Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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HDACI and DAC induce specific epigenetic profile in DLBCL

ABSTRACT: Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoid neoplasm in the world representing 30-40% of all lymphomas. Standard immunochemotherapy (cyclophosphamide, doxorubicin, vincristine, prednisone and rituximab) ensures cure in 60 to 65% of patients, while the rest progress or relapse. While advances have been made in the treatment of DLBCL, especially with the addition of rituximab, it is now apparent that there may be significant differences in prognosis that are related to the cell of origin, and that this disease is heterogeneous and novel treatment options are needed. It has been hypothesized that the combination of HDACI and hypomethylating agents might be a new approach to the treatment of relapsed or refractory DLBCL. This combination is thought to disrupt the transcription repressor complex consisting of methyl binding domain proteins (MBDP) and histone deacetylases (HDACs). We have explored the effect of different HDACI and decitabine combinations in in vitro and in vivo models of DLBCL. These data suggest a class effect, with all four HDACI (panobinostat, belinostat, vorinostat, depsipeptide) synergizing with decitabine in cytotoxicity assay across the spectrum of DLBCL cells. Synergy was validated in a number of other assays including a caspase 3 activation and apoptosis. Furthermore, the combination of panobinostat and decitabine induced markedly increased histone acetylation. The in vitro observations were confirmed in an in vivo murine xenograft experiment with the Ly1 DLBCL line. Genome wide methylation analysis and gene expression profiling demonstrated that the combination of these two epigenetic therapies produced a unique gene expression profile compared to the samples treated with single drugs. These data strongly support the potential therapeutic role of a combinatorial epigenetic platform for the treatment of B-cell lymphomas, in particular in patients with DLBCL. Clearly, future studies will need to focus on integrating the appropriate correlative studies, with an effort to identify and or validate biomarkers of activity with these combinations. The likelihood moving forward is that the mechanism of action of these combinations may vary from disease context to disease context. Genome-wide array findings from these kinds of studies could be expanded to samples from patients on clinical trials to identify novel biomarkers of response, leading to the rational treatment of individual diseases based upon the underlying pathogenesis. We have used single samples from three DLBCL cell lines (biological replicates) which were treated with DMSO, decitabine alone, panobinostat alone and their combination for 48h - total of 12 samples

ORGANISM(S): Homo sapiens

SUBMITTER: Matko Kalac

PROVIDER: E-GEOD-27226 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoid neoplasm in the world representing 30-40% of all lymphomas. Standard immunochemotherapy (cyclophosphamide, doxorubicin, vincristine, prednisone and rituximab) ensures cure in 60 to 65% of patients, while the rest progress or relapse. While advances have been made in the treatment of DLBCL, especially with the addition of rituximab, it is now apparent that there may be significant differences in prognosis that are related to the cell of origin, and that this disease is heterogeneous and novel treatment options are needed. It has been hypothesized that the combination of HDACI and hypomethylating agents might be a new approach to the treatment of relapsed or refractory DLBCL. This combination is thought to disrupt the transcription repressor complex consisting of methyl binding domain proteins (MBDP) and histone deacetylases (HDACs). We have explored the effect of different HDACI and decitabine combinations in in vitro and in vivo models of DLBCL. These data suggest a class effect, with all four HDACI (panobinostat, belinostat, vorinostat, depsipeptide) synergizing with decitabine in cytotoxicity assay across the spectrum of DLBCL cells. Synergy was validated in a number of other assays including a caspase 3 activation and apoptosis. Furthermore, the combination of panobinostat and decitabine induced markedly increased histone acetylation. The in vitro observations were confirmed in an in vivo murine xenograft experiment with the Ly1 DLBCL line. Genome wide methylation analysis and gene expression profiling demonstrated that the combination of these two epigenetic therapies produced a unique gene expression profile compared to the samples treated with single drugs. These data strongly support the potential therapeutic role of a combinatorial epigenetic platform for the treatment of B-cell lymphomas, in particular in patients with DLBCL. Clearly, future studies will need to focus on integrating the appropriate correlative studies, with an effort to identify and or validate biomarkers of activity with these combinations. The likelihood moving forward is that the mechanism of action of these combinations may vary from disease context to disease context. Genome-wide array findings from these kinds of studies could be expanded to samples from patients on clinical trials to identify novel biomarkers of response, leading to the rational treatment of individual diseases based upon the underlying pathogenesis.

