Tofacitinib induces G1 cell-cycle arrest and inhibits tumor growth in Epstein-Barr virus-associated T and natural killer cell lymphoma cells

Abstract

Epstein-Barr virus (EBV) infects not only B cells, but also T cells and natural killer (NK) cells, and is associated with T or NK cell lymphoma. These lymphoid malignancies are refractory to conventional chemotherapy. We examined the activation of the JAK3/STAT5 pathway in EBV-positive and -negative B, T and NK cell lines and in cell samples from patients with EBV-associated T cell lymphoma. We then evaluated the antitumor effects of the selective JAK3 inhibitor, tofacitinib, against these cell lines in vitro and in a murine xenograft model. We found that all EBV-positive T and NK cell lines and patient samples tested displayed activation of the JAK3/STAT5 pathway. Treatment of these cell lines with tofacitinib reduced the levels of phospho-STAT5, suppressed proliferation, induced G1 cell-cycle arrest and decreased EBV LMP1 and EBNA1 expression. An EBV-negative NK cell line was also sensitive to tofacitinib, whereas an EBV-infected NK cell line was more sensitive to tofacitinib than its parental line. Tofacitinib significantly inhibited the growth of established tumors in NOG mice. These findings suggest that tofacitinib may represent a useful therapeutic agent for patients with EBV-associated T and NK cell lymphoma.

Keywords: EBV; cell-cycle arrest; lymphoma; tofacitinib.

Figure 1. Effects of tofacitinib on JAK3/STAT5 pathway components and growth in B, T and NK cell lines

(A) The following cell lines: EBV-negative B cell line (BJAB), EBV-transformed lymphoblastoid cell line (LCL), EBV-negative T cell lines (Jurkat and MOLT4), EBV-positive T cell lines (SNT15 and SNT16), EBV-negative NK cell line (KHYG1), and EBV-positive NK cell lines (KAI3 and SNK6), were treated without (−) or with (+) 1 μM tofacitinib for 24 h and cell lysates were then immunoblotted for the indicated proteins. (B) BJAB, LCL, Jurkat, SNT15, KHYG1 and KAI3 cells were treated with the indicated concentrations of tofacitinib, and viable cells were counted at the indicated times using the trypan blue exclusion test. Values are means ± SE of the results from triplicate experiments. *P < 0.05 as compared with DMSO-treated cells. (C) B, T and NK cell lines were treated with the indicated concentrations of tofacitinib for 72 h. Cell number is shown as the ratio of the cell number in the different treatment groups to DMSO-treated cells. Values are means ± SE of the results from triplicate experiments. *P < 0.05 as compared with Jurkat or MOLT4.

Figure 2. Effects of tofacitinib on JAK3/STAT5 pathway components and growth of IL2-dependent and -independent SNT16 cell lines

(A) IL2-dependent and -independent SNT16 cells were treated with 1 μM tofacitinib for 24 h and cell lysates were immunoblotted for the indicated proteins. (B) SNT16 cells were treated with the indicated concentrations of tofacitinib, and viable cells were counted using the trypan blue exclusion test. Values are means ± SE of the results from triplicate experiments. *P < 0.05 as compared with DMSO-treated cells. (C) SNT16 cells were treated with the indicated concentrations of tofacitinib for 72 h. Cell number is shown as the ratio of the cell number in the different treatment groups to DMSO-treated cells.

Figure 3. Effects of tofacitinib on JAK3/STAT5 pathway components and growth in NKL/TL1 cell lines

(A) NKL and TL1 cell lines were treated with 1 μM tofacitinib for 24 h and cell lysates were immunoblotted for the indicated proteins. (B) The EBV-positive cell line (TL1) and its parental EBV negative cell line (NKL) were treated with the indicated concentrations of tofacitinib, and viable cells were counted using the trypan blue exclusion test. Values are means ± SE of the results from triplicate experiments. *P < 0.05 as compared with DMSO-treated cells. (C) TL1 and NKL cells were treated with the indicated concentrations of tofacitinib for 72 h. Cell number is shown as the ratio of the cell number in the different treatment groups to DMSO-treated cells. Values are means ± SE of the results from triplicate experiments. *P < 0.05 as compared with DMSO-treated cells.

