Contributions
Abstract: S863
Type: Oral Presentation
Presentation during EHA23: On Saturday, June 16, 2018 from 16:15 - 16:30
Location: Room A4
Background
Mixed-phenotype acute leukemia (MPAL) is a rare subtype of leukemia in which there is co-expression of myeloid and lymphoid markers on the same malignant cells. The pathogenesis of these leukemias is unknown and their treatment is challenging. This research is based on our discovery of the recurrent t(8;12)(q13;p13) chromosomal translocation in children with acute leukemia. The translocation generates a transcript in which the PNT domain of the transcriptional repressor ETV6 fuses with the epigenetic-modification domains of the co-activator NCOA2. Remarkably, this genetic event is always associated with acute leukemia co-expressing T-lymphoid and myeloid (T/M) markers and often presents with activating mutations of Notch1.
Aims
We hypothesize that ETV6-NCOA2 (EN2) fusion protein is a major driver of MPAL and of Early T-cell leukemia (ETP) with a T/M phenotype through transcriptional dysregulation.
Methods
To examine whether EN2 directly contributes to leukemogenesis we transduced lineage-negative BM progenitors with EN2 and performed in-vitro and in-vivo assays: methylcellulose replating assay to assess self-renewal; co-cultures on OP9DL4 to examine T-cell differentiation ability and transplantation into C57B/6 mice to examine leukemia development. Cord blood derived CD34+ hematopoietic stem and progenitor cells (HSPCs) were co-transduced with EN2 and a Notch1 allele with a weak activating mutation (L1601PdP) and transplanted into immunodeficient mice. To understand the initiating changes in gene expression we conducted RNA-sequencing of HSPCs transduced with EN2. To reveal the effect of EN2 fusion protein on wt-ETV6 we preformed CO-IP experiments to explore this interaction and luciferase reporter assay to examine changes in transcription.
Results
In-vitro experiments demonstrated enhanced self-renewal ability and T-cell differentiation arrest at the DN1/DN2 stage. C57B/6 mice transplanted with lin- transduced with EN2 cells developed a lymphoma/leukemia malignancy characterized by extra medullary infiltration including spleen, liver, lungs and lymph nodes. Immunophenotypic analysis displayed a mixed T/M phenotype. Several leukemic/lymphoma cells developed spontaneous Notch1 mutations similarly to those found in human EN2 patients. The tumors were propagated in secondary transplantations. Immunodeficient mice transplanted with HSPCs co-expressing EN2 and Notch1-L1601PdP developed leukemia with co-expression of T-cell and myeloid markers. RNA-seq of CD34+ cells transduced with EN2 after five days in culture with myeloid and stem cell cytokines revealed that EN2 induces an early T-cell precursor’s transcriptional program. We detected a significant up-regulation of ETV6 target genes. We hypothesize that EN2 has a dominant negative effect on wt-ETV6 through dimerization with its PNT domain and recruitment of histone acetylase EP300 to ETV6 targets. COIP experiments confirmed that EN2 and ETV6 interacts and Luciferase assay showed that EN2 expression can abolish ETV6's repression.
Conclusion
ETV6-NCOA2 is a driver of T/M MPAL by inducing a T-cell program in early hematopoietic progenitors, possibly by de-repression of ETV6 genes. These findings are consistent with an earlier report on dominant negative mutations in ETV6 in early T-cell ALL, characterized by a T/M phenotype. The development of the first preclinical model of T/M acute leukemia will contribute to the understanding of this disease and may serve as a future model for therapeutics. Furthermore, this research could shed more light on the lineage choice decisions of early hematopoietic progenitors.
Session topic: 1. Acute lymphoblastic leukemia – Biology & Translational Research
Keyword(s): ETV6, Mixed lineage leukemia, Translocation
Abstract: S863
Type: Oral Presentation
Presentation during EHA23: On Saturday, June 16, 2018 from 16:15 - 16:30
Location: Room A4
Background
Mixed-phenotype acute leukemia (MPAL) is a rare subtype of leukemia in which there is co-expression of myeloid and lymphoid markers on the same malignant cells. The pathogenesis of these leukemias is unknown and their treatment is challenging. This research is based on our discovery of the recurrent t(8;12)(q13;p13) chromosomal translocation in children with acute leukemia. The translocation generates a transcript in which the PNT domain of the transcriptional repressor ETV6 fuses with the epigenetic-modification domains of the co-activator NCOA2. Remarkably, this genetic event is always associated with acute leukemia co-expressing T-lymphoid and myeloid (T/M) markers and often presents with activating mutations of Notch1.
Aims
We hypothesize that ETV6-NCOA2 (EN2) fusion protein is a major driver of MPAL and of Early T-cell leukemia (ETP) with a T/M phenotype through transcriptional dysregulation.
Methods
To examine whether EN2 directly contributes to leukemogenesis we transduced lineage-negative BM progenitors with EN2 and performed in-vitro and in-vivo assays: methylcellulose replating assay to assess self-renewal; co-cultures on OP9DL4 to examine T-cell differentiation ability and transplantation into C57B/6 mice to examine leukemia development. Cord blood derived CD34+ hematopoietic stem and progenitor cells (HSPCs) were co-transduced with EN2 and a Notch1 allele with a weak activating mutation (L1601PdP) and transplanted into immunodeficient mice. To understand the initiating changes in gene expression we conducted RNA-sequencing of HSPCs transduced with EN2. To reveal the effect of EN2 fusion protein on wt-ETV6 we preformed CO-IP experiments to explore this interaction and luciferase reporter assay to examine changes in transcription.
Results
In-vitro experiments demonstrated enhanced self-renewal ability and T-cell differentiation arrest at the DN1/DN2 stage. C57B/6 mice transplanted with lin- transduced with EN2 cells developed a lymphoma/leukemia malignancy characterized by extra medullary infiltration including spleen, liver, lungs and lymph nodes. Immunophenotypic analysis displayed a mixed T/M phenotype. Several leukemic/lymphoma cells developed spontaneous Notch1 mutations similarly to those found in human EN2 patients. The tumors were propagated in secondary transplantations. Immunodeficient mice transplanted with HSPCs co-expressing EN2 and Notch1-L1601PdP developed leukemia with co-expression of T-cell and myeloid markers. RNA-seq of CD34+ cells transduced with EN2 after five days in culture with myeloid and stem cell cytokines revealed that EN2 induces an early T-cell precursor’s transcriptional program. We detected a significant up-regulation of ETV6 target genes. We hypothesize that EN2 has a dominant negative effect on wt-ETV6 through dimerization with its PNT domain and recruitment of histone acetylase EP300 to ETV6 targets. COIP experiments confirmed that EN2 and ETV6 interacts and Luciferase assay showed that EN2 expression can abolish ETV6's repression.
Conclusion
ETV6-NCOA2 is a driver of T/M MPAL by inducing a T-cell program in early hematopoietic progenitors, possibly by de-repression of ETV6 genes. These findings are consistent with an earlier report on dominant negative mutations in ETV6 in early T-cell ALL, characterized by a T/M phenotype. The development of the first preclinical model of T/M acute leukemia will contribute to the understanding of this disease and may serve as a future model for therapeutics. Furthermore, this research could shed more light on the lineage choice decisions of early hematopoietic progenitors.
Session topic: 1. Acute lymphoblastic leukemia – Biology & Translational Research
Keyword(s): ETV6, Mixed lineage leukemia, Translocation