burst. Released agents retained cytotoxic capabilities, thus indicating that the incorporation of drugs into PLGA/PEG matrices does not impair molecular structure integrity. CONCLUSIONS: PLGA/PEG microparticle-based matrices can be used for the sustained release of agents that retain cytotoxic capabilities. Combinatorial release of TSA, etoposide, and methotrexate from a single formulation and the development of a novel diffusion chamber that enables the modeling of chemotherapy penetration through brain tissue will be discussed. The outlined proof-of-concept studies will precede local drug-delivery strategies using orthotopic xenograft models. The PLGA/PEG injectable/moldable, self-assembling drug delivery system will be widely applicable for brain tumors and other infiltrative solid tumors for which local control is an essential part of the treatment strategy.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-16. PROLONGED SURVIVAL WITH RHENIUM-186 LIPOSOMES AS CONVECTION-ENHANCED NANOPARTICLE BRACHYTHERAPY IN GLIOMA XENOGRAFTS

Andrew J Brenner ¹, Beth Goins ¹, Ande Bao ¹, Jessica Miller ¹, Abram Trevino ¹, Richard Zuniga ¹, William T Phillips ¹

Abstract

While external beam radiation is an essential component of the current standard treatment of primary brain tumors, its application is limited by toxicity at doses above 80 Gy. Recent studies have suggested that brachytherapy with liposomally encapsulated radionuclides may be of benefit, and we have reported methods to markedly increase the specific activity of ¹⁸⁶Rhenium (¹⁸⁶Re)-liposomes. To better characterize the potential delivery, toxicity, and efficacy of these high specific activity ¹⁸⁶Re-liposomes, we evaluated their intracranial application by convection-enhanced delivery (CED) in an orthotopic U87 glioma rat model. After establishing an optimal volume of 25 µL, we observed focal activity confined to the site of injection over a 96-hour period. Doses of up to 1850 Gy were administered without overt clinical or microscopic evidence of toxicity. Animals treated with ¹⁸⁶Re-liposomes had a median survival of 126 days (95% confidence interval [CI] 78.4-173 days) compared to 49 days (95% CI 44-53 days) in controls. Log-rank analysis between these two groups was highly significant (p = 0.0013) and was even higher when 100 Gy was used as a cutoff (p < 0.0001). Noninvasive luciferase imaging as a surrogate for tumor volume showed that separation in bioluminescence between animals treated with ≤100 Gy was statistically significant by 11 days post-treatment χ²(1, N = 19) = 4.8, p = 0.029. Magnetic resonance imaging also supported this difference in tumor size. Analysis of tumors by histology revealed minimal areas of necrosis and gliosis. These results support the potential efficacy of high specific activity brachytherapy by ¹⁸⁶Re-liposome CED in glioma.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-17. HYALURONAN OLIGOMERS (O-HA) AS ADJUVANT TO RADIOTHERAPY AND CHEMOTHERAPY REGIMENS FOR THERAPY-RESISTANT BRAIN TUMORS

Anne G Gilg ¹, Karen G Bowers ¹, Bryan P Toole ², Bernard L Maria ¹

Abstract

INTRODUCTION: Pediatric malignant gliomas, especially those classified by the World Health Organization as grade IV glioblastomas, are among the most devastating human tumors. Glioblastomas are highly resistant to both radiotherapy and chemotherapy. Recent experiments have shown that a key factor involved in resistance to therapy is the polysaccharide hyaluronan, a major component of the pericellular matrix surrounding glioblastoma cells in vivo. Through interactions with its cell surface receptor, CD44, hyaluronan constitutively regulates receptor tyrosine kinase activation, antiapoptotic pathway activities, and ABC-family drug transporter function. Small hyaluronan oligomers inhibit these activities and consequently suppress the growth of several tumor types in vivo. METHODS: Spheroids were prepared from fresh biopsies of human glioblastomas to enrich for malignant, therapy-resistant cells that closely mirrored the parent tumor. The effect of hyaluronan oligomers on survival of glioblastoma cells isolated in this manner was measured using the WST assay and the clonogenic assay. RESULTS: Preliminary work showed that systemic administration of these oligomers sensitized tumor cells to chemotherapeutic agents and irradiation in vivo. The objective of our studies was to determine whether a combination of hyaluronan oligomers and chemotherapy or irradiation would synergistically inhibit human glioblastoma cell survival. Our results show that hyaluronan oligomers reduce the resistance of human glioblastoma cells to irradiation and chemotherapy. CONCLUSION: Despite advancements in surgical resection, local radiotherapy, and chemotherapy, long-term survival rates of patients with glioblastoma multiforme remain poor. Cancer cells exhibit multiple methods of resistance to therapies, including enhanced drug efflux transporters and increased cell survival signaling receptors. We previously demonstrated that the stability of these cell-surface proteins is modulated by their association with CD44, which is in turn bound to hyaluronan, an extracellular sugar polymer scaffold for CD44 and its cell surface protein partners. Our results suggest that hyaluronan oligomers may be utilized as an adjuvant to standard therapies in patients with malignant brain tumors.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-18. ACQUIRED TEMOZOLOMIDE RESISTANCE IN HUMAN MALIGNANT GLIOMAS: A PROTEOMICS APPROACH AND THE ROLE OF PROLYL 4-HYDROXYLASE, BETA POLYPEPTIDE

Gilberto K Leung ¹, Stella Sun ¹, Stanley T Wong ¹, Xiao Qin Zhang ¹, Jenny K Pu ¹, Wai Man Lui ¹

Abstract

Temozolomide (TMZ) provides significant benefits in the treatment of malignant glioma, but many patients continue to suffer from progressive or recurrent disease because of the intrinsic or acquired resistance of their tumors to TMZ. In this study, we employed proteomic profiling to study changes in global protein expression in TMZ-resistant malignant glioma cells. We also investigated the potential role of one of the protein candidates, prolyl 4-hydroxylase, beta polypeptide (P4HB), that may be involved in the development of acquired TMZ resistance. Human malignant glioma cell lines (D54 and U87) with acquired TMZ resistance were first developed, and their biological behaviors were characterized before protein profiling analysis using 2-dimensional gel electrophoresis. Potential protein spot candidates were identified by MALDI-TOF/TOF. Transient transfection with small interfering RNAs was used to knock down potential candidates to study their biological roles. Our results revealed 15 protein spots that were highly upregulated or downregulated proteins in TMZ-resistant glioma cells. Among these, the protein P4HB was found to be consistently dysregulated in TMZ-resistant D54 sublines when compared to control sublines. Dysregulated expression of P4HB was also confirmed in U87 cells. Transient knockdown of P4HB expression was found to resensitize TMZ-resistant cells to subsequent treatment with TMZ. P4HB is known to play an important role in hypoxia-induced cell death. To our knowledge, this study is the first to describe the roles of P4HB in the regulation of TMZ-induced apoptosis in malignant glioma cells and in the development of acquired TMZ resistance. P4HB is a potential candidate for future investigations of TMZ-induced cytotoxicity and novel therapeutic strategies for the treatment of TMZ-resistant malignant glioma.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-19. DNA METHYLATION AND HDAC INVOLVEMENT IN MEDULLOBLASTOMA THERAPY RESISTANCE AND PROGRESSION

Anna M Marino ¹

Abstract

Epigenetic alterations are potentially reversible by a number of small molecules known as epigenetic drugs. DNA methylation and histone modifications are the most studied epigenetic aberrations. We have combined DNA methylation and histone deacetylase (HDAC) inhibitors, alone and together with other drugs. Our results showed cell cycle arrest, induction of apoptosis, synergistic cell killing after combination with other drugs, and inhibition of clonogenicity. We are now studying alterations in methylation and upregulation of different HDACs in medulloblastoma cell models. Previous results showed synergistic effects after combining treatment with 5-aza-2'-deoxycitidine, 4-phenylbutyrate (an HDAC class 1 inhibitor) and imatinib, and these effects were dose dependent. Our results also showed an inhibition of clonogenicity and cell-cycle arrest after combination treatments, the results varied in the cell lines used. Previously, we checked the cell lines for levels of Gli and beta catenin. Therefore, we aimed at investigating the role of the HDAC class 1 family and DNA methylation in medulloblastoma cell models. We will assay global methylation by employing LUMA bisulfite pyrosequencing (to study promoter methylation) and immunoprecipitation techniques followed by electrophoresis to investigate different members of the HDAC class 1 family before and after treatment with 5-aza-2'deoxycitidine, 4-phenylbutyrate, and RTKi. Our findings warrant strong consideration of HDAC inhibitors and DNA methylation inhibitors in future clinical trials and for a better understanding of the role of HDACs and DNA methylation.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-20. NIGELLA SATIVA AND THYMOQUINONE INDUCE CASPASE-9/3 ACTIVATION AND GLIOBLASTOMA CELL DEATH

Isa M Hussaini ¹, Samson Amos ², Kendra Simpson ², Gerard T Redpath ², Charles Lyons ², Charles Dipierro ²

Abstract

The seeds of Nigella sativa have been used for centuries by various cultures for the treatment of a host of illnesses and symptoms, including asthma, inflammation, and fever. Recently, N. sativa seed oil was shown to exhibit anticonvulsant and anti-prostate cancer activities. We investigated the effect of N. sativa oil in glioblastoma cell lines (U-1242, U251, U-87, U-373, A172 and SNB19) and identified the active component responsible for its anticancer activity. We characterized thymoquinone as the anticancer compound in N. sativa oil. Both N. sativa seed oil (50-500 µg/ml) and thymoquinone (1-20 µM) inhibited, in a dose-dependent manner, glioblastoma multiforme (GBM) cell growth and soft agar colony formation with IC₅₀ values of 260 µg/ml and 6 µM, respectively. In contrast, hydroquinone had no effect on the viability of GBM cell lines at 500 µg/ml. The seed oil and its active component also reduced GBM size and volume in a xenograft mouse model. DNA microarray pharmacogenomic analysis revealed that N. sativa and thymoquinone had no effect on the mRNA levels of apoptosis-associated genes but significantly reduced the level of insulin growth factor-1 receptor mRNA. In contrast, the cell death induced by N. sativa and thymoquinone was accompanied by caspase-9/3 activation and the cleavage of the death substrate poly(ADP-ribose) polymerase (PARP). To evaluate the toxicity of N. sativa and thymoquinone, we assayed their effects on liver enzymes in vivo and found them to be non-toxic at 50 mg/kg. Taken together, these results suggest that N. sativa oil and thymoquinone may be useful alone or in combination with currently used therapeutic agents in the management of malignant astrocytomas.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-21. PRECLINICAL USE OF MILD HYPERTHERMIA TO ENHANCE DRUG DELIVERY TO BRAIN TUMORS

Gerald A Grant ¹, Christy Wilson ¹, Sara Salami ¹, Paolo Macaroni ¹, Shuqin Li ¹, Ji-Young Park ¹, David Needham ¹, Darell Bigner ¹, Mark Dewhirst ¹

