An IκB kinase 2 inhibitor IMD‐0354 suppresses the survival of adult T‐cell leukemia cells

Abstract

Adult T‐cell leukemia (ATL) is a fatal T‐cell malignancy associated with human T‐cell leukemia virus type I infection. The aberrant expression of nuclear factor‐κB (NF‐κB) is considered to contribute to the malignant phenotype and chemo‐resistance of ATL cells. Because of the poor prognosis of ATL, the development of new therapeutic strategies is direly needed. In the present study, we show that an IκB kinase 2 (IKK2) inhibitor, IMD‐0354, efficiently inhibits the survival of CD4⁺ CD25⁺  primary ATL cells and prevents the growth of or induces apoptosis of patient‐derived ATL cell lines. Assays of transcription with integrated forms of reporter genes revealed that IMD‐0354 suppresses NF‐κB‐dependent transcriptional activity. Moreover, the daily administration of IMD‐0354 prevents the growth of tumors in mice inoculated with ATL cells. Our results suggest that targeting IKK2 with a small molecule inhibitor, such as IMD‐0354, is an attractive strategy for the treatment of ATL. (Cancer Sci 2012; 103: 100–106)

Human T‐cell leukemia virus type I (HTLV‐I) is the etiological agent of adult T‐cell leukemia (ATL) and is known to transform T lymphocytes.^{( 1} , ² , ^{3 )} While the majority of infected individuals remain asymptomatic during their lifetime, 2–5% of carriers develop an aggressive form of CD4‐positive T‐cell leukemia after a long latency period of 50–60 years, and another 0.25–3% of carriers develop a chronic neuro‐inflammatory disorder termed HTLV‐I‐associated myelopathy/tropical spastic paraparesis.^{( 4} , ⁵ , ⁶ , ^{7 )} At present, there are few established regimens for ATL treatment, mainly because of the intrinsic resistance to conventional drug therapies.^{( 8 )} Thus, novel therapeutic interventions are urgently required. Although the viral oncoprotein Tax is believed to play a central role in HTLV‐I‐induced leukemogenesis,^{( 9} , ^{10 )} the expression levels of Tax and other viral proteins are extremely low in leukemic cells freshly isolated from ATL patients and ATL‐derived cell lines.^{( 11} , ^{12 )} The tax gene is even mutated or truncated in some ATL cases.^{( 13} , ^{14 )} Thus, Tax might not necessarily be required at later stages of disease progression, and Tax‐independent mechanisms are likely to contribute to the proliferation and resistance to apoptosis of leukemic cells. The constitutive activation of nuclear factor‐κB (NF‐κB) was found in peripheral ATL cells and ATL‐derived cell lines,^{( 15 )} implying important prosurvival roles of NF‐κB in ATL cells. Nuclear factor‐κB activation has long been implicated in association with multiple processes of oncogenesis, including cell‐cycle progression, control of apoptosis, migration, and metastasis.^{( 16 )} Thus, the inhibition of NF‐κB was suggested to be a potential means for cancer therapy in tumors highly expressing NF‐κB.^{( 17 )} We previously reported that a reduction of NF‐κB activity by the forced expression of a dominant‐negative form of IκBα strongly suppresses the survival of ATL cell lines, and that abrogation of NF‐κB activity by a protease inhibitor, ritonavir, significantly prevented the growth of tumors in non‐obese, diabetic/SCID/γc^null (NOG) mice inoculated with primary ATL cells.^{( 18} , ^{19 )}

Nuclear factor‐κB, a dimeric form of the Rel family proteins, is an inducible transcription factor that regulates a wide variety of gene expression, including those encoding cytokines, chemokines, anti‐apoptotic factors, and inducible enzymes.^{( 20 )} Nuclear factor‐κB exists as an inactive form by binding its endogenous inhibitory molecule, IκB. The exposure of cells to stimuli, such as LPS, phorbol ester, or certain inflammatory cytokines, activates the IκB kinase (IKK) complex, and then IκB is rapidly phosphorylated, ubiquitinated, and degraded by the proteasome, releasing NF‐κB into the nucleus to initiate the expression of target genes.^{( 21 )}

IMD‐0354 was originally designed to competitively interrupt the access of ATP to its docking site on IKK2, thereby suppressing the activity of the IKK complex.^{( 22} , ^{23 )} Previously, we and others reported that IMD‐0354 attenuates myocardial ischemia/reperfusion injury and oncogenic proliferation of pancreatic cancer cells.^{( 22} , ^{24 )} In the present study, we examined whether IMD‐0354 is capable of suppressing the viability of ATL cells through the inhibition of NF‐κB activity.

