In a compound screen on melanoma cells, we identified FX-11 as one of the compounds inhibiting the growth of sensitive and Encorafenib/Binimetinib-resistant 624Mel and Wm3248 melanoma cells. FX-11, reported to be a lactate dehydrogenase inhibitor (LDHi), did not decrease the NAD+/NADH ratio, glucose uptake, or lactate secretion of melanoma cells, unlike other LDH inhibitors we used as controls. Instead, FX-11 increased both the oxygen consumption and the extracellular acidification rate of cells in Seahorse assays, behaving similarly to the mitochondrial uncouplers FCCP and BAM15. FX-11 could also be used as an uncoupler in Mito Stress tests. In addition, FX-11 and BAM15 drastically decreased the mitochondrial membrane potential, in contrast to the LDH inhibitors LDH-IN-I and GNE-140. Finally, FX-11 treatment induced an increase in AMPK and acetyl-CoA carboxylase (ACC) phosphorylation and a decrease in phosphorylation of the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), indicative of AMPK activation by decreased ATP/AMP ratio. The observed AMPK activation seems to be involved in FX-11-mediated cell death since knock-down of AMPKα1 reduced cell death. Specific activation of AMPK by MK8722, on the other hand, increased cell death.Taken together, we provide evidence that FX-11 inhibits the growth of melanoma cells, including drug-resistant ones, through an AMPK-dependent mechanism by acting as a mitochondrial uncoupler. Our data do not support that FX-11 acts as an LDH inhibitor.

Resistance of melanoma cells to targeted therapy (BRAF and MEK inhibitors) is a major clinical problem and alternative treatments are sought. We describe the establishment of modular physiologic medium (MPM) and Mel-MPM (which contains additional supplements and sustains the 3D growth of melanoma cells, fibroblasts (NHDFs) and HMEC-1 endothelial cells) as novel resources for melanoma and combine them with a multi-cell-type matrix-embedded 3D culture model to investigate melanoma cell vulnerabilities in a more physiological setting. We made use of the modular nature of MPM to interrogate NEAA dependencies in melanoma cells and we found them to be particularly sensitive to the depletion of C/C. We additionally describe that melanoma cells are less sensitive to ferroptosis inducing compounds when cultured in MPM compared to RPMI and we could attribute this to different components of MPM and Mel-MPM (selenite, B27). Cell death induced by the glutathione peroxidase 4 inhibitor, ML162, had characteristics of ferroptosis or apoptosis depending on cell type, its drug resistance status and the culture medium. Cystine/cysteine starvation and ML162 treatment combinations increased melanoma cell death in 2D, 3D, and also in the complex matrix embedded multi-cell-type-3D system in Organoplates. This underlines the potential of combining metabolism-oriented drug treatments with amino acid starvation conditions, which is of interest in view of future therapeutic approaches to combat melanoma and other cancer types. ### Competing Interest Statement The authors have declared no competing interest.

Many melanoma patients do not respond to therapy or rapidly develop resistance to MAPK pathway inhibitors and immune checkpoint blockade treatments, highlighting the urgent need for additional therapeutic strategies for these patients. To identify compounds that target drug-naive and -resistant melanoma cells (Encorafenib/Binimetinib-resistant), we performed a screen using a metabolism-oriented compound library in different cell culture media and growth conditions (2D and 3D). The efficacy of several compounds varied considerably under changing test conditions, but importantly, we also detected compounds that work across all tested conditions. In general, drug activity was reduced under hypoxia and in 3D spheroids. Thorough validation was performed for drugs showing potency in all conditions with a focus on efficacy in 3D spheroids grown in an in-house physiological culture medium. Using hydrogel matrix-embedded multi-cell type 3D models, we found that FK866, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, is very potently suppressing melanoma cell growth while not affecting the growth of healthy cells such as fibroblasts and endothelial cells. ### Competing Interest Statement The authors have declared no competing interest.

1University of Luxembourg, Life Sciences ResearchUnit—Signal Transduction Laboratory, Belvaux, Luxembourg 2University of Luxembourg, Life Sciences ResearchUnit—Bioinformatics Core Facility, Belvaux, Luxembourg 3UniversityHospitalWürzburg,Medical Clinic II, Division ofHepatology,Würzburg, Germany 4Proteome andGenomeResearchUnit, Department ofOncology, Luxembourg Institute of Health, Luxembourg, Luxembourg 5University of Luxembourg, Life Sciences ResearchUnit—MolecularDiseaseMechanisms Laboratory, Belvaux, Luxembourg Correspondence ClaudeHaan,University of Luxembourg, Life SciencesResearchUnit—Signal Transduction Laboratory, 6, avenueduSwing, L-4367Belvaux, Luxembourg. E-mail: claude.haan@uni.lu Abstract 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-γ-like response in the microarray analyses, with up-regulation of genes involved in antiviral defense, antigen presentation, and immune suppression. Like IFN-γ , 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 observedwithin the different cell lines andwhen comparing IL27 to IFN-γ . 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.

