Béatrice Vayssière

BMP-2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop.

Wnt/β‐catenin signaling has recently been suggested to be involved in bone biology. The precise role of this cascade in osteoblast differentiation was examined. We show that a Wnt autocrine loop mediates the induction of alkaline phosphatase and mineralization by BMP‐2 in pre‐osteoblastic cells.

Deletion of a single allele of the Dkk1 gene leads to an increase in bone formation and bone mass

Wnt/β‐catenin signaling has been proven to play a central role in bone biology. Unexpectedly, the Wnt antagonist Dkk2 is required for terminal osteoblast differentiation and mineralized matrix formation. We show that Dkk1, unlike Dkk2, negatively regulates osteoblast differentiation and bone formation.

A New Member of the Amphiphysin Family Connecting Endocytosis and Signal Transduction Pathways

Src homology 3 (SH3) domains are conserved modules which participate in protein interaction by recognizing proline-rich motifs on target molecules. To identify new SH3-containing proteins, we performed a two-hybrid screen with a proline-rich region of human SOS-1. One of the specific SOS-1 interacting clones that were isolated from a mouse brain cDNA library defines a new protein that was named amphiphysin 2 because of its homology to the previously reported amphiphysin. Amphiphysin 2 is expressed in a number of mouse tissues through multiple RNA transcripts. Here, we report the amino acid sequence of a brain form of amphiphysin2 (BRAMP2) encoded by a 2.5-kilobase mRNA. BRAMP2 associates in vitro with elements of the endocytosis machinery such as α-adaptin and dynamin. On a biosensor surface, the BRAMP2/dynamin interaction appeared to be direct and partly dependent on a proline-rich sequence of dynamin. Association with dynamin was also observed in PC12 cells after cell stimulation with nerve growth factor, suggesting that amphiphysin 2 may be connected to receptor-dependent signaling pathways. This hypothesis is strengthened by the ability of BRAMP2 to interact with the p21 ras exchange factor SOS, in vitro, as a possible point of interconnection between the endocytic and signaling pathways.

Preclinical Characterization of GLPG0634, a Selective Inhibitor of JAK1, for the Treatment of Inflammatory Diseases

The JAKs receive continued interest as therapeutic targets for autoimmune, inflammatory, and oncological diseases. JAKs play critical roles in the development and biology of the hematopoietic system, as evidenced by mouse and human genetics. JAK1 is critical for the signal transduction of many type I and type II inflammatory cytokine receptors. In a search for JAK small molecule inhibitors, GLPG0634 was identified as a lead compound belonging to a novel class of JAK inhibitors. It displayed a JAK1/JAK2 inhibitor profile in biochemical assays, but subsequent studies in cellular and whole blood assays revealed a selectivity of ∼30-fold for JAK1- over JAK2-dependent signaling. GLPG0634 dose-dependently inhibited Th1 and Th2 differentiation and to a lesser extent the differentiation of Th17 cells in vitro. GLPG0634 was well exposed in rodents upon oral dosing, and exposure levels correlated with repression of Mx2 expression in leukocytes. Oral dosing of GLPG0634 in a therapeutic set-up in a collagen-induced arthritis model in rodents resulted in a significant dose-dependent reduction of the disease progression. Paw swelling, bone and cartilage degradation, and levels of inflammatory cytokines were reduced by GLPG0634 treatment. Efficacy of GLPG0634 in the collagen-induced arthritis models was comparable to the results obtained with etanercept. In conclusion, the JAK1 selective inhibitor GLPG0634 is a promising novel therapeutic with potential for oral treatment of rheumatoid arthritis and possibly other immune-inflammatory diseases.

Interaction between LRP5 and Frat1 Mediates the Activation of the Wnt Canonical Pathway

Low density lipoprotein receptor-related protein 5 (LRP5) has been identified as a Wnt co-receptor involved in the activation of the β-catenin signaling pathway. To improve our understanding of the molecular mechanisms by which LRP5 triggers the canonical Wnt signaling cascade, we have screened for potential partners of LRP5 using the yeast two-hybrid system and identified Frat1 as a protein interacting with the cytoplasmic domain of LRP5. We demonstrate here that LRP5/Frat1 interaction is involved in β-catenin nuclear translocation and TCF-1 transcriptional activation. The addition of Wnt3a or overexpression of constitutively active truncated LRP5 (LRP5C) induces Frat1 recruitment to the cell membrane. Overexpression of a dominant negative form of disheveled (Dvl) shows that this protein positively affects LRP5/Frat1 interaction. Furthermore, the fact that dominant negative Dvl does not interfere with LRP5C/Frat1 interaction can explain how LRP5C is capable of acting independently of this major Wnt signaling player. Axin, which has been shown to interact with LRP5 and to be recruited to the membrane through this interaction, was found to co-immunoprecipitate with Frat1 and LRP5. We propose that recruitment of Axin and Frat1 to the membrane by LRP5 leads to both Axin degradation and Frat1-mediated inhibition of glycogen synthase kinase-3. As a consequence, β-catenin is no longer bound to Axin or phosphorylated by glycogen synthase kinase-3, resulting in TCF-1 activation.

