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Research Articles: Therapeutics, Targets, and Development

MAPK-independent impairment of T-cell responses by the multikinase inhibitor sorafenib

Roland Houben, Heike Voigt, Christiane Noelke, Valeska Hofmeister, Juergen C. Becker and David Schrama
DOI: 10.1158/1535-7163.MCT-08-1051 Published February 2009

Abstract

Sorafenib, originally developed as CRAF inhibitor but soon recognized as a multikinase inhibitor, is currently widely tested for the treatment of different cancers either alone or in combination therapy. However, the clinical success, particularly in immunogenic tumors such as melanoma, was less than anticipated. Because T-cell activation is tightly regulated by a multitude of kinases, we scrutinized effects of sorafenib on immune responses. To this end, comprehensive in vitro studies revealed that the presence of sorafenib concentrations comparable with observed plasma levels in patients strongly impairs the activation of T cells. Notably, even established tumor-specific immune responses are influenced by sorafenib. Indeed, ELISPOT data of peripheral blood lymphocytes obtained from melanoma patients vaccinated against survivin show markedly diminished survivin-specific immune responses in the presence of sorafenib. Surprisingly, inhibition of T-cell activation was not associated with reduced extracellular signal-regulated kinase phosphorylation. In fact, on T-cell receptor stimulation phospho-extracellular signal-regulated kinase and phospho-mitogen-activated protein kinase kinase levels were found to be elevated in the presence of sorafenib, showing the complexity of signal transduction events following T-cell receptor stimulation. In conclusion, our data show that T-cell function is sensitive toward the multikinase inhibitor sorafenib in a mitogen-activated protein kinase-independent fashion. This observation has important implications for the use of sorafenib as therapy for immunogenic cancers. [Mol Cancer Ther 2009;8(2):433–40]

Keywords:
  • lymphocytes
  • T cells
  • RAF
  • kinase inhibitor

Introduction

It is widely accepted that cancer cells have acquired a particular set of functional capabilities during their development (1). Among these is the self-sufficiency in growth signals. In this regard, the Cancer Genome Project revealed BRAF to be affected by activating mutations in several cancers in general and in melanoma in particular (2). BRAF, ARAF, and CRAF are the three isoforms of the RAF family of serine/threonine kinases. Active RAF [mitogen-activated protein kinase (MAPK) kinase kinase] phosphorylates and activates MEK (MAPK kinase), which in turn phosphorylates and activates extracellular signal-regulated kinase (ERK) 1/2 (p44/p42 MAPK). Whereas RAF and MEK appear largely restricted to only one class of substrates, ERK targets >70 substrates including membrane, cytoskeletal, cytoplasmic, nuclear, and even mitochondrial proteins (3).

In human tumors, the MAPK pathway is found to be activated not only by RAF mutations but also by altered upstream signaling proteins (4, 5). Therefore, the MAPK pathway appears to be a suitable target for cancer therapy. In this regard, at the time when BRAF mutations in human cancer were revealed, sorafenib (BAY 43-9006), which was developed as a CRAF inhibitor, has already reached early clinical trials (6, 7). Consequently, because sorafenib inhibits mutated BRAF, it was evaluated as a therapeutic agent for melanoma. As monotherapeutic agent, however, it failed to show any efficacy (8). However, sorafenib is inhibitory not only to RAF kinases but also to more distantly related kinases including vascular endothelial growth factor receptors 2 and 3, platelet-derived growth factor receptor, FLT3, and KIT (9). Indeed, the success of sorafenib in the treatment of renal cell carcinoma (10) is primarily attributed to inhibition of angiogenesis by blocking vascular endothelial growth factor and platelet-derived growth factor receptors (11). At present, the multikinase inhibitor sorafenib is evaluated for the treatment of a wide variety of cancers either as monotherapy or in combinations in >100 different clinical trials.1 In this context, it has been suggested that phospho-ERK in peripheral blood lymphocytes (PBL) may be a useful biomarker for measuring and predicting the effects of sorafenib (12, 13). In this respect, a lost ability to stimulate ERK phosphorylation by phorbol myristate acetate (PMA) in PBLs of patients under sorafenib has been reported (14). However, it is very important to note that ERK activation is regarded as one necessary step in the activation of T cells in response to T-cell receptor (TCR) stimulation and particularly for developing and exerting effector function of cytotoxic T cells (15, 16). Therefore, sorafenib might lead to impaired immune responses by affecting MAPK pathway signaling, which is especially undesirable for immunogenic tumors such as melanoma. To test this notion in more detail, we analyzed the effect of sorafenib on the function of T lymphocytes in vitro. To this end, viability/proliferation, activation, and effector functions of T lymphocytes were affected by the presence of physiologic concentrations of sorafenib, concentrations comparable with plasma levels of patients treated with sorafenib. Surprisingly, inhibition of T-cell activation was not associated with reduced ERK phosphorylation, suggesting that target molecules others than RAF mediate this effect.

