Abstract PR01: Modeling clonal hematopoietic disorders in zebrafish using combinatorial mutagenesis and color barcoding
Authors: Avagyan Serine , Henninger Jonathan E. , Mannherz William , Moore Jessica , Zon Leonard I. 
Published: August 2018

Myeloid neoplasms, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), are clonal hematopoietic malignancies arising from mutant hematopoietic stem and progenitor cells (HSPCs). To follow the hematopoietic output of HSPC clones endogenously, we characterized a novel marking strategy with color barcoding, in which each HSPC clone is labeled with a unique fluorescent hue. The transgenic zebrafish line Zebrabow uses a Cre/lox system where stochastic recombination of multiple transgenic insertions, harboring three fluorescent proteins, produces stable and heritable color barcodes in blood stem cells and their mature cell progeny. The tamoxifen-dependent Cre recombinase under blood-specific promoter draculin allows temporal and spatial control of labeling. The result of Zebrabow labeling is a collection of colored blood cells which can be clustered into distinct groups based on their hue. Equipped with this endogenous color barcoding system, we perturbed the hematopoietic system to induce clonal blood disorders by introducing combinations of known genetic mutations found in MDS and AML. Zebrabow embryos at the 1-cell stage were injected with combinations of mutations and HSPC color labeling was performed at 36 to 48 hours post-fertilization, during peak emergence of definitive blood stem cells from the dorsal aorta. The transgenic overexpression of Jak2V617F, FLT3ITD, IDH2R140Q, NPM1c, or KRASG12D under the zebrafish draculin promoter was combined with somatic mutagenesis of endogenous tet2, asxl1, dnmt3a, ezh2, gata2, or tp53 via the CRISPR/Cas9 system. Mosaic mutations in epigenetic factors resulted in the expansion of single color clones contributing &gt;30% of granulocytes in approximately 30% of zebrafish at 8 months post-fertilization. The majority of zebrafish exhibiting clonal expansion harbored frame-shift mutations in asxl1 with &gt;25% variant allele frequency. Combinations of asxl1 or tet2 mutations with overexpression of Jak2V617F or FLT3ITD also led to single color dominance by 3 months post-fertilization. These combinations led to significant expansion of blast-like cells in the zebrafish marrow and spleen with concomitant decrease in myeloid cells but no effect on lymphocyte count. We have successfully utilized a color barcoding system to model clonal hematopoietic disorders ranging from benign clonal expansion to myleoid leukemia via mosaic mutagenesis in an endogenous setting in zebrafish.

This abstract is also being presented as Poster 38.

Citation Format: Serine Avagyan, Jonathan E. Henninger, William Mannherz, Jessica Moore, Leonard I. Zon. Modeling clonal hematopoietic disorders in zebrafish using combinatorial mutagenesis and color barcoding [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR01.