Triazolopyridines as Selective JAK1 Inhibitors: From Hit Identification to GLPG0634

Janus kinases (JAK1, JAK2, JAK3, and TYK2) are involved in the signaling of multiple cytokines important in cellular function. Blockade of the JAK-STAT pathway with a small molecule has been shown to provide therapeutic immunomodulation. Having identified JAK1 as a possible new target for arthritis at Galapagos, the compound library was screened against JAK1, resulting in the identification of a triazolopyridine-based series of inhibitors represented by 3. Optimization within this chemical series led to identification of GLPG0634 (65, filgotinib), a selective JAK1 inhibitor currently in phase 2B development for RA and phase 2A development for Crohns disease (CD).

Interaction of the Grb7 adapter protein with Rnd1, a new member of the Rho family

Grb7 is a member of a family of molecular adapters which are able to contribute positively but also negatively to signal transduction and whose precise roles remain obscure. Rnd1 is a member of the Rho family, but, as opposed to usual GTPases, it is constitutively bound to GTP. We show here that Rnd1 and Grb7 interact, in two‐hybrid assays, in vitro, and in pull‐down experiments performed with SK‐BR3, a breast cancer cell line that overexpresses Grb7. This interaction involves switch II loop of Rnd1, a region crucial for guanine nucleotide exchange in all GTPases, and a Grb7 SH2 domain, a region crucial for Grb7 interaction with several activated receptors. The contribution of the interaction between Rnd1 and Grb7 to their respective functions and properties is discussed.

Expression, purification and functional characterization of Wnt signaling co-receptors LRP5 and LRP6.

Activation of the Wnt signaling cascade plays a pivotal role during development and in various disease states. Wnt signals are transduced by seven-transmembrane Frizzled (Fz) proteins and the single-transmembrane LDL-receptor-related proteins 5 or 6 (LRP5/6). Genetic mutations resulting in a loss or gain of function of LRP5 in humans lead to osteopenia and bone formation, respectively. These findings demonstrate the genetic link between LRP5 signaling and the regeneration of bone mass. Herein we describe for the first time the production and characterization of soluble ectodomains of LRP5 and LRP6, (EC-LRP5, EC-LRP6). We have produced these proteins in amounts that are compatible with both in vitro and cell-based assays to study their binding properties. Purified EC-LRP5 and EC-LRP6 were able to interact with Wnt signaling components Dkk1 and Dkk2 and their functionality was confirmed in cell-based Wnt signaling assays. Hence, tools are now available to explore LRP5/6 interaction with other proteins and to screen for synthetic or natural compounds and biologics that might be novel therapeutics targeting the Wnt pathway.

AB0506 Identification of a Gene Signature and Response Biomarkers in Circulating Leukocytes of RA Patients After Treatment with the JAK1-Selective Inhibitor Filgotinib (GLPG0634)

Background The 4 Janus kinases (JAK1, JAK2, JAK3 and TYK2) are cytoplasmic tyrosine kinases that mediate intracellular signaling of cytokines (e.g. certain interleukins and interferons) and growth factors (e.g. erythropoietin). Filgotinib (formerly GLPG0634) is the first JAK inhibitor that displays a high selectivity for JAK1 over the 3 other JAK family members in biochemical and cellular assays. It showed a favorable safety and efficacy profile in two 4-week Phase 2a studies in rheumatoid arthritis (RA) patients. Objectives To assess the effect of RA disease effects, characterize the disease-relevant treatment responses to filgotinib and identify response biomarkers, we compared the gene expression profile of circulating leukocytes of RA patients, before and after up to 4 weeks of once-daily treatment with 200mg of filgotinib, with healthy volunteers. Methods RA patients participated in the Phase 2a Proof of concept, a randomized, double-blind, placebo-controlled study enrolling 24 patients with insufficient response to MTX. They were orally treated with placebo or 200 mg QD filgotinib for 4 weeks. Blood was sampled in PAXgene tubes at pre-dose and at the last day of treatment. Non-matched healthy volunteers were also sampled. mRNA was extracted, labeled and profiled using Affymetrix U219 micro-arrays. Data analysis was performed in R/BioConductor using linear regression models (limma). Results The leukocyte gene signature of 12 healthy subjects was first compared to the one obtained from the 24 RA patients prior to placebo or filgotinib treatment. Genes showing differential expression compared to healthy subjects allowed for definition of a disease signature, which includes novel players as well as genes already described in RA (TFPI or ITGA2B). Four weeks of treatment with filgotinib impacted the signal levels for 1005 probes (254 downregulated, 751 upregulated) in RA patient samples (p-value <0.05 and absolute log2-fold change >0.5 compared to pre-dose), while the signal levels of only 161 probes (86 down- and 75 upregulated) were impacted to the same extent in the placebo group. Several disease-relevant genes (CETP, CTSD, FSTL1, HTRA1, MYL9) were affected by filgotinib treatment. This was confirmed by qPCR. Response biomarkers could be identified by comparing strong responders and weaker responders based on percentage change in disease activity score 28 (DAS28). Functional classification of these biomarkers showed enrichment towards key RA pathways including regulation of immune cells, neutrophil recruitment, inositol phosphate metabolism and phosphatidylinositol signaling (PI3K). In addition, in the best responders, ECM-receptor interaction and focal adhesion pathways were enriched in the genes affected by filgotinib while in the weaker responders, regulation of actin cytoskeleton and TGF-beta signalling were affected by filgotinib. Conclusions Blood transcriptome analysis showed that 4-week filgotinib treatment partially reverses the disease effect and allows for the identification of treatment response biomarkers in circulating leukocytes of RA patients. Additional information to be obtained from the Darwin studies currently ongoing should provide more insights. Disclosure of Interest M. Ongenaert Grant/research support from: AbbVie, Employee of: Galapagos, S. Dupont Grant/research support from: AbbVie, Employee of: Galapagos, B. Vayssière Grant/research support from: AbbVie, Employee of: Galapagos, L. Meuleners Grant/research support from: AbbVie, Employee of: Galapagos, R. Brys Grant/research support from: AbbVie, Employee of: Galapagos, R. Galien Grant/research support from: AbbVie, Employee of: Galapagos

