Eva Maria Putz

Comparison of three different experimental methods for the assessment of peripheral compartment pharmacokinetics in humans

In many cases the concentration reached in a peripheral effect compartment rather than in plasma determines the clinical outcome of therapy. Therefore, several experimental approaches have been developed for direct assessment of drug kinetics in peripheral compartments. Particularly saliva sampling, skin blister fluid sampling, and in vivo microdialysis are frequently employed for measuring peripheral drug concentrations. However, data derived from these techniques have never been directly compared. In the present study, the tissue kinetics of theophylline were measured following single dose administration simultaneously in cantharides induced skin blisters, saliva and microdialysates of subcutaneous- and skeletal muscle- tissue and compared to plasma concentrations. Theophylline was administered to 9 healthy volunteers as an i.v. infusion of 240 mg. Mean ratio (AUCsaliva/AUCplasma) was 0.63 +/- 0.05, mean ratio (AUCblister/AUCplasma) was 0.69 +/- 0.12, mean ratio (AUCmuscle/AUCplasma) was 0.41 +/- 0.10, mean ratio (AUCsubcutaneous/AUCplasma) was 0.34 +/- 0.07. The time course of the concentration(peripheral)/concentration(plasma)-ratios showed that tissue concentrations obtained by microdialysis were closely correlated to free plasma levels, whereas saliva- and cantharides blister data overestimated the corresponding free plasma concentrations. It is concluded that microdialysis represents a reliable technique for the measurement of unbound peripheral compartment concentrations and is superior to saliva- and skin blister concentration measurements.

STAT5 Is a Key Regulator in NK Cells and Acts as a Molecular Switch from Tumor Surveillance to Tumor Promotion

UNLABELLED Natural killer (NK) cells are tightly regulated by the JAK-STAT signaling pathway and cannot survive in the absence of STAT5. We now report that STAT5-deficient NK cells can be rescued by overexpression of BCL2. Our experiments define STAT5 as a master regulator of NK-cell proliferation and lytic functions. Although NK cells are generally responsible for killing tumor cells, the rescued STAT5-deficient NK cells promote tumor formation by producing enhanced levels of the angiogenic factor VEGFA. The importance of VEGFA produced by NK cells was verified by experiments with a conditional knockout of VEGFA in NK cells. We show that STAT5 normally represses the transcription of VEGFA in NK cells, in both mice and humans. These findings reveal that STAT5-directed therapies may have negative effects: In addition to impairing NK-cell-mediated tumor surveillance, they may even promote tumor growth by enhancing angiogenesis. SIGNIFICANCE The importance of the immune system in effective cancer treatment is widely recognized. We show that the new signal interceptors targeting the JAK-STAT5 pathway may have dangerous side effects that must be taken into account in clinical trials: inhibiting JAK-STAT5 has the potential to promote tumor growth by enhancing NK-cell-mediated angiogenesis.

PAK-dependent STAT5 serine phosphorylation is required for BCR-ABL-induced leukemogenesis

The transcription factor STAT5 (signal transducer and activator of transcription 5) is frequently activated in hematological malignancies and represents an essential signaling node downstream of the BCR-ABL oncogene. STAT5 can be phosphorylated at three positions, on a tyrosine and on the two serines S725 and S779. We have investigated the importance of STAT5 serine phosphorylation for BCR-ABL-induced leukemogenesis. In cultured bone marrow cells, expression of a STAT5 mutant lacking the S725 and S779 phosphorylation sites (STAT5SASA) prohibits transformation and induces apoptosis. Accordingly, STAT5SASA BCR-ABL+ cells display a strongly reduced leukemic potential in vivo, predominantly caused by loss of S779 phosphorylation that prevents the nuclear translocation of STAT5. Three distinct lines of evidence indicate that S779 is phosphorylated by group I p21-activated kinase (PAK). We show further that PAK-dependent serine phosphorylation of STAT5 is unaffected by BCR-ABL tyrosine kinase inhibitor treatment. Interfering with STAT5 phosphorylation could thus be a novel therapeutic approach to target BCR-ABL-induced malignancies.

Loss of STAT3 in murine NK cells enhances NK cell–dependent tumor surveillance

The members of the signal transducer and activator of transcription (STAT) family of transcription factors modulate the development and function of natural killer (NK) cells. NK cell-mediated tumor surveillance is particularly important in the bodys defense against hematological malignancies such as leukemia. STAT3 inhibitors are currently being developed, although their potential effects on NK cells are not clear. We have investigated the function of STAT3 in NK cells with Stat3(Δ/Δ)Ncr1-iCreTg mice, whose NK cells lack STAT3. In the absence of STAT3, NK cells develop normally and in normal numbers, but display alterations in the kinetics of interferon-γ (IFN-γ) production. We report that STAT3 directly binds the IFN-γ promoter. In various in vivo models of hematological diseases, loss of STAT3 in NK cells enhances tumor surveillance. The reduced tumor burden is paralleled by increased expression of the activating receptor DNAM-1 and the lytic enzymes perforin and granzyme B. Our findings imply that STAT3 inhibitors will stimulate the cytolytic activity of NK cells against leukemia, thereby providing an additional therapeutic benefit.

