Christine Le Roy

In the microenvironment, cell interactions are established between different cell types to regulate their migration, survival and activation. β-Catenin is a multifunctional protein that stabilizes cell–cell interactions and regulates cell survival through its transcriptional activity. We used chronic lymphocytic leukemia (CLL) cells as a cellular model to study the role of β-catenin in regulating the adhesion of tumor cells to their microenvironment, which is necessary for tumor cell survival and accumulation. When co-cultured with a stromal cell line (HS-5), a fraction of the CLL cells adhere to stromal cells in a dynamic fashion regulated by the different levels of β-catenin expression. In non-adherent cells, β-catenin is stabilized in the cytosol and translocates into the nucleus, increasing the expression of cyclin D1. In adherent cells, the level of cytosolic β-catenin is low but membrane β-catenin helps to stabilize the adhesion of CLL to stromal cells. Indeed, the overexpression of β-catenin enhances the interaction of CLL with HS-5 cells, suggesting that this protein behaves as a regulator of cell adhesion to the stromal component and of the transcriptional regulation of cell survival. Inhibitors that block the stabilization of β-catenin alter this equilibrium and effectively disrupt the support that CLL cells receive from the cross-talk with the stroma.

Chronic Lymphocytic Leukemia (CLL) is a heterogeneous B cell neoplasm ranging from indolent to rapidly progressive disease. Leukemic cell subsets with regulatory properties evade immune clearance; however, the contribution of such subsets during CLL progression is not completely elucidated. Here, we report that CLL B cells crosstalk with their immune counterparts, notably by promoting the regulatory T (Treg) cell compartment and shaping several helper T (Th) subsets. Among various constitutively- and BCR/CD40-mediated factors secreted, tumour subsets co-express two important immunoregulatory cytokines, IL10 and TGFβ1, both associated with a memory B cell phenotype. Neutralizing secreted IL10 or inhibiting the TGFβ signalling pathway demonstrated that these cytokines are mainly involved in Th- and Treg differentiation/maintenance. In line with the regulatory subsets, we also demonstrated that a CLL B cell population expresses FOXP3, a marker of regulatory T cells. Analysis of IL10, TGFβ1 and FOXP3 positive subpopulations frequencies in CLL samples discriminated 2 clusters of untreated CLL patients that were significantly different in Tregs frequency and time-to-treatment. Since this distinction was pertinent to disease progression, the regulatory profiling provides a new rationale for patient stratification and sheds light on immune dysfunction in CLL.

Spleen Tyrosine Kinase (Syk) plays a crucial role in immune cell signalling and its altered expression or activation are involved in several cancers. Syk activity relies on its phosphorylation status and its multiple phosphorylation sites predict several Syk conformations. In this report, we characterized Syk structural changes according to its phosphorylation/activation status by Surface Enhanced Raman Spectroscopy (SERS). Unphosphorylated/inactive and phosphorylated/active Syk forms were produced into two expression systems with different phosphorylation capability. Syk forms were then analysed by SERS that was carried out in liquid condition on a lithographically designed gold nanocylinders array. Our study demonstrated that SERS signatures of the two Syk forms were drastically distinct, indicating structural modifications related to their phosphorylation status. By comparison with the atomic structure of the unphosphorylated Syk, the SERS peak assignments of the phosphorylated Syk nearest gold nanostructures revealed a differential interaction with the gold surface. We finally described a model for Syk conformational variations according to its phosphorylation status. In conclusion, SERS is an efficient technical approach for studying in vitro protein conformational changes and might be a powerful tool to determine protein functions in tumour cells.

// <![CDATA[ $('.header-date').hide();$('#titleAuthors').hide();$('#abstractHeader').hide(); // ]]> Stéphane Saint Georges1,2, Maude Quettier1,2, Marouane Bouyaba3, Stéphanie Le Coquil1,2, Vanessa Laurienté1,2, Lionel Guittat1,2, Vincent Lévy3, Florence Ajchenbaum-Cymbalista1,2,4, Nadine Varin-Blank1,2, Christine Le Roy1,2,* and Dominique Ledoux1,2,* 1 INSERM U978, Bobigny, France 2 Université Paris 13, Sorbonne Paris Cité, Labex “Inflamex”, Bobigny, France 3 Assistance Publique-Hôpitaux de Paris, Hôpital Avicenne, Unité de Recherche Clinique, Bobigny, France 4 Assistance Publique-Hôpitaux de Paris, Hôpital Avicenne, Service d’Hématologie Biologique, Bobigny, France * These authors are co-seniors authors Correspondence to: Christine Le Roy, email: // <![CDATA[ mail("christine.le-roy","inserm",18,"") // ]]> Nadine Varin-Blank, email: // <![CDATA[ mail("nadine.varin","inserm",18,"") // ]]> Keywords: CLL, lymphadenopathy, B-cell receptor, CXCR4/CXCR5, protein kinase D Received: December 23, 2015 Accepted: April 16, 2016 Published: April 26, 2016

