Molecular characteristics of CTA056, a novel interleukin-2-inducible T-cell kinase inhibitor that selectively targets malignant T cells and modulates oncomirs

Abstract

Interleukin-2-inducible T-cell kinase (Itk) is a member of the Btk (Bruton's tyrosine kinase) family of tyrosine kinases. Itk plays an important role in normal T-cell functions and in the pathophysiology of both autoimmune diseases and T-cell malignancies. Here, we describe the initial characterization of a selective inhibitor, 7-benzyl-1-(3-(piperidin-1-yl)propyl)-2-(4-(pyridin-4-yl)phenyl)-1H-imidazo[4,5-g]quinoxalin-6(5H)-one (CTA056), that was developed through screening a 9600-compound combinatorial solution phase library, followed by molecular modeling, and extensive structure-activity relationship studies. CTA056 exhibits the highest inhibitory effects toward Itk, followed by Btk and endothelial and epithelial tyrosine kinase. Among the 41 cancer cell lines analyzed, CTA056 selectively targets acute lymphoblastic T-cell leukemia and cutaneous T-cell lymphoma. Normal T cells are minimally affected. Incubation of Jurkat and MOLT-4 cells with CTA056 resulted in the inhibition of the phosphorylation of Itk and its effectors including PLC-γ, Akt, and extracellular signal-regulated kinase, as well as the decreased secretion of targeted genes such as interleukin-2 and interferon-γ. Jurkat cells also underwent apoptosis in a dose-dependent manner when incubated with CTA056. The potent apoptosis-inducing potential of CTA056 is reflected by the significant modulation of microRNAs involved in survival pathways and oncogenesis. The in vitro cytotoxic effect on malignant T cells is further validated in a xenograft model. The selective expression and activation of Itk in malignant T cells, as well as the specificity of CTA056 for Itk, make this molecule a potential therapeutic agent for the treatment of T-cell leukemia and lymphoma.

Fig. 1.

Synthetic scheme of CTA056 (A) and molecular modeling studies of binding conformation of CTA056 to the kinase domain of human Itk (B). The docking pocket was created from the crystal structure of the kinase domain of Itk using AutoDock 4.0. Docking was performed using AutoDock Vina 1.0, and the docking results were analyzed using PyMOL 0.99. CTA056 was shown to bind to the ATP-binding pocket in the kinase domain of Itk.

Fig. 2.

Kinase inhibitory activity of CTA056 (A) and inhibition of Itk autophosphorylation by CTA056 (B). Kinase activity was measured using TLC assay. Purified kinase (20 nM), CTA056 (0–10 μM), and the peptide substrate were incubated with [³³P]ATP in a kinase reaction. The resulting product was analyzed on a TLC plate. The relative kinase activity to control was calculated using densitometry. Itk autophosphorylation activity was measured by an in vitro kinase assay. Purified Itk (100 ng) was mixed with CTA056 inhibitor, the ice-cold ATP and hot r-[³³P]ATP in the kinase assay buffer, and the Itk auto-kinase activity was analyzed by filmless autoradiographic analysis (Bio-Rad Laboratories). Columns, mean; bars, S.D., n = 3.

Fig. 3.

Expression profile of Itk in 45 cell lines (41 cancer cell lines and 4 normal cell lines) and CTA056-induced growth inhibition of a panel of cancer cells. The expression profile of Itk was measured using Western blot (A). For growth inhibition, cells were seeded at 5000 cells/well in 96-well plate overnight and were treated with CTA056 (B, 0 and 2 μM; C, 0–10 μM). After 72 h, cell proliferation was measured using the MTT assay. Columns, mean; bars, S.D., n = 3.

Fig. 4.

Itk inhibition and growth inhibition to Jurkat cells of a series of CTA compounds. Kinase activity was measured using TLC assay. Purified Itk (20 nM), CTA compounds (1 μM), and the peptide substrate TSFYGRH were incubated with [³³P]ATP in a kinase reaction. The resulting product was analyzed on a TLC plate. For growth inhibition, cells were seeded at 5000 cells/well in 96-well plate overnight and were treated with CTA compounds (2 μM). After 72 h, cell proliferation was measured using the MTT assay. Columns, mean; bars, S.D., n = 3. CTA056 was shown to inhibit Itk and inhibit the growth of Jurkat cells.

