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Antitumour and immune-adjuvant activities of protein-tyrosine kinase inhibitors
Review
Antitumour and immune-adjuvant activities of protein-tyrosine kinase inhibitors Barbara Seliger1, Chiara Massa1, Brian Rini2, Jennifer Ko3 and Jim Finke2 1
Martin-Luther-Universitaet Halle-Wittenberg, Institute of Medical Immunology, 06112 Halle (Saale), Germany Department of Solid Tumor Oncology, Taussig Cancer Institute 3 Department of Immunology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue (NB30) Cleveland, USA 2
The immunologic approach to tumour therapy is hampered by the development of direct immune escape mechanisms and the induction of an immunosuppressive tumour microenvironment characterised by the expansion of myeloid-derived suppressor cells (MDSCs) and tumour-specific regulatory T cells (Tregs). The implementation of inhibitors targeting protein tyrosine kinases, which are involved in the process of tumour development and angiogenesis, has produced robust clinical responses. The consequences of these compounds on the functionality of immune effector cells have been investigated. This review summarises recent reports on the direct and indirect effects of protein tyrosine kinase inhibitors (TKIs) on the immune system and discusses the application of immunotherapeutic strategies in combination with these inhibitors to improve the efficacy of immune-based therapies. Introduction In the past decade, improvements in our knowledge of the transformation process have allowed the design of ‘tumourtargeting’ therapeutic approaches that can effectively abrogate tumour growth without the accompanying adverse side effects observed in patients undergoing radiation or chemotherapy. Malignant transformation is characterised by alterations in the intracellular signalling pathways that regulate cell proliferation, survival, differentiation and metabolism. Key components in the activation of such pathways are protein kinases that, upon the phosphorylation of target molecules, induce signalling cascades that culminate in the activation of gene transcription and modulation of protein expression or function. Thus, deregulated expression or activity of tyrosine kinases can promote different diseases, including malignancies of the haematopoietic system as well as solid tumours (Box 1). Based on the molecular events responsible for the maintenance of the malignant phenotype, specific inhibitors targeting those signal transduction pathways have been developed and demonstrated to have significant antitumour efficacies. So far, more than 30 protein kinase inhibitors are being used in clinical studies, whereas a series of others are currently being used in preclinical analyses to test their efficacy (e.g. TKI258, Corresponding author: Seliger, B. ([email protected]).
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regorafenib, axitinib, cediranib and brevanib). Second generation TKIs are being developed to manage primary and secondary drug resistance as well as the intolerance of a small group of patients to these drugs. In recent years, a deeper analysis of their mechanisms of action has highlighted broader molecular and cellular targets for these inhibitors, which exhibit not only specific effects on the tumour and its microenvironment, but also affect systemic activity, for example influencing a patient’s immune system. Therefore, in this review we will focus on the effects of TKIs not only on the tumour and its microenvironment, but also on particular cells of the innate and adaptive immune system. In addition, we support the idea of using TKIs to ‘de-bulk’ the primary tumour and induce a more responsive state in the patient to allow an immunemediated elimination of the residual disease. Imatinib, sorafenib and sunitinib: their targets, function and use in the clinic The first kinase inhibitor approved by the FDA was the TKI imatinib (STI571; Glivec, Novartis, Switzerland). This compound inhibits the ABL kinase present in the BCRABL fusion protein characteristic for Philadelphia chromosome positive (Ph+) chronic myeloid leukaemia (CML). After the first clinical trials that resulted in an ‘accelerated approval’ from the FDA [1], further phase III clinical trials confirmed a better response of chronic-phase CML patients to imatinib than to IFN-a treatment with complete haematological response in 95% and 55% of CML patients, respectively [2]. Because imatinib also targets the plateletderived growth factor (PDGF) receptor c-KIT (or CD117), the macrophage colony-stimulating factor (M-CSF) receptor and the fms-like tyrosine-protein kinase 3 (FLT3), it is not only used as first-line therapy (alone or in combination with allogeneic haematopoietic stem cell transplantation {HSCT}) for Ph+ CML, but also for c-KIT+ unresectable, metastatic gastrointestinal stromal tumours (GISTs) [3]. Owing to the clinical success of the imatinib treatment, several drugs have been designed to target metabolic as well as signalling pathways crucial for the initiation and maintenance of the malignant phenotype of other tumour types. Examples are sorafenib and sunitinib, two oral multi-targeted TKIs that block tumour growth, cell proliferation and angiogenesis in vitro and in vivo by targeting tumour and tumour endothelial cells. Both TKIs have been
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Box 1. Tyrosine kinases (TKs) in healthy and transformed cells TKs are involved in signalling pathways, which are essential in the regulation of all aspects of cellular life such as proliferation, differentiation, migration and metabolism. Deregulated activity of TKs is involved in the acquisition of many characteristics required for malignant transformation. For this reason, attempts have been made to identify inhibitors that specifically target molecules responsible for the malignant phenotype. Functionally, TKs can be classified into receptor and non-receptor kinases. The first family includes receptors of growth factors that regulate cell behaviour in response to extracellular stimuli. Structurally, these molecules are composed of an extracellular domain for binding of the specific ligand (i.e. the growth factor), a transmembrane region and the intracellular catalytic domain. Upon ligand binding, the receptors dimerise and undergo conformational changes, which activate the catalytic domain and signalling cascade(s) through the phosphorylation of target substrate(s). The cytoplasmic non-receptor TKs are involved in intracellular signalling and are frequently downstream of RTKs. Their catalytic activity is regulated by their interaction with other intracellular molecules that modify the TK activating it, or that relocate them to the proximity of their targets. The modifications that promote TK-mediated malignant transformation are diverse. Mutations in the catalytic site can produce a constitutively activated enzyme; modification in the ‘regulatory’ domain owing to mutation, deletion or chromosomal recombination can alter the modulation of the catalytic activity. For RTKs, overexpression can favour spontaneous dimerisation and constitutive activation in the absence of a ligand. Moreover, the ectopic production of the ligand by the tumour can induce the abnormal activation of an unaltered signalling pathway.
approved by the health authorities for the treatment of various tumours including advanced/metastatic renal cell carcinoma (RCC). The multikinase inhibitor sorafenib (BAY43-9006, Nexavar, Bayer Healthcare, Germany) targets a variety of receptor tyrosine kinases (RTKs) including c-KIT, the vascular endothelial growth factor (VEGF) receptors, PDGF receptor-b, FLT3 as well as the Raf–MEK–ERK pathway, which is frequently deregulated in solid tumours [4]. Sorafenib treatment resulted in an acceptable tolerability and a prolonged progression-free survival (PFS) compared with a placebo both in cytokine-refractory metastatic RCC [5–7] and hepatocellular carcinoma (HCC) [8,9]. Sunitinib (SU 11248, Sutent, Pfizer, USA) inhibits the RTKs c-KIT, VEGF receptor, PDGF receptor and FLT3. A phase III clinical trial in metastatic RCC demonstrated that sunitinib was superior to standard IFN-a treatment concerning clinical activity, toxicity profile and quality of life. The phase III study met the primary endpoint of PFS at second interim analysis. Follow-up data include PFS of 11 months, 31% objective response rate (ORR) and median overall survival of 26.4 months in metastatic RCC patients treated with sunitinib compared with a 6% ORR and five months PFS for IFN-a treatment [10,11]. In addition, sunitinib has been approved as a second-line therapy of imatinib-refractory GISTs [12]. The improved outcome of patients with advanced disease upon TKI treatment can be explained by the targeting of multiple kinases, which regulate tumour development at various levels (Figure 1). TKIs can directly affect tumour cells by inhibiting their proliferation and/or promoting antiapoptotic pathways [13,14], but they can also affect
Figure 1. ‘Expected’ antitumour effect of TKIs. TKIs can inhibit tumour progression by acting at different levels. Direct effects on tumour cells induce decreased proliferation and/or increased apoptosis. Similar effects on endothelial cells reduce the formation of new vascular and lymphatic vessels, thereby reducing nutrient supply and metastasis formation, respectively. Treatment with TKIs can remove endothelial anergy, thereby allowing a normalisation of the vessel structure and adhesiveness.