Materials and Methods

Cell Culture

PBLs were derived by leukapheresis from vaccine-naive melanoma patients or patients who had received a survivin-based peptide vaccination (17). Following leukapheresis, the cells were frozen and stored in liquid nitrogen. Before stimulation experiments, cells were thawed and cultured for 24 h at 37°C in RPMI 1640 supplemented with 10% FCS, 100 units/mL penicillin, and 0.1 mg/mL streptomycin. Alternatively, for some experiments, PBLs were derived from heparinized blood from healthy donors by density gradient centrifugation using lympholyte (Cedarlane). In addition, purified T cells isolated by magnetic cell sorting were used. To this end, PBLs or leukapheresis products were subjected to the Pan T-Zell Kit I (Miltenyi Biotec) according to the manufacturer's instructions.

Stimulation of T cells was done by incubation with 100 nmol/L PMA (Calbiochem), 10 μg/mL phytohemagglutinin-M (PHA; Calbiochem), or 0.5 μg/mL of α-CD3 (clone UCHT1) and α-CD28 (clone CD28.2) antibodies (Becton Dickinson).

Flow Cytometry

Cells (2 × 105) were pelleted and then resuspended in 50 μL PBS with 1% bovine serum albumin. Fluorescence-labeled specific antibodies or corresponding isotype control antibodies (1 μg) were added followed by incubation on ice for 40 min in the dark. After the cells were washed once with PBS/1% bovine serum albumin, they were incubated with 7-aminoactinomycin D (5 μg/mL) for identification of living cells. After another wash, the cells were analyzed using a Becton Dickinson FACScanto. Gates for lymphocytes detected in the forward/side scatter blot and for 7-aminoactinomycin D-negative cells were intersected. The antibodies used were α-CD3-FITC (clone MEM-57), α-CD25-PE (clone TB-30), α-CD4-FITC, and α-CD8-FITC (all Immunotools). The flow cytometry data presented in the figures are representative of at least three independent experiments.

Western Blot Analysis

For protein analysis, cells were lysed using Laemmli buffer. Cell lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Following blocking for 1 h with PBS containing 0.05% Tween 20 and 5% powdered skim milk, blots were incubated overnight with primary antibody, washed three times with PBS/0.05% Tween 20, and then incubated with the peroxidase-coupled secondary antibody. The bands were detected using a chemiluminescence detection kit (Roche Diagnostics). The antibodies used were α-phospho-p44/42 MAPK (Thr202/Tyr204) clone E10, rabbit polyclonal α-phospho-MEK1/2 (Ser217/Ser221; all Cell Signaling), and α-vimentin clone SP20 (LabVision). The Western blots depicted in the figures are representative of at least three independent experiments.

Proliferation and Viability Measured by the MTS Assay

PBLs derived from a patient by leukapheresis were seeded with 2 × 105 cells per well in 96-well plates. Sorafenib with variable concentrations and/or α-CD3 and α-CD28 (0.5 μg/mL each) were added to the cells. Following an incubation period at 37°C for 3 days, proliferation and cell viability were assessed by the MTS assay (CellTiter 96 AQueous One Solution Cell Proliferation assay; Promega). To this end, 10 μL CellTiter 96 Aqueous One Solution Reagent containing a tetrazolium compound (MTS) were added to each well and the cells were incubated for ∼90 min at 37°C. Metabolically active, viable cells convert MTS into a colored formazan product that was measured in a spectrophotometric microplate reader (Perkin-Elmer) at 493 nm. The MTS assay was done in triplicates with at least two independent experiments leading to similar results.

ELISPOT

The ELISPOT assay used to quantify peptide epitope-specific IFN-releasing effector cells was described previously (18). Briefly, nitrocellulose-bottomed 96-well plates (MultiScreen MAIP N45; Millipore) were coated with an anti-IFN antibody (clone 1-D1K; Mabtech), and nonspecific binding was blocked with X-Vivo (Cambrex Biosciences). Following 7 days of preincubation with a modified HLA-A2-restricted survivin96-104 epitope (Girindus; 10 μmol/L), PBLs were added at different cell densities with or without the specific peptide and with different sorafenib concentrations and were incubated overnight at 37°C. After three washes, the biotinylated detection antibody (7-B6-1-Biotin; Mabtech) was added. Its specific binding was visualized by using alkaline phosphatase-streptavidin together with the respective substrate (Mabtech). The reaction was terminated by washing with water on the appearance of dark purple spots. After drying the plates, spots were counted. The ELISPOT assay was done in triplicates with at least two independent experiments leading to similar results.

