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

Simple Summary Despite remarkable progress in the treatment of acute lymphoblastic leukemia (ALL) in recent years, it remains a significant contributor to pediatric cancer-related deaths. This highlights the urgent need for innovative therapeutic strategies that target the genetic alterations driving ALL. We built a novel zebrafish disease model for ETV6::RUNX1-positive ALL, which harbors secondary lesions in the two commonly mutated genes, pax5, and cdkn2a/b. The introduction of secondary mutations significantly augmented the incidence of disease. This model provides a valuable tool for investigating the etiological role of secondary mutations and facilitating the evaluation of drug sensitivities in the future.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.