scholarly journals Mirs-138 and -424 Control Palmitoylation-Dependent CD95-Mediated Cell Death By Targeting Acyl Protein Thioesterases 1 and 2 in Chronic Lymphocytic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1953-1953
Author(s):  
Valeska Berg ◽  
Marion Rusch ◽  
Nachiket Vartak ◽  
Christian Juengst ◽  
Astrid Schauss ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Protein Transport ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Mrna Level ◽  
Resonance Energy ◽  
Lymphocytic Leukemia ◽  
Luciferase Reporter ◽  
Fluorescence Lifetime Imaging ◽  
Post Translational Modification

Abstract Introduction: Resistance towards CD95-mediated apoptosis is a hallmark of many different malignancies, like it is known from primary chronic lymphocytic leukemia (CLL) cells. Moreover, apoptosis mediated through CD95 is an essential mechanism to eliminate e.g. auto-reactive or virally infected cells. However, its mode of action is still not fully understood. Recently, it could be shown that palmitoylation of CD95 can influence its signaling properties. Nevertheless, the role and regulation of palmitoylated CD95 still needs to be determined. Methods and results: Previously, we could show that miR-138 and -424 are down-regulated in CLL cells. By applying luciferase reporter assays, mutations of the binding sites qRT-PCR and immunoblots after transfection of both miRs, we identified two new target genes, namely acyl protein thioesterase (APT) 1 and 2, which are under control of both miRs and thereby are significantly over-expressed in CLL cells. Interestingly, our data reveal that expression of APTs is already controlled by miRs on mRNA level. This way APT1 is regulated by miR-138 and expression of APT2 is controlled by miR-424. So far, APTs are the only enzymes known to promote de-palmitoylation. Indeed, membrane proteins are significantly less palmitoylated in CLL cells compared to normal B cells as we determined by click-chemistry, which is a non-radioactive method to determine palmitoylated proteins. Importantly, via acyl-biotin exchange assays with subsequent immunoprecipitation of CD95 and fluorescence lifetime imaging microscopy (FLIM) to Foerster resonance energy transfer (FRET) in living cells we identified APTs to directly interact with CD95 to promote de-palmitoylation, thus impairing apoptosis mediated through CD95. As proof of concept APTs were inhibited specifically by siRNAs, miRs-138/-424 or our pharmacological inhibitor Palmostatin B. Thereby we could restore CD95-mediated apoptosis in CLL cells and other cancers, pointing to a central regulatory role of APTs in CD95 apoptosis. Conclusion: The identification of the de-palmitoylation reaction of CD95 by APTs as a miRNA target provides a novel molecular mechanism how malignant cells escape from CD95-mediated apoptosis. Here, we introduce palmitoylation as a novel post-translational modification in CLL. In light of global palmitoylome studies, which show that potentially palmitoylated proteins are involved in all central cellular processes, such as protein transport, survival, migration, apoptosis and B-cell receptor signaling, this emphasizes the importance of palmitoylation and might put it on par with modifications like phosphorylation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Akt Inhibitors Induce Apoptosis in Chronic Lymphocytic Leukemia Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1731-1731
Author(s):  
Mercè de Frias ◽  
Daniel Iglesias-Serret ◽  
Ana M Cosialls ◽  
Llorenç Coll-Mulet ◽  
Antonio F Santidrián ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Therapeutic Option ◽  
Mrna Level ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Healthy Donors ◽  
Induced Apoptosis