Common in ageing patient, the solar lentigo is a macular hyperpigmented skin lesion that results from chronic exposure to ultraviolet irradiations. Despite sharing numerous features with other pigmented spots, the diagnostic of this benign lesion is well characterized at the tissue level. Recent studies shed lights on several factors and their pathogenic mechanisms involved in the development of the solar lentigo. This review summarizes how diverse experimental approaches allowed the identification of several biomarkers, which contribute to a better understanding on the initiation and the maintenance of this pigmentary disorder.

Antigen-driven signals are involved in the pathogenesis and progression of CLL with a particular occurrence within the lymph node microenvironment. We have previously shown that ex-vivo BCR engagement promoted simultaneous down-regulation of CXCR4 and CD62L membrane expressions in CLL cells from patients at risk of disease progression only. Functionally, cells down-regulating CXCR4 and CD62L in response to BCR triggering displayed both a reduction in migration toward CXCL12 and in adhesion to lymphatic endothelial cells. We studied a cohort of 73 previously untreated patients, 36 of whom were IGHV mutated and 37 were IGHV unmutated. The distribution of the extent of CXCR4 down-regulation was identical among CXCR4 responders, whether IGHV mutated (median 49% [Q1:28-Q3:62]) or unmutated (median 43% [Q1:31-Q3:66]). We also studied the expression of another chemokine receptor, CXCR5, which is also expressed at the membrane of CLL cells and plays an important role in the homing and trafficking of lymphocytes to the lymphoid follicles. Sustained antigenic stimulation of CLL cell samples (n=25) promoted down-regulation of CXCR4 and CXCR5 in a strictly correlated manner (r= 0,9) i.e. in the same subsets of cells. The extent of BCR-dependent decrease of the two proteins was strikingly representative of the heterogeneous capacity of the B-CLL cells to respond to BCR engagement in a given patient. Moreover, treatment with the specific PKD inhibitor CID755673 blocked significantly both CXCR4 and CXCR5 BCR-mediated decrease (n=11), demonstrating that PKDs specifically target these 2 molecules, whereas CD62L down-regulation was not significantly affected by the PKD inhibitor. At the functional level, treatment with the PKD inhibitor restored CLL cell migration capacity in response to CXCL12. PKD phosphorylation/activation in response to BCR stimulation, which involvesPI3K-d, was required for CXCR4-phosphorylation and its down-regulation. We then studied the clinical relevance of CXCR4 down-regulation after BCR engagement. The capacity to down-regulate CXCR4 was significantly related to shorter PFS (p=0.043). This cohort allowed exploring the link with IGHV mutational status. 36/37 unmutated IGHV cases had a significant CXCR4 down-regulation after in vitro BCR stimulation, in concordance with the ultimately constant progression of the disease. Conversely, IGHV mutated cases fell into two groups: a) one group of 14/36 patients, with very low (<5%) or no CXCR4 decrease in response to BCR ligation. None of these patients had any lymph node enlargement with a median follow up of 8.4 years [Q1:4.4-Q3:9.9]. Four patients among them required treatment but all for other reasons than nodal progression. b) the other group of 22/36 patients, for which there was a variable but significant CXCR4 down-regulation after BCR engagement. Among these cases, with a median follow up of 9.2 years [Q1:6.4-Q3:10.7], 14 patients developed clinical lymphadenopathy and 7 patients required treatment, all of them for tumor progression. The difference in lymphadenopathy was highly significant between these 2 IGHV mutated groups (p< 0.001). In conclusion, the capacity of cells to down-regulate cell surface chemokine receptors and L-selectin is reflecting the size of the cell subset responding to BCR signaling. Interestingly, BCR signaling capacity to down-regulate CXCR4 is variable among IGHV mutated samples and strongly linked to the presence of lymph nodes as well as disease progression with time. It is tempting to speculate that depending on the BCR-mediated CXCR4/CXCR5 down-regulation capacity, some IGHV mutated patients might not benefit from Btk or PI3K-d inhibitors. Given the crucial importance of down-regulation of these two chemokine receptors after BCR engagement, we suggest that PKD could be promising new therapeutic target in CLL.