Abstract

The inability of chemotherapeutic agents to cross the blood-brain barrier and reach brain tumors at a therapeutic concentration is a major limitation. Methods for applying mild hyperthermia in peripheral tumors has been studied, but the applicability of those methods to enhance drug delivery to brain tumors is novel. Current methods of hyperthermia treatment of brain tumors involve inserting probes or antennae to locally generate heat, a strategy that is invasive and requires monitoring to ensure proper placement. The current work is a feasibility study of the use of a surface-based miniature microwave applicator to focally heat brain tumors through a cranial window to enhance drug delivery to the tumor. We anesthetized 25-28-g CD1 nu/nu mice, placed them in a stereotactic frame, and removed a 4-mm x 6-mm bone flap. Dura was then excised, and 106 D270 human xenograft glioblastoma tumor cells were injected into the right hemisphere and a cranial window was placed. Mild hyperthermia was achieved by applying a specially designed microwave applicator ipsilateral to the tumor after 12-14 days. The power (heat) deposition of the applicator was optimized using a numerical model of the mouse brain and electromagnetic simulation software. Doxorubicin (Dox) was delivered using low temperature sensitive liposomes (LTSLs) that released at 41.5°C, and intravital confocal microscopy was used to visualize the distribution of Dox into the brain tumor. Confocal microscopy showed that Dox released from the LTSLs following mild hyperthermia (4-5°C) penetrated deeper into the tumor tissue compared to Dox alone or under normothermic conditions. The ability to apply mild hyperthermia noninvasively to brain tumors can significantly enhance the delivery of chemotherapeutic agents measured by concentration and depth of penetration. This study shows promise that thermosensitive liposomes can be delivered to brain tumors and that a miniature microwave applicator approach is a feasible option for such heating.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-22. ACTIVE EFFLUX AT THE BLOOD-BRAIN BARRIER LIMITS EFFECTIVE PHARMACOTHERAPY OF MALIGNANT GLIOMA

John Ohlfest ¹, Jose Gallardo ¹, Sagar Argawal ¹, Rajendar Mittapalli ¹, Randy Donelson ¹, William F Elmquist ¹

Abstract

P-glycoprotein (PgP) and breast cancer resistance protein (BCRP) efflux drugs from brain capillary endothelial cells back into the blood. The relevance of active efflux in preventing effective pharmacotherapy of glioma has been questionable because the tight junctions of the blood-brain barrier are disrupted in the tumor core, thereby allowing some systemic drug delivery. We determined the effect of BCRP/PgP expression on the efficacy of drugs being used to treat glioblastoma. Malignant gliomas were induced by brain transfection with PDGF and a short hairpin RNA against P53 in mice that were wild-type (WT) or deficient for both alleles of MDR1 (genes encoding PgP) and BCRP, hereafter referred to as “triple knockout” (TKO) mice. Dasatinib, a kinase inhibitor being tested in clinical trials, or temozolomide was administered orally to mice bearing well-established glioblastoma. TKO mice treated with dasatinib or temozolomide survived over twice as long as WT mice. Microdissection of the tumor core, invasive rim, and normal brain revealed 2-5-fold increases in dasatinib brain concentrations in TKO mice relative to WT mice. Western blotting for phospho(p)-AKT and phospho(p)-Src (a dasatinib target) demonstrated profound inhibition of signaling in the normal brain, tumor rim, and tumor core of TKO mice. In contrast, dasatinib had no effect on p-Src or p-AKT levels in the normal brain and tumor rim and a modest effect in the tumor core of WT mice. These data suggest that both standard and experimental therapies are limited in their efficacy by active efflux in areas of tumor-infiltrated normal brain, which appears to have a relatively intact blood-brain barrier and is the disease that causes recurrence after surgery in the vast majority of patients. This provides a strong impetus for developing dual BCRP/PgP inhibitors, because previous studies have shown that PgP and BCRP have redundant function in many cases.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-23. TARGETED THERAPY FOR BRAFV600E MUTANT MALIGNANT ASTROCYTOMA

Theodore Nicolaides ¹, Sujatmi Hariono ¹, Krister Barkovich ¹, Rintaro Hashizume ¹, David Rowitch ¹, William Weiss ¹, Denise Sheer ², Suzanne Baker ³, Barbara Paugh ³, Todd Waldman ⁴, Huifang Li ⁴, Chris Jones ⁵, Tim Forshew ², David James ¹

Abstract

Malignant astrocytomas are an aggressive group of central nervous system tumors with a poor prognosis. Activating mutations of BRAF (BRAFV600E) have been reported in a subset of these tumors, especially those occurring in children. In this report, we investigated the incidence of BRAFV600E mutation in additional pediatric patient cohorts and examined the effects of pharmacologic blockade of BRAF protein activity in preclinical models of BRAFV600E mutant malignant astrocytoma. BRAFV600E mutations were identified in 3/28 (11%) and 3/30 (10%) grade III and grade IV pediatric malignant astrocytomas, respectively. BRAF protein was overexpressed, relative to normal human astrocytes, in 16 of 20 malignant astrocytoma cell lines, among which BRAFV600E was found in four cases (20%). Among serially propagated grade IV malignant astrocytoma (i.e., glioblastoma multiforme) xenografts, BRAFV600E was identified in 2 of 16 cases (13%), both of which were proneural subtype. When the BRAFV600E specific inhibitor PLX4720 was used, pharmacologic blockade of BRAF revealed preferential antiproliferative activity against BRAFV600E mutant cells in vitro, in contrast to the use of shRNA-mediated knockdown of BRAF, which inhibited cell growth of glioma cell lines regardless of BRAF mutation status. Using an orthotopic xenograft model, we demonstrated that PLX4720 treatment decreased tumor growth and increased overall survival in mice bearing BRAFV600E xenografts, while being ineffective, and possibly tumor promoting, against xenografts with wild-type BRAF. These results support the evaluation of BRAFV600E-specific inhibitors for efficacy in treating BRAFV600E malignant astrocytoma.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-24. INTERFERON-BETA INCREASES THE SENSITIVITY OF STEM-LIKE GLIOMA CELLS TO TEMOZOLOMIDE

Happold Caroline ¹, Roth Patrick ¹, Lamszus Katrin ², Frei Karl ¹, Tabatabai Ghazaleh ¹, Weller Michael ¹

Abstract

Temozolomide is an alkylating chemotherapeutic drug that significantly prolongs the survival of patients with newly diagnosed glioblastoma in combination with surgery and radiotherapy. This effect is strongly associated with the methylation of the O⁶-methyl guanine transferase (MGMT) gene promoter in glioma cells, and in vitro data suggest an additional role for the tumor suppressor protein p53. Prior reports revealed a sensitizing effect of temozolomide by interferon-beta in an MGMT- and p53-dependent manner. Here, we aimed to determine whether this effect is restrained to MGMT- and p53-expressing glioma cells and whether stem-like glioma cells can be targeted by interferon-beta and temozolomide as well. Glioma cells expressing MGMT and p53 as well as transfectants with silencing of either MGMT or p53 were exposed to temozolomide in increasing doses after pre-exposure to interferon-beta and assessed for acute and clonogenic survival. Identical experiments were performed with stem-like glioma cells with positive or negative MGMT/p53 expression. Interferon-beta strongly sensitized glioma cells to temozolomide-induced cell death. This sensitization was independent of p53 since LNT-229 glioma cells with a siRNA-mediated silencing of the p53 gene became more susceptible to temozolomide after prior exposure to interferon-beta, too. Moreover, LN-18 MGMT-positive glioma cells and transfectants with downregulated MGMT were equally sensitized to temozolomide by interferon-beta. Interestingly, these sensitization effects were demonstrated even more clearly in stem-like glioma cells when compared to their adherent counterparts. In summary, we demonstrate that interferon-beta-mediated sensitization to temozolomide is mediated by neither p53 nor MGMT and that interferon-beta also targets stem-like glioma cells, thereby suggesting that interferon-beta might be a powerful adjuvant for enhancing the benefit of temozolomide chemotherapy in glioblastoma.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-25. SPECIFIC TARGETING OF EGF-RECEPTOR IN MALIGNANT GLIOMAS WITH DOUBLE-STRANDED RNA POLY(I:C) AND POLYETHYLENE GLYCOL-LINEAR POLYETHYLENIMINE AS A CARRIER MOLECULE

Valerie Albrecht ¹, Jun Thorsteinsdottir ¹, Ernst Wagner ², Jörg-Christian Tonn ¹, Manfred Ogris ², Christian Schichor ¹

Abstract

INTRODUCTION: Glioblastoma multiforme (GBM) is characterized by an overexpression of the wtEGF receptor (wtEGFR) and its variant vIII (deltaEGFR), which is important for the proliferation, invasion, and angiogenesis of glial tumor cells. Targeted cellular delivery of proapoptotic, immune stimulatory double-stranded RNA polyinosinic:polycytidylic acid (poly[I:C]) via this receptor can be of therapeutic importance and was the aim of this study. METHODS: Tissue samples from glioblastomas and nonneoplastic brain tissue were stained immunohistochemically for wtEGFR/deltaEGFR expression. Additionally, different malignant glioma cell lines (U87, U251, U373, U138, U87wtEGFR, and U87deltaEGFR) as well as primary cells isolated from GBM (primary culture and mesenchymal stem-like cells [gbMSCs]) in early passages (p < 5) were analyzed via immunocytochemistry and flow cytometry. Virus-sized particles (polyplexes) were formed with RNA and a nucleic acid carrier conjugate based on linear polyethylenimine and an epidermal growth factor receptor (EGFR)-targeting ligand. Cells were then exposed to poly(I:C) polyplexes, the respective control poly(I) polyplexes, or no polyplex for up to 48 hours. Inhibition of proliferation was measured with the sulforhodamine-B proliferation assay. RESULTS: As expected, overexpression of EGFR was found in all glioma samples, compared to nonneoplastic brain tissues. All tumor cell lines displayed overexpression of EGFR, except EGFR-negative U138-cells. The primary cultures derived from GBM showed strong positivity for EGFR, whereas other subpopulations (gbMSCs) showed only weak positivity. Endothelial cells displayed no EGFR staining at all. Incubation of the cell lines and isolated cells with poly(I:C) polyplexes resulted in the effective suppression of proliferation, depending on the respective EGFR expression. CONCLUSION: Tumor cell proliferation was effectively inhibited by treatment with the dsRNA-containing polyplexes targeting EGFR in malignant glial tumor cells. These findings render this agent a promising new candidate for potential tumor cell receptor-targeted therapy in GBM.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-26. COMPARATIVE STUDIES OF PLATINUM COMPOUNDS WITH AND WITHOUT RADIATION, THREE ROUTES OF ADMINISTRATION, AND PLATINUM FORMULATIONS FOR GLIOBLASTOMA TREATMENT: IN VIVO EXPERIMENTS

Gabriel Charest ¹, Benoit Paquette ¹, Léon Sanche ¹, David Mathieu ¹, David Fortin ¹

Abstract

OBJECTIVES: Despite recent advances, radiotherapy and chemotherapy protocols only marginally improve the overall survival of patients with glioblastoma (GBM). In our study, the anticancer efficiency with and without radiation combination of five platinum compounds (cisplatin, oxaliplatin, their liposomal formulations, and carboplatin) was tested. The liposomal formulations were included since they could potentially reduce the toxicity of cisplatin and oxaliplatin. We also investigated the tumor drug uptake according to three different routes of administration: intravenous (i.v.), intaarterial (i.a.) by the carotid artery, and i.a. with prior blood-brain barrier disruption (BBBD). The aim of this study was to find a better route of administration, a better chemotherapy formulation, and a better post-administration time to combine ionizing radiation in clinical GBM therapy. METHODS: The tumor F98 glioma implanted in the brain of Fischer rats was used as model to mimic human glioblastoma. To improve the efficiency of chemotherapeutic agents, the BBB was temporary disrupted prior to i.a. administration. RESULTS: Experiments using the i.v. and i.a. route of drug administration were completed. The i.a. route allowed preferential uptake (up to 140x) into the tumor DNA compared to the i.v. administration. Our study confirms that the liposomal formulations allowed the bypass of toxicity and considerably improved the lifespan of the animals. Concomitant i.a. treatment with radiotherapy further extended the mean survival times, with the highest efficiency obtained with carboplatin (47 days) compared to 34 days with radiation only. The same drugs delivered by i.v. administration did not show a significant difference compared to radiation alone. CONCLUSIONS: Our study confirms that the liposomal formulations allowed the bypass of toxicity and considerably improved the lifespan of the animals. I.a. drug administration improves tumor uptake and lifespan considerably compared to i.v. injection.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-27. NOV C-TER: A NOVEL ANTIANGIOGENIC AND ANTITUMOR AGENT

