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Supplementary Figure 5 from Down-regulation of CXCR4 and CD62L in Chronic Lymphocytic Leukemia Cells Is Triggered by B-Cell Receptor Ligation and Associated with Progressive Disease

Supplementary Figure 2 from Down-regulation of CXCR4 and CD62L in Chronic Lymphocytic Leukemia Cells Is Triggered by B-Cell Receptor Ligation and Associated with Progressive Disease

Supplementary Figure 1 from Down-regulation of CXCR4 and CD62L in Chronic Lymphocytic Leukemia Cells Is Triggered by B-Cell Receptor Ligation and Associated with Progressive Disease

Supplementary Table 1 from Down-regulation of CXCR4 and CD62L in Chronic Lymphocytic Leukemia Cells Is Triggered by B-Cell Receptor Ligation and Associated with Progressive Disease

Supplementary Figure 3 from Down-regulation of CXCR4 and CD62L in Chronic Lymphocytic Leukemia Cells Is Triggered by B-Cell Receptor Ligation and Associated with Progressive Disease

// Stephane Saint-Georges 1,2 , Maude Quettier 1,2 , Marouane Bouyaba 3 , Stephanie Le Coquil 1,2 , Vanessa Lauriente 1,2 , Lionel Guittat 1,2 , Vincent Levy 3 , Florence Ajchenbaum-Cymbalista 1,2,4 , Nadine Varin-Blank 1,2 , Christine Le Roy 1,2,* and Dominique Ledoux 1,2,* 1 INSERM U978, Bobigny, France 2 Universite Paris 13, Sorbonne Paris Cite, Labex “Inflamex”, Bobigny, France 3 Assistance Publique-Hopitaux de Paris, Hopital Avicenne, Unite de Recherche Clinique, Bobigny, France 4 Assistance Publique-Hopitaux de Paris, Hopital Avicenne, Service d’Hematologie Biologique, Bobigny, France * These authors are co-senior authors Correspondence to: Christine Le Roy, email: // Nadine Varin-Blank, email: // Keywords : CLL, lymphadenopathy, B-cell receptor, CXCR4/CXCR5, protein kinase D Received : December 23, 2015 Accepted : April 16, 2016 Published : April 26, 2016 Abstract In Chronic Lymphocytic Leukemia (CLL), infiltration of lymph nodes by leukemic cells is observed in patients with progressive disease and adverse outcome. We have previously demonstrated that B-cell receptor (BCR) engagement resulted in CXCR4 down-regulation in CLL cells, correlating with a shorter progression-free survival in patients. In this study, we show a simultaneous down-regulation of CXCR4, CXCR5 and CD62L upon BCR triggering. While concomitant CXCR4 and CXCR5 down-regulation involves PKDs, CD62L release relies on PKC activation. BCR engagement induces PI3K-δ-dependent phosphorylation of PKD2 and 3, which in turn phosphorylate CXCR4 Ser 324/325 . Moreover, upon BCR triggering, PKD phosphorylation levels correlate with the extent of membrane CXCR4 decrease. Inhibition of PKD activity restores membrane expression of CXCR4 and migration towards CXCL12 in BCR-responsive cells in vitro . In terms of pathophysiology, BCR-dependent CXCR4 down-regulation is observed in leukemic cells from patients with enlarged lymph nodes, irrespective of their IGHV mutational status. Taken together, our results demonstrate that PKD-mediated CXCR4 internalization induced by BCR engagement in B-CLL is associated with lymph node enlargement and suggest PKD as a potential druggable target for CLL therapeutics.

