Cerdulatinib (cat. no. S7634; Selleck Chemicals, Houston, TX, USA), JAK inhibitor 1 (cat. no. 420099; Merck Millipore, Burlington, MA, USA), PRT060318 (cat. no. SYN-1204; SYNkinase, Parkville, Australia) and z-VAD-FMK (cat. no. G7232; Promega Corp., Madison, WI, USA) were dissolved in dimethyl sulfoxide (cat. no. 13407-45; Nacalai Tesque, Inc., Kyoto, Japan). Antibodies against SYK (cat. no. 2712), phospho-SYK (Tyr525/526; cat. no. 2710), STAT3 (cat. no. 9132), phospho-STAT3 (Tyr705; cat. no. 9145), STAT5 (cat. no. 9363), phospho-STAT5 (Tyr694; cat. no. 4322), ERK (cat. no. 4695), phospho-ERK (Thr202/Tyr204; cat. no. 4370), phospho-p38 (Thr180/Tyr182; cat. no. 9211), Bcl-xL (cat. no. 2762), Bax (cat. no. 2772), Bak (cat. no. 3814), survivin (cat. no. 2808), AKT (cat. no. 9272), phospho-AKT (Thr308; cat. no. 13038 and Ser473; cat. no. 4060), phospho-IκBα (Ser32/36; cat. no. 9246), IκB kinase (IKK) α (cat. no. 2682), IKKβ (cat. no. 2684), phospho-IKKα/β (Ser176/180 and Ser177/181; cat. no. 2694), cleaved caspase-8 (cat. no. 9496), cleaved caspase-9 (cat. no. 9501), cleaved caspase-3 (cat. no. 9664), cleaved poly(ADP-ribose) polymerase (PARP; cat. no. 9541) and c-FLIP (cat. no. 3210) were from Cell Signaling Technology, Inc. (Beverly, MA, USA). Antibodies against cyclin B1 (cat. no. MS-868), cyclin-dependent kinase (CDK)1 (cat. no. MS-275), p53 (cat. no. MS-738) and actin (cat. no. MS-1295) were from Neomarkers, Inc. (Fremont, CA, USA). Antibodies against p21 (cat. no. MS-891), p27 (cat. no. MS-256) and X-linked inhibitor of apoptosis (XIAP; cat. no. M044-3) were from Medical & Biological Laboratories, Co. (Aichi, Japan). Antibodies against JAK1 (cat. no. sc-277), phospho-JAK1 (Tyr1022/1023; cat. no. sc-16773-R), IκBα (cat. no. sc-371), Mcl-1 (cat. no. sc-819) and c-IAP2 (cat. no. sc-7944) were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

HTLV-1-infected T-cell lines include HTLV-1-transformed T-cell lines and ATLL-derived T-cell lines. HTLV-1-transformed MT-2 and HUT-102, ATLL-derived TL-OmI, and uninfected control Jurkat and CCRF-CEM T-cell lines were grown in RPMI-1640 medium (cat. no. 30264-56, Nacalai Tesque, Inc.) supplemented with 10% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel) and 1% penicillin/streptomycin (cat. no. 09367-34; Nacalai Tesque, Inc.) at 37°C and 5% CO₂ in a humidified incubator. The HUT-102 and Jurkat cells were obtained from Fujisaki Cell Center, Hayashibara Laboratories, Inc. (Okayama, Japan). The MT-2 cell line was kindly provided by Dr Naoki Yamamoto (Tokyo Medical and Dental University, Tokyo, Japan). The TL-OmI and CCRF-CEM cells were provided by Dr Masahiro Fujii (Niigata University, Niigata, Japan). Frozen human peripheral blood mononuclear cells (PBMCs) (cat. no. HC-0001) were obtained from Lifeline Cell Technology (Frederick, MD, USA).

The cells were seeded in triplicate in 96-well flat microtiter plates and treated with cerdulatinib (0.16–10 µM), PRT060318 (0.63–10 µM) and JAK inhibitor 1 (0.63–10 µM) for 24–72 h. Cell growth and cytotoxicity were determined with the water-soluble tetrazolium (WST)-8 uptake assay using a kit (Nacalai Tesque, Inc.) according to the manufacturer's instructions. Briefly, 10 µl of WST-8 reagent (cat. no. 07553-44; Nacalai Tesque, Inc.) was added to each well. After 4 h, WST-8 reduction was measured at 450 nm using a Wallac 1420 Multilabel Counter (PerkinElmer, Inc., Waltham, MA, USA). The absorbance values were normalized to those of the untreated control samples. The 50% inhibitory concentration (IC₅₀) for WST-8 activity was calculated by fitting data points with a logarithmic curve using CalcuSym software (version 2.0; Biosoft, Cambridge, UK).

