Academic literature on the topic 'Calissons'

Abstract Abstract 184 Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature B cells in peripheral blood (PB), bone marrow (BM) and lymph node (LN). Recent studies identified the LN as an important site of tumor cell activation and proliferation (Herishanu et al 2011). Using in vivo labeling of “newly-born” cells with deuterated water (2H2O; heavy water), the proliferation rate of CLL cells was estimated to range between 0.1 to >1% of the clone per day (Messmer et al 2005). Furthermore, a CXCR4dimCD5bright population of CLL cells in the PB contained more deuterium (2H)-labeled DNA and hence “recently-born cells than the CXCR4brightCD5dim population (Calissano et al 2011). Possible explanations for this observation include that CXCR4dimCD5bright cells proliferate more rapidly in the PB or that these cells are recent emigrants from tissues where proliferation occurs. As prior studies were performed on PB cells, the growth rates and characterization of the proliferative fraction of CLL cells in the LN remain unknown. Here we used 2H-labelling to directly compare cellular growth rates in PB, BM, and LN. Patients drank 2H2O for 28 days; on day 13 an excisional LN biopsy and a BM aspirate were obtained. PB samples were obtained at baseline and on days 13 and 28. CLL cells were isolated using positive selection, or sorted based on reciprocal differences in CXCR4 and CD5 density for isolation of “proliferative” (CXCR4dimCD5bright), “intermediate” (CXCR4intCD5int), and “resting” (CXCR4brightCD5dim) fractions of CLL clones. 2H incorporation into the DNA of newly divided cells was measured by mass spectrometry. Raw values were normalized to the 2H2O content in total body water. Cellular growth rates were calculated by dividing the fraction of 2H-labeled cells by the number of days from the start of the labeling period. To date, samples from 6 treatment-naïve CLL patients have been analyzed. On day 13, up to 16%, 9%, and 24% of the CLL cells sampled from PB, BM, and LN, respectively were 2H-labeled. The resulting mean estimated growth rate in % of the clone per day was for PB 0.41 (0.09 – 1.13), for BM 0.41 (0.28 – 0.68), and for LN 0.83 (0.31 – 1.84). The difference in growth rates between PB and LN was statistically significant (P<.02) and on average 2.5 times higher in the LN than in the PB. On day 28, the total fraction of 2H-labeled PB cells had further increased with the calculated growth rate in agreement with the growth rate in PB on day 13. CLL cells in the BM had a mean growth rate of 0.41% (0.28 – 0.68) of the clone per day, which was not significantly different than the growth rate in the PB. In fact in 2 patients the growth rate in the BM was lower than in the PB. The growth rates determined in LN and PB on day 13 were inversely correlated to the lymphocyte doubling time (r=-0.65 by Pearson correlation) and tended to be higher in patients with ZAP70 positive CLL. In keeping with the growth rates measured by 2H labeling, the fraction of Ki67-expressing CLL cells was higher in the LN than in the PB [% cells mean ± SD (PB = 3.8 ± 1.6, LN = 18.4 ± 3.1; P=.005)]. Interestingly, while the average CLL growth rate in LNs by 2H-labeling was 2.5-times the rate in PB, the Ki-67 positive fraction in LNs was 5-times the fraction in the PB, further supporting the view that active clonal proliferation occurs predominantly in the LN, from which recently-born cells enter the PB. Consistent with a prior study of PB CLL cells (Calissano et al 2011), the CXCR4dimCD5bright subset in LN exhibited higher 2H-labeling than the bulk of the clonal cells (intermediate fraction) and the resting fraction. These studies indicate that a CXCR4dimCD5bright subset exists in LN-resident CLL cells and has higher 2H-labeling than the rest of the clone. Specifically, the calculated growth rate of the CXCR4dimCD5bright subset was on average 3.2-times the growth rate of all CLL cells in the LN. Moreover, the data suggest that sampling the PB for newly-born cells is a reliable measure of the degree of proliferation occurring in LNs. Taken together our data show that the proliferation rate of CLL cells is higher in the LN than in the BM and PB and suggest that some of the newborn cells exit the LN within days. A clonal subset of CXCR4dimCD5bright cells is present in both the LN and PB and might harbor the proliferative core of the disease. Disclosures: Gatmaitan: KineMed: Employment. Emson:KineMed: Employment. Chiorazzi:KineMed: Dr. Chiorazzi holds stock options in KineMed, Inc. Other.