Key Points: CD9 expression enhances the CXCL12-induced migration of pre-B leukemic lymphocytes via RAC1 signaling. CD9 influences the chemotactic migration and engraftment of pre-B leukemic cells in NOD/SCID mouse testis.

Introduction and hypothesis : B-acute lymphoblastic leukemia (B-ALL) expressing the fusion transcript ETV6/RUNX1 is one of the most frequent subtype of childhood leukemia. This disease is associated with a good survival rate but 5 to 10% of patients relapse more than five years after treatment most notably in extra-hematopoietic sanctuary sites like testis. CD9, a protein member of the tetraspanin family, has been shown to be under-expressed in ETV6/RUNX1 B-ALL. This protein is involved in hematopoietic stem cell engraftment. Its expression has also been correlated with the risk of metastasis and is associated with a poor clinical outcome in various types of cancer. Our hypothesis is that CD9, through its functional properties on migration and homing, could be a key actor of ETV6/RUNX1 leukemia relapse. The purpose of our study was to investigate the effect of CD9 expression on motility and engrafment of pre-B lymphocytes. Methods and results : We used the human pre-B lymphoblastic REH cell line which express CD9. By lentiviral transduction of small hairpin RNAs targeting CD9 mRNA, we generated REH cells depleted in CD9 protein. We performed engraftment assays in vivo using Nod Scid Gamma immunodeficient mice. Mice injected with REH cells depleted in CD9 had a longer survival rate than mice injected with REH control cells. In vitro analysis of adhesion on fibronectin demonstrated that cellular adhesion is dependent of membrane expression of CD9. As well, the more CD9 is expressed, the higher migration rate in response to CXCL12 chemokine is. F‐actin labelling after CXCL12 stimulation showed also an increased F‐actin polymerization in CD9‐positive cells and the formation of actin extensions where CD9 and actin are colocalized. No modifications in CXCR4 (receptor of CXCL12) internalization has been found in CD9 depleted cell lines, which suggests that CD9 does not affect the receptor itself but the signalling pathway downstream the receptor. We investigated the activation of RAC1 protein, a Rho GTPase known to induce the formation of actin-rich lamellipodia in diverse cellular functions including migration. We demonstrated that cells depleted in CD9 have lost their ability to activate RAC1 in response to CXCL12. The inhibition and the depletion of RAC1 respectively have the same effect as CD9 depletion on the migration in response to CXCL12 in vitro and on the survival in vivo. Finally, we tested the ability of REH cells and REH depleted in CD9 cells to migrate to testis in vivo after xenograft in mouse model. We showed that only CD9 positive-REH cells were found in testis tissue after xenograft. Moreover we tested in vitro, the ability of testis tissue to induce the migration of REH cells in a Boyden chamber model. We showed that testis tissue induced the migration of REH cells and this migration was strongly decreased using either an inhibitor of CXCR4, an inhibitor of RAC1 or an antibody targeting specifically CD9. Conclusion : Our data show that CD9 is a key actor in pre‐B lymphoblasts adhesion, CXCL12 dependant migration and actin remodelling. We show for the first time in pre-B cells a link between CD9, RAC1 pathway and homing in testis which is a preferential site of late relapse. This work is supported by CNRS, University of Rennes 1, University Hospital of Rennes, Ligue Régionale contre le Cancer (sections 22, 35 and 56) (MPA, VG, MBT), Région Bretagne (MPA, MBT, VG), SFR Biosit UMS 3480 (VG, MBT), Association Laurette Fugain (VG).

Only a minority of chronic lymphocytic leukemia (CLL) patients harboring a positive direct antiglobulin test (DAT) will develop autoimmune hemolytic anemia (AIHA). In a single institution cohort of 378 CLL patients, 56 patients (14.8%) had at least one positive DAT during the course of the disease, either at diagnosis or later. We found no relationship between the time of the first positive DAT and overall survival (OS). However, patients with a positive DAT who did not develop AIHA had the same adverse outcome as patients who developed AIHA. Of the patients who were in Binet stage A at diagnosis, those with a positive DAT had a significantly shorter OS, regardless of their IGHV mutational status, however, there was a strong association with VH1‐69. By multivariate analysis, a positive DAT was found to be an independent adverse prognostic factor for OS. Thus, DAT represents a strong adverse prognostic factor and its determination should be repeated during follow‐up. Am. J. Hematol. 90:E5–E8, 2015.