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 pathways exploited by malignant cells. In multiple myeloma (MM), the interleukin-6/JAK/signal transducers and activators of transcription (IL-6/JAK/STAT) pathway has been the focus of research for a number of years and IL-6 has an established role in MM drug resistance. JAKs therefore make a rational drug target for anti-MM therapy. CYT387 is a novel, orally bioavail- able JAK1/2 inhibitor, which has recently been described. This preclinical evaluation of CYT387 for treatment of MM demon- strated that CYT387 was able to prevent IL-6-induced phos- phorylation of STAT3 and greatly decrease IL-6- and insulin-like growth factor-1-induced phosphorylation of AKT and extra- cellular signal-regulated kinase in human myeloma cell lines (HMCL). CYT387 inhibited MM proliferation in a time- and dose- dependent manner in 6/8 HMCL, and this was not abrogated by the addition of exogenous IL-6 (3/3 HMCL). Cell cycling was inhibited with a G2/M accumulation of cells, and apoptosis was induced by CYT387 in all HMCL tested (3/3). CYT387 synergised in killing HMCL when used in combination with the conven- tional anti-MM therapies melphalan and bortezomib. Impor- tantly, apoptosis was also induced in primary patient MM cells (n 6) with CYT387 as a single agent, and again synergy was seen when combined with conventional therapies.
Leukemia (2011) 25, 1891–1899; doi:10.1038/leu.2011.175;
published online 26 July 2011
Keywords: multiple myeloma; CYT387; JAK inhibitors; drug resistance; STAT signalling
Introduction
Multiple myeloma (MM) is an incurable drug-resistant clonal B cell malignant neoplasm localised to the bone marrow. Janus kinases (JAKs) are well-characterised signalling kinases compris- ing four family members JAK1, JAK2, JAK3 and TYK2 that are important in haematological malignancy, as JAK mutations have been shown to contribute to the pathogenesis of both myeloproliferative disorders1–3 and leukaemias.4 JAKs have an established role in signalling for many cells (reviewed by Rane and Reddy5), and in MM, JAKs are activated by a variety of cytokines including interleukin-6 (IL-6),6,7 interferon-a6,8 and epidermal growth factor.6 Many pathways downstream of JAKs are exploited by malignant cells; the role of IL-6 and its subsequent activation of JAK/signal transducers and activators of transcription (STAT) is possibly the most studied pathway in
Correspondence: Professor A Spencer, Myeloma Research Group, 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]
Received 2 June 2011; accepted 21 June 2011; published online 26
July 2011
MM. Some human myeloma cell lines (HMCL) cannot proliferate or survive without exogenous IL-6,9,10 and some conventional drugs are ineffective in the presence of IL-6.11–13 The bone marrow microenvironment, known to provide supportive signals to MM cells, produces IL-6;14 hence, reducing the pro-survival effect of IL-6 may abrogate the drug- resistant phenotype of MM. CD45 is a phenotypic marker expressed by some myeloma cells, which has been reported to influence IL-6 responsiveness.15 It is therefore likely that CD45 expression might affect sensitivity to JAK inhibitors.
CYT387 is a novel JAK inhibitor that can inhibit JAK1, JAK2, JAK3 and TYK2 kinase activity.16,17 The structure and develop- ment of the compound has recently been described.18 Several JAK inhibitors are currently in various stages of development and investigation for use in MM including INCB000020,19 INCB16562,20 AG490,21,22 AZD1480(ref.23) and Pyridone 6,24
or for other haematological malignancies such as WP1066 in AML.25 CYT387 has recently undergone Phase I evaluation, demonstrating safe use of low micromolar concentrations, with no relevant haematological toxicities evident.26 Given the tolerance of CYT387 in patients and the putative role of IL-6 in MM drug resistance, JAK inhibitors are being investigated for 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- in HMCL, which can sensitise them to various other anti-MM treatments. CYT387 can inhibit proliferation and disrupt the cell cycle of MM cells. Finally, CYT387 induces apoptosis as a single agent and synergises with melphalan and bortezomib, when used against either HMCL or primary MM tumour cells.
Materials and methods
Reagents
The JAK1/2 inhibitor CYT387 was kindly provided by YM BioSciences Australia (Melbourne, Victoria, Australia) and dissolved in dimethyl sulfoxide. The proteasome inhibitor bortezomib (Janssen-Cilag, North Ryde, New South Wales, Australia) was reconstituted in saline. The alkylating agent melphalan (Sigma-Aldrich, Sydney, New South Wales, Australia) was dissolved in 0.5% HCl.EtOH. All stock drug solutions were diluted in complete RPMI-1640 culture medium to various concentrations for experimentation.
Cell lines and culture conditions
HMCL LP-1, NCI-H929, OPM2, RPMI-8226 and U266, and the
human stromal cell line HS5, were obtained from the American
Type Culture Collection (Manassas, VA, USA). ANBL-6,
OCI-MY1 and XG-1 were a kind gift from the Winthrop P Rockefeller Cancer Institute (Little Rock, AR, USA). HMCL were grown and treated at densities between 2.0 and 5.0 × 105 cells/
ml in RPMI-1640 media (Gibco, Invitrogen, Mulgrave, Victoria,
Australia) supplemented with 10% heat-inactivated foetal bovine serum (Lonza, Mt Waverley, Victoria, Australia) and 2 mM L-glutamine (Gibco, Invitrogen). IL-6-dependent cell lines were cultured with 2–5 ng/ml IL-6 as required. All cells were cultured in a humidified incubator at 37 1C with 5% CO2. All
HMCL were passaged 24 h before the experimental setup to
ensure high viability and cycling.