Results

Differences in Lymphocyte Signal Transduction following PMA, PHA, and Anti-CD3/CD28 Antibody Stimulation

For in vitro assays, T lymphocytes are commonly stimulated either by PMA, an activator of protein kinase C (19), the lectin PHA, which is thought to work by binding to cell membrane glycoproteins, including the TCR-CD3 complex (20), or the combined application of α-CD3 and α-CD28 antibodies (21). From these three different stimuli, PMA had the most pronounced effect on the activity of the MAPK pathway as measured by an increase in phospho-ERK (Fig. 1 ). PMA stimulation, however, induced the weakest functional T-cell activation as revealed by CD25 expression (Fig. 1). In contrast, both PHA and α-CD3/α-CD28 antibodies evoked only a relatively weak phospho-ERK response but nevertheless resulted in a strong increase in CD25 expression by T lymphocytes (Fig. 1). This pattern was observed for both PBLs and purified T cells (data not shown) from several different patients. Notably, in the experiment depicted in Fig. 1, MAPK pathway activation was maximal within minutes for PMA, whereas PHA and α-CD3/α-CD28 stimulation resulted in a delayed MAPK activation; the maximum of ERK phosphorylation was reached after several hours. This, however, was not consistently observed with PBLs or purified T cells from all patients: for some patients, stronger ERK phosphorylation following PHA or α-CD3/α-CD28 stimulation was observed earlier (Fig. 4D ).

Figure 1.
Figure 1.

CD25 induction and ERK phosphorylation following PMA, PHA, and α-CD3/α-CD28 antibody treatment. PBLs derived by leukapheresis from melanoma patients were incubated with PMA (100 ng/mL), PHA (10 μg/mL), and α-CD3/α-CD28 antibodies (0.5 μg/mL each), respectively. Following the indicated periods, total cell lysates were harvested, subjected to Western blot analysis, and probed with the indicated antibodies. Following 3 d in the presence of the different stimuli, CD25 and CD3 expression was analyzed by flow cytometry. Frequency of CD25+ cells among the CD3+ cells.

Figure 4.
Figure 4.

Differential effects of sorafenib and U0126 on MAPK signaling on TCR stimulation. PBLs derived by leukapheresis from a melanoma patient (A-C) or healthy donor (D) were stimulated with α-CD3 and α-CD28 antibodies in the presence of sorafenib (A and D) or U0126 (B and C). A, B, and D, following the indicated periods, total cell lysates were harvested, subjected to Western blot analysis, and probed with the indicated antibodies. C, after 3 d of stimulation, the cells were double-stained for CD25 with CD4 or CD8 and analyzed by flow cytometry. Percentage of CD25+ cells among the CD4+ and CD8+ cells.

Inhibition of PMA-Stimulated Induction of ERK Phosphorylation and CD25 Expression by Sorafenib

From the analysis of sorafenib-treated patients, it became apparent that steady-state sorafenib plasma levels of up to 10 μg/mL can be achieved by oral administration (14, 22). To date, it is still unclear whether PMA induced ERK phosphorylation in T lymphocytes is impaired in patients treated with sorafenib. Indeed, contradictory reports have been published (13, 14). Therefore, we analyzed the level of ERK phosphorylation in PBLs following PMA stimulation in the presence of 1, 5, and 25 μg/mL sorafenib corresponding to 2.15, 10.75, and 53.75 μmol/L, respectively. After 15 min, PMA-induced ERK phosphorylation was clearly reduced by 5 μg/mL sorafenib, whereas at 25 μg/mL phospho-ERK was similar to unstimulated PBLs, nearly undetectable (Fig. 2A ). This inhibitory effect of sorafenib was even more pronounced at 5 h subsequent to PMA stimulation. Flow cytometry analysis showed that this inhibition of MAPK pathway signaling was indeed associated with an inhibition of T-cell activation: in the presence of 5 μg/mL sorafenib, the PMA-induced increase of the percentage of CD3+ cells expressing CD25 was almost completely blocked (Fig. 2B). Similar results, sorafenib-induced decreased T-cell activation paralleled by inhibition of MAPK pathway signaling following PMA stimulation, were also observed for purified T cells from melanoma patients and healthy control groups (data not shown).

Figure 2.
Figure 2.

PMA-induced effects in T cells are impaired by sorafenib and U0126. PBLs derived by leukapheresis from melanoma patients were stimulated with PMA in the presence of sorafenib (A and B) or U0126 (C) at the indicated concentrations. A, at the indicated time points following addition of PMA, total cell lysates were harvested, subjected to Western blot analysis, and probed with the indicated antibodies. B, following 1 and 3 d of culture under the indicated conditions, the cells were analyzed for CD25 and CD3 expression by flow cytometry. Percentage of CD25+ cells among the CD3+ cells. C, at the indicated time points following addition of PMA, total cell lysates were harvested, subjected to Western blot analysis, and probed with the indicated antibodies.

