The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

The first-in-class inhibitor of ALK, c-MET and ROS1, crizotinib (Xalkori), has shown remarkable clinical efficacy in treatment of ALK-positive non-small cell lung cancer. However, in neuroblastoma, activating mutations in the ALK kinase domain are typically refractory to crizotinib treatment, highlighting the need for more potent inhibitors. The next-generation ALK inhibitor PF-06463922 is predicted to exhibit increased affinity for ALK mutants prevalent in neuroblastoma. We examined PF-06463922 activity in ALK-driven neuroblastoma models in vitro and in vivo In vitro kinase assays and cell-based experiments examining ALK mutations of increasing potency show that PF-06463922 is an effective inhibitor of ALK with greater activity towards ALK neuroblastoma mutants. In contrast to crizotinib, single agent administration of PF-06463922 caused dramatic tumor inhibition in both subcutaneous and orthotopic xenografts as well as a mouse model of high-risk neuroblastoma driven by Th-ALK(F1174L)/MYCN Taken together, our results suggest PF-06463922 is a potent inhibitor of crizotinib-resistant ALK mutations, and highlights an important new treatment option for neuroblastoma patients.

Anaplastic Lymphoma Kinase (ALK) regulates initiation of transcription of MYCN in neuroblastoma cells.

Neuroblastoma is a neural crest-derived embryonal tumour of the postganglionic sympathetic nervous system and a disease with several different chromosomal gains and losses, which include MYCN-amplified neuroblastoma on chromosome 2, deletions of parts of the chromosomes 1p and 11q, gain of parts of 17q and triploidy. Recently, activating mutations of the ALK (Anaplastic Lymphoma Kinase) RTK (Receptor Tyrosine Kinase) gene have been described in neuroblastoma. A meta-analysis of neuroblastoma cases revealed that ALK mutations (49 of 709 cases) in relation to genomic subtype were most frequently observed in MYCN amplified tumours (8.9%), correlating with a poor clinical outcome. MYCN proteins target proliferation and apoptotic pathways, and have an important role in the progression of neuroblastoma. Here, we show that both wild-type and gain-of-function mutants in ALK are able to stimulate transcription at the MYCN promoter and initiate mRNA transcription of the MYCN gene in both neuronal and neuroblastoma cell lines. Further, this stimulation of MYCN gene transcription and de novo MYCN protein expression is abrogated by specific ALK inhibitors, such as crizotinib (PF-2341066), NVP-TAE684, and by small interfering RNA to ALK resulting in a decrease in proliferation rate. Finally, co-transfection of ALK gain-of-function mutations together with MYCN leads to an increase in transformation potential. Taken together, our results indicate that ALK signalling regulates initiation of transcription of the MYCN gene providing a possible explanation for the poor clinical outcome observed when MYCN is amplified together with activated ALK.

No support for a truncated interferon-alpha 17 allele as risk factor for MS.

Cytokines have a central role in multiple sclerosis (MS) pathogenesis and may contribute to the aetiology of MS. A polymorphism in the IFNA17 gene with an allele carrying a pre-mature stop codon has been suggested to convey a 26-fold increased risk for MS. We investigated the possible association between this polymorphism and MS using population-based samples from a genetically well-characterized population. The IFNA17 gene variant was found in 2.8% of 327 MS cases and 3.3% of 698 referents (P = 0.64). Thus, our study does not support an association between the IFNA17 allele and risk for MS.

Successful treatment of restless legs syndrome with an implanted pump for intrathecal drug delivery.

Two patients with incapacitating symptoms from restless legs syndrome, not adequately responding to conventional treatment with dopaminergic drugs, were implanted with a pump device (Isomed) for intrathecal delivery of morphine and bupivacaine. The treatment resulted in total resolution of all symptoms with few side effects.

Interferon-alpha promotes survival of human primary B-lymphocytes via phosphatidylinositol 3-kinase.

Signaling pathways for the antiviral and antiproliferative biological effects of type I interferons (IFN) are well established. In this report we demonstrate a novel signaling pathway for IFN-alpha, as it induced rapid phosphorylation of both PKB/Akt and its substrate forkhead. The PI3-kinase inhibitor LY294002 abolished these phosphorylations. PI3-kinase has been implicated in cell survival mediating its effect through the second messenger PIP3 and the subsequent activation of PKB/Akt. We could show that IFN-alpha inhibited spontaneous apoptosis of primary B-lymphocytes, in the absence of a mitogenic stimulus. This effect was inhibited by LY294002. Thus, our data suggests that IFN-alpha promotes survival of peripheral B-lymphocytes via the PI3-kinase-PKB/Akt pathway. In addition, IFN-alpha stimulation of anti-IgM activated cells resulted in downregulated expression of the cell cycle inhibitor p27/Kip1.