tumour-associated stroma cells, such as endothelial cells that are involved in neoangiogenesis. Indeed, solid tumours such as RCCs secrete proangiogenic factors such as VEGF or basic fibroblast growth factor (bFGF), which promote endothelial cell proliferation and differentiation into new vessels. Owing to the deregulated process that leads to vessel formation, tumour vasculature is characterised by vessels with larger interendothelial junctions, a lack of normal basement membrane and unorganised branches with uneven diameters, which results in abnormal blood flow and tissue pressure. In many preclinical murine studies, sorafenib and sunitinib have demonstrated antitumour activity, resulting in the regression of xenografts of different tumour types, which was directly associated with reduced microvessel density. Moreover, the treatment of RCC-bearing mice with sunitinib resulted in a normalisation of the tumour vasculature and blood flow; at higher concentrations, sunitinib caused tumour vessel destruction and also some alterations of the vasculature of the healthy kidney [15]. The negative impact of TKIs on healthy vasculature is linked to hypertension, a common side effect in patients treated with antiangiogenic therapy [16]. The antiangiogenic activity of VEGF receptor inhibitors might also interfere with lymphatic spread and metastases formation. Indeed, in parallel with vascular neoangiogenesis, tumour cells promote lymphangiogenesis. Cediranib, a novel VEGF receptor-2 and -3 inhibitor, is able to reduce the formation of lymphatic vessels and thereby inhibit the capacity of primary tumour cells to metastasise to the lymph node [17,18]. Patients often develop resistance to TKIs, which might be caused by secondary genetic alterations, such as amplifications or point mutations of their targets, or by the 185
Review activation of alternate signalling pathways [19]. Understanding the molecular mechanisms by which cancer patients develop resistance to TKIs is crucial and is a challenge for achieving long-term disease control [20]. Owing to the heterogeneity of TKI resistance, predicting the efficacy of TKIs is needed to select patients that could experience clinical benefit from this treatment. To identify prediction-related factors, tumour biopsies or serum samples have been collected from patients before treatment and are monitored at the clinical and molecular level [21–24]. To circumvent the development of resistance, it could be advantageous to combine TKIs with other treatment modalities such as immunotherapy. This might be a successful approach because TKIs, like sunitinib, can reduce the immunosuppression characterised by a high frequency of myeloid and T-suppressor cells observed in cancer patients [25] and murine tumour models [26]. Thus, as we will discuss below, the removal of immunosuppressive cells by TKI treatment might not only prevent opportunistic infection but also improve the antitumour effects of immunotherapeutic strategies. Tumour immunotherapy: rationale and problems It is generally accepted that tumour cells can be recognised and destroyed by the immune system. Based on this belief, two main strategies have been investigated to promote the immune-mediated elimination of cancer, namely active and adoptive immunotherapy. Active tumour immunotherapy takes inspiration from preventive vaccination against infectious diseases, where antigens derived from pathogens are injected into healthy individuals to activate their immune system against these pathogens. In addition to the induction of immune effector cells, which fight the disease, a further advantage of this strategy is the development of a pool of antigen-specific memory T cells, which protect against possible relapses. Although several different vaccination strategies have been successfully implemented in preclinical mouse models, the translation of such approaches into the clinical setting has been disappointing; although the effective induction of tumour-specific CD8+ cytotoxic T lymphocytes (CTLs) occurs in patients, long-term survival is not improved [27]. Many different vaccination strategies have been used. In the cases where the tumour antigens are known, single or multiple peptides are injected either ‘naked’ or complexed to carriers such as heat-shock proteins, which can favour targeting to and uptake by antigen presenting cells (APCs). The injection of irradiated and genetically modified whole tumour cells to enhance their immunogenicity has also been implemented to provide the complete antigenic repertoire of the tumour in cases where the specific antigens have not been identified. With an increased knowledge about the role of dendritic cells (DCs) in the crosstalk between adaptive and innate immunity for shaping the immune response, different DC-based protocols have been developed. During the past decade many clinical trials have employed DCs that were expanded and differentiated in vitro and loaded with tumour antigens, either by pulsing them with peptides or whole tumour cell lysates, or by transfection [28–32]. 186
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In adoptive or passive immunotherapy, effector cells are transferred into patients to recognise and kill the tumour, as observed in the graft-vs.-leukaemia effect upon successful HSCT. Because the autologous immune system is not involved in tumour elimination, this strategy cannot provide patients with immunologic memory. However, an advantage of this approach is that the in vitro manipulation of effector cells could increase their antitumour efficacy. For example, autologous tumour-infiltrating lymphocytes (TILs) could be restimulated in vitro with cytokine cocktails or genetically engineered with tumour-specific T cell receptors to induce antitumour specificity [33]. The underlying reasons for the failure or impaired efficacy of immunotherapies might be explained by the immune escape strategies developed by the tumour cells themselves or the establishment of an immunosuppressive status. Indeed, tumours often exhibit a downregulation of HLA class I surface antigens and a lack of co-stimulatory molecules such as B7-1/B7-2 that, together with the expression of inhibitory molecules such as HLA-G or members of the B7-H family, lead to reduced immune cell recognition and/or activation. In addition, abnormalities in the tumour vasculature regarding its structure and adhesiveness provide a further ‘physical’ barrier for the immune cell infiltration of the tumour. The adhesion molecules, intracellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), required for leukocyte adhesion, rolling and extravasation, are often constitutively expressed at lower levels in tumour endothelial cells than healthy tissue endothelium, and are not inducible by inflammatory stimuli, a situation referred to as ‘endothelial anergy’. Another explanation for the failure of immunotherapy is the induction of an immunosuppressive micro-milieu. Indeed, an abnormal myeloid lineage differentiation profile has been demonstrated in tumour patients, which might have possible consequences for their immune competence. In RCC patients, a high frequency of myeloid cells, in particular neutrophilic granulocytes, was found and was associated with a poor prognosis [34,35]. In addition, low frequencies of immunostimulatory DC subsets and an accumulation of circulating MDSCs as well as Tregs have been found in patients with various cancers, including breast, colon, pancreatic, non-small cell lung, head and neck and RCCs; these observations are consistent with an immunosuppressive tumour microenvironment [36–38]. A direct link between MDSCs and the induction of Tregs was demonstrated both in vitro and in murine cancer models in vivo; induction depended on IFN-g and IL-10 but not on nitric oxide (NO) production [39,40]. Based on the involvement of MDSCs and Tregs in the initiation of immunosuppression, attempts to reverse their immunosuppressive activity by decreasing their frequency and function might enhance immune responses as well as the efficacy of immunotherapies. Early studies by Gabrilovich and co-authors [41] demonstrated that in mixed lymphocyte reactions, the function of DCs was impaired in the presence of tumour cell culture supernatants, which could be at least partially reverted by antibodies directed against VEGF [42,43]. In addition to blocking monocyte differentiation into DCs, VEGF has been shown to mediate immunosuppression through the
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Table 1. Effects of TKIs on immune cells Cell type Imatinib
Effect
Setting
References
T cells
Inhibition of proliferation Inhibition of memory, but not naive CD8+ T cell responses Marginal impairment of differentiation
In vitro Mice In vitro
[53] [54,55] [53]
Stimulation to acquire NK stimulatory activity Partial recovery of frequency in peripheral blood Functional recovery of PDC Marginal impairment of maturation Enhancement of APC function
Mice and GIST CML CML In vitro Mice
[60] [53] [59] [53] [58]
In vitro
[49–51]
In vitro
[49]
Monocytes
Reduced proliferation as a result of the inhibition of ERK and lck phosphorylation; induction of apoptosis at high doses; CD4+ are more affected than CD8+ T cells Inhibition of Treg proliferation; induction of apoptosis upon T-cell receptor triggering without IL-2 Reduced, but not significant, lymphocyte numbers in combination therapy with IFN-a Correct differentiation in the presence of VEGF
RCC In vitro
[6] [43]
DCs
Impaired maturation and ability to stimulate T-cell proliferation
In vitro
[46]
Reduced suppressive activity; induction of apoptosis; Reduction of circulating MDSCs Reduced number of circulating Tregs Reduction of Th2-bias Inhibition of IL-12 production if present during differentiation No negative effect if present during maturation Normalisation of circulating MDC1 Increased frequency of mature tumour infiltrating DCs as a result of inhibition of STAT3 activity
In vitro Mice and RCC Mice and RCC RCC In vitro In vitro In vitro Mice
[26,48] [13,25,46,47] [25,46,48] [25,48] [43] [46] [47] [13]
Monocytes DCs
Sorafenib T cells
Sunitinib MDSCs T cells DCs
inhibition of the differentiation and migration of thymic lymphocyte progenitors from bone marrow (BM), resulting in reduced CD4+ and CD8+ T cell numbers. Studies in preclinical mouse models have demonstrated that it is possible to fight cancer with the immune system, but one has to counteract the evasion and suppressive mechanisms that tumours continuously develop during their progression. By the time tumours are detected, many have developed several escape mechanisms, which might negatively interfere with the success of immunotherapies. For this reason, combining immunotherapy with other therapies that can counteract immunosuppression and recover immune responsiveness might be beneficial for patients. Immunomodulatory activity of TKIs There is evidence that different TKIs are able to modulate immune responses. Thus, for the optimised clinical use of these inhibitors, a better understanding of the impact of TKIs and other coadministered therapies on specific antitumour responses as well as on the general immune activity against possible opportunistic infections is required, in particular because TKIs might mediate both beneficial and harmful effects on immune cells [44,45] (Table 1 and Figure 2). Immunomodulatory effects of TKIs on immunosuppressive cells Because MDSCs have been shown to promote tumourdependent angiogenesis and tumour metastases as well as tumour resistance to antiangiogenic drugs [40], the
effect of TKIs on MDSCs was determined in vitro and in vivo in preclinical and clinical settings. Sunitinib induced a significant decrease in MDSC accumulation in mouse models and RCC patients [14,25,46,47]. In vitro assays highlighted increased apoptosis of MDSCs upon treatment of patients’ MDSCs with sunitinib [43] and a reduced capacity to inhibit T cell effector function [26,48]. Some reports indicated that sunitinib administration significantly decreased the frequency of Tregs in vitro and in vivo [25,46,48], whereas the proliferation capacity of preactivated Tregs in response to IL-2 was not altered in vitro [25]. Similarly, no variations in Treg proliferation were found upon sorafenib treatment whether cells were left untreated or stimulated by T cell receptor triggering and IL-2. By contrast, sorafenib dramatically decreased proliferation and induced apoptosis of Tregs stimulated via the T cell receptor in the absence of IL-2 [49]. The numerical reduction of immunosuppressive cells upon sunitinib treatment caused a significant increase in the number of lymphocytes producing IFN-g (Th1 response) and a diminished expression of type-2 cytokines upon in vitro stimulation [25,48]. However, whether the reduction of Tregs and MDSCs or the induction of T cell responses contributes to the therapeutic effects of sunitinib remains to be elucidated, because the reduced frequency of Tregs did not correlate with the reduction in tumour size in RCC patients [25]. In this context, it is noteworthy that the number of MDSCs was significantly reduced in three different murine tumour models treated with sunitinib, although tumour shrinkage was minimal in one model, moderate in a second and dramatically reduced in a third. 187
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Figure 2. Immunologic effects of TKI. In comparison with healthy individuals (a), cancer patients (b) present an accumulation of MDSCs and Tregs that can directly inhibit effector cells. The differentiation of professional presenting cells is impaired, and thereby there is a reduced amount and/or functionality of circulating APCs, which are impaired in their ability to activate effector cells. As a result of this reduced activation and enhanced inhibition of effector cells, cancer patients are in a general immunecompromised status and are unable to counteract tumour growth or protect themselves from other pathologies such as viral or bacterial infections. (c) Treatment with TKIs can affect the development and/or functionality of many immune cells in different directions depending on the particular TKI and target cell population. Imatinib favours the recovery of number and/or functionality of various APC populations, whereas it has contradictory effects on T cells. Sunitinib also favours the functionality of terminally differentiated DCs while inhibiting their differentiation from monocytes. Sunitinib inhibits the accumulation of MDSCs and Tregs, thereby reducing the suppression of immune responses. Sorafenib has an immunosuppressive activity; it inhibits effector cell activation by either acting directly on T cells or affecting DCs in their maturation and interaction with the lymphocytes.
Thus, the decreased frequency of MDSCs was not linked to diminished tumour size. Effects of TKIs on effector T cells In addition to indirect effects based on the removal of immunosuppression, there also exist reports about the direct activities of TKIs on effector T cells. Sorafenib negatively affects T cells; it inhibits CD25 and CD69 expression as well as IL-2 production, reduces or inhibits the phos188
phorylation of key components of the T cell signal transduction pathway such as ERK and lck and causes cell cycle arrest at G0/G1 [50,51]. The sorafenib-mediated reduction in activated T cell proliferation was time- and dose-dependent, resulting in an irreversible induction of apoptosis at concentrations greater than 10 mM. However, Hipp and coauthors [46] suggested that the effect of sorafenib on T cells is indirect because the preincubation of T cells with the drug did not impair successful proliferation in allogeneic DCs.
