Development of Hras-Induced Zebrafish Leukemia Models

Background:Over the last years, the zebrafish has emerged as a versatile novel experimental model for hematopoietic studies. The major genetic pathways have proven conserved between fish and mammalian hematopoiesis. Several oncogenes involved in human leukemia have been successfully overexpressed in a transient fashion in zebrafish embryos. However, despite first encouraging results these experimental models often failed to fully recapitulate human myeloid malignancy, perhaps due to early lethality caused by off-target expression or lack of secondary events necessary for full malignant transformation.

Material and Methods: Here we took advantage of the Gal4/UAS binary system and of existing transgenic lines to overexpress the human oncogenic HRAS gene in zebrafish hematopoietic cells under control of specific promoters (fli.1, pu.1, runx.1). HRAS-transgenic lines were generated and fish followed by microscopy from early development until, if possible, sexual maturity. Hematopoietic cell development was studied at embryonic (fli.1, pu.1), larval (pu.1, runx1) and adult stages (runx1) by in situ hybridization and real-time PCR analysis of hematopoietic gene expression, flow cytometry, immunohistochemistry and/or blood smear morphological assessment.

Results: All HRAS transgenic lines showed hematopoietic abnormalities. However, different phenotypes were observed depending on the promoter driving the oncogene expression. HRAS induction via the early hematopoietic promoter fli.1 affected primitive hematopoiesis inducing a myelo-erythroid proliferation characterized by the expansion of the caudal hematopoietic tissue, enhanced expression of myelo-erythroid genes and delayed erythrocyte maturation (Alghisi et al. 2013). Surprisingly, no obvious effects were noted on the emerging hematopoietic stem cells (HSCs) in the aorta-gonado-mesonephros (AGM) region and studies at later stages were hampered by early lethality of the fish due to vascular defects and cardiac edema. The lethality at early stages was also observed using the myeloid promoter pu.1 that induced the expansion of primitive myeloid cells along with severe developmental defects. In contrast, HRAS expression driven by runx1, a known HSC marker, did not affect primitive hematopoiesis and allowed studies at later developmental stages. Supporting the results obtained with the fli.1 promoter, no alteration was noted in the AGM of runx1-HRAS induced fish. However, at 1 month post-fertilization, flow cytometry analyses revealed a prominent cellular expansion of the hematopoietic stem/progenitor cells in the kidney marrow, the zebrafish definitive hematopoietic compartment. Kidney marrow cytospin preparation and flow cytometry analysis confirmed high numbers of undifferentiated cells indicating that HRAS-overexpression in this model induced stem/progenitor cell proliferation. Interestingly, first analyses suggested that the numbers of differentiated cells might be reduced in these fish, implying that HRAS-induced stem/progenitors have impaired differentiation capacity.

Outlook and conclusion: We are currently further investigating the effects of runx1-driven HRAS on the hematopoietic compartment and generate tools to explore potential cooperation of HRAS with other oncogenes during leukemogenesis. Successfully established zebrafish leukemia models shall be eventually used for identification of therapeutically active compounds in small molecule screens.