Leukemia (2014) 28, 680–689 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu ORIGINAL ARTICLE Histone deacetylase 3 as a novel therapeutic target in multiple myeloma J Minami1,4, R Suzuki1,4, R Mazitschek2, G Gorgun1, B Ghosh2,3, D Cirstea1,YHu1, N Mimura1, H Ohguchi1, F Cottini1, J Jakubikova1, NC Munshi1, SJ Haggarty3, PG Richardson1, T Hideshima1 and KC Anderson1 Histone deacetylases (HDACs) represent novel molecular targets for the treatment of various types of cancers, including multiple myeloma (MM). Many HDAC inhibitors have already shown remarkable antitumor activities in the preclinical setting; however, their clinical utility is limited because of unfavorable toxicities associated with their broad range HDAC inhibitory effects. Isoform- selective HDAC inhibition may allow for MM cytotoxicity without attendant side effects. In this study, we demonstrated that HDAC3 knockdown and a small-molecule HDAC3 inhibitor BG45 trigger significant MM cell growth inhibition via apoptosis, evidenced by caspase and poly (ADP-ribose) polymerase cleavage. Importantly, HDAC3 inhibition downregulates phosphorylation (tyrosine 705 and serine 727) of signal transducers and activators of transcription 3 (STAT3). Neither interleukin-6 nor bone marrow stromal cells overcome this inhibitory effect of HDAC3 inhibition on phospho-STAT3 and MM cell growth. Moreover, HDAC3 inhibition also triggers hyperacetylation of STAT3, suggesting crosstalk signaling between phosphorylation and acetylation of STAT3. Importantly, inhibition of HDAC3, but not HDAC1 or 2, significantly enhances bortezomib-induced cytotoxicity. Finally, we confirm that BG45 alone and in combination with bortezomib trigger significant tumor growth inhibition in vivo in a murine xenograft model of human MM. Our results indicate that HDAC3 represents a promising therapeutic target, and validate a prototype novel HDAC3 inhibitor BG45 in MM. Leukemia (2014) 28, 680–689; doi:10.1038/leu.2013.231 Keywords: multiple myeloma; histone deacetylase 3; STAT3; bortezomib INTRODUCTION The natural product romidepsin is a cyclic tetrapeptide with HDAC A major challenge for molecular targeted therapy in multiple inhibitory activity primarily towards class-I HDACs. Other myeloma (MM) is its genetic complexity and molecular hetero- HDACi based on aminobenzamide biasing elements, such as geneity. Gene transcription within the tumor cell and its mocetinostat (MGCD103) and entinostat (MS275), are highly microenvironment can also be altered by epigenetic modulation specific for HDAC1, 2 and 3. Importantly, clinical trials with non- (i.e., acetylation and methylation) in histones, and inhibition of selective HDACi such as vorinostat combined with bortezomib histone deacetylases (HDACs) has therefore emerged as a novel have shown efficacy in MM, but have attendant fatigue, diarrhea targeted treatment strategy in MM and other cancers.1 HDACs are and thrombocytopenia.5 divided into four classes: class-I (HDAC1, 2, 3 and 8), class-IIa Our preclinical studies characterizing the biologic impact of (HDAC4, 5, 7 and 9), class-IIb (HDAC6 and 10), class-III (SIRT1–7) isoform-selective HDAC6 inhibition in MM, using HDAC6 knock- and class-IV (HDAC11). These classes differ in their subcellular down and HDAC6-selective inhibitor tubacin,6 showed that localization (class-I HDACs are nuclear and class-II enzymes combined HDAC6 and proteasome inhibition triggered dual cytoplasmic), and their intracellular targets. Moreover, recent blockade of aggresomal and proteasomal degradation of studies have identified non-histone targets of HDACs in cancer protein, massive accumulation of ubiquitinated protein and cells associated with various functions including gene expression, synergistic MM cell death. On the basis of these studies, a DNA replication and repair, cell cycle progression, cytoskeletal potent and selective HDAC6 inhibitor ACY-1215 (ref. 7) was reorganization and protein chaperone activity. developed, which is now demonstrating promise and tolerability Several HDAC inhibitors (HDACi) are currently in clinical in phase I/II clinical trials in MM.8 In this study, we similarly development in MM,2 and both vorinostat (SAHA) and determine whether isoform inhibition of class-I HDAC mediates romidepsin (FK228 or FR901228) have already received approval cytotoxicity, without attendant toxicity to normal cells. We define by the Food and Drug Administration for the treatment of the role of HDAC3-selective inhibition in MM cell growth and cutaneous T-cell lymphoma.3 Vorinostat is a hydroxamic acid- survival using both lentiviral HDAC3 knockdown and a novel based HDACi that, like other