Abstract Introduction: Chronic Lymphocytic Leukemia (CLL) is characterized by an accumulation of CD19+ CD5+ CLL cells in the peripheral blood and the lymphoid compartments. The disease is still incurable despite recent advances in the development of novel therapy concepts (e.g. inhibitors of BCR signaling). Although it is not entirely clear how CLL develops, proliferation centers located in the lymph node and the bone marrow are thought to be the crucial niche that provides the surrounding in which CLL cells originate and re-establish the clone after therapy, leading to relapse in almost 100% of CLL patients. In analogous normal lymphoproliferative environments the transcription factor IRF4 plays an important role in controlling B cell maturation, differentiation, proliferation and survival. A SNP in the human IRF4 3’UTR was linked to CLL susceptibility (DiBernardo Nat.Gen. 2008) and in the mouse model germline deletion of IRF4 in all lineages contributed to CLL development in the New Zealand Black and in the VH11 mouse model (Shukla Blood 2013, Ma J.Biol.Chem. 2013). Methods: CLL samples were collected from the peripheral blood of CLL patients according to the Declaration of Helsinki and with written informed consent. IRF4 mRNA expression analyses were performed in purified CD19+ CLL cells using TaqMan Real Time PCR. In vitro studies were performed in soloculture without stimulation or in coculture systems mimicking the lymph node microenvironment, as previously described (Asslaber Br.J.Haematol. 2013). IRF4 knockdown was performed using lipofection and IRF4 specific siRNAs or non-targeting control siRNAs. IRF4 protein expression was measured by intracellular flow cytometry. Proliferation was detected by Ki-67 intracellular staining. All intracellular stainings were performed after gating on viable CD19+ CD5+ CLL cells. Cell death was assessed by Annexin V / 7AAD staining or using a fixable viability dye. Results: Overall, IRF4 mRNA expression was decreased in CLL patients (N=101) when compared to healthy donors (N=8, P=0.04). In the CLL cohort low IRF4 mRNA expression (cut-off determined by ROC analysis and Youden Index calculation) was significantly correlated with earlier time to treatment (median treatment-free survival: 40 month in the IRF4LOW and 69 month in the IRF4HIGH CLL patient group (P<0.01)). This was proved to be independent of mutation state and other CLL risk factors in multivariate analysis. Using in vitro knockdown of IRF4 in solo- and coculture models we found that lowered expression of IRF4 was associated with increased CLL cell survival and abrogated spontaneous apoptosis (N=11, P<0.01). Moreover, we detected a failure of CLL cells to respond to CD40L signals derived from the microenvironment after IRF4 knockdown, which was associated with abrogated proliferation (N=9, P<0.01). Indeed microenvironmental stimuli (e.g. CD40L ligation or addition of activated T cells) were required to maintain IRF4 expression in vitro (N=17, P < 0.001) which also corresponds to our observation that IRF4 positive CLL cells were highly overrepresented in the CXCR4dim CD5brightCLL population (N = 55, P < 0.0001) which is considered to be the proliferative CLL cell fraction emigrated from the lymph node (Calissano Mol. Med. 2011). Conclusion: We demonstrate that low IRF4 expression is an independent negative prognostic marker for CLL progression. In the microenvironmental context IRF4 plays a dual role, with high expression limiting CLL survival on the one hand but being a crucial mediator of CD40L mediated proliferation on the other hand. While our data suggest that IRF4 is upregulated in response to microenvironmental stimuli the association of low IRF4 levels with bad prognosis suggests that IRF4 levels may serve to limit CLL growth by enhancing cell death responses. The exact mechanisms by which IRF4 impacts CLL pathophysiology is currently being investigated in vivo using B-cell specific IRF4-deficient CLL mouse models. