Zebrafish ALL Driven By HumanMYCand a New Technique to Biopsy Living Fish

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer; B-lineage ALL (B-ALL) represents ~85% of cases. Transgenic humanMYC(hMYC), an oncogenic transcription factor in many blood cancers, is known to induce T-ALL in zebrafish. Until recently robust zebrafish B-ALL models did not exist, but we discovered fish of this transgenic line develop B-ALL also. To distinguish T-ALL from B-ALL, we use a transgenic marker,lck:GFP, where T-ALL fluoresce brightly while B-ALL fluoresce dimly. To define B- and T-ALL incidence, we monitored >600 fish by serial fluorescent microscopy and found 100% of fish developed ALL (64% T-ALL, 23% B-ALL, both 13%) by 10 months. To find transcriptional programs active in both ALL types, we performed RNA-seq on 10 T- and 14 B-ALL and compared these to T- and B-ALL induced by murineMyc(mMyc). Unexpectedly, we found B-ALL occur in distinct lineages in these related lines, withhMYCB-ALL expressingighzheavy chains, butmMycB-ALLighm. To investigate glucocorticoid responses, a key agent in ALL, we treated fishin vivowith dexamethasone (DXM).hMYCB-ALL were highly DXM-sensitive, but many recurred rapidly after treatment ceased.

Studies lymphocytes or ALL cells from zebrafish typically require fish euthanization; it would be useful to obtain cells without euthanasia. Thus, we developed biopsy techniques in living fish to obtain lymphocytes from the thymus, kidney-marrow, and even scales. We found B and T cells can be obtained from thymus, with B cells abundant in scales. We can also recover ALL cells from scales of fish with B- or T-ALL. Analyses of normal lymphocytes and ALL cells from these anatomic regions prove that GFP^hi cells express T cell genes, and GFP^lo cells express B cell genes. These novel biopsy methods will permit studies ofex vivosamples from live fish. Overall, these new results and methods expand the utility of zebrafishhMYC-driven ALL, a model of the most important pediatric cancer.