Serial dilutions of JAK inhibitor 1 or PRT060318 alone, or in combination were tested. The combination index (CI) was calculated using CalcuSym software, with CI<1, CI=1 and CI>1 representing synergistic, additive or antagonistic interactions of the two agents, respectively.

Cell cycle distribution was determined by staining with propidium iodide to label nuclear DNA using the CycleTEST Plus DNA Reagent kit (cat. no. 340242; Becton-Dickinson Immunocytometry Systems, San Jose, CA, USA) according to the supplier's instructions. The cells were sorted on an Epics XL flow cytometer (Beckman Coulter, Inc., Brea, CA, USA) and MultiCycle software (version 3.0; Phoenix Flow Systems, San Diego, CA, USA) was used to determine the fraction of cells at each phase of the cell cycle.

The cells were treated with cerdulatinib (2.5–10 µM) for up to 72 h and then permeabilized by incubation on ice for 20 min with 100 µg/ml of digitonin, followed by treatment with phycoerythrin-conjugated anti-APO2.7 antibody (1:10) (cat. no. IM2088; Beckman Coulter, Inc., Marseille, France) for 15 min at room temperature. The apoptotic fraction was separated by flow cytometry. To evaluate changes in nuclear morphology, the cells were stained with 10 µg/ml Hoechst 33342 (cat. no. 346-07951; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) and observed under a DMI6000 microscope (Leica Microsystems, Wetzlar, Germany) (17).

Caspase activity was measured using Colorimetric Caspase Assay kits (cat. nos. 4800, 4805 and 4810, Medical & Biological Laboratories, Co.). Briefly, cell extracts were recovered using the cell lysis buffer supplied with the kit, and caspase-8, -9 and -3 activity was assessed using the specific colorimetric probe. The kits are based on detection of the chromophore ρ-nitroanilide after cleavage from caspase-specific labeled substrates. Colorimetric measurements were made with a Wallac 1420 Multilabel Counter.

The cells were lysed in lysis buffer containing 62.5 mM Tris-HCl (pH 6.8) (cat. no. 35434-21; Nacalai Tesque, Inc.), 2% sodium dodecyl sulfate (SDS) (cat. no. 31607-65; Nacalai Tesque, Inc.), 10% glycerol (cat. no. 17045-65; Nacalai Tesque, Inc.), 6% 2-mercaptoethanol (cat. no. 21438-82; Nacalai Tesque, Inc.) and 0.01% bromophenol blue (cat. no. 021-02911; Wako Pure Chemical Industries, Osaka, Japan). The protein concentration was determined using the DC Protein Assay kit (cat. no. 5000116JA; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Cell lysates (20 µg) were separated by 8–15% SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (cat. no. IPVH00010EMD; Merck KGaA, Darmstadt, Germany) that was probed with primary antibodies (1:1,000). The membranes were blocked with 4% non-fat dry milk (cat. no. 9999; Cell Signaling Technology, Inc.) for 1 h at room temperature. Following incubation with horseradish peroxidase-conjugated secondary anti-mouse (1:1,000) (cat. no. 7076; Cell Signaling Technology, Inc.) or anti-rabbit (1:1,000) (cat. no. 7074; Cell Signaling Technology, Inc.) IgG, immunoreactivity was visualized using enhanced chemiluminescence reagent (cat. no. RPN2232; Amersham Biosciences Corp., Piscataway, NJ, USA).

To assess STAT3, STAT5, nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) activation, we prepared nuclear extracts from the cerdulatinib-treated cells and performed EMSA, as previously described (18). Nuclear extracts with 5 µg of protein were incubated with ³²P-labeled probes. The top strand sequences of the oligonucleotide probes were as follows: 5′-GATCGACATTTCCCGTAAATCG-3′ (STAT3 consensus binding motif derived from the c-fos gene); 5′-GATCAGATTTCTAGGAATTCAAATC-3′ (STAT5 consensus binding motif derived from the CSN2 gene); 5′-GATCCGGCAGGGGAATCTCCCTCTC-3′ (typical NF-κB element of the IL-2RA gene); and 5′-GATCGTGATGACTCAGGTT-3′ (consensus AP-1 element of the IL-8 gene). The oligonucleotide 5′-GATCTGTCGAATGCAAATCACTAGAA-3′ containing the consensus sequence of the octamer (Oct) binding motif was used to evaluate specific binding of the transcription factor Oct-1, which regulates the transcription of a number of housekeeping genes. STAT3, STAT5, NF-κB, AP-1 and Oct-1 binding sites are underlined in the above sequences. It has been reported that NF-κB (p50, RelA and RelB), AP-1 (JunB and JunD), STAT3 and STAT5 specifically bind to the above oligonucleotides in HTLV-1-infected T-cell lines and ATLL cells (8,19).

