Intracranial Tumor Growth Research Articles

ME-13 * LOSS OF SOCS3 IN MYELOID CELLS DELAYS TUMOR GROWTH AND PROLONGS SURVIVAL IN A SYNGENEIC MODEL OF GLIOBLASTOMA

Glioblastoma (GBM), a particularly devastating type of brain tumor, remains a challenging and difficult disease to treat. Despite aggressive therapies, patients diagnosed with GBM have a median survival of 12-16 months. On a cellular level, GBM tumors are extremely heterogeneous, consisting of resident tumor cells, tumor initiating cells, infiltrating immune cells, endothelial cells, and other tumor associated stromal cells, which makes developing targeted therapies a challenge. In GBM, microglia (MG) and macrophages (MP) are the largest population of tumor-infiltrating cells. GBM tumor cells recruit MG/MP via the secretion of chemo-attractants; however, upon arrival at the tumor, the MG/MP adopt an anti-inflammatory phenotype, and in turn accidentally aide in tumor promotion through the secretion of various cytokines. We have previously determined that macrophages with deletion of Suppressor of Cytokine Signaling 3 (SOCS3), the negative regulator of Signal Transducer and Activator of Transcription 3 (STAT3), have an enhanced M1 (pro-inflammatory) response. Herein, we sought to determine the role of SOCS3 in MG/MP functionality in GBM. In this report, we utilized a conditional model in which SOCS3 deletion is restricted to the myeloid cell population. We found that deletion of SOCS3 in the myeloid cell population delays intracranial tumor growth and increases survival of mice bearing orthotopic glioma tumors. Intracranial tumors from mice with SOCS3 deficient myeloid cells have an increase in the number of infiltrating immune cells, including monocytes, neutrophils and macrophages. Lastly, SOCS3 deficient bone marrow derived macrophages display enhanced and prolonged expression of pro-inflammatory M1 cytokines (TNF-α, IL-6) when exposed to tumor cell conditioned medium in vitro. Therefore, loss of SOCS3 in MG/MP in the tumor promotes an inflammatory, immune response early and thus delays tumor growth. These preliminary findings highlight an important role of the STAT3/SOCS3 signaling axis in MG/MP in GBM tumors.

Combined VEGF and CXCR4 antagonism targets the GBM stem cell population and synergistically improves survival in an intracranial mouse model of glioblastoma.

Glioblastoma recurrence involves the persistence of a subpopulation of cells with enhanced tumor-initiating capacity (TIC) that reside within the perivascular space, or niche (PVN). Anti-angiogenic therapies may prevent the formation of new PVN but have not prevented recurrence in clinical trials, suggesting they cannot abrogate TIC activity. We hypothesized that combining anti-angiogenic therapy with blockade of PVN function would have superior anti-tumor activity. We tested this hypothesis in an established intracranial xenograft model of GBM using a monoclonal antibody specific for murine and human VEGF (mcr84) and a Protein Epitope Mimetic (PEM) CXCR4 antagonist, POL5551. When doses of POL5551 were increased to overcome an mcr84-induced improvement in vascular barrier function, combinatorial therapy significantly inhibited intracranial tumor growth and improved survival. Anti-tumor activity was associated with significant changes in tumor cell proliferation and apoptosis, and a reduction in the numbers of perivascular cells expressing the TIC marker nestin. A direct effect on TICs was demonstrated for POL5551, but not mcr84, in three primary patient-derived GBM isolates. These findings indicate that targeting the structure and function of the PVN has superior anti-tumor effect and provide a strong rationale for clinical evaluation of POL5551 and Avastin in patients with GBM.

NEO212, temozolomide conjugated to perillyl alcohol, is a novel drug for effective treatment of a broad range of temozolomide-resistant gliomas.

