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Will the models soon help managers optimize their field intervention strategies to curb the Asian knotweed invasion? This is the challenge that Irstea researchers are taking up by proposing a new modelling tool that will eventually make it possible to describe the growth dynamics of a knotweed massif subjected to different mowing frequencies.; Les modèles permettront-ils prochainement d'aider les gestionnaires à optimiser leurs stratégies d'intervention sur le terrain pour freiner l'invasion par les renouées asiatiques ? C'est le pari que font les chercheurs d'Irstea en proposant un nouvel outil de modélisation qui permettra à terme de décrire la dynamique de croissance d'un massif de renouées soumis à différentes fréquences de fauche.

Ibrutinib (PCI-32765) is a highly potent oral Bruton tyrosine kinase (BTK) inhibitor in clinical development for treating B-cell lymphoproliferative diseases. Patients with chronic lymphocytic leukemia (CLL) often show marked, transient increases of circulating CLL cells following ibrutinib treatments, as seen with other inhibitors of the B-cell receptor (BCR) pathway. In a phase 1 study of ibrutinib, we noted similar effects in patients with mantle cell lymphoma (MCL). Here, we characterize the patterns and phenotypes of cells mobilized among patients with MCL and further investigate the mechanism of this effect. Peripheral blood CD19(+)CD5(+) cells from MCL patients were found to have significant reduction in the expression of CXCR4, CD38, and Ki67 after 7 days of treatment. In addition, plasma chemokines such as CCL22, CCL4, and CXCL13 were reduced 40% to 60% after treatment. Mechanistically, ibrutinib inhibited BCR- and chemokine-mediated adhesion and chemotaxis of MCL cell lines and dose-dependently inhibited BCR, stromal cell, and CXCL12/CXCL13 stimulations of pBTK, pPLCγ2, pERK, or pAKT. Importantly, ibrutinib inhibited migration of MCL cells beneath stromal cells in coculture. We propose that BTK is essential for the homing of MCL cells into lymphoid tissues, and its inhibition results in an egress of malignant cells into peripheral blood. This trial was registered at www.clinicaltrials.gov as #NCT00114738.

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib and the phosphatidylinositol 3-kinase δ (PI3Kδ) inhibitor idelalisib show promising clinical efficacy in the treatment of Waldenstrom macroglobulinemia (WM), a lymphoplasmacytic lymphoma.[1][1]–[3][2] Very recently, ibrutinib became the first

To the editor: Most B-cell malignancies are dependent upon signaling by the B-cell receptor (BCR) and other growth and survival signals provided by the tumor microenvironment. Two recently US Food and Drug Administration–approved drugs that target the BCR signalosome, the Bruton tyrosine kinase (

Personalized medicine requires the identification of unambiguous prognostic and predictive biomarkers to inform therapeutic decisions. Within this context, the management of lymph node-negative breast cancer is the subject of much debate with particular emphasis on the requirement for adjuvant chemotherapy. The identification of prognostic and predictive biomarkers in this group of patients is crucial. Here, we demonstrate by tissue microarray and automated image analysis that the cocaine- and amphetamine-regulated transcript (CART) is expressed in primary and metastatic breast cancer and is an independent poor prognostic factor in estrogen receptor (ER)-positive, lymph node-negative tumors in two separate breast cancer cohorts (n = 690; P = 0.002, 0.013). We also show that CART increases the transcriptional activity of ERα in a ligand-independent manner via the mitogen-activated protein kinase pathway and that CART stimulates an autocrine/paracrine loop within tumor cells to amplify the CART signal. Additionally, we demonstrate that CART expression in ER-positive breast cancer cell lines protects against tamoxifen-mediated cell death and that high CART expression predicts disease outcome in tamoxifen-treated patients in vivo in three independent breast cancer cohorts. We believe that CART profiling will help facilitate stratification of lymph node-negative breast cancer patients into high- and low-risk categories and allow for the personalization of therapy.

