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135 IN VITRO AZACITIDINE CULTURE INDUCES DNA DEMETHYLATION AND INCREASED MRNA-LEVELS IN PRIMARY MDS PROGENITOR CELLS
Poster Presentations – 13th International Symposium on Myelodyspastic Syndromes / Leukemia Research 39 S1 (2015) S1–S166
transplantation (84%). Median age at time of transplantation was 55 years (IQR: 46-60). Median time from diagnosis to transplantation was 11 months (IQR: 7-22). Donor was an HLA matched sibling in 122 (42%) patients and an unrelated donor in the remaining patients. The preferred source of stem cells was peripheral blood stem cells (PB) (78%) followed by bone marrow (19%) and cord blood (3%). Conditioning regimen consisted in a reduced intensity conditioning regimen in the majority of patients (59%) and 58% received an in vivo T depletion.PFS and OS at 3 years were 52% (46-59) and 57% (51-64). Cumulative incidence of grade II-IV acute GVHD and chronic GVHD were 29% (24-34) and 44% (38-50) while non-relapse mortality and relapse rates were 31% (25-37) and 17% (13-22). Multivariate analysis with imputed data set (excluding CB) found the following factors as poor prognostic for PFS: transfusions (Hazard ratio (HR): 0.53, p=0.007), source of stem cells (HR for PB:1.65, p=0.01), donor/recipient CMV status (D/R: -/+, HR: 1.89, p=0.01). Multivariate analysis for OS identified transfusion (HR: 0.65, p=0.098) and ATG according to donor (poor prognostic for non-identical sibling donor without ATG, HR: 2.90, p=0.0004). The second multivariate analysis included original dataset gave similar results. Conclusion: To conclude, outcome after HSCT in patients with lower risk IPSS are better than those observed in higher risk. In favorable transplant condition, expected OS reaches 70% which could be a valid option in some lower risk patients presenting some ‘high risk factors” as poor molecular biology or resistance to agents stimulating erythropoiesis.
135 IN VITRO AZACITIDINE CULTURE INDUCES DNA DEMETHYLATION AND INCREASED MRNA-LEVELS IN PRIMARY MDS PROGENITOR CELLS H. Abdulkadir1, M. Tobiasson1, A. Lennartsson2, S. Katayama3, F. Marabita4, M. Karimi5, Y. Qu5, E. Einarsdottir3, M. Grövdal5, M. Jansson5, A. Ben Azenkoud5, S. Lehmann5, K. Ekwall6, J. Kere3, E. Hellström-Lindberg5, J. Ungerstedt5 1 Center for Hematology and Regenerative Medicine Department of medicine Huddinge division of Hematology, Karolinska Institute, Stockholm, Sweden; 2Department of Biosciences and Nutrition and Center for Innovative Medicine, Karolinska Institute, Stockholm, Sweden; 3Department of Biosciences and Nutrition and Center for Innovative Medicine, Karolinska Institutet, Stockholm, Sweden; 4 Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden; 5Center for Hematology and Regenerative Medicine Department of medicine Huddinge division of Hematology, Karolinska Institutet, Stockholm, Sweden; 6Department of Biosciences and Nutrition and Center for Innovative Medicine 4 Molecular Neurology Research Program University of Helsinki and Folkhälsan Institute of Genetics Helsinki Finland, Karolinska Institutet, Stockholm, Sweden Background: Azacitidine is first-line treatment for patients with higher risk MDS, and reduces DNA methylation in myeloid progenitors. However, the effect of Azacitidine on gene expression remains unclear. Material and methods: We treated CD34+ bone marrow cells from 11 consecutive patients with higher-risk MDS and compared treated and untreated cells with regards to global DNA methylation using the Illumina 450k array, global transcription levels using RNA sequencing, cell growth, and repressive chromatin mark H3K9me3 by ChIP qPCR. Results: Cell growth and viability over 48 h showed little variation between untreated and treated cultures. While no spontaneous change in methylation occurred in the control cultures, gene expression increased with time in culture. Exposure to Azacitidine
S69
for 24 h lead to marked global DNA demethylation, with the majority of the demethylated sites found in open sea regions as defined by the Illumina array. There was an enrichment of demethylated sites in regions that in normal CD34+ cells are associated with heterochromatin and active transcription. We identified 1822 regions differentially methylated in treated versus untreated samples. These regions were enriched for gene ontology pathways involved in differentiation and development. In contrast to previous observations using Affymetrix-based expression arrays, Azacitidine treatment increased transcription in 2136 genes, however, we did not observe gene-specific associations between demethylated genes and increased transcription. With ChIP qPCR for DNA demethylated genes with no increase in transcription upon treatment, we demonstrated an increase in repressive chromatin mark H3K9me3, which would possibly explain the lack of increase in transcription despite DNA demethylation. Conclusions: In vitro Azacitidine treatment induces profound DNA demethylation and a moderate increase in transcription, without clear association between these changes. Comprehensive epigenetic studies including chromatin structure are warranted to elucidate the mechanisms by which DNA demethylated genes are not transcribed, and how increased gene expression is mediated in non demethylated genes.