Review Notably, even established tumour-specific immune responses are negatively influenced by sorafenib through a mechanism independent of the mitogen-activated protein kinase (MAPK) [52]. Sorafenib, but not sunitinib, reversibly inhibited CD8+ T cell responses without affecting the phenotype of the cells. These contrasting results might be because of differences in experimental protocols or the existence of T cell subsets with distinct sensitivities. Indeed, treatment with sorafenib demonstrated that CD4+ T cells seem to be more susceptible than CD8+ T cells to the inhibitory effects of the drug [49]. Similarly, imatinib was reported to inhibit T cell proliferation in vitro [53], whereas in vivo this inhibition was confined to memory CD8+ T cells [54,55]. The results from patients treated with imatinib in combination with allogeneic HSCT are supportive of there being at least a ‘nonnegative’ activity of imatinib on the immune system. Based on these data, many clinical trials are currently being performed using imatinib in combination with active vaccination. These approaches are providing promising results [56]; for example, 73% of CML patients treated with imatinib and vaccinated with a cocktail of BCR-ABL peptides induced peptide-specific T cells with a memory phenotype [57]. Immunomodulatory effects of TKIs on DCs It is well known that DCs, which are the most potent professional APCs, play an important role in regulating immune tolerance and activating the immune system. Studies performed with TKIs in vitro have mainly focused on the differentiation and subsequent maturation of DCs starting from monocytic CD14+ precursors. Sunitinib and sorafenib treatment led to opposite effects depending on the chosen experimental procedures. When monocytes were differentiated in the presence of the drugs, sunitinib specifically reduced the ability of DCs to produce IL-12 upon maturation, although no dramatic changes in their ability to promote the proliferation of allogeneic T cells were reported. Moreover, sunitinib was unable to override the immunosuppressive effect of VEGF on differentiating monocytes, whereas sorafenib was able to restore
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normal differentiation despite the presence of VEGF [43]. Sorafenib treatment before DC maturation resulted in the impaired maturation and acquisition of co-stimulatory activities, whereas the properties of DCs pretreated with sunitinib were comparable with those of untreated control DCs [46]. Treatment with imatinib did not dramatically affect healthy or Ph+ monocytes in their ability to differentiate into stimulatory DCs in vitro [53]. Furthermore, murine BM-derived DCs exhibited an enhanced stimulatory activity in the presence of these drugs [58]. Preliminary data are also available on the effects of TKIs on the different DC populations in vivo. In preclinical models, tumour-infiltrating DCs of mice treated with sunitinib expressed higher levels of co-stimulatory molecules owing to a reduced level of phosphorylation of STAT3 [14]. Sorafenib, but not sunitinib, induced DC apoptosis and attenuated primary T cell responses in vivo [46]. RCC patients treated with sunitinib displayed normalisation in the myeloid lineage with an increase in blood CD1c+ MDC1 that correlated with the clinical outcome [47]. A similar trend in the recovery of terminally differentiated DCs has been described in CML patients treated with imatinib [53]; treatment also resulted in the complete recovery of plasmacytoid dendritic cell (PDC) number and function in patients with complete remission [59]. Moreover, owing to the inhibition of c-KIT, imatinib treatment provided DCs with a natural killer (NK) stimulatory activity in vivo in mice and GIST patients [60]. However, there exists limited information about the extent to which TKIs can alter DC populations in vivo and what impact such effects might have on adaptive immune responses. Thus, further evaluation of the recovery of the DC number and their functionality in vivo in TKI-treated patients is required to evaluate the possibility of combining TKIs with active immunotherapy to target endogenous DCs. Perspectives for combination treatments of TKIs with immunotherapies Therapy with multi-target TKIs could lead to a direct measure for the control of malignant cell growth by inhibiting important signalling pathways involved in the
Figure 3. Effects of antiangiogenic therapy. In healthy tissue (a), vessels are characterised by a complete endothelial layer that can express adhesion molecules and interact with leucocytes. The hierarchical branching of vessels leads to a constant and regular blood flow and pressure. In transformed tissue (b), induced vessels have irregular branches and diameters, which leads to irregular blood flow. The endothelial layer is in an anergic state with regard to adhesion molecule expression and there are gaps between the cells. Treatment with TKIs can lead to an initial ‘normalisation’ of the vasculature, and then to its death. The normalisation happens both at the single-cell level by the removal of endothelial anergy and at the vessel level by the recovery of a hierarchical structure. Further treatment with TKI can induce apoptosis in the endothelial cells, which can collapse the vessel and block the oxygen and nutrient supply.
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Review promotion of tumour proliferation and survival and by indirectly affecting tumour vascularisation, which affects its nutrient supply and possibly the formation of metastases. In addition, TKIs cause changes in the tumour microenvironment, thereby reducing its immunosuppressive activity and shaping the immune cell repertoire. These tumour extrinsic effects of TKIs together with the problem of tumours developing drug resistance support the design of combination therapies that employ TKIs together with chemotherapy or immunotherapeutic approaches. Indeed, the ‘normalising’ activity of antiangiogenic inhibitors can restore normal blood flow through the tumour, which can facilitate drug penetration and revert the endothelial anergy, thereby allowing the extravasation of leukocytes into the tumour. For this reason, the aims of antiangiogenic therapies have shifted from a ‘kill the vessel’ to induce tumour starvation and death strategy to a ‘normalise the vessel’ to improve targeting by other therapeutic approaches strategy [61,62] (Figure 3). Among the different TKIs analysed, sunitinib seems the most suitable for combination strategies with immunotherapy. Indeed, despite sharing many targets with sorafenib, the two compounds display almost opposite activities; whereas sunitinib favours an immune competent status, sorafenib impairs active immune responses. An explanation for these differences might be the distinct potency that each drug has against the various target receptors. The drugs might inhibit different pathways. The significant differences observed in the immune cell profiles between samples collected before and after treatment with the compound, which demonstrate an immune modulatory activity of this drug in cancer patients [47,48], lends hope to the idea of using sunitinib in combination with immunotherapy protocols. However, it is crucial to implement an extensive immunomonitoring protocol in these studies to properly characterise the numbers and functionality of the immune cells resulting from the different treatment modalities. Furthermore, the use of these novel drugs could help scientists and clinicians understand the complexities of the immune system, in particular the interactions between T cells, NK cells, APCs and tumours. This is especially important because these TKIs might not only affect the adaptive immune responses but also the innate immune responses by inhibiting NK cell activity against HLA class I negative tumours [63]. The potential to bidirectionally modulate the host immune responses with clinical doses of TKIs suggests that systematic TKI treatment can be coupled with immunotherapy strategies to provide an efficacious antitumour immune response, and could also be used for inducing therapeutic immunosuppression in cases of allogeneic tissue transplantation or autoimmune diseases [64]. An additional unexpected therapeutic application of TKIs could also be the induction of antiviral immunity, because sorafenib can directly inhibit viral replication through interference with c-Raf signalling [65]. Concluding remarks Preclinical and clinical studies with TKIs have demonstrated antitumour activity as evidenced by the effective 190
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Box 2. Outstanding questions What accounts for the opposing results of the same inhibitor on the same cell type reported by different groups? Are immunosuppressive effects long-lasting or are they lost between the cycles of therapy? Do the immunomodulatory effects of sunitinib contribute to its antitumour properties? How does TKI treatment affect the interplay between the different immune cell populations? How can complete and durable clinical responses be obtained? Do combinations of TKIs and immunotherapies increase the efficacy of therapy?