Primary samples
Primary MM samples were obtained from bone marrow aspirates from relapsed and refractory MM patients, following written informed consent with approval from the Alfred Hospital Research and Ethics Committee, and isolated and treated as previously described.27 Briefly, bone marrow mononuclear cells (BMMC) were isolated with Ficoll-Paque Plus (Amersham Biosciences, Rydalmere, New South Wales, Australia), washed in phosphate-buffered saline (PBS), and red blood cells were lysed with NH4Cl solution (8.29 g/l ammonium chloride,
0.037 g/l ethylene diamine tetraacetic acid, 1 g/l potassium bicarbonate). Cells were then washed again in PBS and quantitated by haemocytometer. BMMC samples were then cultured in complete RPMI-1640 media (as above for HMCL) for 24 h. Subsequently, the BMMC were plated at 5 105 cells/ml and were treated with CYT387 (5–50 mM), alone or (dependant on cell numbers) in combination with bortezomib (5–40 nM) or melphalan (50–200 mM) for 24 and/or 48 h. Drug-induced MM-specific cell apoptosis was then compared with untreated and vehicle controls by staining for CD45 FITC (BD, North Ryde, New South Wales, Australia), CD38 PerCP-Cy5.5 (BD) and Apo 2.7 PE (Immunotech Beckman Coulter, Mt Waverley,
Victoria, Australia) to determine apoptosis in CD45—CD38 þ MM cells. Samples were subsequently analysed by fluores-
cence-activated cell sorting (FACS).
Primary bone marrow stromal cells (BMSC) were also collected from patient BMMC and cells that adhered to the flask after an initial 24 h culture were cultured with continued selection for adherent cells over several passages. Once cells had expanded in culture, they were used in coculture (CC) to stimulate MM cells in parallel to experiments utilising the HS5 stromal cell line.
Western blots
HMCL were treated with CYT387 (1 or 2 mM) for 60 min and then stimulated with 10 ng/ml IL-6 for 15 min. Protein lysates of CYT387-treated and -untreated HMCL were made with radio- immunoprecipitation buffer (50 mM Tris.HCl, pH 7.4, 150 mM NaCl, 1 mM phenylmethanesulfonyl fluoride, 1 mM ethylene diamine tetraacetic acid, 5 mg/ml Aprotinin, 5 mg/ml Leupeptin, 1% Triton X-100, 1% sodium deoxycholate and 0.1% SDS). Briefly, cells were incubated in radioimmunoprecipitation buffer on ice for 30–60 min before being centrifuged at 16 100 g for 20 min at 4 1C and the supernatant collected. Protein concen-
tration was quantified using DC Protein Assay (Bio-Rad,
Gladesville, New South Wales, Australia) as per manufacturer’s instructions. Subsequently, 100 mg of each protein lysate was separated by 10% SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose (Hybond ECL, Amersham Bios- ciences) using the Bio-Rad semi-dry transfer system. Membranes were blocked with 5% skim milk powder 0.1% Tween-20/PBS
for 60 min then incubated with mouse monoclonal anti-
phospho-STAT3 (pY705, Santa Cruz, ThermoFisher Scientific, Scoresby, Victoria, Australia), mouse monoclonal anti-STAT3 (Santa Cruz) or mouse monoclonal anti-a-tubulin (Sigma-
Aldrich) for 1–2 h at room temperature or overnight at 4 1C. The blots were washed three times for 15 min in 0.1% Tween- 20/PBS, then incubated with secondary horseradish peroxidase
tagged antibody (swine anti-rabbit Ig HRP or rabbit anti-mouse Ig HRP (Dako, Campbellfield, Victoria, Australia)) for 1–2 h at room temperature before washing as above. Blots were visualised with Supersignal west pico ECL reagents (Pierce, ThermoFisher Scientific).
Intracellular FACS
Activation of the JAK/STAT, PI3K/AKT and Ras/MAPK pathways was investigated using intracellular flow cytometry to measure the phosphorylation of STAT3 at tyrosine 705 (p-STAT3), AKT at serine 473 (p-AKT), and extracellular signal-regulated kinase
(ERK)1/2 at threonine 202 and tyrosine 204 (p-ERK). HMCL were stimulated alone with 10 ng/ml IL-6±200 ng/ml insulin-like
growth factor-1 (IGF-1) or stimulated in CC with HS5 stromal cells or primary BMSC, with or without CYT387 treatment. For CC, HS5 and primary BMSC were seeded into a 24-well plate at 2 × 10 cells/ml and allowed to establish for 4 h, after which
HMCL prestained with CD38 or CD138 FITC (BD) were added.
MM cells were stimulated alone (10 ng/ml IL-6, or 5 ng/ml IL-6 and 100 ng/ml IGF-1), or in CC (direct CC with stroma or transwell CC with stroma), with or without either 60 min of
CYT387 pretreatment or 15 min CYT387 co-treatment. After stimulation±treatment, MM cells were harvested and fixed with 2% paraformaldehyde for 10–30 min, washed, then permeabi-
lised with methanol overnight. Methanol was washed off and the cells were resuspended in p-STAT3 PE (BD), p-AKT PE (BD) or p-ERK (BD) and stained for 45–60 min at room temperature. Unbound antibody was washed off and the cells resuspended in 2% foetal bovine serum PBS and acquired by FACS.