Fig. 5.

Induction of apoptosis of Jurkat cells after treatment with CTA056. Jurkat cells were seeded at 10⁶ cells/ml (2 ml) in a six-well plate and treated with CTA056 at the indicated concentrations for 24 h. Cell-cycle arrest was analyzed using PI staining (A). Apoptosis was analyzed using Annexin-V-FITC apoptosis detection kit (Abcam, Inc.) (B). Columns, mean; bars, S.D., n = 3. 125, 250, 500, and 1000 nM are significantly different from control (*, p < 0.05, one-way ANOVA with Tukey's test for pairwise comparison).

Fig. 6.

Inhibition of Itk phosphorylation in Jurkat cells after treatment with CTA056. Itk phosphorylation in Jurkat cells was examined using Western blot (A). Cells were seeded at 10⁶ cells/ml (5 ml) and treated with CTA056 at the indicated concentrations for 24 h. Before harvest, cells were pretreated with pervanadate for 15 min. pItk, Itk, and actin levels were measured using the corresponding antibodies through Western blot. B, Jurkat cells were pretreated with CTA056 30 min and then incubated with anti-CD3, anti-CD28 monoclonal antibodies (10 μg/ml), and protein G (10 μg/ml). At 30 s, 1 min, and 5 min, the stimulation was stopped. The cells were then permeabilized and stained with a phospho-Itk-specific antibody. One of three similar experiments is depicted. Values are averages of triplicate samples.

Fig. 7.

Inhibition of cell signaling in Jurkat and MOLT-4 cells after treatment with CTA056. Cells were seeded at 10⁶ cells/ml (5 ml) and pretreated with 0.5 μM CTA056 for 30 min. Cells were then stimulated with anti-CD3, anti-CD28 monoclonal antibodies (10 μg/ml), and protein G (10 μg/ml) for 1 min. pPLC-γ1, PLC-γ1, pERK, ERK, pSrc, Src, pAkt, and Akt levels were measured using the corresponding antibodies through Western blot. One of three similar experiments is depicted.

Fig. 8.

Inhibition of IL-2 and IFN-γ mRNA level (A) and inhibition of IL-2 (B) and IFN-γ (C) release of Jurkat cells after treatment with CTA056. For mRNA levels, Jurkat cells were pretreated with CTA056 0.5 μM for 30 min and then stimulated with anti-CD3 and anti-CD28 antibodies (1 μg/ml) for 6 h. RNA was isolated and the mRNA levels were measured using real-time PCR. Supernatants were then harvested and IL-2 was quantified using a sandwich ELISA. For interferon-γ release assay, Jurkat cells were pretreated with CTA056 (0, 50, and 500 nM) or methotrexate (MTX) for 30 min then stimulated with recombinant human IL-2 and anti-CD3 antibody (1 μg/ml) for 6 h. IFN-γ was quantified using the enzyme-linked immunosorbent spot assay. Values are averages of triplicate samples (columns, mean; bars, S.D.). CTA056 treatment significantly decreased IL-2 and IFN-γ secretion from Jurkat cells compared with control. *, p < 0.05, one-way ANOVA with Tukey's test for pairwise comparison.

Fig. 9.

Induction of tumor suppressive miRNAs and decrease of oncogenic miRNAs in Jurkat cells after treatment of 0.5 μM CTA056 for 8 h. RNA was isolated and miRNA profile was examined using miRNA array. The fold change between treatment and control was indicated. Green columns, tumor suppressive miRNAs; red columns, oncogenic miRNAs.

Fig. 10.

Inhibition of MOLT-4 xenograft tumor growth by CTA056. Ten million MOLT-4 cells were injected subcutaneously to nude mice. The tumors were grown to the indicated size, and the mice were randomly divided into two groups (eight mice per group). The control group was treated with vehicle. The treatment group was treated with CTA056 at 5 mg/kg twice a week with intratumoral injection. The tumor size (A) and body weight (B) were measured once a week. Marks, mean; bars, S.D.; n = 8.