Patients with glioblastoma multiforme (GBM), a malignant primary brain tumor, inevitably develop resistance to standard-of-care chemotherapy, temozolomide. This study explores the effects of the novel agent NEO212, a conjugate of temozolomide to perillyl alcohol, on temozolomide-resistant gliomas. NEO212 was tested for cytotoxic activity on three human temozolomide-resistant glioma cell lines, which were resistant to temozolomide based on overexpression of the base excision repair (BER) pathway, mismatch repair (MMR) deficiency, or overexpression of O(6) methyl-guanine-DNA methyltransferase (MGMT). BER expression was evaluated by Western blotting and PARP activity. MMR deficiency was determined by Western blotting and microsatellite instability. MGMT overexpression was evaluated by Western blotting and O(6)-benzylguanine (O(6)BG) inhibition. For in vivo evaluation of NEO212, temozolomide-resistant glioma cells were implanted into immune-incompetent mice, and NEO212 was administered. NEO212, at equimolar concentrations of temozolomide, was more cytotoxic for temozolomide-resistant cells than temozolomide and not toxic to normal cells. NEO212-induced cell death in temozolomide-resistant glioma cells was independent of such mechanisms of resistance as high levels of MGMT, MMR deficiencies, or overexpression of BER proteins. NEO212 functions as a DNA alkylating agent, similar to temozolomide; however, this novel conjugate is unique for it may induce endoplasmic reticulum (ER) stress and inhibits autophagy. In vivo studies show that NEO212 reduces intracranial tumor growth and increases animal survival without significant toxicity. These results demonstrate that NEO212 is an effective drug against malignant gliomas that can be used for a broad range of newly diagnosed and temozolomide-resistant gliomas.

A Novel Temozolomide–Perillyl Alcohol Conjugate Exhibits Superior Activity against Breast Cancer Cells In Vitro and Intracranial Triple-Negative Tumor Growth In Vivo

There is no effective therapy for breast cancer that has spread to the brain. A major roadblock is the blood-brain barrier (BBB), which prevents the usual breast cancer drugs from effectively reaching intracranial metastases. The alkylating agent temozolomide (TMZ) is able to penetrate the BBB and has become the gold standard for chemotherapeutic treatment of glioblastoma. However, when it was tested in clinical trials for activity against brain metastases of breast cancer, the results were mixed and ranged from "encouraging activity" to "no objective responses." In an effort to generate an agent with greater activity against intracranial breast metastases, we synthesized a TMZ analog where the natural product perillyl alcohol (POH) was covalently linked to TMZ's amide functionality. The resulting novel compound, called TMZ-POH (T-P), displayed greatly increased anticancer activity in a variety of breast cancer cell lines, inclusive of TMZ-resistant ones. It caused DNA damage and cell death much more efficiently than its parental compound TMZ, because linkage with POH increased its biologic half-life and thus provided greater opportunity for placement of cytotoxic DNA lesions. In an intracranial mouse tumor model with triple-negative breast cancer, T-P revealed considerably greater therapeutic efficacy than TMZ, where a single cycle of treatment extended median survival benefit from 6 days (in the case of TMZ) to 28 days. At the same time, T-P seemed to be well tolerated by the animals. Thus, T-P may have potential as a novel therapy for brain-targeted breast cancer metastases.

Inhibition of Notch signaling alters the phenotype of orthotopic tumors formed from glioblastoma multiforme neurosphere cells but does not hamper intracranial tumor growth regardless of endogene Notch pathway signature

Background Brain cancer stem-like cells (bCSC) are cancer cells with neural stem cell (NSC)-like properties found in the devastating brain tumor glioblastoma multiforme (GBM). bCSC are proposed a central role in tumor initiation, progression, treatment resistance and relapse and as such present a promising target in GBM research. The Notch signaling pathway is often deregulated in GBM and we have previously characterized GBM-derived bCSC cultures based on their expression of the Notch-1 receptor and found that it could be used as predictive marker for the effect of Notch inhibition. The aim of the present project was therefore to further elucidate the significance of Notch pathway activity for the tumorigenic properties of GBM-derived bCSC.Methods Human-derived GBM xenograft cells previously established as NSC-like neurosphere cultures were used. Notch inhibition was accomplished by exposing the cells to the gamma-secretase inhibitor DAPT prior to gene expression analysis and intracranial injection into immunocompromised mice.Results By analyzing the expression of several Notch pathway components, we found that the cultures indeed displayed different Notch pathway signatures. However, when DAPT-treated neurosphere cells were injected into the brain of immunocompromised mice, no increase in survival was obtained regardless of Notch pathway signature and Notch inhibition. We did however observe a decrease in the expression of the stem cell marker Nestin, an increase in the proliferative marker Ki-67 and an increased number of abnormal vessels in tumors formed from DAPT-treated, high Notch-1 expressing cultures, when compared with the control.Conclusion Based on the presented results we propose that Notch inhibition partly induces differentiation of bCSC, and selects for a cell type that more strongly induces angiogenesis if the treatment is not sustained. However, this more differentiated cell type might prove to be more sensitive to conventional therapies.