Sorafenib Inhibits the Activation of T Lymphocytes by TCR Stimulation

Because PMA leads only to a limited activation of T cells, we next investigated whether sorafenib inhibits also more “physiologic” T-cell stimulation by compounds acting on the TCR complex at the plasma membrane. Notably, the induction of CD25 expression in T cells by combined treatment with α-CD3 and α-CD28 antibodies was largely reduced by the presence of 5 μg/mL sorafenib (Fig. 3A and B ). Moreover, this effect was independent both of the source of lymphocytes, healthy donors or melanoma patients, or the fact that PBL or purified T cells were used. A very similar reduction was observed by the presence of sorafenib on stimulation with PHA (data not shown). After 3 days of culture with a concentration of 25 μg/mL sorafenib, there were no CD25+ T cells detectable (Fig. 3B). This observation could be ascribed to an extensive loss of events in the lymphocyte gate correlating with a strong increase in 7-aminoactinomycin D-positive cells, which indicates massive cell death (data not shown). Moreover, by means of the MTS assay, we show that viability and/or proliferation of PBLs are affected by sorafenib. It is important to note that this was observed both in the absence and in the presence of an activating stimulus (Fig. 3C). These effects were most prominent at 25 μg/mL sorafenib but were also observed at lower concentrations, indicating that sorafenib affects not only the T-cell activation but also the vitality of lymphocytes at concentrations corresponding to those observed in the plasma of patients under therapy.

Figure 3.
Figure 3.

Sorafenib inhibits CD25 induction in α-CD3/α-CD28 antibody-stimulated T cells and affects viability/proliferation of PBLs. PBLs derived by leukapheresis from a melanoma patient were incubated for 3 d in the presence (stim.) or absence (unstim.) of α-CD3 and α-CD28 antibody in medium containing sorafenib. Following 3 d of culture, cells were double-stained for CD25 and CD4 or CD25 and CD8 and analyzed by flow cytometry. A, representative flow cytometry dot blots of cells stained for CD25 and CD4. B, frequency of CD25+ cells among CD4+ and CD8+ cells, respectively. Percentage of top right quadrant divided by the sum of right quadrants. C, following a 3-d culture under the indicated conditions, proliferation and cell viability were assessed by the MTS assay. This assay measures the conversion of MTS into a colored formazan product by metabolically active cells. The extinction is measured at 493 nm in spectrophotometric microplate reader. Mean of triplicates.

Elevated MAPK Pathway Activity on TCR Stimulation in the Presence of Sorafenib

Because impairment of T-cell activation by PMA stimulation correlated with decreased MAPK pathway activity, we also analyzed the effect of sorafenib on ERK phosphorylation after TCR stimulation. Surprisingly, sorafenib at 1 and 5 μg/mL, which showed an inhibitory effect on CD25 induction on stimulation (Fig. 3B), did not lead to reduced ERK phosphorylation. In fact, phospho-ERK signals following α-CD3/α-CD28 antibody stimulation were increased in the presence of 1 and 5 μg/mL sorafenib (Fig. 4A and D); the same unexpected observation was made for PHA stimulation of both PBLs and purified T cells obtained from either healthy donors or melanoma patients (data not shown). Western blot analysis for MEK phosphorylation further revealed that this MAPK pathway-activating effect of sorafenib can be observed already directly downstream of the RAF kinases because also MEK phosphorylation was increased by 1 and 5 μg/mL sorafenib (Fig. 4D).

In contrast to sorafenib, the MEK-specific inhibitor U0126 inhibited MAPK pathway activity and ERK phosphorylation in both α-CD3/α-CD28 antibody and PHA-stimulated PBLs (Fig. 4B; data not shown for PHA). Notably, 1 and 5 μg/mL sorafenib and 1 and 5 μmol/L U0126, although affecting ERK phosphorylation quite differently in TCR-stimulated PBLs, displayed a very similar degree of inhibition of ERK phosphorylation in PMA-stimulated PBLs (Fig. 2A and C).

The inhibition of the induction of CD25 expression in TCR-stimulated T cells by U0126 (Fig. 4C; data not shown for PHA) suggests that this process critically depends on active MAPK pathway signaling. For sorafenib, however, MAPK pathway inactivation seems not to be the mode of action to inhibit T-cell activation induced by receptor stimulation.

Sorafenib Inhibits Tumor Antigen-Specific T-Cell Responses

All stimuli used thus far were global; they were activating the whole T-cell population unspecifically. Because sorafenib is intended to be used for the treatment of cancer, we further scrutinized whether sorafenib would also affect ongoing specific immune responses directed against tumor antigens. This situation can be simulated by activation of T cells with peptides of tumor-associated antigens presented by antigen-presenting cells. To this end, PBLs from patients who have received a survivin-based peptide vaccination were analyzed in ELISPOT assays using the respective survivin peptides for MHC class I-restricted stimulation (17, 23). Each spot (Fig. 5A ) represents peptide-reactive, IFN-γ-producing T cells. In the presence of 1 and 5 μg/mL sorafenib, the number of survivin-reactive T cells was already markedly reduced (Fig. 5A). Accordingly, the frequency of CD25-expressing cells on peptide stimulation was also diminished by sorafenib (Fig. 5B). Notably, frequencies of CD25-expressing cells on specific antigen stimulations are in the expected range, which is substantially lower than observed for global stimulation.

