Epigenetic Therapy Restores Normal Hematopoiesis in a Zebrafish Model

Leukemia (2015) 29, 2086–2097 © 2015 Macmillan Publishers Limited All rights reserved 0887-6924/15 www.nature.com/leu ORIGINAL ARTICLE Epigenetic therapy restores normal hematopoiesis in a zebrafish model of NUP98–HOXA9-induced myeloid disease AP Deveau1,2,15, AM Forrester1,2,15, AJ Coombs2,3, GS Wagner2,3, C Grabher4,5, IC Chute6, D Léger6, M Mingay7, G Alexe4,8, V Rajan1,2, R Liwski9,10, M Hirst7,11, K Steigmaier4,8, SM Lewis1,6,12,13, AT Look4 and JN Berman1,2,10,14 Acute myeloid leukemia (AML) occurs when multiple genetic aberrations alter white blood cell development, leading to hyperproliferation and arrest of cell differentiation. Pertinent animal models link in vitro studies with the use of new agents in clinical trials. We generated a transgenic zebrafish expressing human NUP98–HOXA9 (NHA9), a fusion oncogene found in high-risk AML. Embryos developed a preleukemic state with anemia and myeloid cell expansion, and adult fish developed a myeloproliferative neoplasm (MPN). We leveraged this model to show that NHA9 increases the number of hematopoietic stem cells, and that oncogenic function of NHA9 depends on downstream activation of meis1, the PTGS/COX pathway and genome hypermethylation through the DNA methyltransferase, dnmt1. We restored normal hematopoiesis in NHA9 embryos with knockdown of meis1 or dnmt1, as well as pharmacologic treatment with DNA (cytosine-5)-methyltransferase (DNMT) inhibitors or cyclo-oxygenase (COX) inhibitors. DNMT inhibitors reduced genome methylation to near normal levels. Strikingly, we discovered synergy when we combined sub-monotherapeutic doses of a histone deacetylase inhibitor plus either a DNMT inhibitor or COX inhibitor to block the effects of NHA9 on zebrafish blood development. Our work proposes novel drug targets in NHA9-induced myeloid disease, and suggests rational therapies by combining minimal doses of known bioactive compounds. Leukemia (2015) 29, 2086–2097; doi:10.1038/leu.2015.126 INTRODUCTION in vivo studies to identify novel gene collaborators or to verify new Current treatment strategies for acute myeloid leukemia (AML) are drug discoveries. aggressive and use nonspecific cytotoxic drugs.1,2 These regimens In mouse models and human cell culture, overexpression of have led to modest improvements in survival over the last 10 either HOXA9 or NHA9 increases the number of hematopoietic stem cells (HSCs), suppresses myeloid differentiation and main- years, but are often associated with substantial morbidity and risk 10–13 of induction-related death.3,4 In the current genomic era, efforts tains survival of progenitor cells. NHA9 induces a preleukemic are underway to develop more rational therapeutic strategies by myeloproliferative neoplasm (MPN) in mouse models, in contrast to the myelodysplastic syndrome (MDS) seen in humans harboring targeting specific molecular abnormalities with small molecule NHA9.14 Co-overexpression of Hoxa9 and Meis1 in mice produces inhibitors improving efficacy and reducing toxicity. reduced latency over Hoxa9 alone (3 vs 8 months).15 Meis1 One particularly high-risk group of patients harbor the NUP98- overexpression also accelerates the progression to overt AML in HOXA9 (NHA9) fusion oncogene. NHA9 results from a t(7;11)(p15; NHA9-expressing mice, but maintains the preleukemic MPN p15) chromosomal translocation that fuses nucleoporin 98 kDa phase.16 Thus, HOXA9 and NHA9 appear to rely on multiple (NUP98) to homeobox A9 (HOXA9), an essential gene for 5 6 collaborating mutations to induce AML. However, other than hematopoiesis, leading to overexpression of the latter. Over MEIS1, few candidate gene or pathways have been identified. 80% of human AML cases show overexpression of HOXA9, and The zebrafish, Danio rerio, has been firmly established as a frequent upregulation of its cofactor, myeloid ecotropic integra- reliable in vivo tool for modeling human leukemia,17,18 with ease 7 tion site 1 (MEIS1). HOXA9 overexpression is the single most of genetic interrogation and inherent capacity for drug important predictor of treatment failure using traditional screening.19,20 We previously created a transgenic zebrafish chemotherapy.8 Moreover, the NHA9 oncogene confers inferior expressing human NHA9 downstream of a Cre/lox-inducible prognosis in de novo and treatment-related AML, and progression cassette under the 9.1-kb zebrafish spi1 myeloid promoter.21 to blast crisis in chronic myelogenous leukemia (CML).9 Targeted Nearly 25% of adult NHA9 fish develop MPN between 19 and therapies are needed, but there has been a relative paucity of 23 months of age. In embryos, NHA9 disrupts early blood 1Department of Microbiology & Immunology, Dalhousie University, Halifax, Nova Scotia, Canada; 2Department of