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 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 retinoblastoma-pathway 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 drugs moderately increased αvβ3 integrin levels and viral infection in responding but not in non-responding GSCs. LBH589 moderately increased late viral gene expression, however, virus titration revealed diminished viral progeny production by both HDACi, Scriptaid augmented caspase-3/7 activity, LC3B conversion, p62 and phospho-p70S6K consumption, as well as LDH levels. LBH589 increased LDH and phospho-p70S6K consumption. Responsiveness correlated with expression of various Rb-pathway genes and integrins. Combination treatments induced limited toxicity to human astrocytes. Conclusion: LBH589 and Scriptaid combined with Delta24-RGD revealed synergistic anti-tumor activity in a subset of GSCs. Both HDACi moderately augmented viral infection and late gene expression, but slightly reduced progeny production. The drugs differentially activated multiple cell death pathways. The limited toxicity on astrocytes supports further evaluation of the proposed combination therapies

Affected cell death mechanisms in HDACi/Delta24-RGD treatment.

<p>(A) The cells of GS102, GS224 and GS289 cells were concomitantly treated with HDACi and Delta24-RGD. Caspase-3/7 activity was measured using caspase-3/7 apoptosis assay and the IncuCyte system and counts/well are shown *Indicates significance at p<0.05 for single treatments compared to non-treated controls ± standard deviation; and **/*** Indicates significance at p<0.05 for the combination treatments compared to controls (**) and the two single agent treatments (***). (B) The cells of GS102 were concomitantly treated with HDACi and Delta24-RGD. After 72 hours, the cells were harvested and LC3BI/II, p62 and phospho-p70SK levels were determined by Western blot analysis. Beta-actin was detected as a loading control. (C) The cells of GS102, GS224 and GS289 cells were concomitantly treated with Scriptaid (upper panel, grey) or LBH589 (lower panel, white) and Delta24-RGD. The LDH levels were determined using the CytoTox-One Assay reagent at five days post-treatment. Fluorescence was measured with the Tecan reader. The LDH levels are presented as percentage of non-treated controls ± standard deviation, corrected for viability. * Indicates significance at p<0.05 for the combination treatments compared to controls (**) and the two single agent treatments(***).</p

Delta24-RGD combined with various HDACi in patient-derived GSC cultures.

<p>(A-E) The eight patient-derived GSC cultures that were treated according to a simultaneous treatment schedules of HDACi and Delta24-RGD at indicated concentrations and MOIs, respectively. The treatments were added concomitantly to the cells. Results are shown for one dose of drug and one dose of oncolytic virus, for Scriptaid (A), LBH589 (B), SAHA(C), VPA (D) and MS275 (E). Response was defined as a reduction in viability, which was significantly different from both mono-treatments (p<0.05), and is indicated in the graph by the blue bars (responders). If not meeting these criteria, the culture was defined as resistant (red bars). The results are displayed by the mean viability % ± standard deviations of triplicates. *Indicates significance of combination treatment compared to HDACi or Delta24-RGD alone, p<0.05.</p

The effects of HDACi on viral transgene expression, replication and production.

<p>(A) GS 102, GS224 and GS289 cells were concomitantly treated with LBH589 or Scriptaid and Delta24-RGD-GFP virus. In a time-lapse, fluorescence intensity per mm² and counts per well were measured and analyzed using the IncuCyte system and Image J, respectively. The results were plotted graphically as means ± standard deviation. *Indicates significance of the combination treatments compared to Delta24-RGD alone (p<0.05). (B) Representative fluorescence images acquired by the IncuCyte system at 96 hours are shown of GS102, GS224 and GS289 (magnification 10X). (C) Cells of the responsive GSCs GS102 and GS224 (blue), and cells of the non-responsive GSC cultures GS79 and GS245 (red) were concomitantly treated with Delta24-RGD and LBH589 or Scriptaid. The viral titers in the cell extracts that were obtained at 48h and 96h post-treatment are shown. *Indicates significant difference in the viral titers of Delta24-RGD alone compared to the combination treatments with HDACi and Delta24-RGD (p<0.05).</p

Increased Ad.luc.RGD infection and integrin levels by HDACi treatment in responding patient-derived GSC cultures.

