TRANSCRIPTION-COUPLED GENETIC INSTABILITY MARKS ACUTE LYMPHOBLASTIC LEUKEMIA STRUCTURAL VARIATION HOTSPOTS
(Abstract release date: 05/19/16)
EHA Library. Heinäniemi M. 06/12/16; 135292; S798
Prof. Merja Heinäniemi
Contributions
Contributions
Abstract
Abstract: S798
Type: Oral Presentation
Presentation during EHA21: On Sunday, June 12, 2016 from 08:00 - 08:15
Location: Hall C11
Background
Next generation sequencing technologies have drawn attention to the analysis of genetic alterations across different cancer genomes. Precursor leukemias are unique in that they often harbor structural variations (SV) and have relatively few mutations. Instead, the maturing lymphoid cells are vulnerable to off-target effects downstream of the recombination activating genes (RAG) and activation-induced deaminase (AID, encoded by the AICDA gene) activity that is required for immune gene rearrangement.
Aims
Earlier in vitro studies on DNA binding and genome-wide binding profiles of RAGs have shed light on the mechanisms how this complex is recruited to DNA; however these studies have been carried out using normal cells or mouse models that limit their integration with patient WGS data. More recently, a functional role of transcription in genomic instability has begun to emerge: AID can generate off-target DNA breaks at loci that harbor highly active enhancers and display convergent transcription (transcription from both strands) in lymphomas. The present study represents a systematic investigation of SVs detected in acute pre-B-cell leukemia (pre-B-ALL) in the context of global transcriptional activity in leukemic cells.
Methods
RNA polymerases engaged into primary transcription across the genome can be measured using Global-Run-On sequencing (GRO-seq). Therefore, this method is ideally suited to distinguish features of transcription at SV sites. Transcriptional activity was assayed using GRO-seq from ALL cells representing seven different pre-B-ALL cytogenetic subtypes, and jointly analyzed with public WGS data from the ETV6-RUNX1 (51 cases), high hyperdiploid (16 cases), hypodiploid (20 cases) and MLL-rearranged (22 cases at diagnosis and 2 relapses) subtypes of precursor B-ALL. Pre-B-ALL patient expression profiles (N=1,382), B-lymphoid cell chromatin conformation capture (HiC), RNA polymerase ChIP-seq and DNAse hypersensitivity (DNAse-seq) data were used as additional data.
Results
The active transcriptional profiles from ALL patients revealed striking similarity at SV sites to AID off-target lesions reported in lymphomas. Based on pre-B-ALL transcriptomes, high AICDA levels were particularly prevalent in sample clusters that corresponded to high-risk ALL cases and 62% were cytogenetically classified to the subtype “other”. The highest level of AICDA expression was presented by a relapsed ALL case with high expression already at diagnosis. SV with RAG recognition motifs (RSS motif) also associated strongly with features of transcriptional activity. We could show that elevated levels of convergent transcription that typically occurs at intragenic enhancers positively correlated with detected R-loop levels (needed for AID recruitment) and number of RNA polymerase stalling events at breakpoints. The wide stalling regions identified correlated with highly accessible DNA regions. Accordinly, RNA polymerase stalling sites detected at TSS regions with breakpoints harboring the RAG recognition motif were significantly wider than stalling sites found at TSS regions with no breakpoints, indicating that RNA polymerase stalling could mediate RAG access to its cleavage sites.
Conclusion
The results support a model in which transcriptional features (convergence of transcription and Pol2 stalling), through altering access to DNA, could act to guide secondary DNA lesions and therefore leukemia progression. Our results position RAG among complexes involved in transcription-coupled genomic instability. Moreover, we present evidence of expression of AICDA in high-risk / cytogenetic class “other” pre-B-ALL cases.
Session topic: ALL Biology - Transcriptional dysregulation
Keyword(s): Acute lymphoblastic leukemia, Genetic instability
Type: Oral Presentation
Presentation during EHA21: On Sunday, June 12, 2016 from 08:00 - 08:15
Location: Hall C11
Background
Next generation sequencing technologies have drawn attention to the analysis of genetic alterations across different cancer genomes. Precursor leukemias are unique in that they often harbor structural variations (SV) and have relatively few mutations. Instead, the maturing lymphoid cells are vulnerable to off-target effects downstream of the recombination activating genes (RAG) and activation-induced deaminase (AID, encoded by the AICDA gene) activity that is required for immune gene rearrangement.
Aims
Earlier in vitro studies on DNA binding and genome-wide binding profiles of RAGs have shed light on the mechanisms how this complex is recruited to DNA; however these studies have been carried out using normal cells or mouse models that limit their integration with patient WGS data. More recently, a functional role of transcription in genomic instability has begun to emerge: AID can generate off-target DNA breaks at loci that harbor highly active enhancers and display convergent transcription (transcription from both strands) in lymphomas. The present study represents a systematic investigation of SVs detected in acute pre-B-cell leukemia (pre-B-ALL) in the context of global transcriptional activity in leukemic cells.
