NUP98-HOXA9 drives High-Risk Myeloid Disease in Zebrafish – An In Vivo platform to Study Interacting Genes and Perform Drug Discovery,

Abstract

Abstract 3622High-risk forms of acute myeloid leukemia (AML) remain fatal for 40% of patients, both due to refractory disease and toxicity from traditional chemotherapeutic agents. Using zebrafish, we aim to discover new or repurposed drugs to combat high-risk leukemia that are less toxic and achieve better survival for human patients.The NUP98-HOXA9 (NHA9) mutation is a hallmark of high-risk AML, characterized by a failure of granulocyte maturation and increased cell division. The zebrafish is a robust model to study blood development and leukemia, with the inherent capacity to easily perform drug screens in embryos. We created an AML disease model using transgenic zebrafish that harbor the NHA9 mutation (Forrester et al., Br J Haematol, Aug 2 Epub). We are using these living models of cancer to study the genetic interactions that promote high-risk leukemia.Our model has already provided novel insights into the direct mechanisms of NHA9 oncogenesis. In response to DNA-damage, NHA9 failed to initiate cell cycle arrest and apoptosis, likely through upregulation of bcl2 rather than direct suppression of tumor protein 53 (tp53) signaling. Upon exposure to 16 Gy ionizing radiation, wild-type embryos displayed a 4.16-fold decrease of phospho-histone-H3 (pH3)-labeled cells, whereas NHA9 embryos only suffered a 1.63-fold decrease (relative difference of 2.55-fold, P <0.005). We also found that NHA9 blocked the mitochondrial apoptosis response, with loss of caspase-3 activation (6.31-fold decrease, P <0.005). Both whole mount in situ hybridization (WISH) and qRT-PCR detected upregulation of proto-oncogenes, bcl2 and bcl2l1/bcl-xL in NHA9 embryos (3.79-fold, P <0.00005, and 2.07-fold, P <0.0005, respectively), particularly in the hematopoietic posterior blood island (PBI), but normal expression levels of canonical p53 target genes, bbc3/puma and cdkn1a/p21waf1/cip1.NHA9 also perturbs blood cell development. In embryos, NHA9 produced a loss of red blood cells (gata1a, decreased expression 19.34-fold by qRT-PCR, P <0.00005) and an increase of immature myeloid cells (spi1/pu.1, increased expression 2.79-fold by WISH, P <0.005), but no change in cell division (by BrdU incorporation and pH3 labeling). Between 19 to 23 months of age, nearly 25% of NHA9 fish developed a myeloproliferative neoplasm (MPN) in the kidney, which is the site of blood development in adult zebrafish. Affected kidneys show invasion of proliferating myeloid cells that stain positive with periodic acid-Schiff (PAS). This pathology is similar to NHA9 mice, which develop a long latency, polyclonal MPN, prior to onset of AML. However, frank leukemia was not observed in our NHA9 fish, suggesting the need for additional genetic “hits” to achieve malignant progression. We are investigating the interactions of NHA9 with β-catenin and Meis1.The cell division gene, β-catenin, has recently proven important for driving AML in mice, but co-operation with NHA9 is unknown. We are contrasting ‘stimulated’ fish (NHA9 + β-catenin, using 20 μM prostaglandin E2 [PGE2]) with ‘inhibited’ fish (NHA9 – β-catenin, using 20 μM indomethacin) to measure the combined effects of NHA9 and β-catenin on cell division and blood development. Similarly, overexpression of Meis1 accelerates the onset of NHA9 -induced leukemia in mice, but the underlying mechanism is unknown. We show that morpholino (MO) knockdown of zebrafish meis1 limits the myeloproliferative effects in NHA9 embryos by blocking myeloid cell development in the PBI (83% decreased expression of the myeloid marker, l-plastin /lcp1). Microarray analysis on these various cohorts of embryos will help to identify a subset of genes that are regulated by NHA9, β-catenin, and Meis1 together. These studies will help determine how normal blood cells are transformed into leukemia cells.Our ultimate goal is to use these NHA9 fish to discover new drugs that combat high-risk leukemia. Our model provides an excellent opportunity to perform chemical library screens for compounds that restore normal levels of red blood cells (measured by in situ for gata1a) in NHA9 embryos. We will present results of screening with compounds contained in the Biomol ICCB 480 library. Promising candidates may represent future therapies against NHA9 in human patients with AML. Disclosures:No relevant conflicts of interest to declare.