Multi-kinase inhibitor C1 triggers mitotic catastrophe of glioma stem cells mainly through MELK kinase inhibition.

Glioblastoma multiforme (GBM) is a highly lethal brain tumor. Due to resistance to current therapies, patient prognosis remains poor and development of novel and effective GBM therapy is crucial. Glioma stem cells (GSCs) have gained attention as a therapeutic target in GBM due to their relative resistance to current therapies and potent tumor-initiating ability. Previously, we identified that the mitotic kinase maternal embryonic leucine-zipper kinase (MELK) is highly expressed in GBM tissues, specifically in GSCs, and its expression is inversely correlated with the post-surgical survival period of GBM patients. In addition, patient-derived GSCs depend on MELK for their survival and growth both in vitro and in vivo. Here, we demonstrate evidence that the role of MELK in the GSC survival is specifically dependent on its kinase activity. With in silico structure-based analysis for protein-compound interaction, we identified the small molecule Compound 1 (C1) is predicted to bind to the kinase-active site of MELK protein. Elimination of MELK kinase activity was confirmed by in vitro kinase assay in nano-molar concentrations. When patient-derived GSCs were treated with C1, they underwent mitotic arrest and subsequent cellular apoptosis in vitro, a phenotype identical to that observed with shRNA-mediated MELK knockdown. In addition, C1 treatment strongly induced tumor cell apoptosis in slice cultures of GBM surgical specimens and attenuated growth of mouse intracranial tumors derived from GSCs in a dose-dependent manner. Lastly, C1 treatment sensitizes GSCs to radiation treatment. Collectively, these data indicate that targeting MELK kinase activity is a promising approach to attenuate GBM growth by eliminating GSCs in tumors.

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 µm in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

Abstract P6-11-06: Efficacy of carboplatin alone or with ABT888 in an intracranial murine model of BRCA-mutated, basal-like, triple negative breast cancer (TNBC)

Abstract Background: TNBC frequently spreads to the central nervous system and is associated with poor outcome. Twenty percent of women with TNBC harbor a BRCA mutation(mut). BRCA dysfunction impairs repair of DNA double-strand breaks by homologous recombination(HR) and sensitizes tumor cells to DNA-damaging cytotoxic agents. Emerging evidence suggests that TNBC responds to platinums(Pt) and drugs potentiating efficiency of DNA damage, such as PARP inhibitors; combination therapy may enhance DNA damage and induce additional cytotoxicity. Recognizing that Pt and many PARP inhibitors penetrate the blood brain barrier(BBB), we evaluated the efficacy of carboplatin(Carbo) alone and with the PARP inhibitor, ABT888(ABT), in murine intracranial(IC) TNBC models characterized by BRCA status. Methods: Athymic (nu/nu) mice(10wks) were inoculated intracerebrally with 2×105 SUM149(BRCA1mut, pTEN-; basal) cells expressing luciferase. On day14, tumor-bearing mice began treatment with Control(PBS IP weekly), Carbo(50mg/kg IP weekly) and ABT(25mg/kg OG daily) as single agents, or in combination. Survival and IC tumor growth via bioluminescence were compared by treatment. Net PAR levels(PARpg/ml/100ug total protein) were measured in IC tumor tissue(ELISA pharmacodynamic assay) 14days post ABT. Presence of Pt DNA adducts was measured in IC tumor tissue following Carbo+/-ABT by ICP-MS. Survival and IC tumor growth via bioluminescence of the MDA-MB468(BRCAnon-mut, pTEN-; basal) IC model treated with the same regimens were assessed. Results: In the SUM149 TNBC IC model, Carbo+/-ABT resulted in improved survival compared to control (Table). Single agent ABT did not yield a survival benefit over control. Combination Carbo/ABT showed a modest advantage in survival compared to Carbo alone (p = 0.28). IC tumor mean net PAR levels ± SEM were significantly lower in ABT-treated animals compared to control (182.1 ± 91.3 vs 825.7 ± 174.7, p = 0.0043). Addition of ABT did not impair Carbo BBB penetration; Mean ± SEM Pt-DNA adducts were detected in IC tumor after administration of Carbo and Carbo/ABT (0.91±0.13 and 1.46±0.03 pgPt/ugDNA). In contrast to the SUM149 IC model, there was no survival improvement for the MDA-MB468 IC model treated with Carbo+/-ABT compared to control(p = 0.81). SUM149(BRCA1mut)MDA-MB468(BRCA1non-mut)TreatmentNMedian Survival (95%CI), dayspValueNMedian Survival (95%CI), daysControl1736(34-40)N/A535(29-57)ABT1039(30-46)p = 0.22544(33-57)Carbo1658(47-67)p<.0001537(33-54)Carbo/ABT1764(59-75)p<.0001537(33-50) Conclusions: This study shows Pt analogues, specifically Carbo, represent a promising treatment strategy for TNBC brain metastases with BRCA dysfunction. Improved survival relies on the ability of Pt analogues to penetrate the BBB and higher cytotoxicity may be due to compromised HR in TNBC as compared to models with functional BRCA. Although inhibitory activity of ABT was detected in IC tumors, we did not observe a significant additive effect of ABT to Carbo when used in combination. Confirmatory studies in additional IC TNBC models, including claudin-low subtype, as well as mechanistic studies evaluating DNA damage via γH2AX foci and differential gene expression in response to treatment are ongoing. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-11-06.