Figure 5.
Figure 5.

Sorafenib inhibits antigen-specific T-cell stimulation. A, following a 7-day in vitro stimulation with the survivin peptide, the given cell numbers of PBLs were subjected to the ELISPOT assay in the presence of sorafenib. Exemplary wells are depicted with each spot representing a survivin-specific T cells. The number of specific spots, the difference between spots counted with peptide and without, respectively, are presented as bar graphs. B, PBLs derived by leukapheresis from a melanoma patient receiving survivin peptide vaccination therapy were stimulated for 3 d with a modified HLA-A2 restricted survivin96-104 epitope in the presence of different concentrations of sorafenib. Flow cytometry dot blots of cells stained for CD25 and CD4.

Discussion

Sorafenib, which has been already clinically tested since 2000 for the treatment of different cancers, is a multikinase inhibitor with high activity toward RAF kinases, vascular endothelial growth factor receptors, platelet-derived growth factor receptors, FLT3, and KIT (9, 24). Indeed, sorafenib is very efficient in the treatment of advanced renal cell and hepatocellular carcinoma, which consequently led to its approval by the Food and Drug Administration for therapy of this malignancies (10, 25). In contrast, sorafenib monotherapy for treatment of melanoma failed to show any efficacy (8). This was unexpected because melanoma is the tumor harboring the highest frequency of activating BRAF mutations (2) and BRAF mutant cancer cell lines have been shown to be exceptionally sensitive to inhibition of the MAPK pathway (26). However, melanoma is known to be an immunogenic tumor, and disease stabilization as well as cases of spontaneous regression have been attributed to the patients immune system being capable of controlling tumor growth (27, 28). Therefore, we speculated whether the failure of sorafenib in the treatment of melanoma might be due to an immunosuppressive activity counterbalancing the direct antitumor activity of sorafenib, if sorafenib directly affects T-cell function. Noteworthy, in combination therapy with chemotherapeutics, sorafenib seems to add a therapeutic efficacy only for patients with brain metastases, patients harboring tumors at an immunoprivileged site (29, 30). Indeed, our data show that the presence of sorafenib at concentrations corresponding to plasma levels observed in sorafenib-treated patients inhibits the activation of T cells. This inhibition was shown as reduced CD25 expression in response to either protein kinase C activation by PMA or TCR stimulation with PHA or α-CD3/α-CD28 antibodies. Notably, this reduced activity became also evident on a functional level, a reduced antigen-specific IFN-γ production in the presence of sorafenib. These results are in agreement with several recently published findings. Zhao et al. showed that sorafenib inhibits activation and proliferation of lymphocytes in vitro (31). Moreover, using a mouse model with picryl chloride-induced contact dermatitis, sorafenib inhibited the extent of the induced allergic reaction in vivo. Indeed, voices were raised against the use of sorafenib for the treatment of immunogenic tumors or the use of sorafenib in combination with immunotherapeutic approaches (32). Hipp et al. show that immune responses are affected by sorafenib due to an impairment of the immunostimulatory capacity of dendritic cells. However, in contrast to our findings and those of Zhao et al., these authors report that the proliferation properties of isolated CD3+ T cells are not affected by sorafenib. The reasons for the observed difference remains elusive. One explanation, however, are the different experimental settings: Hipp et al. measured T-cell proliferation either after pretreatment of T cells with sorafenib in a mixed lymphocyte reaction or in the presence of artificial antigen-presenting cells. Thus, sorafenib was either absent during the proliferation phase of the T cells and/or stimulation conditions were different.

The measure of PMA-induced ERK phosphorylation in PBLs has been suggested to be a useful biomarker for measuring and predicting the effects of sorafenib (12). In this respect, Tong et al. reported that ERK phosphorylation on PMA treatment was not affected in PBLs from sorafenib-treated patients (13); others, however, observed a complete loss of phospho-ERK inducibility on sorafenib treatment (14). Our presented data support the latter finding because sorafenib concentrations comparable with patient's plasma levels efficiently inhibited ERK phosphorylation on PMA treatment. However, although PMA induced high levels of ERK phosphorylation, its effect on functional T-cell activation is rather limited, suggesting that it is not the ideal agent to study functional consequences of sorafenib on T lymphocytes. In contrast, PHA and α-CD3/α-CD28 antibody treatment activates T cells very efficiently. Notably, this activation was also impaired in the presence of even low concentrations of sorafenib. Surprisingly, however, this inhibition of T-cell activation was accompanied by an increase of ERK phosphorylation. Hence, inhibition of ERK phosphorylation seems not to be an adequate biomarker to monitor sorafenib effects on T-cell stimulation, because its outcome largely depends on the nature of the T-cell stimulus.