Abstract Abstract 1731 Poster Board I-757 Phosphatidylinositol-3-kinase (PI3K)/Akt pathway has been described to be critical in the survival of chronic lymphocytic leukemia (CLL) cells. Here, we have analyzed the effect of two selective chemical inhibitors of Akt (Akti-1/2 and A-443654) in the survival of CLL cells. We studied by cytometric analysis the cytotoxic effects of Akt inhibitors on peripheral B and T lymphocytes from patients with CLL and from healthy donors. Both inhibitors induced apoptosis in CLL cells in a dose-dependent manner. Moreover, B cells from CLL samples were more sensitive to Akt inhibitors than T cells from CLL samples, and B or T cells from healthy donors. Survival factors for CLL cells, such as IL-4 and SDF-1a, were not able to block the apoptosis induced by both Akt inhibitors. We studied the changes induced by Akti-1/2 and A-443654 at mRNA level by performing reverse transcriptase multiplex ligation–dependent probe amplification (RT-MLPA). Akti-1/2 did not induce any change in the mRNA expression profile of genes involved in apoptosis, while A-443654 induced some changes, including an increase in NOXA and PUMA mRNA levels, suggesting the existence of additional targets for A-443654. We also studied the changes induced by both Akt inhibitors in some BCL-2 protein family members on CLL cells by Western blot. Both inhibitors induced an increase in PUMA and NOXA protein levels, and a decrease in MCL-1 protein level. Moreover, Akti-1/2 and A-443654 induced apoptosis irrespective of TP53 status. These results demonstrate that Akt inhibitors induce apoptosis of CLL cells and might be a new therapeutic option for the treatment of CLL. Disclosures No relevant conflicts of interest to declare.

Serum IgM Activates BCR Signaling Pathways and Increases Survival of Chronic Lymphocytic Leukemia B Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4131-4131
Author(s):  
Stefania Gobessi ◽  
Binu K Sasi ◽  
Luca Laurenti ◽  
Dimitar G Efremov
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Signaling Pathways ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Direct Interaction ◽  
Leukemic Cell ◽  
Fold Increase ◽  
Lymphocytic Leukemia ◽  
Bcr Signaling

Abstract Serum IgM would be expected to bind chronic lymphocytic leukemia B cells through two different mechanisms. The first mechanism is via interactions between the immunoglobulin heavy chain CDR3 of the leukemic B cell receptors (BCRs) and internal epitopes located in the FR2 and FR3 regions of serum IgM molecules, analogous to the recently identified cell-autonomous BCR-BCR interaction. The latter interaction represents a general feature of human CLL BCRs and was recently shown to be positively selected during leukemia development in the Eμ-TCL1 transgenic murine model. The second mechanism is by binding of serum IgM to the recently identified Fc receptor for IgM (FcμR), which is overexpressed on CLL B cells. In the present study we investigated the consequences of the interaction between serum IgM and CLL cells. Incubation of CLL cells with Alexa488-conjugated human IgM resulted in strong cell surface labeling, confirming that IgM binds to CLL cells. Binding was substantially inhibited by preculture of CLL cells with Fcμ, suggesting that IgM interacts with CLL B cells primarily through the FcμR. To investigate whether IgM also binds to the leukemic BCRs, we analyzed activation of downstream BCR signaling pathways and expression of a well-defined set of BCR-target genes (Herishanu Y et al, Blood. 2011;117:563-74) in CLL cells cultured in the presence or absence of purified IgM. After three hours in culture with polyclonal or monoclonal human IgM, 5 of the 7 investigated BCR target genes (OAS3, RGS1, GFI1, CCND2 and KLF4) showed a 2- to 9-fold increase with respect to unstimulated CLL cells, whereas the remaining two genes (EGR1 and EGR2) were not induced. The induced BCR target genes were also upregulated to an equal or even greater extent by Fcμ, suggesting that these effects are primarily or exclusively caused by binding of IgM to the FcμR. Analysis of downstream signaling events, such as SYK and ERK phosphorylation, also showed similar induction by IgM and Fcμ. However, intracellular Ca2+ flux was induced to a substantially greater extent with IgM, suggesting that certain effects are mediated by a direct interaction between serum IgM and the leukemic cell BCRs. Since co-ligation of the FcμR was recently shown to enhance the survival of anti-IgM-stimulated murine B lymphocytes (Ouchida R et al, J Immunol. 2015;194:3096-101), we investigated the consequences of IgM binding on CLL cell survival. CLL cells from 18 patients were cultured with or without purified human IgM for 72 hours and then analyzed by Annexin V/PI staining. A modest but significant increase in the percentage of viable CLL cells was observed in the presence of IgM (percentage of viable CLL cells without IgM: 40.5±17.8; with IgM: 43.8±18.4; P =0.016), which was replicated in a smaller series of samples cultured with Fcμ (n=12, percentage of viable CLL cells without Fcμ: 41.1±17.8; with Fcμ: 49.5±15.6; P =0.019). Altogether, these data suggest that binding of serum IgM results in activation of prosurvival pathways in CLL cells and that this effect is most likely mediated by co-triggering the FcμR and BCR. Disclosures No relevant conflicts of interest to declare.

