Abstract:
Acute myeloid leukaemia (AML) is an aggressive malignancy, characterised by increased proliferation a block of differentiation resulting in an increase of immature haematopoietic cell in the bone marrow. Without treatment AML patients die within a few days or weeks. AML is caused by somatically acquired genetic lesions including chromosomal translocations and mutations. The CALM/AF10 fusion, which is the result of the t(10;11)(p13;q14) translocation, is found in about 1% of AML patients. Patients with CALM/AF10 have a very poor prognosis. In this study, transgenic zebrafish with four different CALM/AF10 minimal fusion transgenic constructs were generated (R:CA/MF-p-GFP, R:CA/MF-p-mCherry, R:CA/MF-I-GFP, R:CA/MF-I-mCherry [R:CA/MF]). All transgenic constructs express the CALM/AF10 minimal fusion gene under the control of the Runx1+23 promoter/enhancer. F0, F1 and F2 transgenic fish developed aggressive leukaemia with a median latency of about one year. The F0 R:CA/MF fish showed signs of distress or sickness included fish being less active and sitting at the bottom of tanks, not chasing food, bleeding, with some protrusion around the abdomen, heart area and eye region and F1 and F2 generations of R:CA/MF zebrafish developed tumours around the peritoneal cavity or head and eye region. Flow cytometric analysis of the whole kidney marrow of leukaemic zebrafish showed predominately expansion of the precursors and myeloid cell compartments. Whole mount RNA in-situ hybridization of 1-3 dpf transgenic embryos showed overexpression of myeloid and stem cells marker genes in haematopoietic cells. The leukaemic cells from F0 R:CA/MF-p-GFP zebrafish could be serially transplanted into secondary and tertiary recipients. Furthermore, whole exome analysis of 6 leukaemias detected potentially somatic mutations in 14 genes. Mutations in some of the mutated genes like in rnf38, mhc1zba, vcan-a, ugt5b4, cyp2k2, and ms4a17a.8 might be cooperating driver mutations. In addition, a preliminary transcriptome analysis using RNASeq revealed that genes overexpressed in the leukemic cells were enriched in the KRAS and MTOR oncogenic pathway gene sets. To our knowledge, our CALM/AF10 zebrafish leukaemia model is the only zebrafish AML model which has been shown to be serially transplantable, and also the only zebrafish leukaemia model in which potentially cooperating somatic mutations have been identified using whole exome sequencing.