Xiaoyang Qi ¹, Franck Cuttitta ², Zhengtao Chu ¹, Jerome Celerier ³, Jihane Pakradouni ³, Olivier Rixe ⁴

Abstract

NOV (nephroblastoma overexpressed) C-terminal sequence (C-Ter) is the carboxy-terminal sequence (170 aa) of the NOV/CCN3 protein. NOV/CCN3 (357 aa), a member of the CCN family, is secreted by quiescent cells as endothelium and is involved in the regulation of various cellular functions, including angiogenesis, proliferation, differentiation, survival, adhesion, and migration. It activates the Notch signal and inhibits VEGF₁₆₅ and FGF signals through the cysteine-rich domain at the C-Ter. Inhibition of the proliferation and tumorigenicity of glioma cells by NOV/CCN3 has been previously reported. Here, we report the antiangiogenic and antitumor activities of NOV C-Ter. In vitro, NOV C-Ter inhibited porcine aortic endothelial tube formation and HUVEC and human endothelial cell growth. NOV C-Ter demonstrated a differential cytotoxic effect in several glioblastoma cell lines, including X-12, U373, and Gli36. In vivo, NOV C-Ter showed antitumor activity in an orthotopic glioma model using human X-12 cells. Nude mice (10 mice per group) received intracranial X-12 cells, followed by NOV C-Ter treatment (i.v. injection), r PBS (i.v. injection), or bevacizumab (i.p. injection). NOV C-Ter provided a significant overall survival (OS) benefit over the control (PBS) and bevacizumab arms: the median OS times were 100 and 152 days for PBS and bevacizumab groups, respectively, while it was not reached after 186 days in the NOV C-Ter group. No significant difference in body weight was observed between the NOV C-Ter and PBS control groups. In summary, NOV C-Ter demonstrated antiangiogenic and cytotoxic effects. NOV C-Ter could represent an effective future therapeutic strategy in glioblastoma.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-28. CDK4/6 INHIBITION FOR THE TREATMENT OF ATYPICAL TERATOID/RHABDOID TUMORS

Rintaro Hashizume ¹, Ashley Gragg ¹, Sabine Muller ¹, Anuradha Banerjee ¹, Joanna Phillips ¹, Michael Prados ¹, Daphne Haas-Kogan ¹, Nalin Gupta ¹, David James ¹

Abstract

Atypical teratoid/rhabdoid tumors (AT/RTs) are highly aggressive embryonal pediatric central nervous system (CNS) tumors. The development of novel, effective therapeutic approaches for treating AT/RTs has been hindered by their infrequent occurrence, resulting in limited opportunities for studying this cancer. INI1/hSNF5 inactivation is the initiating event in ATRT development. INI1/hSNF5-mediated tumor suppression occurs through the induction of G1 arrest by the repression of cyclin D1-CDK4/6, activation of cyclin-dependent kinase inhibitors p16INK4a and p21CIP, and pRb. These observations suggest that disruption of the cyclinD1-CDK4/6-p16INK4a-Rb growth regulatory axis is critical for achieving high-grade malignancy in tumors initiated from the loss of INI1/hSNF5 function. To characterize the components of the cyclinD1-CDK4/6-p16INK4a-Rb signaling pathway, we conducted Western blot analysis. AT/RT cells expressed Rb, cyclinD1, and CDK4/6 but lacked expression of p16INK4a. A selective CDK4/6 inhibitor, PD-0332991, showed dose-dependent growth inhibition in AT/RT cells with IC50 values of 0.71 ± 0.08 µM (BT12) and 0.58 ± 0.18 µM (BT16) and induced G1 cell cycle arrest. Clonogenic survival assays revealed dose-dependent antiproliferative effects in AT/RT cells. Finally, we investigated the antitumor activity of PD-0332991 alone and in combination with radiation in human AT/RT intracranial xenografts. PD-0332991 showed antitumor activity and significant survival benefit (p < 0.0001). Radiation therapy inhibited tumor growth in a dose-dependent manner. Early in vivo studies suggest an additive effect of the combination of PD-0332991 and radiation therapy. Our results indicate that 1) AT/RT cell lines express Rb, cyclinD1, and CDK4/6 but lack the cyclin-dependent kinase inhibitor p16INK4a; 2) a CDK4/6 inhibitor, PD-0332991, induces dose-dependent growth inhibition and G1 cell cycle arrest in AT/RT cell lines; 3) in vivo treatment with PD-0332991 or radiation indicates antitumor activity against orthotopic AT/RTs, resulting in increased survival; and 4) targeting CDK4/6 is a rational therapy for AT/RTs and should be considered for its potential to enhance tumor response to irradiation.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-29. NARCICLASINE, AN OLD COMPOUND WITH NOVEL MECHANISMS OF ACTION, IS A PROMISING WEAPON AGAINST BRAIN TUMORS

Lefranc Florence ¹, Van Goietsenoven Gwendoline ², Mathieu Véronique ², Kiss Robert ²

Abstract

INTRODUCTION: Narciclasine extracted from daffodil, an Amaryllidaceae isocarbostyril controlling plant growth by means of actin cytoskeleton modulation, could represent a powerful new weapon targeting the Achilles' heel of brain tumors. METHODS: The in vitro effects of narciclasine on cell proliferation, morphology, actin cytoskeleton organization, and the Rho/ROCK/LIMK/cofilin pathway have been determined. In vivo, we used human glioblastoma models (Hs683 and primary culture), primary culture of human melanoma brain metastases, and a non-small-cell pulmonary cancer implanted into the brain of immunodeficient mice. RESULTS: Narciclasine displayed potent in vitro antitumor activity below 100 nM in the NCI 60 cancer cell line panel. It was far less toxic in normal than in cancer cells. The compound was proapoptotic at high concentrations ( ≥ 1 µM) to cancer cells of epithelial origin but not to glioma cells. In glioma cells, which are naturally resistant to apoptosis, narciclasine impaired the organization of the actin cytoskeleton at concentrations that were antiproliferative (IC₅₀ values of 30-90 nM) through the activation of the ROCK/LIMK/cofilin pathway. The same features were observed in melanoma cells that were also resistant to apoptosis. Treatment of glioma cells with narciclasine at 50-100 nM caused an increase of unpolarized cells with strong stress fibers and focal adhesions all around. Narciclasine impaired protein synthesis in cancer cells through the inhibition of the eEF1A elongation factor and also affected actin cytoskeleton organization. The treatment of human glioblastoma orthotopic xenograft- and melanoma metastasis orthotopic xenograft-bearing immunocompromized mice with nontoxic doses of narciclasine significantly increased their survival. Narciclasine antitumor effects were of the same magnitude as those conferred by temozolomide. CONCLUSION: Specific and selective targeted delivery approaches should be envisaged to move narciclasine into preclinical trials of patients with brain malignancies, including gliomas and brain metastases.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-30. INFLUENCE OF BEVACIZUMAB ON THE TARGETED DELIVERY OF TYROSINE KINASE INHIBITORS AND TEMOZOLOMIDE IN PRIMARY GBM XENOGRAFT MODELS

Sagar Agarwal ¹, Rajendar K Mittapalli ¹, Ling Cen ², Brett L Carlson ², William F Elmquist ¹, Jann N Sarkaria ²

Abstract

Combinations of bevacizumab with tyrosine kinase inhibitors (TKIs) are being studied in clinical trials for recurrent glioblastoma multiforme (GBM). Since antiangiogenic therapy can restore blood-brain barrier (BBB) integrity, we hypothesized that bevacizumab might actually impair the delivery of TKIs in GBM. To this end, we investigated the in vivo action of two important BBB efflux pumps, p-glycoprotein (Pgp) and breast cancer resistance protein (BCRP), on the brain distribution of erlotinib and dasatinib. The brain-to-plasma ratios in wild-type FVB mice were 0.02 ± 0.02 and 0.04 ± 0.03 for erlotinib and dasatinib, respectively, whereas the same ratios in triple-knockout mice (TKO; mdr1a/b−/− and BCRP1−/−) lacking efflux pump activity were 0.62 ± 0.27 and 0.65 ± 0.33, respectively, which indicates that active efflux transport is an important mechanism limiting the brain distribution of these TKIs. To test whether bevacizumab affects brain tumor accumulation, we treated mice with intracranial GBM10 xenografts with bevacizumab or placebo for 1 week and then co-administered dasatinib and temozolomide (TMZ). At 1 hour post-dose, mice were euthanized and the brain-to-plasma ratio of the tumor-bearing hemisphere was compared between groups. The mean tumor-to-plasma ratio for TMZ following bevacizumab pretreatment was significantly reduced compared to placebo (0.61 ± 0.23 vs. 0.38 ± 0.07, respectively; p = 0.04). A similar effect was observed with dasatinib (1.34 ± 1.11 vs. 0.39 ± 0.35, respectively; p = 0.07). A refined experiment using fluorescence-aided dissection of GBM12 xenografts is currently being analyzed for the interaction between bevacizumab and erlotinib. In an efficacy evaluation in GBM12, erlotinib or bevacizumab monotherapy significantly extended survival compared to placebo (p < 0.0001), but there was no evidence of additive benefits with the combination (p = 0.33, compared to erlotinib alone) (median survival times: placebo, 27 days; erlotinib, 47 days; bevacizumab, 39 days; and erlotinib + bevacizumab, 35 days). Collectively, these data suggest that bevacizumab therapy may compromise the achievable intratumoral levels of concurrently administered drugs, which may limit the efficacy of combination therapy.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-31. LEVERAGING GABRA5 EXPRESSION IN CLINICALLY AGGRESSIVE MEDULLOBLASTOMAS AS A NOVEL THERAPEUTIC TARGET

Soma Sengupta ¹, Shyamal D Weeraratne ¹, Sundari Rallapalli ², Vladimir Amani ¹, Jessica Pierre-Francois ¹, Natalia Teider ¹, Alexander Rotenberg ¹, James Cook ², Scott L Pomeroy ¹, Frances Jenses ¹, Yoon-Jae Cho ¹

Abstract

INTRODUCTION: Neural tumors have distinct genetic signatures indicative of their cellular and/or developmental origins. Thus, they often express factors such as neurotransmitter receptors that are important in the function and/or maintenance of their lineage. Although neurotransmitter receptors have been well-defined in normal neurophysiological, developmental, and pharmacological settings, their importance in the maintenance and progression of brain tumors and, importantly, the effect of their targeting in brain cancers remain obscure. In a recent large-scale genomic analysis of primary medulloblastomas, we demonstrated high levels of GABRA5 expression in a particularly aggressive molecular subtype of medulloblastoma. Here, we hypothesize that modulation of GABA receptor activity in these tumors could alter their survival characteristics and provide a novel treatment strategy for individuals diagnosed with this subtype of medulloblastoma. METHODS: We treated medulloblastoma cell lines expressing GABRA5 with GABA receptor agonists, such as GABA and QHii066, and nonspecific GABA receptor antagonists, such as picrotoxin. We then monitored the effects of such treatment using standard assays for proliferation, cell cycle analysis, apoptosis, and differentiation. Our results revealed a potent decrease in medulloblastoma cell survival through agonism of GABA receptors in a GABRA5-dependent manner. In particular, treatment with QHii066, a highly specific GABRA5 agonist, resulted in the largest decrease in medulloblastoma cell survival. Furthermore, QHii066 markedly increased the radiosensitivity and chemosensitivity of medulloblastoma cells in a GABRA5-dependent manner. CONCLUSION: We have leveraged the expression of GABRA5 in clinically aggressive medulloblastomas for therapeutic targeting. Specific agonism of GABRA5 receptors with QHii066 results in decreased medulloblastoma cell survival and markedly increases the efficacy of chemotherapy and radiotherapy. Thus, our results provide a novel approach to the treatment and management of this highly lethal medulloblastoma subtype.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-32. ELECTROPORATION-INDUCED BBB DISRUPTION AND TISSUE DAMAGE DEPICTED BY MRI

