ABNORMAL INTRAGENIC TRANSCRIPTIONAL ACTIVATION IN MLL-AF9 AND KAT6A-CREBBP ACUTE MYELOID LEUKEMIA (AML)
(Abstract release date: 05/19/16)
EHA Library. Marneth A. 06/10/16; 133163; P175
Ms. Anna Marneth
Contributions
Contributions
Abstract
Abstract: P175
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
The mutated transcription factors AML1-ETO, CBFB-MYH11 and CEBPA cause unique pathogenic gene expression profiles (GEPs) in AML. Previously, we identified an AML subgroup with a unique GEP characterized by outlier high expression of the BRE gene. Most cases with this unique GEP harbor the oncogenic transcriptional regulator MLL-AF9. However, since ~50% of adult MLL-AF9+ AML cases does not exhibit this unique GEP nor outlier high BRE expression, we postulated that another pathogenic mechanism besides the MLL-AF9 translocation causes this unique GEP.
Aims
We aimed at characterizing outlier high BRE expression and the associated unique GEP in MLL-AF9+ AML, whether these phenomena were exclusive to MLL-AF9+ AML and whether they associated with known oncogenic pathways.
Methods
AML samples were subjected to RNA-sequencing (n=4), H3K4me3 (n=7) and/or H3K27ac (n=3) ChIP-sequencing. Informed consent was obtained from all subjects prior to this study. 5’ Rapid Amplification of cDNA Ends (RACE) was performed to characterize the 5’ end of BRE transcripts. BRE gene expression was measured by qRT-PCR. Expression of an alternate BRE transcript was normalized to PBGD expression and a control sample without outlier high BRE expression. A normalized value >500 was considered high. Ingenuity pathway analyses were performed on the top 200 genes positively associated with BRE expression in MLL-AF9+ AML.
Results
RNA-seq and qRT-PCR of MLL-AF9+ patient samples showed a marked increase in BRE expression starting from exon 5, exclusively in samples with outlier high BRE expression. H3K4me3 and H3K27ac ChIP-sequencing revealed clear marks in BRE intron 4, 3 kb upstream of BRE exon 5, only in AML cases with outlier high BRE expression. To demonstrate that this region relates to active transcription, we performed 5’ RACE. A new transcript starting in BRE intron 4 near the H3K4me3/H3K27ac marks was identified, with sequences spliced to BRE exon 5. A qRT-PCR specific for the alternate BRE transcript showed that it was frequently expressed in adult MLL-AF9 samples and in samples with the KAT6A-CREBBP fusion gene (18/35, median expression high 8580, low 0.6). This is in line with published data showing that part of MLL-AF9+ and KAT6A-CREBBP+ samples share a common GEP. Also childhood MLL-rearranged and KAT6A-CREBBP+ AML samples frequently expressed the alternate BRE transcript (31/43, median expression high 8172, low 26.9). Alternate BRE expression was generally low in patients with other types of AML (n=42, 1.4) and non-AML hematological malignancies (n=10, 0.4) or normal CD34+ and bone marrow cells (n=14, 0.3). A polymorphism in BRE intron 5 revealed that alternate BRE transcription initiation is bi-allelic. Importantly, analysis of genome-wide H3K4me3 marks and RNA-seq data uncovered 98 potential intragenic transcriptional activation sites, several of which are in genes correlating with high BRE expression. Ingenuity pathway analyses using the top 200 genes positively associated with BRE expression, identified potential pathogenic pathways including p53 and protein kinase A signaling (p<0.03).
Conclusion
Alternate transcription initiation in the ALK gene was recently identified as oncogenic mechanism causing melanoma (Wiesner et al., Nature, 2015). Our data show genome-wide alternate transcription initiation as new phenomenon in MLL-AF9+ and KAT6A-CREBBP+ AML cases that exhibit a unique GEP. Potential oncogenic pathways were identified in these AML subgroups. Further studies investigating the molecular mechanisms contributing to alternate transcription initiation in AML are warranted.
Session topic: Acute myeloid leukemia - Biology 1
Keyword(s): 11q23, AML, Gene expression profile, MLL
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
The mutated transcription factors AML1-ETO, CBFB-MYH11 and CEBPA cause unique pathogenic gene expression profiles (GEPs) in AML. Previously, we identified an AML subgroup with a unique GEP characterized by outlier high expression of the BRE gene. Most cases with this unique GEP harbor the oncogenic transcriptional regulator MLL-AF9. However, since ~50% of adult MLL-AF9+ AML cases does not exhibit this unique GEP nor outlier high BRE expression, we postulated that another pathogenic mechanism besides the MLL-AF9 translocation causes this unique GEP.
Aims
We aimed at characterizing outlier high BRE expression and the associated unique GEP in MLL-AF9+ AML, whether these phenomena were exclusive to MLL-AF9+ AML and whether they associated with known oncogenic pathways.