Project description:Background: Epigenetic modifications such as methylation silencing of genes with CpG-island-associated promoters is frequently observed in cancer. Studies regarding the implications of epigenetic modifications in osteosarcoma (OS) have been limited. The epigenetic drug decitabine is a potential re-activator of silenced genes through de-methylation, and is currently undergoing clinical trials for cancer treatment. No study to date has utilized decitabine to modify gene expression in OS-derived cells to identify gene-specific methylation targets that may have therapeutic importance. The objective of this study was to measure the response of the OS cell line, U-2OS, to decitabine treatment both in vitro and in vivo. Results: Genome-wide screening was used to distinguish decitabine-dependent changes in gene expression in U-2OS and to identify responsive loci with de-methylated CpG regions. U-2OS xenografts were established in the sub-renal capsule of immune-deficient mice to study the effect of decitabine in vivo on tumor growth and differentiation. Genome-wide screening of U-2OS cells in vitro revealed that decitabine treatment at low-dosage (1 ÂµM) caused a significant induction (p<0.0025) in the expression of 88 genes, 13 had a â?¥2-fold change, 11 of which had CpG-island-associated promoters. Interestingly, 6 of these 11 were pro-apoptotic genes. 1 ÂµM decitabine resulted in a significant induction of cell death in U-2OS cells in vitro (p<0.05). Decitabine treatment also reduced tumor volume size significantly (p<0.05) in U-2OS in vivo at a 2.5 mg/kg dose. Histological analysis of U-2OS xenograft sections revealed a lower mitotic activity (p<0.0001), a higher bone matrix production (p<0.0001), and higher apoptosis (p = 0.0329) after treatment. The 6 pro-apoptotic genes were also induced to â?¥2-fold in vivo. Quantitative Methylation Pyrosequencing (Pyro Q-CpG) showed that 4 pro-apoptotic genes had CpG-island DNA de-methylation as a result of treatment in vitro and in vivo. Conclusion: Our data provide new insights regarding the use of epigenetic modifiers in OS, and have important implications for therapeutic trials involving demethylation drugs. The findings of this study suggest an epigenetic mechanism, whereby OS tumor cells silence expression of pro-apoptotic genes through promoter-CpG methylation, to maintain high proliferation capacity and continuous growth. Experiment Overall Design: Total RNA was extracted using the RNeasy kit (Qiagen, Germany) from duplicate experiments of U-2OS cells at Day3 after treatment with 1 ÂµM decitabine or medium alone (control). In each experiment RNA yields were pooled from two independent cultures per treatment arm to minimize experimental noise. For each case, 10 Âµg of RNA was labeled and hybridized to the Affymetrix HG-U133A GeneChips using the manufacturerâ??s protocol (Affymetrix, Santa Clara, CA) by the Center of Applied Genomics at the Hospital for Sick Children (Toronto, Canada). Data were extracted using the Microarray Suite (MAS) version 5.0 (Affymetrix) and linearly scaled to achieve an average intensity of 150 across each chip. The candidate gene list obtained from the MAS 5.0-extracted data was selected by eliminating genes that were not present in at least one experiment. The arrays were subjected to a pair wise comparison using MAS 5.0, with signal intensities from the no-treatment cells as the baseline. The statistical significance for the change of expression for each probe set between the decitabine treated and control was calculated by the MAS5.0 software. The criteria for gene selection for real-time expression validation analysis was based on the statistically significant up-regulation (p<0.0025) and fold change of â?¥ 2 for expression after decitabine treatment. The gene list was annotated based on the NetAffx data-base (http://www.affymetrix.com/analysis /index.affx) and further verified using the Human Genome Browser data base (http://genome.ucsc.edu/)

Project description:Neuroblastoma is the most common extra-cranial malignancy in childhood and accounts for approximately 15% of childhood cancer deaths. Amplification of N-Myc in neuroblastoma is associated with aggressive disease and predicts for poor prognosis. Novel therapeutic approaches are therefore essential to improving patient outcomes in this setting. The histone deacetylases are known to interact with N-Myc and regulate numerous cellular processes, including differentiation in neuroblastoma. We therefore investigated the antitumor activity of the histone deacetylase inhibitor, panobinostat in the setting of N-Myc amplified neuroblastoma using the TH-MYCN murine model. Continuous treatment of tumor bearing TH-MYCN transgenic mice with panobinostat for 9 weeks led to a significant improvement in survival as compared to mice treated with vehicle, or continuous treatment with panobinostat for a three week period. Panobinostat induced rapid tumor regression with no regrowth observed during the treatment period. Tumor response was associated with an initial apoptosis phenotype mediated via up-regulation of BMF and BIM. When treated was stopped at three weeks 100% of mice relapsed with aggressive neuroblastoma. Differentiation of neuroblastoma into benign ganglioneuroma, with a characteristic increase in S100 expression and reduction of N-Myc expression, was observed in mice treated for nine weeks, resulting in a sustained remission on 90% of mice treated. RNA-sequencing analysis of tumors from treated animals confirmed significant up-regulation of gene pathways associated with apoptosis and differentiation as early as 24 hours after treatment. Together our data demonstrate the potential of panobinostat as a therapeutic strategy for high-risk neuroblastoma patients. Eight homozygous TH-MYCN mice bearing neuroblastomas were treated with either 5mg/kg panobinostat (4 animals) or vehicle (4 animals) for 24hr (two doses) and tumours harvested 4hr post the second dose.

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HDACI and DAC induce specific epigenetic profile in DLBCL

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