CP-690,550, a therapeutic agent, inhibits cytokine-mediated Jak3 activation and proliferation of T cells from patients with ATL and HAM/TSP

Abstract

The retrovirus, human T-cell-lymphotrophic virus-1 (HTLV-I) is the etiologic agent of adult T-cell leukemia (ATL) and the neurological disorder HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-I-encoded protein tax constitutively activates interleukin-2 (IL-2), IL-9, and IL-15 autocrine/paracrine systems that in turn activate the Jak3 (Janus kinase 3)/STAT5 (signal transducers and activators of transcription 5) pathway, suggesting a therapeutic strategy that involves targeting Jak3. We evaluated the action of the Jak3 inhibitor CP-690,550 on cytokine dependent ex vivo proliferation that is characteristic of peripheral blood mononuclear cells (PBMCs) from select patients with smoldering or chronic subtypes of ATL, or from those with HAM/TSP whose PBMCs are associated with autocrine/paracrine pathways that involve the production of IL-2, IL-9, IL-15, and their receptors. CP-690,550 at 50 nM inhibited the 6-day ex vivo spontaneous proliferation of PBMCs from ATL and HAM/TSP patients by 67.1% and 86.4%, respectively. Furthermore, CP-690,550 inhibited STAT5 phosphorylation in isolated ATL T cells ex vivo. Finally, in an in vivo test of biological activity, CP-690,550 treatment of mice with a CD8 T-cell IL-15-transgenic leukemia that manifests an autocrine IL-15/IL-15Rα pathway prolonged the survival duration of these tumor-bearing mice. These studies support further evaluation of the Jak3 inhibitor CP-690,550 in the treatment of select patients with HTLV-I-associated ATL and HAM/TSP.

Figure 1

CP-690,550 inhibited the proliferation of the cytokine-dependent NK92 cell line mediated by cytokines that signal through Jak3/STAT5 but not by cytokines that use other pathways. (A) After cytokine starvation for 24 hours, NK92 cells were stimulated by the addition of human IL-2 for 48 hours with and without the addition of serially increasing concentrations of CP-690,550. ³H-thymidine was added during the last 6 hours of the cultures. Cells were then harvested and analyzed for ³H-thymidine incorporation. (B) Cytokine-starved NK92 cells were stimulated with human IL-2, combined IL-6/IL-6R, or IL-12 for 48 hours with and without serially increasing concentrations of CP-690,550. (C) The NK92 cells were treated as those in panel B with and without a single 50nM dose of CP-690,550 (CP). Data are presented as means ± SD (A-C) and are representative of 3 independent experiments. (D) After cytokine starvation for 24 hours, NK92 cells were stimulated with 30 ng/mL of IL-2, 100 ng/mL of combined IL-6/IL-6R, or 100 ng/mL of IL-12 for 1 hour with and without the addition of CP-690,550 (CP). The cell lysates were immunoblotted with an anti–phospho-STAT5 monoclonal antibody and an anti-STAT5 antibody. β-Actin was used as an input control. Data are representative of 3 independent experiments.

Figure 2

Inhibition of cytokine-mediated proliferation of 32Dβ cells by CP-690,550 showed specificity for the γc/Jak3/STAT5-signaling pathway. (A) After cytokine starvation for 24 hours, 32Dβ cells were stimulated with human IL-2 or murine IL-3 for 48 hours with and without CP-690,550. ³H-thymidine was added during the last 6 hours of the cultures. Cells were then harvested and analyzed for ³H-thymidine incorporation. (B) After cytokine starvation for 24 hours, 32Dβ cells were stimulated with 1 μg/mL of human IL-2 or 10 ng/mL of murine IL-3 for 2 hours with and without the addition of CP-690,550. The cell lysates were immunoblotted with an anti–phospho-STAT5 monoclonal antibody and an anti-STAT5 antibody. β-Actin was used as an input control. Data are presented as means ± SD (A) and are representative of 3 independent experiments.

Figure 3

CP-690,550 inhibited the 6-day ex vivo spontaneous proliferation of PBMCs from patients with ATL and with HAM/TSP. The PBMCs from a patient with ATL (A) or a patient with HAM/TSP (B) were cultured ex vivo for 6 days with and without CP-690,550 or with 10 μg/mL of antibody when a single antibody directed against the cytokine IL-2, IL-9, or IL-15 was used or with their combination (5 μg/mL each). ³H-thymidine was added during the last 6 hours of the cultures. Cells were then harvested and analyzed for ³H-thymidine incorporation. The percentage of inhibition of proliferation with CP-690,550 addition was determined for 12 patients with ATL (C) or 9 patients with HAM/TSP (D). The value was calculated as: % inhibition = (cpm of proliferation without CP − cpm of proliferation with CP)/(cpm of proliferation without CP) × 100. Black bars indicate the mean inhibition percentage.

