Glioblastoma cells have increased capacity to repair radiation-induced DNA damage after migration to the olfactory bulb.

BACKGROUND: The invasive nature of GBM combined with the diversity of brain microenvironments creates the potential for a topographic heterogeneity in GBM radioresponse. Investigating the mechanisms responsible for a microenvironment-induced differential GBM response to radiation may provide insights into the molecules and processes mediating GBM radioresistance. METHODS: Using a model system in which human GBM stem-like cells implanted into the right striatum of nude mice migrate throughout the right hemisphere (RH) to the olfactory bulb (OB), the radiation-induced DNA damage response was evaluated in each location according to γH2AX and 53BP1 foci and cell cycle phase distribution as determined by flow cytometry and immunohistochemistry. RNAseq was used to compare transcriptomes of tumor cells growing in the OB and the RH. Protein expression and neuron-tumor interaction were defined by immunohistochemistry and confocal microscopy. RESULTS: After irradiation, there was a more rapid dispersal of γH2AX and 53BP1 foci in the OB versus in the RH, indicative of increased double strand break repair capacity in the OB and consistent with the OB providing a radioprotective niche. With respect to the cell cycle, by 6 h after irradiation there was a significant loss of mitotic tumor cells in both locations suggesting a similar activation of the G2/M checkpoint. However, by 24 h post-irradiation there was an accumulation of G2 phase cells in the OB, which continued out to at least 96 h. Transcriptome analysis showed that tumor cells in the OB had higher expression levels of DNA repair genes involved in non-homologous end joining and genes related to the spindle assembly checkpoint. Tumor cells in the OB were also found to have an increased frequency of soma-soma contact with neurons. CONCLUSION: GBM cells that have migrated to the OB have an increased capacity to repair radiation-induced double strand breaks and altered cell cycle regulation. These results correspond to an upregulation of genes involved in DNA damage repair and cell cycle control. Because the murine OB provides a source of radioresistant tumor cells not evident in other experimental systems, it may serve as a model for investigating the mechanisms mediating GBM radioresistance.

Detection of metabolic change in glioblastoma cells after radiotherapy using hyperpolarized 13 C-MRI.

Dynamic nuclear polarization (DNP) of 13 C-labeled substrates enables the use of magnetic resonance imaging (MRI) to monitor specific enzymatic reactions in tumors and offers an opportunity to investigate these differences. In this study, DNP-MRI chemical shift imaging with hyperpolarized [1-13 C] pyruvate was conducted to evaluate the metabolic change in glycolytic profiles after radiation of two glioma stem-like cell-derived gliomas (GBMJ1 and NSC11) and an adherent human glioblastoma cell line (U251) in an orthotopic xenograft mouse model. The DNP-MRI showed an increase in Lac/Pyr at 6 and 16 h after irradiation (18% ± 4% and 14% ± 3%, respectively; mean ± SEM) compared with unirradiated controls in GBMJ1 tumors, whereas no significant change was observed in U251 and NSC11 tumors. Metabolomic analysis likewise showed a significant increase in lactate in GBMJ1 tumors at 16 h. An immunoblot assay showed upregulation of lactate dehydrogenase-A expression in GBMJ1 following radiation exposure, consistent with DNP-MRI and metabolomic analysis. In conclusion, our preclinical study demonstrates that the DNP-MRI technique has the potential to be a powerful diagnostic method with which to evaluate GBM tumor metabolism before and after radiation in the clinical setting.

Translation Initiation Machinery as a Tumor Selective Target for Radiosensitization.

