The Raf Kinase Inhibitor Sorafenib Inhibits JAK-STAT Signal Transduction in Human Immune Cells

Abstract

Sorafenib is an oral multikinase inhibitor that was originally developed as a Raf kinase inhibitor. We hypothesized that sorafenib would also have inhibitory effects on cytokine signaling pathways in immune cells. PBMCs from normal donors were treated with varying concentrations of sorafenib and stimulated with IFN-α or IL-2. Phosphorylation of STAT1 and STAT5 was measured by flow cytometry and confirmed by immunoblot analysis. Changes in IFN-α- and IL-2-stimulated gene expression were measured by quantitative PCR, and changes in cytokine production were evaluated by ELISA. Cryopreserved PBMCs were obtained from cancer patients before and after receiving 400 mg sorafenib twice daily. Patient PBMCs were thawed, stimulated with IL-2 or IFN-α, and evaluated for phosphorylation of STAT1 and STAT5. Pretreatment of PBMCs with 10 μM sorafenib decreased STAT1 and STAT5 phosphorylation after treatment with IFN-α or IL-2. This inhibitory effect was observed in PBMCs from healthy donors over a range of concentrations of sorafenib (5-20 μM), IL-2 (2-24 nM), and IFN-α (10(1)-10(6) U/ml). This effect was observed in immune cell subsets, including T cells, B cells, NK cells, regulatory T cells, and myeloid-derived suppressor cells. Pretreatment with sorafenib also inhibited PBMC expression of IFN-α- and IL-2-regulated genes and inhibited NK cell production of IFN-γ, RANTES, MIP1-α, and MIG in response to IFN-α stimulation. PBMCs from patients receiving sorafenib therapy showed decreased responsiveness to IL-2 and IFN-α treatment. Sorafenib is a Raf kinase inhibitor that could have off-target effects on cytokine-induced signal transduction in immune effector cells.

FIGURE 1

Phosphorylation of STAT1 and STAT5 is Confirmed by Flow Cytometry and Immunoblotting. Freshly isolated PBMCs were treated with multiple concentrations of sorafenib for 30 minutes and then stimulated with 8 nM IL-2, 10⁵ U/mL IFN-α, 10 ng/mL IFN-γ, or 10 ng/mL IL-6 for 15 minutes at 37°C. Phosphorylated STAT5 and STAT1 were measured by intracellular flow cytometry (a) and confirmed by immunoblotting whole cell lysates for pSTAT5, pSTAT1, STAT5, and STAT1. Phosphorylation of STAT3 was also evaluated by immunoblot analysis (b). Data are representative of two independent experiments with similar results.

FIGURE 2

Sorafenib Treated PBMCs Stimulated with Cytokines Show Differences in Specific Fluorescence When Measuring PSTAT1 and pSTAT5 Levels via Flow Cytometry Versus Control DMSO. Freshly isolated PBMCs were treated with DMSO or 10 μM sorafenib for 30 minutes and then stimulated with increasing doses of IL-2, IFN-α, or IFN-γ for 15 minutes at 37°C. Phosphorylated STAT5 or STAT1 levels were measured by intracellular flow cytometry and the results expressed as specific fluorescence (Fsp = Ft - Fb) (a, b, c). PBMCs were treated with DMSO or 10 μM sorafenib for 30 minutes. The media was changed and the PBMCs were allowed to rest for varying amounts of time before being stimulated with 8 nM IL-2 for 15 minutes at 37°C. Phosphorylated STAT5 was measured by intracellular flow cytometry. Data are expressed as specific fluorescence (d). Data shown are the mean ±SEM of three (a,b,c) or five independent experiments (d). There was a statistically significant difference between DMSO and sorafenib treatment (a-d) (p < 0.05).

FIGURE 3

qPCR and ELISA Results Show Differences in Gene Expression and Cytokine Production for IFN-α and IL-2 Responsive Genes After Treatment with Sorafenib. Freshly isolated PBMCs were treated with 10 μM sorafenib for 30 minutes and stimulated with either 10⁵ U/ml IFN-α or 8 nM IL-2 for 4 hours at 37°C. Total RNA was isolated from treated cells and converted to cDNA. Real-Time PCR was performed for the IFN-α responsive genes OAS1 (a), IFIT2 (b), CXCL10 (c), CD69 (d) and a cytokine-inducible negative regulator of cytokine signaling (SOCS1) (e). The IL-2 responsive genes PIM-1, CIS, and interferon-gamma (IFN-γ) (f) were also examined. Induction of gene expression was calculated relative to β-actin andGAPDH. Data shown are representative of two independent experiments with similar results (c, d, and e) or one independent experiment (a, b, and f). The mean and standard deviation of triplicate determination are shown for all panels. Freshly isolated NK cells were co-cultured with K562 cells, pre-treated with 20 μM sorafenib and then stimulated with 10³ U/ml IFN-α. Cytokine production of IFN-γ, RANTES, MIP1-α, and MIG was evaluated by ELISA (g). Data shown are representative of two experiments with similar results. The mean and standard deviation of triplicate determination are shown for all panels.