Methods
RNA polymerases engaged into primary transcription across the genome can be measured using Global-Run-On sequencing (GRO-seq). Therefore, this method is ideally suited to distinguish features of transcription at SV sites. Transcriptional activity was assayed using GRO-seq from ALL cells representing seven different pre-B-ALL cytogenetic subtypes, and jointly analyzed with public WGS data from the ETV6-RUNX1 (51 cases), high hyperdiploid (16 cases), hypodiploid (20 cases) and MLL-rearranged (22 cases at diagnosis and 2 relapses) subtypes of precursor B-ALL. Pre-B-ALL patient expression profiles (N=1,382), B-lymphoid cell chromatin conformation capture (HiC), RNA polymerase ChIP-seq and DNAse hypersensitivity (DNAse-seq) data were used as additional data.
Results
The active transcriptional profiles from ALL patients revealed striking similarity at SV sites to AID off-target lesions reported in lymphomas. Based on pre-B-ALL transcriptomes, high AICDA levels were particularly prevalent in sample clusters that corresponded to high-risk ALL cases and 62% were cytogenetically classified to the subtype “other”. The highest level of AICDA expression was presented by a relapsed ALL case with high expression already at diagnosis. SV with RAG recognition motifs (RSS motif) also associated strongly with features of transcriptional activity. We could show that elevated levels of convergent transcription that typically occurs at intragenic enhancers positively correlated with detected R-loop levels (needed for AID recruitment) and number of RNA polymerase stalling events at breakpoints. The wide stalling regions identified correlated with highly accessible DNA regions. Accordinly, RNA polymerase stalling sites detected at TSS regions with breakpoints harboring the RAG recognition motif were significantly wider than stalling sites found at TSS regions with no breakpoints, indicating that RNA polymerase stalling could mediate RAG access to its cleavage sites.
Conclusion
The results support a model in which transcriptional features (convergence of transcription and Pol2 stalling), through altering access to DNA, could act to guide secondary DNA lesions and therefore leukemia progression. Our results position RAG among complexes involved in transcription-coupled genomic instability. Moreover, we present evidence of expression of AICDA in high-risk / cytogenetic class “other” pre-B-ALL cases.
Session topic: ALL Biology - Transcriptional dysregulation
Keyword(s): Acute lymphoblastic leukemia, Genetic instability
Abstract: S798
Type: Oral Presentation
Presentation during EHA21: On Sunday, June 12, 2016 from 08:00 - 08:15
Location: Hall C11
Background
Next generation sequencing technologies have drawn attention to the analysis of genetic alterations across different cancer genomes. Precursor leukemias are unique in that they often harbor structural variations (SV) and have relatively few mutations. Instead, the maturing lymphoid cells are vulnerable to off-target effects downstream of the recombination activating genes (RAG) and activation-induced deaminase (AID, encoded by the AICDA gene) activity that is required for immune gene rearrangement.
Aims
Earlier in vitro studies on DNA binding and genome-wide binding profiles of RAGs have shed light on the mechanisms how this complex is recruited to DNA; however these studies have been carried out using normal cells or mouse models that limit their integration with patient WGS data. More recently, a functional role of transcription in genomic instability has begun to emerge: AID can generate off-target DNA breaks at loci that harbor highly active enhancers and display convergent transcription (transcription from both strands) in lymphomas. The present study represents a systematic investigation of SVs detected in acute pre-B-cell leukemia (pre-B-ALL) in the context of global transcriptional activity in leukemic cells.
Methods
RNA polymerases engaged into primary transcription across the genome can be measured using Global-Run-On sequencing (GRO-seq). Therefore, this method is ideally suited to distinguish features of transcription at SV sites. Transcriptional activity was assayed using GRO-seq from ALL cells representing seven different pre-B-ALL cytogenetic subtypes, and jointly analyzed with public WGS data from the ETV6-RUNX1 (51 cases), high hyperdiploid (16 cases), hypodiploid (20 cases) and MLL-rearranged (22 cases at diagnosis and 2 relapses) subtypes of precursor B-ALL. Pre-B-ALL patient expression profiles (N=1,382), B-lymphoid cell chromatin conformation capture (HiC), RNA polymerase ChIP-seq and DNAse hypersensitivity (DNAse-seq) data were used as additional data.
