Modulators of hematopoietic stem cell specification using zebrafish embryo cultures

Abstract

Hematopoietic stem cells (HSCs) have been used clinically to reconstitute the bone marrow, but half of all patients lack an optimally matched donor marrow. HSCs derived from induced pluripotent stem cells (iPSCs) hold great promise for treating these patients, but our understanding of the pathways involved in HSC formation is incomplete. A zebrafish blastomere culture screening system was adapted to find modulators of the hematopoietic stem and progenitor cell markers runx1 and c-myb to identify new pathways that specify and expand HSCs. Transgenic zebrafish embryos were dissociated and treated with 3,840 independent compounds, and after two days in culture, fluorescence was read out. Retinoic acid agonists were potent suppressors of c-myb:GFP expression in the zebrafish embryo cell cultures. Translocations that drive overexpression of the oncogenic transcription factor MYB are molecular hallmarks of adenoid cystic carcinoma (ACC), a fatal salivary gland tumor with no effective therapy. Since MYB translocations in ACC retain MYB regulatory regions, transcriptional suppression of MYB by retinoic acid would be potentially useful in treating ACC. Patient derived xenograft studies in mice showed an average tumor size inhibition after 28 days of treatment across three primary ACC models that were among the highest levels for any drugs tested in these xenograft models. These findings identify an important role of retinoic acid in MYB regulation and represent a potential new effective therapy for ACC. 21 chemicals were also identified that induced runx1 expressing cell expansion in vitro, including runx1-CBFβ inhibitors. HSC formation from hemogenic endothelium is dependent on a heterodimeric transcription factor composed of runx1 and core binding factor β. The runx1-CBFβ inhibitor Ro5-3335 increased the hematopoietic induction of CD34+CD45+ cells from human iPSCs. A cross-species approach involving zebrafish and mammalian systems thus offers complementary methodologies to engineer HSCs for therapy and demonstrates transient runx1-CBFβ inhibition to enhance HSC specification. Chemical factors revealed in our zebrafish culture system establish pathways that can be useful targets in oncogenic events and to promote iPSC-derived HSC expansion.