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 3868 Chronic lymphocytic leukemia (CLL) has been historically considered an accumulative disease. However, recent studies demonstrate the relevance of proliferation centers found in lymphoid organs (lymph nodes [LN] and bone marrow [BM]),which provide survival and proliferation signals to CLL cells. In CLL, immunohistochemical analyses show that survivin is mainly expressed in CLL cells that are actively proliferating in proliferation centers. Interestingly, increased expression of survivin in peripheral blood CLL cells correlates with worse response to treatment. YM155 (Astellas®), a survivin suppressant, has demonstrated promising antitumoral activity in a wide variety of human cancer cell lines and xenograft models, and nowadays it is being evaluated in several phase II clinical trials. As survivin is usually expressed in proliferating cells, herein we tested the effects of YM155 on CLL cells co-cultured in a system mimicking the conditions that they are encountering in the proliferation centers. Primary CLL cells from 20 patients were co-cultured with BM stromal cells along with CD40 ligand and oligodeoxynucleotides-CpG. Optimal proliferative response was observed after 48 hours of co-culture (mean % Ki-67 expression, 1.52±0.06 in cells in suspension vs. 11.78±6.73 in co-culture, p<0.001). Therefore, CLL cells were subsequently treated with YM155 and/or fludarabine at this time point. By Western Blot, increasing survivin expression in co-culture, was completely abrogated by treatment with YM155 50nM for 24 hours. Induction of apoptosis was assessed by flow cytometry (FC) measurement of annexin V and propidium iodide (PI). Mean LD50 of YM155 was 430.4 nM (95%CI 227.3–815.3 nM) for CLL cells cultured in suspension, whereas it was 7.7 times lower (56.25nM, 95%CI 39.71–79.7 nM) in proliferative conditions, revealing that apoptotic cell death induced by inhibition of survivin is mainly affecting proliferating CLL cells. Unlike treatment with YM155, CLL cells treated with 5μg/ml fludarabine showed significantly higher viability in co-culture than in suspension (normalized mean of viability: 60.2%±16.9% vs. 23.4%±7.1%, p<0.001), thus confirming the microenvironment-induced chemoresistance. Of note, the addition of 50nM YM155 overcame this chemoresistance by significantly increasing fludarabine-induced cytotoxicity (normalized mean of viability: 42.1%±7.6% for fludarabine vs. 5.2%±1.9% for the combination; p<0.05). Prior to any treatment, 98.54%±0.12% of cells in suspension were arrested at G0/G1, whereas the co-culture increased the number of CLL cells in S/G2/M phase from 0.89%±0.31% on day 0 to 44.96%±23% at 72 hours (p=0.05). The addition of fludarabine, YM155, or the combination of both drugs after 48 hours of co-culture induced a significant G1 arrest at 72 hours (% cells in G1 phase: control, 55.04%±13.28%; fludarabine, 88.26%±7.94%; YM155, 81.61%±8.47%; combination, 90.75±4.68; p<0.01). Finally, YM155 treatment caused a drastic reduction of 23.31%±9.52% in Ki67 expression, whereas the combination of YM155 with fludarabine caused a significant decrease of 49.64%±4.12% (p<0.001), an effect that fludarabine alone could not achieve. Following this, we also analyzed the effect of YM155 on the proliferative compartment defined by Calissano, C et al (Mol Med, 2011; CD5high/CXCR4dim). Firstly, we observed that the co-culture induced an increase in this fraction (from 3.28%±1.12% to 9.66%±2.83%, p<0.05). Treatment with YM155 alone or combined with fludarabine reduced this proliferative fraction (control: 13.29%±4.21%; YM155: 5.82%±1.24%[p<0.05]; combination: 4.12%±1.14%[p<0.01]), whereas the proportion of the non-proliferative compartments where not affected by these treatments. Altogether, these data show that reproducing signals from the microenvironment in ex vivo co-cultures can promote proliferation and protect CLL cells from apoptosis induced by cytotoxic agents such as fludarabine. In this setting, YM155 treatment is able to efficiently overcome microenvironment-mediated cell protection and proliferation and has specific effect on actively proliferating CLL cells. Combination experiments revealed that YM155 strongly potentiates fludarabine cytotoxicity, especially in proliferating CLL cells chemoresistants to fludarabine alone. Finally, these results open the door to future clinical trials inhibiting survivin in CLL. Disclosures: No relevant conflicts of interest to declare.