Pediatrics, IWK Health Centre, Halifax, Nova Scotia, Canada; 3Department of Marine Biology, Dalhousie University, Halifax, Nova Scotia, Canada; 4Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; 5Institute of Toxicology & Genetics, Karlsruhe Institute of Technology, Karlsruhe, Germany; 6Department of Biology, University of New Brunswick, Saint John, New Brunswick, Canada; 7Department of Microbiology & Immunology, Centre for High-Throughput Biology, University of British Columbia, Vancouver, British Columbia, Canada; 8Harvard Medical School, Boston, MA, USA; 9Department of Pathology, Queen Elizabeth II Health Science Centre, Halifax, Nova Scotia, Canada; 10Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada; 11Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, British Columbia, Canada; 12Department of Chemistry & Biochemistry, Université de Moncton, Moncton, New Brunswick, Canada; 13Atlantic Cancer Research Institute, Moncton, New Brunswick, Canada and 14Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada. Correspondence: Dr JN Berman, Berman Lab N103, Department of Pediatrics, Life Sciences Research Institute, Dalhousie University, 1348 Summer Street, Halifax, Nova Scotia B3H 4R2, Canada. E-mail: [email protected] 15These authors contributed equally to this work. Received 21 November 2014; revised 7 April 2015; accepted 22 April 2015; accepted article preview 28 May 2015; advance online publication, 3 July 2015 Epigenetic therapy blocks NHA9-mediated disease AP Deveau et al 2087 development with a gain in myeloid cells at the expense of Quantitative reverse-transcription PCR erythroid cells, with evidence of arrested myeloid differentiation. Embryos were heat-shocked at 24 h.p.f., grown to 28 h.p.f., then stabilized Here we show that NHA9 cooperates with zebrafish meis1 and with RNAlater (Ambion; Carlsbad, CA, USA). RNA was extracted using expands the number of early HSCs, which confirms the shared RNeasy Mini Kit, cDNA synthesized using QuantiTect Reverse Transcription features of our fish with previously studied mammalian models. Kit and quantitative reverse-transcription PCR (qRT-PCR) performed using We leveraged the blood developmental phenotypes in transgenic QuantiFast SYBR Green PCR Kit (QIAGEN; Valencia, CA, USA) with fi Stratagene Mx3000P QPCR thermocycler (Agilent; Santa Clara, CA, USA). zebra sh and found that NHA9 upregulates ptgs2 (prostaglandin Quantification used the 2 − ΔΔCt method (details in Supplementary synthase 2), which encodes a cyclo-oxygenase (COX) enzyme Methods). All gene expression studies included a minimum of three 22,23 isoform. Blocking this pathway with COX inhibitors restores independent experiments. normal hematopoiesis in our NHA9 embryos, which is in keeping with recent studies in AML1-ETO zebrafish20 and Hoxa9;Meis1 24 Fluorescence-activated cell sorting mice. Furthermore, using an unbiased microarray-based strat- Groups of 30–50 embryos were dissociated to a single cell suspension egy, we discovered that NHA9 activity depends on the over- (described by Covassin et al., 2009) and analyzed by FACSAriaIII (BD expression of DNA (cytosine-5)-methyltransferase 1 (dnmt1), which Biosciences; Mississauga, ON, Canada) at 4 °C. GFP+ stem cells were excited reveals a direct link between NHA9 and epigenetic regulation for using 488-nm emission and gated for the unique ‘GFP-low’ group. the first time. Finally, we demonstrate the use of synergetic drug Statistical analysis was done using the student’s t-test. combinations by combining submonotherapeutic doses of DNMT or COX inhibitors with histone deacetylase (HDAC) inhibitors to Drug treatments restore normal blood development in NHA9 embryos. Embryos were treated with either 0.3% dimethylsulfoxide (DMSO), or indicated doses of decitabine (DAC), zebularine (Zeb), valproic acid sodium salt (VPA), trichostatin A (TSA), indomethacin (Indo) or NS-398 (all MATERIALS AND METHODS compounds from Sigma-Aldrich; St Louis, MO, USA). Details of dose – fi optimization can be found in Supplementary Methods. Groups of 25 30 Zebra sh husbandry embryos were arrayed into six-well plates with 6 ml pure E3 medium. Zebrafish were maintained, bred and developmentally staged according to Compounds were added, plates were immediately heat-shocked, and then Westerfield.25 The Dalhousie University Animal Care Committee approved incubated at 28 °C until fixation. use of zebrafish (protocol no. 13–129). AB zebrafish were used as wild-type (WT) controls. Reporter lines Tg(cd41::eGFP) and Tg(runx1::eGFP) were gifts Microarray experiments from Drs Robert Handin (Brigham and Women’s Hospital, Boston, MA, USA) and Phil Crosier (University of Auckland, NZ), respectively. RNA was extracted using Trizol+RNeasy Mini Kit, then reverse

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