Novel Intracranial Xenografts Of CNS Lymphoma Implicate a Role For Cereblon As a Mediator Of Lenalidomide Efficacy

BackgroundCNS manifestations of aggressive non-Hodgkin Lymphoma are associated with serious morbidity and adverse prognosis. Primary CNS lymphomas (PCNSL) exhibit a dichomatous growth pattern, either dissemination within brain, typical at presentation, and/or leptomeningeal spread, common at relapse. Elucidation of the mechanistic basis of CNS lymphoma progression as well as drug resistance requires preclinical models that recapitulate their pathogenesis. MethodsWe developed a novel method to derive cell lines of CNS lymphoma that recapitulate disease phenotypes upon intracranial implantation into mice. We are applying genomics, in vitrochemotaxis, preclinical testing of targeted therapies and neuroimaging to evaluate mechanisms of invasion and resistance. ResultsWe developed 7 CNS lymphoma cell lines; 6 DLBCL (all ABC-type), 1 Burkitt; 5 from secondary CNS lymphoma (SCNSL), and 2 from PCNSL, of which 1 was treatment naïve. Intracranial implantation of lymphoma cells from these tumours within NSG mice provides a reproducible model to dissect the pathogenesis of CNS lymphomas. PCNSL specimens were 10X more efficient in CNS dissemination than SCNSL. High resolution array-CGH demonstrated that intracranial tumour growth was associated with retention of genomic aberrations of the original tumours (e.g. del 6q, gains on 12, etc) and that these were maintained with serial passage in vivo. CNS-infiltrative lymphomas expressed significantly increased levels of MMP-7 and RGS-13 transcripts compared to lymphomas that did not infiltrate brain, while osteopontin and cathepsin D expression by lymphoma cells did not correlate with CNS invasion. Targeted shRNA-mediated knockdown of RGS-13 was performed using lentiviral infection and resulted in significant delay of CNS lymphoma growth in vivo in a xenograft model but had no effect on lymphoma proliferation in culture. Therapeutic response to lenalidomide, minus and plus rituximab, was recapitulated in RAG-/- mice, despite deficient T-cell function and correlated with baseline relative cereblon expression, as quantified using a highly specific immunohistochemical assay. The emergence of resistance to lenalidomide in human CNS lymphoma xenografts also correlated with loss of cereblon protein expression, supporting a role for cereblon in the efficacy of lenalidomide in CNS lymphomas. Notably, significant cereblon protein expression by lymphoma cells was detected by immunohistochemistry in 12/22 diagnostic specimens of aggressive CNS lymphoma. Metabolic imaging of model CNS lymphomas using magnetic resonance spectroscopy demonstrated significant intratumoural lactate production in the microenvironment, detectable before evidence of aberrant T2 signal and reduced diffusion. Lenalidomide reduced tumour expression of lactate dehydrogenase and lactate, as well as RGS-13, consistent with anti-proliferative as well as anti-invasive effects. ConclusionsTo the best of our knowledge we have developed the first panel of patient-derived CNS lymphoma cell lines. We have used these to generate intracranial xenografts that provide a highly reproducible model system to dissect key elements of CNS lymphoma pathogenesis, leading to the elucidation of mechanisms of CNS lymphoma growth and invasion as well as resistance. Our results support a direct, T-cell independent effect of lenalidomide on CNS lymphoma growth and invasion which may be cereblon-dependent. Additional studies are needed to define the role of cereblon as a biomarker and mediator of lenalidomide efficacy in CNS lymphomas. In addition, we are using these models to identify novel genomic and metabolic aberrations predictive of early resistance to lenalidomide and other targeted therapies. Supported by the Lymphoma Research Foundation, Leukemia and Lymphoma Society, and by NIH R01CA139-83-01A1. Disclosures:Heise:Celgene: Employment, Equity Ownership. Rubenstein:Celgene: Research Funding; Genentech: Research Funding. Off Label Use: use of lenalidomide in CNS lymphoma.

