Contributions
Abstract: S862
Type: Oral Presentation
Presentation during EHA23: On Saturday, June 16, 2018 from 16:00 - 16:15
Location: Room A4
Background
Insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. Previously, we have shown that transposon based genetic screening can overcome the challenge of pinpointing cancer drivers that are not mutated, but are among the thousands of transcriptionally, epigenetically, or posttranslationally dysregulated genes in cancer. Although the majority of transposon insertions are detected in intergenic regions, the consequence of these insertions for tumor development has not yet been investigated systematically.
Aims
We used PiggyBac for the identification of novel cancer genes, but also to decipher the role of non-coding regulatory elements in leukemogenesis and lymphomagenesis.
Methods
Using PiggyBac transposon mutagenesis in mice, we performed genome-wide surveys for cancer drivers and regulatory elements across distinct hematologic malignancies (n=256 cancers), including T cell, B cell, and myeloid neoplasms.
In addition, using intergenic knockout mouse models, we validated these newly identified regulatory elements in vivo.
Results
In line with previous studies more than 90% of RosaPB;ATP2 mice developed hematopoietic malignancies. The subgroup of T cell lymphoblastic lymphomas/ T cell acute lymphoblastic leukemia (T-LBL/T-ALL, n=53) demonstrated a significantly reduced survival compared to other subgroups or single-transgenic control mice. Using semi-quantitative transposon insertion site sequencing (Friedrich et al., Nature Protocols 2017) we found genes already known to be important in T cell malignancies (e.g., Notch1, Ikzf1, Pten, Myb) as well as potential novel candidate genes. Moreover, systematic genome-wide analyses of intergenic insertions identified cancer-relevant regulatory regions, including high-coverage insertions on chromosome 12 downstream of Bcl11b in the T-LBL/T-ALL subgroup. Two of these intergenic common insertion sites show an overlap with markers for open chromatin such as H3K27ac, H3K4me1, and DNase hypersensitivity sites confirming their potential role as regulatory elements. Knockout of two such intergenic regions (105 kb and 1 Mb) in novel mouse models resulted in lymphoma and solid cancer development in 30-40% of cases. Whereas the 105 kb knockout mice (n=148) showed a diverse tumor spectrum, the knockout of the 1 Mb region (n=47 mice) mainly led to the development of lymphomas (70% T-LBL/T-ALL). Consistent with this observation, the mRNA expression of Bcl11b was more prominently reduced in mice with the knockout of the 1 Mb region, thereby indicating a role of this region in Bcl11b regulation and T-LBL/T-ALL development. Importantly, the common insertion sites found downstream of Bcl11b share over 80% sequence similarity to the concordant human region and might therefore also play a role in human BCL11B regulation.
Conclusion
Taken together, our studies give comprehensive novel insights into the coding and regulatory landscapes in malignant hematopoiesis and show that an intergenic knockout model can recapitulate the phenotype of the transposon mice.
Session topic: 1. Acute lymphoblastic leukemia – Biology & Translational Research
Keyword(s): Mouse model, Screening, T-ALL
Abstract: S862
Type: Oral Presentation
Presentation during EHA23: On Saturday, June 16, 2018 from 16:00 - 16:15
Location: Room A4
Background
Insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. Previously, we have shown that transposon based genetic screening can overcome the challenge of pinpointing cancer drivers that are not mutated, but are among the thousands of transcriptionally, epigenetically, or posttranslationally dysregulated genes in cancer. Although the majority of transposon insertions are detected in intergenic regions, the consequence of these insertions for tumor development has not yet been investigated systematically.
Aims
We used PiggyBac for the identification of novel cancer genes, but also to decipher the role of non-coding regulatory elements in leukemogenesis and lymphomagenesis.
Methods
Using PiggyBac transposon mutagenesis in mice, we performed genome-wide surveys for cancer drivers and regulatory elements across distinct hematologic malignancies (n=256 cancers), including T cell, B cell, and myeloid neoplasms.
In addition, using intergenic knockout mouse models, we validated these newly identified regulatory elements in vivo.
Results
In line with previous studies more than 90% of RosaPB;ATP2 mice developed hematopoietic malignancies. The subgroup of T cell lymphoblastic lymphomas/ T cell acute lymphoblastic leukemia (T-LBL/T-ALL, n=53) demonstrated a significantly reduced survival compared to other subgroups or single-transgenic control mice. Using semi-quantitative transposon insertion site sequencing (Friedrich et al., Nature Protocols 2017) we found genes already known to be important in T cell malignancies (e.g., Notch1, Ikzf1, Pten, Myb) as well as potential novel candidate genes. Moreover, systematic genome-wide analyses of intergenic insertions identified cancer-relevant regulatory regions, including high-coverage insertions on chromosome 12 downstream of Bcl11b in the T-LBL/T-ALL subgroup. Two of these intergenic common insertion sites show an overlap with markers for open chromatin such as H3K27ac, H3K4me1, and DNase hypersensitivity sites confirming their potential role as regulatory elements. Knockout of two such intergenic regions (105 kb and 1 Mb) in novel mouse models resulted in lymphoma and solid cancer development in 30-40% of cases. Whereas the 105 kb knockout mice (n=148) showed a diverse tumor spectrum, the knockout of the 1 Mb region (n=47 mice) mainly led to the development of lymphomas (70% T-LBL/T-ALL). Consistent with this observation, the mRNA expression of Bcl11b was more prominently reduced in mice with the knockout of the 1 Mb region, thereby indicating a role of this region in Bcl11b regulation and T-LBL/T-ALL development. Importantly, the common insertion sites found downstream of Bcl11b share over 80% sequence similarity to the concordant human region and might therefore also play a role in human BCL11B regulation.
Conclusion
Taken together, our studies give comprehensive novel insights into the coding and regulatory landscapes in malignant hematopoiesis and show that an intergenic knockout model can recapitulate the phenotype of the transposon mice.
Session topic: 1. Acute lymphoblastic leukemia – Biology & Translational Research
Keyword(s): Mouse model, Screening, T-ALL