Acute Leukaemia (AL) is a genetically heterogeneous disease caused by somatic mutations and chromosomal translocations. Translocations can lead to the formation of fusion genes such as the MLL-AF9 fusion, which results from the t (9;11)(p22;q23). MLL-AF9 is one of the most common fusions involving the MLL (KMT2A) gene. MLLAF9 leukemias have an intermediate to poor prognosis. The MLL-AF9 fusion is frequently used to establish murine leukemia models. However, to date, no MLL-AF9 leukemia model in zebrafish has been reported.
Our aim was to establish a transgenic zebrafish leukemia model using the MLL/AF9
We cloned two transgenic constructs (pTol2-Runx1+23_MLL-AF9_IRES-EGFP and pTol2-Runx1+23_MLL-AF9_IRES-mCherry). The Runx1+23 murine promoter was used to target MLL-AF9 expression to zebrafish hematopoietic stem cells, while EGFP or mCherry were used as fluorescent markers. The final transgenic constructs were co-injected with Tol2 transposase mRNA into fertilized embryos at the one cell stage. The embryos were sorted 24 hours post fertilization based on the expression of the heart marker.
About 40% of the transgenic embryos died in the first week after injection but the remaining 60% reached adulthood. After 6 to 12 months, 30% (30 out of 100) fish showed signs of disease. They became less active, sat at the bottom of the tank and, in some cases, they had splayed eyes. When this happened, the fish were euthanized and dissected. The autopsies showed enlarged kidneys, splenomegaly and in some cases a pale spleen. Histological sections revealed increased cellularity of the kidney and liver with massive infiltration of hematopoietic cells in these organs. In flow cytometry, kidney marrow cells from the transgenic fish showed a different forward scatter (FSC) and side scatter (SSC) profile compared to that of the normal zebrafish kidney marrow cells. The FSC/SSC profile in the transgenic F0 zebrafish showed an expansion of the hematopoietic progenitor cell population. These findings strongly suggest that the fish had developed a leukemia-like disease. We performed whole mount in situ hybridization (WISH) with early hematopoietic markers in 24 hpf embryos and detected reduced expression of gata1, myb and tal1as well as increased expression of lyz in transgenic embryos. Currently, transplantation experiments are being performed to determine whether the disease is transplantable which is a sign of leukemia.
Although our flow cytometry, histology and WISH data strongly suggest that we have established an MLL/AF9-driven leukemia model in zebrafish, additional experiments are needed to further characterize our model and confirm the malignant nature of the observed disease.
Our MLL/AF9 zebrafish leukemia model will be a helpful tool to study leukemia biology and test new therapeutic strategies.