Results
The active transcriptional profiles from ALL patients revealed striking similarity at SV sites to AID off-target lesions reported in lymphomas. Based on pre-B-ALL transcriptomes, high AICDA levels were particularly prevalent in sample clusters that corresponded to high-risk ALL cases and 62% were cytogenetically classified to the subtype “other”. The highest level of AICDA expression was presented by a relapsed ALL case with high expression already at diagnosis. SV with RAG recognition motifs (RSS motif) also associated strongly with features of transcriptional activity. We could show that elevated levels of convergent transcription that typically occurs at intragenic enhancers positively correlated with detected R-loop levels (needed for AID recruitment) and number of RNA polymerase stalling events at breakpoints. The wide stalling regions identified correlated with highly accessible DNA regions. Accordinly, RNA polymerase stalling sites detected at TSS regions with breakpoints harboring the RAG recognition motif were significantly wider than stalling sites found at TSS regions with no breakpoints, indicating that RNA polymerase stalling could mediate RAG access to its cleavage sites.
Conclusion
The results support a model in which transcriptional features (convergence of transcription and Pol2 stalling), through altering access to DNA, could act to guide secondary DNA lesions and therefore leukemia progression. Our results position RAG among complexes involved in transcription-coupled genomic instability. Moreover, we present evidence of expression of AICDA in high-risk / cytogenetic class “other” pre-B-ALL cases.
Session topic: ALL Biology - Transcriptional dysregulation
Keyword(s): Acute lymphoblastic leukemia, Genetic instability
Type: Oral Presentation
Presentation during EHA21: On Sunday, June 12, 2016 from 08:00 - 08:15
Location: Hall C11
Background
Next generation sequencing technologies have drawn attention to the analysis of genetic alterations across different cancer genomes. Precursor leukemias are unique in that they often harbor structural variations (SV) and have relatively few mutations. Instead, the maturing lymphoid cells are vulnerable to off-target effects downstream of the recombination activating genes (RAG) and activation-induced deaminase (AID, encoded by the AICDA gene) activity that is required for immune gene rearrangement.
Aims
Earlier in vitro studies on DNA binding and genome-wide binding profiles of RAGs have shed light on the mechanisms how this complex is recruited to DNA; however these studies have been carried out using normal cells or mouse models that limit their integration with patient WGS data. More recently, a functional role of transcription in genomic instability has begun to emerge: AID can generate off-target DNA breaks at loci that harbor highly active enhancers and display convergent transcription (transcription from both strands) in lymphomas. The present study represents a systematic investigation of SVs detected in acute pre-B-cell leukemia (pre-B-ALL) in the context of global transcriptional activity in leukemic cells.
Methods
RNA polymerases engaged into primary transcription across the genome can be measured using Global-Run-On sequencing (GRO-seq). Therefore, this method is ideally suited to distinguish features of transcription at SV sites. Transcriptional activity was assayed using GRO-seq from ALL cells representing seven different pre-B-ALL cytogenetic subtypes, and jointly analyzed with public WGS data from the ETV6-RUNX1 (51 cases), high hyperdiploid (16 cases), hypodiploid (20 cases) and MLL-rearranged (22 cases at diagnosis and 2 relapses) subtypes of precursor B-ALL. Pre-B-ALL patient expression profiles (N=1,382), B-lymphoid cell chromatin conformation capture (HiC), RNA polymerase ChIP-seq and DNAse hypersensitivity (DNAse-seq) data were used as additional data.
Results
The active transcriptional profiles from ALL patients revealed striking similarity at SV sites to AID off-target lesions reported in lymphomas. Based on pre-B-ALL transcriptomes, high AICDA levels were particularly prevalent in sample clusters that corresponded to high-risk ALL cases and 62% were cytogenetically classified to the subtype “other”. The highest level of AICDA expression was presented by a relapsed ALL case with high expression already at diagnosis. SV with RAG recognition motifs (RSS motif) also associated strongly with features of transcriptional activity. We could show that elevated levels of convergent transcription that typically occurs at intragenic enhancers positively correlated with detected R-loop levels (needed for AID recruitment) and number of RNA polymerase stalling events at breakpoints. The wide stalling regions identified correlated with highly accessible DNA regions. Accordinly, RNA polymerase stalling sites detected at TSS regions with breakpoints harboring the RAG recognition motif were significantly wider than stalling sites found at TSS regions with no breakpoints, indicating that RNA polymerase stalling could mediate RAG access to its cleavage sites.
Conclusion
The results support a model in which transcriptional features (convergence of transcription and Pol2 stalling), through altering access to DNA, could act to guide secondary DNA lesions and therefore leukemia progression. Our results position RAG among complexes involved in transcription-coupled genomic instability. Moreover, we present evidence of expression of AICDA in high-risk / cytogenetic class “other” pre-B-ALL cases.
Session topic: ALL Biology - Transcriptional dysregulation
Keyword(s): Acute lymphoblastic leukemia, Genetic instability
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