Towards improving the efficacy of radiotherapy, one approach is to target the molecules and processes mediating cellular radioresponse. Along these lines, translational control of gene expression has been established as a fundamental component of cellular radioresponse, which suggests that the molecules participating in this process (i.e., the translational machinery) can serve as determinants of radiosensitivity. Moreover, the proteins comprising the translational machinery are often overexpressed in tumor cells suggesting the potential for tumor specific radiosensitization. Studies to date have shown that inhibiting proteins involved in translation initiation, the rate-limiting step in translation, specifically the three members of the eIF4F cap binding complex eIF4E, eIF4G, and eIF4A as well as the cap binding regulatory kinases mTOR and Mnk1/2, results in the radiosensitization of tumor cells. Because ribosomes are required for translation initiation, inhibiting ribosome biogenesis also appears to be a strategy for radiosensitization. In general, the radiosensitization induced by targeting the translation initiation machinery involves inhibition of DNA repair, which appears to be the consequence of a reduced expression of proteins critical to radioresponse. The availability of clinically relevant inhibitors of this component of the translational machinery suggests opportunities to extend this approach to radiosensitization to patient care.

Detection of glioblastoma intratumor heterogeneity in radiosensitivity using patient-derived neurosphere cultures.

PURPOSE: Glioblastoma (GBM) is characterized by extensive clonal diversity suggesting the presence of tumor cells with varying degrees of treatment sensitivity. Radiotherapy is an integral part of glioblastoma treatment. Whether GBMs are comprised of spatially distinct cellular populations with uniform or varying degrees of radiosensitivity has not been established. METHODS: Spatially distinct regions of three GBMs (J3, J7 and J14) were resected and unique cell lines were derived from each region. DNA from cell lines, corresponding tumor fragments, and patient blood was extracted for whole exome sequencing. Variants, clonal composition, and functional implications were compared and analyzed with superFreq and IPA. Limiting dilution assays were performed on cell lines to measure intrinsic radiosensitivity. RESULTS: Based on WES, cell lines generated from different regions of the same tumor were more closely correlated with their tumor of origin than the other GBMs. Variant and clonal composition comparisons showed that cell lines from distinct tumors displayed increasing levels of ITH with J3 and J14 having the lowest and highest, respectively. The radiosensitivities of the cell lines generated from the J3 tumor were similar as were those generated from the J7 tumor. However, the radiosensitivities of the 2 cell lines generated from the J14 tumor (J14T3 and J14T6) were significantly different with J14T6 being more sensitive than J14T3. CONCLUSION: Data suggest a tumor dependent ITH in radiosensitivity. The existence of ITH in radiosensitivity may impact not only the initial therapeutic response but also the effectiveness of retreatment protocols.

MGMT inhibition regulates radioresponse in GBM, GSC, and melanoma.

Radiotherapy is the standard treatment for glioblastoma (GBM), but the overall survival rate for radiotherapy treated GBM patients is poor. The use of adjuvant and concomitant temozolomide (TMZ) improves the outcome; however, the effectiveness of this treatment varies according to MGMT levels. Herein, we evaluated whether MGMT expression affected the radioresponse of human GBM, GBM stem-like cells (GSCs), and melanoma. Our results indicated a correlation between MGMT promoter methylation status and MGMT expression. MGMT-producing cell lines ACPK1, GBMJ1, A375, and MM415 displayed enhanced radiosensitivity when MGMT was silenced using siRNA or when inhibited by lomeguatrib, whereas the OSU61, NSC11, WM852, and WM266-4 cell lines, which do not normally produce MGMT, displayed reduced radiosensitivity when MGMT was overexpressed. Mechanistically lomeguatrib prolonged radiation-induced γH2AX retention in MGMT-producing cells without specific cell cycle changes, suggesting that lomeguatrib-induced radiosensitization in these cells is due to radiation-induced DNA double-stranded break (DSB) repair inhibition. The DNA-DSB repair inhibition resulted in cell death via mitotic catastrophe in MGMT-producing cells. Overall, our results demonstrate that MGMT expression regulates radioresponse in GBM, GSC, and melanoma, implying a role for MGMT as a target for radiosensitization.

Targeting PRMT5 enhances the radiosensitivity of tumor cells grown in vitro and in vivo.

