THE DNA METHYLATION INHIBITORS AZACITIDINE AND DECITABINE ACTIVATE UNIQUE AND COMMON TRANSCRIPTIONAL PATHWAYS THAT INCLUDE POTENTIAL NOVEL MARKERS OF RESPONSE
(Abstract release date: 05/19/16)
EHA Library. Clarke K. 06/09/16; 132754; E1205
Dr. Kathryn Clarke
Contributions
Contributions
Abstract
Abstract: E1205
Type: Eposter Presentation
Background
Myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) patients have poor tolerability of standard chemotherapies and an unmet need for novel therapeutic approaches. Epigenetic silencing of genes can occur via hypermethylation or the recruitment of histone deacetylases (HDACs), with these changes being reversible making them prime targets for therapy. Agents that inhibit DNA methylation have proven successful in the treatment of haematological malignancies and have shown promise by increasing the tolerability for MDS and AML patients, however the underlying mechanism of action remains to be fully elucidated.
Aims
In this report, we have studied the molecular and cellular consequences of treating AML and MDS derived cell lines with Azacitidine as a single agent as well as performing a small meta-analysis comparing Azacitidine an Decitabine gene expression with the intention of finding potential rational combination partners.
Methods
LINE-1 pyrosequencing was used to measure global methylation levels and western blotting used as a measure of protein expression. Cytospins were performed and slides were stained with Wright-Giemsa to assess the morphological effect on OCI-AML3 and SKM-1 cells. Gene expression profiling was performed using Affymetrix U133 plus 2.0 arrays and results were analysed using Partek Genomics Suite. CHI3L1 methylation was determined using pyrosequencing with primers designed against the promoter, intron and exon. Survival analysis examined TGCA dataset of 183 clinically annotated adult cases of de novo AML, whilst the local dataset consisted of 228 clinically annotated cases of MDS and AML.
Results
Azacitidine decreased viability and LINE-1 DNA methylation with a concomitant reduction in DNMT1 protein expression. Morphological assessment of treated OCI-AML3 and SKM-1 cells displayed an increase the proportion of macrophage cells – indicative of an induction of differentiation. Gene expression profiling using Affymetrix U133 plus 2.0 arrays identified 173 differentially expressed probesets following treatment with pathway analysis using DAVID identifying the immune system and an inflammatory response, which was also observed with network analysis using STRING. Overlap with Decitabine gene expression analysis highlighted 138 probesets that were common to both drugs and supported the role of the immune system in response to demethylating agents including S100A8/A9, HLA and CHI3L1. CHI3L1 was up-regulated with both agents and an increase in gene expression correlated to a loss of promoter, intron and exon DNA methylation. Survival analysis in locally and publically available datasets of stratified AML patients recognised that CHI3L1 could be used as a potential marker of response in patients with good cytogenetics (p = <0.02) compared to those in other subtypes (intermediate - ns; poor – ns).
Conclusion
These analyses give further insight into the mechanism of action of both Azacitidine and Decitabine in AML/MDS cell lines 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): Methylation, Microarray analysis
Type: Eposter Presentation
Background
Myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) patients have poor tolerability of standard chemotherapies and an unmet need for novel therapeutic approaches. Epigenetic silencing of genes can occur via hypermethylation or the recruitment of histone deacetylases (HDACs), with these changes being reversible making them prime targets for therapy. Agents that inhibit DNA methylation have proven successful in the treatment of haematological malignancies and have shown promise by increasing the tolerability for MDS and AML patients, however the underlying mechanism of action remains to be fully elucidated.
Aims
In this report, we have studied the molecular and cellular consequences of treating AML and MDS derived cell lines with Azacitidine as a single agent as well as performing a small meta-analysis comparing Azacitidine an Decitabine gene expression with the intention of finding potential rational combination partners.
Methods
LINE-1 pyrosequencing was used to measure global methylation levels and western blotting used as a measure of protein expression. Cytospins were performed and slides were stained with Wright-Giemsa to assess the morphological effect on OCI-AML3 and SKM-1 cells. Gene expression profiling was performed using Affymetrix U133 plus 2.0 arrays and results were analysed using Partek Genomics Suite. CHI3L1 methylation was determined using pyrosequencing with primers designed against the promoter, intron and exon. Survival analysis examined TGCA dataset of 183 clinically annotated adult cases of de novo AML, whilst the local dataset consisted of 228 clinically annotated cases of MDS and AML.
Results
Azacitidine decreased viability and LINE-1 DNA methylation with a concomitant reduction in DNMT1 protein expression. Morphological assessment of treated OCI-AML3 and SKM-1 cells displayed an increase the proportion of macrophage cells – indicative of an induction of differentiation. Gene expression profiling using Affymetrix U133 plus 2.0 arrays identified 173 differentially expressed probesets following treatment with pathway analysis using DAVID identifying the immune system and an inflammatory response, which was also observed with network analysis using STRING. Overlap with Decitabine gene expression analysis highlighted 138 probesets that were common to both drugs and supported the role of the immune system in response to demethylating agents including S100A8/A9, HLA and CHI3L1. CHI3L1 was up-regulated with both agents and an increase in gene expression correlated to a loss of promoter, intron and exon DNA methylation. Survival analysis in locally and publically available datasets of stratified AML patients recognised that CHI3L1 could be used as a potential marker of response in patients with good cytogenetics (p = <0.02) compared to those in other subtypes (intermediate - ns; poor – ns).
