ABT-888 enhances cytotoxic effects of temozolomide independent of MGMT status in serum free cultured glioma cells

Background: The current standard of care for Glioblastoma Multiforme (GBM) consists of fractionated focal irradiation with concomitant temozolomide (TMZ) chemotherapy. A promising strategy to increase the efficacy of TMZ is through interference with the DNA damage repair machinery, by poly(ADP-ribose) polymerase protein inhibition(PARPi). The o

A Systematic Comparison Identifies an ATP-Based Viability Assay as Most Suitable Read-Out for Drug Screening in Glioma Stem-Like Cells

Serum-free culture methods for patient-derived primary glioma cultures, selecting for glioma stem-like cells (GSCs), are becoming the gold standard in neurooncology research. These GSCs can be implemented in drug screens to detect patient-specific responses, potentially bridging the translational gap to personalized medicine. Since numerous compounds are available, a rapid and reliable readout for drug efficacies is required. This can be done using approaches that measure viability, confluency, cytotoxicity, or apoptosis. To determine which assay is best suitable for drug screening, 10 different assays were systematically tested on established glioma cell lines and validated on a panel of GSCs. General applicability was assessed using distinct treatment modalities, being temozolomide, radiation, rapamycin, and the oncolytic adenovirus Delta24-RGD. The apoptosis and cytotoxicity assays did not unequivocally detect responses and were excluded from further testing. The NADH- and ATP-based viability assays revealed comparable readout for all treatments; however, the latter had smaller standard deviations and direct readout. Importantly, drugs that interfere with cell metabolism require alternative techniques such as confluency monitoring to accurately measure treatment effects. Taken together, our data suggest that the combination of ATP luminescence assays with confluency monitoring provides the most specific and reproducible readout for drug screening on primary GSCs

The Sequence of Delta24-RGD and TMZ Administration in Malignant Glioma Affects the Role of CD8(+)T Cell Anti-tumor Activity

The conditionally replicating oncolytic adenovirus Delta24-RGD (Ad) is currently under investigation in clinical trials for glioblastoma, including in combination with temozolomide (TMZ), the standard chemotherapy for this tumor. Previously, we showed that the efficacy of Delta24-RGD in a murine model is primarily dependent on the virus-induced anti-tumor immune response. As observed with most chemotherapies, TMZ has pronounced immune-modulating effects. Here, we studied the combined effects of these treatments in a murine glioma model. In vitro, we observed a synergistic activity between Delta24-RGD and TMZ. In vivo, C57BL/6 mice bearing intracranial GL261 tumors were treated with TMZ for 5 days either prior to intratumoral Delta24-RGD injection (TMZ/Ad) or post virus injection (Ad/TMZ). Notably, the Ad/TMZ regimen led to similar tumoral CD8+ T cell influx as the virus-only treatment, but increased the ability of CD8+ T cells to specifically recognize the tumor cells. This was accompanied by improved survival. The TMZ/Ad regimen also improved survival significantly compared to controls, but not compared to virus alone. In this group, the influx of dendritic cells is impaired, followed by a significantly lower number of tumor-infiltrating CD8+ T cells and no recognition of tumor cells. Depletion of either CD4+ T cells or CD8+ T cells impaired the efficacy of Delta24-RGD, underscoring the role of these cells in therapeutic activity of the virus. Overall, we show that the addition of TMZ to Delta24-RGD treatment leads to a significant increase in survival and that the order of sequence of these treatments affects the CD8+T cell anti-tumor activity

The in vivo therapeutic efficacy of the oncolytic adenovirus Delta24-RGD is mediated by tumor-specific immunity