Longitudinal MRI Evaluation of Intracranial Development and Vascular Characteristics of Breast Cancer Brain Metastases in a Mouse Model

Longitudinal MRI was applied to monitor intracranial initiation and development of brain metastases and assess tumor vascular volume and permeability in a mouse model of breast cancer brain metastases. Using a 9.4T system, high resolution anatomic MRI and dynamic susceptibility contrast (DSC) perfusion MRI were acquired at different time points after an intracardiac injection of brain-tropic breast cancer MDA-MB231BR-EGFP cells. Three weeks post injection, multifocal brain metastases were first observed with hyperintensity on T2-weighted images, but isointensity on T1-weighted post contrast images, indicating that blood-tumor-barrier (BTB) at early stage of brain metastases was impermeable. Follow-up MRI revealed intracranial tumor growth and increased number of metastases that distributed throughout the whole brain. At the last scan on week 5, T1-weighted post contrast images detected BTB disruption in 160 (34%) of a total of 464 brain metastases. Enhancement in some of the metastases was only seen in partial regions of the tumor, suggesting intratumoral heterogeneity of BTB disruption. DSC MRI measurements of relative cerebral blood volume (rCBV) showed that rCBV of brain metastases was significantly lower (mean = 0.89±0.03) than that of contralateral normal brain (mean = 1.00±0.03; p<0.005). Intriguingly, longitudinal measurements revealed that rCBV of individual metastases at early stage was similar to, but became significantly lower than that of contralateral normal brain with tumor growth (p<0.05). The rCBV data were concordant with histological analysis of microvascular density (MVD). Moreover, comprehensive analysis suggested no significant correlation among tumor size, rCBV and BTB permeability. In conclusion, longitudinal MRI provides non-invasive in vivo assessments of spatial and temporal development of brain metastases and their vascular volume and permeability. The characteristic rCBV of brain metastases may have a diagnostic value.

Tumor-Specific Activation of the C-JUN/MELK Pathway Regulates Glioma Stem Cell Growth in a p53-Dependent Manner

Accumulated evidence suggests that glioma stem cells (GSCs) may contribute to therapy resistance in high-grade glioma (HGG). Although recent studies have shown that the serine/threonine kinase maternal embryonic leucine-zipper kinase (MELK) is abundantly expressed in various cancers, the function and mechanism of MELK remain elusive. Here, we demonstrate that MELK depletion by shRNA diminishes the growth of GSC-derived mouse intracranial tumors in vivo, induces glial fibrillary acidic protein (+) glial differentiation of GSCs leading to decreased malignancy of the resulting tumors, and prolongs survival periods of tumor-bearing mice. Tissue microarray analysis with 91 HGG tumors demonstrates that the proportion of MELK (+) cells is a statistically significant indicator of postsurgical survival periods. Mechanistically, MELK is regulated by the c-Jun NH(2)-terminal kinase (JNK) signaling and forms a complex with the oncoprotein c-JUN in GSCs but not in normal progenitors. MELK silencing induces p53 expression, whereas p53 inhibition induces MELK expression, indicating that MELK and p53 expression are mutually exclusive. Additionally, MELK silencing-mediated GSC apoptosis is partially rescued by both pharmacological p53 inhibition and p53 gene silencing, indicating that MELK action in GSCs is p53 dependent. Furthermore, irradiation of GSCs markedly elevates MELK mRNA and protein expression both in vitro and in vivo. Clinically, recurrent HGG tumors following the failure of radiation and chemotherapy exhibit a statistically significant elevation of MELK protein compared with untreated newly diagnosed HGG tumors. Together, our data indicate that GSCs, but not normal cells, depend on JNK-driven MELK/c-JUN signaling to regulate their survival, maintain GSCs in an immature state, and facilitate tumor radioresistance in a p53-dependent manner.