PRMT5 is a widely expressed arginine methyltransferase that regulates processes involved in tumor cell proliferation and survival. In the study described here, we investigated whether PRMT5 provides a target for tumor radiosensitization. Knockdown of PRMT5 using siRNA enhanced the radiosensitivity of a panel of cell lines corresponding to tumor types typically treated with radiotherapy. To extend these studies to an experimental therapeutic setting, the PRMT5 inhibitor LLY-283 was used. Exposure of the tumor cell lines to LLY-283 decreased PRMT5 activity and enhanced their radiosensitivity. This increase in radiosensitivity was accompanied by an inhibition of DNA double-strand break repair as determined by γH2AX foci and neutral comet analyses. For a normal fibroblast cell line, although LLY-283 reduced PRMT5 activity, it had no effect on their radiosensitivity. Transcriptome analysis of U251 cells showed that LLY-283 treatment reduced the expression of genes and altered the mRNA splicing pattern of genes involved in the DNA damage response. Subcutaneous xenografts were then used to evaluate the in vivo response to LLY-283 and radiation. Treatment of mice with LLY-283 decreased tumor PRMT5 activity and significantly enhanced the radiation-induced growth delay. These results suggest that PRMT5 is a tumor selective target for radiosensitization.

Revisiting Concurrent Radiation Therapy, Temozolomide, and the Histone Deacetylase Inhibitor Valproic Acid for Patients with Glioblastoma-Proteomic Alteration and Comparison Analysis with the Standard-of-Care Chemoirradiation.

BACKGROUND: Glioblastoma (GBM) is the most common brain tumor with an overall survival (OS) of less than 30% at two years. Valproic acid (VPA) demonstrated survival benefits documented in retrospective and prospective trials, when used in combination with chemo-radiotherapy (CRT). PURPOSE: The primary goal of this study was to examine if the differential alteration in proteomic expression pre vs. post-completion of concurrent chemoirradiation (CRT) is present with the addition of VPA as compared to standard-of-care CRT. The second goal was to explore the associations between the proteomic alterations in response to VPA/RT/TMZ correlated to patient outcomes. The third goal was to use the proteomic profile to determine the mechanism of action of VPA in this setting. MATERIALS AND METHODS: Serum obtained pre- and post-CRT was analyzed using an aptamer-based SOMAScan® proteomic assay. Twenty-nine patients received CRT plus VPA, and 53 patients received CRT alone. Clinical data were obtained via a database and chart review. Tests for differences in protein expression changes between radiation therapy (RT) with or without VPA were conducted for individual proteins using two-sided t-tests, considering p-values of <0.05 as significant. Adjustment for age, sex, and other clinical covariates and hierarchical clustering of significant differentially expressed proteins was carried out, and Gene Set Enrichment analyses were performed using the Hallmark gene sets. Univariate Cox proportional hazards models were used to test the individual protein expression changes for an association with survival. The lasso Cox regression method and 10-fold cross-validation were employed to test the combinations of expression changes of proteins that could predict survival. Predictiveness curves were plotted for significant proteins for VPA response (p-value < 0.005) to show the survival probability vs. the protein expression percentiles. RESULTS: A total of 124 proteins were identified pre- vs. post-CRT that were differentially expressed between the cohorts who received CRT plus VPA and those who received CRT alone. Clinical factors did not confound the results, and distinct proteomic clustering in the VPA-treated population was identified. Time-dependent ROC curves for OS and PFS for landmark times of 20 months and 6 months, respectively, revealed AUC of 0.531, 0.756, 0.774 for OS and 0.535, 0.723, 0.806 for PFS for protein expression, clinical factors, and the combination of protein expression and clinical factors, respectively, indicating that the proteome can provide additional survival risk discrimination to that already provided by the standard clinical factors with a greater impact on PFS. Several proteins of interest were identified. Alterations in GALNT14 (increased) and CCL17 (decreased) (p = 0.003 and 0.003, respectively, FDR 0.198 for both) were associated with an improvement in both OS and PFS. The pre-CRT protein expression revealed 480 proteins predictive for OS and 212 for PFS (p < 0.05), of which 112 overlapped between OS and PFS. However, FDR-adjusted p values were high, with OS (the smallest p value of 0.586) and PFS (the smallest p value of 0.998). The protein PLCD3 had the lowest p-value (p = 0.002 and 0.0004 for OS and PFS, respectively), and its elevation prior to CRT predicted superior OS and PFS with VPA administration. Cancer hallmark genesets associated with proteomic alteration observed with the administration of VPA aligned with known signal transduction pathways of this agent in malignancy and non-malignancy settings, and GBM signaling, and included epithelial-mesenchymal transition, hedgehog signaling, Il6/JAK/STAT3, coagulation, NOTCH, apical junction, xenobiotic metabolism, and complement signaling. CONCLUSIONS: Differential alteration in proteomic expression pre- vs. post-completion of concurrent chemoirradiation (CRT) is present with the addition of VPA. Using pre- vs. post-data, prognostic proteins emerged in the analysis. Using pre-CRT data, potentially predictive proteins were identified. The protein signals and hallmark gene sets associated with the alteration in the proteome identified between patients who received VPA and those who did not, align with known biological mechanisms of action of VPA and may allow for the identification of novel biomarkers associated with outcomes that can help advance the study of VPA in future prospective trials.