C.B-17/Icr-severe combined immune deficient (SCID) mice are a useful model for investigating the proliferative and tumorigenic potential of HTLV-1-infected T-cell lines. HUT-102 cells (1×10⁷/0.2 ml RPMI-1640 medium) were subcutaneously transplanted into the postauricular region of 5-week-old female SCID mice (Kyudo, Co., Tosu, Japan) on day 0. The mouse body weight was 16.6–18.3 g upon purchase. Animal cages were maintained at a temperature of 24°C and a humidity of 60%. The mice were fed a standard rodent diet (CE-2; CLEA Japan, Inc., Tokyo, Japan) and water ad libitum. The mice were divided into 2 groups (n=5, each). Cerdulatinib was solubilized in 0.5% methylcellulose (cat. no. 133–17815; Wako Pure Chemical Industries) and administered at a dose of 35 mg/kg by oral gavage 5 times a week. The treatment was continued for up to 28 days starting from the day after cell inoculation. The control group received the vehicle (0.3 ml of 0.5% methylcellulose) only. The tumor diameter was measured weekly with a shifting caliper and tumor volume was calculated using the ellipsoid volume formula (π/6 × length × width × height) (20). Body weight was also measured weekly. The mice were sacrificed on day 28. Tumors were collected and their weight was measured. Experimental procedures and animal care were in compliance with the Guidelines for Animal Experimentation of University of The Ryukyus (Nishihara, Japan) and were approved by the Animal Care and Use Committee of University of The Ryukyus (reference no. A2016088).

Tumor specimens were collected from mice, fixed in formalin (Wako Pure Chemical Industries) solution, dehydrated through a graded ethanol series (Japan Alcohol Selling Co., Tokyo, Japan) and embedded in paraffin (cat. no. 09620; Sakura Finetek Japan Co., Tokyo, Japan). The specimens were cut into sections that were stained with hematoxylin and eosin (H&E; cat. nos. 234-12 and 1159350025; Merck KGaA) for histological examination. DNA fragmentation was analyzed with the TUNEL assay using a commercial kit (cat. no. 11684817910; Roche Applied Science, Penzberg, Germany) according to the manufacturer's instructions. Cells were examined under a light microscope (Axioskop 2 Plus) with an Achroplan 40×/0.65 lens (both from Zeiss, Hallbergmoos, Germany). Images were acquired with an AxioCam 503 color camera and AxioVision LE64 software (Zeiss GmbH, Jena, Germany).

Results are expressed as the means ± standard deviation (SD). The Student's t-test or ANOVA with the Tukey-Kramer test were used to compare the means of 2 groups or >2 groups, respectively. Differences were considered statistically significant at P<0.05.

Using the WST-8 assay, we first examined the cytotoxicity of various concentrations of cerdulatinib in HTLV-1-infected T-cell lines up to 72 h after treatment (Fig. 1A). Cerdulatinib decreased WST-8 activity (and thus, cell viability) in a dose- and time-dependent manner in all HTLV-1-infected T-cell lines. Notably, the uninfected Jurkat and CCRF-CEM T-cell lines were resistant to the cytotoxic effects of cerdulatinib. The effects of cerdulatinib on PBMCs from a healthy donor were less pronounced (Fig. 1B). The IC₅₀ values at 24 h were 3.5 µM for the MT-2, 7.3 µM for the HUT-102 and 8.4 µM for the TL-OmI cells, as compared to 62.9 µM for the normal PBMCs. In the Jurkat and CCRF-CEM cells, the IC₅₀ value was not attained with the administered doses. We compared the effects of cerdulatinib with those of two reference compounds, the SYK-selective inhibitor, PRT060318, and the pan-JAK inhibitor, JAK inhibitor 1, on the MT-2 and HUT-102 cells, and found that cerdulatinib decreased cell viability more potently than either single agent (Fig. 1, compare parts A and C). As PRT060318 or JAK inhibitor 1 decreased the viability of HTLV-1-infected T-cell lines, we further tested the combination of the two agents to determine whether they acted synergistically to suppress the viability of HTLV-1-infected T-cells. The CI was used to determine whether the effect of the combined treatment was synergistic, additive or antagonistic. Instead of a simple additive effect, the combination of PRT060318 and JAK inhibitor 1 exerted a synergistic cytotoxic effect on HUT-102 cells, with the optimal CI of 0.609 (Fig. 2).

