Laurent Vallat

Spleen Tyrosine Kinase Is Overexpressed and Represents a Potential Therapeutic Target in Chronic Lymphocytic Leukemia

B-cell receptor signaling contributes to apoptosis resistance in chronic lymphocytic leukemia (CLL), limiting the efficacy of current therapeutic approaches. In this study, we investigated the expression of spleen tyrosine kinase (SYK), a key component of the B-cell receptor signaling pathway, in CLL and its role in apoptosis. Gene expression profiling identified enhanced expression of SYK and downstream pathways in CLL compared with healthy B cells. Immunoblotting showed increased expression and phosphorylation of SYK, PLCgamma(2), signal transducers and activators of transcription 3, and extracellular signal regulated kinase 1/2 in CLL compared with healthy B cells, suggesting enhanced activation of these mediators in CLL. SYK inhibitors reduced phosphorylation of SYK downstream targets and induced apoptosis in primary CLL cells. With respect to prognostic factors, SYK inhibitors exerted stronger cytotoxic effects in unmutated and ZAP70(+) cases. Cytotoxic effects of SYK inhibitors also associated with SYK protein expression, potentially predicting response to therapy. Combination of fludarabine with SYK Inhibitor II or R406 increased cytotoxicity compared with fludarabine therapy alone. We observed no stroma-contact-mediated drug resistance for SYK inhibitors as described for fludarabine treatment. CD40 ligation further enhanced efficacy of SYK inhibition. Our data provide mechanistic insight into the recently observed therapeutic effects of the SYK inhibitor R406 in CLL. Combination of SYK inhibitors with fludarabine might be a novel treatment option particularly for CLL patients with poor prognosis and should be further evaluated in clinical trials.

A unique proteomic profile on surface IgM ligation in unmutated chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is characterized by a highly variable clinical course with 2 extreme subsets: indolent, ZAP70(-) and mutated immunoglobulin heavy chain gene (M-CLL); and aggressive, ZAP70(+) and unmutated immunoglobulin heavy chain (UM-CLL). Given the long-term suspicion of antigenic stimulation as a primum movens in the disease, the role of the B-cell receptor has been extensively studied in various experimental settings; albeit scarcely in a comparative dynamic proteomic approach. Here we use a quantitative 2-dimensional fluorescence difference gel electrophoresis technology to compare 48 proteomic profiles of the 2 CLL subsets before and after anti-IgM ligation. Differentially expressed proteins were subsequently identified by mass spectrometry. We show that unstimulated M- and UM-CLL cells display distinct proteomic profiles. Furthermore, anti-IgM stimulation induces a specific proteomic response, more pronounced in the more aggressive CLL. Statistical analyses demonstrate several significant protein variations according to stimulation conditions. Finally, we identify an intermediate form of M-CLL cells, with an indolent profile (ZAP70(-)) but sharing aggressive proteomic profiles alike UM-CLL cells. Collectively, this first quantitative and dynamic proteome analysis of CLL further dissects the complex molecular pathway after B-cell receptor stimulation and depicts distinct proteomic profiles, which could lead to novel molecular stratification of the disease.

Expression and proliferative effect of hemokinin-1 in human B-cells.

Tachykinins are a family of structurally related peptides, including substance P (SP), hemokinin-1 (HK-1), neurokinin A (NKA), and neurokinin B. SP and NKA have been shown to modulate hematopoiesis and rat/mouse HK-1 has been found to be involved in the survival and differentiation of mouse B-cells. This study was designed to assess the expression of tachykinins with a focus on human HK-1 (hHK-1) in human B lymphocytes and the role of these peptides in cell differentiation, apoptosis and proliferation. Expression of tachykinin and tachykinin receptor mRNA was determined quantitatively in human B lymphoproliferative malignancies and compared to normal B-cells. Expression of hHK-1 and NK(1) receptor, but not SP, was detected in human B-lymphocytes, and was up-regulated in B-lymphocytes from chronic lymphocytic leukemia and non-Hodgkins lymphoma, while it was down-regulated in acute lymphoblastic leukemia. Moreover, hHK-1, in contrast to SP, was able to induce proliferation of human pre-B lymphocytes through a NK(1) receptor-independent mechanism. These data suggest a role for hHK-1 in normal and pathological B lymphopoiesis, and open the door to a better understanding of the physiopathological mechanisms leading to lymphoproliferative malignancies.