Materials and Methods

Patient samples.  Peripheral blood mononuclear cells (PBMCs) from ATL patients were obtained with informed consent at Imamura Bun‐in Hospital (Kagoshima, Japan), and supplied through the Joint Study on Predisposing Factors of ATL Development. The diagnosis of ATL was based on clinical features, hematological findings, and the presence of the HTLV‐I proviral genome in the leukemia cells and antibodies to ATL‐associated antigens in serum. The use of PBMCs from ATL patients for research purposes was approved by the institutional review board of each institute. Peripheral blood was also obtained from healthy donors under informed consent. Peripheral blood mononuclear cells were purified by density gradient separation with Ficoll–Plaque PLUS (GE Healthcare Bio‐Sciences AB, Uppsala, Sweden), washed twice with 1× PBS containing 2% FBS, and maintained in RPMI‐1640 supplemented with 10% FBS, 100 units/mL penicillin G, and 100 μg/mL streptomycin sulfate. Cell viability was determined by MTT assay, as described previously.^{( 25 )}

Cells and reagents.  Jurkat, Molt4, and CEM are HTLV‐I‐free human T‐lymphoblastic leukemia cell lines. MT‐2 and SLB‐1 are HTLV‐I‐infected T‐cell lines established in vitro. ATL‐43Tb(−), ATL‐55T(−), ED40515(−), and TL‐Om1 are T‐cell lines of leukemic cell origin established from ATL patients. The clonal origin of HUT102 is an HTLV‐I‐infected cell derived from a lymph node biopsy sample of a patient with cutaneous T‐cell lymphoma. Cells were cultured in RPMI‐1640, conditioned as described earlier. DMSO was purchased from Wako Pure Chemical Industries (Osaka, Japan). IMD‐0354 was kindly provided from Institute of Medicinal Molecular Design (IMMD) Inc. (Tokyo, Japan). In all cases, the final DMSO concentration was 0.1%. Experiments were performed with cell lines utilizing logarithmically‐growing cells (3–5 × 10⁵/mL) or with PBMCs at an approximate cell density of 1 × 10⁶/mL. Cell viability was determined by MTT assay. A proteasome inhibitor, MG132, was purchased from Peptide Institute Inc. (Osaka, Japan).

Plasmids.  The lentivirus vector CS‐κB‐R2.2 was described previously.^{( 26 )} For the construction of pCS‐EF‐1α‐Rluc‐puro (pCERp), the elongation factor (EF)‐1α promoter sequence from the pEF/myc/cyto vector (Invitrogen, Carlsbad, CA, USA) was inserted between the KpnI and SalI sites of the pGL4.84 vector (Promega, Madison, WI, USA), generating pGL4‐EF‐1α‐Rluc‐puroR (pGERp). The entire transcription cassette was then excised from pGERp by KpnI and XhoI digestion, and transferred to the KpnI and XhoI sites of pCS‐short3(−)‐MCS.^{( 26 )} The resultant plasmid was designated as pCERp. For the production of lentivirus vectors, 293T cells were cotransfected with a lentivirus vector, together with the pCMVΔR8.2 packaging construct and pHCMV‐VSV‐G (kind gifts from Dr I.S.Y. Chen, UCLA, Los Angeles, CA, USA), using the calcium phosphate precipitation method. Culture supernatants were collected 60 h after transfection and filtrated.

Western blot analysis.  For the preparation of whole cell extracts, the cells were suspended in RIPA buffer with protease inhibitors, as described previously.^{( 25 )} Extracts were cleared by centrifugation. Whole cell extracts were fractionated on 12% SDS–PAGE and transferred onto Immobilon membranes (Millipore Corporation, Billerica, MA, USA). The primary antibodies to phospho‐IκBα (Cell Signaling, Danvers, MA, USA), IκBα (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and α‐tubulin (Sigma‐Aldrich, St Louis, MO, USA) were used. Blots were revealed with an enhanced chemiluminescence detection system (Perkin Elmer, Waltham, MA, USA). All the experiments were repeated at least twice.