Conditional IFNAR1 ablation reveals distinct requirements of Type I IFN signaling for NK cell maturation and tumor surveillance

Mice with an impaired Type I interferon (IFN) signaling (IFNAR1- and IFNβ-deficient mice) display an increased susceptibility toward v-ABL-induced B-cell leukemia/lymphoma. The enhanced leukemogenesis in the absence of an intact Type I IFN signaling is caused by alterations within the tumor environment. Deletion of Ifnar1 in tumor cells (as obtained in Ifnar1f/f CD19-Cre mice) failed to impact on disease latency or type. In line with this observation, the initial transformation and proliferative capacity of tumor cells were unaltered irrespective of whether the cells expressed IFNAR1 or not. v-ABL-induced leukemogenesis is mainly subjected to natural killer (NK) cell-mediated tumor surveillance. Thus, we concentrated on NK cell functions in IFNAR1 deficient animals. Ifnar1-/- NK cells displayed maturation defects as well as an impaired cytolytic activity. When we deleted Ifnar1 selectively in mature NK cells (by crossing Ncr1-iCre mice to Ifnar1f/f animals), maturation was not altered. However, NK cells derived from Ifnar1f/f Ncr1-iCre mice showed a significant cytolytic defect in vitro against the hematopoietic cell lines YAC-1 and RMA-S, but not against the melanoma cell line B16F10. Interestingly, this defect was not related to an in vivo phenotype as v-ABL-induced leukemogenesis was unaltered in Ifnar1f/f Ncr1-iCre compared with Ifnar1f/f control mice. Moreover, the ability of Ifnar1f/f Ncr1-iCre NK cells to kill B16F10 melanoma cells was unaltered, both in vitro and in vivo. Our data reveal that despite the necessity for Type I IFN in NK cell maturation the expression of IFNAR1 on mature murine NK cells is not required for efficient tumor surveillance.

PI3Kδ Is Essential for Tumor Clearance Mediated by Cytotoxic T Lymphocytes

Background PI3Kδ is a lipid kinase of the phosphoinositide 3-kinase class 1A family and involved in early signaling events of leukocytes regulating proliferation, differentiation and survival. Currently, several inhibitors of PI3Kδ are under investigation for the treatment of hematopoietic malignancies. In contrast to the beneficial effect of inhibiting PI3Kδ in tumor cells, several studies reported the requirement of PI3Kδ for the function of immune cells, such as natural killer and T helper cells. Cytotoxic T lymphocytes (CTLs) are essential for tumor surveillance. The scope of this study is to clarify the potential impact of PI3Kδ inhibition on the function of CTLs with emphasis on tumor surveillance. Principal Findings PI3Kδ-deficient mice develop significantly bigger tumors when challenged with MC38 colon adenocarcinoma cells. This defect is accounted for by the fact that PI3Kδ controls the secretory perforin-granzyme pathway as well as the death-receptor pathway of CTL-mediated cytotoxicity, leading to severely diminished cytotoxicity against target cells in vitro and in vivo in the absence of PI3Kδ expression. PI3Kδ-deficient CTLs express low mRNA levels of important components of the cytotoxic machinery, e.g. prf1, grzmA, grzmB, fasl and trail. Accordingly, PI3Kδ-deficient tumor-infiltrating CTLs display a phenotype reminiscent of naïve T cells (CD69lowCD62Lhigh). In addition, electrophysiological capacitance measurements confirmed a fundamental degranulation defect of PI3Kδ−/− CTLs. Conclusion Our results demonstrate that CTL-mediated tumor surveillance is severely impaired in the absence of PI3Kδ and predict that impaired immunosurveillance may limit the effectiveness of PI3Kδ inhibitors in long-term treatment.

Identification of an Indispensable Role for Tyrosine Kinase 2 in CTL-Mediated Tumor Surveillance

We showed previously that Tyk2(-/-) natural killer cells lack the ability to lyse leukemic cells. As a consequence, the animals are leukemia prone. Here, we show that the impaired tumor surveillance extends to T cells. Challenging Tyk2(-/-) mice with EL4 thymoma significantly decreased disease latency. The crucial role of Tyk2 for CTL function was further characterized using the ovalbumin-expressing EG7 cells. Tyk2(-/-) OT-1 mice developed EG7-induced tumors significantly faster compared with wild-type (wt) controls. In vivo assays confirmed the defect in CD8(+) cytotoxicity on Tyk2 deficiency and clearly linked it to type I IFN signaling. An impaired CTL activity was only observed in IFNAR1(-/-) animals but not on IFNgamma or IL12p35 deficiency. Accordingly, EG7-induced tumors grew faster in IFNAR1(-/-) and Tyk2(-/-) but not in IFNgamma(-/-) or IL12p35(-/-) mice. Adoptive transfer experiments defined a key role of Tyk2 in CTL-mediated tumor surveillance. In contrast to wt OT-1 cells, Tyk2(-/-) OT-1 T cells were incapable of controlling EG7-induced tumor growth.