Introduction. Altered metabolism is one of the hallmarks of cancer. CLL cells circulate between peripheral blood (PB) and lymph nodes (LN) which necessitates high metabolic plasticity. In LN, CLL cells receive proliferative and pro-survival signals from surrounding cells, and become metabolically activated. However, detailed insight into the altered metabolism of LN CLL and how this may be related to therapeutic responses is lacking. As it is technically difficult to obtain direct insight into CLL LN metabolism, we have applied a two-tiered strategy. By using PB samples taken from patients before/after treatment with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib (IBR), which drives CLL cells out of the LN, combined with in vitro re-stimulation of TME signals, we indirectly mapped the metabolism of CLL in their TME, as well as the effects of IBR treatment. We hypothesized that the overlapping/distinct metabolites affected by IBR and in vitro stimulations would reflect the actual CLL metabolism in LN. Methods. PB samples were obtained from 7 CLL patients before or after 3 months of ibrutinib treatment. These paired samples were in vitro stimulated via CD40 and B cell receptor (BCR), which are potential key signals within the tumour microenvironment (TME). Seahorse extracellular flux (ECF) analyses, expression of activation markers (CD95, pS6 by FACS), RNA was isolated for expression of Myc (major driver of metabolic reprogramming) and its target genes, and metabolomics by mass-spec was performed. Results. ECF analyses showed that in comparison to BCR stimulated PB CLL cells, stimulation by CD40 resulted in a high increase of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). A prominent effect on OXPHOS and glycolytic activity was confirmed in direct LN samples, and indirectly by marker analyses in LN emigrants using CXCR4/CD5 staining [1]. Subsequent metabolomics analyses showed that metabolic reprogramming following CD40 or BCR stimulation revealed both shared and distinct responses. The affected metabolic pathways, predicted by significantly changed metabolites, were compared in a pairwise fashion; upregulated by CD40 and BCR but downregulated by IBR, respectively. The results demonstrated 5 upregulated pre-defined pathways (KEGG) by both CD40 and BCR triggering: purine metabolism, Warburg effect, lysine degradation, glucose-alanine cycle and glutamate metabolism. In contrast, the following pathways indicated the two signals had distinct functions on regulating metabolism: CD40 signalling mostly regulates amino acid metabolism, tricarboxylic acid cycle (TCA) and mitochondrial metabolism related to oxidative phosphorylation (OXPHOS) and energy production. BCR signalling mainly involves glucose and glycerol metabolism, which are usually related to biosynthesis. CLL cells from IBR-treated patients showed enhanced BCR responsiveness, in line with the increased in surface IgM expression upon IBR [2]. In contrast, IBR treatment suppressed in vitro CD40 activation, which was accompanied by a lower CD40 expression. Metabolomics analyses also demonstrated that CD40 responses decreased but BCR response increased after IBR. Additionally, analyses of Myc and its target genes showed that they are induced after BCR as well as CD40 stimulation. Effects of IBR on Myc (target) expression were variable for BCR and reduced for CD40 stimulation. Conclusions. In vivo IBR treatment suppresses CD40 expression and activation and enhances BCR responsiveness. Metabolic changes of CLL in LN are recapitulated by these two signals, while IBR treatment shows opposite effects, together providing indirect insight into the LN metabolism. In LN, CD40 may play a prominent role to enhance most of the key metabolic pathways, particularly OXPHOS. This is the first study to describe the metabolic network of CLL cells in LN, and the long-term effects of IBR may yield new clues to therapy response and resistance. References 1. Calissano, Carlo, et al. "Intraclonal complexity in chronic lymphocytic leukemia: fractions enriched in recently born/divided and older/quiescent cells." Molecular Medicine 17.11 (2011): 1374-1382. 