Reverse-engineering the genetic circuitry of a cancer cell with predicted intervention in chronic lymphocytic leukemia

Cellular behavior is sustained by genetic programs that are progressively disrupted in pathological conditions—notably, cancer. High-throughput gene expression profiling has been used to infer statistical models describing these cellular programs, and development is now needed to guide orientated modulation of these systems. Here we develop a regression-based model to reverse-engineer a temporal genetic program, based on relevant patterns of gene expression after cell stimulation. This method integrates the temporal dimension of biological rewiring of genetic programs and enables the prediction of the effect of targeted gene disruption at the system level. We tested the performance accuracy of this model on synthetic data before reverse-engineering the response of primary cancer cells to a proliferative (protumorigenic) stimulation in a multistate leukemia biological model (i.e., chronic lymphocytic leukemia). To validate the ability of our method to predict the effects of gene modulation on the global program, we performed an intervention experiment on a targeted gene. Comparison of the predicted and observed gene expression changes demonstrates the possibility of predicting the effects of a perturbation in a gene regulatory network, a first step toward an orientated intervention in a cancer cell genetic program.

Reduced DICER1 Expression Bestows Rheumatoid Arthritis Synoviocytes Proinflammatory Properties and Resistance to Apoptotic Stimuli.

While the regulatory role of individual microRNAs (miRNAs) in rheumatoid arthritis (RA) is well established, the role of DICER1 in the pathogenesis of the disease has not yet been investigated. The purpose of this study was to analyze the expression of factors involved in miRNA biogenesis in fibroblast‐like synoviocytes (FLS) from RA patients and to monitor the arthritis triggered by K/BxN serum transfer in mice deficient in the Dicer gene (Dicerd/d).

Cascade: a R package to study, predict and simulate the diffusion of a signal through a temporal gene network

SUMMARY Temporal gene interactions, in response to environmental stress, form a complex system that can be efficiently described using gene regulatory networks. They allow highlighting the more influential genes and spotting some targets for biological intervention experiments. Despite that many reverse engineering tools have been designed, the Cascade package is an integrated solution adding several new and original key features such as the ability to predict changes in gene expressions after a biological perturbation in the network and graphical outputs that allow monitoring the spread of a signal through the network. AVAILABILITY AND IMPLEMENTATION The R package Cascade is available online at http://www-math.u-strasbg.fr/genpred/spip.php?rubrique4.

A1.71 Reduced dicer expression correlates with rheumatoid arthritis

Background and Objectives A large body of evidence supports the role of microRNAs (miRNAs) in inflammatory diseases. Concerning rheumatoid arthritis (RA), several miRNAs exhibiting deregulated expression pattern were identified in various fluids (serum or synovial) or different cell types (macrophages, synoviocytes). The potential interest of these molecules as biomarkers is increasingly considered, but their precise mode of action still remains largely hypothetical because the identification of their physiological mRNA targets is challenging. To evaluate the overall importance of miRNAS in the pathogenesis of RA, we investigated the expression of genes involved in miRNA biogenesis in FLS isolated from healthy donors and RA patients. We also used a mouse mutant line carrying a hypomorphic mutation in the Dicer gene to monitor the impact of reduced miRNA maturation on experimental arthritis triggered by K/BxN serum transfer. Materials and Methods Human FLS were isolated from synovial tissues from RA and OA patients and healthy controls after informed consent. Mice FLS were isolated from Dicer d/d and littermates controls. IL-6 release was measured in culture supernatants and following RNA extraction using TRIzol and reverse transcription, real-time quantitative RT-qPCR was performed. Handling of mice and procedures were in accordance with the French Law for the Protection of Laboratory Animals and reviewed by the Regional Ethical Committee for Animal Experimentation (CREMEAS) of the Strasbourg University (authorisation number A-67-345). Serum collected from 9-week old arthritic K/BxN mice was used to trigger arthritis in adult Dicer d/d and + / + mice by two successive i.p. injection (150 μL). The articular arthritis index was visually determined on a 0–4 scale (0 = no swelling or erythema, 4 = excessive edema with joint rigidity). Results Dicer is the only gene involved in the miRNA biogenesis pathway showing significantly reduced expression in FLS isolated from RA patients compared to controls or OA. Similarly, mutant mice (Dicer d/d) with reduced Dicer expression exhibit more severe arthritis symptoms upon K/BxN serum transfer. Conclusions miRNAs are important players to keep pro inflammatory molecules under tight control. Mutations globally affecting mature miRNAs production represent a previously unidentified risk factor for RA.