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 associated with immune escape of cancer. Interestingly, differential expression of these genes was 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 T(H)1 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 pre-stimulationmimicking a situation occurring, for example, in IL6-secreting tumors or in tumor inflammation-induced cachexiacan 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 IL27 with blocking antibodies against PD-L1 or/and 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.

Interleukin-27 (IL-27) is a type-I-cytokine predominantly secreted by activated macrophages and dendritic cells. Best described are the effects of IL-27 on T-cells and on innate immune cells. Although IL-27 can have pro-inflammatory effects, many studies also suggest that IL-27 is immunosuppressive. IL-27 is composed of the two non-covalently linked subunits p28 and EBI3 (Epstein-Barr virus induced gene 3) and signals via the WSX-1 and gp130 cytokine receptors; it can be grouped into the IL-6/IL-12 superfamily of cytokines. We here report that a variety of non-hematopoietic cells respond to IL-27. When comparing signaling of IL-27 to the one of IL-6-type cytokines (STAT3-dominated) and Interferon-gamma (STAT1-dominated), we observe that IL-27 elicits responses similar to Interferon-gamma.

Aberrant activation of oncogenic kinases is frequently observed in human cancers, but the underlying mechanism and resulting effects on global signaling are incompletely understood. Here, we demonstrate that the oncogenic FIP1L1-PDGFRα kinase exhibits a significantly different signaling pattern compared to its PDGFRα wild type counterpart. Interestingly, the activation of primarily membrane-based signal transduction processes (such as PI3-kinase- and MAP-kinase- pathways) is remarkably shifted toward a prominent activation of STAT factors. This diverging signaling pattern compared to classical PDGF-receptor signaling is partially coupled to the aberrant cytoplasmic localization of the oncogene, since membrane targeting of FIP1L1-PDGFRα restores activation of MAPK- and PI3K-pathways. In stark contrast to the classical cytokine-induced STAT activation process, STAT activation by FIP1L1-PDGFRα does neither require Janus kinase activity nor Src kinase activity. Furthermore, we investigated the mechanism of STAT5 activation via FIP1L1-PDGFRα in more detail and found that STAT5 activation does not involve an SH2-domain-mediated binding mechanism. We thus demonstrate that STAT5 activation occurs via a non-canonical activation mechanism in which STAT5 may be subject to a direct phosphorylation by FIP1L1-PDGFRα.

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.

The Janus kinase 2 mutant V617F occurs with high frequency in myeloproliferative neoplasms. Further mutations affecting the Janus kinase family have been discovered mostly in leukaemias and in myeloproliferative neoplasms. Owing to their involvement in neoplasia, inflammatory diseases and in the immune response, Janus kinases are promising targets for kinase inhibitor therapy in these disease settings. Various quantitative assays including two newly developed screening assays were used to characterize the function of different small-molecule compounds in cells expressing Jak2V617F. A detailed comparative analysis of different Janus kinase inhibitors in our quantitative assays and the subsequent characterization of additional activities demonstrated for the first time that the most potent Jak2 inhibitor in our study, CEP701, also targets Aurora kinases. CEP701 shows a unique combination of both activities which is not found in other compounds also targeting Jak2. Furthermore, colony forming cell assays showed that Janus kinase 2 inhibitors preferentially suppressed the growth of erythroid colonies, whereas inhibitors of Aurora kinases preferentially blocked myeloid colony growth. CEP701 demonstrated a combined suppression of both colony types. Moreover, we show that combined application of a Janus and an Aurora kinase inhibitor recapitulated the effect observed for CEP701 but might allow for more flexibility in combining both activities in clinical settings, e.g. in the treatment of myeloproliferative neoplasms. The newly developed screening assays are high throughput compatible and allow an easy detection of new compounds with Janus kinase 2 inhibitory activity.