Conclusion
These analyses give further insight into the mechanism of action of both Azacitidine and Decitabine in AML/MDS cell lines 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): Methylation, Microarray analysis
Abstract: E1205
Type: Eposter Presentation
Background
Myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) patients have poor tolerability of standard chemotherapies and an unmet need for novel therapeutic approaches. Epigenetic silencing of genes can occur via hypermethylation or the recruitment of histone deacetylases (HDACs), with these changes being reversible making them prime targets for therapy. Agents that inhibit DNA methylation have proven successful in the treatment of haematological malignancies and have shown promise by increasing the tolerability for MDS and AML patients, however the underlying mechanism of action remains to be fully elucidated.
Aims
In this report, we have studied the molecular and cellular consequences of treating AML and MDS derived cell lines with Azacitidine as a single agent as well as performing a small meta-analysis comparing Azacitidine an Decitabine gene expression with the intention of finding potential rational combination partners.
Methods
LINE-1 pyrosequencing was used to measure global methylation levels and western blotting used as a measure of protein expression. Cytospins were performed and slides were stained with Wright-Giemsa to assess the morphological effect on OCI-AML3 and SKM-1 cells. Gene expression profiling was performed using Affymetrix U133 plus 2.0 arrays and results were analysed using Partek Genomics Suite. CHI3L1 methylation was determined using pyrosequencing with primers designed against the promoter, intron and exon. Survival analysis examined TGCA dataset of 183 clinically annotated adult cases of de novo AML, whilst the local dataset consisted of 228 clinically annotated cases of MDS and AML.
Results
Azacitidine decreased viability and LINE-1 DNA methylation with a concomitant reduction in DNMT1 protein expression. Morphological assessment of treated OCI-AML3 and SKM-1 cells displayed an increase the proportion of macrophage cells – indicative of an induction of differentiation. Gene expression profiling using Affymetrix U133 plus 2.0 arrays identified 173 differentially expressed probesets following treatment with pathway analysis using DAVID identifying the immune system and an inflammatory response, which was also observed with network analysis using STRING. Overlap with Decitabine gene expression analysis highlighted 138 probesets that were common to both drugs and supported the role of the immune system in response to demethylating agents including S100A8/A9, HLA and CHI3L1. CHI3L1 was up-regulated with both agents and an increase in gene expression correlated to a loss of promoter, intron and exon DNA methylation. Survival analysis in locally and publically available datasets of stratified AML patients recognised that CHI3L1 could be used as a potential marker of response in patients with good cytogenetics (p = <0.02) compared to those in other subtypes (intermediate - ns; poor – ns).
Conclusion
These analyses give further insight into the mechanism of action of both Azacitidine and Decitabine in AML/MDS cell lines 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): Methylation, Microarray analysis
Type: Eposter Presentation
Background
Myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) patients have poor tolerability of standard chemotherapies and an unmet need for novel therapeutic approaches. Epigenetic silencing of genes can occur via hypermethylation or the recruitment of histone deacetylases (HDACs), with these changes being reversible making them prime targets for therapy. Agents that inhibit DNA methylation have proven successful in the treatment of haematological malignancies and have shown promise by increasing the tolerability for MDS and AML patients, however the underlying mechanism of action remains to be fully elucidated.
Aims
In this report, we have studied the molecular and cellular consequences of treating AML and MDS derived cell lines with Azacitidine as a single agent as well as performing a small meta-analysis comparing Azacitidine an Decitabine gene expression with the intention of finding potential rational combination partners.
Methods
LINE-1 pyrosequencing was used to measure global methylation levels and western blotting used as a measure of protein expression. Cytospins were performed and slides were stained with Wright-Giemsa to assess the morphological effect on OCI-AML3 and SKM-1 cells. Gene expression profiling was performed using Affymetrix U133 plus 2.0 arrays and results were analysed using Partek Genomics Suite. CHI3L1 methylation was determined using pyrosequencing with primers designed against the promoter, intron and exon. Survival analysis examined TGCA dataset of 183 clinically annotated adult cases of de novo AML, whilst the local dataset consisted of 228 clinically annotated cases of MDS and AML.
Results
Azacitidine decreased viability and LINE-1 DNA methylation with a concomitant reduction in DNMT1 protein expression. Morphological assessment of treated OCI-AML3 and SKM-1 cells displayed an increase the proportion of macrophage cells – indicative of an induction of differentiation. Gene expression profiling using Affymetrix U133 plus 2.0 arrays identified 173 differentially expressed probesets following treatment with pathway analysis using DAVID identifying the immune system and an inflammatory response, which was also observed with network analysis using STRING. Overlap with Decitabine gene expression analysis highlighted 138 probesets that were common to both drugs and supported the role of the immune system in response to demethylating agents including S100A8/A9, HLA and CHI3L1. CHI3L1 was up-regulated with both agents and an increase in gene expression correlated to a loss of promoter, intron and exon DNA methylation. Survival analysis in locally and publically available datasets of stratified AML patients recognised that CHI3L1 could be used as a potential marker of response in patients with good cytogenetics (p = <0.02) compared to those in other subtypes (intermediate - ns; poor – ns).
Conclusion
These analyses give further insight into the mechanism of action of both Azacitidine and Decitabine in AML/MDS cell lines 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): Methylation, Microarray analysis
{{ help_message }}
{{filter}}