BACKGROUND: Multiple myeloma is a hematologic malignancy characterized by a clonal expansion of malignant plasma cells in the bone marrow, which is accompanied by the development of osteolytic lesions and/or diffuse osteopenia. The intricate bi-directional interaction with the bone marrow microenvironment plays a critical role in sustaining the growth and survival of myeloma cells during tumor progression. Identification and functional analysis of the (adhesion) molecules involved in this interaction will provide important insights into the pathogenesis of multiple myeloma.DESIGN AND METHODS: Multiple myeloma cell lines and patients' samples were analyzed for expression of the adhesion molecule N-cadherin by immunoblotting, flow cytometry, immunofluorescence microscopy, immunohistochemistry and expression microarray. In addition, by means of blocking antibodies and inducible RNA interference we studied the functional consequence of N-cadherin expression for the myeloma cells, by analysis of adhesion, migration and growth, and for the bone marrow microenvironment, by analysis of osteogenic differentiation.RESULTS: The malignant plasma cells in approximately half of the multiple myeloma patients, belonging to specific genetic subgroups, aberrantly expressed the homophilic adhesion molecule N-cad-herin. N-cadherin-mediated cell-substrate or homotypic cell-cell adhesion did not contribute to myeloma cell growth in vitro. However, N-cadherin directly mediated the bone marrow localization/retention of myeloma cells in vivo, and facilitated a close interaction between myeloma cells and N-cadherin-positive osteoblasts. Furthermore, this N-cadherin-mediated interaction contributed to the ability of myeloma cells to inhibit osteoblastogenesis.CONCLUSIONS: Taken together, our data show that myeloma cells frequently display aberrant expression of N-cadherin and that N-cadherin mediates the interaction of myeloma cells with the bone marrow microenvironment, in particular the osteoblasts. This N-cadherin-mediated interaction inhibits osteoblast differentiation and may play an important role in the pathogenesis of myeloma bone disease.

Fourier-transform pulsed-field-gradient NMR measurements have been used to analyse the diffusion of some n-alkanes (methane, n-butane and n-pentane) in zeolite ZSM-5. The intention of the NMR study was to compare results obtained by molecular dynamics calculations and by uptake measurements, using the same systems. Methane clearly exhibits a bi-exponential spin-echo attenuation. This indicates two types of diffusion: intracrystalline diffusion and long-range diffusion, which is a combination of inter- and intra-crystalline diffusion. In the case of small crystals the diffusion of methane into the macropores between the crystals dominates the decay. However, as expected, for larger crystals, the contribution of intracrystalline (or micropore) diffusion increases significantly. From the curves, the coefficient of intracrystalline diffusion of methane in ZSM-5 has been determined (3.8 × 10–9 m2 s–1 at 25 °C). The NMR methane diffusion data are in good agreement with values obtained by molecular dynamics calculations. Subsequent NMR measurements of n-butane and n-pentane diffusion in ZSM-5 indicate that the diffusion decreases sharply with increasing chain length of the hydrocarbons (11 × 10–11 and 4.4 × 10–11 m2 s–1, respectively, at 25 °C and 20 kPa loading). To allow for a comparison with the diffusivities obtained independently by other techniques, the concentration dependence of the NMR self-diffusion coefficient of n-pentane in ZSM-5 was determined and was found to decrease with increasing sorbate concentration. In addition, from the temperature dependence of the diffusion rates the activation energies for the n-butane and n-pentane diffusion in ZSM-5 have been determined (8.4 and 12.6 kJ mol–1 at 20 kPa, respectively). The PFG NMR and MD results for the diffusion of light n-alkanes in ZSM-5 have also been compared with relevant diffusion data from the literature (obtained using other techniques, i.e. uptake methods, ZLC, MT). The microscopic self-diffusivity (from PFG NMR and MD) differs systematically by ca. two orders of magnitude from the much slower, macroscopic diffusion observed by uptake, ZLC and MT methods. On the other hand, there is satisfactory agreement between the self-diffusivity of n-butane obtained with PFG NMR and the transport diffusivity of the same system measured using the frequency response method.