reduction in tumour progression. The evaluation of the molecular and cellular mechanisms of TKI activity has highlighted a broad spectrum of biological pathways and cell types that are affected by the compounds, suggesting that TKIs could be combined with other therapies, such as immunotherapy. In a setting of adoptive immunotherapy, the TKI-mediated removal of suppression at the tumour site together with better blood flow can be enough to allow transferred effector cells to invade the tumour and perform their killing activity. In a setting of active immunotherapy, in which the autologous effector cells have to be primed against the tumour, a more complete re-establishment of an immune competent status will be needed. To evaluate whether this second strategy can be applied, patients undergoing TKI therapy should undergo comprehensive immunomonitoring, which considers the number of suppressive versus effector cells and their functionality. Indeed, most of the ‘functional’ studies that have been performed to date test cells in vitro with a short exposure to the inhibitors and do not test cells taken from patients who have undergone cycle(s) of TKI treatment, which can have more profound effects on cellular differentiation. Finally, another important aspect that remains to be evaluated is the optimal level of rescue of immunosuppression so that, upon active immunisation, excessive autoimmunity does not become a problem (Box 2). References 1 Cohen, M.H. et al. (2002) Report from the FDA: approval summary for imatinib mesylate capsules in the treatment of chronic myelogenous leukemia. Clin. Cancer Res. 8, 935–942 ´ Brien, S. et al. (2003) Imatinib compared with interferon and low-dose 2 O cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N. Engl. J. Med. 348, 994–1004 3 Dagher, R. et al. (2002) Report from the FDA: approval summary: imatinib mesylate in the treatment of metastatic and/or unresectable malignant gastrointestinal stromal tumors. Clin. Cancer Res. 8, 3034– 3038 4 Adnane, L. et al. (2006) Sorafenib (BAY 43-9006, Nexavar), a dualaction inhibitor that targets RAF/MEK/ERK pathway in tumor cells and tyrosine kinases VEGFR/PDGFR in tumor vasculature. Methods Enzymol. 407, 597–612 5 Ratain, M.J. et al. (2006) Phase II placebo-controlled randomized discontinuation trial of sorafenib in patients with metastatic renal cell carcinoma. J. Clin. Oncol. 24, 2505–2512 6 Escudier, B. et al. (2007) Phase I trial of sorafenib in combination with IFN alpha-2a in patients with unresectable and/or metastatic renal cell carcinoma or malignant melanoma. Clin. Cancer Res. 13, 1801–1809 7 Escudier, B. et al. (2007) Sorafenib in advanced clear-cell renal-cell carcinoma. N. Engl. J. Med. 356, 125–134
Review 8 Abou-Alfa, G.K. et al. (2006) Phase II study of sorafenib in patients with advanced hepatocellular carcinoma. J. Clin. Oncol. 24, 4293– 4300 9 Llovet, J.M. et al. (2008) Sorafenib in advanced hepatocellular carcinoma. N. Engl. J. Med. 359, 378–390 10 Motzer, R.J. et al. (2006) Sunitinib in patients with metastatic renal cell carcinoma. JAMA. 295, 2516–2524 11 Motzer, R.J. et al. (2007) Sunitinib versus interferon alpha in metastatic renal-cell carcinoma. N. Engl. J. Med. 356, 115–124 12 Demetri, G.D. et al. (2006) Efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumour after failure of imatinib a randomized controlled trial. Lancet 368, 1329–1338 13 Liu, L. et al. (2006) Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res. 66, 11851– 11858 14 Xin, H. et al. (2009) Sunitinib inhibition of Stat3 induces renal cell carcinoma tumor cell apoptosis and reduces immunosuppressive cells. Cancer Res. 69, 2506–2513 15 Hillman, G.G. et al. (2009) Dynamic contrast-enhanced magnetic resonance imaging of vascular changes induced by sunitinib in papillary renal cell carcinoma xenograft tumors. Neoplasia 11, 910–920 16 Van der Veldt, A.A. et al. (2010) Reduction in skin microvascular density and changes in vessel morphology in patients treated with sunitinib. Anticancer drugs DOI: 10.10197/CAD.0b013e3283359c79 (http://journals.lww.com/anti-cancerdrugs/pages/default.aspx) 17 Heckman, C.A. et al. (2008) The tyrosine kinase inhibitor cediranib blocks ligand-induced vascular endothelial growth factor receptor-3 activity and lymphangiogenesis. Cancer Res. 68, 4754–4762 18 Padera, T.P. et al. (2008) Differential response of primary tumor versus lymphatic metastasis to VEGFR-2 and VEGFR-3 kinase inhibitors cediranib and vandetanib. Mol. Cancer Ther. 7, 2272–2279 19 Quinta´s-Cardama, A. et al. (2009) Mechanisms of primary and secondary resistance to imatinib in chronic myeloid leukemia. Cancer Control: Journal of the Moffitt Cancer Center 16 (2), 122– 131 20 Zhang, W.W. et al. (2009) Predictors of primary imatinib resistance in chronic myelogenous leukemia are distinct from those in secondary imatinib resistance. J. Clin. Oncol. 27, 3642–3649 21 Perez-Gracia, J.L. et al. (2009) Identification of TNF-alpha and MMP-9 as potential baseline predictive serum markers of sunitinib activity in patients with renal cell carcinoma using a human cytokine array. Br. J. Cancer 101, 1876–1883 22 Hirsch, F.R. et al. (2009) Predictive value of EGFR and HER2 overexpression in advanced non-small-cell lung cancer. Oncogene 28 (Suppl 1), S32–37 23 McWeeney, S.K. et al. (2009) A gene expression signature of CD34+ cells to predict major cytogenetic response in chronic phase chronic myeloid leukemia patients treated with imatinib. Blood 115, 315– 325 24 Dahabreh, I.J. et al. (2010) Somatic EGFR mutation and gene copy gain as predictive biomarkers for response to tyrosine kinase inhibitors in non-small cell lung cancer. Clin. Cancer Res. 16, 291–303 25 Finke, J.H. et al. (2008) Sunitinib reverses type-1 immune suppression and decreases T-regulatory cells in renal cell carcinoma patients. Clin. Cancer Res. 14, 6674–6682 26 Ozao-Choy, J. et al. (2009) The novel role of tyrosine kinase inhibitor in the reversal of immune suppression and modulation of tumor microenvironment for immune-based cancer therapies. Cancer Res. 69, 2514–2522 27 Gouttefangeas, C. et al. (2007) Immunotherapy of renal cell carcinoma. Cancer Immunol Immunother. 56, 117–128 28 Hoeltl, L. et al. (1999) Cellular and humoral immune responses in patients with metastatic renal cell carcinoma after vaccination with antigen pulsed dendritic cells. J. Urol. 161, 777–782 29 Su, Z. et al. (2003) Immunological and clinical responses in metastatic renal cancer patients vaccinated with tumor RNA-transfected dendritic cells. Cancer Res. 63, 2127–2133 30 Bleumer, I. et al. (2007) Preliminary analysis of patients with progressive renal cell carcinoma vaccinated with CA9-peptide-pulsed mature dendritic cells. J. Immunother. 30, 116–122 31 Berntsen, A. et al. (2008) Therapeutic dendritic cell vaccination of patients with metastatic renal cell carcinoma: a clinical phase 1/2 trial. J. Immunother. 31, 771–780
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32 Asemissen, A.M. and Brossart, P. (2009) Vaccination strategies in patients with renal cell carcinoma. Cancer Immunol Immunother. 58, 1169–1174 33 Rosenberg, S.A. et al. (2008) Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat. Rev. Cancer 8, 299–308 34 Blay, J.Y. et al. (1997) Role of interleukin-6 in the paraneoplastic inflammatory syndrome associated with renal-cell carcinoma. Int. J. Cancer. 72, 424–430 35 Choudeiri, T.K. et al. (2007) Clinical factors associated with outcome in patients with metastatic clear-cell renal cell carcinoma treated with vascular endothelial growth factor-targeted therapy. Cancer 110, 543–550 36 Young, M.R. and Lathers, D.M. (1999) Myeloid progenitor cells mediate immune suppression in patients with head and neck cancers. Int. J. Immunopharmacol. 21, 241–252 37 Almand, B. et al. (2001) Increased production of immature myeloid cells in cancer patients: a mechanism of immunosuppression in cancer. J. Immunol. 166, 678–689 38 Pan, P.Y. et al. (2008) Reversion of immune tolerance in advanced malignancy: modulation of myeloid-derived suppressor cell development by blockade of stem-cell factor function. Blood 111, 219–228 39 Bronte, V. et al. (2001) Tumor-induced immune dysfunctions caused by myeloid suppressor cells. J. Immunother. 24, 431–446 40 Ko, J.S. et al. (2009) Myeloid-derived suppressor cells: a novel therapeutic target. Curr. Oncol. Rep. 11, 87–93 41 Gabrilovich, D. et al. (1996) Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells. Nat. Med. 2, 1096–1103 42 Gabrilovich, D. et al. (1998) Vascular endothelial growth factor inhibits the development of dendritic cells and dramatically affects the differentiation of multiple hematopoietic lineages in vivo. Blood 92, 4150–4166 43 Alfaro, C. et al. (2009) Influence of bevacizumab, sunitinib and sorafenib as single agents or in combination on the inhibitory effects of VEGF on human dendritic cell differentiation from monocytes. Br. J. Cancer 100, 1111–1119 44 Mohty, M. et al. (2005) Imatinib: the narrow line between immune tolerance and activation. Trends Mol. Med. 11, 397–402 45 Seggewiss, R. et al. (2008) Immunomodulatory effects of imatinib and second-generation tyrosine kinase inhibitors on T cells and dendritic cells: an update. Cytotherapy 10, 633–641 46 Hipp, M.M. et al. (2008) Sorafenib, but not sunitinib, affects function of dendritic cells and induction of primary immune responses. Blood 111, 5610–5620 47 van Cruijsen, H. et al. (2008) Sunitinib-induced myeloid lineage redistribution in renal cell cancer patients: CD1c+ dendritic cell frequency predicts progression-free survival. Clin. Cancer Res. 14, 5884–5892 48 Ko, J.S. et al. (2009) Sunitinib mediates reversal of myeloid-derived suppressor cell accumulation in renal cell carcinoma patients. Clin. Cancer Res. 15, 2148–2157 49 Molhoek, K.R. et al. (2009) Apoptosis of CD4(+)CD25(high) T cells in response to Sirolimus requires activation of T cell receptor and is modulated by IL-2. Cancer Immunol Immunother. 58, 867–876 50 Hilger, R.A. et al. (2004) Correlation of ERK-phosphorylation and toxicities in patients treated with the Raf kinase inhibitor BAY 439006. Int. J. Clin. Pharmacol. Ther. 42, 648–649 51 Zhao, W. et al. (2008) Sorafenib inhibits activation of human peripheral blood T cells by targeting LCK phosphorylation. Leukemia. 22, 1226–1233 52 Houben, R. et al. (2009) MAPK-independent impairment of T-cell responses by the multikinase inhibitor sorafenib. Mol. Cancer Ther. 8, 433–440 53 Boissel, N. et al. (2006) Imatinib mesylate minimally affects bcr-abl+ and normal monocyte-derived dendritic cells but strongly inhibits T cell expansion despite reciprocal dendritic cell-T cell activation. J. Leukoc. Biol. 79, 747–756 54 Mumprecht, S. et al. (2006) Imatinib mesylate selectively impairs expansion of memory cytotoxic T cells without affecting the control of primary viral infections. Blood 108, 3406–3413 55 Sinai, P. et al. (2007) Imatinib mesylate inhibits antigen-specific memory CD8 T cell responses in vivo. J. Immunol. 178, 2028–2037
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Review 56 Pinilla-Ibarz, J. et al. (2009) The biological basis for immunotherapy in patients with chronic myelogenous leukemia. Cancer Control 16, 141–152 57 Rojas, J.M. (2007) Clinical evaluation of BCR-ABL peptide immunisation in chronic myeloid leukaemia: results of the EPIC study. Leukemia. 21, 2287–2295 58 Wang, H. et al. (2005) Imatinib mesylate (STI-571) enhances antigenpresenting cell function and overcomes tumor-induced CD4+ T-cell tolerance. Blood 105, 1135–1143 59 Mohty, M. et al. (2004) Imatinib and plasmacytoid dendritic cell function in patients with chronic myeloid leukemia. Blood 103, 4666–4668 60 Borg, C. et al. (2004) Novel mode of action of c-Kit tyrosine kinase inhibitors leading to NK cell-dependent antitumor effects. J. Clin. Invest. 114, 379–388
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Trends in Molecular Medicine Vol.16 No.4 61 Bellone, M. et al. (2008) Vascular targeting, chemotherapy and active immunotherapy: teaming up to attack cancer. Trends Immunol. 29, 235–241 62 Griffioen, A.W. (2008) Anti-angiogenesis: making the tumor vulnerable to the immune system. Cancer Immunol Immunother. 57, 1553–1558 63 Fraser, C.K. et al. (2009) Dasatinib inhibits recombinant viral antigenspecific murine CD4+ and CD8+ T-cell responses and NK-cell cytolytic activity in vitro and in vivo. Exp. Hematol. 37, 256–265 64 Louvet, C. et al. (2008) Tyrosine kinase inhibitors reverse type 1 diabetes in nonobese diabetic mice. Proc. Natl. Acad. Sci. U. S. A. 105, 18895–18900 65 Himmelsbach, K. et al. (2009) New aspects of an anti-tumor drug: sorafenib efficiently inhibits HCV replication. Gut. 58, 1644–1653
Antitumour and immune-adjuvant activities of protein-tyrosine kinase inhibitors Barbara Seliger1, Chiara Massa1, Brian Rini2, Jennifer Ko3 and Jim Finke2 1
Martin-Luther-Universitaet Halle-Wittenberg, Institute of Medical Immunology, 06112 Halle (Saale), Germany Department of Solid Tumor Oncology, Taussig Cancer Institute 3 Department of Immunology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue (NB30) Cleveland, USA 2
The immunologic approach to tumour therapy is hampered by the development of direct immune escape mechanisms and the induction of an immunosuppressive tumour microenvironment characterised by the expansion of myeloid-derived suppressor cells (MDSCs) and tumour-specific regulatory T cells (Tregs). The implementation of inhibitors targeting protein tyrosine kinases, which are involved in the process of tumour development and angiogenesis, has produced robust clinical responses. The consequences of these compounds on the functionality of immune effector cells have been investigated. This review summarises recent reports on the direct and indirect effects of protein tyrosine kinase inhibitors (TKIs) on the immune system and discusses the application of immunotherapeutic strategies in combination with these inhibitors to improve the efficacy of immune-based therapies. Introduction In the past decade, improvements in our knowledge of the transformation process have allowed the design of ‘tumourtargeting’ therapeutic approaches that can effectively abrogate tumour growth without the accompanying adverse side effects observed in patients undergoing radiation or chemotherapy. Malignant transformation is characterised by alterations in the intracellular signalling pathways that regulate cell proliferation, survival, differentiation and metabolism. Key components in the activation of such pathways are protein kinases that, upon the phosphorylation of target molecules, induce signalling cascades that culminate in the activation of gene transcription and modulation of protein expression or function. Thus, deregulated expression or activity of tyrosine kinases can promote different diseases, including malignancies of the haematopoietic system as well as solid tumours (Box 1). Based on the molecular events responsible for the maintenance of the malignant phenotype, specific inhibitors targeting those signal transduction pathways have been developed and demonstrated to have significant antitumour efficacies. So far, more than 30 protein kinase inhibitors are being used in clinical studies, whereas a series of others are currently being used in preclinical analyses to test their efficacy (e.g. TKI258, Corresponding author: Seliger, B. ([email protected]).