Genetic deficiency of Jak3 leads to abrogation of signal transduction through the common gamma chain (γc) and thus to immunodeficiency suggesting that specific inhibition of Jak3 kinase may result in immunosuppression. Jak1 cooperates with Jak3 in signaling through γc-containing receptors. Unexpectedly, a Jak3-selective inhibitor was less efficient in abolishing STAT5 phosphorylation than pan-Jak inhibitors. We therefore explored the roles of Jak1 and Jak3 kinase functionality in signaling using a reconstituted system. The presence of kinase-inactive Jak1 but not kinase-inactive Jak3 resulted in complete abolishment of STAT5 phosphorylation. Specific inhibition of the "analog-sensitive" mutant AS-Jak1 but not AS-Jak3 by the ATP-competitive analog 1NM-PP1 abrogated IL-2 signaling, corroborating the data with the selective Jak3 inhibitor. Jak1 thus plays a dominant role over Jak3 and these data challenge the notion that selective ATP-competitive Jak3 kinase inhibitors will be effective.

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 (>95 % in polycythemia vera (PV), >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.

CRISPR-Cas9 proteins function within bacterial immune systems to target and destroy invasive DNA and have been harnessed as a robust technology for genome editing. Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9, providing an efficient off switch for Cas9-based applications. Here, we show that two Acrs, AcrIIC1 and AcrIIC3, inhibit Cas9 by distinct strategies. AcrIIC1 is a broad-spectrum Cas9 inhibitor that prevents DNA cutting by multiple divergent Cas9 orthologs through direct binding to the conserved HNH catalytic domain of Cas9. A crystal structure of an AcrIIC1-Cas9 HNH domain complex shows how AcrIIC1 traps Cas9 in a DNA-bound but catalytically inactive state. By contrast, AcrIIC3 blocks activity of a single Cas9 ortholog and induces Cas9 dimerization while preventing binding to the target DNA. These two orthogonal mechanisms allow for separate control of Cas9 target binding and cleavage and suggest applications to allow DNA binding while preventing DNA cutting by Cas9.

The Janus kinases, Jaks, constitutively associate with the cytoplasmic region of cytokine receptors and play an important role in a multitude of biological processes. Jak2 dysfunction has been implicated in myeloproliferative diseases and leukemia. Although Jaks were studied extensively for many years, the molecular mechanism of Jak activation upon cytokine stimulation of cells is still incompletely understood. In this study, we investigated the importance of an unusual insertion located within the kinase domain in Jak2. We found that the deletion of this insertion, which we named the Jak-specific insertion (JSI), totally abrogates Jak2 autophosphorylation. We further point mutated four residues within the JSI that are conserved in all Jak family members. Three of these mutants showed abrogated or reduced autophosphorylation, whereas the fourth displayed increased autophosphorylation. We found that the phosphorylation state of these mutants is not influenced by other domains of the kinase. Our data further suggest that the JSI is not required for the negative regulation of kinase activity by the suppressor of cytokine signaling proteins, SOCS. Most importantly, we show that mutations in this region differentially affect IFN-γ and erythropoietin signal transduction. Taken together, the dramatic effects on the phosphorylation status of Jak2 as well as the differential effects on the signaling via different cytokines highlight the importance of this unusual region for the catalytic activity of Jaks.

The interleukin‐6–type cytokine oncostatin M (OSM) acts via the Janus kinase/signal transducer and activator of transcription pathway as well as via activation of mitogen‐activated protein kinases and is known to critically regulate processes such as liver development and regeneration, hematopoiesis, and angiogenesis, which are also determined by hypoxia with the hypoxia‐inducible factor 1α (HIF1α) as a key component. Here we show that treatment of hepatocytes and hepatoma cells with OSM leads to an increased protein level of HIF1α under normoxic and hypoxic conditions. Furthermore, the OSM‐dependent HIF1α increase is mediated via Janus kinase/signal transducer and activator of transcription 3 and mitogen‐activated protein kinase kinase/extracellular signal‐regulated kinase 1/2 pathways. OSM‐mediated HIF1α up‐regulation did not result from an increase in HIF1α protein stability but from increased transcription from the HIF1α gene. In addition, we show that the OSM‐induced HIF1α gene transcription and the resulting enhanced HIF1α protein levels are important for the OSM‐dependent vascular endothelial growth factor and plasminogen activator inhibitor 1 gene induction associated with several diseases. Conclusion: HIF1α levels increase significantly after treatment of hepatocytes and hepatoma cells with OSM, and HIF1α contributes to OSM downstream signaling events, pointing to a cross‐talk between cytokine and hypoxia signaling in processes such as liver development and regeneration. (HEPATOLOGY 2009.)