Proliferation and viability assays
The viability and proliferation of CYT387-treated HMCL and – untreated/vehicle controls were determined using various methods as described previously.28 Proliferation was measured first using Celltiter 96 AQeous one solution cell proliferation assay MTS reagent (Promega, South Sydney, New South Wales, Australia) on a panel of eight HMCL. Cells were cultured at
2.0 105 cells/ml in 100 ml fresh media in 96-well plates for 24,
48 and 72 h with CYT387 (0.1–5 mM). 20 ml of MTS reagent (Promega) was added for the final 4 h of treatment, and the plates were read at 490 nm using a Fluostar Optima plate reader (BMG Labtech, Mornington, Victoria, Australia). The viable cell numbers of a panel of five HMCL that were treated with CYT387, with and without IL-6 co-treatment, was also measured by trypan blue staining and haemocytometer count.
The HMCL NCI-H929, OCI-MY1 and U266 were then selected for further analysis. Apoptosis of CYT387-treated cells was assessed by FACS with Annexin-V and propidium iodide (PI) staining. HMCL were treated for 24 or 72 h with 1 or 5 mM CYT387, then harvested and washed in Annexin Buffer (0.01 HEPES, 0.14 M NaCl, 2.5 mM CaCl2, pH 7.4) and stained with Annexin-V FITC (Biosource) made up in Annexin Buffer, for 30 min at room temperature. Unbound antibody was then washed off with Annexin Buffer and cells were resuspended in Annexin Buffer with 62.5 ng/ml PI (Sigma-Aldrich) and analysed by FACS.
For the synergy experiments, HMCL were treated with CYT387 in combination with bortezomib or melphalan for 24 and 48 h before being harvested, and resuspended in FACS Buffer (0.5% heat-inactivated foetal bovine serum in PBS) supplemented with 62.5 ng/ml PI (Sigma-Aldrich). Cells were immediately analysed by FACS. The proportion of PI-positive cells was quantitated by subtracting the background death of untreated cells. Single drug-treated cells were compared with combination-treated cells, and synergism was calculated using Calcusyn software (Biosoft, Cambridge, UK).
Cell cycling
The effect of CYT387 treatment on HMCL cell cycling was measured after 24 and 72 h. CYT387 (0.5, 1 or 5 mM)-treated and
-untreated HMCL were harvested, washed in PBS and resus- pended in 100 ml PBS. Cells were fixed with 1 ml of cold 70% ethanol while being vortexed. Tubes were stored at —20 1C until
analysis. Once all samples were collected, tubes were
centrifuged at 500 g for 10 min, the supernatant was carefully removed and the cells were washed in 5 ml PBS. After the final wash, cells were resuspended in 250–500 ml of PI/RNase staining buffer (BD) and incubated in the dark at room temperature for 15 min before being analysed by FACS.
Data analysis
All FACS data was acquired on a BD FACSCalibur and data analysis done using Flowjo 7.6. Software (Treestar, Ashland, OR, USA). All statistical analysis was done using GraphPad Prism 5.03 software (La Jolla, CA, USA).
Results
JAK/STAT signalling is inhibited by CYT387
IL-6 signalling through the JAK/STAT pathway is well charac- terised in MM cells, with binding of IL-6 to its receptor inducing JAK2 to phosphorylate STAT3. The ability of CYT387 to inhibit JAK2 was first confirmed by measuring the level of STAT3 phosphorylation by western blotting and FACS. HMCL (NCI- H929 and U266) were incubated with CYT387 for 1 h before being stimulated with IL-6 for 15 min to induce p-STAT3. CYT387 (1–2 mM) inhibited the phosphorylation of STAT3 in IL-6 stimulated samples as demonstrated by FACS (Figure 1a) and confirmed by western blotting (Figure 1b). As seen in Figures 1a and b, CYT387 was also able to reduce constitutive levels of p-STAT3 in U266 cells, as well as p-STAT3 induced by exogenous IL-6. Overall, the total STAT3 protein was unaffected by IL-6 stimulation or CYT387 treatment.
Given the importance of the bone marrow microenvironment in MM growth and survival, it was important to establish if CYT387 could similarly modulate signalling in MM cells in CC with BMSC. This was done with both immortalised BMSC (HS5) and primary patient BMSC. In each case, 15 min of CC with BMSC (with or without contact) was able to induce phosphor- ylation of STAT3 in the MM cells, whereas contemporaneous treatment with CYT387 dramatically reduced the amount of p-STAT3 in the HMCL (Figure 1c and Supplementary Figure 1A and 1B), thus, demonstrating that CYT387 is able to prevent STAT3 activation in MM cells induced by soluble factors within the bone marrow microenvironment, as well as the contact- mediated signalling provided to MM by BMSC.