inhibitors of this class including small-molecule HDAC3-selective inhibitor BG45. Within class-I panobinostat (LBH589) and belinostat (PXD101), are generally HDACs, our results show that HDAC3 represents a promising non-selective with activity against class-I, -II and -IV HDACs.4 therapeutic target in MM, and that combined HDAC3 and 1Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA; 2Center for Systems Biology, Massachusetts General Hospital, Boston, MA, USA and 3Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. Correspondence: Dr KC Anderson, Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute-Harvard Medical School, 450 Brookline Avenue, Mayer 557, Boston, MA 02215, USA. E-mail: [email protected] 4These authors contributed equally to this work. Received 3 April 2013; revised 22 July 2013; accepted 25 July 2013; accepted article preview online 5 August 2013; advance online publication, 6 September 2013 HDAC3 is a novel target in myeloma J Minami et al 681 proteasome inhibition mediates synergistic cytotoxicity. Our of transcription 3 (STAT3), -phospho-STAT3 (p-STAT3) (tyrosine 705), -p- studies provide the preclinical rationale for derived clinical trials STAT3 (serine 727), -p21, -Janus kinase 2 (JAK2), -acetylated-lysine and using HDAC3-selective inhibitors to both enhance MM cytotoxicity antiphosphorylated-tyrosine antibodies (Abs; Cell Signaling Technology, and improve tolerability. Beverly, MA, USA). For immunoprecipitation, MM cells were lysed with Nonidet P-40 buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 5 mM ethylenediaminetetraacetic acid, 5 mM NaF, 2 mM Na3VO4,1mM MATERIALS AND METHODS phenylmethylsulfonyl fluoride, 5 mg/ml leupeptin and 5 mg/ml aprotinin). Reagents Whole-cell lysates were incubated with anti-STAT3, -JAK2 and -green Non-selective HDACi LBH589 (panobinostat) and MS275 (entinostat), as fluorescent protein Abs for 2 h at 4 1C, and then incubated with Protein A/G well as HDAC6-selective inhibitor tubastatin-A were purchased from PLUS-Agarose (Santa Cruz Biotechnology, Dallas, TX, USA) overnight at Selleck Chemicals (Houston, TX, USA). Bortezomib was also obtained from 4 1C. Anti-green fluorescent protein Ab served as a control. Immune Selleck Chemicals. BG45 (N-(2-aminophenyl)pyrazine-2-carboxamide) and complexes were analyzed by immunoblotting with anti-STAT3, -JAK2, - Merck60 (4-acetamido-N-(2-amino-5-(thiophen-2-yl)phenyl)benzamide) acetylated-lysine and -phosphorylated-tyrosine Abs. (PMID: 18182289) were synthesized in-house (Massachusetts General Hospital, Cambridge, MA, USA). Human recombinant interleukin (IL)-6 Transfection of short hairpin RNA was purchased from R & D Systems (Minneapolis, MN, USA). HDAC1, HDAC2 and HDAC3 pLKO.1 short hairpin RNA (shRNA) vectors were obtained from the RNA Interference Screening Facility at the Cells Dana-Farber Cancer Institute. Recombinant lentivirus was produced and RPMI8226 and U266 human MM cell lines, as well as human embryonic infection of MM cells was performed as described previously.11 kidney 293T cells, were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). MM.1S cells were kindly provided by Synthesis of a small-molecule HDAC3 inhibitor BG45 Dr Steven Rosen (Northwestern University, Chicago, IL, USA). Interleukin (IL)-6-dependent INA-6 cell line was obtained from Dr Renate Burger The procedure to generate BG45 is demonstrated in Supplementary (University of Kiel, Kiel, Germany). Melphalan- (LR5) and doxorubicin- Figure S2A. resistant (RPMI-DOX40) cells were kindly provided by Dr William Dalton (Lee Moffitt Cancer Center, Tampa, FL, USA). OPM1 and OPM2 cells were Synthesis of tert-butyl (2-aminophenyl)carbamate (2) obtained from Dr Edward Thompson (University of Texas Medical Branch, To a stirring solution of benzene-1,2-diamine (1.0 g, 9.247 mmol) and Galveston, TX, USA). MM cell lines were maintained in RPMI 1640 medium 4-dimethylminopyridine (50 mg) in tetrahydrofuran (20 ml), a solution of (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% fetal bovine di-tert-butyl dicarbonate (1.009 g, 4.6236 mmol) in dichloromethane (20 ml) serum, 2 mML-glutamine (Invitrogen, Carlsbad, CA, USA), 100 U/ml was added dropwise at room temperature. The reaction mixture was penicillin and 100 U/ml streptomycin (Invitrogen). The 293T cells were evaporated in a rotary evaporator and purified by column chromatography maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) using hexane and ethylacetate solvent mixture (80:20) to obtain the supplemented with 10% fetal bovine serum, 100 U/ml penicillin and desired mono-Boc protected compound 2 (0.380 g, 20% yield). 100 mg/ml streptomycin (Invitrogen).