AB0494 Filgotinib (GLPG0634), a Selective JAK1 Inhibitor, Shows Similar PK and PD Profiles in Japanese and Caucasian Healthy Volunteers

Background Filgotinib is an oral, selective Janus kinase 1 (JAK1) inhibitor combining a favorable safety profile and clinical efficacy in patients after 4 weeks of dosing with rheumatoid arthritis (RA) with a rapid onset of action. Its once-daily oral administration is supported by the long half-life of an active metabolite with the same JAK1 selectivity profile as the parent compound. Given ethnicity may cause for differences in drug metabolism and pharmacokinetics (PK), and pharmacodynamics (PD) resulting in variability in clinical response, filgotinib was studied in Japanese healthy volunteers to evaluate the potential for different dosing requirements compared to Caucasians. Objectives Compare the PK, PD and safety of filgotinib in Japanese to Caucasian healthy volunteers at 200 mg filgotinib. Methods In a single-center Phase 1 study, 2 panels of 10 Japanese (1st and 2nd generation residing outside Japan for less than 5 years) and 10 Caucasian healthy volunteers received once daily 200 mg filgotinib or placebo for 10 days. The PK of filgotinib and its active metabolite were evaluated, and the overall PD of the two moieties was assessed in whole blood using ex vivo IL-6 induced phosphorylation of STAT1 (pSTAT1) as biomarker for JAK1 activity and GM-CSF induced phosphorylation of STAT5 (pSTAT5) for JAK2 activity. Standard safety assessments were performed throughout the study duration. Results Steady state in filgotinib plasma concentrations was reached in 2 days in both Japanese and Caucasian volunteers with half-lives of 6 and 11 hours, respectively. In both ethnic groups, the active metabolite showed plasma concentrations well exceeding those of filgotinib with half-life values ranging of 17-20 hours and overall exposures for filgotinib and its metabolite being the same between the populations. In both ethnic groups, IL-6 induced pSTAT1 was inhibited over the entire 24 hour post-dosing period, with maximum inhibition observed between 1 and 5 hours post dose. No relevant inhibition of JAK2 activity was observed in Japanese and Caucasian healthy volunteers confirming the JAK1 over JAK2 selectivity of filgotinib. In Japanese healthy volunteers, filgotinib was generally safe and well tolerated with no relevant differences in safety profile as compared to Caucasian healthy volunteers. Conclusions Filgotinib showed comparable PK, PD and safety profiles in Japanese and Caucasian healthy volunteers. The similarity in the PK and PD response suggests that there are no relevant differences among the groups in drug metabolism or sensitivity to selective inhibition of JAK1. Thus, these data support that filgotinib could be safely administered without dose adjustment to Japanese RA patients. Disclosure of Interest F. Namour Shareholder of: GALAPAGOS, Employee of: GALAPAGOS, B. Vayssière Employee of: GALAPAGOS, R. Galien Shareholder of: GALAPAGOS, Employee of: GALAPAGOS, L. Fagard Employee of: GALAPAGOS, A. Van der Aa Shareholder of: GALAPAGOS, Employee of: GALAPAGOS, P. Harrison Shareholder of: GALAPAGOS, Employee of: GALAPAGOS, C. Tasset Shareholder of: GALAPAGOS, Employee of: GALAPAGOS