Several studies provide evidences for mantle cell lymphoma (MCL) cell survival relying on B-cell receptor (BCR)-mediated signalling pathways, whereas the nature of this activation is unknown. Significant progress in MCL treatment is achieved through therapies targeting BCR-associated kinases, i.e., Ibrutinib and Fostamatinib, inhibitors of BTK and SYK, respectively. Our study addresses survival signals emanating from the BCR or the tumour environment and how inhibiting BCR signalling effectors might impact these survival signals. We found that BTK was constitutively activated and that SYK phosphorylation was highly increased and sustained upon BCR activation of primary MCL cells. Moreover, MCL cells from leukaemic patients secreted high amount of IL-1β, IL-6, IL-8 and CCL5. Activation of the BCR induced (i) cell survival, (ii) STAT3 activation and (iii) increased autocrine secretion of IL-1β, IL-6, IL-8, CCL5, IL-10, TNFα and VEGF. Specific inhibition of BTK by Ibrutinib or SYK by Fostamatinib (R406) reversed these protective effects and decreased both basal and BCR-induced autocrine cytokine secretions associated with STAT3 phosphorylation. Interestingly, targeting BTK and SYK prevented and inhibited BCR-induced MCL cell adhesion to human bone marrow stromal cells (HMSCs) in short- and long-term co-culture. We demonstrated that BCR-induced survival relies on autocrine secretion of IL-1β, TNFα and CCL5 that might facilitate adhesion of MCL cells to HMSC. Treatment with Ibrutinib or Fostamatinib blocked the chemotactic signal thus increasing apoptosis.

Matrix metalloproteinases (MMPs) are thought to play an important role in skeletal muscle cell growth and differentiation. In view of the MMP inducing function of EMMPRIN/CD147, its role in myogenic cell differentiation was investigated. EMMPRIN level increased during differentiation of both rat primary myoblasts derived from satellite cells and mouse C2.7 myogenic cells and was associated with an alteration in its molecular forms. In parallel, expression of pro-MMP-9 gradually decreased and that of pro-MMP-2 and active MMP-2 increased. While small interfering RNA (siRNA) inhibition of EMMPRIN expression accelerated cell differentiation, exogenously added recombinant EMMPRIN inhibited differentiation by an MMP-mediated mechanism, as the MMP inhibitor marimastat abrogated EMMPRIN's effect. Our results further suggest that EMMPRIN regulates differentiation through an MMP activation of transforming growth factor beta (TGFβ), a known inhibitor of myoblast's differentiation, as the increased activation and signaling of TGFβ by EMMPRIN was attenuated in the presence of marimastat. EMMPRIN inhibition may thus represent a novel strategy in the treatment of muscular degenerative disorders. J. Cell. Physiol. 226: 141–149, 2010. © 2010 Wiley-Liss, Inc.

We recently demonstrated that stromal cell-derived factor-1 (SDF-1/CXCL12) forms complexes with CXCR4, but also with syndecan-4 expressed by human primary lymphocytes and macrophages, and HeLa cells. We also suggested that syndecan-4 behaves as a SDF-1-signaling molecule. Here, we demonstrate that SDF-1 strongly accelerates the shedding of syndecan-4 ectodomains and to a lesser extent that of syndecan-1 from HeLa cells. The fact that this acceleration was not inhibited by the CXCR4 antagonist AMD3100, anti-CXCR4 mAb 12G5, and CXCR4 gene silencing suggests its CXCR4-independence. Pre-treating the cells with heparitinases I, III, or with the protein kinase C (PKC) inhibitor, bisindolylmaleimide, significantly inhibited this accelerated shedding, which suggests the involvement of both cell-surface heparan sulfate and PKC transduction pathway. In contrast, Map Kinase or NF-kappaB pathway inhibitors had no effect. Moreover, SDF-1 increases the matrix metalloproteinase-9 (MMP-9) mRNA level as well as MMP-9 activity in HeLa cells, and MMP-9 silencing by RNA interference strongly decreases the syndecan-1 and -4 ectodomain shedding accelerated by SDF-1. Finally, SDF-1 also accelerates in a CXCR4-independent manner, the shedding of syndecan-1 and -4 from human primary macrophages, which is significantly inhibited by anti-MMP-9 antibodies. This strongly indicates the role of MMP-9 in these events occurring in both a tumoral cell line and in human primary macrophages. Because MMP-9 plays a crucial role in extracellular matrix degradation during cancer cell metastasis and invasion, and shed ectodomains of syndecans may likely be involved in tumor cell proliferation, these data further indicate the multiplicity of the roles played by SDF-1 on tumor cell biology.