The oncolytic adenovirus Delta24-RGD represents a new promising therapeutic agent for patients with a malignant glioma and is currently under investigation in clinical phase I/II trials. Earlier preclinical studies showed that Delta24-RGD is able to effectively lyse tumor cells, yielding promising results in various immune-deficient glioma models. However, the role of the immune response in oncolytic adenovirus therapy for glioma has never been explored. To this end, we assessed Delta24-RGD treatment in an immune-competent orthotopic mouse model for glioma and evaluated immune responses against tumor and virus. Delta24-RGD treatment led to long-term survival in 50% of mice and this effect was completely lost upon administration of the immunosuppressive agent dexamethasone. Delta24-RGD enhanced intra-tumoral infiltration of F4/80+ macrophages, CD4+ and CD8+ T-cells, and increased the local production of pro-inflammatory cytokines and chemokines. In treated mice, T cell responses were directed to the virus as well a

The BCL-2 inhibitor obatoclax overcomes resistance to histone deacetylase inhibitors SAHA and LBH589 as radiosensitizers in patient-derived glioblastoma stem-like cells

Glioblastoma has shown resistance to histone deacetylase inhibitors (HDACi) as radiosensitizers in cultures with Bcl-XL over-expression. We study the efficacy of SAHA/RTx and LBH589/RTx when manipulating Bcl-2 family proteins using the Bcl-2 inhibitor Obatoclax in patient-derived glioblastoma stem-like cell (GSC) cultures. GSC cultures in general have a deletion in phosphatase and tensin homolog (PTEN). Synergy was determined by the Chou Talalay method. The effects on apoptosis and autophagy were studied by measuring caspase-3/7, Bcl-XL, Mcl-1 and LC3BI/II proteins. The relation between treatment response and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status, recurrence and gene expression levels of the tumors were studied. Obatoclax synergized with SAHA and LBH589 and sensitized cells to HDACi/RTx. Over 50% of GSC cultures were responsive to Obatoclax with either single agent. Combined with HDACi/RTx treatment, Obatoclax increased caspase-3/7 and inhibited Bcl-2 family proteins Bcl-XL and Mcl-1 more effectively than other treatments. Genes predictive for treatment response were identified, including the F-box/WD repeat-containing protein-7, which was previously related to Bcl-2 inhibition and HDACi sensitivity. We emphasize the functional relation between Bcl-2 proteins and radiosensitization by HDACi and provide a target for increasing responsiveness in glioblastoma by using the Bcl-2 inhibitor Obatoclax

The HDAC inhibitors scriptaid and LBH589 combined with the oncolytic virus Delta24-RGD exert enhanced anti-tumor efficacy in patient-derived glioblastoma cells

Background: A phase I/II trial for glioblastoma with the oncolytic adenovirus Delta24-RGD was recently completed. Delta24-RGD conditionally replicates in cells with a disrupted retinoblastomapathway and enters cells via αvβ3/5 integrins. Glioblastomas are differentially sensitive to Delta24-RGD. HDAC inhibitors (HDACi) affect integrins and share common cell death pathways with Delta24-RGD. We studied the combination treatment effects of HDACi and Delta24-RGD in patient-derived glioblastoma stem-like cells (GSC), and we determined the most effective HDACi. Methods: SAHA, Valproic Acid, Scriptaid, MS275 and LBH589 were combined with Delta24-RGD in fourteen distinct GSCs. Synergy was determined by Chou Talalay method. Viral infection and replication were assessed using luciferase and GFP encoding vectors and hexon-titration assays. Coxsackie adenovirus receptor and αvβ3 integrin levels were determined by flow cytometry. Oncolysis and mechanisms of cell death were studied by viability, caspase-3/7, LDH and LC3B/p62, phospho-p70S6K. Toxicity was studied on normal human astrocytes. MGMT promotor methylation status, TCGA classification, Rb-pathway and integrin gene expression levels were assessed as markers of responsiveness. Results: Scriptaid and LBH589 acted synergistically with Delta24-RGD in approximately 50% of the GSCs. Both dr

The Sequence of Delta24-RGD and TMZ Administration in Malignant Glioma Affects the Role of CD8+T Cell Anti-tumor Activity

