Leukemia (2012) 26, 1517 --1526 & 2012 Macmillan Publishers Limited All rights reserved 0887-6924/12 www.nature.com/leu ORIGINAL ARTICLE Preclinical activity of LBH589 alone or in combination with chemotherapy in a xenogeneic mouse model of human acute lymphoblastic leukemia A Vilas-Zornoza1,13, X Agirre1,13, G Abizanda1,2, C Moreno3, V Segura4, A De Martino Rodriguez5, ES Jose´ -Eneriz1, E Miranda1, JI Martı´n-Subero6, L Garate1, MJ Blanco-Prieto7, JA Garcı´a de Jalo´n5, P Rio8, J Rifo´n2, JC Cigudosa9, JA Martinez-Climent1, J Roma´n-Go´ mez10, MJ Calasanz11, JM Ribera12 and F Pro´ sper1,2 Histone deacetylases (HDACs) have been identified as therapeutic targets due to their regulatory function in chromatin structure and organization. Here, we analyzed the therapeutic effect of LBH589, a class I--II HDAC inhibitor, in acute lymphoblastic leukemia (ALL). In vitro, LBH589 induced dose-dependent antiproliferative and apoptotic effects, which were associated with increased H3 and H4 histone acetylation. Intravenous administration of LBH589 in immunodeficient BALB/ c-RAG2À/ÀgcÀ/À mice in which human-derived T and B-ALL cell lines were injected induced a significant reduction in tumor growth. Using primary ALL cells, a xenograft model of human leukemia in BALB/c-RAG2À/ÀgcÀ/À mice was established, allowing continuous passages of transplanted cells to several mouse generations. Treatment of mice engrafted with T or B-ALL cells with LBH589 induced an in vivo increase in the acetylation of H3 and H4, which was accompanied with prolonged survival of LBH589-treated mice in comparison with those receiving vincristine and dexamethasone. Notably, the therapeutic efficacy of LBH589 was significantly enhanced in combination with vincristine and dexamethasone. Our results show the therapeutic activity of LBH589 in combination with standard chemotherapy in pre-clinical models of ALL and suggest that this combination may be of clinical value in the treatment of patients with ALL. Leukemia (2012) 26, 1517--1526; doi:10.1038/leu.2012.31 Keywords: LBH589; acute lymphoblastic leukemia; epigenetics; histone; mouse model INTRODUCTION ALL suggests that several genetic lesions need to act in concert 1 Although it is tempting to consider acute lymphoblastic leukemia to induce overt leukemia. (ALL) as a curable disease because of the remarkable improve- The classical view of cancer as a genetic disease has been ment in the cure rates observed in recent years, the prognosis of challenged by the clear demonstration that epigenetic modifica- relapsed patients is still dismal.1 Almost 80% of children tions can alter gene expression by mechanisms that do not affect diagnosed with ALL become cured with modern risk-adapted the DNA sequence itself. Epigenetics has an important role in the therapies. However, 460% of adult patients will eventually pathogenesis and prognosis of various tumors. The hypermethyla- relapse and most of them will succumb to their disease. This tion of DNA promoters and changes in histone modification underlies the need for new therapeutic options for resistant patterns are the most frequently described abnormalities in tumor patients. The role of recurrent chromosomal translocations in the cells.6 Along with others, we have extensively demonstrated that pathogenesis of ALL has been clearly established2 providing not patients with ALL frequently show an abnormal hypermethylation only important prognostic information but also guiding the of DNA promoter of tumor-suppressor genes, microRNAs (miRNAs) development of new treatments such as the use of tyrosine- or genes involved in tumorigenic pathways such as WNT or kinase inhibitors (imatinib or dasatinib) in patients with t(9;22).3 Ephrin-Eph pathway, and that these changes are associated with Other genetic alterations such as the overexpression of FLT3 prognosis of the disease.7--12 receptor tyrosine kinase in mixed-lineage leukemia gene-rear- Among the various epigenetic modifiers, histone deacetylases ranged or hyperdiploid B-ALL4 or the presence of NOTCH1- inhibitors (HDACi) have emerged as promising drugs for the activating mutations in T-cell ALL are attractive candidates for treatment of a number of tumors.13 HDACis are a class of targeted therapies with FLT3 or NOTCH inhibitors (g-secretase anticancer agents that inhibit deacetylation of histones and inhibitors).5 Indeed, the current view of the pathogenesis of non-histone cellular proteins, inducing hyperacetylation and an 1Oncology Division, University of Navarra, Pamplona, Spain; 2Hematology Service, Clı´nica Universidad de Navarra, University of Navarra, Pamplona, Spain; 3Department of Immunology, University of Navarra, Pamplona, Spain; 4Department of Bioinformatics, Foundation for Applied Medical