Morphological analysis.  T‐cell lines were treated with 2.5 μM of IMD‐0354 or 0.1% DMSO for 24 h, washed with 1× PBS, and fixed with 1% formaldehyde in 1× PBS at room temperature for 15 min. After centrifugation, the cells were resuspended in 1× PBS containing RNase A (0.1 mg/mL), and stained with Hoechst 33342 (0.2 μg/mL; Invitrogen, Carlsbad, CA, USA) at room temperature for 15 min in a dark place. The cells were then washed with 1× PBS, and microscopic images of stained cells were obtained.

Cell‐cycle analysis.  T‐cell lines and PBMCs were treated with 2.5 μM IMD‐0354 or 0.1% DMSO for 24 h, washed with 1× PBS, and fixed in PBS with 70% ethanol at room temperature for 15 min. After centrifugation, the cells were resuspended in 1× PBS containing RNase A (0.1 mg/mL) and stained with 100 μg/mL propidium iodide for 10 min in a dark place. Cell suspensions were analyzed on FACSCalibur, using CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA).

Flow cytometry.  The population of CD4 and CD25‐double positive cells in the PBMCs from ATL patients was evaluated by flow cytometric analysis, utilizing CD4‐R‐phycoerythrin, CD25‐fluorescein isothiocyanate (Dako, Carpinteria, CA, USA), and 7‐aminoactinomycin D (7‐AAD; BD Biosciences Pharmingen, San Diego, CA, USA). After staining with the antibodies and 7‐AAD in staining buffer (1× PBS containing 5% FBS and 0.1% sodium azide) for 30 min on ice, the cells were washed with cold 1× PBS containing 2% FBS and applied for FACS analysis.

Dual luciferase assay.  Two hundred microliters of T‐cell cultures (3 × 10⁵/mL) carrying two integrated lentivirus vectors (CS‐κB‐R2.2 and pCERp) were treated with several concentrations of IMD‐0354 or 0.1% DMSO for 24 h in a 96‐well plate with a round bottom. Plates were centrifuged at 760g for 3 min, and 180 μL of the supernatant was removed. Five microliters of 5× passive lysis buffer (Promega) was added to each well, and a half amount of total lysates was transferred to a white‐wall 96‐well plate (Corning, Corning, NY, USA), according to the manufacturer’s instructions. The luminescence activities were detected by the GloMax Multi Luminescence System (Promega). Firefly luciferase activity was normalized on the basis of Renilla luciferase activity, whose value was expressed as relative light unit. Each experiment was repeated at least three times, and the results are expressed as mean ± SD.

Caspase 3/7 assay.  Peripheral blood mononuclear cells (5 × 10⁵/mL) were treated with increasing concentrations of IMD‐0354 or 0.1% DMSO for 24 h in a 96‐well plate. After treatment, 10 μL of cell suspension was mixed with 10 μL of Caspase‐Glo 3/7 reagent (Promega), and luciferase activity was determined after 30 min of incubation, as described for the dual luciferase assay.

Mice.  Non‐obese, diabetic/SCID/γc^null mice were purchased from the Center Institute for Experimental Animals (Kawasaki, Japan). The mice were maintained under specific pathogen‐free conditions at the Experimental Animal Center of Tokyo Medical and Dental University (Tokyo, Japan). The ethical review board of the university approved the experimental protocol. Mice were anesthetized with ether, and ATL cells (1 × 10⁷) were inoculated subcutaneously in the postauricular region of the NOG mice. All mice were sacrificed 30 days after inoculation with ATL cells, and the weight of each tumor was measured.