CYT387 inhibits PI3K/AKT and Ras/MAPK signalling
JAK signalling kinases are involved in many cellular pathways, which led us to investigate the effect of CYT387 on IL-6 and IGF-1-induced PI3K/AKT, and Ras/MAPK signalling in NCI- H929, OCI-MY1 and U266 cells (Figure 1d). OCI-MY1 showed distinct p-AKT activation after IL-6 and IGF-1 stimulation that was significantly reduced by CYT387 co-treatment. IL-6 and IGF-1 stimulation induced p-ERK in U266 cells, which was significantly inhibited by CYT387. The levels of p-AKT and p-ERK showed only a small increase in response to IL-6 and IGF-1 stimulation in NCI-H929. IGF-1 can also induce JAK/ STAT signalling,29,30 because of this, we confirmed that CYT387 co-treatment was able to significantly inhibit p-STAT3 induced by simultaneous IL-6 and IGF-1 stimulation in all three HMCL (Supplementary Figure 1C).
CYT387 inhibits proliferation in HMCL
Given that CYT387 can inhibit important signalling pathways in HMCL, we next investigated the effect of CYT387 on prolifera- tion and cycling of the HMCL. The effect of CYT387 (0.1–5 mM) on cell proliferation/viability was measured by MTS assay at 24,
48 and 72 h (Figure 2a) on a panel of eight HMCL (IL-6 non-responsive, predominantly CD45 phenotypeFLP-1, NCI- H929, OPM2, and RPMI-8226, and IL-6 responsive, predomi- nantly CD45 phenotypeFANBL-6, OCI-MY1, U266 and XG-1). Six of the eight HMCL had a time- and dose-dependent response to CYT387, with inhibition in some HMCL within 24 h. At 72 h, NCI-H929 and XG-1 were the most sensitive to CYT387 treatment, demonstrating that CYT387 can be a potent inhibitor of both IL-6-responsive and non-responsive HMCL.
Proliferation of HMCL cultured with CYT387 over 72 h was also assessed by quantitation of viable cell number by haemo- cytometer (Figure 2b). Three diverse HMCL (NCI-H929, OCI-MY1 and U266) representing both the IL-6-responsive and non-responsive phenotype were selected to determine the specific effect of CYT387 on absolute cell number. Because of the obvious relationship between IL-6 signalling and the inhibition of JAK2 by CYT387, the proliferation of three HMCL was measured with the addition of IL-6 and/or CYT387. The three HMCL proliferated well in complete media with or without supplementation with 10 ng/ml IL-6, and CYT387 (0.5–1 mM) was able to reduce the proliferation of HMCL in culture even in the presence of IL-6. A single dose of CYT387 inhibited cell proliferation and resulted in a reduction in absolute cell number by 50% in NCI-H929 (1 mM), 50% in OCI-MY1 (0.5 mM) and 44% in U266 (1 mM) after 72 h, compared with untreated controls.
Treatment of HMCL with CYT387 results in an accumulation of cells in G2/M phase of the cell cycle The anti-proliferative effects of CYT387 were further charac-
terised by evaluating the cell cycle of HMCL treated with CYT387. HMCL (NCI-H929, OCI-MY1 and U266) treated with
CYT387 (0.5–5 mM) showed a marked accumulation of cells in the G2/M phase of the cell cycle. This was most pronounced in NCI-H929 cells, in which there was a 1.5 fold increase in G2/M in cells treated with 1 mM CYT387 and a two fold increase in cells treated with 5 mM CYT387 (Figure 2c), compared with untreated or vehicle-treated controls after 24 and 72 h treatment. An additional polyploid population was found in the 5 mM CYT387 treated samplesFsuggesting that CYT387 treatment causes further aberration from the normal cell cycle of MM cells. This correlated well with the CYT387-induced cytostasis seen in Figure 2b.
Figure 1 CYT387 prevents signalling downstream of IL-6 or CC stimulation. HMCL were incubated with or without CYT387 (1–2 mM) for 1 h before stimulation with 10 ng/ml IL-6 for 15 min. Cells were then harvested and p-STAT3 (pY705) was measured. (a) By intracellular FACS with the geometric mean fluorescence intensity measured and graphed (n 3, mean±s.e., stimulated cells±CYT387 were analysed using a one-way ANOVA with Tukey post-test *Po0.05, **Po 0.01). (b) By western blot for p-STAT3 (pY705), total STAT3 and a-tubulin as a loading control. (c) p- STAT3 was also induced in HMCL using IL-6, direct CC with HS5 immortalised BMSC or primary BMSC or transwell (TW) ‘soluble only’ CC with HS5. HMCL were fluorescently labelled with CD38 or CD138 and stimulated for 15 min with or without co-treatment with 2 mM CYT387. Representative plots of NCI-H929, (n 3, for NCI-H929, OCI-MY1 and U266). (d) NCI-H929, OCI-MY1 and U266 cells were starved overnight and stimulated with 5 ng/ml IL-6 and 100 ng/ml IGF-1 for 15 min, with or without co-treatment with 2 mM CYT387. p-AKT (pS473) and p-ERK1/2 (pT202/pY204) were measured by intracellular FACS with the geometric mean fluorescence intensity normalised to the untreated (UT) control and averaged (n ¼ 4, mean±s.e., stimulated cells±CYT387 were analysed using a one-way ANOVA with Tukey post-test *Po0.05).
CYT387 induces apoptosis in HMCL
After demonstrating the potent effect of CYT387 on cell signalling and proliferation, we next investigated the induction of apoptosis by Annexin-V/PI FACS staining in three HMCL (NCI-H929, OCI-MY1 and U266). An increased proportion of apoptotic cells was detected in all three HMCL, which was most evident in NCI-H929 (Figure 3a), with a 21 and 52% increase in apoptotic cells detected at 24 and 72 h, respectively, after treatment with 5 mM CYT387 (Figure 3b). The toxicity of CYT387 (0.5–10 mM) on normal donor peripheral blood
mononuclear cells was also investigated after 72 h, with only low levels of apoptosis seen (mean 14% death after 72 h with 5 mM CYT387, data not shown).