Matrix metalloproteinases (MMPs) play an important role in cancer metastasis. Here, we investigated the effect of fibroblast growth factor-2 (FGF-2) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the secretion of type IV collagenases (MMP-2, MMP-9) in breast cancer MCF-7 cells. As shown by gelatin zymography, both FGF-2 and TPA stimulated the secretion of MMP-9 in MCF-7 cells while they did not change the level of MMP-2 secretion. Signaling cascade studies indicated that both FGF-2 and TPA induced Ras activation, c-Raf phosphorylation, mitogen-activated protein kinase/ERK kinase (MEK(1/2)) phosphorylation, and extracellular signal-regulated kinase (ERK(1/2)) phosphorylation. The FGF-2- and TPA-induced MMP-9 secretion was significantly inhibited by transient transfection of MCF-7 cells with dominant negative Ras (Ras-N17) and by treatment with MEK(1/2) inhibitor PD98059. A pan-protein kinase C (PKC) inhibitor, GF109203X, was found to totally abolish the FGF-2- and TPA-induced MMP-9 secretion and ERK(1/2) phosphorylation. Use of isoform-specific PKC inhibitors such as Rotllerin and Gö6976 suggested, moreover, that the PKC-delta isoform is a likely component of FGF-2 and TPA trophic signaling. These results demonstrated that FGF-2 and TPA induce MMP-9 secretion in MCF-7 cells mainly through PKC-dependent activation of the Ras/ERK(1/2) signaling pathway.

Several studies provide evidences for mantle cell lymphoma (MCL) cell survival relying on B-cell receptor (BCR)-mediated signalling pathways, whereas the nature of this activation is unknown. Significant progress in MCL treatment is achieved through therapies targeting BCR-associated kinases, i.e., Ibrutinib and Fostamatinib, inhibitors of BTK and SYK, respectively. Our study addresses survival signals emanating from the BCR or the tumour environment and how inhibiting BCR signalling effectors might impact these survival signals. We found that BTK was constitutively activated and that SYK phosphorylation was highly increased and sustained upon BCR activation of primary MCL cells. Moreover, MCL cells from leukaemic patients secreted high amount of IL-1β, IL-6, IL-8 and CCL5. Activation of the BCR induced (i) cell survival, (ii) STAT3 activation and (iii) increased autocrine secretion of IL-1β, IL-6, IL-8, CCL5, IL-10, TNFα and VEGF. Specific inhibition of BTK by Ibrutinib or SYK by Fostamatinib (R406) reversed these protective effects and decreased both basal and BCR-induced autocrine cytokine secretions associated with STAT3 phosphorylation. Interestingly, targeting BTK and SYK prevented and inhibited BCR-induced MCL cell adhesion to human bone marrow stromal cells (HMSCs) in short- and long-term co-culture. We demonstrated that BCR-induced survival relies on autocrine secretion of IL-1β, TNFα and CCL5 that might facilitate adhesion of MCL cells to HMSC. Treatment with Ibrutinib or Fostamatinib blocked the chemotactic signal thus increasing apoptosis.

Some synthetic dextran derivatives that mimic the action of heparin/heparan sulfate were shown to promote in vivo tissue repair when added alone to wounds. These biofunctional mimetics were therefore designated as “regenerating agents” in regard to their in vivo properties.In vitro, these biopolymers were able to protect various heparin-binding growth factors against proteolytic degradation as well as to inhibit the enzymatic activity of neutrophil elastase. In the present work, different dextran derivatives were tested for their capacity to inhibit the enzymatic activity of human plasmin. We show that dextran containing carboxymethyl, sulfate as well as benzylamide groups (RG1192 compound), was the most efficient inhibitor of plasmin amidolytic activity. The inhibition of plasmin by RG1192 can be classified as tight binding hyperbolic noncompetitive. One molecule of RG1192 bound 20 molecules of plasmin with a K i of 2.8 × 10− 8 m. Analysis with an optical biosensor confirmed the high affinity of RG1192 for plasmin and revealed that this polymer equally binds plasminogen with a similar affinity (K d = 3 × 10− 8 m). Competitive experiments carried out with 6-aminohexanoic acid and kringle proteolytic fragments identified the lysine-binding site domains of plasmin as the RG1192 binding sites. In addition, RG1192 blocked the generation of plasmin from Glu-plasminogen and inhibited the plasmin-mediated proteolysis of fibronectin and laminin. Data from the present in vitroinvestigation thus indicated that specific dextran derivatives can contribute to the regulation of plasmin activity by impeding the plasmin generation, as a result of their binding to plasminogen and also by directly affecting the catalytic activity of the enzyme.

