Leukemia (2011) 25, 1891–1899 & 2011 Macmillan Publishers Limited All rights reserved 0887-6924/11 www.nature.com/leu ORIGINAL ARTICLE The novel JAK inhibitor CYT387 suppresses multiple signalling pathways, prevents proliferation and induces apoptosis in phenotypically diverse myeloma cells KA Monaghan1,2, T Khong1,2, CJ Burns3 and A Spencer1,2 1Myeloma Research Group, Malignant Haematology and Stem Cell Transplantation, Alfred Hospital, Melbourne, Victoria, Australia; 2Australian Centre for Blood Diseases, Department of Clinical Haematology, Monash University, Melbourne, Victoria, Australia and 3YM BioSciences Australia, Melbourne, Victoria, Australia Janus kinases (JAKs) are involved in various signalling MM. Some human myeloma cell lines (HMCL) cannot pathways exploited by malignant cells. In multiple myeloma proliferate or survive without exogenous IL-6,9,10 and some (MM), the interleukin-6/JAK/signal transducers and activators of conventional drugs are ineffective in the presence of IL-6.11–13 transcription (IL-6/JAK/STAT) pathway has been the focus of The bone marrow microenvironment, known to provide research for a number of years and IL-6 has an established role 14 in MM drug resistance. JAKs therefore make a rational drug supportive signals to MM cells, produces IL-6; hence, target for anti-MM therapy. CYT387 is a novel, orally bioavail- reducing the pro-survival effect of IL-6 may abrogate the drug- able JAK1/2 inhibitor, which has recently been described. This resistant phenotype of MM. CD45 is a phenotypic marker preclinical evaluation of CYT387 for treatment of MM demon- expressed by some myeloma cells, which has been reported to strated that CYT387 was able to prevent IL-6-induced phos- influence IL-6 responsiveness.15 It is therefore likely that CD45 phorylation of STAT3 and greatly decrease IL-6- and insulin-like growth factor-1-induced phosphorylation of AKT and extra- expression might affect sensitivity to JAK inhibitors. cellular signal-regulated kinase in human myeloma cell lines CYT387 is a novel JAK inhibitor that can inhibit JAK1, JAK2, (HMCL). CYT387 inhibited MM proliferation in a time- and dose- JAK3 and TYK2 kinase activity.16,17 The structure and develop- dependent manner in 6/8 HMCL, and this was not abrogated by ment of the compound has recently been described.18 Several the addition of exogenous IL-6 (3/3 HMCL). Cell cycling was JAK inhibitors are currently in various stages of development and inhibited with a G2/M accumulation of cells, and apoptosis was investigation for use in MM including INCB000020,19 induced by CYT387 in all HMCL tested (3/3). CYT387 synergised 20 21,22 (ref.23) 24 in killing HMCL when used in combination with the conven- INCB16562, AG490, AZD1480 and Pyridone 6, tional anti-MM therapies melphalan and bortezomib. Impor- or for other haematological malignancies such as WP1066 in tantly, apoptosis was also induced in primary patient MM cells AML.25 CYT387 has recently undergone Phase I evaluation, (n ¼ 6) with CYT387 as a single agent, and again synergy was demonstrating safe use of low micromolar concentrations, with seen when combined with conventional therapies. no relevant haematological toxicities evident.26 Given the Leukemia (2011) 25, 1891–1899; doi:10.1038/leu.2011.175; tolerance of CYT387 in patients and the putative role of IL-6 published online 26 July 2011 Keywords: multiple myeloma; CYT387; JAK inhibitors; drug in MM drug resistance, JAK inhibitors are being investigated for resistance; STAT signalling their potential use as a single agent or in combination therapy for MM. Furthermore, preliminary in vitro data has demon- strated the potential of JAK/STAT inhibition to sensitise MM cells to conventional therapies.20,22 Here we show that the JAK inhibitor CYT387 can modulate IL-6-stimulated signalling with- Introduction in HMCL, which can sensitise them to various other anti-MM treatments. CYT387 can inhibit proliferation and disrupt the cell Multiple myeloma (MM) is an incurable drug-resistant clonal cycle of MM cells. Finally, CYT387 induces apoptosis as a single B cell malignant neoplasm localised to the bone marrow. Janus agent and synergises with melphalan and bortezomib, when kinases (JAKs) are well-characterised signalling kinases compris- used against either HMCL or primary MM tumour cells. ing four family members JAK1, JAK2, JAK3 and TYK2 that are important in haematological malignancy, as JAK mutations have Materials and methods been shown to contribute to the pathogenesis of both 1–3 4 myeloproliferative disorders and leukaemias. JAKs have an Reagents established role in signalling for many cells (reviewed by Rane 5 The JAK1/2 inhibitor CYT387 was kindly provided by YM and Reddy ), and in MM, JAKs are activated by a variety of BioSciences Australia (Melbourne, Victoria, Australia) and cytokines including interleukin-6 (IL-6),6,7 interferon-a6,8 and 6 dissolved in dimethyl sulfoxide. The