Zebrafish Tools To Combat Acute Myeloid Leukaemia – The Nucleoporin 98 kiloDalton—Homeobox A9 (NUP98-HOXA9) Fusion Oncogene Drives Myeloproliferation By Upregulating DNA (Cytosine-5-)-Methyltransferase 1 (dnmt1)

Abstract

We need to better understand the genetic mechanisms that transform normal blood cells into acute myeloid leukaemia (AML). Overall survival is improving for children and adults with AML, but traditional therapies can be very toxic and high-risk forms of disease remain fatal for 2 out of 5 patients. One high-risk indicator is homeobox A9 (HOXA9), a gene necessary for normal blood development. HOXA9 is overexpressed in 80% of AML cases, especially as part of the NUP98-HOXA9 (NHA9) mutation. Our research goal is to improve survival for human AML caused by NHA9, by identifying new contributing genes and less toxic drugs. To do this, a new animal model of disease is required. The zebrafish, Danio rerio, was chosen as a reliable in vivo tool to study leukaemia, thanks to its conserved genetics and cell biology. Compared to mice, zebrafish also support rapid chemical and genetic screening, which is a tremendous asset. We created mutant zebrafish carrying the human NHA9 mutation. We found that 23% of NHA9 adult fish developed a myeloproliferative neoplasm (MPN) by 19 to 23 months of life, which highlights the role of mutant HOXA9 in myeloid disease. In addition, ~80% of NHA9 embryos displayed defects in early blood development. NHA9 decreased gata1a erythroid expression and increased spi1 myeloid expression, which matches the myeloproliferation in adult fish. Of note, the HOX co-factor, MEIS1, is also overexpressed in human AML, and zebrafish meis1 gene knockdown inhibited myeloproliferation in NHA9 embryos. I then leveraged this myeloproliferation phenotype in NHA9 embryos to examine new contributing genes and drugs. Microarray analysis identified high levels of zebrafish dnmt1, a gene that regulates blood cell maturation via epigenetic DNA methylation. Our preliminary evidence suggests that NHA9 is inhibited by dnmt1 gene knockdown or treatment with Decitabine, a demethylating agent. We also looked at the Wnt/?-catenin pathway, which is often hijacked to promote AML. We blocked myeloproliferation in NHA9 embryos by targeting Wnt/?-catenin with Indomethacin, a COX inhibitor. Finally, I transplanted human leukaemia cells into zebrafish for testing new drugs, called prodigiosenes. These drugs reduced the proliferation of leukaemia cells, and were reasonably non-toxic to embryos.