Molecular profiling indicates orthotopic xenograft of glioma cell lines simulate a subclass of human glioblastoma.

Cell line models have been widely used to investigate glioblastoma multiforme (GBM) pathobiology and in the development of targeted therapies. However, GBM tumours are molecularly heterogeneous and how cell lines can best model that diversity is unknown. In this report, we investigated gene expression profiles of three preclinical growth models of glioma cell lines, in vitro and in vivo as subcutaneous and intracerebral xenografts to examine which cell line model most resembles the clinical samples. Whole genome DNA microarrays were used to profile gene expression in a collection of 25 high-grade glioblastomas, and comparisons were made to profiles of cell lines under three different growth models. Hierarchical clustering revealed three molecular subtypes of the glioblastoma patient samples. Supervised learning algorithm, trained on glioma subtypes predicted the intracerebral cell line model with one glioma subtype (r = 0.68; 95% bootstrap CI -0.41, 0.46). Survival analysis of enriched gene sets (P < 0.05) revealed 19 biological categories (146 genes) belonging to neuronal, signal transduction, apoptosis- and glutamate-mediated neurotransmitter activation signals that are associated with poor prognosis in this glioma subclass. We validated the expression profiles of these gene categories in an independent cohort of patients from 'The Cancer Genome Atlas' project (r = 0.62, 95% bootstrap CI: -0.42, 0.43). We then used these data to select and inhibit a novel target (glutamate receptor) and showed that LY341595, a glutamate receptor specific antagonist, could prolong survival in intracerebral tumour-implanted mice in combination with irradiation, providing an in vivo cell line system of preclinical studies.

CX-5461 induces radiosensitization through modification of the DNA damage response and not inhibition of RNA polymerase I.

Increased ribosome biogenesis is a distinguishing feature of cancer cells, and small molecule inhibitors of ribosome biogenesis are currently in clinical trials as single agent therapy. It has been previously shown that inhibiting ribosome biogenesis through the inhibition of nuclear export of ribosomal subunits sensitizes tumor cells to radiotherapy. In this study, the radiosensitizing potential of CX-5461, a small molecule inhibitor of RNA polymerase I, was tested. Radiosensitization was measured by clonogenic survival assay in a panel of four tumor cell lines derived from three different tumor types commonly treated with radiation. 50 nM CX-5461 radiosensitized PANC-1, U251, HeLa, and PSN1 cells with dose enhancement factors in the range of 1.2-1.3. However, 50 nM CX-5461 was not sufficient to inhibit 45S transcription alone or in combination with radiation. The mechanism of cell death with the combination of CX-5461 and radiation occurred through mitotic catastrophe and not apoptosis. CX-5461 inhibited the repair and/or enhanced the initial levels of radiation-induced DNA double strand breaks. Understanding the mechanism of CX-5461-induced radiosensitization should be of value in the potential application of the CX-5461/radiotherapy combination in cancer treatment.

Inhibition of the Translation Initiation Factor eIF4A Enhances Tumor Cell Radiosensitivity.