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regorafenib, axitinib, cediranib and brevanib). Second generation TKIs are being developed to manage primary and secondary drug resistance as well as the intolerance of a small group of patients to these drugs. In recent years, a deeper analysis of their mechanisms of action has highlighted broader molecular and cellular targets for these inhibitors, which exhibit not only specific effects on the tumour and its microenvironment, but also affect systemic activity, for example influencing a patient’s immune system. Therefore, in this review we will focus on the effects of TKIs not only on the tumour and its microenvironment, but also on particular cells of the innate and adaptive immune system. In addition, we support the idea of using TKIs to ‘de-bulk’ the primary tumour and induce a more responsive state in the patient to allow an immunemediated elimination of the residual disease. Imatinib, sorafenib and sunitinib: their targets, function and use in the clinic The first kinase inhibitor approved by the FDA was the TKI imatinib (STI571; Glivec, Novartis, Switzerland). This compound inhibits the ABL kinase present in the BCRABL fusion protein characteristic for Philadelphia chromosome positive (Ph+) chronic myeloid leukaemia (CML). After the first clinical trials that resulted in an ‘accelerated approval’ from the FDA [1], further phase III clinical trials confirmed a better response of chronic-phase CML patients to imatinib than to IFN-a treatment with complete haematological response in 95% and 55% of CML patients, respectively [2]. Because imatinib also targets the plateletderived growth factor (PDGF) receptor c-KIT (or CD117), the macrophage colony-stimulating factor (M-CSF) receptor and the fms-like tyrosine-protein kinase 3 (FLT3), it is not only used as first-line therapy (alone or in combination with allogeneic haematopoietic stem cell transplantation {HSCT}) for Ph+ CML, but also for c-KIT+ unresectable, metastatic gastrointestinal stromal tumours (GISTs) [3]. Owing to the clinical success of the imatinib treatment, several drugs have been designed to target metabolic as well as signalling pathways crucial for the initiation and maintenance of the malignant phenotype of other tumour types. Examples are sorafenib and sunitinib, two oral multi-targeted TKIs that block tumour growth, cell proliferation and angiogenesis in vitro and in vivo by targeting tumour and tumour endothelial cells. Both TKIs have been
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Box 1. Tyrosine kinases (TKs) in healthy and transformed cells TKs are involved in signalling pathways, which are essential in the regulation of all aspects of cellular life such as proliferation, differentiation, migration and metabolism. Deregulated activity of TKs is involved in the acquisition of many characteristics required for malignant transformation. For this reason, attempts have been made to identify inhibitors that specifically target molecules responsible for the malignant phenotype. Functionally, TKs can be classified into receptor and non-receptor kinases. The first family includes receptors of growth factors that regulate cell behaviour in response to extracellular stimuli. Structurally, these molecules are composed of an extracellular domain for binding of the specific ligand (i.e. the growth factor), a transmembrane region and the intracellular catalytic domain. Upon ligand binding, the receptors dimerise and undergo conformational changes, which activate the catalytic domain and signalling cascade(s) through the phosphorylation of target substrate(s). The cytoplasmic non-receptor TKs are involved in intracellular signalling and are frequently downstream of RTKs. Their catalytic activity is regulated by their interaction with other intracellular molecules that modify the TK activating it, or that relocate them to the proximity of their targets. The modifications that promote TK-mediated malignant transformation are diverse. Mutations in the catalytic site can produce a constitutively activated enzyme; modification in the ‘regulatory’ domain owing to mutation, deletion or chromosomal recombination can alter the modulation of the catalytic activity. For RTKs, overexpression can favour spontaneous dimerisation and constitutive activation in the absence of a ligand. Moreover, the ectopic production of the ligand by the tumour can induce the abnormal activation of an unaltered signalling pathway.
approved by the health authorities for the treatment of various tumours including advanced/metastatic renal cell carcinoma (RCC). The multikinase inhibitor sorafenib (BAY43-9006, Nexavar, Bayer Healthcare, Germany) targets a variety of receptor tyrosine kinases (RTKs) including c-KIT, the vascular endothelial growth factor (VEGF) receptors, PDGF receptor-b, FLT3 as well as the Raf–MEK–ERK pathway, which is frequently deregulated in solid tumours [4]. Sorafenib treatment resulted in an acceptable tolerability and a prolonged progression-free survival (PFS) compared with a placebo both in cytokine-refractory metastatic RCC [5–7] and hepatocellular carcinoma (HCC) [8,9]. Sunitinib (SU 11248, Sutent, Pfizer, USA) inhibits the RTKs c-KIT, VEGF receptor, PDGF receptor and FLT3. A phase III clinical trial in metastatic RCC demonstrated that sunitinib was superior to standard IFN-a treatment concerning clinical activity, toxicity profile and quality of life. The phase III study met the primary endpoint of PFS at second interim analysis. Follow-up data include PFS of 11 months, 31% objective response rate (ORR) and median overall survival of 26.4 months in metastatic RCC patients treated with sunitinib compared with a 6% ORR and five months PFS for IFN-a treatment [10,11]. In addition, sunitinib has been approved as a second-line therapy of imatinib-refractory GISTs [12]. The improved outcome of patients with advanced disease upon TKI treatment can be explained by the targeting of multiple kinases, which regulate tumour development at various levels (Figure 1). TKIs can directly affect tumour cells by inhibiting their proliferation and/or promoting antiapoptotic pathways [13,14], but they can also affect
Figure 1. ‘Expected’ antitumour effect of TKIs. TKIs can inhibit tumour progression by acting at different levels. Direct effects on tumour cells induce decreased proliferation and/or increased apoptosis. Similar effects on endothelial cells reduce the formation of new vascular and lymphatic vessels, thereby reducing nutrient supply and metastasis formation, respectively. Treatment with TKIs can remove endothelial anergy, thereby allowing a normalisation of the vessel structure and adhesiveness.
