Recombinant Interleukin-24 Lacks Apoptosis-Inducing Properties in Melanoma Cells

IL-24, also known as melanoma differentiation antigen 7 (mda-7), is a member of the IL-10 family of cytokines and is mainly produced by Th2 cells as well as by activated monocytes. Binding of IL-24 to either of its two possible heterodimeric receptors IL-20R1/IL-20R2 and IL-22R/IL-20R2 activates STAT3 and/or STAT1 in target tissues such as lung, testis, ovary, keratinocytes and skin. To date, the physiological properties of IL-24 are still not well understood but available data suggest that IL-24 affects epidermal functions by increasing proliferation of dermal cells. In stark contrast to its “normal” and physiological behaviour, IL-24 has been reported to selectively and efficiently kill a vast variety of cancer cells, especially melanoma cells, independent of receptor expression and Jak-STAT signalling. These intriguing properties have led to the development of adenovirally-expressed IL-24, which is currently being evaluated in clinical trials. Using three different methods, we have analysed a large panel of melanoma cell lines with respect to IL-24 and IL-24 receptor expression and found that none of the investigated cell lines expressed sufficient amounts of functional receptor pairs and therefore did not react to IL-24 stimulation with Jak/STAT activation. Results for three cell lines contrasted with previous studies, which reported presence of IL-24 receptors and activation of STAT3 following IL-24 stimulation. Furthermore, evaluating four different sources and modes of IL-24 administration (commercial recombinant IL-24, bacterially expressed GST-IL-24 fusion protein, IL-24 produced from transfected Hek cells, transiently over-expressed IL-24) no induction or increase in cell death was detected when compared to appropriate control treatments. Thus, we conclude that the cytokine IL-24 itself has no cancer-specific apoptosis-inducing properties in melanoma cells

The PD-L1- and IL6-mediated dampening of the IL27/STAT1 anticancer responses are prevented by a-PD-L1 or a-IL6 antibodies

Interleukin-27 (IL27) is a type-I cytokine of the IL6/IL12 family and is predominantly secreted by activated macrophages and dendritic cells.We show that IL27 induces STAT factor phosphorylation in cancerous cell lines of different tissue origin. IL27 leads to STAT1 phosphorylation and recapitulates an IFN- -like response in the microarray analyses, with up-regulation of genes involved in antiviral defense, antigen presentation, and immune suppression. Like IFN- , IL27 leads to an up-regulation of TAP2 and MHC-I proteins, which mediate increased tumor immune clearance. However, both cytokines also upregulate proteins such as PD-L1 (CD274) and IDO-1, which are associatedwith immune escape of cancer. Interestingly, differential expression of these geneswas observed within the different cell lines and when comparing IL27 to IFN- . In coculture experiments of hepatocellular carcinoma (HCC) cells with peripheral blood mononuclear cells, pre-treatment of the HCC cells with IL27 resulted in lowered IL2 production by anti-CD3/-CD28 activated T-lymphocytes. Addition of anti-PD-L1 antibody, however, restored IL2 secretion. The levels of other TH1 cytokines were also enhanced or restored upon administration of anti-PD-L1. In addition, we show that the suppression of IL27 signaling by IL6-type cytokine prestimulation— mimicking a situation occurring, for example, in IL6-secreting tumors or in tumor inflammation–induced cachexia—can be antagonized by antibodies against IL6-type cytokines or their receptors. Therapeutically, the antitumor effects of IL27 (mediated, e.g., by increased antigen presentation) might thus be increased by combining IL27with blocking antibodies against PD-L1 or/and IL6-type cytokines

Crosstalk between different family members: IL27 recapitulates IFNγ responses in HCC cells, but is inhibited by IL6-type cytokines

Interleukin-27 (IL27) is a type-I-cytokine of the IL6/IL12 family predominantly secreted by activated macrophages and dendritic cells. In the liver, IL27 expression was observed to be upregulated in patients with hepatitis B, and sera of hepatocellular carcinoma (HCC) patients contain significantly elevated levels of IL27 compared to healthy controls or patients with hepatitis and/or liver cirrhosis. In this study, we show that IL27 induces STAT1 and STAT3 phosphorylation in 5 HCC lines and 3 different types of non-transformed liver cells. We were especially interested in the relevance of the IL27-induced STAT3 activation in liver cells. Thus, we compared the IL27 responses with those induced by IFNγ (STAT1-dominated response) or IL6-type cytokines (IL6, hyper-IL6 (hy-IL6) or OSM) (STAT3-dominated response) by microarray analysis and find that in HCC cells, IL27 induces an IFNγ-like, STAT1-dependent transcriptional response, but we do not find an effective STAT3-dependent response. Validation experiments corroborate the finding from the microarray evaluation. Interestingly, the availability of STAT1 seems critical in the shaping of the IL27 response, as the siRNA knock-down of STAT1 revealed the ability of IL27 to induce the acute-phase protein γ-fibrinogen, a typical IL6 family characteristic. Moreover, we describe a crosstalk between the signaling of IL6-type cytokines and IL27: responses to the gp130-engaging cytokine IL27 (but not those to IFNs) can be inhibited by IL6-type cytokine pre-stimulation, likely by a SOCS3-mediated mechanism. Thus, IL27 recapitulates IFNγ responses in liver cells, but differs from IFNγ by its sensitivity to SOCS3 inhibition

Combined Inhibition of Janus and Aurora Kinase Effectively Suppresses Proliferation of JAK2 V617F-expressing Cells

