Leukemia (2007) 21, 2519–2526 & 2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00 www.nature.com/leu ORIGINAL ARTICLE Biological pathways and in vivo antitumor activity induced by Atiprimod in myeloma P Neri1,2,3, P Tassone1,2,3, M Shammas1, H Yasui2, E Schipani4, RB Batchu1, S Blotta1,2,3, R Prabhala1, L Catley2, M Hamasaki2, T Hideshima2, D Chauhan2, GS Jacob5, D Picker5, S Venuta3, KC Anderson2 and NC Munshi1,2 1Jerome Lipper Multiple Myeloma Center, Department of Adult Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA; 2Boston VA Healthcare System, Department of Medicine, Harvard Medical School, MA, USA; 3Department of Experimental and Clinical Medicine, University of ‘Magna Græcia’ and Cancer Center, Catanzaro, Italy; 4Endocrine Unit, Massachusetts General Hospital, Boston, MA, USA and 5Callisto Pharmaceuticals Inc., New York, NY, USA Atiprimod (Atip) is a novel oral agent with anti-inflammatory tion.7,8 Atip inhibits the inflammatory response and preserves properties. Although its in vitro activity and effects on signaling bone integrity in murine models of rheumatoid arthritis (RA),9–12 in multiple myeloma (MM) have been previously reported, here targets macrophages, inhibits phospholipase A and C in rat we investigated its molecular and in vivo effects in MM. Gene 13,14 expression analysis of MM cells identified downregulation of alveolar macrophages and exhibits antiproliferative and 15–17 genes involved in adhesion, cell-signaling, cell cycle and bone antiangiogenic activities in human cancer models. Impor- morphogenetic protein (BMP) pathways and upregulation of tantly, we have previously reported that Atip inhibits MM cell genes implicated in apoptosis and bone development, follow- growth, induces caspase-mediated apoptosis, blocks the phos- ing Atip treatment. The pathway analysis identified integrin, phorylation of Jak2/STAT3 and NF-kB, and downregulates the TGF-b and FGF signaling as well as Wnt/b-catenin, IGF1 and 16,17 cell-cycle regulation networks as being most modulated by Atip antiapoptotic proteins BCL-2, Bcl-Xl and Mcl-1 in MM cells. treatment. We further evaluated its in vivo activity in three To further explore the anti-MM potential of Atip, we here mouse models. The subcutaneous model confirmed its in vivo investigate the specific signaling pathways perturbated by Atip activity and established its dose; the SCID-hu model using INA- in IL-6 dependent (INA-6) as well as independent (OPM1) MM 6 cells, confirmed its ability to overcome the protective effects cells, and evaluate the in vivo activity of this agent in three of BM milieu; and the SCID-hu model using primary MM cells different SCID mouse models of human MM that provides a reconfirmed its activity in a model closest to human disease. Finally, we observed reduced number of osteoclasts and preclinical framework for its clinical evaluation in MM patients. modulation of genes related to BMP pathways. Taken together, these data demonstrate the in vitro and in vivo antitumor activity of Atip, delineate potential molecular targets triggered Materials and methods by this agent, and provide a preclinical rational for its clinical evaluation in MM. Drug Leukemia (2007) 21, 2519–2526; doi:10.1038/sj.leu.2404912; Atip was provided by Callisto Pharmaceuticals Inc. (New York, published online 20 September 2007 Keywords: atiprimod; multiple myeloma; SCID-hu model; NY, USA) as powder, and was dissolved in PBS (GIBCO, Grand experimental treatment; gene expression profile; bone effects Island, NY, USA) at a final concentration of 10 mM and then stored in aliquots at À201 until use, to avoid multiple freeze– thaw cycles. Introduction MM cell lines and primary patients cells OPM1 and INA-6 human MM cell lines were kindly provided by Multiple myeloma (MM) is a B-cell malignancy characterized by Dr Edward Thompson (University of Texas Medical Branch, clonal accumulation of malignant plasma cells in bone marrow Galveston, TX, USA) and Burger18 (University of Erlangen- (BM). The BM microenvironment plays a critical role in Nuernberg, Erlangen, Germany), respectively. The IL-6 inde- promoting MM cell growth, survival, migration and develop- pendent OPM1 was cultured in RPMI 1640 media (GIBCO) ment of drug resistance.1 These findings have provided a containing 10% fetal bovine serum, 2 mML-glutamine (GIBCO), framework for novel therapeutic strategies targeting both tumor À1 À1 1,2 100 U ml penicillin and 100 mgml streptomycin (GIBCO). cells and their BM microenvironment. However, despite The IL-6 dependent INA-6 cell line was cultured in the presence recent advances in the understanding of MM biology and the of 2.5 ng mlÀ1 of human recombinant IL-6 (R&D Systems Inc., availability of new agents, such as thalidomide,3 revlimid4 and 5 6 Minneapolis, MN, USA). BM aspirates were obtained from MM bortezomib, which have improved responses and survival, patients following informed consent, subjected to Ficoll– MM remains incurable and