Abstract 4433: DW-MR imaging as a predictive biomarker of radiosensitization of GBM through targeted inhibition of checkpoint kinases.

Abstract Purpose: The inherent treatment resistance of glioblastoma (GBM) can involve multiple mechanisms including checkpoint kinase (Chk1/2) mediated increased DNA repair capability which can attenuate the effects of genotoxic chemotherapies and radiation. The goal of this study was to evaluate DW-MRI as a biomarker for Chk1/2 inhibitors in combination with radiation for enhancement of treatment efficacy in GBM. Experimental Design: We evaluated a specific small molecule inhibitor of Chk1/2, AZD7762, in combination with radiation using in vitro human cell lines and in vivo using a genetically engineered GBM mouse model. Diffusion-weighted and T1-contrast MRI were used to follow treatment effects on intracranial tumor cellularity and growth rates, respectively. Results: AZD7762 inhibited clonal proliferation in a panel of GBM cell lines and increased radiosensitivity in p53-mutated GBM cell lines to a greater extent compared to p53-wild type cells. In vivo efficacy of AZD7762 demonstrated a dose-dependent inhibitory effect on GBM tumor growth rate and a reduction in tumor cellularity based on DW-MRI scans along with enhancement of radiation efficacy. Conclusion: DW-MRI was found to be a useful imaging biomarker for the detection of radiosensitization through inhibition of checkpoint kinases. Chk1/2 inhibition resulted in antiproliferative activity, prevention of DNA-damage induced repair, and radiosensitization in preclinical GBM tumor models, both in vitro and in vivo. The effects were found to be maximal in p53-mutated GBM cells. These results provide the rationale for integration of DW-MRI in clinical translation of Chk1/2 inhibition with radiation for the treatment of GBM. Citation Format: Terence Williams, Stefanie Galbán, Fei Li, Kevin Heist, Craig Galbán, Theodore S. Lawrence, Eric C. Holland, Tami Thomae, Thomas Chenevert, Alnawaz Rehemtulla, Brian D. Ross. DW-MR imaging as a predictive biomarker of radiosensitization of GBM through targeted inhibition of checkpoint kinases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4433. doi:10.1158/1538-7445.AM2013-4433

Abstract LB-359: Inhibition of DNA repair by the dual PI3K/mTOR inhibitor NVP-BEZ235 as a novel radiosensitization strategy for glioblastoma.

Abstract Glioblastomas (GBM) are deadly brain tumors with extremely poor prognosis. The standard of care for GBM is tumor resection followed by treatment with ionizing radiation (IR) and the DNA alkylating agent temozolomide. As GBMs are highly radioresistant, it is crucial to identify effective radiosensitizers for improving therapeutic outcome. We find that NVP-BEZ235, a dual PI3K/mTOR inhibitor that is in clinical trials, potently inhibits both ATM and DNA-PKcs, the two major kinases that respond to IR-induced DNA double-strand breaks (DSBs). Consequently, this drug blocks non-homologous end joining (NHEJ) as well as homologous recombination (HR), the two major pathways of DSB repair. We find that low concentrations of NVP-BEZ235 (100 nM) can block DSB repair in a panel of human GBM lines resulting in significant radiosensitization. This radiosensitizing effect is more potent than that seen with much higher concentrations (10 μm) of current inhibitors of DNA-PKcs or ATM that are being optimized for clinical testing. In order to examine the effects of NVP-BEZ235 in vivo, we sub-cutaneously injected mice with radioresistant U87 glioma cells ectopically expressing EGFRvIII and treated with clinically-relevant doses of the drug. We find that NVP-BEZ235 blocks the activation of both DNA-PKcs and ATM in irradiated tumors resulting in significant attenuation of DSB repair. Tumors treated with a combination of IR and NVP-BEZ235 exhibit elevated DSBs and reduced Ki67 positivity compared to tumors treated with only IR or NVP-BEZ235. Consequently, a combination of IR and NVP-BEZ235 results in dramatic inhibition of tumor growth, in contrast to almost no inhibition with either treatment modality alone. Finally, we established that NVP-BEZ235 can cross the blood-brain barrier and therefore examined its effectiveness in an orthotopic brain tumor model. We find that NVP-BEZ235 given with IR can attenuate the growth of intracranial tumors generated from U87-EGFRvIII cells, thereby extending survival of tumor-bearing mice. Importantly, NVP-BEZ235 also sensitizes subcutaneous and intracranial tumors to temozolomide by attenuating the repair of secondary (replication-associated) DSBs that are generated upon temozolomide treatment. In sum, this study provides support for the potential use of NVP-BEZ235 as a radio- and chemo-sensitizer for improving the outcome of GBM therapy. Citation Format: Carlos Gil Del Alcazar, Molly Gillam, Nozomi Tomimatsu, Xiaohuan Gao, Bipasha Mukherjee, Sandeep Burma. Inhibition of DNA repair by the dual PI3K/mTOR inhibitor NVP-BEZ235 as a novel radiosensitization strategy for glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-359. doi:10.1158/1538-7445.AM2013-LB-359