CLL progression and chemoresistance can result from signals from the lymph node (LN) microenvironment and from acquired aberrations in the DNA damage repair (DDR) pathway. Clinical targeting of kinases upstream in the B cell receptor (BCR) activation pathway, such as Btk or PI3Kδ results in egress of cells from the LN microenvironment [1,2]. Such prolonged lymphocytosis during kinase-inhibitor treatment appears to pose no clinical disadvantage [3]. However, it enhances the chance of accumulating resistance-inducing mutations, and therefore drugs that combine LN egress with direct cytoxicity could provide an improved therapeutic strategy for CLL. The mTOR complex, consisting of mTOR1 and 2, is the main downstream kinase of the PI3K/Akt pathway and contributes to proliferation and survival. DNA-PK is a kinase required for non-homologous end joining (NHEJ) of the DNA repair pathway. Inhibitors of crucial components of the DDR pathway might be active in CLL, especially in patients harboring mutations in DNA repair molecules such as Ataxia telangiectasia mutated (ATM). In this study the potency of a novel dual mTOR1,2 and DNA-PK inhibitor (CC-115) was studied in primary CLL samples of different prognostic subgroups with respect to induction of cytotoxicity, and inhibition of adhesion, CD40-mediated chemoresistance and proliferation. In vitro, CC-115 inhibited mTOR1 and 2 and also affected the DDR reflected by inhibition of irradiation-induced γH2AX, not only in ATM-mutated but also in ATM-wild type CLL cells. CC-115 showed induction of caspase-dependent cell killing (IC50=0.625µM) which was more robust than selective kinase inhibitors (table 1), irrespective of p53 or ATM status. This cytotoxic effect was not observed in the T cells from CLL patients. BCR-mediated adhesion to fibronectin [4] was inhibited by CC-115 to a similar extent as PI3Kδ inhibitor (idelalisib) (table 1). CD40-mediated chemoresistance [5] could be reverted completely by CC-115 while more specific inhibitors had only modest effects. CLL proliferation induced by CD40L+IL-21 treatment [6] was completely blocked by both CC-115 and a dual mTOR1,2 inhibitor but not by inhibitor of the more upstream kinase PI3Kδ (table 1). In conclusion, these data show that CC-115 induces direct cytotoxicity and inhibits several clinically relevant biological features of CLL, and provide a rationale for clinical trials with CC-115 in CLL patients. [1] Hoellenriegel J, Meadows SA, Sivina M et al. The phospoinositide 3’-kinase delta inhibitor, CAL-101, inhibits B-cell receptor signaling and chemokine networks in chronic lymphocytic leukemia. Blood 2011;(118):3603-3612 [2] Burger JA. Bruton’s Tyrosine Kinase (BTK) Inhibitors in Clinical Trials. Curr Hematol Malig Rep (2014) 9:44–49 [3] Herman SE, et al. Ibrutinib-induced lymphocytosis in patients with chronic lymphocytic leukemia: correlative analyses from a phase II study. Leukemia. 2014 Apr 4. doi: 10.1038/leu.2014.122. [Epub ahead of print] [4] de Rooij MF, Kuil A, Geest CR et al. The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia. Blood 2012;(119):2590-2594. [5] Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocitic leukemia cells correlates with survival capacity. Blood 2007;(109):1660-1668. [6] Pascutti MF, Jak M, Tromp JM et al. IL-21 and CD40L signals from autologous T cells can induce antigen-independent proliferation of CLL cells. Blood 2013;(122):3010-3019.Smit LA, Hallaert DY, Spijker R et al. Table 1. The effect of the mTOR1,2 + DNA-PK inhibitor (CC-115), mTOR1,2 inhibitor (CC-214), DNA-PK inhibitor (NU7441) and PI3Kδ inhibitor (idelalisib) on CLL cells in functional assays. CC-115 CC-214 NU7441 Idelalisib (CAL-101) Target mTOR1,2 DNA-PK mTOR1,2 DNA-PK PI3Kδ Cytotoxicity (IC50) 0.625 µ M >10µM >10µM >10µM Inhibition of adhesion 40%** 0% 22% 48%** Activation Inhibition of CD40-mediated resistance to fludarabine (6.25 µ M) 92%** 42%* n.d. 21% Inhibition of CD40L+IL21 induced proliferation 98%** 98%* 28% 51%** n.d. = not done The one sample T test was used to determine the significance of differences between means of treated samples and normalized values of untreated samples (100%). * p Disclosures Kersten: Celgene: Research Funding. Kater:Celgene: Research Funding.

