INTEGRATED ANALYSIS OF BOTH BIOLOGICAL AND MOLECULAR EFFECTS OF THE EPIGENETIC MODIFYING AGENT ROMIDEPSIN IN MDS/AML
(Abstract release date: 05/19/16)
EHA Library. Clarke K. 06/09/16; 132755; E1206
Dr. Kathryn Clarke
Contributions
Contributions
Abstract
Abstract: E1206
Type: Eposter Presentation
Background
Myelodysplastic syndrome (MDS) patients have poor tolerability of standard chemotherapies and an unmet need for novel therapeutic approaches. Epigenetic silencing of genes can occur via the recruitment of histone deacetylases (HDACs) with these changes being reversible, making them prime targets for therapy. Agents that inhibit HDACs (HDACi) have proven successful in certain malignancies and have shown promise by increasing the tolerability for MDS patients
Aims
In this report, we aimed to study the molecular and cellular consequences of treating AML and MDS derived cell lines with Romidepsin as a single agent, which could ultimately provide a rationale for combination therapy.
Methods
Cell Titre Glo® was used to measure viability, alongside HDAC Glo® assay to quantitate HDAC activity. Western blotting was performed to determine protein expression levels and flow cytometry using propidium iodide staining was used to measure cell cycle. Affymetrix U133 plus 2.0 arrays were used for gene expression analysis in conjunction with DAVID and STRING for pathway and network analysis. The Illumina Next-Seq 500 was used in-house to perform ChIP-Seq experiments.
Results
Preliminary analysis identified a candidate dose, time and cell line to be carried forward for gene expression profiling and ChIP-Seq analysis based on the ability of Romidepsin to affect proliferation, HDAC activity, acetylation and cell cycle.Microarray analysis indicated that 484 probesets were significantly (p<0.05) up-regulated compared to only 3 down-regulated after 24 hour treatment with 1.5 nM Romidepsin. Pathway and network analysis, using STRING and DAVID identified that ROS, inflammation and activation of mitochondrial stress pathways were associated with Romidepsin treatment. Matched samples from Romidepsin treated, and untreated SKM-1 cells were used for ChIP-Seq analysis of H3K9ac, H3K9me and H3K9me3 chromatin marks which provided information on positional enrichment and enabled an integrated transcriptomic-epigenetic mapping approach, thus identifying specific Romidepsin-induced gene-expression networks. Overlap of differential enrichment of each of the aforementioned marks with the microarray data has highlighted that increases in acetylation share the highest degree of overlap with gene expression.
Conclusion
These analyses give further insight into the mode of action of Romidepsin by identifying molecular gene and pathway targets that may be primed for novel therapeutic combinations for the treatment of elderly patients with AML or high risk MDS.
Session topic: E-poster
Keyword(s): Chromatin, Epigenetic, Microarray analysis
Type: Eposter Presentation
Background
Myelodysplastic syndrome (MDS) patients have poor tolerability of standard chemotherapies and an unmet need for novel therapeutic approaches. Epigenetic silencing of genes can occur via the recruitment of histone deacetylases (HDACs) with these changes being reversible, making them prime targets for therapy. Agents that inhibit HDACs (HDACi) have proven successful in certain malignancies and have shown promise by increasing the tolerability for MDS patients
Aims
In this report, we aimed to study the molecular and cellular consequences of treating AML and MDS derived cell lines with Romidepsin as a single agent, which could ultimately provide a rationale for combination therapy.
Methods
Cell Titre Glo® was used to measure viability, alongside HDAC Glo® assay to quantitate HDAC activity. Western blotting was performed to determine protein expression levels and flow cytometry using propidium iodide staining was used to measure cell cycle. Affymetrix U133 plus 2.0 arrays were used for gene expression analysis in conjunction with DAVID and STRING for pathway and network analysis. The Illumina Next-Seq 500 was used in-house to perform ChIP-Seq experiments.
Results
Preliminary analysis identified a candidate dose, time and cell line to be carried forward for gene expression profiling and ChIP-Seq analysis based on the ability of Romidepsin to affect proliferation, HDAC activity, acetylation and cell cycle.Microarray analysis indicated that 484 probesets were significantly (p<0.05) up-regulated compared to only 3 down-regulated after 24 hour treatment with 1.5 nM Romidepsin. Pathway and network analysis, using STRING and DAVID identified that ROS, inflammation and activation of mitochondrial stress pathways were associated with Romidepsin treatment. Matched samples from Romidepsin treated, and untreated SKM-1 cells were used for ChIP-Seq analysis of H3K9ac, H3K9me and H3K9me3 chromatin marks which provided information on positional enrichment and enabled an integrated transcriptomic-epigenetic mapping approach, thus identifying specific Romidepsin-induced gene-expression networks. Overlap of differential enrichment of each of the aforementioned marks with the microarray data has highlighted that increases in acetylation share the highest degree of overlap with gene expression.