In human breast cancer MCF-7 and MCF-7ras cells, we demonstrated that whereas insulin had a mitogenic effect on both cell lines, fibroblast growth factor-2 (FGF-2) had opposite effects, stimulating MCF-7 and inhibiting MCF-7ras cell proliferation. The inhibitory signal induced by FGF-2 was related to sustained mitogen-activated protein kinase (MAPK) activation in MCF-7ras cells, while transient MAPK activation was associated with MCF-7 cell proliferation. FGF-2 was further used in combination with insulin or cAMP. In MCF-7 cells, insulin and cAMP reversed the mitogenic effect of FGF-2. In MCF-7ras cells, insulin did not modify the inhibitory effect of FGF-2, but cAMP markedly enhanced it. These effects were also associated with an increased level and duration of MAPK activation. PD98056 abolished the effect of FGF-2 on DNA synthesis in both cell lines, demonstrating that the dual effect of FGF-2 on cell proliferation is dependent on the activity of the extracellular-signal-regulated kinase 1 and 2 (ERK1/2) signalling pathway.

Progressive cases of B-cell chronic lymphocytic leukemia (CLL) are frequently associated with lymphadenopathy, highlighting a critical role for signals emanating from the tumor environment in the accumulation of malignant B cells. We investigated on CLL cells from 30 untreated patients the consequence of B-cell receptor (BCR) triggering on the membrane expression of CXCR4 and CD62L, two surface molecules involved in trafficking and exit of B-lymphocytes from lymph nodes. BCR stimulation promoted a strictly simultaneous down-regulation of CXCR4 and CD62L membrane expression to a variable extent. The variable BCR-dependent decrease of the two proteins was strikingly representative of the heterogeneous capacity of the CLL cells to respond to BCR engagement in a given patient. Functionally, cells down-regulating CXCR4 and CD62L in response to BCR engagement displayed a reduction in both migration toward CXCL12 and adhesion to lymphatic endothelial cells. Remarkably, the ability of CLL cells to respond to BCR ligation was correlated with unfavorable prognostic markers and short progression-free survival. In conclusion, BCR signaling promotes decrease of CXCR4 and CD62L membrane expression in progressive cases only. These results are consistent with the hypothesis that BCR-mediated signaling pathways favor accumulation of a proliferative pool within the lymph nodes of progressive CLL cases. [Cancer Res 2009;69(16):6387–95]

Bisphosphonates are extensively used in the treatment of patients with metastasis-induced osteolysis. The major drawback in the efficacy of all bisphosphonates lies in their high hydrophilic nature, which results in poor membrane permeability and low availability for soft tissues. A reasonable approach to overcome these problems consists in masking one or more ionizable groups of bisphosphonates, notably by esterification of the hydroxyl functions. We have previously shown that the novel non-nitrogen-containing bisphosphonate BP7033 inhibited angiogenesis and growth of primary tumors in nude mice. The present study focuses on the dimethyl-esterified analog of this compound (Me-BP7033). In-vitro, Me-BP7033 inhibited proliferation of human carcinoma A431 cells as well as their invasive activity based on a transwell invasion assay. in-vivo, administration of Me-BP7033 (0.3 mg/kg) twice a week for 5 weeks inhibited the tumor growth of A431 cells xenografted in nude mice by 65%. Immunostaining of endothelial cells (ECs) in tumor sections revealed that Me-BP7033 inhibited the intratumor ECs density by 60%. The in-vivo anti-angiogenic properties of Me-BP7033 were also demonstrated in an in-vivo angiogenesis assay showing that Me-BP7033 reduced the vascular endothelial growth factor-stimulated infiltration of ECs in a Matrigel plug by 70%. In summary, we demonstrated for the first time that a diesterified bisphosphonate exhibited in vivo both anti-tumoral and anti-angiogenic activities with no apparent sign of toxic effects. These new diesterified compounds, which could display enhanced bioavailability and pharmacokinetics, thus represent interesting candidates for therapeutic applications such as cancer treatment.

