EXPLORING THE ROLE OF WNT10B AS LEUKEMIA DRIVER IN ZEBRAFISH MODEL

ABSTRACT Acute myeloid leukemia (AML) is a heterogeneous clonal disorder characterized by a misregulation in the differentiation program of myeloid progenitor cells that proliferate abnormally and arrest in their maturation. In the effort to reveal the mechanism that confers self-renewal potential to Leukemia Stem Cells (LSCs), Wnt pathway came out as a candidate. The Wnt pathway has a central role in the hematopoiesis regulating the hematopoietic stem cells (HSCs) self-renewal. A recent study showed an increase of WNT proteins (i.e. WNT10B) expression and release within the microenvironment in both leukemic blasts and stromal-like cells of AML patients, indicating its possible autocrine/paracrine involvement in the bone marrow microenvironment, and suggesting that the regenerative WNT signaling is a stem cell-associated function altered in AML stem cell fraction. The aim of this work is to elucidate the role of WNT10B in AML generating an animal model in which this signaling is disregulated.
The striking similarities of zebrafish and human hematopoiesis make this fish an excellent model for elucidating physiologic and pathological mechanisms. Furthermore, various hematopoietic zebrafish mutants, mimicking the human hematopoietic system diseases, have been generated. wnt10b was transiently upregulated in zebrafish embryos and effects of such misregulation on embryonic hematopoiesis were analyzed. In-situ hybridization assays, performed using myeloid and HSCs markers (pu.1 and scl, respectively), and in vivo observation of transgenic fish lines expressing fluorescent proteins in erythroid progenitors and mature neutrophils - Tg(gata1:dsRed) and Tg(mpx:EGFP) - demonstrated an abnormal accumulation of HSCs, myeloid and erythroid progenitors in the embryo hematopoietic tissue, at the expense of more differentiated cells. Moreover, wnt10b overexpression affects the migration of myeloid cells in the ALM (Anterior Lateral Mesoderm). Taken together, these results support the notion of an involvement of wnt10b in vertebrate hematopoiesis.
A plasmid construct with zebrafish wnt10b under the control of runx1 (a pivotal gene in definitive hematopoiesis) promoter and flanked by trasposase-responsive elements has been generated; injection of this plasmid in zebrafish embryos together with trasposase mRNA will lead to the generation of a transgenic line in which wnt10b expression can be induced only in definitive HSCs. Future plans include the temporal control of the transgene expression through the insertion in the plasmid of a lox-P cassette flanking a STOP signal and the outcross of the resulting transgenic line with a line expressing Cre recombinase under the control of hsp70 (heat-shock protein) promoter. This model will provide the chance to better understand the Wnt-mediated early mechanisms that lead to leukemia development.