One of the first signaling events that occur on TCR stimulation is the activation of the two Src family kinases LCK and FYN (15). These subsequently phosphorylate several tyrosines within the immunoreceptor tyrosine-based activation motif of the TCR ζ chain. The phospho-immunoreceptor tyrosine-based activation motifs serve as docking sites for the tyrosine kinase ZAP70. On recruitment, ZAP70 gets phosphorylated on several tyrosine residues by two mechanisms: autophosphorylation and transphosphorylation by LCK (33). Among the residues that get phosphorylated in ZAP70, Tyr319 exerts a pivotal role for TCR-mediated activation of T cells (34, 35): phospho-Tyr319 serves as a binding site for LCK, which is critical for sustained immunoreceptor tyrosine-based activation motif phosphorylation (36). Consequently, LCK and ZAP70 phosphorylation are possible targets of sorafenib in the context of inhibition of T-cell activation. Indeed, it has recently been shown that sorafenib treatment results in reduced LCK phosphorylation in response to lymphocyte stimulation (31). However, the author's conclusion that this might be causal for reduced lymphocyte activation is not convincing because phosphorylation of LCK Tyr505, which is a known inhibitory site (37), was measured. The same authors presented evidence for reduced ZAP70 Tyr319 phosphorylation in the presence of sorafenib. We made a similar observation in some of our experiments but were not able to reproduce this result reliably (data not shown). To date, the available information on the inhibitory capacity of sorafenib on the kinase activity of LCK, which is responsible for ZAP70 phosphorylation, are not consistent: information distributed by the manufacturing company states that the IC50 value for LCK is >2,000 times higher than for CRAF.2 Nevertheless, it was recently shown that LCK activity was reduced to <30% at a concentration of 10 μmol/L sorafenib (38). Furthermore, the multikinase inhibitor sorafenib may affect additional pathways apart from the LCK/ZAP70 cascade translating TCR-mediated stimuli resulting in T-cell activation. These downstream events include activation of Ras and Rho family GTPases, the different MAPK cascades, phosphatidylinositol 3-kinase, protein kinase C, and the nuclear factor-κB pathway, activation of phospholipase C-γ1 and calcium signaling, which are all supposed to contribute to transcriptional activation of genes such as those encoding CD25 (39).

For the MAPK pathway-activating effect of sorafenib observed after PHA or CD3/CD28 stimulation, inhibition of phosphatidylinositol 3-kinase/Akt signaling may also serve as explanation as recently suggested (32). These authors reported increased ERK phosphorylation in lipopolysaccharide-stimulated dendritic cells in response to sorafenib and discussed inhibition of phosphatidylinositol 3-kinase/Akt signaling as possible mechanism because phosphorylation by Akt is known to inhibit RAF (40). Akt, however, is at least no direct target of sorafenib (38). Importantly, in lymphocytes, the MAPK pathway-activating effect of sorafenib was only observed after TCR-mediated stimuli and not after PMA stimulation. The principal difference between PMA and PHA or CD3/CD28 stimulation is that the protein kinase C activator PMA is acting on a protein that directly phosphorylates RAF, whereas the other more physiologic stimuli act via cell membrane receptors and therefore involve more complex signaling events regulating the MAPK pathway. In this respect, it is known that TCR signaling not only is positively regulating the MAPK pathway by activating RAF (41) but also involves a second set of signals down-regulating the MAPK pathway (15). For example, the dual-specificity phosphatase VHR, one of several MAPK-specific phosphatases, is activated (42). However, because we showed that MEK phosphorylation is also increased, the activation of the MAPK pathway occurs upstream of ERK. Hence, there must be additional modes of action that may include also RAF-activating events.

In summary, we show that sorafenib profoundly affects signal transduction during T-cell activation. Notably, the nature of these effects largely depends on the kind of T-cell stimulus. Nevertheless, for all tested stimuli, PMA, PHA, α-CD3/α-CD28 antibodies, or peptide-specific stimulation, the activity of lymphocytes is decreased by sorafenib. Thus, the differences in clinical benefits of sorafenib for the treatment of different malignancies may at least in part be ascribed to varying degrees of immunosurveillance for the respective tumors.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Susanne Schueler and Katharina Meder for excellent technical assistance.

Footnotes

  • ↵1 http://www.clinicaltrials.gov

  • ↵2 http://www.nexavar.nl/pdf/Brochure_voor_artsen.pdf

  • Grant support: DFG grant KFO124 (R. Houben and J.C. Becker).

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    • Accepted November 25, 2008.
    • Received January 24, 2008.
    • Revision received November 7, 2008.