Brief intervention for male heavy drinkers in routine general practice: a three-year randomized controlled study.

The aim of this research was to evaluate the effectiveness of long-term brief intervention in routine general practice. In five primary care out-patient clinics in a Finnish town, 296 male early-phase heavy drinkers consulting a general practitioner (GP) for various reasons were identified. Control group C (n = 88) was informed of the risks of drinking after the screening and were advised at the subsequent feedback about 2 weeks later to reduce their drinking. Groups A (n = 109) and B (n = 99) were offered in addition seven and three brief intervention sessions, respectively. All GPs took part, whether or not they indicated a special interest. The main outcome measures were differences between beginning and end-point at 3 years in self-reported alcohol consumption, mean corpuscular volume (MCV), and serum carbohydrate-deficient transferrin, aspartate aminotransferase, alanine aminotransferase and gamma-glutamyltransferase. There were no statistically significant differences between study groups A, B and C in mean changes in outcome measures. Within all the groups, MCV decreased. Depending on the outcome measure used and the study group analysed, clinically significant reduction of drinking was found in 25-53% of the subjects. In routine general practice, giving additional sessions of brief intervention may not be as effective as in special research conditions. Factors reducing the effectiveness of brief intervention programmes should be investigated, so that primary health care staff can be better supported in their efforts.

Effect of suramin on human interferon alpha binding to cell receptors.

Radioiodinated human recombinant interferon alpha 88 (125I-HuIFN-alpha 88) binds to high affinity receptors of IFN sensitive Daudi cells (Burkitt lymphoma cell line). Suramin, a low molecular weight (1429), polyanionic compound at concentrations 105-175 microM completely abolished 125I-IFN-alpha 88 binding to Daudi cells at low temperature (4 degrees C). At 37 degrees C, however, its effect was only partial and depended on incubation time. Suramin also dissociated IFN-alpha 88-receptor complexes but, upon incubation of cells with IFN at 37 degrees C IFN-receptor complexes, became gradually less sensitive to suramin action. Dissociation by suramin IFN-alpha 88-receptor complexes prevented induction of (2-5)A synthetase activity and inhibited down-regulation of IFN receptors on Daudi cells. We suppose that the first reaction which represents IFN binding to the surface receptors is inhibited and dissociated by suramin, but when IFN is transferred to a tight activation complexes on the cell membrane or internalized, such complexes cannot be dissociated by suramin.

The cytoplasmic C-terminal domain but not the N-terminal domain of latent membrane protein 1 of Epstein-Barr virus is essential for B cell activation.

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) is essential for EBV-induced immortalization of human primary B cells, transforms rodent fibroblasts and induces the up-regulation of several B cell activation markers when transiently expressed in primary B cells. The biochemical function of LMP-1 is unclear and limited information is available on the involvement of different domains of the protein in B cell activation. The present study describes the characterization of LMP-1 N- and C-terminal deletion mutants in terms of their cell surface distribution and ability to induce activation markers in primary human B cells and in the type I Burkitt's lymphoma cell line DG75. The C-terminal deletion mutant was detected by immunofluorescence via antibodies targeted against an eight amino acid FLAG epitope substituted for the entire predicted cytoplasmic C-terminal domain. Our findings show that N-terminal deletion mutants of LMP-1 are unable to attain their usual patched distribution on the plasma membrane but retain the ability to activate B cells. In contrast, the C-terminal deletion mutant shows the same patched cell surface distribution as wild-type LMP-1 but is unable to activate B cells. The patched distribution of LMP-1 in the plasma membrane is therefore not sufficient nor necessary for the induction of B cell activation and the results rule out patching as a direct mechanism in LMP-1-induced activation. This is the first study addressing the role of the LMP-1 C-terminal domain in lymphocytes and our results show that this domain is essential for B cell activation and therefore likely to be important for the early events of B cell immortalization by EBV.

Characterization of monoclonal anti-DNA antibodies and visualization of binding to chromosomes and cell nuclei.

Monoclonal antibodies against DNA from two hybridoma cell lines were produced and characterized. One had specificity for single stranded (ss) DNA with some cross-reactivity to RNA, while the other was specific for both single (ss) and double stranded (ds) DNA. The latter ds and ss DNA-binding antibody was used as a model for analysing the distribution of the epitope in chromosomes and cell nuclei. A linear correlation between antibody binding and propidium iodide counterstaining was found on flow cytometric analysis of suspended chromosomes. Immunofluorescence of rat myoblast cells showed a speckled distribution of the antibody in the nucleus with a variability between the cells. Using electron microscopy to visualize antibody binding with gold particles, codistribution with uranyl acetate staining of leucocytes was found. These results suggested that the antibody preferentially binds to condensed chromatin in cells and chromosomes.