Figure 4

CP-690,550 inhibited ATL and HAM/TSP PBMC 6-day culture supernatant–mediated NK92-cell proliferation. After cytokine starvation for 24 hours, NK92 cells were stimulated with a 6-day ex vivo culture supernatant of PBMCs from an ATL patient (A) or a HAM/TSP patient (B) for 48 hours. Antibodies directed against the cytokines IL-2, IL-9, or IL-15 or CP-690,550 were added to the plates 1 hour prior to cell seeding. ³H-thymidine was added during the last 6 hours of the cultures. Cells were then harvested and analyzed for ³H-thymidine incorporation. The percentage of inhibition of proliferation with CP-690,550 was determined for 11 ATL culture supernatants (C) or 8 HAM/TSP culture supernatants (D). Black bars indicate the mean percentage of inhibition.

Figure 5

CP-690,550 inhibited the activation of the Jak3/STAT5 pathway present in patients ATL cells. (A) CP-690,550 inhibited the phosphorylation status of STAT5 in isolated T cells from patients with ATL. The T cells from a patient with ATL and those from a normal control were immediately isolated from the freshly separated PBMCs and lysed with cell lysis buffer; alternatively, the T cells were isolated from PBMCs cultured for 2 days with and without 50nM CP-690,550. Cell lysates were immunoblotted with anti–phospho-STAT5 monoclonal antibody and anti–STAT5 antibody. β-actin was used as an input control. (B) CFSE staining of CD3^LowCD25⁺ lymphocytes was used to monitor cell division. ATL PBMCs were labeled with CFSE at day 0, and then cultured with (CP) or without (−) CP-690,550 for 6 days. FACS analysis of CD3^LowCD25⁺CFSE triple-positive ATL cells were performed on day 6. Normal donor PBMCs (Cont) were used as a control. Because normal PBMCs proliferated under stimulation of anti-CD3/CD28 and ATL PBMCs spontaneously proliferated regardless of such stimulation, the nonstimulated normal and ATL PBMCs were chosen for analysis.

Figure 6

CP-690,550 showed therapeutic efficacy in a mouse model of B1 IL-15–transgenic CD8 T-cell leukemia that manifested an autocrine IL-15/IL-15Rα loop that acts through Jak3/STAT5. (A) Serum levels of human IL-15 from the human IL-15–transgenic CD8 T-cell leukemia–bearing mice after inoculation of tumor cells. (B) Serum levels of mouse IL-15Rα from the IL-15–transgenic CD8 T-cell leukemia–bearing mice after inoculation of tumor cells. (C) Kaplan-Meier analysis demonstrating CP-690,550 prolongation of the survival of mice bearing the IL-15–transgenic CD8 T-cell leukemia. Mice in the control group received PEG300 by subcutaneous pump for 14 days. Mice in the CP-690,550-pre group (CP-pre) received a pump infusion with CP-690,550 at 30 mg/kg/d for 14 days that was placed into the mice 2 days before tumor cell inoculation. Mice in the CP-690,550-post group (CP-post) received a pump infusion with CP-690,550 at 30 mg/kg/d for 14 days that was placed into the mice 1 day after inoculation of the leukemic cells. Mice in the anti–human IL-15 monoclonal antibody M111 group received M111 at 100 μg intraperitoneally weekly for 2 weeks starting at 3 days after inoculation of the leukemic cells. (D) CP-690,550 inhibited the phosphorylation status of Jak3/STAT5 in splenocytes from IL-15–transgenic CD8 T-cell leukemia–bearing mice. Mice in the tumor-only group received PEG300 by a subcutaneous pump for 14 days. Mice in the CP-690,550 group (CP) received a pump infusion with CP-690,550 at 30 mg/kg/d for 14 days that was placed into the mice 1 day after inoculation of the leukemic cells. Mice in the M111 group received M111 at 100 μg intraperitoneally weekly for 2 weeks starting at 3 days after inoculation of the leukemic cells. Mice in the normal group (no tumor, no treatment) were used as a control. The splenic cells were separated 13 days after tumor cell inoculation and checked for the phosphorylation status of STAT5. *P < .00001.