Reduced immunosuppressive properties of axitinib in comparison with other tyrosine kinase inhibitors

Abstract

The multikinase inhibitors sunitinib, sorafenib, and axitinib have an impact not only on tumor growth and angiogenesis, but also on the activity and function of immune effector cells. In this study, a comparative analysis of the growth inhibitory properties and apoptosis induction potentials of tyrosine kinase inhibitors on T cells was performed. Tyrosine kinase inhibitor treatment resulted in a dramatic decrease in T cell proliferation along with distinct impacts on the cell cycle progression. This was at least partially associated with an enhanced induction of apoptosis although triggered by distinct apoptotic mechanisms. In contrast to sunitinib and sorafenib, axitinib did not affect the mitochondrial membrane potential (Δψm) but resulted in an induction or stabilization of the induced myeloid leukemia cell differentiation protein (Mcl-1), leading to an irreversible arrest in the G2/M cell cycle phase and delayed apoptosis. Furthermore, the sorafenib-mediated suppression of immune effector cells, in particular the reduction of the CD8(+) T cell subset along with the down-regulation of key immune cell markers such as chemokine CC motif receptor 7 (CCR7), CD26, CD69, CD25, and CXCR3, was not observed in axitinib-treated immune effector cells. Therefore, axitinib rather than sorafenib seems to be suitable for implementation in complex treatment regimens of cancer patients including immunotherapy.

Keywords: Apoptosis; Axitinib; Immunology; Immunosuppression; Immunotherapy; Sorafenib; Sunitinib; T Cell; Targeted Therapy; Tyrosine Kinase Inhibitor.

Figures

FIGURE 1.

TKI-mediated inhibition of proliferation and viability of immune cells. PBMCs from healthy donors were prepared by Ficoll density gradient centrifugation and stimulated directly with anti-CD3 and anti-CD28 antibodies (mouse anti-human; 1 μg/ml each; BD Biosciences). Jurkat cells were used without stimulation. Cells were grown for 6 days or 72 h in the absence or presence of various concentrations of TKI (PBMCs, 0–20 μm; Jurkat, 0–50 μm) and analyzed via flow cytometry using covalent 5,6-carboxyfluorescein diacetate succinimidyl ester staining (Invitrogen) (A, PBMCs; C, Jurkat cells) or via XTT assay using the XTT Cell Proliferation Kit (Roche Applied Science) (B, PBMCs; D, Jurkat cells) according to the manufacturer's protocol. Proliferation (A and C) and viability (B and D) are expressed as mean percentage (%) ±S.D. (error bars) based on five independent experiments. The right panel in each segment of Fig. 1 highlights the effects of physiologic TKI concentrations on viability/proliferation, respectively.

FIGURE 2.

Sorafenib, but not axitinib, reduced the frequency of specific immune cell subpopulations and the expression levels of specific immune cell markers. PBMCs from healthy donors were prepared by Ficoll density gradient centrifugation and stimulated directly with anti-CD3 and anti-CD28 antibodies (mouse anti-human; 1 μg/ml each; BD Biosciences) and IL-2 (100 units/ml). Cells were grown for 72 h in the absence or presence of 5 μm TKI and analyzed via flow cytometry for lymphocyte markers using seven-color staining (A). Cells were fixed and analyzed by flow cytometry for Tregs (B) and after intracellular staining for IFNγ expression (C). Data are represented as mean percentage (%) ±S.D. (error bars) based on five independent experiments. A, TKI-induced reduction in the percentage of CD8+ T cells as wells as CCR7+, CD26+, and CXCR3+ cells within the total CD4+ and CD8+ T cell population and CD26+ or CD69+ cells within the total NK cell population. B, TKI-induced reduction in the percentage of peripheral blood CD4+ CD25+ FOXP3+ T cells within the total CD4+ T cell population. C, TKI-induced reduction in the percentage of IFNγ-producing cells within the total CD4+ T cell population. *, p < 0.05 (t test).

FIGURE 3.