Conclusion
These analyses give further insight into the mode of action of Romidepsin by identifying molecular gene and pathway targets that may be primed for novel therapeutic combinations for the treatment of elderly patients with AML or high risk MDS.
Session topic: E-poster
Keyword(s): Chromatin, Epigenetic, Microarray analysis
Abstract: E1206
Type: Eposter Presentation
Background
Myelodysplastic syndrome (MDS) patients have poor tolerability of standard chemotherapies and an unmet need for novel therapeutic approaches. Epigenetic silencing of genes can occur via the recruitment of histone deacetylases (HDACs) with these changes being reversible, making them prime targets for therapy. Agents that inhibit HDACs (HDACi) have proven successful in certain malignancies and have shown promise by increasing the tolerability for MDS patients
Aims
In this report, we aimed to study the molecular and cellular consequences of treating AML and MDS derived cell lines with Romidepsin as a single agent, which could ultimately provide a rationale for combination therapy.
Methods
Cell Titre Glo® was used to measure viability, alongside HDAC Glo® assay to quantitate HDAC activity. Western blotting was performed to determine protein expression levels and flow cytometry using propidium iodide staining was used to measure cell cycle. Affymetrix U133 plus 2.0 arrays were used for gene expression analysis in conjunction with DAVID and STRING for pathway and network analysis. The Illumina Next-Seq 500 was used in-house to perform ChIP-Seq experiments.
Results
Preliminary analysis identified a candidate dose, time and cell line to be carried forward for gene expression profiling and ChIP-Seq analysis based on the ability of Romidepsin to affect proliferation, HDAC activity, acetylation and cell cycle.Microarray analysis indicated that 484 probesets were significantly (p<0.05) up-regulated compared to only 3 down-regulated after 24 hour treatment with 1.5 nM Romidepsin. Pathway and network analysis, using STRING and DAVID identified that ROS, inflammation and activation of mitochondrial stress pathways were associated with Romidepsin treatment. Matched samples from Romidepsin treated, and untreated SKM-1 cells were used for ChIP-Seq analysis of H3K9ac, H3K9me and H3K9me3 chromatin marks which provided information on positional enrichment and enabled an integrated transcriptomic-epigenetic mapping approach, thus identifying specific Romidepsin-induced gene-expression networks. Overlap of differential enrichment of each of the aforementioned marks with the microarray data has highlighted that increases in acetylation share the highest degree of overlap with gene expression.
Conclusion
These analyses give further insight into the mode of action of Romidepsin by identifying molecular gene and pathway targets that may be primed for novel therapeutic combinations for the treatment of elderly patients with AML or high risk MDS.
Session topic: E-poster
Keyword(s): Chromatin, Epigenetic, Microarray analysis
Type: Eposter Presentation
Background
Myelodysplastic syndrome (MDS) patients have poor tolerability of standard chemotherapies and an unmet need for novel therapeutic approaches. Epigenetic silencing of genes can occur via the recruitment of histone deacetylases (HDACs) with these changes being reversible, making them prime targets for therapy. Agents that inhibit HDACs (HDACi) have proven successful in certain malignancies and have shown promise by increasing the tolerability for MDS patients
Aims
In this report, we aimed to study the molecular and cellular consequences of treating AML and MDS derived cell lines with Romidepsin as a single agent, which could ultimately provide a rationale for combination therapy.
Methods
Cell Titre Glo® was used to measure viability, alongside HDAC Glo® assay to quantitate HDAC activity. Western blotting was performed to determine protein expression levels and flow cytometry using propidium iodide staining was used to measure cell cycle. Affymetrix U133 plus 2.0 arrays were used for gene expression analysis in conjunction with DAVID and STRING for pathway and network analysis. The Illumina Next-Seq 500 was used in-house to perform ChIP-Seq experiments.
Results
Preliminary analysis identified a candidate dose, time and cell line to be carried forward for gene expression profiling and ChIP-Seq analysis based on the ability of Romidepsin to affect proliferation, HDAC activity, acetylation and cell cycle.Microarray analysis indicated that 484 probesets were significantly (p<0.05) up-regulated compared to only 3 down-regulated after 24 hour treatment with 1.5 nM Romidepsin. Pathway and network analysis, using STRING and DAVID identified that ROS, inflammation and activation of mitochondrial stress pathways were associated with Romidepsin treatment. Matched samples from Romidepsin treated, and untreated SKM-1 cells were used for ChIP-Seq analysis of H3K9ac, H3K9me and H3K9me3 chromatin marks which provided information on positional enrichment and enabled an integrated transcriptomic-epigenetic mapping approach, thus identifying specific Romidepsin-induced gene-expression networks. Overlap of differential enrichment of each of the aforementioned marks with the microarray data has highlighted that increases in acetylation share the highest degree of overlap with gene expression.
Conclusion
These analyses give further insight into the mode of action of Romidepsin by identifying molecular gene and pathway targets that may be primed for novel therapeutic combinations for the treatment of elderly patients with AML or high risk MDS.
Session topic: E-poster
Keyword(s): Chromatin, Epigenetic, Microarray analysis
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