Generation of a Zebrafish Knock-In Model Recapitulating Childhood ETV6::RUNX1-Positive B-Cell Precursor Acute Lymphoblastic Leukemia.

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 ETV6RUNX1 (ER) 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 ER leukemia using CRISPR/Cas9 to introduce the human RUNX1 gene into zebrafish etv6 intron 5, resulting in ER 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 ER 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 ER line significantly increased the incidence in leukemia. Transcriptomics revealed that the ER;pax5mut leukemias exclusively represented B-lineage disease. This novel ER zebrafish model faithfully recapitulates human disease and offers a valuable tool for a more detailed analysis of disease biology in this subtype.