DW-MRI as a Predictive Biomarker of Radiosensitization of GBM through Targeted Inhibition of Checkpoint Kinases

PURPOSE: The inherent treatment resistance of glioblastoma (GBM) can involve multiple mechanisms including checkpoint kinase (Chk1/2)-mediated increased DNA repair capability, which can attenuate the effects of genotoxic chemotherapies and radiation. The goal of this study was to evaluate diffusion-weighted magnetic resonance imaging (DW-MRI) as a biomarker for Chk1/2 inhibitors in combination with radiation for enhancement of treatment efficacy in GBM. EXPERIMENTAL DESIGN: We evaluated a specific small molecule inhibitor of Chk1/2, AZD7762, in combination with radiation using in vitro human cell lines and in vivo using a genetically engineered GBM mouse model. DW-MRI and T1-contrast MRI were used to follow treatment effects on intracranial tumor cellularity and growth rates, respectively. RESULTS: AZD7762 inhibited clonal proliferation in a panel of GBM cell lines and increased radiosensitivity in p53-mutated GBM cell lines to a greater extent compared to p53 wild-type cells. In vivo efficacy of AZD7762 demonstrated a dose-dependent inhibitory effect on GBM tumor growth rate and a reduction in tumor cellularity based on DW-MRI scans along with enhancement of radiation efficacy. CONCLUSION: DW-MRI was found to be a useful imaging biomarker for the detection of radiosensitization through inhibition of checkpoint kinases. Chk1/2 inhibition resulted in antiproliferative activity, prevention of DNA damage-induced repair, and radiosensitization in preclinical GBM tumor models, both in vitro and in vivo. The effects were found to be maximal in p53-mutated GBM cells. These results provide the rationale for integration of DW-MRI in clinical translation of Chk1/2 inhibition with radiation for the treatment of GBM.

Prospective Safety Surveillance of GH-Deficient Adults: Comparison of GH-Treated vs Untreated Patients

Context:In clinical practice, the safety profile of GH replacement therapy for GH-deficient adults compared with no replacement therapy is unknown.Objective:The objective of this study was to compare adverse events (AEs) in GH-deficient adults who were GH-treated with those in GH-deficient adults who did not receive GH replacement.Design and Setting:This was a prospective observational study in the setting of US clinical practices.Patients and Outcome Measures:AEs were compared between GH-treated (n = 1988) and untreated (n = 442) GH-deficient adults after adjusting for baseline group differences and controlling the false discovery rate. The standardized mortality ratio was calculated using US mortality rates.Results:After a mean follow-up of 2.3 years, there was no significant difference in rates of death, cancer, intracranial tumor growth or recurrence, diabetes, or cardiovascular events in GH-treated compared with untreated patients. The standardized mortality ratio was not increased in either group. Unexpected AEs (GH-treated vs untreated, P ≤ .05) included insomnia (6.4% vs 2.7%), dyspnea (4.2% vs 2.0%), anxiety (3.4% vs 0.9%), sleep apnea (3.3% vs 0.9%), and decreased libido (2.1% vs 0.2%). Some of these AEs were related to baseline risk factors (including obesity and cardiopulmonary disease), higher GH dose, or concomitant GH side effects.Conclusions:In GH-deficient adults, there was no evidence for a GH treatment effect on death, cancer, intracranial tumor recurrence, diabetes, or cardiovascular events, although the follow-up period was of insufficient duration to be conclusive for these long-term events. The identification of unexpected GH-related AEs reinforces the fact that patient selection and GH dose titration are important to ensure safety of adult GH replacement.