CD52 is a molecular target in advanced systemic mastocytosis

Advanced systemic mastocytosis (SM) is an aggressive hematopoietic neoplasm with poor prognosis and short survival times. So far, no curative therapy is available for affected patients. We have identified the cell surface antigen CD52 (CAMPATH‐1) as a molecular target expressed abundantly on the surface of primary neoplastic mast cells (MCs) in patients with advanced SM. In contrast, neoplastic MCs of patients with indolent SM and normal MCs expressed only low levels or did not express CD52. To study the mechanisms of CD52 expression and the value of this antigen as a potential therapeutic target, we generated a human MC cell line, designated MCPV‐1, by lentiviral immortalization of cord blood‐derived MC progenitor cells. Functional studies revealed that activated RAS profoundly promotes surface expression of CD52. The CD52‐targeting antibody alemtuzumab induced cell death in CD52+ primary neoplastic MCs obtained from patients with SM as well as in MCPV‐1 cells. NSG mice xenotransplanted with MCPV‐1 cells survived significantly longer after treatment with alemtuzumab (median survival: 31 d untreated vs. 46 d treated; P=0.0012). We conclude that CD52 is a novel marker and potential therapeutic target in neoplastic MCs in patients with advanced SM.—Hoermann, G., Blatt, K., Greiner, G., Putz, E. M., Berger, A., Herrmann, H., Cerny‐Reiterer, S., Gleixner, K. V., Walz, C., Hoetzenecker, K., Müllauer, L., Reiter, A., Sotlar, K., Sexl, V., Valent, P., Mayerhofer, M. CD52 is a molecular target in advanced systemic mastocytosis. FASEB J. 28, 3540–3551 (2014). www.fasebj.org

In vivo tumor surveillance by NK cells requires TYK2 but not TYK2 kinase activity

Tyrosine kinase 2 (TYK2) is a Janus kinase (JAK) that is crucially involved in inflammation, carcinogenesis and defense against infection. The cytotoxic activity of natural killer (NK) cells in TYK2-deficient (Tyk2−/−) mice is severely reduced, although the underlying mechanisms are largely unknown. Using Tyk2−/− mice and mice expressing a kinase-inactive version of TYK2 (Tyk2K923E), we show that NK cell function is partly independent of the enzymatic activity of TYK2. Tyk2−/− and Tyk2K923E NK cells develop normally in the bone marrow, but the maturation of splenic Tyk2−/− NK cells (and to a lesser extent of Tyk2K923E NK cells) is impaired. In contrast, the production of interferon γ (IFNγ) in response to interleukin 12 (IL-12) or to stimulation through NK cell-activating receptors strictly depends on the presence of enzymatically active TYK2. The cytotoxic activity of Tyk2K923E NK cells against a range of target cells in vitro is higher than that of Tyk2−/− NK cells. Consistently, Tyk2K923E mice control the growth of NK cell-targeted tumors significantly better than TYK2-deficient mice, showing the physiological relevance of the finding. Inhibitors of TYK2s kinase activity are being developed for the treatment of inflammatory diseases and cancers, but their effects on tumor immune surveillance have not been investigated. Our finding that TYK2 has kinase-independent functions in vivo suggests that such inhibitors will leave NK cell mediated tumor surveillance largely intact and that they will be suitable for use in cancer therapy.

Loss of STAT3 in Lymphoma Relaxes NK Cell-Mediated Tumor Surveillance

The transcription factors and proto-oncogenes STAT3 and STAT5 are highly activated in hematological malignancies and represent promising therapeutic targets. Whereas the importance of STAT5 as tumor promoter is beyond doubt, the role of STAT3 in hematological cancers is less well understood. Both, enforced as well as attenuated expression of STAT3 were reported in hematopoietic malignancies. Recent evidence implicates STAT3 as key player for tumor immune surveillance as it both mediates the production of and response to inflammatory cytokines. Here we investigated the effects of STAT3 deletion in a BCR/ABL-induced lymphoma model, which is tightly controlled by natural killer (NK) cells in vivo. Upon STAT3 deletion tumor growth is significantly enhanced when compared to STAT3-expressing controls. The increased tumor size upon loss of STAT3 was accompanied by reduced NK cell infiltration and decreased levels of the cytokine IFN-γ and the chemokine RANTES. Upon transplantation into NK cell-deficient mice differences in lymphoma size were abolished indicating that STAT3 expression in the tumor cells controls NK cell-dependent tumor surveillance. Our findings indicate that STAT3 inhibition in lymphoma patients will impair NK cell-mediated tumor surveillance, which needs to be taken into account when testing STAT3 inhibitors in preclinical or clinical trials.