Results

IMD‐0354 impairs the viability of primary ATL cells.  We first investigated the effect of IMD‐0354 on PBMCs isolated from ATL patients or healthy donors. The cells were treated with various concentrations (0.1–3 μM) of IMD‐0354 or 0.1% DMSO for 48 h, and the viability of the cells was examined by MTT assay (Fig. 1a). A total of 1 μM IMD‐0354 significantly reduced the viability of PBMCs from ATL patients, while PBMCs from healthy donors remained viable with 3 μM IMD‐0354. The average IC₅₀ values of IMD‐0354 against PBMCs from ATL patients and healthy donors were 2.87 and >3.0, respectively. The surface phenotype of ATL cells is reportedly characterized by the expression of the CD4 and CD25 antigens.^{( 27} , ²⁸ , ^{29 )} FACS analyses revealed that treatment with IMD‐0354 reduced the fraction of CD4⁺ CD25⁺  ATL cells (Fig. 1b). Statistical analyses showed that the ratios of CD4⁺ CD25⁺  cells/CD4⁻CD25⁻ cells were lower for IMD‐0354‐treated cells than for DMSO‐treated cells (Fig. 1c). In addition, IMD‐0354 treatment of PBMCs from ATL patients resulted in a significant increase in the subG1 fraction (Fig. 1d). Indeed, treatment with more than 2.5 μM of IMD‐0354 significantly induced caspase 3/7 activation in PBMCs from ATL patients free from anti‐cancer drug treatment, but not in those from healthy donors or from ATL patients under chemotherapy (Fig. 1e). These results indicate that IMD‐0354 selectively impairs the viability of CD4⁺ CD25⁺  primary ATL cells in vitro.

Figure 1.

 IMD‐0354 inhibits the survival of CD4⁺ CD25⁺  primary adult T‐cell leukemia (ATL) cells. (a) Approximately 1 × 10⁶ cells/mL of peripheral blood mononuclear cells (PBMCs) derived from ATL patients and healthy donors were treated with 0.1–3 μM IMD‐0354 for 48 h, and then subjected to MTT assay. Relative cell viability, in comparison with control cells (treated with DMSO only, arbitrarily set at 1), is shown. Average IC₅₀ values of IMD‐0354 against PBMCs are also shown. (b) FACS analysis of the CD4‐ and CD25‐positive cells in PBMCs from ATL patient no. 15 before and after treatment with IMD‐0354 (1 μM) or control DMSO. (c) IMD‐0354 reduced the population of CD4⁺ CD25⁺  ATL cells. Ratio of CD4⁺ CD25⁺ /CD4⁻CD25⁻ cells was analyzed with PBMCs from ATL patients by flow cytometry, as shown in (b). Values were calculated with the following equation: relative ratio = ([no. CD4⁺ CD25⁺  cells on day 2/no. CD4⁻CD25⁻ cells on day 2]/(no. CD4⁺ CD25⁺  cells on day 0/CD4⁻CD25⁻ cells on day 0). (d) Peripheral blood mononuclear cells were treated as described in (b), and subjected to flow cytometric analysis to examine cells in the subG1 fraction following IMD‐0354 treatment. Each value represents cell number in the subG1 fraction of the IMD‐0354‐treated sample relative to that of DMSO‐treated sample. (e) Caspase 3/7 activation was determined after treatment of PBMCs with 2.5 μM of IMD‐0354 for 24 h. The fold caspase 3/7 activation in comparison to the control cells treated with DMSO is shown. (i) untreated ATL patients (n = 5); (ii) ATL patients under chemotherapy (n = 7); (iii) healthy donors (n = 3). *P < 0.05 and **P < 0.01 versus DMSO‐treated samples. n.s., not significant.