The negative effect of cerdulatinib on cell viability (as determined using the WST-8 assay) may result from cell growth inhibition, death or both. To clarify the mechanisms underlying the inhibition of cell growth by cerdulatinib, we evaluated cell cycle distribution in the MT-2, HUT-102 and TL-OmI cells treated with 2.5 and 5 µM cerdulatinib for 24 h. Treatment with cerdulatinib led to a significant accumulation of cells in the G₂/M-phase, with concurrent decreases in the G₁- and/or S-phase fractions (Fig. 3). These results indicated that cerdulatinib inhibits cell proliferation by targeting the G₂/M checkpoint and blocking cell cycle progression.

We then examined the effect of cerdulatinib on cell death. A morphological analysis using Hoechst staining revealed chromatin condensation and nuclear fragmentation in the cells cultured with cerdulatinib (Fig. 4A). APO2.7 immunolabeling indicated that apoptosis was induced by cerdulatinib in a concentration- and time-dependent manner (Fig. 4B). To investigate the role of caspases in the response to cerdulatinib, cell lysates were examined by western blot analysis using specific antibodies. The induction of apoptosis of the HUT-102 cells at increasing concentrations of cerdulatinib was accompanied by the cleavage of caspase-8, -9 and -3, and its substrate PARP (Fig. 5A). We also examined the enzymatic activity of caspase-8, -9 and -3, as this is not always associated with pro-caspase processing, and found the significant activation of all three proteins in the HUT-102 and TL-OmI cells following 72 h of treatment with 10 µM cerdulatinib (Fig. 5B). These results suggested that cerdulatinib induces apoptosis via the activation of extrinsic and intrinsic pathways. Pre-treatment with the pan-caspase inhibitor, z-VAD-FMK, partly abrogated cerdulatinib-induced cytotoxicity (Fig. 5C), suggesting that the latter occurs through caspase-dependent apoptosis.

To determine whether cerdulatinib inhibits the SYK and JAK/STAT signaling pathways, we evaluated the phosphorylation status of SYK and JAK-STAT in the HUT-102 cells treated with cerdulatinib by western blot analysis, and found that SYK, JAK1, STAT3 and STAT5 phosphorylation was decreased (Fig. 6). SYK is associated with various immunoreceptors (21) and is thought to act upstream of phosphoinositide 3-kinase (PI3K)-AKT-mediated IKK-IκBα activation (21–23). In addition, JAK inhibition has been reported to suppress IL-2-mediated T-cell growth and STAT, AP-1 and ERK activation by IL-2 (24). In this study, we found that cerdulatinib blocked the phosphorylation of AKT, IKKα/β, IκBα and ERK, but not that of p38 (Fig. 6), and nuclear extracts obtained from the cerdulatinib-treated cells exhibited a reduced DNA binding of STAT3, STAT5, NF-κB and AP-1, but not Oct-1 (Fig. 7A). Thus, SYK and JAK/STAT, as well as downstream signaling pathways were effectively inhibited by cerdulatinib.

To further clarify the molecular mechanisms underlying the growth inhibitory and cytotoxic effects of cerdulatinib on HTLV-1-infected T-cell lines, we evaluated the expression of proteins associated with cell cycle progression and apoptosis. The results of western blot analysis revealed that cerdulatinib treatment increased the expression of p21 and p27, and decreased that of CDK1, cyclin B1, Bcl-xL, survivin, XIAP and c-FLIP (Fig. 7B and C). However, it had no effect on the levels of Mcl-1, c-IAP2, and the pro-apoptotic proteins, Bak and Bax. As p21 and p27 expression is modulated by p53, we also examined p53 expression, but found no obvious changes in the level of this protein in the cerdulatinib-treated cells.

We examined the in vivo efficacy of cerdulatinib in a mouse xenograft model of ATLL. HUT-102 cells were subcutaneously transplanted into SCID mice, which were then orally administered the vehicle or 35 mg/kg cerdulatinib 5 times weekly. The volume and weight of the transplanted tumors were lower in the mice treated with cerdulatinib than in the control group (Fig. 8A and B). Cerdulatinib was well tolerated, as it did not cause significant weight loss or other clinical manifestations (Fig. 8C). Tumor tissue from the mice treated with cerdulatinib examined by H&E staining (Fig. 8D, left panel) exhibited evidence of apoptosis, including cytoplasmic condensation, chromatin hyperchromatism and condensation, and nuclear fragmentation. Apoptosis in the tumor tissue from the cerdulatinib-treated mice was confirmed by TUNEL assay (Fig. 8D, right panel), which was consistent with our in vitro findings. These results demonstrated the effectiveness of cerdulatinib in controlling ATLL progression in vivo.