Oncogenic HOXA9 Inhibits Cellular Apoptosis Induced by Ionizing Radiation (IR) in Transgenic Zebrafish
Authors: Boyd Ellen R , Forrester A. Michael , Grabher Clemens , Kai Fui Boon , Daas Sahar , Look A. Thomas , Berman Jason N 
Published: April 2020

Abstract
Acute myeloid leukemia (AML) is characterized by the failure of terminal cellular differentiation of the myeloid lineage, and retains a significant rate of mortality despite aggressive therapy. One key oncogenic candidate, HOXA9 – belonging to the highly-conserved HOX family of developmental transcription factors – is frequently overexpressed in AML. In particular, this may result from the rare, but recurrent t(7;11)(p15;p15) chromosomal translocation, yielding the chimeric transcription factor NUP98-HOXA9. This translocation event is associated with a poor prognosis and elucidating the molecular activity of this fusion oncogene will enable the development of targeted therapeutics. Despite established mammalian models of AML, specific transcriptional targets of HOXA9 remain poorly defined. The zebrafish, Danio rerio, possesses orthologous blood cells to humans, making it a reliable model of vertebrate hematopoiesis and a promising tool for studying the molecular mechanisms underlying AML. The zebrafish has many advantages over traditional mouse models: chiefly, the ex utero development and transparency of zebrafish embryos allow for ease of manipulation, phenotypic analysis, and high throughput screening. We have demonstrated that ubiquitous overexpression of the human NUP98-HOXA9 oncogene in germline transgenic zebrafish embryos protects against apoptosis induced by ionizing radiation (IR) – a hallmark of oncogenesis. Acridine orange staining was indicative of decreased apoptosis in live transgenic embryos compared with wild type (WT) controls. Using whole mount immunofluorescent labeling, we similarly demonstrated that IR-treated transgenic embryos have a significant reduction in levels of activated caspase-3, a downstream effector protein in the conserved caspase cascade. By comparison, only a mild reduction in IR-induced apoptosis and activated caspase-3 was observed in a second transgenic zebrafish line overexpressing murine native Hoxa9, suggesting a gradient of impact on cell survival. Taken together, these findings implicate a block in caspase-3-dependent apoptosis as a mechanism underlying HOXA9-mediated oncogenesis in vivo and provide an opportunity to perform chemical modifier screens to identify agents that restore a WT phenotype. Whole mount RNA in situ hybridization for expression of pro-apoptotic genes, bad and puma, and anti-apoptotic genes, bcl2 and bcl-xL, will provide further insight into the effects of oncogenic HOXA9 on the caspase cascade. Similarly, rescue of caspase-3 activation and IR-induced apoptosis is underway via microinjection of bad and puma mRNA. To achieve robust and continuous myeloid-specific expression of oncogenic HOXA9 and partners, new transgenic strategies using both the Tol2 transposon-based Gateway cloning system and Cre/lox-induction are being employed. These lines will enable evaluation of myeloid-specific apoptosis, cell cycle regulation and cellular maturation to corroborate our findings in the ubiquitously-expressing lines, providing further insight into leukemogenic mechanisms and the potential to identify novel targeted therapies to improve the outcome in human AML.