A fundamental component of cellular radioresponse is the translational control of gene expression. Because a critical regulator of translational control is the eukaryotic translation initiation factor 4F (eIF4F) cap binding complex, we investigated whether eIF4A, the RNA helicase component of eIF4F, can serve as a target for radiosensitization. Knockdown of eIF4A using siRNA reduced translational efficiency, as determined from polysome profiles, and enhanced tumor cell radiosensitivity as determined by clonogenic survival. The increased radiosensitivity was accompanied by a delayed dispersion of radiation-induced γH2AX foci, suggestive of an inhibition of DNA double-strand break repair. Studies were then extended to (-)-SDS-1-021, a pharmacologic inhibitor of eIF4A. Treatment of cells with the rocaglate (-)-SDS-1-021 resulted in a decrease in translational efficiency as well as protein synthesis. (-)-SDS-1-021 treatment also enhanced the radiosensitivity of tumor cell lines. This (-)-SDS-1-021-induced radiosensitization was accompanied by a delay in radiation-induced γH2AX foci dispersal, consistent with a causative role for the inhibition of double-strand break repair. In contrast, although (-)-SDS-1-021 inhibited translation and protein synthesis in a normal fibroblast cell line, it had no effect on radiosensitivity of normal cells. Subcutaneous xenografts were then used to evaluate the in vivo response to (-)-SDS-1-021 and radiation. Treatment of mice bearing subcutaneous xenografts with (-)-SDS-1-021 decreased tumor translational efficiency as determined by polysome profiles. Although (-)-SDS-1-021 treatment alone had no effect on tumor growth, it significantly enhanced the radiation-induced growth delay. These results suggest that eIF4A is a tumor-selective target for radiosensitization.

Grand rounds at the National Institutes of Health: HDAC inhibitors as radiation modifiers, from bench to clinic.

Glioblastoma multiforme (GBM) is the most common and aggressive malignant brain tumour. Patients afflicted with this disease unfortunately have a very poor prognosis, and fewer than 5% of patients survive for 5 years from the time of diagnosis. Therefore, improved therapies to treat this disease are sorely needed. One such class of drugs that have generated great enthusiasm for the treatment of numerous malignancies, including GBM, is histone deacetylase (HDAC) inhibitors. Pre-clinical data have demonstrated the efficacy of various HDAC inhibitors as anticancer agents, with the greatest effects shown when HDAC inhibitors are used in combination with other therapies. As a result of encouraging pre-clinical data, numerous HDAC inhibitors are under investigation in clinical trials, either as monotherapies or in conjunction with other treatments such as chemotherapy, biologic therapy or radiation therapy. In fact, two actively studied HDAC inhibitors, vorinostat and depsipeptide, were recently approved for the treatment of refractory cutaneous T cell lymphoma. In this review, we first present a patient with GBM, and then discuss the pathogenesis, epidemiology and current treatment options of GBM. Finally, we examine the translation of pre-clinical studies that have demonstrated HDAC inhibitors as potent radiosensitizers in in vitro and in vivo models, to a phase II clinical trial combining the HDAC inhibitor, valproic acid, along with temozolomide and radiation therapy for the treatment of GBM.

Radiosensitization of glioma cells by modulation of Met signalling with the hepatocyte growth factor neutralizing antibody, AMG102.

The hepatocyte growth factor (HGF)/Met signalling pathway is up-regulated in many cancers, with downstream mediators playing a role in DNA double strand break repair. Previous studies have shown increased radiosensitization of tumours through modulation of Met signalling by genetic methods. We investigated the effects of the anti-HGF monoclonal antibody, AMG102, on the response to ionizing radiation in a model of glioblastoma multiforme in vitro and in vivo. Radiosensitivity was evaluated in vitro in the U-87 MG human glioma cell line. Met activation was measured by Western blot, and the effect on survival following radiation was evaluated by clonogenic assay. Mechanism of cell death was evaluated by apoptosis and mitotic catastrophe assays. DNA damage was quantitated by γH2AX foci and neutral comet assay. Growth kinetics of subcutaneous tumours was used to assess the effects of AMG102 on in vivo tumour radiosensitivity. AMG102 inhibited Met activation after irradiation. An enhancement of radiation cell killing was shown with no toxicity using drug alone. Retention of γH2AX foci at 6 and 24 hrs following the drug/radiation combination indicated an inhibition of DNA repair following radiation, and comet assay confirmed DNA damage persisting over the same duration. At 48 and 72 hrs following radiation, a significant increase of cells undergoing mitotic catastrophe was seen in the drug/radiation treated cells. Growth of subcutaneous tumours was slowed in combination treated mice, with an effect that was greater than additive for each modality individually. Modulation of Met signalling with AMG102 may prove a novel radiation sensitizing strategy. Our data indicate that DNA repair processes downstream of Met are impaired leading to increased cell death through mitotic catastrophe.