We previously reported a simple and efficient one-pot procedure for synthesis of 1-hydroxymethylene-1,1-bisphosphonic acids (HMBP). According to this method, we synthesized a series of new aromatic HMBP and investigated structure-activity relationships by evaluating their anti-proliferative activity against A431 human tumor cell line. Our results showed that the introduction of an extra methylene group in a pyridyl-containing R2 side chain increased 100-fold the anti-proliferative activity of the HMBP. In contrast, this chemical modification did not modify the anti-proliferative activity of compounds substituted with a phenyl-containing R2 side chain. Para-substitution of the phenyl ring with various groups markedly influenced the HMBP activity, the order of potency (brominechlorinefluorine = none) closely matching the atomic volume of the substituted group. Moreover, changes in the substitution position of the bromine group also affected the anti-proliferative activity, the more potent activity being obtained with para-substitution of the phenyl ring. In conclusion, this structure-activity study led us to identify the new aromatic HMBP [(4-Bromo-phenyl)-hydroxy-phosphono-methyl]-phosphonic acid as a potent in vitro anti-proliferative molecule against tumor cell lines (IC50 value of 9.5 x 10(-5) M). Interestingly, this compound can be further easily esterified on its phosphonic acid functions according to our chemical method and, thus, represents a potential candidate for the development of new esterified HMBP with enhanced pharmacokinetics.

Bisphosphonates (BP) are powerful inhibitors of bone resorption and are widely used in the treatment of patients with metastasis-induced osteolysis. In the present study, we show that a novel non-nitrogen-containing BP (BP7033) that exhibits antitumor activity is a potent inhibitor of both in vivo and in vitro angiogenesis. When administered to mice, BP7033 inhibited tumoral angiogenesis (65% at 0.06mg/injection) as well as tumor growth (65% at 0.006mg/injection) in a tumor model of A431 cells xenografted in nude mice, with no sign of toxicity. Additionally, in vivo angiogenesis induced by vascular endothelial growth factor-containing Matrigel implants was reduced by 90% in the presence of BP7033 (0.6mg/plug). In vitro, BP7033 inhibited proliferation of human umbilical vein endothelial cells (HUVEC) (IC(50) value 3x10(-4) M) and completely prevented the formation of capillary-like tubules by HUVEC in Matrigel. Moreover, treatment of A431 cells by BP7033 induced an inhibition of Ras processing and a decrease in the secretion of both vascular endothelial growth factor and matrix metalloproteinase-2, two well-known stimulators of the proliferation and migration of endothelial cells. These findings indicate that this new BP compound has marked antiangiogenic properties and thus represents a promising candidate for treatment of malignant diseases with an angiogenic component.

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The heparin affin regulatory peptide (HARP) growth factor, also known as pleiotrophin, is a developmentally regulated protein that displays biological functions during cell growth and differentiation. To study the physiological role of this protein, we investigated the cellular distribution of HARP mRNA and protein in the resting human mammary gland. In situ hybridization histochemistry revealed that HARP mRNA was localized in alveolar myoepithelial cells, whereas alveolar epithelial cells were negative. In the stroma, HARP mRNA was localized in endothelial cells and smooth muscle cells of blood vessels. Interestingly, HARP protein and mRNA were not always co-localized. HARP protein immunocytochemistry staining was observed in an area including both alveolar myoepithelial and epithelial cells, although epithelial cells do not express HARP transcript. In contrast, the distribution of HARP protein is parallel to that of HARP mRNA in endothelial and vascular smooth muscle cells. In the light of these results, the putative role of HARP in controlling the proliferation and/or differentiation of the different mammary cell types is proposed and discussed.