CYT387 synergises with other anti-myeloma agents in HMCL
To assess CYT387 as part of a combination therapy, NCI-H929, OCI-MY1 and U266 were treated with a range of doses of CYT387, bortezomib and melphalan to establish dose effect
Figure 2 CYT387 inhibits HMCL proliferation. (a) CYT387 inhibits HMCL in a time- and dose-dependant manner. IL-6 phenotype HMCL (ANBL-6, OCI-MY1, U266 and XG-1) and non-IL-6 phenotype HMCL (LP-1, NCI-H929, OPM2 and RPMI-8226) were cultured for 24, 48 and 72 h untreated (UT), with CYT387 (0.1, 0.5, 1, 2.5 or 5 mM) or with vehicle (dimethyl sulfoxide). Cell proliferation was then determined by MTS assay (72 h data shown, n 3, mean±s.e.). (b) Treatment with CYT387 inhibits myeloma cell proliferation even in the presence of IL-6. Absolute cell numbers of viable cells were determined by haemocytometer counts of HMCL cultured alone (UT) with IL-6 (10 ng/ml), with CYT387 (0.5–1 mM), or with IL-6 and
CYT387. Culture with CYT387 greatly decreased the proliferation of the HMCL over 72 h (treatment at time 0 only). Results represent the mean of three independent experiments±s.e. (c) CYT387 prevents cell cycling. HMCL were treated with CYT387 (1 mM or 5 mM) for 24 and 72 h then they were harvested and fixed, and cell cycle analysed by FACS. Representative cell cycle plots of NCI-H929 UT or 5 mM CYT387 for 24 or 72 h, with mean of four independent experiments±s.e. of cycling cells in G2/M phase of the cell cycle.
curves for each drug before combining with CYT387 and measuring the synergy using Calcusyn software. Dose effect curves generated for each compound (melphalan and bortezo- mib data not shown) show CYT387 (0.5–10 mM) induced apoptosis in three HMCL in a time- and dose-dependent manner (Figure 4a). CYT387 displayed synergism with both bortezomib (1–4 nM) and melphalan (25–50 mM), but with variations in the degree of synergy seen with different drug dosages and analysis timepoints (Figure 4b). No distinct pattern of specific drug dose, timepoint or cell line was evident. CYT387 was also combined with the second-generation proteasome inhibitor NPI-0052, as well as other conventional therapies cisplatin, dexamethasone and etoposide, which showed mixed synergistic and antagonis- tic results (data not shown).
CYT387 induces apoptosis as a single agent and synergises with bortezomib and melphalan in primary MM cells
The effect of CYT387 on ex-vivo primary MM cells was also investigated, both as a single agent and as part of combination therapy. MM patient BMMC were cultured with various doses of CYT387 (5–50 mM) for 24 and 48 h, after which the
CD38 CD45 MM cell populations were assessed for apoptosis by flow cytometry. Six patients were treated and apoptosis was seen in between 5 and 59% of MM cells treated with 20 mM CYT387 after 48 h (Figure 5a). The effect of CYT387 (10–20 mM), in combination with melphalan (50–200 mM) and bortezomib (10–40 nM), was also investigated. CYT387 was seen to synergise with melphalan in two of three patients and synergy was also observed with bortezomib in some patients/doses (Figure 5b).
Discussion
Multiple lines of evidence have confirmed the role of IL-6/JAK/ STAT signalling in MM, including experiments demonstrating the IL-6 dependence of some MM cells, the upregulation of proliferation of MM cells with IL-6, the inhibition of drug- induced apoptosis by IL-6, and most important to this investigation, the direct induction of apoptosis in MM cells by inhibition of the IL-6/JAK/STAT pathway. These data and the abundance of IL-6 in the bone marrow microenvironment makes JAK/STAT signalling a rational target for inhibition with new chemotherapeutics and is supported by preliminary in vitro
Figure 3 CYT387 induces apoptosis in HMCL. (a) Representative Annexin-V and PI plots of NCI-H929. UT, vehicle (dimethyl sulfoxide) treated, 24 h 5 mM CYT387 treatment and 72 h 5 mM CYT387 treatment.
(b) Proportion of apoptotic (Annexin-V or PI ) cells after CYT387
treatment compared with UT. Data shown is the mean of four independent experiments±s.e.
studies that have demonstrated MM cell apoptosis induction via siRNA targeting of the JAK/STAT pathway.31 Furthermore, the signal transduction role of JAKs in other important pathways (reviewed by Rane and Reddy5) and the pro-survival effects of JAK mutations in other haematological malignancies1–4 have already demonstrated the therapeutic potential of JAK inhibition. The therapeutic challenge is to inhibit MM cells in the presence of IL-6 or BMSC. Here, we have expanded upon previous work evaluating JAK inhibition in MM by studying a broader range of HMCL, by demonstrating that CYT387 can inhibit JAK/STAT activation in the context of CC models and by demonstrating the impact of CYT387 on primary MM tumour cells.