novel treatments are urgently Hypaque density gradient centrifugation and mononuclear cells needed. Atiprimod (Atip) is an Azaspirane cationic amphiphilic were separated and suspended in RPMI 1640 media (GIBCO) compound (N-N-diethyl-8,8-dipropyl-2-azaspiro [4.5] decane- containing 20% fetal bovine serum, 2 mML-glutamine (GIBCO), 2-propanamine), with anti-inflammatory activity in a number of 100 U mlÀ1 penicillin and 100 mgmlÀ1 streptomycin (GIBCO). experimental models of autoimmune disease and transplanta- Correspondence: Dr NC Munshi, Jerome Lipper Multiple Myeloma Cell proliferation assay Center, Department of Adult Oncology, Dana-Farber Cancer Institute, DNA synthesis was measured by tritiated thymidine uptake [3H- 44 Binney Street, Boston, MA 02115, USA. E-mail: [email protected] TdR] (NEN Life Science Products, Boston, MA, USA). Briefly, 4 Received 29 April 2007; revised 17 July 2007; accepted 20 July 2007; MM cells (2  10 cells per well) were incubated in 96-well published online 20 September 2007 culture plates (Costar, Cambrige, MA, USA) in the presence of Biological pathways and in vivo anti-MM activity of Atiprimod P Neri et al 2520 different concentrations of Atip for 24 or 48 h at 37 1C. Cells USA), which is able to explore the most relevant networks of were pulsed with 3H-TdR (0.5 mCi (0.185 MBq) per well), gene interactions from experimental data. A cutoff of 2 was set harvested onto glass filter with an automatic cell harvester to identify genes with significant regulation (Focus Genes, (Cambridge Technology, Cambridge, MA, USA) and uptake Ingenuity Pathways analysis, 2003 release, Ingenuity Systems). measured using the LKB b-plate scintillation counter (Wallac, The use of this stringent statistical threshold of 2, comparing Gaithersburg, MD, USA). All experiments were performed in treated versus control MM cells resulted in the identification of triplicates. 1357 genes for INA-6 and 1372 for OPM1. The identified genes were mapped to all networks available in the Ingenuity database and were then ranked by score. The score is the probability that Colorimetric survival assay a collection of genes equal to or greater than the number in a Colorimetric assays were also performed to evaluate cell network could be achieved by chance alone. Genes with survival using a tetrazolium assay (CellTiter 96 Non-Radioactive maximum score are the most significantly modulated. Pathways Cell Proliferation Assay; Promega, Madison, WI, USA). MM cells with the higher score of 38 for INA-6 and 37 for OPM1 have 4 (1  10 per well) were incubated in 96-well plates in 100 ml been used to form a composite network, representing the RPMI media containing 10% fetal bovine serum, L-glutamine underlying biology of the process. and antibiotics and treated as indicated. At the end of each treatment, cells were incubated with 150 ml dye solution and incubated at 37 1C for 4 h. A solubilization/stop solution was In vivo activity: animals then added to each well under vigorous pipetting to dissolve the CB-17 SCID-mice were obtained from Taconic (Germantown, formazan crystals. Absorbance readings at a wavelength of a NY, USA), maintained and monitored in our animal research 570 nm were obtained with a spectrophotometer (Molecular facility. All animal studies were conducted according to Devices, Sunnyvale, CA, USA), and cell viability was estimated protocols approved by the Institutional Animal Care and Use as percentage of untreated controls. Committee. Animals were killed when their tumors reached 2 cm in diameter or when paralysis or major compromise in Gene expression and microarray data analysis their quality of life occurred. INA-6 and OPM1 cells (2  106) were exposed to Atip at the IC dose for 24 h. Total RNA was isolated utilizing an ‘Rneasy’ 50 Human MM xenograft murine model kit (Qiagen Inc., Valencia, CA, USA) and gene expression profile CB 17 SCID-mice were subcutaneously (s.c.) inoculated in the was evaluated using the HG-U133A array chip (Affymetrix, interscapular area with 2.5  106 OPM1 cells in 100 ml of RPMI- Santa Clara, CA, USA), representing approximately 33 000 1640 medium. When the tumor was measurable, approximately human genes. GeneChip arrays were scanned on a Gene Array 3 weeks after the MM cells injection, mice were treated with Scanner (Affymetrix). Normalization of arrays and calculation of Atip intraperitoneally (i.p.) at 20, 30 and 50 mg kgÀ1 on alternate expression value were performed using the DNA-Chip Analy- days for 7 days. Tumor size was measured every 3 days in two zer.19 The Invariant Set Normalization method was used to dimensions using an electronic caliper, and the tumor volume normalize arrays at probe level to make them comparable, and was calculated using the following formula: V ¼ 0.5a  b2, the model-based method was used for probe selection and to where a and b are the long and short diameter of the tumor, compute expression values.19 These expression levels were respectively.