Research, University of Navarra, Pamplona, Spain; 5Department of Animal Pathology, Veterinary Faculty, University of Zaragoza, Zaragoza, Spain; 6Department of Anatomic Pathology, Pharmacology and Microbiology, University of Barcelona, Barcelona, Spain; 7Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Navarra, Pamplona, Spain; 8Centro de Investigaciones Energe´ticas, Medioambientales y Tecnolo´gicas (CIEMAT), Madrid, Spain; 9Molecular Cytogenetics Group, Centro Nacional Investigaciones Oncolo´gicas (CNIO), Madrid, Spain; 10Hematology Department, Reina Sofia Hospital, Maimonides Institute for Biomedical Research, Cordoba, Spain; 11Department of Genetics, University of Navarra, Pamplona, Spain and 12Hematology Service, Institut Catala` d’Oncologia, Hospital Universitari Germans Trias i Pujol, Universitat Auto`noma de Barcelona, Badalona, Barcelona, Spain. Correspondence: Dr F Pro´sper, Hematology and Cell Therapy, Clı´nica Universidad de Navarra, Avda, Pı´o XII 36, Pamplona 31008, Spain. E-mail: [email protected] 13These authors are equal first authors. Received 25 April 2011; revised 12 December 2011; accepted 30 January 2012; accepted article preview online 6 February 2012; advance online publication, 16 March 2012 LBH589 in ALL A Vilas-Zornoza et al 1518 ‘open chromatin’ configuration. In cancer, such hyperacetylation analyzed on Affymetrix GeneChip Human mapping 250K SNP arrays (Santa is associated with a greater transcriptional activity of silenced Clara, CA, USA) capable of genotyping on average 250 000 single- tumor-suppressor genes. LBH589 (also called Panobinostat) is an nucleotide polymorphisms according to the manufacturer’s protocol. HDACi characterized as a pan-deacetylase inhibitor with activity Microarray data were analyzed for determination of both total and against both class I and II HDACs. This drug has demonstrated a allelic-specific copy numbers using the Genotyping Console software significant activity against different tumors such as myeloma, (Affymetrix, Santa Clara, CA, USA) as previously described.17 acute myelogenous leukemia and T-cell lymphomas as well as 14,15 breast, prostate, colon and pancreatic cancer. DNA methylation array analysis In this study, we investigate the role of the HDACi LBH589 using A HumanMethylation27 BeadChip (Illumina, Inc., San Diego, CA, USA) was in vitro and in vivo models of ALL, and demonstrate that treatment used to quantify DNA methylation. The panel is developed to quantify the of ALL cells with LBH589 induces a prolonged survival in a mouse methylation status of 27 578 CpG sites located within the proximal model of human ALL. Also, we show that this effect is significantly promoter of 14 475 well-annotated genes from the consensus coding improved when combined with currently active drugs used to sequence project as well as known cancer genes and miRNAs. The protocol treat ALL, providing the basis for using this combination in was performed according to the manufacturer’s instructions. The patients with ALL. methylation status of a CpG was determined by the beta value calculation, which ranges from 0 for unmethylated CpGs to 1 for completely methylated CpGs. MATERIALS AND METHODS Human samples and cell lines Gene expression array analysis Six ALL-derived cell lines TOM-1, REH, 697, s.e.m., TANOUE and MOLT-4, Total RNA from primary cells and tissues was extracted with Ultraspec were purchased from the DSMZ (Deutche Sammlung von Microorganis- (Biotecx, Houston, TX, USA) following the manufacturer’s instructions after men und Zellkulturen GmbH, Braunschweig, Germany). Cell lines were tissue lysates were prepared using a mechanical tissue homogenization by maintained in culture in RPMI 1640 medium supplemented with 10% fetal ultraturrax (IKA, Wilmington, DE, USA). RNA was quantified using NanoDrop bovine serum and with 1% penicillin--streptomycin and 2% HEPES (Gibco- Specthophotometer (NanoDrop Technologies). Gene expression analysis BRL, Verviers, Belgium) at 37 1C in a humid atmosphere containing 5% CO . 2 was performed using GeneChip Human Gene 1.0 ST array (Affymetrix) To generate the mouse model, bone marrow (BM) or peripheral blood (PB) following the manufacturer’s instructions. Microarray data were normalized mononuclear cells were obtained at diagnosis from patients with ALL after and analyzed as previously described.18 signed informed consent had been obtained from the patient or the patient’s guardians, in accordance with the Declaration of Helsinki. TaqMan low-density arrays Reagent and cell drug treatment A TaqMan Low-Density Array A v2.1 (Applied Biosystems, Foster City, CA, USA) was used for 377 human miRNA expression assays after treatment. A The HDACi LBH589 was provided by Novartis Pharmaceuticals (East total concentration of 500 ng of