FIGURE 4

Differences in pSTAT1 and pSTAT5 Levels Between Control Treated and Sorafenib Treated Cells via Flow Cytometry. Freshly isolated PBMCs were treated with DMSO or 10 μM of sorafenib for 30 minutes at 37°C and stimulated with 8 nM IL-2 or 10⁵ U/ml IFN-α for 15 minutes at 37°C. Phosphorylated STAT5 specific fluorescence (a) was measured by intracellular flow cytometry within the CD3, CD4, CD8, and CD56 immune subsets. Phosphorylated STAT1 specific fluorescence was measured by intracellular flow cytometry within the CD3, CD4, CD8, CD21, CD14, CD56 and CD34 immune subsets (b). CD4+ (c) and CD8+ T cells (d) (central, effector, naïve) were purified from fresh leukopacks and pre-treated for 30 minutes with 20 μM sorafenib, stimulated for 15 minutes with 8 nM IL-2 or 10⁵ MU/mL IFN-α, and levels of activated STAT1 or STAT5 (respectively) were measured by intracellular flow cytometry. A similar experiment was conducted with NK cells (tolerant, regulatory, cytotoxic) (e). Raw gating examples are shown in Supplementary Figures S3 (A-B). There was a statistically significant difference between DMSO and sorafenib treatment (a-e) (p < 0.05). Proliferation of stimulated CD4+ T cells is indicated by the presence of daughter peaks on the histogram which represents the CFSE labeled cells become more dilute with each generation. Pre-treatment of CD4+ T cells with sorafenib (10 or 20 μM) led to marked inhibition of proliferation . Identical results were obtained with CD8+ T cells (f). NK-92 proliferation was inhibited by 97% with sorafenib pre-treatment versus a vehicle control (g). T cells were co-cultured for 48 hours with 10 μM or 20 μM sorafenib, stimulated with CD3/CD28 beads, and IFN-γ levels in culture supernatants were measured at 72 hours by ELISA (h). There was a statistically significant difference between DMSO and 20 μM sorafenib treatment (p<0.0002). Data shown are the mean ±SEM of three (a, b, c, d, and e) or two (f, and h) independent experiments. A similar experiment was conducted with NK-92 cells stimulated with IL-2 plus IL-15 (i). Data shown are the results of one independent experiment (i).

FIGURE 5

Sorafenib Decreases Cytokine Signaling in Immune Suppressive Cells. Freshly isolated PBMCs were treated with DMSO or 10 μM of sorafenib for 30 minutes at 37°C and stimulated with 8 nM IL-2 or 10⁵ U/ml IFN-α for 15 minutes at 37°C. The specific fluorescence of pSTAT5 (a) and pSTAT1 (b) were measured in the CD4+CD25+ subset. There was a statistically significant difference between DMSO and sorafenib treatment (a-b) (p < 0.05). Murine MDSCs were treated with DMSO or 10 μM of sorafenib for 30 minutes at 37°C and stimulated with PBS, 10³ U/ml IFN-α or 10⁴ U/ml IFN-α for 15 minutes at 37°C. Phosphorylated STAT1 specific fluorescence was measured by intracellular flow cytometry. There was a statistically significant difference between DMSO and sorafenib treatment (c) (p< 0.001). MDSCs isolated from RCC patients were treated with DMSO or 10 μM of sorafenib for 30 minutes at 37°C and stimulated with PBS, 10³ U/ml IFN-α or 10⁴ U/ml IFN-α for 15 minutes at 37°C. Phosphorylated STAT1 specific fluorescence was measured by intracellular flow cytometry, and the figure shows the data from one representative patient. Other patients demonstrated a range of 14-66% decrease in phosphorylation of STAT1 following sorafenib treatment compared to DMSO but the existence of inter-patient variability led to the results not being statistically significant (d). Data shown are the mean ±SEM of five (c), three (d) or two (a, b) independent experiments.

FIGURE 6

pSTAT1 Differences in Sorafenib Versus Control Conditions on Several Cell Lines Measured Via Flow Cytometry. B cell lymphoma cell lines 697 and Ramos, renal cell carcinoma cell lines Caki and SK-RC-45, and melanoma cell lines A374 and Hs294t were treated with DMSO or 10 μM of sorafenib for 30 minutes at 37°C and then stimulated with 10⁵ U/ml IFN-α for 15 minutes at 37°C. Phosphorylated STAT1 levels were measured by intracellular flow cytometry. Data are expressed as specific fluorescence (* p<0.05). Data shown are the mean ±SEM of three independent experiments.

FIGURE 7

Patient Levels of pSTAT1 and pSTAT5 During Therapy Measured Via Flow Cytometry. Cryopreserved PBMCs isolated from patients prior to therapy and 8 and 16 weeks after the initiation of sorafenib therapy were stimulated with 8 nM IL-2 or 10⁵ U/mL IFN-α. Phosphorylated STAT5 (a) and STAT1 (b) levels were measured by intracellular flow cytometry. Data were log transformed and are expressed as specific fluorescence (* p=0.02). Data shown are the mean of four independent experiments.