The conditionally replicating oncolytic adenovirus Delta24-RGD (Ad) is currently under investigation in clinical trials for glioblastoma, including in combination with temozolomide (TMZ), the standard chemotherapy for this tumor. Previously, we showed that the efficacy of Delta24-RGD in a murine model is primarily dependent on the virus-induced anti-tumor immune response. As observed with most chemotherapies, TMZ has pronounced immune-modulating effects. Here, we studied the combined effects of these treatments in a murine glioma model. In vitro, we observed a synergistic activity between Delta24-RGD and TMZ. In vivo, C57BL/6 mice bearing intracranial GL261 tumors were treated with TMZ for 5 days either prior to intratumoral Delta24-RGD injection (TMZ/Ad) or post virus injection (Ad/TMZ). Notably, the Ad/TMZ regimen led to similar tumoral CD8+ T cell influx as the virus-only treatment, but increased the ability of CD8+ T cells to specifically recognize the tumor cells. This was accompanied by improved survival. The TMZ/Ad regimen also improved survival significantly compared to controls, but not compared to virus alone. In this group, the influx of dendritic cells is impaired, followed by a significantly lower number of tumor-infiltrating CD8+ T cells and no recognition of tumor cells. Depletion of either CD4+ T cells or CD8+ T cells impaired the efficacy of Delta24-RGD, underscoring the role of these cells in therapeutic activity of the virus. Overall, we show that the addition of TMZ to Delta24-RGD treatment leads to a significant increase in survival and that the order of sequence of these treatments affects the CD8+T cell anti-tumor activity

TP53 mutated glioblastoma stem-like cell cultures are sensitive to dual mTORC1/2 inhibition while resistance in TP53 wild type cultures can be overcome by combined inhibition of mTORC1/2 and Bcl-2

Background: Glioblastoma is the most malignant tumor of the central nervous system and still lacks effective treatment. This study explores mutational biomarkers of 11 drugs targeting either the RTK/Ras/PI3K, the p53 or the Rb pathway using 25 patient-derived glioblastoma stem-like cell cultures (GSCs). Results: We found that TP53 mutated GSCs were approximately 3.5 fold more sensitive to dual inhibition of mammalian target of rapamycin complex 1 and 2 (mTORC1/2) compared to wild type GSCs. We identified that Bcl-2(Thr56/Ser70) phosphorylation contributed to the resistance of TP53 wild type GSCs against dual mTORC1/2 inhibition. The Bcl-2 inhibitor ABT-263 (navitoclax) increased sensitivity to the mTORC1/2 inhibitor AZD8055 in TP53 wild type GSCs, while sensitivity to AZD8055 in TP53 mutated GSCs remained unchanged. Conclusion: Our data suggest that Bcl-2 confers resistance to mTORC1/2 inhibitors in TP53 wild type GSCs and that combined inhibition of both mTORC1/2 and Bcl-2 is worthwhile to explore further in TP53 wild type glioblastomas, whereas in TP53 mutated glioblastomas dual mTORC1/2 inhibitors should be explored

The in vivo therapeutic efficacy of the oncolytic adenovirus Delta24-RGD is mediated by tumor-specific immunity.

The oncolytic adenovirus Delta24-RGD represents a new promising therapeutic agent for patients with a malignant glioma and is currently under investigation in clinical phase I/II trials. Earlier preclinical studies showed that Delta24-RGD is able to effectively lyse tumor cells, yielding promising results in various immune-deficient glioma models. However, the role of the immune response in oncolytic adenovirus therapy for glioma has never been explored. To this end, we assessed Delta24-RGD treatment in an immune-competent orthotopic mouse model for glioma and evaluated immune responses against tumor and virus. Delta24-RGD treatment led to long-term survival in 50% of mice and this effect was completely lost upon administration of the immunosuppressive agent dexamethasone. Delta24-RGD enhanced intra-tumoral infiltration of F4/80+ macrophages, CD4+ and CD8+ T-cells, and increased the local production of pro-inflammatory cytokines and chemokines. In treated mice, T cell responses were directed to the virus as well as to the tumor cells, which was reflected in the presence of protective immunological memory in mice that underwent tumor rechallenge. Together, these data provide evidence that the immune system plays a vital role in the therapeutic efficacy of oncolytic adenovirus therapy of glioma, and may provide angles to future improvements on Delta24-RGD therapy