CLL cells are highly dependent on B- cell receptor (BCR) signaling and on stimuli from the microenvironment for survival and proliferation. New drugs targeting PI3K downstream of BCR signaling have emerged as promising treatment options for patients with CLL. Among four PI3K catalytic subunits, the PI3Kd isoform is crucial for downstream BCR signaling, but the relative importance of the PI3Kα isoform in CLL is less clear. Impressive clinical activity of idelalisib in CLL and indolent NHL patients was recently reported. Idelalisib, a PI3Kd specific inhibitor, inhibits chemotaxis and adhesion of leukemia cells, resulting in rapid lymphocytosis followed by a decrease in lymphadenopathy. However, idelalisib has no direct impact on leukemic cell survival [1], raising the potential risk of residual clones responsible for the development of resistance. In this study, we evaluated the impact of a pan-class I PI3K inhibitor (SAR245409/XL765), a PI3Kα-specific inhibitor (BYL719) and a PI3Kd specific inhibitor (idelalisib) on PI3K/mTOR signaling, apoptosis, cell adhesion, CD40-induced survival and proliferation in primary patient derived leukemic cells. Phosphorylation of the downstream effector of mTOR, S6RP, was completely blocked by SAR245409 but not by BYL719 or idelalisib. SAR245409 induced apoptosis in unstimulated CLL cells (IC50= 0.86µM) in contrast to BYL719 or idelalisib (IC50 >10µM), demonstrating that targeting multiple PI3K isoforms is required to completely block the PI3K/Akt/mTOR pathway (table 1). Importantly, SAR245409 also induced apoptosis in p53 or ATM dysfunctional CLL samples. SAR245409, as well as idelalisib, and in contrast to BYL719 completely inhibited BCR-mediated adhesion to fibronectin [2]. Similarly, SAR245409 inhibited CD40L-mediated survival [3], and induced upregulation of the pro-apoptotic protein BIM. All 3 PI3K inhibitors inhibited CD40 ligation + IL-21-mediated CLL proliferation [4]. This study revealed that the pan-class I PI3K inhibitor SAR245409 is more cytotoxic to primary CLL cells than PI3Kα or PI3Kd specific inhibitors. Furthermore, combined inhibition of PI3Kα and d can block signaling pathways that are critical for CLL survival, adhesion and proliferation in the LN microenvironment (see table 1). This work provides a rationale for the evaluation of SAR245409 in CLL patients either as monotherapy or in combination therapies. [1] Hoellenriegel et al. The phospoinositide 3'-kinase delta inhibitor, CAL-101, inhibits B-cell receptor signaling and chemokine networks in chronic lymphocytic leukemia. Blood 2011;(118):3603-3612 [2] de Rooij et al. The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia. Blood 2012;(119):2590-2594. [3] Smit et al. Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocitic leukemia cells correlates with survival capacity. Blood 2007;(109):1660-1668. [4] Pascutti et al. IL-21 and CD40L signals from autologous T cells can induce antigen-independent proliferation of CLL cells. Blood 2013;(122):3010-3019. Table 1. The effect of the PI3Kd inhibitor idelalisib, PI3Kα inhibitor BYL719 or pan PI3K inhibitor SAR245409 on CLL cells in functional assays PI3Kd inhibitor PI3Kα inhibitor pan PI3K inhibitor Cytotoxicity (IC50)1 >10µM >10µM 0.86µM Inhibition of adhesion2 48%** 21% 43%** Activation Inhibition of CD40L-induced survival3 14% 0% 54%* Inhibition of CD40L+IL21 induced proliferation4 47%* 35%* 51%* 1 CLL cells were incubated with 0.001-10 μM idelalisib (n=18), BYL719 (n=6) or SAR245409 (n=28) for 48 hours. Viability was assessed by DiOC6/PI staining.2 CLLcells pretreated with 1 µM idelalisib, BYL719, or SAR245409 were stimulated with α-IgM and allowed to adhere to fibronectin-coated surfaces (n=5). 3 CLL cells were cultured on fibroblast expressing CD40L in the absence or presence of 1 µM of idelalisib, BYL719, or SAR245409 for 3 days. Apoptosis was assessed by DiOC6/PI staining (n=8).4 CFSE labelledCLL cells were cultured on fibroblast expressing CD40L with IL-21 and co-treated with 1 µM idelalisib, BYL719, or SAR245409. After 4 days, CFSE was measured by FACS (n=11)2-4 The one sample T test was used to determine the significance of differences between means of treated samples and normalized values of untreated samples (100%). * p Disclosures Egile: Sanofi: Employment. Kersten:Sanofi: Research Funding. Kater:Sanofi: Research Funding.