It has been hypothesised that in the earlier stages of MM, the malignant cells are predominantly CD45 þ , whereas in more advanced, drug-resistant disease, CD45— MM cells predomi- nate.32 Moreover, CD45— MM cells are considered less IL-6 responsive and express fewer IL-6 receptors than CD45 MM
cells.15 Other studies of JAK inhibition have focused predomi- nantly on CD45 þ IL-6 responsive HMCL, and although this is a
logical focus for preliminary investigation, it must be stressed
that such target cell populations represents only a subset of MM cells. Furthermore, patients may demonstrate mixed populations of both CD45— and CD45 þ MM cells.15 Importantly, we have
demonstrated the effectiveness of CYT387 against NCI-H929, a
HMCL considered to have an IL-6 non-responsive phenotype, suggesting that CYT387 will be effective against a range of MM phenotypes, whereas others20 have reported only limited success against CD45 MM, using alternative JAK inhibitors. Our data demonstrating synergy between CYT387 and bortezo- mib, or melphalan against several HMCL and primary MM tumour cells confirms previously published work.20
Available data suggests that the inhibition of JAK in MM cells may have downstream effects other than the direct inhibition of the JAK/STAT pathway. The importance of IL-6 in MM cell survival has been well characterised in terms of the JAK/STAT pathway, but there is now increasing evidence of IL-6-induced PI3K/AKT activation19,33 and Ras/MAPK activation21,33–36 in various HMCL. Given the established importance of both the PI3K/AKT and Ras/MAPK pathways in addition to the JAK/STAT pathway in MM, small molecule inhibitors that could modulate all three could have enormous clinical potential. Investigating the effects of JAK inhibition on the PI3K/AKT and Ras/MAPK pathways has yielded contrasting results. AZD1480 and AG490 have been shown to reduce IL-6-induced activation of Ras/ MAPK,21,23 but AG490 in the hands of others showed no decrease in IL-6-induced Ras/MAPK activation.34 The JAK inhibitor INCB20 could inhibit IL-6-induced Ras/MAPK activa- tion in MM.1S cells, but did not affect constitutive Ras/MAPK activation in INA-6 cells.19 The inhibition of JAK with AG490 could also abrogate AKT activation in phosphatase and tensin homolog-mutated OPM2 MM cells.33 Similarly, INCB20 could also inhibit IL-6-induced p-AKT, but had no effect on IGF-1- induced p-AKT in INA-6 cells.19 Variability in the available data may be the result of differences in inhibitors and heterogeneity amongst HMCL that are commonly studied. In our study, there was a significant reduction in IL-6 and IGF-1-induced p-ERK in U266 cells, as well as a dramatic reduction of IL-6 and IGF-1- induced p-AKT in OCI-MY1 cells, supporting the data of others that suggests JAK inhibition may have broader anti-MM activity than would be initially expected. The inhibition of IL-6-induced JAK/STAT and PI3K/AKT signalling may also result in a reduction in IL-6 receptor expression on the surface of MM cells,33 which could also lead to a reduction in the pro-survival effects of IL-6.
The profound anti-proliferative effect of CYT387 on various HMCL after a single dose is an important demonstration of its effectiveness. Furthermore, the ability of CYT387 to inhibit HMCL growth even in the presence of exogenous IL-6, which has been shown many times as a mediator of drug resistance11–13 is noteworthy. This significant effect of JAK inhibition may be the result of HMCL having some dependence on JAK/STAT for proliferative signals, or more likely, the involvement and subsequent inhibition of alterative signalling pathways men- tioned above. The effect on proliferation is also seen in the cell cycling analysis, which demonstrates that CYT387 can prevent cell cycling. Also of interest was the additional polyploidy population (8n) induced by CYT387 treatment, which may suggest a role for JAK signalling leading to cell cycle regulation or may be the result of CYT387 inhibition of other kinases involved in cell cycle, such as aurora B, aurora C, cyclin A or cyclin B as described by Pardanani et al.17 A direct or downstream effect of JAK inhibitors on cell cycling proteins is supported by studies on other JAK inhibitors, Scuto et al.23 found AZD1480 inhibited cyclin D2 in two HMCL.
Figure 4 CYT387 synergises with melphalan and bortezomib in HMCL. (a) Dose effect curves of NCI-H929, OCI-MY1 and U266 after 24 and 48 h treatment CYT387 treatment (0.5–10 mM) as determined by the proportion of PI cells minus background death (untreated). Mean of four independent experiments±s.e. (b) CYT387 (1–5 mM) in combination with melphalan (25–50 mM) or bortezomib (1–4 nM) shows synergism. Synergy
was measured using a combination index calculated by Calcusyn software, where values less than 1 represent synergism, plotted against the fraction of cells killed with various dose/ratios of the drugs. Synergy is seen between melphalan and CYT387 at a range of doses/ratios/cell lines and timepoints. Bortezomib and CYT387 demonstrated synergistic or nearly additive in 18/24 combinations. Synergism was calculated from dose effect curves of the mean of four independent experiments.
Figure 5 CYT387 induces apoptosis in primary samples as a single agent or in combination with melphalan and bortezomib. (a) Proportion of apoptotic (Apo 2.7 ) CD38 CD45 primary patient myeloma cells after 48 h CYT387 treatment (n 6). (b) Synergy between CYT387 (10–20 mM) and melphalan (50–200 mM) or bortezomib (10–40 nM) after 24 h treatment as determined by Calcusyn software in primary patient CD38 þ CD45— cells.