tumour-associated stroma cells, such as endothelial cells that are involved in neoangiogenesis. Indeed, solid tumours such as RCCs secrete proangiogenic factors such as VEGF or basic fibroblast growth factor (bFGF), which promote endothelial cell proliferation and differentiation into new vessels. Owing to the deregulated process that leads to vessel formation, tumour vasculature is characterised by vessels with larger interendothelial junctions, a lack of normal basement membrane and unorganised branches with uneven diameters, which results in abnormal blood flow and tissue pressure. In many preclinical murine studies, sorafenib and sunitinib have demonstrated antitumour activity, resulting in the regression of xenografts of different tumour types, which was directly associated with reduced microvessel density. Moreover, the treatment of RCC-bearing mice with sunitinib resulted in a normalisation of the tumour vasculature and blood flow; at higher concentrations, sunitinib caused tumour vessel destruction and also some alterations of the vasculature of the healthy kidney [15]. The negative impact of TKIs on healthy vasculature is linked to hypertension, a common side effect in patients treated with antiangiogenic therapy [16]. The antiangiogenic activity of VEGF receptor inhibitors might also interfere with lymphatic spread and metastases formation. Indeed, in parallel with vascular neoangiogenesis, tumour cells promote lymphangiogenesis. Cediranib, a novel VEGF receptor-2 and -3 inhibitor, is able to reduce the formation of lymphatic vessels and thereby inhibit the capacity of primary tumour cells to metastasise to the lymph node [17,18]. Patients often develop resistance to TKIs, which might be caused by secondary genetic alterations, such as amplifications or point mutations of their targets, or by the 185
Review activation of alternate signalling pathways [19]. Understanding the molecular mechanisms by which cancer patients develop resistance to TKIs is crucial and is a challenge for achieving long-term disease control [20]. Owing to the heterogeneity of TKI resistance, predicting the efficacy of TKIs is needed to select patients that could experience clinical benefit from this treatment. To identify prediction-related factors, tumour biopsies or serum samples have been collected from patients before treatment and are monitored at the clinical and molecular level [21–24]. To circumvent the development of resistance, it could be advantageous to combine TKIs with other treatment modalities such as immunotherapy. This might be a successful approach because TKIs, like sunitinib, can reduce the immunosuppression characterised by a high frequency of myeloid and T-suppressor cells observed in cancer patients [25] and murine tumour models [26]. Thus, as we will discuss below, the removal of immunosuppressive cells by TKI treatment might not only prevent opportunistic infection but also improve the antitumour effects of immunotherapeutic strategies. Tumour immunotherapy: rationale and problems It is generally accepted that tumour cells can be recognised and destroyed by the immune system. Based on this belief, two main strategies have been investigated to promote the immune-mediated elimination of cancer, namely active and adoptive immunotherapy. Active tumour immunotherapy takes inspiration from preventive vaccination against infectious diseases, where antigens derived from pathogens are injected into healthy individuals to activate their immune system against these pathogens. In addition to the induction of immune effector cells, which fight the disease, a further advantage of this strategy is the development of a pool of antigen-specific memory T cells, which protect against possible relapses. Although several different vaccination strategies have been successfully implemented in preclinical mouse models, the translation of such approaches into the clinical setting has been disappointing; although the effective induction of tumour-specific CD8+ cytotoxic T lymphocytes (CTLs) occurs in patients, long-term survival is not improved [27]. Many different vaccination strategies have been used. In the cases where the tumour antigens are known, single or multiple peptides are injected either ‘naked’ or complexed to carriers such as heat-shock proteins, which can favour targeting to and uptake by antigen presenting cells (APCs). The injection of irradiated and genetically modified whole tumour cells to enhance their immunogenicity has also been implemented to provide the complete antigenic repertoire of the tumour in cases where the specific antigens have not been identified. With an increased knowledge about the role of dendritic cells (DCs) in the crosstalk between adaptive and innate immunity for shaping the immune response, different DC-based protocols have been developed. During the past decade many clinical trials have employed DCs that were expanded and differentiated in vitro and loaded with tumour antigens, either by pulsing them with peptides or whole tumour cell lysates, or by transfection [28–32]. 186
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In adoptive or passive immunotherapy, effector cells are transferred into patients to recognise and kill the tumour, as observed in the graft-vs.-leukaemia effect upon successful HSCT. Because the autologous immune system is not involved in tumour elimination, this strategy cannot provide patients with immunologic memory. However, an advantage of this approach is that the in vitro manipulation of effector cells could increase their antitumour efficacy. For example, autologous tumour-infiltrating lymphocytes (TILs) could be restimulated in vitro with cytokine cocktails or genetically engineered with tumour-specific T cell receptors to induce antitumour specificity [33]. The underlying reasons for the failure or impaired efficacy of immunotherapies might be explained by the immune escape strategies developed by the tumour cells themselves or the establishment of an immunosuppressive status. Indeed, tumours often exhibit a downregulation of HLA class I surface antigens and a lack of co-stimulatory molecules such as B7-1/B7-2 that, together with the expression of inhibitory molecules such as HLA-G or members of the B7-H family, lead to reduced immune cell recognition and/or activation. In addition, abnormalities in the tumour vasculature regarding its structure and adhesiveness provide a further ‘physical’ barrier for the immune cell infiltration of the tumour. The adhesion molecules, intracellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), required for leukocyte adhesion, rolling and extravasation, are often constitutively expressed at lower levels in tumour endothelial cells than healthy tissue endothelium, and are not inducible by inflammatory stimuli, a situation referred to as ‘endothelial anergy’. Another explanation for the failure of immunotherapy is the induction of an immunosuppressive micro-milieu. Indeed, an abnormal myeloid lineage differentiation profile has been demonstrated in tumour patients, which might have possible consequences for their immune competence. In RCC patients, a high frequency of myeloid cells, in particular neutrophilic granulocytes, was found and was associated with a poor prognosis [34,35]. In addition, low frequencies of immunostimulatory DC subsets and an accumulation of circulating MDSCs as well as Tregs have been found in patients with various cancers, including breast, colon, pancreatic, non-small cell lung, head and neck and RCCs; these observations are consistent with an immunosuppressive tumour microenvironment [36–38]. A direct link between MDSCs and the induction of Tregs was demonstrated both in vitro and in murine cancer models in vivo; induction depended on IFN-g and IL-10 but not on nitric oxide (NO) production [39,40]. Based on the involvement of MDSCs and Tregs in the initiation of immunosuppression, attempts to reverse their immunosuppressive activity by decreasing their frequency and function might enhance immune responses as well as the efficacy of immunotherapies. Early studies by Gabrilovich and co-authors [41] demonstrated that in mixed lymphocyte reactions, the function of DCs was impaired in the presence of tumour cell culture supernatants, which could be at least partially reverted by antibodies directed against VEGF [42,43]. In addition to blocking monocyte differentiation into DCs, VEGF has been shown to mediate immunosuppression through the
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Table 1. Effects of TKIs on immune cells Cell type Imatinib
Effect
Setting
References
T cells
Inhibition of proliferation Inhibition of memory, but not naive CD8+ T cell responses Marginal impairment of differentiation
In vitro Mice In vitro
[53] [54,55] [53]
Stimulation to acquire NK stimulatory activity Partial recovery of frequency in peripheral blood Functional recovery of PDC Marginal impairment of maturation Enhancement of APC function
Mice and GIST CML CML In vitro Mice
[60] [53] [59] [53] [58]
In vitro
[49–51]
In vitro
[49]
Monocytes
Reduced proliferation as a result of the inhibition of ERK and lck phosphorylation; induction of apoptosis at high doses; CD4+ are more affected than CD8+ T cells Inhibition of Treg proliferation; induction of apoptosis upon T-cell receptor triggering without IL-2 Reduced, but not significant, lymphocyte numbers in combination therapy with IFN-a Correct differentiation in the presence of VEGF
RCC In vitro
[6] [43]
DCs
Impaired maturation and ability to stimulate T-cell proliferation
In vitro
[46]
Reduced suppressive activity; induction of apoptosis; Reduction of circulating MDSCs Reduced number of circulating Tregs Reduction of Th2-bias Inhibition of IL-12 production if present during differentiation No negative effect if present during maturation Normalisation of circulating MDC1 Increased frequency of mature tumour infiltrating DCs as a result of inhibition of STAT3 activity
In vitro Mice and RCC Mice and RCC RCC In vitro In vitro In vitro Mice
[26,48] [13,25,46,47] [25,46,48] [25,48] [43] [46] [47] [13]
Monocytes DCs
Sorafenib T cells
Sunitinib MDSCs T cells DCs
inhibition of the differentiation and migration of thymic lymphocyte progenitors from bone marrow (BM), resulting in reduced CD4+ and CD8+ T cell numbers. Studies in preclinical mouse models have demonstrated that it is possible to fight cancer with the immune system, but one has to counteract the evasion and suppressive mechanisms that tumours continuously develop during their progression. By the time tumours are detected, many have developed several escape mechanisms, which might negatively interfere with the success of immunotherapies. For this reason, combining immunotherapy with other therapies that can counteract immunosuppression and recover immune responsiveness might be beneficial for patients. Immunomodulatory activity of TKIs There is evidence that different TKIs are able to modulate immune responses. Thus, for the optimised clinical use of these inhibitors, a better understanding of the impact of TKIs and other coadministered therapies on specific antitumour responses as well as on the general immune activity against possible opportunistic infections is required, in particular because TKIs might mediate both beneficial and harmful effects on immune cells [44,45] (Table 1 and Figure 2). Immunomodulatory effects of TKIs on immunosuppressive cells Because MDSCs have been shown to promote tumourdependent angiogenesis and tumour metastases as well as tumour resistance to antiangiogenic drugs [40], the
effect of TKIs on MDSCs was determined in vitro and in vivo in preclinical and clinical settings. Sunitinib induced a significant decrease in MDSC accumulation in mouse models and RCC patients [14,25,46,47]. In vitro assays highlighted increased apoptosis of MDSCs upon treatment of patients’ MDSCs with sunitinib [43] and a reduced capacity to inhibit T cell effector function [26,48]. Some reports indicated that sunitinib administration significantly decreased the frequency of Tregs in vitro and in vivo [25,46,48], whereas the proliferation capacity of preactivated Tregs in response to IL-2 was not altered in vitro [25]. Similarly, no variations in Treg proliferation were found upon sorafenib treatment whether cells were left untreated or stimulated by T cell receptor triggering and IL-2. By contrast, sorafenib dramatically decreased proliferation and induced apoptosis of Tregs stimulated via the T cell receptor in the absence of IL-2 [49]. The numerical reduction of immunosuppressive cells upon sunitinib treatment caused a significant increase in the number of lymphocytes producing IFN-g (Th1 response) and a diminished expression of type-2 cytokines upon in vitro stimulation [25,48]. However, whether the reduction of Tregs and MDSCs or the induction of T cell responses contributes to the therapeutic effects of sunitinib remains to be elucidated, because the reduced frequency of Tregs did not correlate with the reduction in tumour size in RCC patients [25]. In this context, it is noteworthy that the number of MDSCs was significantly reduced in three different murine tumour models treated with sunitinib, although tumour shrinkage was minimal in one model, moderate in a second and dramatically reduced in a third. 187
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Figure 2. Immunologic effects of TKI. In comparison with healthy individuals (a), cancer patients (b) present an accumulation of MDSCs and Tregs that can directly inhibit effector cells. The differentiation of professional presenting cells is impaired, and thereby there is a reduced amount and/or functionality of circulating APCs, which are impaired in their ability to activate effector cells. As a result of this reduced activation and enhanced inhibition of effector cells, cancer patients are in a general immunecompromised status and are unable to counteract tumour growth or protect themselves from other pathologies such as viral or bacterial infections. (c) Treatment with TKIs can affect the development and/or functionality of many immune cells in different directions depending on the particular TKI and target cell population. Imatinib favours the recovery of number and/or functionality of various APC populations, whereas it has contradictory effects on T cells. Sunitinib also favours the functionality of terminally differentiated DCs while inhibiting their differentiation from monocytes. Sunitinib inhibits the accumulation of MDSCs and Tregs, thereby reducing the suppression of immune responses. Sorafenib has an immunosuppressive activity; it inhibits effector cell activation by either acting directly on T cells or affecting DCs in their maturation and interaction with the lymphocytes.
Thus, the decreased frequency of MDSCs was not linked to diminished tumour size. Effects of TKIs on effector T cells In addition to indirect effects based on the removal of immunosuppression, there also exist reports about the direct activities of TKIs on effector T cells. Sorafenib negatively affects T cells; it inhibits CD25 and CD69 expression as well as IL-2 production, reduces or inhibits the phos188
phorylation of key components of the T cell signal transduction pathway such as ERK and lck and causes cell cycle arrest at G0/G1 [50,51]. The sorafenib-mediated reduction in activated T cell proliferation was time- and dose-dependent, resulting in an irreversible induction of apoptosis at concentrations greater than 10 mM. However, Hipp and coauthors [46] suggested that the effect of sorafenib on T cells is indirect because the preincubation of T cells with the drug did not impair successful proliferation in allogeneic DCs.