Abstract Abstract 2813 Background: A somatic point mutation in the Janus kinase 2 gene (JAK2) leading to the expression of the JAK2 V617F mutant occurs with high frequency in myeloproliferative neoplasm (MPN) patients (&gt;95 % in polycythemia vera (PV), &gt;50 % in essential thrombocythemia (ET) and primary myelofibrosis (PMF)). It confers constitutive activity to the kinase and results in cytokine hypersensitivity and a proliferative advantage of hematopoietic progenitor cells. These findings suggest that inhibiting JAK2 V617F may be therapeutically beneficial. Several JAK2 inhibitors are currently in clinical trials for the treatment of MPN, and first results show clinical improvements for PMF patients. However, since approximately 50 % of ET and PMF patients do not carry an activating mutation in JAK2, we speculate that the inhibition of signaling proteins other than JAK2 or in combination with JAK2 inhibition could be beneficial for these patients. Methods: We characterized compounds from different chemical classes, which previously have been published to be JAK(2) inhibitors. These compounds were compared in several assays using primary CD34+ cells from PV patients positive for the JAK2 V617F mutation and/or the JAK2 V617F-bearing cell line HEL. We used (quantitative) Western blot detections, in vitro kinase assays, proliferation assays, cell size measurements, cell cycle analyses and colony forming cell (CFC) assays to analyze the efficacy of the different inhibitors. Moreover, the IC50 values of the compounds were determined. Results: In total 15 published JAK2 inhibitors have been characterized in detail. As monitored in an in vitro kinase assay and by Western blot detection of phosphorylated signaling proteins, several compounds previously described as JAK(2) inhibitors did not target JAK2 V617F. However, some compounds, which turned out not to inhibit JAKs, showed growth-inhibitory effects on JAK2 V617F-positive cells. Such compounds could be used in combination with a specific JAK inhibitor in order to achieve beneficial effects on suppression of cell proliferation and induction of apoptosis. We could demonstrate that the combined application of a JAK inhibitor together with an Aurora kinase inhibitor was most promising: application of both Janus and Aurora kinase inhibitors in proliferation assays and CFC assays demonstrated a more effective suppression of growth than achieved by respective single treatments. Interestingly, we observed in the CFC assay that a JAK2 inhibitor seems to preferentially suppress the growth of erythroid colonies, while an Aurora kinase inhibitor preferentially blocks myeloid colony growth. Conclusion: Here we present a comparative analysis and a detailed biochemical characterization of numerous compounds from different chemical classes, all supposed to be JAK(2) inhibitors. We confirmed JAK(2) inhibitory activity for several compounds but not for all. In addition, we identified some compounds, which effectively inhibited the proliferation of JAK2 V617F-bearing cells without targeting JAK2. Thus, combined inhibition of JAK2 and other kinases may represent a promising therapeutic strategy. In particular, we suggest that a combination of Janus and Aurora kinase inhibitors might be beneficial for the treatment of MPN patients. Disclosures: No relevant conflicts of interest to declare. </jats:sec

SOCS-mediated downregulation of mutant Jak2 (V617F, T875N and K539L) counteracts cytokine-independent signaling.

Recently, mutations in the gene of Janus kinase 2 (Jak2) were discovered in patients suffering from chronic myeloproliferative disorders (MPD) and leukemia. As suppressors of cytokine signaling (SOCS) proteins are potent feedback inhibitors of Jak-mediated signaling, we investigated their role in signal transduction through constitutively active Jak2 mutants. We selected two mutants, Jak2-V617F and Jak2-K539L, found in patients with MPDs and Jak2-T875N identified in acute megakaryoblastic leukemia. We found SOCS family members to be induced through Jak2-V617F in human leukemia cell lines expressing the mutant allele and in stable HEK transfectants inducibly expressing constitutively active Jak2 mutants. SOCS proteins were recruited to the membrane and bound to the constitutively active Jaks. In contrast to wild-type Jak2, the mutant proteins were constitutively ubiquitinated and degraded through the proteasome. Taken together, we show a SOCS-mediated downregulation of the constitutively active, disease-associated mutant Jak2 proteins. Furthermore, a threshold level of mutant Jak expression has to be overcome to allow full cytokine-independent constitutive activation of signaling proteins, which may explain progression to homozygocity in MPDs as well as gene amplification in severe phenotypes and leukemia

JAK2-V617F-induced MAPK activity is regulated by PI3K and acts synergistically with PI3K on the proliferation of JAK2-V617F-positive cells.

The identification of a constitutively active JAK2 mutant, namely JAK2-V617F, was a milestone in the understanding of Philadelphia chromosome-negative myeloproliferative neoplasms. The JAK2-V617F mutation confers cytokine hypersensitivity, constitutive activation of the JAK-STAT pathway, and cytokine-independent growth. In this study we investigated the mechanism of JAK2-V617F-dependent signaling with a special focus on the activation of the MAPK pathway. We observed JAK2-V617F-dependent deregulated activation of the multi-site docking protein Gab1 as indicated by constitutive, PI3K-dependent membrane localization and tyrosine phosphorylation of Gab1. Furthermore, we demonstrate that PI3K signaling regulates MAPK activation in JAK2-V617F-positve cells. This cross-regulation of the MAPK pathway by PI3K affects JAK2-V617F-specific target gene induction, erythroid colony formation, and regulates proliferation of JAK2-V617F-positive patient cells in a synergistically manner