The induction of apoptosis in MM cells in primary marrow CCs using clinically relevant low-micromolar doses is a critical demonstration of the potential of CYT387. In contrast, other
studies inhibiting IL-6 signalling have failed to demonstrate any convincing evidence of apoptosis when MM cells were treated in the presence of BMSC.35 The investigators suggested that this
might represent MM cell independence from IL-6 in the
presence of BMSC. Our findings with CYT387 could be interpreted as either refuting the latter or alternatively being consistent with the capacity of JAK signalling inhibition to interrupt non-IL6-mediated survival pathways. Consistent with the latter was that the primary MM samples treated with CYT387
were representative of autologous whole marrow CCs with the CD38 þ CD45— MM cells making up between 4 and 67% of treated cells. Therefore, the demonstration that CYT387 was able to induce apoptosis of these heavily treated MM cells was
particularly encouraging.
Our pre-clinical evaluation of CYT387 has demonstrated promising anti-myeloma effects of an orally bioavailable compound already through phase I clinical trials. We show the inhibition of important signalling pathways in MM cells, the subsequent reduction in cell viability, proliferation and corre- sponding inhibition of the cell cycle. Furthermore, we present specific evidence of the induction of apoptosis in both primary MM cells and HMCL. The synergy demonstrated by CYT387 with common myeloma therapies bortezomib and melphalan make it a very attractive compound for further study in the clinic.
Conflict of interest
Katherine A Monaghan, Tiffany Khong and Andrew Spencer declare no potential conflict of interest. Christopher J Burns is an employee of YM BioSciences, and holds equity in the company.
Acknowledgements
CYT387 was kindly provided by YM BioSciences, Australia. This study was supported by part funding from the Multiple Myeloma Research Foundation.
References
1 Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005; 352: 1779–1790.
2 Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer cell 2005; 7: 387–397.
3 Scott LM, Tong W, Levine RL, Scott MA, Beer PA, Stratton MR et al. JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis. N Engl J Med 2007; 356: 459–468.
4 Mullighan CG, Zhang J, Harvey RC, Collins-Underwood JR, Schulman BA, Phillips LA et al. JAK mutations in high-risk childhood acute lymphoblastic leukemia. Proc Natl Acad SciUSA 2009; 106: 9414–9418.
5 Rane SG, Reddy EP. Janus kinases: components of multiple signaling pathways. Oncogene 2000; 19: 5662–5679.
6 French JD, Walters DK, Jelinek DF. Transactivation of gp130 in myeloma cells. J Immunol 2003; 170: 3717–3723.
7 Berger LC, Hawley TS, Lust JA, Goldman SJ, Hawley RG. Tyrosine phosphorylation of JAK-TYK kinases in malignant plasma cell lines growth-stimulated by interleukins 6 and 11. Biochem Biophys Res Commun 1994; 202: 596–605.
8 Gomez-Benito M, Balsas P, Carvajal-Vergara X, Pandiella A, Anel A, Marzo I et al. Mechanism of apoptosis induced by IFN- alpha in human myeloma cells: role of Jak1 and Bim and potentiation by rapamycin. Cell Signal 2007; 19: 844–854.
9 Jelinek DF, Ahmann GJ, Greipp PR, Jalal SM, Westendorf JJ, Katzmann JA et al. Coexistence of aneuploid subclones within a myeloma cell line that exhibits clonal immunoglobulin gene
rearrangement: clinical implications. Cancer Res 1993; 53:
5320–5327.
10 Zhang XG, Gaillard JP, Robillard N, Lu ZY, Gu ZJ, Jourdan M et al. Reproducible obtaining of human myeloma cell lines as a model for tumor stem cell study in human multiple myeloma. Blood 1994; 83: 3654–3663.
11 Cheung WC, Van Ness B. The bone marrow stromal microenvir- onment influences myeloma therapeutic response in vitro. Leukemia 2001; 15: 264–271.
12 Moreaux J, Legouffe E, Jourdan E, Quittet P, Reme T, Lugagne C et al. BAFF and APRIL protect myeloma cells from apoptosis induced by interleukin 6 deprivation and dexamethasone. Blood 2004; 103: 3148–3157.
13 Perez LE, Parquet N, Shain K, Nimmanapalli R, Alsina M, Anasetti C et al. Bone marrow stroma confers resistance to Apo2 ligand/TRAIL in multiple myeloma in part by regulating c-FLIP. J Immunol 2008; 180: 1545–1555.
14 Klein B, Zhang XG, Jourdan M, Content J, Houssiau F, Aarden L et al. Paracrine rather than autocrine regulation of myeloma-cell growth and differentiation by interleukin-6. Blood 1989; 73: 517–526.
15 Hata H, Xiao H, Petrucci MT, Woodliff J, Chang R, Epstein J. Interleukin-6 gene expression in multiple myeloma: a character- istic of immature tumor cells. Blood 1993; 81: 3357–3364.
16 Tyner JW, Bumm TG, Deininger J, Wood L, Aichberger KJ, Loriaux MM et al. CYT387, a novel JAK2 inhibitor, induces hematologic responses and normalizes inflammatory cytokines in murine myeloproliferative neoplasms. Blood 2010; 115: 5232–5240.