136 ROLE OF THE TRANSCRIPTION FACTOR CTCF IN THE PATHOGENESIS OF MYELODYSPLASTIC SYNDROMES C. Brunetti1, C.F. Minervini1, P. Orsini1, L. Anelli1, A. Zagaria1, N. Coccaro1, G. Tota1, A. Minervini1, P. Casieri1, A. Cellamare1, C. Cumbo1, L. Impera1, A. Ricco1, G. Specchia1, F. Albano1 1 Department of Emergency and Organ Transplantation (D.E.T.O.) Hematology Section, University of Bari, Bari, Italy Myelodysplastic syndromes (MDS) are neoplastic hematologic disorders characterized by ineffective hematopoiesis. Cytopenia can be observed in all three myeloid lineages suggesting the involvement of multipotent, immature, hematopoietic cell in the pathogenesis of the disease. Also, MDS is considered a “preleukemic” status due to a significantly elevated risk of developing an overt acute leukemia. Genetic and epigenetic abnormalities, critically altering cell proliferation and differentiation of stem and progenitor cells, have been found to be associated with MDS. Aberrant differentiation in MDS can often be traced to abnormal DNA methylation, consisting in losses and gains of DNA methylation both genome-wide and at specific loci. In the attempt of unravelling a maturation defect driver event linked to MDS epigenetics, we studied the role of CCCTC-binding factor (CTCF), a master gene in the epigenetic regulation, already known to be involved in hematopoiesis. We investigated CTCF genomic binding landscape in bone marrow-derived CD34+ cells from two high-risk MDS patients by chromatin immunoprecipitation (ChIP) sequencing (Illumina HiSeq). Differential binding analysis (DiffBind R package) revealed 131 sites enriched in MDS patients as compared to healthy donors (HD) (fold-change≥|2|, FDR≤0.1). To explore the relationship between CTCF-binding sites and its putative targets, we identified genes mapping upstream and downstream our differentially enriched genomic regions (GREAT version 2.0.2). Interestingly, we found that the 303 genes associated to CTCF-enriched binding sites in patients were mainly involved in basic hematopoietic stem cell functions. Publically available microarray (GSE19429) and RNA-Seq (GSE63569) datasets were selected in order to identify a subgroup of CTCF-target genes also differentially expressed in MDS patients (fold-change≥|1.5|, FDR≤0.25). Ninety-eight and 18 genes
transplantation (84%). Median age at time of transplantation was 55 years (IQR: 46-60). Median time from diagnosis to transplantation was 11 months (IQR: 7-22). Donor was an HLA matched sibling in 122 (42%) patients and an unrelated donor in the remaining patients. The preferred source of stem cells was peripheral blood stem cells (PB) (78%) followed by bone marrow (19%) and cord blood (3%). Conditioning regimen consisted in a reduced intensity conditioning regimen in the majority of patients (59%) and 58% received an in vivo T depletion.PFS and OS at 3 years were 52% (46-59) and 57% (51-64). Cumulative incidence of grade II-IV acute GVHD and chronic GVHD were 29% (24-34) and 44% (38-50) while non-relapse mortality and relapse rates were 31% (25-37) and 17% (13-22). Multivariate analysis with imputed data set (excluding CB) found the following factors as poor prognostic for PFS: transfusions (Hazard ratio (HR): 0.53, p=0.007), source of stem cells (HR for PB:1.65, p=0.01), donor/recipient CMV status (D/R: -/+, HR: 1.89, p=0.01). Multivariate analysis for OS identified transfusion (HR: 0.65, p=0.098) and ATG according to donor (poor prognostic for non-identical sibling donor without ATG, HR: 2.90, p=0.0004). The second multivariate analysis included original dataset gave similar results. Conclusion: To conclude, outcome after HSCT in patients with lower risk IPSS are better than those observed in higher risk. In favorable transplant condition, expected OS reaches 70% which could be a valid option in some lower risk patients presenting some ‘high risk factors” as poor molecular biology or resistance to agents stimulating erythropoiesis.