Methods
AML samples were subjected to RNA-sequencing (n=4), H3K4me3 (n=7) and/or H3K27ac (n=3) ChIP-sequencing. Informed consent was obtained from all subjects prior to this study. 5’ Rapid Amplification of cDNA Ends (RACE) was performed to characterize the 5’ end of BRE transcripts. BRE gene expression was measured by qRT-PCR. Expression of an alternate BRE transcript was normalized to PBGD expression and a control sample without outlier high BRE expression. A normalized value >500 was considered high. Ingenuity pathway analyses were performed on the top 200 genes positively associated with BRE expression in MLL-AF9+ AML.
Results
RNA-seq and qRT-PCR of MLL-AF9+ patient samples showed a marked increase in BRE expression starting from exon 5, exclusively in samples with outlier high BRE expression. H3K4me3 and H3K27ac ChIP-sequencing revealed clear marks in BRE intron 4, 3 kb upstream of BRE exon 5, only in AML cases with outlier high BRE expression. To demonstrate that this region relates to active transcription, we performed 5’ RACE. A new transcript starting in BRE intron 4 near the H3K4me3/H3K27ac marks was identified, with sequences spliced to BRE exon 5. A qRT-PCR specific for the alternate BRE transcript showed that it was frequently expressed in adult MLL-AF9 samples and in samples with the KAT6A-CREBBP fusion gene (18/35, median expression high 8580, low 0.6). This is in line with published data showing that part of MLL-AF9+ and KAT6A-CREBBP+ samples share a common GEP. Also childhood MLL-rearranged and KAT6A-CREBBP+ AML samples frequently expressed the alternate BRE transcript (31/43, median expression high 8172, low 26.9). Alternate BRE expression was generally low in patients with other types of AML (n=42, 1.4) and non-AML hematological malignancies (n=10, 0.4) or normal CD34+ and bone marrow cells (n=14, 0.3). A polymorphism in BRE intron 5 revealed that alternate BRE transcription initiation is bi-allelic. Importantly, analysis of genome-wide H3K4me3 marks and RNA-seq data uncovered 98 potential intragenic transcriptional activation sites, several of which are in genes correlating with high BRE expression. Ingenuity pathway analyses using the top 200 genes positively associated with BRE expression, identified potential pathogenic pathways including p53 and protein kinase A signaling (p<0.03).
Conclusion
Alternate transcription initiation in the ALK gene was recently identified as oncogenic mechanism causing melanoma (Wiesner et al., Nature, 2015). Our data show genome-wide alternate transcription initiation as new phenomenon in MLL-AF9+ and KAT6A-CREBBP+ AML cases that exhibit a unique GEP. Potential oncogenic pathways were identified in these AML subgroups. Further studies investigating the molecular mechanisms contributing to alternate transcription initiation in AML are warranted.
Session topic: Acute myeloid leukemia - Biology 1
Keyword(s): 11q23, AML, Gene expression profile, MLL
Abstract: P175
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
The mutated transcription factors AML1-ETO, CBFB-MYH11 and CEBPA cause unique pathogenic gene expression profiles (GEPs) in AML. Previously, we identified an AML subgroup with a unique GEP characterized by outlier high expression of the BRE gene. Most cases with this unique GEP harbor the oncogenic transcriptional regulator MLL-AF9. However, since ~50% of adult MLL-AF9+ AML cases does not exhibit this unique GEP nor outlier high BRE expression, we postulated that another pathogenic mechanism besides the MLL-AF9 translocation causes this unique GEP.
Aims
We aimed at characterizing outlier high BRE expression and the associated unique GEP in MLL-AF9+ AML, whether these phenomena were exclusive to MLL-AF9+ AML and whether they associated with known oncogenic pathways.
Methods
AML samples were subjected to RNA-sequencing (n=4), H3K4me3 (n=7) and/or H3K27ac (n=3) ChIP-sequencing. Informed consent was obtained from all subjects prior to this study. 5’ Rapid Amplification of cDNA Ends (RACE) was performed to characterize the 5’ end of BRE transcripts. BRE gene expression was measured by qRT-PCR. Expression of an alternate BRE transcript was normalized to PBGD expression and a control sample without outlier high BRE expression. A normalized value >500 was considered high. Ingenuity pathway analyses were performed on the top 200 genes positively associated with BRE expression in MLL-AF9+ AML.
Results
RNA-seq and qRT-PCR of MLL-AF9+ patient samples showed a marked increase in BRE expression starting from exon 5, exclusively in samples with outlier high BRE expression. H3K4me3 and H3K27ac ChIP-sequencing revealed clear marks in BRE intron 4, 3 kb upstream of BRE exon 5, only in AML cases with outlier high BRE expression. To demonstrate that this region relates to active transcription, we performed 5’ RACE. A new transcript starting in BRE intron 4 near the H3K4me3/H3K27ac marks was identified, with sequences spliced to BRE exon 5. A qRT-PCR specific for the alternate BRE transcript showed that it was frequently expressed in adult MLL-AF9 samples and in samples with the KAT6A-CREBBP fusion gene (18/35, median expression high 8580, low 0.6). This is in line with published data showing that part of MLL-AF9+ and KAT6A-CREBBP+ samples share a common GEP. Also childhood MLL-rearranged and KAT6A-CREBBP+ AML samples frequently expressed the alternate BRE transcript (31/43, median expression high 8172, low 26.9). Alternate BRE expression was generally low in patients with other types of AML (n=42, 1.4) and non-AML hematological malignancies (n=10, 0.4) or normal CD34+ and bone marrow cells (n=14, 0.3). A polymorphism in BRE intron 5 revealed that alternate BRE transcription initiation is bi-allelic. Importantly, analysis of genome-wide H3K4me3 marks and RNA-seq data uncovered 98 potential intragenic transcriptional activation sites, several of which are in genes correlating with high BRE expression. Ingenuity pathway analyses using the top 200 genes positively associated with BRE expression, identified potential pathogenic pathways including p53 and protein kinase A signaling (p<0.03).