Bench to bedside radiosensitizer development strategy for newly diagnosed glioblastoma.

Glioblastoma is the most common primary brain malignancy and carries with it a poor prognosis. New agents are urgently needed, however nearly all Phase III trials of GBM patients of the past 25 years have failed to demonstrate improvement in outcomes. In 2019, the National Cancer Institute Clinical Trials and Translational Research Advisory Committee (CTAC) Glioblastoma Working Group (GBM WG) identified 5 broad areas of research thought to be important in the development of new herapeutics for GBM. Among those was optimizing radioresponse for GBM in situ. One such strategy to increase radiation efficacy is the addition of a radiosensitizer to improve the therapeutic ratio by enhancing tumor sensitivity while ideally having minimal to no effect on normal tissue. Historically the majority of trials using radiosensitizers have been unsuccessful, but they provide important guidance in what is required to develop agents more efficiently. Improved target selection is essential for a drug to provide maximal benefit, and once that target is identified it must be validated through pre-clinical studies. Careful selection of appropriate in vitro and in vivo models to demonstrate increased radiosensitivity and suitable bioavailability are then necessary to prove that a drug warrants advancement to clinical investigation. Once investigational agents are validated pre-clinically, patient trials require consistency both in terms of planning study design as well as reporting efficacy and toxicity in order to assess the potential benefit of the drug. Through this paper we hope to outline strategies for developing effective radiosensitizers against GBM using as models the examples of XPO1 inhibitors and HDAC inhibitors developed from our own lab.

In Vitro Methods for the Study of Glioblastoma Stem-Like Cell Radiosensitivity.

Ionizing radiation is a critical component of glioblastoma (GBM) therapy. Recent data have implicated glioblastoma stem-like cells (GSCs) as determinants of GBM development, maintenance, and treatment response. Understanding the response of GSCs to radiation should thus provide insight into the development of improved GBM treatment strategies. Towards this end, in vitro techniques for the analysis of GSC radiosensitivity are an essential starting point. One such method, the clonogenic survival assay has been adapted to assessing the intrinsic radiosensitivity of GSCs and is described here. As an alternative method, the limiting dilution assay is presented for defining the radiosensitivity of GSC lines that do not form colonies or only grow as neurospheres. In addition to these cellular strategies, we describe γH2AX foci analysis, which provides a surrogate marker for radiosensitivity at the molecular level. Taken together, the in vitro methods presented here provide tools for defining intrinsic radiosensitivity of GSCs and for testing agents that may enhance GBM radioresponse.

The Radiosensitizing Effect of AZD0530 in Glioblastoma and Glioblastoma Stem-Like Cells.

AZD0530, a potent small-molecule inhibitor of the Src kinase family, is an anticancer drug used in the treatment of various cancers. In the case of glioblastoma (GBM), where resistance to radiotherapy frequently occurs, Src kinase is known as one of the molecules responsible for imparting radioresistance to GBM. Thus, we evaluated the effect of AZD0530 on the radiosensitivity of human GBM cells and human glioblastoma stem-like cells (GSCs). We show that Src activity of GBM and GSC is increased by radiation and inhibited by AZD0530, and using clonogenic assays, AZD0530 enhances the radiosensitivity of GBM and GSCs. Also, AZD0530 induced a prolongation of radiation-induced γH2AX without specific cell cycle and mitotic index changes, suggesting that AZD0530-induced radiosensitization in GBM cells and GSCs results from the inhibition of DNA repair. In addition, AZD0530 was shown to inhibit the radiation-induced EGFR/PI3K/AKT pathway, which is known to promote and regulate radioresistance and survival of GBM cells by radiation. Finally, mice bearing orthotopic xenografts initiated from GBM cells were then used to evaluate the in vivo response to AZD0530 and radiation. The combination of AZD0530 and radiation showed the longest median survival compared with any single modality. Thus, these results show that AZD0530 enhances the radiosensitivity of GBM cells and GSCs and suggest the possibility of AZD0530 as a clinical radiosensitizer for treatment of GBM.