proteasome inhibitor epidermal growth factor. Many pathways downstream of JAKs bortezomib (Janssen-Cilag, North Ryde, New South Wales, are exploited by malignant cells; the role of IL-6 and its Australia) was reconstituted in saline. The alkylating agent subsequent activation of JAK/signal transducers and activators of melphalan (Sigma-Aldrich, Sydney, New South Wales, Australia) transcription (STAT) is possibly the most studied pathway in was dissolved in 0.5% HCl.EtOH. All stock drug solutions were diluted in complete RPMI-1640 culture medium to various Correspondence: Professor A Spencer, Myeloma Research Group, concentrations for experimentation. Malignant Haematology and Stem Cell Transplantation, Alfred Hospital and Australian Centre for Blood Diseases, Monash University, AMREP, Commercial Road, Prahran, Victoria, 3181, Australia. E-mail: [email protected] Cell lines and culture conditions Received 2 June 2011; accepted 21 June 2011; published online 26 HMCL LP-1, NCI-H929, OPM2, RPMI-8226 and U266, and the July 2011 human stromal cell line HS5, were obtained from the American Anti-myeloma activity of the JAK inhibitor CYT387 KA Monaghan et al 1892 Type Culture Collection (Manassas, VA, USA). ANBL-6, for 60 min then incubated with mouse monoclonal anti- OCI-MY1 and XG-1 were a kind gift from the Winthrop P phospho-STAT3 (pY705, Santa Cruz, ThermoFisher Scientific, Rockefeller Cancer Institute (Little Rock, AR, USA). HMCL were Scoresby, Victoria, Australia), mouse monoclonal anti-STAT3 grown and treated at densities between 2.0 and 5.0 Â 105 cells/ (Santa Cruz) or mouse monoclonal anti-a-tubulin (Sigma- ml in RPMI-1640 media (Gibco, Invitrogen, Mulgrave, Victoria, Aldrich) for 1–2 h at room temperature or overnight at 4 1C. Australia) supplemented with 10% heat-inactivated foetal The blots were washed three times for 15 min in 0.1% Tween- bovine serum (Lonza, Mt Waverley, Victoria, Australia) and 20/PBS, then incubated with secondary horseradish peroxidase 2mML-glutamine (Gibco, Invitrogen). IL-6-dependent cell lines tagged antibody (swine anti-rabbit Ig HRP or rabbit anti-mouse were cultured with 2–5 ng/ml IL-6 as required. All cells were Ig HRP (Dako, Campbellfield, Victoria, Australia)) for 1–2 h at cultured in a humidified incubator at 37 1C with 5% CO2. All room temperature before washing as above. Blots were HMCL were passaged 24 h before the experimental setup to visualised with Supersignal west pico ECL reagents (Pierce, ensure high viability and cycling. ThermoFisher Scientific). Primary samples Intracellular FACS Primary MM samples were obtained from bone marrow Activation of the JAK/STAT, PI3K/AKT and Ras/MAPK pathways aspirates from relapsed and refractory MM patients, following was investigated using intracellular flow cytometry to measure written informed consent with approval from the Alfred Hospital the phosphorylation of STAT3 at tyrosine 705 (p-STAT3), AKT at Research and Ethics Committee, and isolated and treated as serine 473 (p-AKT), and extracellular signal-regulated kinase previously described.27 Briefly, bone marrow mononuclear cells (ERK)1/2 at threonine 202 and tyrosine 204 (p-ERK). HMCL were (BMMC) were isolated with Ficoll-Paque Plus (Amersham stimulated alone with 10 ng/ml IL-6±200 ng/ml insulin-like Biosciences, Rydalmere, New South Wales, Australia), washed growth factor-1 (IGF-1) or stimulated in CC with HS5 stromal in phosphate-buffered saline (PBS), and red blood cells were cells or primary BMSC, with or without CYT387 treatment. For lysed with NH Cl solution (8.29 g/l ammonium chloride, 4 CC, HS5 and primary BMSC were seeded into a 24-well plate at 0.037 g/l ethylene diamine tetraacetic acid, 1 g/l potassium 2 Â 105 cells/ml and allowed to establish for 4 h, after which bicarbonate). Cells were then washed again in PBS and HMCL prestained with CD38 or CD138 FITC (BD) were added. quantitated by haemocytometer. BMMC samples were then MM cells were stimulated alone (10 ng/ml IL-6, or 5 ng/ml IL-6 cultured in complete RPMI-1640 media (as above for HMCL) for and 100 ng/ml IGF-1), or in CC (direct CC with stroma or 24 h. Subsequently, the BMMC were plated at 5 Â 105 cells/ml transwell CC with stroma), with or without either 60 min of and were treated with CYT387 (5–50 mM), alone or (dependant CYT387 pretreatment or 15 min CYT387 co-treatment. After on cell numbers) in combination with bortezomib (5–40 nM)or stimulation±treatment, MM cells were harvested and fixed with melphalan (50–200 mM) for 24 and/or 48 h. Drug-induced 2% paraformaldehyde for 10–30 min, washed, then permeabi- MM-specific cell apoptosis was then compared with untreated lised with methanol overnight. Methanol was washed off and and vehicle controls by staining for CD45 FITC (BD, North the cells were resuspended in p-STAT3 PE (BD), p-AKT PE (BD) Ryde, New South Wales, Australia), CD38 PerCP-Cy5.5 (BD) or p-ERK (BD) and stained for 45–60 min at room temperature.