Conclusion
Alternate transcription initiation in the ALK gene was recently identified as oncogenic mechanism causing melanoma (Wiesner et al., Nature, 2015). Our data show genome-wide alternate transcription initiation as new phenomenon in MLL-AF9+ and KAT6A-CREBBP+ AML cases that exhibit a unique GEP. Potential oncogenic pathways were identified in these AML subgroups. Further studies investigating the molecular mechanisms contributing to alternate transcription initiation in AML are warranted.
Session topic: Acute myeloid leukemia - Biology 1
Keyword(s): 11q23, AML, Gene expression profile, MLL
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
The mutated transcription factors AML1-ETO, CBFB-MYH11 and CEBPA cause unique pathogenic gene expression profiles (GEPs) in AML. Previously, we identified an AML subgroup with a unique GEP characterized by outlier high expression of the BRE gene. Most cases with this unique GEP harbor the oncogenic transcriptional regulator MLL-AF9. However, since ~50% of adult MLL-AF9+ AML cases does not exhibit this unique GEP nor outlier high BRE expression, we postulated that another pathogenic mechanism besides the MLL-AF9 translocation causes this unique GEP.
Aims
We aimed at characterizing outlier high BRE expression and the associated unique GEP in MLL-AF9+ AML, whether these phenomena were exclusive to MLL-AF9+ AML and whether they associated with known oncogenic pathways.
Methods
AML samples were subjected to RNA-sequencing (n=4), H3K4me3 (n=7) and/or H3K27ac (n=3) ChIP-sequencing. Informed consent was obtained from all subjects prior to this study. 5’ Rapid Amplification of cDNA Ends (RACE) was performed to characterize the 5’ end of BRE transcripts. BRE gene expression was measured by qRT-PCR. Expression of an alternate BRE transcript was normalized to PBGD expression and a control sample without outlier high BRE expression. A normalized value >500 was considered high. Ingenuity pathway analyses were performed on the top 200 genes positively associated with BRE expression in MLL-AF9+ AML.
Results
RNA-seq and qRT-PCR of MLL-AF9+ patient samples showed a marked increase in BRE expression starting from exon 5, exclusively in samples with outlier high BRE expression. H3K4me3 and H3K27ac ChIP-sequencing revealed clear marks in BRE intron 4, 3 kb upstream of BRE exon 5, only in AML cases with outlier high BRE expression. To demonstrate that this region relates to active transcription, we performed 5’ RACE. A new transcript starting in BRE intron 4 near the H3K4me3/H3K27ac marks was identified, with sequences spliced to BRE exon 5. A qRT-PCR specific for the alternate BRE transcript showed that it was frequently expressed in adult MLL-AF9 samples and in samples with the KAT6A-CREBBP fusion gene (18/35, median expression high 8580, low 0.6). This is in line with published data showing that part of MLL-AF9+ and KAT6A-CREBBP+ samples share a common GEP. Also childhood MLL-rearranged and KAT6A-CREBBP+ AML samples frequently expressed the alternate BRE transcript (31/43, median expression high 8172, low 26.9). Alternate BRE expression was generally low in patients with other types of AML (n=42, 1.4) and non-AML hematological malignancies (n=10, 0.4) or normal CD34+ and bone marrow cells (n=14, 0.3). A polymorphism in BRE intron 5 revealed that alternate BRE transcription initiation is bi-allelic. Importantly, analysis of genome-wide H3K4me3 marks and RNA-seq data uncovered 98 potential intragenic transcriptional activation sites, several of which are in genes correlating with high BRE expression. Ingenuity pathway analyses using the top 200 genes positively associated with BRE expression, identified potential pathogenic pathways including p53 and protein kinase A signaling (p<0.03).
Conclusion
Alternate transcription initiation in the ALK gene was recently identified as oncogenic mechanism causing melanoma (Wiesner et al., Nature, 2015). Our data show genome-wide alternate transcription initiation as new phenomenon in MLL-AF9+ and KAT6A-CREBBP+ AML cases that exhibit a unique GEP. Potential oncogenic pathways were identified in these AML subgroups. Further studies investigating the molecular mechanisms contributing to alternate transcription initiation in AML are warranted.
Session topic: Acute myeloid leukemia - Biology 1
Keyword(s): 11q23, AML, Gene expression profile, MLL
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