We previously reported that B-cell receptor (BCR) signaling promoted a concomitant decrease of CXCR4 and CD62L membrane expression only in progressive cases of chronic lymphocytic leukemia (CLL). Recently, Quiroga and colleagues raised some challenging issues and pointed out discrepancies between the two reports. In their letter, Quiroga and colleagues questioned our BCR stimulation protocol. Several studies, including ours, reported that soluble anti-immunoglobulin M (IgM) resulted in apoptosis, whereas immobilized anti-IgM or F(ab9)2 fragment both sustained cell survival (1–3). Hence, the increased cell survival following soluble F(ab9)2 exposure observed by Quiroga and colleagues is rather surprising. We showed that downregulation of CXCR4 was associated with a reduced migration toward CXCL12. Our results are in agreement with numerous reports in various cell types including CLL cells (4). Quiroga and colleagues reported a similar CXCR4 decrease but associated to an unexplained increased chemotaxis toward CXCL12. They relied on data from normal B cells isolated from the bone marrow, hypothesizing that several CXCR4 variants confer differential responsiveness to CXCL12. We considered studying BCR stimulation in the context of lymph nodes to be more relevant: (i) BCR stimulation occurs mainly in lymph nodes; (ii) a higher score of BCR-activated targets in lymph nodes versus bone marrow was recently found (5). Therefore, using a lymph node endothelial cell line in our adhesion assays seems a more physiological model than a bone marrow stromal cell line. Quiroga and colleagues reported a decrease of CXCR4 associated with an increase of CD62L upon BCR engagement. In contrast, we found a systematically coordinated downregulation of both CXCR4 and CD62L occurring in the same subset of cells. We thus believe that our experimental data, done on a delineated subset of cells of 30 untreated patients, are stronger than the results expressed as the mean value of a whole cohort as provided by Quiroga and colleagues. Finally, their inhibition experiment of Syk, the major BCR effector, restores CXCR4 and CD62L levels of expression. This result is in line with our data and is in agreement with the clinical observation on patients treated by the Syk inhibitor R788 showing an initial increase in circulating lymphocytes associated to a decrease in lymph node size, indicative of an exit of CLL cells from the lymph nodes. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.

We have examined the effect of human basic fibroblast growth factor (bFGF) on the proliferation of human neuroblastoma cells with normal and enhanced MYCN oncogene expression. bFGF stimulated the proliferation of the neuroblastoma cells with enhanced, but not normal, MYCN expression. Both cell species express FGFR-1, but not FGFR-2, receptors and both harbor FGF receptor species of Mr 145.000, but they differ in their pattern of lower and higher-molecular weight FGF receptor species. Our results demonstrate that enhanced MYCN expression confers to neuroblastoma cells the ability to respond to bFGF, possibly by inducing functional FGF receptors. This mechanism may contribute to the advanced malignant phenotype of human neuroblastomas with enhanced MYCN expression.

Fibroblast growth factor 2 (FGF2) has been shown to be an autocrine growth factor in human breast epithelial cells HBL100. Here we studied the effects of one dextran derivative (CMDB7) on this autocrine loop. CMDB7 caused a dose-dependent decrease of HBL100 growth in serum-free medium. [3H]thymidine uptake in HBL100 cells and Balbc/3T3 cells by exogenous FGF2 was inhibited by CMDB7. Receptor binding assays with radio-iodinated FGF2, IGF1, EGF showed that CMDB7 only displaced the binding of125I-FGF2 in a dose dependent manner. Scatchard analysis revealed that the presence of CMDB7 reduced 78% and 82 % FGF2 binding capacity to its high and low affinity receptors respectively without altering the affinites of binding sites. These results suggest that CMDB7 exert its antiproliferative action on HBL100 cells by interfering with FGF2 autocrine loop.

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 ( 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. Disclosures Levy: Roche: Honoraria; Janssen: Honoraria; Gilead: Honoraria. Cymbalista:Janssen: Honoraria, Research Funding; Gilead: Honoraria; Roche: Honoraria; Karyopharm: Honoraria.