17 Pardanani A, Lasho T, Smith G, Burns CJ, Fantino E, Tefferi A. CYT387, a selective JAK1/JAK2 inhibitor: in vitro assessment of kinase selectivity and preclinical studies using cell lines and primary cells from polycythemia vera patients. Leukemia 2009; 23: 1441–1445.
18 Burns CJ, Bourke DG, Andrau L, Bu X, Charman SA, Donohue AC et al. Phenylaminopyrimidines as inhibitors of Janus kinases (JAKs). Bioorg Med Chem Lett 2009; 19: 5887–5892.
19 Burger R, Le Gouill S, Tai YT, Shringarpure R, Tassone P, Neri P et al. Janus kinase inhibitor INCB20 has antiproliferative and apoptotic effects on human myeloma cells in vitro and in vivo. Mol Cancer Ther 2009; 8: 26–35.
20 Li J, Favata M, Kelley JA, Caulder E, Thomas B, Wen X et al. INCB16562, a JAK1/2 selective inhibitor, is efficacious against multiple myeloma cells and reverses the protective effects of cytokine and stromal cell support. Neoplasia (New York, NY) 2010; 12: 28–38.
21 De Vos J, Jourdan M, Tarte K, Jasmin C, Klein B. JAK2 tyrosine kinase inhibitor tyrphostin AG490 downregulates the mitogen- activated protein kinase (MAPK) and signal transducer and activator of transcription (STAT) pathways and induces apoptosis in myeloma cells. Br J Haematol 2000; 109: 823–828.
22 Alas S, Bonavida B. Inhibition of constitutive STAT3 activity sensitizes resistant non-Hodgkin’s lymphoma and multiple mye- loma to chemotherapeutic drug-mediated apoptosis. Clin Cancer Res 2003; 9: 316–326.
23 Scuto A, Krejci P, Popplewell L, Wu J, Wang Y, Kujawski M et al. The novel JAK inhibitor AZD1480 blocks STAT3 and FGFR3 signaling, resulting in suppression of human myeloma cell growth and survival. Leukemia 2011; 25: 538–550.
24 Pedranzini L, Dechow T, Berishaj M, Comenzo R, Zhou P, Azare J et al. Pyridone 6, a pan-Janus-activated kinase inhibitor, induces growth inhibition of multiple myeloma cells. Cancer Res 2006; 66: 9714–9721.
25 Ferrajoli A, Faderl S, Van Q, Koch P, Harris D, Liu Z et al. WP1066 disrupts Janus kinase-2 and induces caspase-dependent apoptosis in acute myelogenous leukemia cells. Cancer Res 2007; 67: 11291–11299.
26 Pardanani A, George G, Lasho T, Hogan WJ, Litzow MR, Begna K et al. A Phase I/II Study of CYT387, An Oral JAK-1/2 Inhibitor, In Myelofibrosis: Significant Response Rates In Anemia, Spleno- megaly, and Constitutional Symptoms. Blood; 52nd ASH Annual Meeting and Exposition 6/12/2010; Orlando, Florida, USA, 116. Abstract 460. p. 206.
27 Khong T, Sharkey J, Spencer A. The effect of azacitidine on interleukin-6 signaling and nuclear factor-kappaB activation and its in vitro and in vivo activity against multiple myeloma. Haematologica 2008; 93: 860–869.
28 Monaghan K, Khong T, Smith G, Spencer A. CYT997 Causes
apoptosis in human multiple myeloma. Invest New Drugs 2011;
29: 232–238.
29 Zong CS, Chan J, Levy DE, Horvath C, Sadowski HB, Wang LH. Mechanism of STAT3 activation by insulin-like growth factor I receptor. J Biol Chem 2000; 275: 15099–15105.
30 Gual P, Baron V, Lequoy V, Van Obberghen E. Interaction of Janus kinases JAK-1 and JAK-2 with the insulin receptor and the insulin- like growth factor-1 receptor. Endocrinology 1998; 139: 884–893.
31 Chatterjee M, Stuhmer T, Herrmann P, Bommert K, Dorken B, Bargou RC. Combined disruption of both the MEK/ERK and the IL-6R/STAT3 pathways is required to induce apoptosis of multiple myeloma cells in the presence of bone marrow stromal cells. Blood 2004; 104: 3712–3721.
32 Kumar S, Rajkumar SV, Kimlinger T, Greipp PR, Witzig TE. CD45 expression by bone marrow plasma cells in multiple
myeloma: clinical and biological correlations. Leukemia 2005;
19: 1466–1470.
33 Thabard W, Collette M, Mellerin MP, Puthier D, Barille S, Bataille R et al. IL-6 upregulates its own receptor on some human myeloma cell lines. Cytokine 2001; 14: 352–356.
34 Puthier D, Bataille R, Amiot M. IL-6 up-regulates mcl-1 in human myeloma cells through JAK / STAT rather than ras/MAP kinase pathway. Eur J Immunol 1999; 29: 3945–3950.
35 Chatterjee M, Honemann D, Lentzsch S, Bommert K, Sers C, Herrmann P et al. In the presence of bone marrow stromal cells human multiple myeloma cells become independent of the IL-6/ gp130/STAT3 pathway. Blood 2002; 100: 3311–3318.
36 Ferlin M, Noraz N, Hertogh C, Brochier J, Taylor N, Klein B. Insulin-like growth factor induces the survival and proliferation of myeloma cells through an interleukin-6-independent transduction pathway. Br J Haematol 2000; 111: 626–634.
Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)