References

  1. ↵
    Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57–70.
    OpenUrlCrossRefPubMed
  2. ↵
    Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature 2002;417:949–54.
    OpenUrlCrossRefPubMed
  3. ↵
    Chen Z, Gibson TB, Robinson F, et al. MAP kinases. Chem Rev 2001;101:2449–76.
    OpenUrlCrossRefPubMed
  4. ↵
    Adjei AA. Blocking oncogenic Ras signaling for cancer therapy. J Natl Cancer Inst 2001;93:1062–74.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Sridhar SS, Hedley D, Siu LL. RAF kinase as a target for anticancer therapeutics. Mol Cancer Ther 2005;4:677–85.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Strumberg D, Voliotis D, Moeller JG, et al. Results of phase I pharmacokinetic and pharmacodynamic studies of the RAF kinase inhibitor BAY 43-9006 in patients with solid tumors. Int J Clin Pharmacol Ther 2002;40:580–1.
    OpenUrlCrossRefPubMed
  7. ↵
    Becker JC, Kirkwood JM, Agarwala SS, Dummer R, Schrama D, Hauschild A. Molecularly targeted therapy for melanoma: current reality and future options. Cancer 2006;107:2317–27.
    OpenUrlCrossRefPubMed
  8. ↵
    Eisen T, Ahmad T, Flaherty KT, et al. Sorafenib in advanced melanoma: a phase II randomised discontinuation trial analysis. Br J Cancer 2006;95:581–6.
    OpenUrlCrossRefPubMed
  9. ↵
    Gridelli C, Maione P, Del Gaizo F, et al. Sorafenib and sunitinib in the treatment of advanced non-small cell lung cancer. Oncologist 2007;12:191–200.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Escudier B, Eisen T, Stadler WM, et al. Sorafenib in advanced clear-cell renal-cell carcinoma. N Engl J Med 2007;356:125–34.
    OpenUrlCrossRefPubMed
  11. ↵
    Brugarolas J. Renal-cell carcinoma—molecular pathways and therapies. N Engl J Med 2007;356:185–7.
    OpenUrlCrossRefPubMed
  12. ↵
    Strumberg D, Clark JW, Awada A, et al. Safety, pharmacokinetics, and preliminary antitumor activity of sorafenib: a review of four phase I trials in patients with advanced refractory solid tumors. Oncologist 2007;12:426–37.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Tong FK, Chow S, Hedley D. Pharmacodynamic monitoring of BAY 43-9006 (sorafenib) in phase I clinical trials involving solid tumor and AML/MDS patients, using flow cytometry to monitor activation of the ERK pathway in peripheral blood cells. Cytometry B Clin Cytom 2006;70:107–14.
    OpenUrlPubMed
  14. ↵
    Strumberg D, Richly H, Hilger RA, et al. Phase I clinical and pharmacokinetic study of the novel RAF kinase and vascular endothelial growth factor receptor inhibitor BAY 43-9006 in patients with advanced refractory solid tumors. J Clin Oncol 2005;23:965–72.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Mustelin T, Tasken K. Positive and negative regulation of T-cell activation through kinases and phosphatases. Biochem J 2003;371:15–27.
    OpenUrlCrossRefPubMed
  16. ↵
    Andersen MH, Schrama D, Thor SP, Becker JC. Cytotoxic T cells. J Invest Dermatol 2006;126:32–41.
    OpenUrlCrossRefPubMed
  17. ↵
    Wobser M, Keikavoussi P, Kunzmann V, Weininger M, Andersen MH, Becker JC. Complete remission of liver metastasis of pancreatic cancer under vaccination with a HLA-A2 restricted peptide derived from the universal tumor antigen survivin. Cancer Immunol Immunother 2006;55:1294–8.
    OpenUrlCrossRefPubMed
  18. ↵
    Schrama D, Andersen MH, Terheyden P, et al. Oligoclonal T-cell receptor usage of melanocyte differentiation antigen-reactive T cells in stage IV melanoma patients. Cancer Res 2001;61:493–6.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Touraine JL, Hadden JW, Touraine F, Hadden EM, Estensen R, Good RA. Phorbol myristate acetate: a mitogen selective for a T-lymphocyte subpopulation. J Exp Med 1977;145:460–5.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Kay JE. Mechanisms of T lymphocyte activation. Immunol Lett 1991;29:51–4.
    OpenUrlCrossRefPubMed
  21. ↵
    Trickett A, Kwan YL. T cell stimulation and expansion using anti-CD3/CD28 beads. J Immunol Methods 2003;275:251–5.
    OpenUrlCrossRefPubMed
  22. ↵
    Awada A, Hendlisz A, Gil T, et al. Phase I safety and pharmacokinetics of BAY 43-9006 administered for 21 days on/7 days off in patients with advanced, refractory solid tumours. Br J Cancer 2005;92:1855–61.
    OpenUrlCrossRefPubMed
  23. ↵
    Otto K, Andersen MH, Eggert A, et al. Lack of toxicity of therapy-induced T cell responses against the universal tumour antigen survivin. Vaccine 2005;23:884–9.
    OpenUrlCrossRefPubMed
  24. ↵