Physiologic oxygen concentration enhances the stem-like properties of CD133+ human glioblastoma cells in vitro.

In vitro investigations of tumor stem-like cells (TSC) isolated from human glioblastoma (GB) surgical specimens have been done primarily at an atmospheric oxygen level of 20%. To determine whether an oxygen level more consistent with in situ conditions affects their stem cell-like characteristics, we compared GB TSCs grown under conditions of 20% and 7% oxygen. Growing CD133(+) cells sorted from three GB neurosphere cultures at 7% O(2) reduced their doubling time and increased the self-renewal potential as reflected by clonogenicity. Furthermore, at 7% oxygen, the cultures exhibited an enhanced capacity to differentiate along both the glial and neuronal pathways. As compared with 20%, growth at 7% oxygen resulted in an increase in the expression levels of the neural stem cell markers CD133 and nestin as well as the stem cell markers Oct4 and Sox2. In addition, whereas hypoxia inducible factor 1alpha was not affected in CD133(+) TSCs grown at 7% O(2), hypoxia-inducible factor 2alpha was expressed at higher levels as compared with 20% oxygen. Gene expression profiles generated by microarray analysis revealed that reducing oxygen level to 7% resulted in the up-regulation and down-regulation of a significant number of genes, with more than 140 being commonly affected among the three CD133(+) cultures. Furthermore, Gene Ontology categories up-regulated at 7% oxygen included those associated with stem cells or GB TSCs. Thus, the data presented indicate that growth at the more physiologically relevant oxygen level of 7% enhances the stem cell-like phenotype of CD133(+) GB cells.

HDAC inhibitors in cancer care.

The epigenetic control of gene expression has been shown to play an important role in cancer initiation, progression, and resistance. Thus, agents that modify the epigenetic environment of tumors will likely be an important addition to the anticancer arsenal. Specifically, there is much interest in modulating histone acetylation using a new class of drugs, histone deacetylase (HDAC) inhibitors. Preclinical data have demonstrated the efficacy of various HDAC inhibitors as anticancer agents, with the greatest effects shown when HDAC inhibitors are used in combination with other therapies. As a result of encouraging preclinical data, numerous HDAC inhibitors are being investigated in clinical trials either as monotherapies or in conjunction with other treatments such as chemotherapy, biologic therapy, or radiation therapy. In fact, vorinostat and depsipeptide, two actively studied HDAC inhibitors, were recently approved for the treatment of refractory cutaneous T-cell lymphoma. Although the use of HDAC inhibitors has generated great enthusiasm, a significant amount of work still needs to be done in order to understand their mechanisms of action, as well as to determine the appropriate patient characteristics and subsets of cancer for which HDAC inhibitors hold the most potential for effective treatment.

In vitro and in vivo radiosensitization of glioblastoma cells by the poly (ADP-ribose) polymerase inhibitor E7016.