IMD‐0354 affects the viability of HTLV‐I‐infected T‐cell lines.  Among the HTLV‐I‐infected cell lines, MT‐2 and SLB‐1 cells were established in vitro by coculture with PBMCs derived from ATL patients, while HUT102, ATL‐43Tb(−), ED40515(−), ATL‐55T(−), and TL‐Om1 cells were derived from PBMCs of ATL patients. The cells were cultured in the presence of varying concentrations (0.1–3 μM) of IMD‐0354 for 24, 48, and 72 h, and examined for cell growth by MTT assay. As shown in Figure 2(a), IMD‐0354 efficiently impaired the growth of the HTLV‐I‐infected cell lines in a dose‐dependent manner. The growth of HTLV‐I‐free Jurkat and Molt4 T‐cell lines was also affected. FACS analyses revealed that brief treatment with IMD‐0354 induced a quick and robust increase in the subG1 population of ATL‐43Tb(−) cells, but not of MT‐2 cells or PBMCs from a healthy donor (Fig. 2b), while longer exposure of MT‐2 cells to IMD‐0354 finally induced cell death (data not shown). Rapid increase in the subG1 fraction similar to IMD‐0354‐treated ATL‐43Tb(−) cells was observed in the IMD‐0354‐treated HUT102 and ATL55(−) cells, but not in the IMD‐0354‐treated ED40515(−), TL‐Om1, and SLB‐1 cells (data not shown). Consistently, nuclei of ATL‐43Tb(−) and ATL‐55T(−) cells became fragmented by 24 h treatment with IMD‐0354, while those of MT‐2 and Jurkat cells did not with short‐term treatment (Fig. 2c). These two phenotypes in response to IMD‐0354 treatment correlated with the restoration of cell growth following removal of IMD‐0354 from culture medium. The HTLV‐I‐infected and HTLV‐I‐free cell lines were cultured in the presence of 2.5 μM IMD‐0354 for 18 h, and then refed with IMD‐0354‐free fresh medium. While Jurkat and Molt4 cells restored their growth soon after the removal of IMD‐0354, the ATL‐43Tb(−) and ATL‐55T(−) cells failed to proliferate and eventually died (Fig. 2d). The growth of MT‐2 cells after the removal of IMD‐0354 was greatly delayed. Control treatment with DMSO did not affect the growth of any cell line (data not shown).

Figure 2.

 IMD‐0354 suppresses the viability of adult T‐cell leukemia (ATL) cell lines. (a) Dose‐dependent effect of IMD‐0354 on the growth of ATL‐derived (ATL‐43Tb[−], ATL‐55T[−], ED40515, and TL‐Om1) human T‐cell leukemia virus type I (HTLV‐I)‐infected, and HTLV‐I‐free T‐cell lines. Cells (3 × 10⁵ cells/mL) were cultured with 0.1–3 μM IMD‐0354 for 48 h. Cell growth was assessed by MTT assay, and is expressed as a ratio of viable cells treated with the indicated concentrations of IMD‐0354 to those with DMSO. Data are expressed as the mean ± SD of three independent experiments. (b) Adult T‐cell leukemia‐43Tb(−) cells, MT‐2 cells and peripheral blood mononuclear cells (PBMCs) from a healthy donor were treated with 2.5 μM IMD‐0354 for 24 h. DNA content of the treated cells was analyzed by flow cytometry following propidium iodide staining. DMSO was used as the negative control. (c) Adult T‐cell leukemia‐43(−), ATL‐55T(−), MT‐2, and Jurkat cells were treated with 2.5 μM IMD‐0354 for 24 h. Phase contrast and fluorescent images were obtained following nuclear staining with Hoechst33342. DMSO was used as the negative control. (d) ATL‐43Tb(−), ATL‐55T(−), MT‐2, Jurkat, and Molt4 cells were pretreated with 2.5 μM IMD‐0354 for 18 h. Cells were refed at the 0 h time point with IMD‐03524‐free fresh medium, and cultured for the indicated period of time. Cell number was assessed by the trypan blue dye exclusion assay. Results are expressed as the relative numbers of viable cells to untreated cells at −18 h.

IMD‐0354 inhibits NF‐κB activation in ATL cells.  To assess how IMD‐0354 influences NF‐κB‐dependent transcriptional activity, we developed a lentivirus vector‐mediated reporter gene assay system. The HTLV‐I‐infected cell lines were simultaneously infected with lentivirus vectors carrying either a NF‐κB‐responsive promoter‐driven firefly luciferase gene or constitutive EF‐1α promoter‐driven Renilla luciferase gene. The integrated genes were stably expressed in infected cells for more than 1 month (data not shown). Cells transduced with the above vectors were then treated with various concentrations (0.625–10  μM) of IMD‐0354 for 24 h and subjected to dual luciferase assay. Figure 3(a) shows that IMD‐0354 decreased NF‐κB‐dependent transcriptional activity in these cell lines in a dose‐dependent manner. Western blot analyses revealed that the phosphorylation of IκBα was impaired by the treatment of HTLV‐I‐infected cells with 2.5 μM of IMD‐0354, and that a proteasome inhibitor, MG132, did not restore the phosphorylation of IκBα in these cell lines, except for HUT102, which indeed had a reduced amount of phosphorylated IκBα with 2.5 μM of IMD‐0354 and MG132, but expressed it at levels comparable to the control with MG132 and higher concentrations of IMD‐0354 (Fig. 3b and data not shown). These results suggest that IMD‐0354 inhibited the transcriptional activity of NF‐κB.