Mohammad Hjouj ¹, David Last ², David Guez ², Dianne Daniels ², Jacob Lavee ², Boris Rubinsky ¹, Yael Mardor ²

Abstract

Electroporation (EP), based on the delivery of electric pulses, is known to induce plasma membrane permeability and focal tissue ablation. Here, we demonstrate the application of EP for obtaining transient blood-brain barrier (BBB) disruption. We injected 33 rats with Gd-DOTA and treated them (for <1 minute) with G25 electrodes implanted 6 mm deep into the striata. Magnetic resonance imaging (MRI) was acquired at 30 minutes and 1, 3, 8, and 14 days posttreatment. BBB disruption was depicted as clear enhancement on T1 MRI. Early treatment effects and late irreversible damage were clearly depicted on T2, fluid attenuated inversion recovery, and short T1 inversion recovery MRI. Treatment effects increased in the first 24 hours by a factor of 2.5 ± 0.6 and then decreased/stabilized by day 14. Hemorrhages depicted early on GEMRI preceded permanent damage. Diffusion weighted MRI at 30 minutes showed no significant effects. Eight rats treated with two electrodes positioned on both sides of the midline, demonstrated large BBB disruption volumes (227.3 ± 60.9mm³) with significant brain toxicity and mortality within 24 hours. Eighteen rats were treated with one intracranial electrode and another external electrode: disruption of 6 treated at 300 V/600 V was 27.2 ± 4.4/98.7 ± 11.0 mm³ with T2 damage of 20.5 ± 3.3/61.1 ± 9.6mm³, 50.3 ± 7.4/167.2 ± 33.2mm³, and 11.3 ± 4.6/56.7 ± 9.7mm³ at 30 minutes, day 1, and day 14, respectively. At 250 V, disruption was 21.4 ± 1.9 mm³ with no damage in 5 of 6 treated rats. Adding the 300 + 600 V data resulted in significant correlation (r² = 0.75, p < 0.0003) between BBB disruption and permanent damage. Disruption was still noticed at day 3 but was reversed by day 8 in most rats. Seven additional rats were treated with two electrodes in the same hemisphere at 300-400 V. These rats showed increased disruption volumes, 69.1 ± 7.6 mm³, with compatible damage 41.2 ± 7.0mm³, in agreement with the correlation presented above. These results demonstrate the feasibility of applying this minimally invasive rapid treatment for transient BBB disruption with/without permanent damage and applying MRI for planning/monitoring disruption volume/shape by optimizing electrode positions and treatment parameters.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-33. INCREASED EFFICACY OF NANOLIPOSOMAL TOPOTECAN VIA ANTIBODY-MEDIATED EGFR-TARGETING

Laura P Serwer ¹, Charles O Noble ², Karine Michaud ¹, Daryl C Drummond ², Tomoko Ozawa ¹, Yu Zhou ¹, James D Marks ¹, Krystof Bankiewicz ¹, John W Park ¹, David James ¹

Abstract

INTRODUCTION: Nanoliposomal topotecan (nLS-TPT) has been shown to have antitumor activity when administered directly to brain tumors by convection-enhanced delivery (CED). As a DNA topoisomerase I inhibitor, topotecan (TPT) must be internalized to have a cytotoxic effect. Increasing cellular internalization may be one way of increasing the antitumor activity of nLS-TPT. Toward this aim, we attached an epidermal growth factor receptor (EGFR)-specific antibody to the nLS-TPT surface (nLS-TPT-EGFR). The aim of this study was to determine whether EGFR targeting increases rates of TPT-nLS internalization in vitro and whether nLS-TPT-EGFR offers superior efficacy compared to nLS-TPT in vivo. METHODS: Internalization of liposomes was determined using both high-performance liquid chromatography and flow cytometry to test for the presence of TPT and fluorescently labeled liposomes, respectively. In vivo efficacy was assessed in three distinct mouse models of glioblastoma: U87 cells that overexpress EGFRvIII (U87vIII), a subcutaneously propagated xenograft model that expresses EGFRvIII (GBM39), and an additional xenograft model that does not express EGFR (GBM43). All cell sources were modified to express firefly luciferase, allowing for the noninvasive determination of intracranial tumor growth. Mice were treated with either nLS-TPT or nLS-TPT-EGFR via CED. RESULTS: Internalization studies showed a highly significant increase in the rate of internalization of nLS-TPT-EGFR when compared to nLS-TPT in all the cells that expressed EGFR. In vivo studies in both EGFR-expressing models showed a substantial benefit of nLS-TPT-EGFR treatment compared to nLS-TPT treatment. Studies in the U87vIII model demonstrated that the efficacy of nLS-TPT-EGFR is dose-dependent, with the effects of targeting most prominent at higher dose levels. In cell sources that do not express EGFR (GBM43), nLS-TPT-EGFR showed no benefit over nLS-TPT. CONCLUSION: Our results support the use of nLS-TPT-EGFR against glioblastomas that overexpress EGFR by showing that EGFR targeting improves nLS-TPT internalization which in turn delays tumor growth and prolongs survival.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-34. INTRANASAL PERILLYL ALCOHOL FOR THE TREATMENT OF MALIGNANT GLIOMAS: PRECLINICAL VALIDATION

Weijun Wang ¹, Heeyeon Cho ², Michael Weintraub ¹, Niyati Jhaveri ³, Shering Torres ¹, Nicos Petasis ⁴, Axel H Schonthal ⁵, Stan G Louie ⁶, Florence M Hofman ³, Thomas C Chen ²

Abstract

Perillyl alcohol (POH), a monoterpene purified from trees, has been administered intranasally in patients with recurrent malignant gliomas, with good tolerance and no long-term central nervous system or systemic side effects. Six-month progression-free survival rates for recurrent malignant gliomas have been documented at 50% in phase II trials in Brazil. To better understand the mechanism of POH action against malignant gliomas, we performed in vitro and in vivo intranasal studies. In vitro studies demonstrated that POH is effective against both temozolomide (TMZ)-sensitive and TMZ-resistant gliomas. POH induced endoplasmic reticulum (ER) stress, leading to increased expression of glucose-regulated protein-78 (GRP78), activating transcription factor 3 (ATF3), and C/EBP-homologous protein. POH alone and/or in combination with other ER stress-inducing drugs (i.e., 2,5-dimethyl-celecoxib, nelfinavir, orTMZ) induces apoptosis in both TMZ-sensitive and TMZ-resistant glioma cells and in glioma cancer stem cells. Functional inhibition of glioma secretion of vascular endothelial growth, interleukin-8, and transforming growth factor beta was documented via Western blotting. To demonstrate that intranasal delivery of POH is effective in treating intracranial gliomas, luciferase-positive U251 TMZ-resistant glioma cells were stereotactically implanted into the right frontal lobe of nude mice. Intranasal delivery of POH was well tolerated and demonstrated loss of luciferase positivity in the implanted tumor cells. Our data demonstrate that POH is an effective anti-glioma agent that can be administered intranasally as an anti-glioma therapy.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-35. TARGETING THE TUMOR HETEROGENEITY IN GLIOBLASTOMA: A CHIMERIC ANTIGEN RECEPTOR MOLECULE MEDIATES BISPECIFIC ACTIVATION AND TARGETING OF T LYMPHOCYTES

Zakaria Grada ¹, Meenakshi Hegde ¹, Donald R Schaffer ¹, Alexia Ghazi ¹, Tiara Byrd ¹, Gianpietro Dotti ¹, Winfried Wels ¹, Helen E Heslop ¹, Stephen Gottschalk ¹, Matthew Baker ¹, Nabil Ahmed ¹

Abstract

The downregulation or mutation of target antigens is a common tactic creating antigen loss escape variants. Targeting multiple antigens on tumor cells, simultaneously, could offset this escape mechanism and possibly allow for simultaneous targeting of the tumor and elements of its microenvironment. We used protein structure and docking platforms to construct a chimeric antigen receptor (CAR) specific for CD19 and HER2. Although CD19 and HER2 are not naturally coexpressed in normal or malignant mammalian cells, using them allowed us to distinctly test the bispecific functionality of this CAR. This “chimeric” CAR molecule (C-CAR) was expressed in CD3/CD28-activated T cells by retroviral transduction. The functionality of C-CAR-expressing bispecific T cells was tested in cytotoxicity and cytokine release assays. Modeled structures and docking produced complexes with favorable interaction of the C-CAR and the published CD19 and HER2 sequences. The sequence of the C-CAR exodomain was confirmed using restriction enzyme digestion and single nucleotide sequencing. T cells expressed both the CD19 and the HER2 scFv as judged by flow cytometry analysis. In cytotoxicity assays, C-CAR-transduced T cells recognized and killed both CD19- and HER2-positive tumor cell targets. Soluble HER2 protein blocked tumor cell lysis in a HER2 protein-dependent manner. Similarly, CD19-blocking antibodies inhibited CD19 killing in an antibody concentration-dependent manner. C-CAR-grafted T cells secreted both interferon-gamma and interleukin-2 in coculture with CD19- and HER2-positive tumor cells. CD19-negative and HER2-negative target cells were not lysed and induced no cytokine release. This novel CAR confers bispecific effector functions to T cells. T cells targeting two antigens simultaneously have the potential to improve current T cell therapy approaches for cancer by allowing for the targeting of multiple antigens expressed by the tumor or its microenvironment.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-36. AMG 595, A NOVEL ANTIBODY DRUG CONJUGATE APPROACH FOR TARGETING EGFRVIII-EXPRESSING GLIOBLASTOMA

Kevin J Hamblett ¹, Carl J Kozlosky ¹, Hua Liu ¹, Sophia Siu ¹, Taruna Arora ¹, Marc W Retter ¹, Katherine Matsuda ¹, John S Hill ¹, William C Fanslow ¹

Abstract

Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively active mutant of EGFR in which exons 2-7 have been deleted. EGFRvIII is present in 30% to 50% of glioblastoma cases and is not significantly expressed in normal tissues. A fully human antibody was developed against EGFRvIII using XenoMouse technology. AMG 595 is a specific antibody drug conjugate that targets cells expressing EGFRvIII; AMG 595 is composed of a fully human anti-EGFRvIII specific antibody conjugated to the maytansinoid DM1 via a non-cleavable linker. AMG 595 exhibits potent in vitro cell growth inhibition against EGFRvIII-expressing cells. The efficacy of AMG 595 was evaluated in three EGFRvIII-expressing xenograft models. The dose response of AMG 595 was assessed in mice bearing subcutaneous U251 EGFRvIII-expressing xenografts, and regressions were observed at multiple dose levels. The efficacy of AMG 595 was also evaluated in subcutaneous xenografts of D317, a human tumor model that was derived from a patient tumor that endogenously expressed EGFRvIII. Treatment of D317 subcutaneous xenografts with AMG 595 induced metaphase arrest as evinced by increased levels of phosphohistone H3, leading to tumor regression. AMG 595 was also efficacious in an orthotopic model of D317. A phase I clinical trial with AMG 595 in patients with EGFRvIII-expressing glioblastoma is planned to begin in 2011.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-37. TARGETED DELIVERY OF GOLD NANOPARTICLES TO THE INVASIVE FRONT OF MALIGNANT GLIOMA USING TRANSCRANIAL MAGNETIC RESONANCE IMAGE-GUIDED FOCUSED ULTRASOUND