2. Drennan, Samantha, et al. "Ibrutinib therapy releases leukemic surface IgM from antigen drive in chronic lymphocytic leukemia patients." Clinical Cancer Research 25.8 (2019): 2503-2512. Disclosures Forconi: AbbVie: Honoraria, Other: Fees for cosulting or advisory role, received travel and expenses, Speakers Bureau; Janssen: Honoraria, Other: Fees for cosulting or advisory role, received travel and expenses, Speakers Bureau; Roche: Honoraria; Novartis: Honoraria; Menarini: Other: Fees for cosulting or advisory role; Astra Zeneca: Other: Fees for cosulting or advisory role; Gilead: Research Funding. Kater:Roche: Research Funding; Abbvie: Research Funding; Genentech: Research Funding; Celgene: Research Funding; Janssen: Research Funding. Eldering:Janssen: Research Funding; Celgene: Research Funding; Genentech: Research Funding.

Abstract The aim of this study was to investigate the role of microenvironmental interactions in the regulation of CD20 expression, since down-modulation of CD20 in the context of immune niches would be a straightforward explanation for the stroma-mediated rituximab resistance described by us and others (Mraz et al., BJH, 2011; Buchner et al., BJH, 2010; Marquez et al., BJH, 2015). Firstly, we co-cultured primary CLL cells with the bone marrow stromal cell line HS-5 for 24/48/72 hrs and analyzed CD20 surface expression by flow cytometry. Suprisingly, CD20 levels were significantly up-regulated (P=0.03) on CLL cells co-cultured with HS-5 in comparison to CLL cells cultured on plastic. The changes in CD20 expression levels are likely to play a direct role in microenvironmental interactions and especially BCR signaling in immune niches (Uchida et al., Int Immunol, 2004). To test this hypothesis we assessed the CD20 expression on CLL cell populations defined according to CXCR4 and CD5 levels. The low-level surface expression of chemokine receptor CXCR4 and high-level expression of activation molecule CD5 can characterize B cells that have recently exited the lymph nodes (Calissano et al., Mol Med, 2011). CXCR4dim CD5bright cells had ~2-fold higher surface CD20 expression when compared to CXCR4bright CD5dim CLL cells circulating in the blood of the same patient for a longer time (P<0.0001, N=21) and CD20 expression clearly decreased with the transition of CLL cells from CXCR4dim to CXCR4bright together with the decrease of CD5 expression. Further, sorted CXCR4dim CD5bright CLL cells had also ~2 times higher CD20 mRNA expression (P=0.002, N=8) suggesting that changes in CD20 expression were due to changes in its mRNA levels rather than a surface modulation. This led us to hypothesize that CXCR4/SDF-1 is directly implicated in CD20 regulation. Indeed, CLL cells treated with SDF-1α (CXCL12), a ligand for CXCR4 which is produced by HS-5, up-regulated surface CD20 (P=0.03). We also observed that CLL cells with higher expression of CD20 had higher levels of surface Ig (P=0.0015, N=12) and this was coupled with higher responsiveness to BCR crosslinking with anti-IgM as measured by calcium flux (P=0.005). Therefore, we investigated the effect of ibrutinib on CD20 levels as ibrutinib is a BCR-signaling pathway inhibitor and prevents CLL cells from responding to microenvironmental stimuli, including chemokine signaling. We observed that treatment of CLL cells with