135 IN VITRO AZACITIDINE CULTURE INDUCES DNA DEMETHYLATION AND INCREASED MRNA-LEVELS IN PRIMARY MDS PROGENITOR CELLS H. Abdulkadir1, M. Tobiasson1, A. Lennartsson2, S. Katayama3, F. Marabita4, M. Karimi5, Y. Qu5, E. Einarsdottir3, M. Grövdal5, M. Jansson5, A. Ben Azenkoud5, S. Lehmann5, K. Ekwall6, J. Kere3, E. Hellström-Lindberg5, J. Ungerstedt5 1 Center for Hematology and Regenerative Medicine Department of medicine Huddinge division of Hematology, Karolinska Institute, Stockholm, Sweden; 2Department of Biosciences and Nutrition and Center for Innovative Medicine, Karolinska Institute, Stockholm, Sweden; 3Department of Biosciences and Nutrition and Center for Innovative Medicine, Karolinska Institutet, Stockholm, Sweden; 4 Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden; 5Center for Hematology and Regenerative Medicine Department of medicine Huddinge division of Hematology, Karolinska Institutet, Stockholm, Sweden; 6Department of Biosciences and Nutrition and Center for Innovative Medicine 4 Molecular Neurology Research Program University of Helsinki and Folkhälsan Institute of Genetics Helsinki Finland, Karolinska Institutet, Stockholm, Sweden Background: Azacitidine is first-line treatment for patients with higher risk MDS, and reduces DNA methylation in myeloid progenitors. However, the effect of Azacitidine on gene expression remains unclear. Material and methods: We treated CD34+ bone marrow cells from 11 consecutive patients with higher-risk MDS and compared treated and untreated cells with regards to global DNA methylation using the Illumina 450k array, global transcription levels using RNA sequencing, cell growth, and repressive chromatin mark H3K9me3 by ChIP qPCR. Results: Cell growth and viability over 48 h showed little variation between untreated and treated cultures. While no spontaneous change in methylation occurred in the control cultures, gene expression increased with time in culture. Exposure to Azacitidine
S69
for 24 h lead to marked global DNA demethylation, with the majority of the demethylated sites found in open sea regions as defined by the Illumina array. There was an enrichment of demethylated sites in regions that in normal CD34+ cells are associated with heterochromatin and active transcription. We identified 1822 regions differentially methylated in treated versus untreated samples. These regions were enriched for gene ontology pathways involved in differentiation and development. In contrast to previous observations using Affymetrix-based expression arrays, Azacitidine treatment increased transcription in 2136 genes, however, we did not observe gene-specific associations between demethylated genes and increased transcription. With ChIP qPCR for DNA demethylated genes with no increase in transcription upon treatment, we demonstrated an increase in repressive chromatin mark H3K9me3, which would possibly explain the lack of increase in transcription despite DNA demethylation. Conclusions: In vitro Azacitidine treatment induces profound DNA demethylation and a moderate increase in transcription, without clear association between these changes. Comprehensive epigenetic studies including chromatin structure are warranted to elucidate the mechanisms by which DNA demethylated genes are not transcribed, and how increased gene expression is mediated in non demethylated genes.
136 ROLE OF THE TRANSCRIPTION FACTOR CTCF IN THE PATHOGENESIS OF MYELODYSPLASTIC SYNDROMES C. Brunetti1, C.F. Minervini1, P. Orsini1, L. Anelli1, A. Zagaria1, N. Coccaro1, G. Tota1, A. Minervini1, P. Casieri1, A. Cellamare1, C. Cumbo1, L. Impera1, A. Ricco1, G. Specchia1, F. Albano1 1 Department of Emergency and Organ Transplantation (D.E.T.O.) Hematology Section, University of Bari, Bari, Italy Myelodysplastic syndromes (MDS) are neoplastic hematologic disorders characterized by ineffective hematopoiesis. Cytopenia can be observed in all three myeloid lineages suggesting the involvement of multipotent, immature, hematopoietic cell in the pathogenesis of the disease. Also, MDS is considered a “preleukemic” status due to a significantly elevated risk of developing an overt acute leukemia. Genetic and epigenetic abnormalities, critically altering cell proliferation and differentiation of stem and progenitor cells, have been found to be associated with MDS. Aberrant differentiation in MDS can often be traced to abnormal DNA methylation, consisting in losses and gains of DNA methylation both genome-wide and at specific loci. In the attempt of unravelling a maturation defect driver event linked to MDS epigenetics, we studied the role of CCCTC-binding factor (CTCF), a master gene in the epigenetic regulation, already known to be involved in hematopoiesis. We investigated CTCF genomic binding landscape in bone marrow-derived CD34+ cells from two high-risk MDS patients by chromatin immunoprecipitation (ChIP) sequencing (Illumina HiSeq). Differential binding analysis (DiffBind R package) revealed 131 sites enriched in MDS patients as compared to healthy donors (HD) (fold-change≥|2|, FDR≤0.1). To explore the relationship between CTCF-binding sites and its putative targets, we identified genes mapping upstream and downstream our differentially enriched genomic regions (GREAT version 2.0.2). Interestingly, we found that the 303 genes associated to CTCF-enriched binding sites in patients were mainly involved in basic hematopoietic stem cell functions. Publically available microarray (GSE19429) and RNA-Seq (GSE63569) datasets were selected in order to identify a subgroup of CTCF-target genes also differentially expressed in MDS patients (fold-change≥|1.5|, FDR≤0.25). Ninety-eight and 18 genes