Review Notably, even established tumour-specific immune responses are negatively influenced by sorafenib through a mechanism independent of the mitogen-activated protein kinase (MAPK) [52]. Sorafenib, but not sunitinib, reversibly inhibited CD8+ T cell responses without affecting the phenotype of the cells. These contrasting results might be because of differences in experimental protocols or the existence of T cell subsets with distinct sensitivities. Indeed, treatment with sorafenib demonstrated that CD4+ T cells seem to be more susceptible than CD8+ T cells to the inhibitory effects of the drug [49]. Similarly, imatinib was reported to inhibit T cell proliferation in vitro [53], whereas in vivo this inhibition was confined to memory CD8+ T cells [54,55]. The results from patients treated with imatinib in combination with allogeneic HSCT are supportive of there being at least a ‘nonnegative’ activity of imatinib on the immune system. Based on these data, many clinical trials are currently being performed using imatinib in combination with active vaccination. These approaches are providing promising results [56]; for example, 73% of CML patients treated with imatinib and vaccinated with a cocktail of BCR-ABL peptides induced peptide-specific T cells with a memory phenotype [57]. Immunomodulatory effects of TKIs on DCs It is well known that DCs, which are the most potent professional APCs, play an important role in regulating immune tolerance and activating the immune system. Studies performed with TKIs in vitro have mainly focused on the differentiation and subsequent maturation of DCs starting from monocytic CD14+ precursors. Sunitinib and sorafenib treatment led to opposite effects depending on the chosen experimental procedures. When monocytes were differentiated in the presence of the drugs, sunitinib specifically reduced the ability of DCs to produce IL-12 upon maturation, although no dramatic changes in their ability to promote the proliferation of allogeneic T cells were reported. Moreover, sunitinib was unable to override the immunosuppressive effect of VEGF on differentiating monocytes, whereas sorafenib was able to restore
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normal differentiation despite the presence of VEGF [43]. Sorafenib treatment before DC maturation resulted in the impaired maturation and acquisition of co-stimulatory activities, whereas the properties of DCs pretreated with sunitinib were comparable with those of untreated control DCs [46]. Treatment with imatinib did not dramatically affect healthy or Ph+ monocytes in their ability to differentiate into stimulatory DCs in vitro [53]. Furthermore, murine BM-derived DCs exhibited an enhanced stimulatory activity in the presence of these drugs [58]. Preliminary data are also available on the effects of TKIs on the different DC populations in vivo. In preclinical models, tumour-infiltrating DCs of mice treated with sunitinib expressed higher levels of co-stimulatory molecules owing to a reduced level of phosphorylation of STAT3 [14]. Sorafenib, but not sunitinib, induced DC apoptosis and attenuated primary T cell responses in vivo [46]. RCC patients treated with sunitinib displayed normalisation in the myeloid lineage with an increase in blood CD1c+ MDC1 that correlated with the clinical outcome [47]. A similar trend in the recovery of terminally differentiated DCs has been described in CML patients treated with imatinib [53]; treatment also resulted in the complete recovery of plasmacytoid dendritic cell (PDC) number and function in patients with complete remission [59]. Moreover, owing to the inhibition of c-KIT, imatinib treatment provided DCs with a natural killer (NK) stimulatory activity in vivo in mice and GIST patients [60]. However, there exists limited information about the extent to which TKIs can alter DC populations in vivo and what impact such effects might have on adaptive immune responses. Thus, further evaluation of the recovery of the DC number and their functionality in vivo in TKI-treated patients is required to evaluate the possibility of combining TKIs with active immunotherapy to target endogenous DCs. Perspectives for combination treatments of TKIs with immunotherapies Therapy with multi-target TKIs could lead to a direct measure for the control of malignant cell growth by inhibiting important signalling pathways involved in the
Figure 3. Effects of antiangiogenic therapy. In healthy tissue (a), vessels are characterised by a complete endothelial layer that can express adhesion molecules and interact with leucocytes. The hierarchical branching of vessels leads to a constant and regular blood flow and pressure. In transformed tissue (b), induced vessels have irregular branches and diameters, which leads to irregular blood flow. The endothelial layer is in an anergic state with regard to adhesion molecule expression and there are gaps between the cells. Treatment with TKIs can lead to an initial ‘normalisation’ of the vasculature, and then to its death. The normalisation happens both at the single-cell level by the removal of endothelial anergy and at the vessel level by the recovery of a hierarchical structure. Further treatment with TKI can induce apoptosis in the endothelial cells, which can collapse the vessel and block the oxygen and nutrient supply.
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Review promotion of tumour proliferation and survival and by indirectly affecting tumour vascularisation, which affects its nutrient supply and possibly the formation of metastases. In addition, TKIs cause changes in the tumour microenvironment, thereby reducing its immunosuppressive activity and shaping the immune cell repertoire. These tumour extrinsic effects of TKIs together with the problem of tumours developing drug resistance support the design of combination therapies that employ TKIs together with chemotherapy or immunotherapeutic approaches. Indeed, the ‘normalising’ activity of antiangiogenic inhibitors can restore normal blood flow through the tumour, which can facilitate drug penetration and revert the endothelial anergy, thereby allowing the extravasation of leukocytes into the tumour. For this reason, the aims of antiangiogenic therapies have shifted from a ‘kill the vessel’ to induce tumour starvation and death strategy to a ‘normalise the vessel’ to improve targeting by other therapeutic approaches strategy [61,62] (Figure 3). Among the different TKIs analysed, sunitinib seems the most suitable for combination strategies with immunotherapy. Indeed, despite sharing many targets with sorafenib, the two compounds display almost opposite activities; whereas sunitinib favours an immune competent status, sorafenib impairs active immune responses. An explanation for these differences might be the distinct potency that each drug has against the various target receptors. The drugs might inhibit different pathways. The significant differences observed in the immune cell profiles between samples collected before and after treatment with the compound, which demonstrate an immune modulatory activity of this drug in cancer patients [47,48], lends hope to the idea of using sunitinib in combination with immunotherapy protocols. However, it is crucial to implement an extensive immunomonitoring protocol in these studies to properly characterise the numbers and functionality of the immune cells resulting from the different treatment modalities. Furthermore, the use of these novel drugs could help scientists and clinicians understand the complexities of the immune system, in particular the interactions between T cells, NK cells, APCs and tumours. This is especially important because these TKIs might not only affect the adaptive immune responses but also the innate immune responses by inhibiting NK cell activity against HLA class I negative tumours [63]. The potential to bidirectionally modulate the host immune responses with clinical doses of TKIs suggests that systematic TKI treatment can be coupled with immunotherapy strategies to provide an efficacious antitumour immune response, and could also be used for inducing therapeutic immunosuppression in cases of allogeneic tissue transplantation or autoimmune diseases [64]. An additional unexpected therapeutic application of TKIs could also be the induction of antiviral immunity, because sorafenib can directly inhibit viral replication through interference with c-Raf signalling [65]. Concluding remarks Preclinical and clinical studies with TKIs have demonstrated antitumour activity as evidenced by the effective 190
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Box 2. Outstanding questions What accounts for the opposing results of the same inhibitor on the same cell type reported by different groups? Are immunosuppressive effects long-lasting or are they lost between the cycles of therapy? Do the immunomodulatory effects of sunitinib contribute to its antitumour properties? How does TKI treatment affect the interplay between the different immune cell populations? How can complete and durable clinical responses be obtained? Do combinations of TKIs and immunotherapies increase the efficacy of therapy?