ibrutinib in vitro resulted in CD20 down-modulation (P<0.001). The levels of CD20 in CLL cells were also strongly down-modulated in samples obtained before vs. during ibrutinib treatment of CLL patients in vivo (pre- ibrutinib vs. day 15 and week 5/12, N=8; P=0.02, P=0.01, P=0.07, respectively). These data suggest that a reduction of the target antigen (CD20) is not the cause for rituximab resistance in the context of immune niches, and the sustained activity of ibrutinib and rituximab combinations in clinical trials (Burger et al., Lancet Oncol, 2014) suggests that ibrutinib has other mechanisms that allow for rituximab efficacy. We focused on the regulation of anti-apoptotic molecules, namely Mcl1, since that was shown to protect from rituximab-induced apoptosis and to be rapidly down-modulated by rituximab infusion in vivo (Byrd et al., Blood, 2002; Hussain et al., Clin Cancer Res, 2007). Indeed, we observed higher levels of Mcl1 mRNA in the CXCR4dim CD5bright CLL subpopulation (N=8, P=0.04), and in CLL cells co-cultured with stromal cells. Importantly, Mcl1 was down-regulated after ibrutinib treatment both in vitro and in ibrutinib treated patients. Conclusion: Microenvironmental interactions up-regulate CD20 expression in CLL B cells through the CXCR4/SDF-1 axis, and the ibrutinib-mediated impairment of microenvironmental interactions down-regulates CD20 expression. This study reveals a novel regulation of CD20 levels in the context of immune niches, which has important implications for CD20-targeting antibodies and the use of BCR-inhibitors in combination. Supported by: SoMoPro II-no.4SGA8684 (cofinanced by the European Union and the South-Moravian Region); NGS-PTL(306242); EHA Fellowship award; IGA MZ CR NT11218-6/2010; MUNI/A/1180/2014; CZ.1.05/1.1.00/02.0068; G.P. is a city of Ostrava scholarship holder. #G.P. and V.S. contributed equally to this study. contact: marek.mraz@email.cz Disclosures Mayer: Janssen: Research Funding. Davids:Genentech: Other: ad board; Pharmacyclics: Consultancy; Janssen: Consultancy.

Le cyanure étant un des agents les plus toxiques sur notre planète, il est crucial de le détecter avec précision surtout dans l’industrie alimentaire. Les travaux présentés dans cette thèse portent sur le développement de nouveaux détecteurs colorimétriques du cyanure. Dans un premier temps des benzoquinonemonoimines ont été synthétisées et leurs propriétés optiques investiguées (en version ligand libre et complexes). Nous nous sommes ensuite intéressés à la synthèse de macrocycles de type azacalixarènes, dont l’ajout de cyanure en solution permet l’obtention de complexes de Meisenheimer stables. Le travail s’est ensuite focalisé sur la synthèse de dérivés m-dinitro-benzènes comportant différents groupements donneurs et accepteurs dont leurs propriétés optiques ont été étudiées pour la détection colorimétrique du cyanure. Dans une dernière partie, nous avons étudié la teneur en cyanure des Calissons d’Aix
As cyanide is one of the most toxic agents on our planet, it is crucial to detect it accurately, especially in the food industry. The work presented in this thesis concerns the development of new colorimetric cyanide detectors. First, benzoquinonemonoimines were synthesized and their optical properties investigated (in free ligand and complex version). We then focused on the synthesis of azacalixarene-type macrocycles, for which the addition of cyanide in solution gives stable Meisenheimer complexes. The work then focused on the synthesis of m-dinitro-benzenes derivatives incorporating different donor and acceptor groups whose optical properties were studied for the colorimetric detection of cyanide. In the last part, we investigated the cyanide content of Calissons d´Aix