Roles for Endothelin Receptor B and BCL2A1 in Spontaneous CNS Metastasis of Melanoma

Metastatic spread of melanoma to the central nervous system (CNS) is a common and devastating manifestation of disease progression, which, despite its clinical importance, remains poorly understood with respect to underlying molecular mechanisms. Using a recently developed preclinical model of spontaneous melanoma CNS metastasis, we have identified alterations in expression of endothelin receptor B (EDNRB) as a potential factor that influences brain metastatic potential. Induced overexpression of this gene mediated enhanced overall metastatic disease, and resulted in an increased incidence of spontaneous CNS metastases. In contrast, the overexpression of other highlighted genes, such as BCL2A1, did not affect the incidence of CNS metastases but nevertheless appears to facilitate intracranial tumor growth. The prometastatic effect in the CNS associated with EDNRB appears to be mediated by the interaction with its ligands resulting in enhanced tumor cell proliferation and thus intracranial melanoma growth. That EDNRB contributes to melanoma metastasis is underscored by the fact that its therapeutic inhibition by the EDNRB-specific inhibitor A192621 translated into improved outcomes when treating mice with either visceral metastases or intracranial tumors. The identification of an influential role of EDNRB in CNS melanoma spontaneous metastasis may provide both a target for therapeutic intervention as well as a potential prognostic marker for patients having an increased predisposition for incidence of CNS melanoma metastases.

Fibulin-3 Promotes Glioma Growth and Resistance through a Novel Paracrine Regulation of Notch Signaling

Malignant gliomas are highly invasive and chemoresistant brain tumors with extremely poor prognosis. Targeting of the soluble factors that trigger invasion and resistance, therefore, could have a significant impact against the infiltrative glioma cells that are a major source of recurrence. Fibulin-3 is a matrix protein that is absent in normal brain but upregulated in gliomas and promotes tumor invasion by unknown mechanisms. Here, we show that fibulin-3 is a novel soluble activator of Notch signaling that antagonizes DLL3, an autocrine inhibitor or Notch, and promotes tumor cell survival and invasion in a Notch-dependent manner. Using a strategy for inducible knockdown, we found that controlled downregulation of fibulin-3 reduced Notch signaling and led to increased apoptosis, reduced self-renewal of glioblastoma-initiating cells, and impaired growth and dispersion of intracranial tumors. In addition, fibulin-3 expression correlated with expression levels of Notch-dependent genes and was a marker of Notch activation in patient-derived glioma samples. These findings underscore a major role for the tumor extracellular matrix in regulating glioma invasion and resistance to apoptosis via activation of the key Notch pathway. More importantly, this work describes a noncanonical, soluble activator of Notch in a cancer model and shows how Notch signaling can be reduced by targeting tumor-specific accessible molecules in the tumor microenvironment.

913 Effect of Gemcitabine on the Growth of Intracranial Tumors in Mice

913 Effect of Gemcitabine on the Growth of Intracranial Tumors in Mice

Dysgeusia as a Presenting Complaint of Vestibular Schwannoma

Vestibular schwannoma (VS) is a benign primary intracranial tumor growth arising from Schwann cells of the myelin sheath on the vestibular nerve. It usually arises within the internal auditory meatus and extends into the posterior fossa to occupy the space within the cerebellopontine angle (CPA). Due to local compressive effect within the CPA, surrounding cranial nerves (5, 6, 9, 10) can be affected. The facial nerve (7) is often spared.

Induction of brain tumor stem cell apoptosis by FTY720: a potential therapeutic agent for glioblastoma

FTY720 is a sphingosine analogue that down regulates expression of sphingosine-1-phosphate receptors and causes apoptosis of multiple tumor cell types, including glioma cells. This study examined the effect of FTY720 on brain tumor stem cells (BTSCs) derived from human glioblastoma (GBM) tissue. FTY720 treatment of BTSCs led to rapid inactivation of ERK MAP kinase, leading to upregulation of the BH3-only protein Bim and apoptosis. In combination with temozolomide (TMZ), the current standard chemotherapeutic agent for GBM, FTY720 synergistically induced BTSC apoptosis. FTY720 also slowed growth of intracranial xenograft tumors in nude mice and augmented the therapeutic effect of TMZ, leading to enhanced survival. Furthermore, the combination of FTY720 and TMZ decreased the invasiveness of BTSCs in mouse brains. FTY720 is known to cross the blood-brain barrier and recently received Food and Drug Administration approval for treatment of relapsing multiple sclerosis. Thus, FTY720 is an excellent potential therapeutic agent for treatment of GBM.