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Updated: Jul 7, 2025
Author Spotlight: Identification and Isolation of Quiescent Leukemia Stem Cells from Zebrafish T-ALL
Published on: July 19, 2024
Generation of a Zebrafish Knock-In Model Recapitulating Childhood ETV6::RUNX1-Positive B-Cell Precursor Acute
Veronika Zapilko1, Sanni Moisio2, Mataleena Parikka3
1Tampere Center for Child, Adolescent and Maternal Health Research, Faculty of Medicine and Health Technology, Tampere University, 33100 Tampere, Finland.
Insights
A new zebrafish model for ETV6::RUNX1 (E::R) leukemia was created. Mimicking secondary mutations significantly increased leukemia incidence, offering insights into B-cell precursor acute lymphoblastic leukemia (pB-ALL) development.
Area of Science:
- Hematology
- Genetics
- Developmental Biology
Background:
- Approximately 25% of pediatric B-cell precursor acute lymphoblastic leukemia (pB-ALL) cases involve the t(12;21) translocation, forming the ETV6::RUNX1 (E::R) fusion gene.
- The E::R fusion arises prenatally, but overt leukemia is rare, suggesting secondary genetic events are crucial for disease development.
- The precise role of these secondary mutations in leukemogenesis and treatment resistance remains largely unknown.
Purpose of the Study:
- To develop a novel zebrafish model for E::R-positive pB-ALL that accurately recapitulates human disease.
- To investigate the impact of specific secondary mutations on leukemia development in the context of the E::R fusion.
- To utilize this model for further analysis of E::R leukemia biology.
Main Methods:
- CRISPR/Cas9 gene editing was used to create a zebrafish model expressing the human E::R fusion gene under the control of the endogenous etv6 promoter.
- GFP reporter expression confirmed correct fusion protein localization in zebrafish embryos.
- Targeted mutations in pax5 and cdkn2a/b genes were introduced into the E::R zebrafish line to mimic secondary mutations.
- Transcriptomic analysis was performed on leukemic samples.
Main Results:
- The E::R zebrafish model successfully expressed the fusion protein and showed an expansion of the progenitor cell pool.
- Leukemia incidence in the E::R model was low (2%), but significantly increased upon introduction of pax5 and cdkn2a/b mutations.
- Transcriptomic data confirmed that E::R;pax5mut leukemias were exclusively of B-lineage origin.
- The model demonstrated faithful recapitulation of key aspects of human E::R pB-ALL.
Conclusions:
- The developed zebrafish model is a valuable tool for studying E::R-positive pB-ALL.
- Secondary mutations in genes like PAX5 and CDKN2A/B play a critical role in the development of E::R-driven leukemia.
- This model provides a platform for dissecting the molecular mechanisms underlying leukemia initiation and progression in this subtype.
Abstract:
Approximately 25% of children with B-cell precursor acute lymphoblastic leukemia (pB-ALL) harbor the t(12;21)(p13;q22) translocation, leading to the ETV6::RUNX1 (E::R) fusion gene. This translocation occurs in utero, but the disease is much less common than the prevalence of the fusion in newborns, suggesting that secondary mutations are required for overt leukemia. The role of these secondary mutations remains unclear and may contribute to treatment resistance and disease recurrence. We developed a zebrafish model for E::R leukemia using CRISPR/Cas9 to introduce the human RUNX1 gene into zebrafish etv6 intron 5, resulting in E::R fusion gene expression controlled by the endogenous etv6 promoter. As seen by GFP fluorescence at a single-cell level, the model correctly expressed the fusion protein in the right places in zebrafish embryos. The E::R fusion expression induced an expansion of the progenitor cell pool and led to a low 2% frequency of leukemia. The introduction of targeted pax5 and cdkn2a/b gene mutations, mimicking secondary mutations, in the E::R line significantly increased the incidence in leukemia. Transcriptomics revealed that the E::R;pax5mut leukemias exclusively represented B-lineage disease. This novel E::R zebrafish model faithfully recapitulates human disease and offers a valuable tool for a more detailed analysis of disease biology in this subtype.