reduction in tumour progression. The evaluation of the molecular and cellular mechanisms of TKI activity has highlighted a broad spectrum of biological pathways and cell types that are affected by the compounds, suggesting that TKIs could be combined with other therapies, such as immunotherapy. In a setting of adoptive immunotherapy, the TKI-mediated removal of suppression at the tumour site together with better blood flow can be enough to allow transferred effector cells to invade the tumour and perform their killing activity. In a setting of active immunotherapy, in which the autologous effector cells have to be primed against the tumour, a more complete re-establishment of an immune competent status will be needed. To evaluate whether this second strategy can be applied, patients undergoing TKI therapy should undergo comprehensive immunomonitoring, which considers the number of suppressive versus effector cells and their functionality. Indeed, most of the ‘functional’ studies that have been performed to date test cells in vitro with a short exposure to the inhibitors and do not test cells taken from patients who have undergone cycle(s) of TKI treatment, which can have more profound effects on cellular differentiation. Finally, another important aspect that remains to be evaluated is the optimal level of rescue of immunosuppression so that, upon active immunisation, excessive autoimmunity does not become a problem (Box 2). References 1 Cohen, M.H. et al. (2002) Report from the FDA: approval summary for imatinib mesylate capsules in the treatment of chronic myelogenous leukemia. Clin. Cancer Res. 8, 935–942 ´ Brien, S. et al. (2003) Imatinib compared with interferon and low-dose 2 O cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N. Engl. J. Med. 348, 994–1004 3 Dagher, R. et al. (2002) Report from the FDA: approval summary: imatinib mesylate in the treatment of metastatic and/or unresectable malignant gastrointestinal stromal tumors. Clin. Cancer Res. 8, 3034– 3038 4 Adnane, L. et al. (2006) Sorafenib (BAY 43-9006, Nexavar), a dualaction inhibitor that targets RAF/MEK/ERK pathway in tumor cells and tyrosine kinases VEGFR/PDGFR in tumor vasculature. Methods Enzymol. 407, 597–612 5 Ratain, M.J. et al. (2006) Phase II placebo-controlled randomized discontinuation trial of sorafenib in patients with metastatic renal cell carcinoma. J. Clin. Oncol. 24, 2505–2512 6 Escudier, B. et al. (2007) Phase I trial of sorafenib in combination with IFN alpha-2a in patients with unresectable and/or metastatic renal cell carcinoma or malignant melanoma. Clin. Cancer Res. 13, 1801–1809 7 Escudier, B. et al. (2007) Sorafenib in advanced clear-cell renal-cell carcinoma. N. Engl. J. Med. 356, 125–134
Review 8 Abou-Alfa, G.K. et al. (2006) Phase II study of sorafenib in patients with advanced hepatocellular carcinoma. J. Clin. Oncol. 24, 4293– 4300 9 Llovet, J.M. et al. (2008) Sorafenib in advanced hepatocellular carcinoma. N. Engl. J. Med. 359, 378–390 10 Motzer, R.J. et al. (2006) Sunitinib in patients with metastatic renal cell carcinoma. JAMA. 295, 2516–2524 11 Motzer, R.J. et al. (2007) Sunitinib versus interferon alpha in metastatic renal-cell carcinoma. N. Engl. J. Med. 356, 115–124 12 Demetri, G.D. et al. (2006) Efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumour after failure of imatinib a randomized controlled trial. Lancet 368, 1329–1338 13 Liu, L. et al. (2006) Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res. 66, 11851– 11858 14 Xin, H. et al. (2009) Sunitinib inhibition of Stat3 induces renal cell carcinoma tumor cell apoptosis and reduces immunosuppressive cells. Cancer Res. 69, 2506–2513 15 Hillman, G.G. et al. (2009) Dynamic contrast-enhanced magnetic resonance imaging of vascular changes induced by sunitinib in papillary renal cell carcinoma xenograft tumors. Neoplasia 11, 910–920 16 Van der Veldt, A.A. et al. (2010) Reduction in skin microvascular density and changes in vessel morphology in patients treated with sunitinib. Anticancer drugs DOI: 10.10197/CAD.0b013e3283359c79 (http://journals.lww.com/anti-cancerdrugs/pages/default.aspx) 17 Heckman, C.A. et al. (2008) The tyrosine kinase inhibitor cediranib blocks ligand-induced vascular endothelial growth factor receptor-3 activity and lymphangiogenesis. Cancer Res. 68, 4754–4762 18 Padera, T.P. et al. (2008) Differential response of primary tumor versus lymphatic metastasis to VEGFR-2 and VEGFR-3 kinase inhibitors cediranib and vandetanib. Mol. Cancer Ther. 7, 2272–2279 19 Quinta´s-Cardama, A. et al. (2009) Mechanisms of primary and secondary resistance to imatinib in chronic myeloid leukemia. Cancer Control: Journal of the Moffitt Cancer Center 16 (2), 122– 131 20 Zhang, W.W. et al. (2009) Predictors of primary imatinib resistance in chronic myelogenous leukemia are distinct from those in secondary imatinib resistance. J. Clin. Oncol. 27, 3642–3649 21 Perez-Gracia, J.L. et al. (2009) Identification of TNF-alpha and MMP-9 as potential baseline predictive serum markers of sunitinib activity in patients with renal cell carcinoma using a human cytokine array. Br. J. Cancer 101, 1876–1883 22 Hirsch, F.R. et al. (2009) Predictive value of EGFR and HER2 overexpression in advanced non-small-cell lung cancer. Oncogene 28 (Suppl 1), S32–37 23 McWeeney, S.K. et al. (2009) A gene expression signature of CD34+ cells to predict major cytogenetic response in chronic phase chronic myeloid leukemia patients treated with imatinib. Blood 115, 315– 325 24 Dahabreh, I.J. et al. (2010) Somatic EGFR mutation and gene copy gain as predictive biomarkers for response to tyrosine kinase inhibitors in non-small cell lung cancer. Clin. Cancer Res. 16, 291–303 25 Finke, J.H. et al. (2008) Sunitinib reverses type-1 immune suppression and decreases T-regulatory cells in renal cell carcinoma patients. Clin. Cancer Res. 14, 6674–6682 26 Ozao-Choy, J. et al. (2009) The novel role of tyrosine kinase inhibitor in the reversal of immune suppression and modulation of tumor microenvironment for immune-based cancer therapies. Cancer Res. 69, 2514–2522 27 Gouttefangeas, C. et al. (2007) Immunotherapy of renal cell carcinoma. Cancer Immunol Immunother. 56, 117–128 28 Hoeltl, L. et al. (1999) Cellular and humoral immune responses in patients with metastatic renal cell carcinoma after vaccination with antigen pulsed dendritic cells. J. Urol. 161, 777–782 29 Su, Z. et al. (2003) Immunological and clinical responses in metastatic renal cancer patients vaccinated with tumor RNA-transfected dendritic cells. Cancer Res. 63, 2127–2133 30 Bleumer, I. et al. (2007) Preliminary analysis of patients with progressive renal cell carcinoma vaccinated with CA9-peptide-pulsed mature dendritic cells. J. Immunother. 30, 116–122 31 Berntsen, A. et al. (2008) Therapeutic dendritic cell vaccination of patients with metastatic renal cell carcinoma: a clinical phase 1/2 trial. J. Immunother. 31, 771–780
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32 Asemissen, A.M. and Brossart, P. (2009) Vaccination strategies in patients with renal cell carcinoma. Cancer Immunol Immunother. 58, 1169–1174 33 Rosenberg, S.A. et al. (2008) Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat. Rev. Cancer 8, 299–308 34 Blay, J.Y. et al. (1997) Role of interleukin-6 in the paraneoplastic inflammatory syndrome associated with renal-cell carcinoma. Int. J. Cancer. 72, 424–430 35 Choudeiri, T.K. et al. (2007) Clinical factors associated with outcome in patients with metastatic clear-cell renal cell carcinoma treated with vascular endothelial growth factor-targeted therapy. Cancer 110, 543–550 36 Young, M.R. and Lathers, D.M. (1999) Myeloid progenitor cells mediate immune suppression in patients with head and neck cancers. Int. J. Immunopharmacol. 21, 241–252 37 Almand, B. et al. (2001) Increased production of immature myeloid cells in cancer patients: a mechanism of immunosuppression in cancer. J. Immunol. 166, 678–689 38 Pan, P.Y. et al. (2008) Reversion of immune tolerance in advanced malignancy: modulation of myeloid-derived suppressor cell development by blockade of stem-cell factor function. Blood 111, 219–228 39 Bronte, V. et al. (2001) Tumor-induced immune dysfunctions caused by myeloid suppressor cells. J. Immunother. 24, 431–446 40 Ko, J.S. et al. (2009) Myeloid-derived suppressor cells: a novel therapeutic target. Curr. Oncol. Rep. 11, 87–93 41 Gabrilovich, D. et al. (1996) Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells. Nat. Med. 2, 1096–1103 42 Gabrilovich, D. et al. (1998) Vascular endothelial growth factor inhibits the development of dendritic cells and dramatically affects the differentiation of multiple hematopoietic lineages in vivo. Blood 92, 4150–4166 43 Alfaro, C. et al. (2009) Influence of bevacizumab, sunitinib and sorafenib as single agents or in combination on the inhibitory effects of VEGF on human dendritic cell differentiation from monocytes. Br. J. Cancer 100, 1111–1119 44 Mohty, M. et al. (2005) Imatinib: the narrow line between immune tolerance and activation. Trends Mol. Med. 11, 397–402 45 Seggewiss, R. et al. (2008) Immunomodulatory effects of imatinib and second-generation tyrosine kinase inhibitors on T cells and dendritic cells: an update. Cytotherapy 10, 633–641 46 Hipp, M.M. et al. (2008) Sorafenib, but not sunitinib, affects function of dendritic cells and induction of primary immune responses. Blood 111, 5610–5620 47 van Cruijsen, H. et al. (2008) Sunitinib-induced myeloid lineage redistribution in renal cell cancer patients: CD1c+ dendritic cell frequency predicts progression-free survival. Clin. Cancer Res. 14, 5884–5892 48 Ko, J.S. et al. (2009) Sunitinib mediates reversal of myeloid-derived suppressor cell accumulation in renal cell carcinoma patients. Clin. Cancer Res. 15, 2148–2157 49 Molhoek, K.R. et al. (2009) Apoptosis of CD4(+)CD25(high) T cells in response to Sirolimus requires activation of T cell receptor and is modulated by IL-2. Cancer Immunol Immunother. 58, 867–876 50 Hilger, R.A. et al. (2004) Correlation of ERK-phosphorylation and toxicities in patients treated with the Raf kinase inhibitor BAY 439006. Int. J. Clin. Pharmacol. Ther. 42, 648–649 51 Zhao, W. et al. (2008) Sorafenib inhibits activation of human peripheral blood T cells by targeting LCK phosphorylation. Leukemia. 22, 1226–1233 52 Houben, R. et al. (2009) MAPK-independent impairment of T-cell responses by the multikinase inhibitor sorafenib. Mol. Cancer Ther. 8, 433–440 53 Boissel, N. et al. (2006) Imatinib mesylate minimally affects bcr-abl+ and normal monocyte-derived dendritic cells but strongly inhibits T cell expansion despite reciprocal dendritic cell-T cell activation. J. Leukoc. Biol. 79, 747–756 54 Mumprecht, S. et al. (2006) Imatinib mesylate selectively impairs expansion of memory cytotoxic T cells without affecting the control of primary viral infections. Blood 108, 3406–3413 55 Sinai, P. et al. (2007) Imatinib mesylate inhibits antigen-specific memory CD8 T cell responses in vivo. J. Immunol. 178, 2028–2037
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Review 56 Pinilla-Ibarz, J. et al. (2009) The biological basis for immunotherapy in patients with chronic myelogenous leukemia. Cancer Control 16, 141–152 57 Rojas, J.M. (2007) Clinical evaluation of BCR-ABL peptide immunisation in chronic myeloid leukaemia: results of the EPIC study. Leukemia. 21, 2287–2295 58 Wang, H. et al. (2005) Imatinib mesylate (STI-571) enhances antigenpresenting cell function and overcomes tumor-induced CD4+ T-cell tolerance. Blood 105, 1135–1143 59 Mohty, M. et al. (2004) Imatinib and plasmacytoid dendritic cell function in patients with chronic myeloid leukemia. Blood 103, 4666–4668 60 Borg, C. et al. (2004) Novel mode of action of c-Kit tyrosine kinase inhibitors leading to NK cell-dependent antitumor effects. J. Clin. Invest. 114, 379–388
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Trends in Molecular Medicine Vol.16 No.4 61 Bellone, M. et al. (2008) Vascular targeting, chemotherapy and active immunotherapy: teaming up to attack cancer. Trends Immunol. 29, 235–241 62 Griffioen, A.W. (2008) Anti-angiogenesis: making the tumor vulnerable to the immune system. Cancer Immunol Immunother. 57, 1553–1558 63 Fraser, C.K. et al. (2009) Dasatinib inhibits recombinant viral antigenspecific murine CD4+ and CD8+ T-cell responses and NK-cell cytolytic activity in vitro and in vivo. Exp. Hematol. 37, 256–265 64 Louvet, C. et al. (2008) Tyrosine kinase inhibitors reverse type 1 diabetes in nonobese diabetic mice. Proc. Natl. Acad. Sci. U. S. A. 105, 18895–18900 65 Himmelsbach, K. et al. (2009) New aspects of an anti-tumor drug: sorafenib efficiently inhibits HCV replication. Gut. 58, 1644–1653