    Wilhelm S, Carter C, Lynch M, et al. Discovery and development of sorafenib: a multikinase inhibitor for treating cancer. Nat Rev Drug Discov 2006;5:835–44.
    OpenUrlCrossRefPubMed
  25. ↵
    Pang RW, Poon RT. From molecular biology to targeted therapies for hepatocellular carcinoma: the future is now. Oncology 2007;72 Suppl 1:30–44.
    OpenUrlCrossRefPubMed
  26. ↵
    Solit DB, Garraway LA, Pratilas CA, et al. BRAF mutation predicts sensitivity to MEK inhibition. Nature 2006;439:358–62.
    OpenUrlCrossRefPubMed
  27. ↵
    Enk AH, Becker JC, Schuler G. Immunotherapy of malignant melanoma—basic principles and novel therapeutic approaches. J Dtsch Dermatol Ges 2006;4:635–45.
    OpenUrlCrossRefPubMed
  28. ↵
    Woods GM, Malley RC, Muller HK. The skin immune system and the challenge of tumour immunosurveillance. Eur J Dermatol 2005;15:63–9.
    OpenUrlPubMed
  29. ↵
    Agarwala SS, Keilholz U, Hogg D, et al. Randomized phase III study of paclitaxel plus carboplatin with or without sorafenib as second-line treatment in patients with advanced melanoma. J Clin Oncol (Meet Abstr) 2007;25:8510.
    OpenUrl
  30. ↵
    Amaravadi R, Schuchter LM, McDermott DF, et al. Updated results of a randomized phase II study comparing two schedules of temozolomide in combination with sorafenib in patients with advanced melanoma. J Clin Oncol (Meet Abstr) 2007;25:8527.
    OpenUrl
  31. ↵
    Zhao W, Gu YH, Song R, Qu BQ, Xu Q. Sorafenib inhibits activation of human peripheral blood T cells by targeting LCK phosphorylation. Leukemia 2008;22:1226–33.
    OpenUrlCrossRefPubMed
  32. ↵
    Hipp MM, Hilf N, Walter S, et al. Sorafenib, but not sunitinib, affects function of dendritic cells and induction of primary immune responses. Blood 2008;111:5610–20.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Neumeister EN, Zhu Y, Richard S, Terhorst C, Chan AC, Shaw AS. Binding of ZAP-70 to phosphorylated T-cell receptor ζ and η enhances its autophosphorylation and generates specific binding sites for SH2 domain-containing proteins. Mol Cell Biol 1995;15:3171–8.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Di Bartolo V, Mege D, Germain V, et al. Tyrosine 319, a newly identified phosphorylation site of ZAP-70, plays a critical role in T cell antigen receptor signaling. J Biol Chem 1999;274:6285–94.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Williams BL, Irvin BJ, Sutor SL, et al. Phosphorylation of Tyr319 in ZAP-70 is required for T-cell antigen receptor-dependent phospholipase C-γ1 and Ras activation. EMBO J 1999;18:1832–44.
    OpenUrlAbstract
  36. ↵
    Pelosi M, Di Bartolo V, Mounier V, et al. Tyrosine 319 in the interdomain B of ZAP-70 is a binding site for the Src homology 2 domain of Lck. J Biol Chem 1999;274:14229–37.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Chow LM, Fournel M, Davidson D, Veillette A. Negative regulation of T-cell receptor signalling by tyrosine protein kinase p50csk. Nature 1993;365:156–60.
    OpenUrlCrossRefPubMed
  38. ↵
    Bain J, Plater L, Elliott M, et al. The selectivity of protein kinase inhibitors: a further update. Biochem J 2007;408:297–315.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Nel AE. T-cell activation through the antigen receptor. Part 1. Signaling components, signaling pathways, and signal integration at the T-cell antigen receptor synapse. J Allergy Clin Immunol 2002;109:758–70.
    OpenUrlCrossRefPubMed
  40. ↵
    Zimmermann S, Moelling K. Phosphorylation and regulation of RAF by Akt (protein kinase B). Science 1999;286:1741–4.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Liu B, Carle KW, Whisler RL. Reductions in the activation of ERK and JNK are associated with decreased IL-2 production in T cells from elderly humans stimulated by the TCR/CD3 complex and costimulatory signals. Cell Immunol 1997;182:79–88.
    OpenUrlCrossRefPubMed
  42. ↵
    Alonso A, Rahmouni S, Williams S, et al. Tyrosine phosphorylation of VHR phosphatase by ZAP-70. Nat Immunol 2003;4:44–8.
    OpenUrlCrossRefPubMed
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Molecular Cancer Therapeutics: 8 (2)
February 2009
Volume 8, Issue 2

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MAPK-independent impairment of T-cell responses by the multikinase inhibitor sorafenib
Roland Houben, Heike Voigt, Christiane Noelke, Valeska Hofmeister, Juergen C. Becker and David Schrama
Mol Cancer Ther February 1 2009 (8) (2) 433-440; DOI: 10.1158/1535-7163.MCT-08-1051
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