Roberto J Diaz ¹, Arnold Etame ¹, O'Reilly Meaghan ², Todd Mainprize ², Christian Smith ¹, Kullervo Hynynen ², James Rutka ¹

Abstract

INTRODUCTION: We previously demonstrated the enhanced uptake of 50-nm gold nanoparticles into normal rat brain parenchyma after blood-brain barrier (BBB) opening with transcranial magnetic resonance image (MRI)-guided focused ultrasound (TcMRgFUS). Gold nanoparticles have therapeutic potential, as these particles may be used for thermotherapy or conjugated to small molecule inhibitors, antibodies, standard chemotherapeutic agents, or small interfering RNAs. We hypothesized that TcMRgFUS could be used to open the BBB at the margin of an intra-axial tumor, thereby allowing the targeted delivery 50-nm polyethylene glycol-coated gold nanoparticles (AuNPs) into the brain parenchyma at the tumor invasive front. METHODS: We inoculated 9L rat gliosarcoma cells (160,000 cells per animal) into the right frontal lobe of 8 Wistar rats. Eight days after inoculation, the rats were anesthetized, tail vein catheters were placed, and intracranial imaging with a 1.5 Tesla magnetic resonance imaging (MRI) system was performed. In rats with visible tumor, AuNPs (∼ 14 mg AuCl mass/kg animal weight) were administered via tail vein followed by microbubbles at sonication. Focused ultrasound was directed to the tumor margin using four sonication points spaced in a box surrounding the tumor and administered with peak rarefaction pressure of ∼0.23 MPa (551.5 kHz transducer). Control animals with visible tumor received the AuNPs and microbubbles but not TcMRgFUS. BBB opening was confirmed by contrast-enhanced T1-weighted MRI in three axial planes. Silver augmentation localized AuNPs on histology. RESULTS: Of the 8 rats, 5 developed visible tumors on MRI. Parenchymal distribution of AuNPs at the invasive tumor margin was observed when TcMRgFUS was applied (n = 2) but was absent in nonsonicated tumors (n = 2). CONCLUSION: MRI-guided transcranial focused ultrasound can be used to target the entry of circulating AuNPs into the brain parenchyma at the invasive front of an intra-axial tumor. This technique holds promise for the delivery of targeted AuNPs to human brain tumors.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-38. ENHANCEMENT OF ONCOLYTIC VIRUS THERAPY BY AN ANGIOGENESIS INHIBITOR, ATN-224

Jason Pradarelli ¹, Ji Young Yoo ¹, Azeem Kaka ¹, Christopher Alvarez-Breckenridge ¹, Quintin Pan ¹, E Antonio Chiocca ¹, Theodoros Teknos ¹, Balveen Kaur ¹

Abstract

Oncolytic viruses (OVs) are genetically modified viruses specifically designed to destroy cancerous cells only. Within the tumor microenvironment, the angiogenic and inflammatory responses following OV inoculation have significantly limited the efficacy of oncolytic herpes simplex virus (oHSV) in clinical trials. Physiological levels of copper support angiogenesis and can inhibit the replication of wild-type HSV. Here, we tested whether OV efficacy could be enhanced by ATN-224, a second-generation analog of ammonium tetrathiomolybdate (TM), a copper-chelating agent approved by the U.S. Food and Drug Administration for the treatment of Wilson's disease and currently under investigation as an antiangiogenic and antineoplastic agent in clinical trials. Under serum concentration of copper, both OV replication and glioma cell killing were significantly inhibited (P < 0.001). ATN-224 treatment rescued this copper-mediated inhibition of OV replication and cytotoxicity in vitro (P < 0.01). Antitumor efficacy was evaluated in vivo using two xenograft glioma models. First, mice implanted with subcutaneous U251T3 gliomas were treated with PBS, ATN-224, OV, or OV + ATN224 (n = 10). Average tumor size in the OV + ATN224 group was significantly smaller than in the group treated with OV alone (21.51 vs. 153.93 mm³, P = 0.0383). Next, mice implanted with intracranial U87deltaEGFR gliomas were treated as previously described (n = 8). Kaplan-Meier analysis revealed greater mean survival duration in the OV + ATN224 group than in the group treated with OV alone (43.875 days vs. 24.000 days). To test for a biological explanation for this enhanced efficacy, we evaluated U251T3 tumors from OV-treated mice receiving ATN-224 or PBS cotreatment. Analysis revealed that ATN-224-treated tumors had significantly increased viral presence in vivo compared to PBS-treated tumors. This result was observed when OV was administered both intratumorally (P < 0.05) and intravenously. Therefore, the reduced tumor growth and increased survival previously shown in vivo might be associated with enhanced viral replication. Collectively, our results strongly suggest that the cotreatment of ATN-224 with OV can significantly improve the poor efficacy profile of conventional clinical oncolytic virotherapy.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-39. ARE TOPOISOMERASE INHIBITORS AN ALTERNATIVE TREATMENT OPTION FOR THERAPY-RESISTANT BRAIN TUMORS?

Sang Y Lee ¹, Becky Slagle-Webb ¹, Jonas M Sheehan ¹, James R Connor ¹

Abstract

In the course of evaluating the impact of hemochromatosis gene polymorphisms on cancer cell phenotype, we found both a neuroblastoma and astrocytoma cell line that were resistant to chemotherapy (e.g., temozolomide) and radiation treatment. Using these cell lines, we used our Drug Discovery core to identify a compound that was cytotoxic to these cells but not to human primary astrocytes. This compound is an analog of thiobarbituric acid. We previously reported that this compound is effective in both subcutaneous and intracranial mouse tumor models and has a good safety profile. The mechanism of action of the lead compound has topoisomerase inhibition activity. The lead compound inhibited topoisomerase II-alpha activity with an IC₅₀ of around 5.0 µg/ml. At concentrations higher than 3.15 or 6.3 µg/ml, the lead compound completely inhibited topoisomerase II-alpha-mediated DNA relaxation or topoisomerase II-alpha-catalyzed kDNA decatenation. However, the lead compound did not inhibit topoisomerase I activity. To determine whether topoisomerase inhibitors would be useful in the treatment of temozolomide-resistant tumors, we tested other topoisomerase inhibitors. We found that doxorubicin was cytotoxic to the therapy-resistant astrocytomas. Testing of additional compounds is in progress. These data suggest that topoisomerase inhibitors may be an alternative treatment option for temozolomide- and radiation-resistant astrocytomas.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-40. A BIOSTABLE SYNTHETIC KININ DERIVATIVE ACTING ON B1 RECEPTORS INDUCES SELECTIVE BLOOD TUMOR BARRIER PERMEABILITY IN THE F98 GLIOMA RAT MODEL

Jerome Cote ¹, Martin Lepage ¹, Fernand Gobeil Jr ¹, David Fortin ¹

Abstract

INTRODUCTION: Treatment of brain tumors with chemotherapy is limited mostly because the blood-brain barrier (BBB) prevents the delivery of drugs to migrating tumor cells. The objective of this study was to characterize BBB permeabilization induced by a synthetic stable kinin B1 receptor agonist (NG29) in F98 glioma-implanted Fischer rats using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). METHODS: The DCE-MRI study was performed with a bolus injection of different sized contrast agents (CAs), Gd-DTPA (0.5 kDa) and Gadomer (17 kDa) (0.1 mmol/kg via the caudal vein). A second MRI session was performed with the same CAs immediately following the BBB disruption procedure, which consisted of an infusion of NG29 (0.1 ml/minute; 2.5-50 nmol/kg/minute) alone or in combination with the B1R antagonist R892 (20 nmol/kg/minute), B2R antagonist HOE140 (20 nmol/kg/minute), nitric oxide synthase inhibitor L-NA (5 mg/kg), or cyclooxygenase inhibitor meclofenamate (5 mg/kg) intraarterially (right external carotid) or intravenously (caudal). Postcontrast T1-weighted images were analyzed for the presence or absence of contrast enhancement within and surrounding the tumor area and mathematically processed to yield a CA distribution volume. RESULTS: Our results suggest that the intracarotid infusion of NG29, but not LDBK, modulated topographic uptake profiles of both CAs within rat glioma and brain tissue surrounding the tumor, as observed by increase of both CADV and mean Gd concentration in the implanted hemisphere. The latter effect was not seen in normal brain parenchyma from the ipsilateral and contralateral hemispheres and was negated by coinfusion of excess B1R antagonist R892 or cyclooxygenase inhibitor meclofenamate, but not by B2R antagonist HOE140 or by nitric oxide synthase inhibitor L-NA. The BBB permeabilizing effect of NG29 lasted less than 2 hours and did not affect blood pressure, heartbeat, or arterial blood gases. CONCLUSION: NG29 enhances tumor BBB permeability through B1R in a prostanoid-dependent fashion.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-41. DETERMINING DRUG SENSITIVITY OF PATIENT-DERIVED GLIOMA CELL CULTURES

Anne Kleijn ¹, Rutger Balvers ¹, Jenneke Kloezeman ¹, Clemens Dirven ¹, Martine Lamfers ¹, Sieger Leenstra ¹

Abstract

In recent years, an enormous number of antineoplastic agents have been developed that may benefit patients with glioma. Considering the number of agents, it is impossible to test all compounds in clinical trials. Therefore, a rapid in vitro screening assay on primary patient-derived glioma cultures can help identify promising compounds. To assess the potential of a new compound, a reliable, fast read-out of the treatment effect on the glioma cells is necessary. This can be done using cell proliferation, cell viability, cytotoxicity, or apoptosis assays that are colorimetric-, fluorescence-, or luminescence-based. To select the optimal assay for this drug screening, we compared several assays in read-out after treating glioma cell lines and primary cultures with three distinct types of treatment: temozolomide (TMZ), irradiation (RT), and the kinase inhibitor rapamycin. The glioma cell lines U373 and T98, which are known to be sensitive to TMZ and RT, were used, and the effect of treatment was measured using apoptosis assays (caspase 3/7-, 8- and 9-based), the clonogenic and crystal violet assay, several cell proliferation and viability assays (nicotinamide adenine dinucleotide [NADH]-, adenosine triphosphate [ATP]-, and live-cell protease-based) and cytotoxicity assays (lactase dehydrogenase- and dead-cell protease-based). The apoptosis and cytotoxicity assays did not unequivocally detect treatment effects and were excluded from further testing. The remaining assays were further tested in primary cultures. The clonogenic and crystal violet assays were technically not feasible in these cultures. The NADH-based (colorimetric) and ATP-based (luminescent) viability assays showed similar results; however, the latter had smaller standard deviations and direct read-out. Importantly, drugs that interfere with cell metabolism (such as glycolysis inhibitors) require alternative techniques, such as cell counting or confluency monitoring to accurately measure treatment effects. Therefore, we consider the combination of ATP luminescence assays with confluency monitoring as the best read-out for drug screening in primary patient-derived glioma cultures.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-42. NF1-DEFICIENCY DEFINES A SUBGROUP OF GBM SENSITIVE TO CLINICALLY AVAILABLE MEK INHIBITORS

