Cooperative effects of Janus and Aurora kinase inhibition by CEP701 in cells expressing Jak2V617F.

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.

Palmitoylation of MPP1 (membrane-palmitoylated protein 1)/p55 is crucial for lateral membrane organization in erythroid cells.

S-Acylation of proteins is a ubiquitous post-translational modification and a common signal for membrane association. The major palmitoylated protein in erythrocytes is MPP1, a member of the MAGUK family and an important component of the ternary complex that attaches the spectrin-based skeleton to the plasma membrane. Here we show that DHHC17 is the only acyltransferase present in red blood cells (RBC). Moreover, we give evidence that protein palmitoylation is essential for membrane organization and is crucial for proper RBC morphology, and that the effect is specific for MPP1. Our observations are based on the clinical cases of two related patients whose RBC had no palmitoylation activity, caused by a lack of DHHC17 in the membrane, which resulted in a strong decrease of the amount of detergent-resistant membrane (DRM) material. We confirmed that this loss of detergent-resistant membrane was due to the lack of palmitoylation by treatment of healthy RBC with 2-bromopalmitic acid (2-BrP, common palmitoylation inhibitor). Concomitantly, fluorescence lifetime imaging microscopy (FLIM) analyses of an order-sensing dye revealed a reduction of membrane order after chemical inhibition of palmitoylation in erythrocytes. These data point to a pathophysiological relationship between the loss of MPP1-directed palmitoylation activity and perturbed lateral membrane organization.

Hypoxia-inducible factor 1alpha is up-regulated by oncostatin M and participates in oncostatin M signaling.

UNLABELLED: 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 1alpha (HIF1alpha) as a key component. Here we show that treatment of hepatocytes and hepatoma cells with OSM leads to an increased protein level of HIF1alpha under normoxic and hypoxic conditions. Furthermore, the OSM-dependent HIF1alpha 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 HIF1alpha up-regulation did not result from an increase in HIF1alpha protein stability but from increased transcription from the HIF1alpha gene. In addition, we show that the OSM-induced HIF1alpha gene transcription and the resulting enhanced HIF1alpha protein levels are important for the OSM-dependent vascular endothelial growth factor and plasminogen activator inhibitor 1 gene induction associated with several diseases. CONCLUSION: HIF1alpha levels increase significantly after treatment of hepatocytes and hepatoma cells with OSM, and HIF1alpha contributes to OSM downstream signaling events, pointing to a cross-talk between cytokine and hypoxia signaling in processes such as liver development and regeneration.

LUMI-PCR: an Illumina platform ligation-mediated PCR protocol for integration site cloning, provides molecular quantitation of integration sites.

BACKGROUND: Ligation-mediated PCR protocols have diverse uses including the identification of integration sites of insertional mutagens, integrating vectors and naturally occurring mobile genetic elements. For approaches that employ NGS sequencing, the relative abundance of integrations within a complex mixture is typically determined through the use of read counts or unique fragment lengths from a ligation of sheared DNA; however, these estimates may be skewed by PCR amplification biases and saturation of sequencing coverage. RESULTS: Here we describe a modification of our previous splinkerette based ligation-mediated PCR using a novel Illumina-compatible adapter design that prevents amplification of non-target DNA and incorporates unique molecular identifiers. This design reduces the number of PCR cycles required and improves relative quantitation of integration abundance for saturating sequencing coverage. By inverting the forked adapter strands from a standard orientation, the integration-genome junction can be sequenced without affecting the sequence diversity required for cluster generation on the flow cell. Replicate libraries of murine leukemia virus-infected spleen samples yielded highly reproducible quantitation of clonal integrations as well as a deep coverage of subclonal integrations. A dilution series of DNAs bearing integrations of MuLV or piggyBac transposon shows linearity of the quantitation over a range of concentrations. CONCLUSIONS: Merging ligation and library generation steps can reduce total PCR amplification cycles without sacrificing coverage or fidelity. The protocol is robust enough for use in a 96 well format using an automated liquid handler and we include programs for use of a Beckman Biomek liquid handling workstation. We also include an informatics pipeline that maps reads, builds integration contigs and quantitates integration abundance using both fragment lengths and unique molecular identifiers. Suggestions for optimizing the protocol to other target DNA sequences are included. The reproducible distinction of clonal and subclonal integration sites from each other allows for analysis of populations of cells undergoing selection, such as those found in insertional mutagenesis screens.

Author Correction: Subclonal mutation selection in mouse lymphomagenesis identifies known cancer loci and suggests novel candidates.

The original version of this Article contained an error in the hyperlink for the online repository http://mulvdb.org which was incorrectly given as http://mulv.lms.mrc.ac.uk. This has been corrected in both the PDF and HTML versions of the Article.

Subclonal mutation selection in mouse lymphomagenesis identifies known cancer loci and suggests novel candidates.

Determining whether recurrent but rare cancer mutations are bona fide driver mutations remains a bottleneck in cancer research. Here we present the most comprehensive analysis of murine leukemia virus-driven lymphomagenesis produced to date, sequencing 700,000 mutations from >500 malignancies collected at time points throughout tumor development. This scale of data allows novel statistical approaches for identifying selected mutations and yields a high-resolution, genome-wide map of the selective forces surrounding cancer gene loci. We also demonstrate negative selection of mutations that may be deleterious to tumor development indicating novel avenues for therapy. Screening of two BCL2 transgenic models confirmed known drivers of human non-Hodgkin lymphoma, and implicates novel candidates including modifiers of immunosurveillance and MHC loci. Correlating mutations with genotypic and phenotypic features independently of local variance in mutation density also provides support for weakly evidenced cancer genes. An online resource http://mulv.lms.mrc.ac.uk allows customized queries of the entire dataset.