Figure 3.

 IMD‐0354 inhibits constitutive nuclear factor‐κB (NF‐κB) activation. (a) Adult T‐cell leukemia (ATL)‐43Tb(−), HUT102, and MT‐2 cells were infected with the two lentivirus vectors encoding either an NF‐κB‐dependent promoter‐driven firefly luciferase gene or a constitutively‐active elongation factor (EF)‐1α promoter‐driven Renilla luciferase gene. Pools of lentivirus‐infected cells were treated with various concentrations (0.625–10 μM) of IMD‐0354 or 0.1% DMSO for 24 h, and subjected to dual luciferase assay. Each firefly luciferase activity was normalized with Renilla luciferase activity. Relative light units (RLU) in comparison to control cells (treated with DMSO only) are shown. Data are expressed as mean ± SD of three independent experiments. P‐values are versus control. (b) Jurkat, ATL‐43Tb(−), and HUT102 cells were treated with IMD‐0354 (2.5 μM) or DMSO for 48 h. In parallel, these cells were treated with or without MG‐132 (20 μM) 4 h before extraction of whole‐cell lysates. Thirty micrograms of lysates were subjected to immunoblotting with antiphosphorylated IκBα, anti‐IκBα, or anti‐α‐tubulin antibodies.

IMD‐0354 suppresses tumorigenicity of ATL cells in mice.  The ATL‐43Tb(−) cells were subcutaneously inoculated in the postauricular region of NOG mice. IMD‐0354 (5 mg/kg) was administered daily intraperitoneally for 4 weeks from 1 day after cell inoculation. The mice were then killed, and the weight of tumors was measured. Figure 4(a) shows that an intraperitoneal injection of IMD‐0354 suppressed the growth of the tumor. Statistical analyses revealed significant suppression of tumor growth by IMD‐0354 (P < 0.01) (Fig. 4b). Moreover, local injection of IMD‐0354 at sites close to that of cell inoculation almost completely inhibited tumor formation (Fig. 4c,d). Of note, we did not observe any deleterious effect of IMD‐0354 on the mice, such as body weight loss or dermatitis. Thus, IMD‐0354 successfully inhibited the neoplastic growth of ATL‐43Tb(−) cells in vivo.

Figure 4.

 IMD‐0354 suppresses the growth of adult T‐cell leukemia (ATL) cells in a non‐obese, diabetic/SCID/γc^null mouse model. Mice were injected subcutaneously with ATL‐43Tb(−) cells (1 × 10⁷ cells) in the post‐auricular region. (a) One day after inoculation of the cells, the mice were intraperitoneally administered either 1% DMSO or IMD‐0354 (5 mg/kg) in 1× PBS every day for 30 days. Representative photographs of mice and tumors formed 31 days after cell inoculation are shown. (b) Statistical analysis of the tumor weights obtained after intraperitoneal injection. (c) After cell inoculation, either 1% DMSO or IMD‐0354 (5 mg/kg) in 1× PBS was administered to mice at sites close to those of cell inoculation every day for 30 days. (d) Statistical analysis of the tumor weights obtained after local injection. Each result was obtained from six different mice. Tumor formation in mice was evaluated 31 days after cell inoculation. Means are shown with SD. P‐values are versus DMSO control. *P < 0.01, t‐test.