Wendy See ¹, I-Li Tan ¹, Theodore Nicolaides ¹, Russell Pieper ¹

Abstract

The Ras pathway is aberrantly activated in most glioblastomas (GBMs) and signals downstream through the RAS/RAF/MEK/ERK and PI3-kinase pathways to promote growth and survival. In the mesenchymal subset of GBMs, Ras activation is associated with mutations and/or deletions of NF1, a negative regulator of Ras signaling. Because NF1-deficient acute myeloid leukemias are sensitive to MEK inhibitors, we reasoned that NF1-deficient GBMs may represent a similarly targetable population. To test this idea, we screened a panel of GBM cell lines, and immunoblot analysis revealed that 4 of 18 were NF1-deficient. Treatment with clinically available MEK inhibitors caused a dramatic decrease in cell growth (PD0325901 IC50 ≤100 nM, AZD6244 IC50 = 300 nM) in 2 of 4 NF1-deficient GBM cell lines and in the case of LN229 cells induced apoptosis, suppressed the growth of intracranial xenografts in mice, and enhanced animal survival. Re-expression of GFP-tagged full-length NF1 dramatically decreased proliferation in the NF1-deficient, MEK inhibitor-sensitive LN229 cells but not in the NF1-deficient, MEK inhibitor-insensitive U251 cells, thereby suggesting that MEK inhibitor sensitivity correlates with dependency on NF1-regulated pathways for growth. The dual PI3-kinase/mammalian target of rapamycin inhibitor PI-103 did not confer or enhance sensitivity to MEK inhibitors in NF1-deficient cells, and PTEN status did not correlate with MEK inhibitor sensitivity, thus suggesting that the growth of MEK inhibitor-sensitive GBM cells is not dependent on PI3-kinase signaling. Basal levels of p-ERK, the downstream effector of MEK, however, were elevated in LN229 clones selected for MEK inhibitor resistance, whereas levels of p-AKT, a downstream effector of the PI3-kinase pathway, were unchanged, further supporting the role of RAS/RAF/MEK/ERK pathway signaling in the growth of NF1-deficient GBM cells. Together, these data show that the subset of NF1-deficient GBMs dependent on RAS/RAF/MEK/ERK signaling are uniquely sensitive to MEK inhibitors and may be ideal targets for MEK inhibitor-based clinical trials.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-43. ONCOLYTIC ADENOVIRUS DELTA-24-RGD INDUCES CELL LYSIS THROUGH AUTOPHAGY AND AUTOPHAGY-TRIGGERED CASPASE ACTIVITY

Hong Jiang ¹, Erin White ¹, Christian I Ríos-Vicil ², Wai-Kwan A Yung ¹, Candelaria Gomez-Manzano ¹, Juan Fueyo ¹

Abstract

Oncolytic adenoviruses such as Delta-24-RGD are promising therapies for patients with brain tumors. Clinical trials have shown that the potency of these cancer-selective adenoviruses should be increased to optimize therapeutic efficacy. One potential strategy is to increase the efficiency of adenovirus-induced cell lysis, the mechanism of which has not been clearly described. In this study, we provide the first report that autophagy plays a role in adenovirus-induced cell lysis. At the late stage after adenovirus infection, numerous autophagic vacuoles accompany the disruption of cellular structure, leading to cell lysis. The virus induces a complete autophagic process from autophagosome initiation to its turnover through fusion with lysosomes, although the formation of autophagosomes is sufficient for virally induced cell lysis. Importantly, downmodulation of autophagy genes with siRNA rescues the infected cells from being lysed by the virus, indicating that autophagy plays a role in the process. Moreover, autophagy triggers caspase activity via the extrinsic FADD/caspase 8 pathway, which also contributes to adenovirus-mediated cell lysis. Collectively, our findings implicate autophagy and caspase activation as part of the mechanism for cell lysis induced by adenoviruses and suggest that manipulation of the process is a potential strategy for optimizing the clinical efficacy of oncolytic adenoviruses. Since the FADD/caspase 8 pathway is usually inactivated in gliomas, an oncolytic adenovirus with the backbone of Delta-24-RGD, which expresses caspase 8 under the control of adenoviral major late promoter, is being developed in the laboratory. We expect this new version of Delta-24-RGD will display higher efficacy in lysing glioma cells, resulting in better prognosis for patients with glioma.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-44. ARE XENOGRAFT STUDIES OF ONCOLYTIC VIRAL THERAPY MISLEADING US? DETERMINING THE BARRIERS TO MYXOMA VIRUS TREATMENT IN SYNGENEIC MURINE MODELS OF GLIOMA

Franz J Zemp ¹, Brienne A McKenzie ¹, Xueqing Lun ¹, Grant McFadden ², Peter A Forsyth ³

Abstract

Initial trials using oncolytic virotherapy (OV) for malignant gliomas (MGs) have demonstrated their safety but have not shown significant clinical responses. Myxoma virus (MYXV) has produced tremendous results in mouse orthotopic xenografts, in some instances “curing” animals with a single intracranial injection. However, when translating these studies into syngeneic orthotopic grafts in rodent glioma models, we have not observed any treatment efficacy with MYXV alone. Our studies utilize primary glioma lines derived from a spontaneous murine glioma model (NF1^+/−TP53^+/− mice). These grafts accurately recapitulate the genetic and histologic appearance of human MGs in C57Bl/6 mice. Several of these lines display exquisite susceptibility to MYXV replication and oncolysis in vitro but do not show any replication or efficacy in vivo, with complete viral clearance occurring within ∼5 days posttreatment. Rapid viral elimination suggests involvement of CNS innate immune responses, and this was confirmed using immunodeficient RAG1^−/- mice. We have demonstrated that resistance is independent of type-I interferon, and our in vivo studies suggest that the tumors were already in an antiviral state prior to MYXV administration. Flow cytometry data demonstrated that these tumors contain resident microglial populations that rise rapidly with blood-derived macrophages in response to MYXV treatment. We are currently investigating whether these tumor-resident and/or -recruited microglia/macrophages play a role in mediating in vivo resistance. To this end, we are utilizing an inducible system to specifically ablate these populations (CD11b::DTR) and knockout mice to inhibit their recruitment (CXCR3^−/- and CCR2^−/- mice). Our studies with these models are designed to measure the effect of these immunocytes on both treatment resistance and overall gliomagenesis. These studies will allow us to understand the barriers to OV therapy for MGs and allow us to direct the genetic and/or pharmacological manipulation of the tumor microenvironment to maximize the potential of this therapy.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-46. WEE1 IS A PROMISING MOLECULAR TARGET FOR THE TREATMENT OF PEDIATRIC HIGH-GRADE GLIOMAS

Sabine Mueller ¹, Xiaodong Yang ¹, Rintaro Hashizume ¹, Ashley Gragg ¹, Ivan Smirnov ¹, Micheal Prados ¹, David C James ¹, Joanna J Phillips ¹, Mitchel S Berger ¹, David H Rowitch ¹, Nalin Gupta ¹, Daphne H Haas-Kogan ¹

Abstract

INTRODUCTION: High-grade gliomas (HGGs) are one of the most aggressive pediatric brain tumors. Targeting key cell cycle-regulating kinases such as G2/M check-point inhibitor WEE1 has become an attractive therapeutic approach, particularly as cell cycle inhibitors have become accessible for clinical evaluation. We investigated the role of WEE1 inhibitor MK-1775 alone and in combination with radiation for the treatment of pediatric HGGs. METHODS: Gene expression analysis was performed in 37 pediatric gliomas (3 grade I, 10 grade II, 11 grade III, and 13 grade IV) and 8 control samples using the Agilent 4x44K array. WEE1 expression was also investigated using immunohistochemistry (IHC). Clonogenic survival assays were performed to assess the radiosensitizing effects of MK-1775. In vivo activity of MK-1775 in combination with radiation was assessed in an orthotopic model of pediatric HHG. RESULTS: WEE1 was overexpressed in pediatric HGGs and increasing expression significantly correlated with increasing malignant grade (p = 0.007 for grade 3 + 4 vs. 1 + 2). Overexpression revealed by array analysis correlated with protein overexpression revealed by IHC (Spearman's p = 0.49; p = 0.048). Clonogenic survival assay results showed an MK-1775 dose-dependent antiproliferative effect in each of 5 glioma cell lines. MK-1775 displayed IC50 values of 50-100 nM as a single agent and enhanced antitumor effects when combined with radiation. Combined MK-1775 and radiation conferred significant survival benefit in vivo, compared to radiation (p = 0.0061) or MK-1775 (p = 0.0009) monotherapy. CONCLUSION: WEE1 is overexpressed in pediatric HGGs and correlates with increasing malignancy grade. The WEE1 inhibitor MK-1775 shows antiproliferative effects in all HGG cell lines tested, independent of p53 status. In vivo combination of MK-1775 with radiation confers a survival advantage compared to either therapy alone. Our results advance WEE1 as a promising target for the treatment of pediatric HGGs and highlight the WEE1 inhibitor MK-1775 as a promising new therapeutic agent.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-47. THE ADDITION OF SUNITINIB TO RADIOTHERAPY DELAYS TUMOR GROWTH IN A MURINE MODEL OF GLIOBLASTOMA

Randy D'Amico ¹, Liang Lei ¹, Benjamin Kennedy ¹, Steven S Rosenfeld ¹, Peter Canoll ¹, Jeffrey N Bruce ¹

Abstract

Recent preclinical studies have suggested that treating glioblastomas (GBMs) with a combination of targeted chemotherapy and radiotherapy may enhance the antitumor effects of both therapies. However, the details of how such combined therapies should be administered and the effects that these treatments have on glioma growth and progression are poorly understood. Our lab has generated a mouse glioma model that uses a PDGF-IRES-Cre-expressing retrovirus to infect adult glial progenitors in mice carrying conditional deletions of PTEN and p53. This model produces tumors with the histological features of GBM with 100% penetrance, making it a powerful system for testing novel preclinical treatments. Sunitinib is an orally active, small molecule inhibitor of multiple receptor tyrosine kinases critical for tumor growth and angiogenesis, including PDGF receptors. Treating our PDGF-driven mouse model with either sunitinib or radiation alone had a modest but significant effect on survival. Coadministration of sunitinib and radiation caused further delay of tumor growth and enhanced antitumor activity compared with individual monotherapies. However, tumors recurred in 100% of animals, suggesting that tumors were adapting to the therapy. Histological analysis of treated tumors revealed a reduction in vascular proliferation and a marked increase in brain invasion in animals treated with sunitinib alone or in combination with radiation. An additional study combining sunitinib with higher doses of radiation revealed fatal toxicity in animals receiving combination therapy, even though the monotherapies were well tolerated. These results show that the rational addition of sunitinib to radiotherapy may enhance the therapeutic antitumor effects of either therapy alone. However, additional studies are needed to identify the mechanisms by which gliomas evolve and evade these combined therapies. Furthermore, there is an enhanced risk of toxicity associated with combined therapy that must be considered in the design of future clinical studies.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-48. THE ROLE OF p63 IN THE RESPONSE OF MEDULLOBLASTOMA CELLS TO GENOTOXIC STRESS

Vidya Gopalakrishnan ¹, Chandramallika Das ¹, Pete Taylor ¹, Ramakrishna Kommagani ², Xiaohua Su ¹, Dolly Aguilera ¹, Alexandra Thomas ¹, Johannes Wolff ³, Elsa Flores ¹, Madhavi Kadakia ²