<p>(A) Responsive GSC cultures (blue, GS102, GS224 and GS289) and resistant cultures (red, GS79, GS245 and GS249) were treated with LBH589 (LBH, 15nM) or Scriptaid (Scr, 1.5μM) and concomitantly infected with the luciferase expressing vector Ad.luc.RGD. Luciferase expression was measured after 24 hours post-infection. The results are presented as relative luciferase units (RLU)/100 μL lysate. *Indicates significance of combination treatment compared to virus alone (p<0.05). (B) The cells of three responders (left, GS102, GS224 and GS289) and two non-responders (right, GS79 and GS245) were treated with LBH589 (blue) and Scriptaid (red) as indicated. After 6 hours (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127058#pone.0127058.s003" target="_blank">S3 Fig</a>) and 24 hours (shown) the cells were harvested and the integrin αvβ3 (left) and CAR (right) levels were determined by flow cytometry analysis.</p

Overview of the patient-derived GSC cultures tested for combination treatment.

<p>Fourteen patient-derived GSC cultures were tested for the responsiveness to the five HDACi and Delta-24-RGD. The MGMT promoter methylation status and the molecular TCGA characterizations are depicted. The enhancement factors are shown for the various treatment modalities if EF >1, and if there was a significant difference of the combination treatment compared to the monotreatments (p<0.05). NR indicates non-responsiveness to the proposed combination treatment. Legends: UM = unmethylated; M = methylated; CLA = classical; NEU = neural; PRO = proneural; MES = mesenchymal; ND = not determined.</p><p>Overview of the patient-derived GSC cultures tested for combination treatment.</p

The effects of HDACi/Delta24-RGD on normal human astrocytes.

<p>(A) Normal human astrocytes were concomitantly treated with a dose range of HDACi and Delta24-RGD and monitored for morphological changes using the IncuCyte imaging system. Representative images at day five are depicted (10X magnification) for the tested conditions. (B) Viability of the cells was measured at day five post-treatment using the CellTiter-Glo assay, and the results are shown as percentage of non-treated control cells ± standard deviation. *Indicates significance at p<0.05 of the treated cells compared to the non-treated control cells.</p

Scriptaid and LBH589 synergize with Delta24-RGD oncolysis in patient-derived GSC cultures.

<p>(A) Chou Talalay assays were performed for Scriptaid (μM) and Delta24-RGD (MOI) to determine synergy in the GSCs GS102, GS224 and GS289. The treatments were added concomitantly to the cells. The results were depicted as viable fraction (0–100) compared to non-treated controls ± standard deviation for three replicates. *Indicates significant difference between combination and single agent treatments alone at p<0.05 level. (B) Chou Talalay assays were performed for LBH589 (nM) and Delta24-RGD (MOI) to determine synergy in the responsive GSCs GS102, GS224 and GS289. The treatments were added concomitantly to the cells. The results were depicted as viable fraction compared to non-treated controls ± standard deviation for three replicates. *Indicates significant difference between combination and single agent treatments alone at p<0.05 level. (C) The combination indices (CI) for both drugs and Delta24-RGD were calculated and shown in the same graph per GSC. A CI of < 1 indicates synergy between the two agents.</p

Evolution of DNA repair defects during malignant progression of low-grade gliomas after temozolomide treatment

Contains fulltext : 155226.pdf (publisher's version ) (Closed access)Temozolomide (TMZ) increases the overall survival of patients with glioblastoma (GBM), but its role in the clinical management of diffuse low-grade gliomas (LGG) is still being defined. DNA hypermethylation of the O (6) -methylguanine-DNA methyltransferase (MGMT) promoter is associated with an improved response to TMZ treatment, while inactivation of the DNA mismatch repair (MMR) pathway is associated with therapeutic resistance and TMZ-induced mutagenesis. We previously demonstrated that TMZ treatment of LGG induces driver mutations in the RB and AKT-mTOR pathways, which may drive malignant progression to secondary GBM. To better understand the mechanisms underlying TMZ-induced mutagenesis and malignant progression, we explored the evolution of MGMT methylation and genetic alterations affecting MMR genes in a cohort of 34 treatment-naive LGGs and their recurrences. Recurrences with TMZ-associated hypermutation had increased MGMT methylation compared to their untreated initial tumors and higher overall MGMT methylation compared to TMZ-treated non-hypermutated recurrences. A TMZ-associated mutation in one or more MMR genes was observed in five out of six TMZ-treated hypermutated recurrences. In two cases, pre-existing heterozygous deletions encompassing MGMT, or an MMR gene, were followed by TMZ-associated mutations in one of the genes of interest. These results suggest that tumor cells with methylated MGMT may undergo positive selection during TMZ treatment in the context of MMR deficiency