Figure 4. Effects of tofacitinib on apoptosis and cell-cycle arrest in T and NK cell lines

(A, B) T and NK cell lines were treated with DMSO or 5 μM tofacitinib for 48 h, and apoptosis was evaluated by Annexin V-7AAD staining using flow cytometry. Values are means ± SE of the results from duplicate or triplicate experiments. *P < 0.05 as compared with DMSO-treated cells. (C) T and NK cell lines were treated with 5 μM tofacitinib for 48 h, and cell lysates were immunoblotted for caspase-3 and PARP. (D) T and NK cell lines were treated with DMSO or 5 μM tofacitinib for 24 h, following which they were fixed and stained with propidium iodide. Cell cycle profiles were assessed using flow cytometry. Values are means ± SE of the results from triplicate experiments. *P < 0.05 as compared with DMSO-treated cells. (E) T and NK cell lines were treated with 5 μM tofacitinib for 24 h and cell lysates were immunoblotted for the indicated proteins.

Figure 5. Effects of tofacitinib on the expression of EBV-encoded genes in EBV-positive T and NK cell lines

(A) EBV-positive T cell lines (SNT13, SNT15, and SNT16) and EBV-positive NK cell lines (KAI3 and SNK6) were treated with 5 μM tofacitinib and harvested at 0, 24 and 48 h to evaluate gene expression using real-time RT-PCR. BZLF1 is an immediate early gene and gp350/220 is a late gene in the lytic infection cycle of EBV. LMP1 and EBNA1 are latent EBV genes. β2-Microglobulin was used as an internal control and as a reference gene for relative quantification and was assigned an arbitrary value of 1 (10°). Values are geometric means ± SE of the results from six replicate experiments. *P < 0.05 as compared with DMSO-treated cells. (B) SNT13, SNT15, SNT16, KAI3 and SNK6 cell lines were treated with 5 μM tofacitinib for 48 h, and cell lysates were immunoblotted for LMP1, EBNA1 and BZLF1. Actin was blotted as a loading control.

Figure 6. Effects of tofacitinib on tumor cell growth and proliferation in the murine xenograft model

(A) Tofacitinib inhibited the growth of subcutaneous xenograft tumors in NOG mice. SNT15 cells (2 ×10⁶ cells per flank) were subcutaneously inoculated into the flanks of mice. On the day of tumor inoculation, treatment was started using subcutaneous mini-osmotic pumps and tofacitinib (30 mg/kg/day) was administered for 4 weeks. Tumor size was quantified twice a week. *P < 0.05; n = 6 mice for each group. (B) Representative images of tumor-bearing mice and removed spleens are shown after 4 weeks of treatment with tofacitinib or vehicle (control). The hematoxylin/eosin-stained section shows tumor infiltration into the subcutaneous lesion wall. EBER in situ hybridization shows infiltration into the subcutaneous lesion wall and spleen, respectively.

Figure 7. Effects of tofacitinib on JAK3/STAT5 pathway components and growth in EBV-infected cells isolated from patients with EBV-associated T cell lymphoma

(A) γδ T cells and peripheral blood mononuclear cells (PBMCs) were treated with 1 μM tofacitinib for 24 h and cell lysates were immunoblotted for the indicated proteins. (B) γδ T cells isolated from patients with EBV-associated T cell lymphoma were treated with the indicated concentrations of tofacitinib, and viable cells were counted using the trypan blue exclusion test. *P < 0.05 as compared with DMSO-treated cells. (C) PBMCs isolated from a patient with EBV-associated T cell lymphoma or from a healthy donor (control) were treated with the indicated concentrations of tofacitinib, and viable cells were counted using the trypan blue exclusion test. Values are means ± SE of the results from triplicate experiments using patient samples. *P < 0.05 as compared with DMSO-treated cells.