Abstract

OBJECTIVES: Medulloblastoma is the most common malignant brain tumor of childhood. Chemotherapy and other treatment modalities are often unsuccessful in children with disease relapse. Efforts to improve therapeutic outcomes must be based on a better understanding of tumor cell response to genotoxic agents. The tumor suppressor protein p53 plays an important role in the cellular response to genotoxic stress; however, the pathway is frequently inactivated in medulloblastoma as in other cancers. The goal of this study was to determine the role of p63 and p73 in the response of medulloblastoma cells to the topoisomerase inhibitor etoposide. METHODS: We used knockdown and overexpression studies to examine the role of p63 and p73 in the response of medulloblastoma cells to etoposide. We also evaluated the ability of histone deacetylase inhibitors to modulate the expression of these genes and sensitize tumor cells to etoposide. RESULTS: Here, we show that etoposide treatment elicited a variable increase in p63 or p73 gene expression in medulloblastoma cells with defective p53 activity, and their knockdown abrogated etoposide-induced apoptosis in these cells. The histone deacetylase inhibitor valproic acid enhanced etoposide-dependent apoptosis that was associated with the upregulation of specific isoforms of p63. Specifically, levels of TAp63-gamma were elevated upon exposure to etoposide, whereas valproic acid treatment caused an increased in both TAp63-gamma and deltaNp63-gamma levels. Surprisingly, changes in histone acetylation were observed only at the promoter directing the expression of deltaNp63-gamma. Constitutive expression of TAp63-gamma and deltaNp63-gamma in medulloblastoma cells promoted apoptosis in the absence of drug treatment. CONCLUSION: Our work provides the first demonstration of a role of p63 in the response of medulloblastoma cells to apoptosis. Importantly, we show that deltaNp63-gamma has proapoptotic activity, a finding that contrasts the findings of studies in other cancers that have suggested a prosurvival function of deltaNp63 isoforms.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-49. ENHANCING DRUG DELIVERY FOR BORON NEUTRON CAPTURE THERAPY OF GLIOBLASTOMA WITH FOCUSED ULTRASOUND

Ryan Alkins ¹, Peter Broderson ¹, Rana Sodhi ¹, Kullervo Hynynen ¹

Abstract

Boron neutron capture therapy (BNCT) is a binary treatment, whereby a boron-10-containing drug that preferentially accumulates in tumor cells is administered to a patient, who is subsequently irradiated with thermal neutrons. The boron-10 captures a neutron then undergoes alpha decay, leading to nonrepairable damage within a cell diameter of the event. Thus, BNCT is highly selective if boron-10 can be delivered in sufficient quantity to tumor cells while sparing healthy cells. Previous studies of rodent brain tumors have shown that intra-carotid administration, pharmacological blood-brain barrier (BBB) disruption, and prolonged infusion of the amino acid analogue L-4-boronophenylalanine, complexed with fructose (BPA-f), lead to significantly higher drug accumulation in tumors. However, these drug administration algorithms are impractical in human patients. More recent studies showed that transcranial magnetic resonance imaging (MRI)-guided focused ultrasound can be used to noninvasively and reversibly open the BBB with the aid of timed intravenous microbubble injections. Using a 9L gliosarcoma tumor model in Fisher 344 rats, we delivered single and multiple sonications, with simultaneous intravenous microbubble injection, to the region of the tumor with a 558-kHz ultrasound transducer using MRI guidance. Animals then received a 2-hour intravenous BPA-f infusion and were euthanized 1 hour following completion. Boron determination was carried out on cryogenically prepared sections using imaging time-of-flight secondary ion mass spectrometry. The ratio of boron in tumor tissue to normal brain tissue was increased by a factor of approximately 2 by focused ultrasound compared to non-ultrasound-treated tumors receiving the same dose of BPA-f. There was no evidence of treatment-related tissue injury in frozen sections stained with hematoxylin and eosin. Transcranial focused ultrasound BBB disruption offers a promising means of optimizing the delivery of BPA-f over other more invasive drug delivery protocols, particularly when the latter have demonstrated improved survival in animal models.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-50. TARGETING GLIOBLASTOMA METABOLISM WITH A NOVEL ARSENIC-BASED METABOLIC INHIBITOR, PENAO

Sylvia A Chung ¹, Kerrie L McDonald ¹, Han Shen ¹, Bryan W Day ², Brett W Stringer ², Terrance Johns ³, Stéphanie Decollogne ⁴, Charlie Teo ¹, Philip J Hogg ⁴, Pierre J Dilda ⁴

Abstract

Tumors reorganize the metabolic steps used by normal tissues for glucose utilization and adenosine triphosphate (ATP) production. Tumor cells are heavily dependent on glycolysis for adenosine triphosphate (ATP), whereas normal tissues rely mostly on oxidative phosphorylation. This aberrant metabolism allows for tumor survival when oxygen is limited and poisons the extracellular environment with acid, thereby facilitating invasion and metastasis. PENAO, (4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid) is a mitochondrial toxin that inactivates inner-membrane adenine nucleotide translocase (ANT). PENAO blocks ANT delivery of ATP to mitochondria-bound hexokinase II, thus inhibiting tumor metabolism of glucose, and triggers the mitochondrial permeability transition pore, resulting in proliferation arrest and then apoptotic cell death. We tested the cytostatic efficacy of PENAO in a panel of 5 established glioblastoma cell lines and 4 glioma neural stem cell lines that differed in tumorigenicity, drug resistance, invasion, and DNA repair. PENAO demonstrated impressive antiproliferative activity compared to carboplatin and temozolomide, drugs commonly used to treat glioblastoma. The half-maximal inhibitory concentration values for PENAO were 1-10 µM for established cell lines and 0.5-2 µM for primary glioma cells. The increase in the antiproliferative activity of PENAO compared to carboplatin ranged from 9- to 171-fold. PENAO is a substrate for multidrug resistance proteins 1 and 2, and inhibition of this transporter enhanced PENAO antiproliferative activity 2- to 8-fold. Concomitant inhibition of cellular glutathione synthesis, which is required for PENAO export, increased PENAO efficacy from 14- to 100-fold in established cell lines and 2- to 20-fold in primary glioma cells. In conclusion, PENAO is a potent inhibitor of glioblastoma cell proliferation. This activity was superior to that observed with standard chemotherapeutic agents and warrants further in vivo investigation.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-51. SIZE OF POLYMER NANOPARTICLES INFLUENCES THERAPEUTIC EFFICACY WHEN ADMINISTERED BY CONVECTION-ENHANCED DELIVERY

Toral R Patel ¹, Jiangbing Zhou ¹, Joseph M Piepmeier ², W Mark Saltzman ³

Abstract

INTRODUCTION: Systemic therapies for malignant gliomas are limited by the blood-brain barrier. Convection-enhanced delivery (CED) of nanoparticles is a promising technique for local drug delivery, but standard-sized polymer nanoparticles do not distribute to clinically relevant volumes in the brain. Thus, we previously developed a novel method for fabricating ultrasmall polymer nanoparticles that can be delivered via CED to volumes five times larger than standard-sized formulations. Here, we report the in vitro and in vivo efficacy of drug-loaded ultrasmall polymer nanoparticles. METHODS: A novel, single-emulsion, solvent-evaporation process was used to fabricate paclitaxel-loaded poly(lactide-co-glycolide) nanoparticles. Nanoparticles of two different sizes were produced: ultrasmall (<100 nm) and standard (>100 nm). Paclitaxel loading and controlled release were determined using high-performance liquid chromatography. In vitro efficacy was evaluated via MTT assay. To determine in vivo efficacy, we administered either drug-loaded nanoparticles, control nanoparticles, or free drug via CED to nude rats bearing U87-derived intracranial gliomas. RESULTS: Scanning electron microscopy confirmed that we produced ultrasmall (mean diameter: 71.3 nm) and standard-sized (mean diameter: 147.2 nm) nanoparticles. High drug-loading was achieved for both ultrasmall and standard-sized nanoparticles (encapsulation efficiency: 61% and 62%, respectively). Controlled-release studies demonstrated that both formulations released the drug in a slow, sustained fashion (∼75% release over 28 days). Following in vitro treatment of U87 cells with either free drug or drug-loaded nanoparticles, MTT assay at 6 days revealed an IC50 of 63 nM for paclitaxel-loaded nanoparticles and 305 nM for free paclitaxel. In vivo evaluation demonstrated that median survival was significantly improved (46 days) in animals treated with paclitaxel-loaded ultrasmall nanoparticles compared to all other groups (standard-sized nanoparticles: 38 days, free drug: 30 days, blank/unloaded nanoparticles: 31 days, no treatment: 27 days; p < 0.05). CONCLUSION: CED of ultrasmall, drug-loaded, polymer nanoparticles is more effective against intracranial gliomas than CED of standard-sized polymer nanoparticles. This delivery system can be readily translated into clinical practice.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-52. INHIBITION OF ACTIVE EFFLUX AT THE BBB IMPROVES DELIVERY AND EFFICACY OF ERLOTINIB IN AN ORTHOTOPIC XENOGRAFT MODEL OF GLIOMA

Michael A Vogelbaum ¹, Sagar Agarwal ², Pooja Manchanda ¹, John R Ohlfest ², William F Elmquist ²

Abstract

Several molecularly targeted antitumor agents are substrates for active efflux transporters, such as p-glycoprotein (Pgp) and breast cancer resistance protein (BCRP), and we have shown that this significantly restricts their delivery across the blood-brain barrier (BBB). The objective of this study was to demonstrate the influence of active efflux at the BBB on the delivery of erlotinib in an orthotopic rat xenograft model of glioma. Athymic nude rats bearing orthotopic human gliomas (U87MG) with overexpression of wild-type epidermal growth factor receptor (EGFR) were treated with erlotinib at 20 mg/kg/day for 3 days. The rats were euthanized 30 minutes after the last treatment and perfused with saline, following which blood, normal brain tissue, and tumors were harvested. Erlotinib concentrations were determined using liquid chromatography-mass spectrometry, and EGFR phosphorylation was evaluated using Western blot analysis. A subset of rats was also treated with the Pgp/BCRP dual inhibitor GF120918 30 minutes prior to erlotinib treatment. Delivery of erlotinib across the BBB was significantly limited by the BBB with tissue concentrations significantly lower than plasma. The tissue-to-plasma ratio was 0.63 ± 0.28 in the tumor core, 0.16 ± 0.08 in the brain surrounding the tumor, and 0.14 ± 0.07 in the contralateral hemisphere. Treatment with GF120918 significantly enhanced erlotinib distribution into the tumor core (twofold), the brain surrounding the tumor (sixfold), and normal brain tissue (sixfold). Western blot analysis showed no change in EGFR phosphorylation when erlotinib was administered alone. The influence of GF120918 on erlotinib-induced EGFR dephosphorylation will be presented. These results show that the BBB is intact in areas adjacent to the tumor core and can significantly restrict erlotinib delivery to such areas. This restriction in drug delivery can be overcome by an inhibitor of active drug transporters, such as GF120918. Enhancing drug delivery to the tumor core and invasive tumor cells can be an important strategy for improving the efficacy of molecularly targeted therapy of glioma.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-53. GENE EXPRESSION PROFILING REVEALS FREQUENT UPREGULATION OF MGMT IN ACQUIRED TEMOZOLOMIDE-RESISTANT GLIOBLASTOMA XENOGRAFTS

Gaspar J Kitange ¹, Ann C Mladek ¹, Brett L Carlson ¹, Mark A Schroeder ¹, Jenny L Pokorny ¹, Jann N Sarkaria ¹