Discussion

In the present study, we demonstrated that a low dose of IMD‐0354 efficiently subverts CD4⁺ CD25⁺  primary ATL cells without apparent toxicity to control cells (Fig. 1). Accordingly, caspase activation was demonstrated in PBMCs from untreated ATL patients. One reason for the failure to detect caspase activation in PBMCs from ATL patients under chemotherapy would be the paucity of CD4‐ and CD25‐positive cells; on average, only 2.9% of PBMCs for ATL patients under chemotherapy and 42.1% for those untreated were positive in CD4 and CD25. In this study, we developed a new dual luciferase assay system employing lentivirus vectors. Lentivirus infection enabled us to introduce the reporter genes quite efficiently and stably into the suspension cells, overcoming the difficulty and toxicity of transfection by conventional means, such as electroporation and lipofection. As a result, the dual luciferase assay clearly demonstrated that IMD‐0354 reduced the transcriptional activity of NF‐κB in HTLV‐I‐infected cells in a dose‐dependent manner, which was consistent with the results of the Western blot analyses (Fig. 3). The reason for the substantial expression of phosphorylated IκBα in HUT102 cells treated with IMD‐0354 and MG132 remains unknown. It should be noted, however, that NF‐κB‐dependent transcription and IκBα phosphorylation were both suppressed by IMD‐0354 in HUT102 cells in the absence of MG132. In addition, the growth of ATL cells inoculated in NOG mice was effectively inhibited by IMD‐0354 without deleterious side‐effects (Fig. 4). The animals employed in this study are not suitable to see its immunological effects, but depending on the mode of administration of the drug, certain immunological side effects could potentially be encountered in immune‐competent hosts, and we envisage that immunological complications could be minimized by means of intermittent administration of the drug. These results strongly support the idea that treatment with a small molecular‐weight NF‐κB inhibitor, such as IMD‐0354, could be an effective means to eradicate neoplastic cells from ATL patients. No IKK2 inhibitor is known to be on the way to clinical application at present,^{( 30 )} except for IMD‐1041, a prodrug of IMD‐0354 for oral administration. For the IMD‐1041 capsule, a phase 1 study was completed, and proof‐of‐concept studies have been or are being conducted with administration for up to 12 weeks. IMD‐1041 proved to be rapidly converted into IMD‐0354 as a sole metabolite. Sufficient safety was confirmed, with very few adverse events ranked as mild, and there were no abnormalities in hematology and blood chemistry. Regarding the efficacy of IMD‐1041, potent antitissue remodeling and anti‐inflammatory actions were observed with significantly reduced expression of plasminogen activator inhibitor‐1 and inflammatory cytokines.^{( 31 )}

MTT assays demonstrated that IMD‐0354 suppressed the growth of all the T‐cell lines examined, but the response of cells differed depending on the cell lines. The ATL‐43Tb(−), ATL‐55T(−), and HUT102 cells underwent apoptosis after short‐term exposure to IMD‐0354, whereas ED40515(−), TL‐Om1, MT‐2, SLB‐1, and Jurkat cells stopped cell‐cycle progression and remained viable (Fig. 2). Moreover, the cell fates did not correlate with impaired NF‐κB‐dependent transcription or IκBα phosphorylation, which were observed in all of the tested HTLV‐I‐infected cells exposed to IMD‐0354. These results suggest that other molecular mechanisms, such as interleukin‐6/signal transduction and activator of transcription (STAT)‐, activator protein 1 (AP‐1)‐, or Rho GTPase‐mediated signaling,^{( 32} , ^{33 )} might contribute to the lack of immediate apoptosis induction after IMD‐0354 treatment. This also suggests that the suppression of only one signaling pathway might not be sufficient to induce apoptosis in some ATL cells. Indeed, many reports point to the importance of combined treatment of cancer cells with an NF‐κB inhibitor and other chemotherapeutic agents, expecting synergistic antitumor effects.^{( 34} , ^{35 )} Optimal combinations of IMD‐0354 with other anticancer agents should be further explored. Of note, treatment with IMD‐0354 alone suppressed the proliferation of not only ATL cell lines, but also HTLV‐I‐infected cell lines established by infection in vitro, which express the viral oncoprotein, Tax, suggesting that IMD‐0354 could eventually prevent the emergence of leukemic cell clones in asymptomatic carriers,^{( 36 )} and also be useful in controlling NF‐κB activity of HTLV‐I antigen‐expressing cells in human T‐cell leukemia virus type I‐associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients.^{( 37 )}

In conclusion, IMD‐0354 effectively suppressed the survival of primary and established ATL cells in vitro and in vivo. These results raise the possibility of small molecular‐weight IKK inhibitors, such as IMD‐0354, as a novel therapeutic agent for ATL treatment. Further investigations will be required for establishing a novel regimen to improve the prognosis of ATL.

Disclosure Statement

Akiko Itai is employed by IMMD and holds more than 5% of the stocks of Institute of Medicinal Molecular Design (IMMD) Inc. Susumu Muto is employed by IMMD. The other authors have no conflict of interest.