International audience; A rapid and simple microwave-assisted digestion method with alkaline solution (tetramethylammonium hydroxide or methanolic KOH solution) for speciation analysis of inorganic mercury and methylmercury (MeHg+) in biological tissues was developed. Extracts with quantitative recoveries of mercury species after microwave-assisted alkaline dissolution of the sample were directly analysed by an automated on-line hyphenated system incorporating aqueous hydride generation, cryogenic trapping, gas chromatography and detection by atomic absorption spectrometry. Optimum conditions allow sample throughput to be controlled by the instrumental analysis time (?10 min per sample) and not by the sample preparation step. At a power of 40-60 W, sample preparation time is only 2-4 min, which is much faster than previous literature methods. The proposed method was validated by the analysis of three biological certified reference materials, CRM 463, DORM-1 and TORT-1, and one BCR sample from an interlaboratory study, Tuna Fish 2. The detection limit of the overall procedure was found to be 3 ng per gram of pulverised dried biological tissue, for both labile Hg2+ and MeHg+. After alkaline extraction, a mean recovery of 102% with a relative standard deviation of 7% was obtained for methylmercury concentrations ranging from 0.128 to 3.464 ?g g-1 as Hg in the four reference fish tissues mentioned above. Total mercury, calculated as the sum of determined inorganic mercury and methylmercury, was also in agreement with the certified values. The methylmercury extraction efficiency of five extractants, viz., nitric, hydrochloric and acetic acid, and tetramethylammonium hydroxide and methanolic KOH solution, the temperature profile of these extractants under microwave irradiation and the behaviour and stability of methylmercury in a focused microwave field were also investigated.

Abstract 3900 In the lymph node (LN) microenvironment, chronic lymphocytic leukemia (CLL) cells are protected from apoptosis by upregulation of anti-apoptotic proteins. In vitro, this can be mimicked via CD40-stimulation of CLL cells, which also provides resistance to various chemotherapeutics. Novel drugs that target kinases involved in B cell signalling, including the broad spectrum kinase inhibitor dasatinib, are currently in clinical development for CLL. We have shown previously that dasatinib prevents CD40-mediated anti-apoptotic changes in CLL (Hallaert et Blood 2008). However, the kinase(s) involved remain unidentified. Here, we coupled dasatinib to an affinity matrix and pulled down its targets from CD40-stimulated CLL cells. By mass-spectrometry and Western blotting, Abl and Btk were identified as dominant targets of dasatinib. Functional analysis revealed that CD40-mediated anti-apoptotic signals and drug-resistance could be overcome both by dasatinib and the Abl inhibitor imatinib, but not by the novel Btk inhibitor PCI-32765 (ibrutinib), whereas BCR- and chemokine-controlled adhesion could be abolished by dasatinib and ibrutinib, but not by imatinib. Thus, dasatinib combines two key aspects that are clinically relevant: inhibition of Abl overrides chemoprotective survival signals, whereas inhibition of Btk impairs integrin-mediated adhesion of CLL cells in the microenvironmental niche. This combined inhibition of Abl and Btk was put to an initial test in an open-label phase 2 trial of dasatinib combined with fludarabine in twenty refractory CLL patients. As might be expected based on the in vitro data, reductions in lymph node size were observed in most patients. A LN reduction of ≥20% provided a significant improved PFS (256 days) and OS (510 days) as compared to non-responders (80 days and 158 days respectively). Details of the clinical study will be presented separately. In conclusion, in agreement with in vitro molecular studies, dasatinib seems to have clinical efficacy in heavily pretreated refractory CLL patients. Combined, these data encourage further studies on a broad-spectrum kinase inhibitor like dasatinib in combination with other classes of drugs in relapsed and refractory CLL. Disclosures: No relevant conflicts of interest to declare.