Abstract

Resistance to temozolomide (TMZ) is a main reason for the limited life expectancy of patients with newly diagnosed glioblastoma (GBM). Accordingly, there is a significant interest in understanding the mechanisms modulating TMZ resistance in GBM. In this study, we subjected 5 primary flank GBM xenograft lines to in vivo selection to establish TMZ-resistant lines. The orthotopically evaluated survival benefit associated with TMZ therapy was significantly greater in the parental lines than in the derivative TMZ-resistant lines (median relative survival: parental lines, 3.6 vs. TMZ-resistant lines, 1.2; p = 0.008). Microarray profiling of gene expression comparing parental lines to TMZ-resistant lines demonstrated upregulation of O6-methyguanine-DNA-methyltransferase (MGMT) in 3 of the 5 TMZ-resistant xenografts, and resistance in vitro could be reversed by O6-benzylguanine. Increased MGMT was observed in 6 of the 13 additionally generated TMZ-resistant lines. To test whether upregulation of MGMT is a reproducible tumor-specific event, we independently developed 10 TMZ-resistant lines from a single tumor (GBM12). Intriguingly, MGMT was upregulated in only 2 of the 10 newly established GBM12 TMZ-resistant lines. MGMT was upregulated regardless of the methylation status of the parental tumors. Instead, chromatin immunoprecipitation analysis of the paired methylated GBM12 lines revealed an association between MGMT upregulation and elevated acetylation of lysine 9 of histone H3 (H3K9-ac), reduced di-methylation (H3K9-me2), and a loss of MeCP2 recruitment to the MGMT promoter. These chromatin changes were coupled with increased MGMT promoter binding of the transcription factors SP1, NF-kB, p300, and c-JUN. In contrast, in the paired lines from GBM22, a methylated tumor without a change in MGMT, both the parental and TMZ-resistant lines had low acetylation and high methylation of H3K9 within the MGMT promoter. These findings suggest a heterogeneous development of acquired TMZ resistance in GBM, which includes MGMT re-expression driven by the epigenetic changes within the histone code.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-54. INNOVATIVE BIOTHERAPY FOR BRAIN TUMORS: MYXOMA VIRUS SYNERGIZES WITH NATURAL KILLER CELLS TO CLEAR MALIGNANT GLIOMAS

Henry Ogbomo ¹, Xueqing Lun ¹, Jiqing Zhang ¹, Grant McFadden ², Christopher Mody ³, Peter Forsyth ⁴

Abstract

Innovative biotherapeutic approaches for experimental malignant gliomas (MGs) and brain tumor stem cells (BTSCs) include using oncolytic viruses such as Myxoma virus (Myxv) or immune cells such as natural killer (NK) cells. In this study, we combined the individual strengths of NK cells and Myxv to treat MGs with a view of overcoming the potential limitations posed when each is used as monotherapy. All MGs (U87, U251, U118) and BTSCs (25EF and 48EF) investigated expressed varying levels of NK ligands, including inhibitory MHC-I, activating ULBP1-3, MICA/B, and co-activating nectin-2 (poliovirus receptor). Infection of MGs and BTICs with Myxv resulted in the downregulation of MHC-I expression both on the cell surface and intracellularly. No effect was observed on the expression of NK activating ligands except for the downregulation of nectin-2. The downregulation of MHC-I and nectin-2 by Myxv was specifically mediated by the M153 gene of Myxv, as loss of M153 restored MHC-I and nectin-2 expression. Subsequently, NK cell killing of MGs was enhanced in Myxv-infected cells. Loss of M153 inhibited NK lysis of MGs. In vivo, Myxv synergized with NK cells to accelerate tumor clearance in luciferase-expressing U87 cells in a mouse intracranial model. Combined intratumoral treatment with Myxv and NK of established U87 tumor cells accelerated tumor clearance when compared to single treatment with Myxv or NK. Tumors in 66% of mice (4 out of 6 mice) were cleared or significantly reduced within 14 days following combination treatment with Myxv and NK. No significant effect was observed in mice treated with Myxv or NK alone within the same time frame.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-55. BRaf INHIBITOR PLX4720 IS A RADIOSENSITIZER IN HUMAN MALIGNANT ASTROCYTOMAS EXPRESSING THE BRaf V600E MUTATION

Tina Dasgupta ¹, Xiaodong Yang ¹, Rintaro Hashizume ¹, Ashley Gragg ¹, Michael Prados ¹, Theodore Nicolaides ¹, C David James ¹, Daphne Haas-Kogan ¹

Abstract

INTRODUCTION: Radiation (RT) is the most effective adjuvant treatment of pediatric malignant astrocytomas, but cure remains elusive. BRaf mutation V600E and its downstream mediator, MEK, are critical to the pathogenesis of 10% to 20% of pediatric malignant astrocytomas. Our objective was to determine whether PLX4720, a specific inhibitor of BRafV600E, enhances the activity of RT against human malignant astrocytomas in vitro and in vivo. METHODS: IC50 values for PLX4720 with and without RT were determined using clonogenic assays. Phospho-MEK was determined using Western blot analysis. In vivo studies utilized orthotopic xenografts of BRafV600E astrocytoma cells modified to express firefly luciferase. Mice were randomized to receive control solvent, PLX4720, XRT, or a combination thereof. RESULTS: Cell lines DBTRG-05MG, AM-38[DH1], KG-1-C, and NMC-G-1 were heterozygous for BRafV600E; GS2, GBM2, and GBM9 were homozygous for wildtype (WT) BRaf. IC50 values of PLX4720 in the BRafV600E cell lines were 2-10 micromolar; in BRaf WT cell lines, IC50 values were not reached even with concentrations above 20 micromolar. Clonogenic studies showed combinations of RT and PLX4720 were more cytotoxic than either modality alone in BRafV600E lines but not in any of the BRaf WT lines. Biochemically, a PLX4720 concentration-dependent decrease in phospho-MEK expression was observed in BRafV600E cell lines but not in WT BRaf cells. Preliminary in vivo data of BRafV600E astrocytomas suggest prolonged survival in mice treated with PLX and RT compared to controls. CONCLUSIONS: Patient tumor-derived malignant astrocytoma cell lines harboring BRafV600E mutations are inhibited by PLX4720. Radiation-induced cytotoxicity is enhanced by the addition of PLX4720. The antineoplastic effects of PLX4720, with or without RT, are paralleled by PLX4720 concentration-dependent inhibition of MEK phosphorylation in cell lines harboring the BRafV600E mutation. Preliminary in vivo data of murine orthotopic intracranial xenografts support these observations. The combination of RT and PLX4720 appears to be effective in the treatment of pediatric malignant astrocytomas with BRafV600E mutations.

Neuro Oncol. 2011 Nov;13(Suppl 3):iii107–iii120.

ET-56. TUMOR-SELECTIVE DELIVERY OF DOXORUBICIN: A TOP2A INHIBITOR AS EFFECTIVE THERAPY FOR MPNST

Achuthamangalam B Madhankumar ¹, Becky Slagle Webb ¹, Annie Park ¹, Kimberly Harbaugh ¹, Jonas Sheehan ¹, James R Connor ¹

Abstract

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive tumors with a poor prognosis and predominantly affect individuals with hereditary neurofibromatosis type 1disorder. Topoisomerase II-alpha (TOP2A) has been reported to be elevated significantly in most MPNSTs. In this work, we demonstrate our ability to deliver doxorubicin, a TOP2A inhibitor to this tumor through an interleukin (IL)-13-conjugated liposome carrying doxorubicin (IL-13-Lip-Dox) based on the observation that IL-13 receptors are expressed in NF-1 cell lines and tissues. We screened Schwannomatosis and NF-1 tissues obtained via biopsy for IL-13 receptor expression using immunostaining and Western blot analysis. Subsequently, we obtained a peripheroneural cancer tissue array and compared IL-13 receptor expression. Immunoblot analysis was performed on well-established NF-1 cell lines such as sNF96.2, sNF94.3, and ST88-14. These cell lines were treated with IL-13-Lip-Dox, and the cytotoxic effect was measured by cell proliferation assay. The majority of the NF-1 tissues were found be positive for IL13Ralpha-2 receptor as confirmed by immunostaining of the tissues and Western blots of the tissue cell lysates. Immunohistochemical analysis of the NF-1 cells treated with IL-13-Lip-Dox demonstrated IL-13 receptor expression on the cell membrane and the ability of the IL-13-Lip-Dox to deliver the doxorubicin in the intracellular region. Western blots with the NF-1 cell lysates indicated the expression of IL-13 receptor at variable levels. The functional significance of this observation is clear from the cytotoxic effect of the IL-13-Lip-Dox on the NF-1 cells. In conclusion, increased expression of IL-13 receptor in MPNST cells and tissues demonstrates the suitability of the IL-13-Lip-Dox to target this soft-tissue sarcoma for therapy. Effective intracellular delivery of doxorubicin in the NF-1 cells indicates the potential of this delivery system to overcome the drug resistance associated with MPNSTs and to inhibit TOP2A to sensitize them to therapy.

PERMALINK

PRECLINICAL EXPERIMENTAL THERAPEUTICS AND PHARMACOLOGY

ET-01. EXPLORING NEW DRUGS THAT CAN AFFECT THE VIABILITY AND TRANSFORMATION OF CHILDHOOD MEDULLOBLASTOMAS

Helena Pavel

Norbert Ajeawung

Robert Faure

Donald Poirier

Deepak Kamnasaran

Abstract

ET-02. DISCOVERY OF A NEW CLASS OF MICROTUBULE-BINDING DRUGS THAT SIGNIFICANTLY AFFECT THE VIABILITY AND TRANSFORMATION OF HUMAN GLIOMA STEM CELLS

Norbert Ajeawung

Harish Joshi

Deepak Kamnasaran

Abstract

ET-03. A CHEMICAL GENETIC SCREEN IDENTIFIES A NOVEL DRUG THAT TARGETS STEROID BIOGENESIS AND RECEPTOR SIGNALING LEADING TO GROWTH INHIBITION OF PEDIATRIC MALIGNANT ASTROCYTOMA CELL LINES

Donald Poirier

Norbert Ajeawung

Deepak Kamnasaran

Abstract

ET-04. COMBINED MYXOMA VIROTHERAPY WITH RAPAMYCIN TARGETING BRAIN TUMOR-INITIATING CELLS

Xueqing Lun

Franz Zemp

Beichen Sun

Owen Stechishin

Artee Luchman

John J Kelly

Samuel Weiss

Mark G Hamilton

Gregory Cairncross

Donna L Senger

John Bell

Grant McFadden

Peter A Forsyth

Abstract

ET-05. SAFE AND EFFECTIVE NONVIRAL DNA DELIVERY TO TREAT GLIOBLASTOMA-DERIVED BRAIN CANCER STEM CELLS

Stephany Y Tzeng

Hugo Guerrero-Cazares

Elliott E Martinez

Noah P Young

Joel C Sunshine

Alfredo Quinones-Hinojosa

Jordan J Green

Abstract

ET-06. PRECLINICAL STUDIES REVEAL MECHANISMS OF RESPONSE AND ADAPTATION OF PRONEURAL GLIOBLASTOMA TO SUNITINIB

Liang Lei

Randy D'Amico

Julia Sisti

Richard Leung

Adam M Sonabend

Paolo Guarnieri

Steven S Rosenfeld

Jeffrey N Bruce

Peter Canoll

Abstract

ET-07. COMPARISON OF VERUBULIN ACTIVITY USING DIFFERENT DOSING SCHEDULES IN XENOGRAFT MODELS

Vijay R Baichwal

Leslie Reeves

Bradford L Chad

Kenton H Zavitz

Andrew P Beelen

Gary G Mather

Robert O Carlson

Abstract

ET-08. MECHANISMS THAT DETERMINE THE EFFICACY OF TWO PROTEASOME INHIBITORS, BORTEZOMIB AND MARIZOMIB, IN GLIOBLASTOMA

Christa Manton

Joya Chandra

Abstract

ET-09. MIBEFRADIL, A NOVEL THERAPY FOR GLIOBLATOMA MULTIFORME: INTERLACED THERAPY IN A MURINE MODEL

Stephen T Keir

David A Reardon

Julia R Saling

Lloyd S Gray

Darell D Bigner

Henry S Friedman

Abstract

ET-10. ONCOLYTIC THERAPEUTIC POTENCY OF FARMINGTON VIRUS AND MODIFIED MARABA VIRUS IN IMMUNOCOMPETENT INTRACRANIAL GLIOMA MODELS AND IN MICE BEARING HUMAN BRAIN TUMOR INITIATING CELLS MODELS

Jiqing Zhang

Jan Brun

Henry Ogbomo

Franz Zemp