Decreased expression of T cell activation markers (CD25 and CD69) in TKI-treated T cells. PBMCs from healthy donors were prepared by Ficoll density gradient centrifugation and stimulated directly with phytohemagglutinin M (PHA-M) (4 μg/ml) and IL-2 (100 units/ml). Cells were grown for 72 h in the absence or presence of 0–5 μm TKI and analyzed via flow cytometry for the activation markers CD25 and CD69 within the total CD3+ T cell population of PBMCs. Data are represented as mean percentage (%) ±S.D. (error bars) based on five independent experiments.

FIGURE 4.

TKI-mediated apoptosis induction in Jurkat cells and T cells isolated from PBMC.A, quantitative analysis of Jurkat cell apoptosis by annexin V- and propidium iodide-double stained flow cytometry after 72 h of incubation in the presence of various concentrations of TKI (0–20 μm) or DMSO. B, C, and D, TKIs induce caspase-8-, -9-, and -3-dependent apoptosis in Jurkat cells. Caspase-8 (B) and -9 (C) activities were measured using the Caspase-Glo-8 and -9 assays (Promega) as described under “Experimental Procedures” after 72 h of incubation in the presence of various concentrations of TKI (0–20 μm) or DMSO. Data are represented as mean (luminescenceTKI/luminescenceDMSO) ±S.D. (error bars) based on three independent experiments performed in triplicate. Caspase-3 activation (D) was assessed after 72 h in the absence and presence of TKI (0–10 μm) via flow cytometry. E and F, quantitative analysis of apoptosis of T cells isolated from PBMCs. The assays were performed as described above after 72 h of phytohemagglutinin M (PHA-M) stimulation and incubation in the presence of various concentrations of TKI (0–20 μm) or DMSO. All data are represented as mean percentage (%) ±S.D. (error bars) based on three independent experiments performed in triplicate. 7AAD, 7-aminoactinomycin D.

FIGURE 5.

Sunitinib and sorafenib, but not axitinib, induced loss of mitochondrial potential.A and B, alterations of the Δψm were determined via flow cytometry after treatment of Jurkat cells (A) or T cells isolated from PBMCs (B) with TKI (0–10 μm) for 72 h as described under “Experimental Procedures.” The number provided within the given profile highlights the percentage of cells that emit only green fluorescence, thereby indicating a depolarized mitochondrial membrane. A representative set of three individual experiments with similar results is shown. C and D, TKI-dependent increase in the green/red fluorescence intensity ratio of Jurkat cells (C) or isolated T cells (D). Sunitinib and sorafenib treatment increased the green/red ratio compared with DMSO controls as well as axitinib treatment, indicating mitochondrial membrane depolarization and therefore the loss of the Δψm. Data are expressed as mean of 520/590-nm emission spectrum ±S.D. (error bars) based on three independent experiments.

FIGURE 6.

Axitinib, but not sunitinib or sorafenib, resulted in an up-regulation of Mcl-1. Jurkat cells (A) or T cells isolated from PBMCs (B) were exposed to DMSO, sunitinib, sorafenib, or axitinib (0–10 μm) for 72 h, cells were pelleted and lysed, and 50 μg of protein were separated by SDS-PAGE. Blots were probed with anti-Bcl-xL or anti-Mcl-1 antibodies, and equivalent loading and transfer were ensured using an anti-GAPDH mAb. The graphs show the band intensities ±S.D. (error bars) of Bcl-xL and Mcl-1 corrected for the intensity of the GAPDH standard based on three independent experiments.

FIGURE 7.

Axitinib, but not sunitinib or sorafenib, induced G2/M arrest. Cells were treated with 0.5 μm axitinib for 0–48 h and analyzed as outlined under “Experimental Procedures.” Representative flow cytometry profiles (A) illustrate the absence of G2/M arrest in cells treated with DMSO for 48 h and the progressive accumulation of cells in G2/M at 12, 24, 36, and 48 h after axitinib treatment. Data are additionally expressed as cells in the G0/G1, G2/M, or S phases of the cell cycle in DMSO- (B) or axitinib-treated (C) Jurkat cells. Cells were subjected to quantitative real time RT-PCR analysis to determine mRNA levels of CCNB1 (D) using oligo-(dT)18-primed cDNA. Mean expressions of hypoxanthine-guanine phosphoribosyltransferase and peptidylprolyl isomerase A were used for normalization. Data are represented as mean percentage (%) ±S.D. (error bars) based on three independent experiments. PE, phycoerythrin.