PURPOSE: Poly (ADP-ribose) polymerase (PARP) inhibitors are undergoing clinical evaluation for cancer therapy. Because PARP inhibition has been shown to enhance tumor cell sensitivity to radiation, we investigated the in vitro and in vivo effects of the novel PARP inhibitor E7016. EXPERIMENTAL DESIGN: The effect of E7016 on the in vitro radiosensitivity of tumor cell lines was evaluated using clonogenic survival. DNA damage and repair were measured using gammaH2AX foci and neutral comet assay. Mitotic catastrophe was determined by immunostaining. Tumor growth delay was evaluated in mice for the effect of E7016 on in vivo (U251) tumor radiosensitivity. RESULTS: Cell lines exposed to E7016 preirradiation yielded an increase in radiosensitivity with dose enhancement factors at a surviving fraction of 0.1 from 1.4 to 1.7. To assess DNA double-strand breaks repair, gammaH2AX measured at 24 hours postirradiation had significantly more foci per cell in the E7016/irradiation group versus irradiation alone. Neutral comet assay further suggested unrepaired double-strand breaks with significantly greater DNA damage at 6 hours postirradiation in the combination group versus irradiation alone. Mitotic catastrophe staining revealed a significantly greater number of cells staining positive at 24 hours postirradiation in the combination group. In vivo, mice treated with E7016/irradiation/temozolomide had an additional growth delay of six days compared with the combination of temozolomide and irradiation. CONCLUSIONS: These results indicate that E7016 can enhance tumor cell radiosensitivity in vitro and in vivo through the inhibition of DNA repair. Moreover, enhanced growth delay with the addition of E7016 to temozolomide and radiotherapy in a glioma mouse model suggests a potential role for this drug in the treatment of glioblastoma multiforme.

Radiosensitizers in the temozolomide era for newly diagnosed glioblastoma.

Glioblastoma (GBM) is a challenging diagnosis with almost universally poor prognosis. Though the survival advantage of postoperative radiation (RT) is well established, around 90% of patients will fail in the RT field. The high likelihood of local failure suggests the efficacy of RT needs to be improved to improve clinical outcomes. Radiosensitizers are an established method of enhancing RT cell killing through the addition of a pharmaceutical agent. Though the majority of trials using radiosensitizers have historically been unsuccessful, there continues to be interest with a variety of approaches having been employed. Epidermal growth factor receptor inhibitors, histone deacetylase inhibitors, antiangiogenic agents, and a number of other molecularly targeted agents have all been investigated as potential methods of radiosensitization in the temozolomide era. Outcomes have varied both in terms of toxicity and survival, but some agents such as valproic acid and bortezomib have demonstrated promising results. However, reporting of results in phase 2 trials in newly diagnosed GBM have been inconsistent, with no standard in reporting progression-free survival and toxicity. There is a pressing need for investigation of new agents; however, nearly all phase 3 trials of GBM patients of the past 25 years have demonstrated no improvement in outcomes. One proposed explanation for this is the selection of agents lacking sufficient preclinical data and/or based on poorly designed phase 2 trials. Radiosensitization may represent a viable strategy for improving GBM outcomes in newly diagnosed patients, and further investigation using agents with promising phase 2 data is warranted.

Improving Radiation Response in Glioblastoma Using ECO/siRNA Nanoparticles Targeting DNA Damage Repair.

Radiation therapy is a mainstay in the standard of care for glioblastoma (GBM), thus inhibiting the DNA damage response (DDR) is a major strategy to improve radiation response and therapeutic outcomes. Small interfering RNA (siRNA) therapy holds immeasurable potential for the treatment of GBM, however delivery of the siRNA payload remains the largest obstacle for clinical implementation. Here we demonstrate the effectiveness of the novel nanomaterial, ECO (1-aminoethylimino[bis(N-oleoylcysteinylaminoethyl) propionamide]), to deliver siRNA targeting DDR proteins ataxia telangiectasia mutated and DNA-dependent protein kinase (DNApk-cs) for the radiosensitzation of GBM in vitro and in vivo. ECO nanoparticles (NPs) were shown to efficiently deliver siRNA and silence target protein expression in glioma (U251) and glioma stem cell lines (NSC11, GBMJ1). Importantly, ECO NPs displayed no cytotoxicity and minimal silencing of genes in normal astrocytes. Treatment with ECO/siRNA NPs and radiation resulted in the prolonged presence of γH2AX foci, indicators of DNA damage, and increased radiosensitivity in all tumor cell lines. In vivo, intratumoral injection of ECO/siDNApk-cs NPs with radiation resulted in a significant increase in survival compared with injection of NPs alone. These data suggest the ECO nanomaterial can effectively deliver siRNA to more selectively target and radiosensitize tumor cells to improve therapeutic outcomes in GBM.