scholarly journals Elevated FANCA Expression Marks a Worse Prognosis in Chronic Lymphocytic Leukemia. Evidence of a New Role of FANCA in the Stability of p53 By a Neddylation-Related Mechanism

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5541-5541
Author(s):  
Carlos Pipaon ◽  
Sara Bravo Navas ◽  
Iñigo Romon ◽  
Eulogio Conde ◽  
Lucrecia Yanez San Segundo
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Chronic Lymphocytic Leukemia ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Clinical Manifestations ◽  
Lymphocytic Leukemia ◽  
Worse Prognosis

Abstract Background: Chronic lymphocytic leukemia (CLL) is the most common hematological malignancy in western countries. It is characterized by a failure in the mechanisms of apoptosis that leads to an accumulation of mature B cells in peripheral blood, bone marrow and lymphoid organs. With exceptions, CLL is considered incurable and some patients show a worse prognosis related to the expression of certain cytogenetic or molecular markers such as p53 dysfunction (17p13 deletion or TP53 inactivation), 11q23 deletion, unmutated IgVH and the presence of a complex karyotype. FANC proteins have been related to chromosomal instability and alterations in the mechanisms of p53 activation, control of cell cycle and apoptosis. Germline mutations in any of the 20 FANC genes known so far generates Fanconi Anemia, a syndrome characterized by a extraordinary proneness to cell apoptosis leading to a progressive bone marrow aplasia and pancytopenia. Some FANCA proteins aggregate in response to DNA damage forming the FANCcore complex that mediates the monoubiquitination of FANCD2 and FANCI, thus activating the mechanism to repair stalled replication forks. In addition, individual FANC proteins have been involved in functions out of the FANCcore. This is the case of FANCA, that has recently been involved in the neddylation of CXCR5 and beta-2-microglobulin, processes reported to be altered in CLL. Hypothesis: Given the fragmentary information connecting FANC proteins with cellular processes altered in CLL, like apoptosis, cell cycle or neddylation, we hypothesize a role of these proteins in the diagnosis or prognosis of CLL. Methods: We analyzed the expression of 5 FANC genes in a cohort of 160 patients of CLL by quantitative RT-PCR. Statistical analysis were carried out to establish relations between FANC genes deregulation and clinical manifestations. We also investigated the role of the FANCA gene in primary circulating B-lymphocytes from CLL patients by either gain- or loss-of-function approaches. Results: Our data identified a group of CLL patients with high expression of FANCA in peripheral B-CLL cells, and we stablished its relationship with the deletion of 11q23 and a worse prognosis. When we investigated the molecular mechanisms of this bad prognosis, we observed a reduction in the mRNA expression of two p53 target genes, p21 and ∆Np73, in CLL primary cells transfected with FANCA. Luciferase studies demonstrated an impairment of p53 function by FANCA. Moreover, we obtained evidence of a cooperation between FANCA and the NEDD8-interacting protein NUB1L in the destabilization of p53. Conclusion: These results point to FANCA as a bad prognosis marker in CLL and unveils a new role of this protein aside from its role in the FA-BRCA DNA repair pathway. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Impact of Chlorambucil and Valproic Acid on Cell Viability, Apoptosis and Expression of p21, HDM2, BCL2 and MCL1 Genes in Chronic Lymphocytic Leukemia

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1088
Author(s):  
Katarzyna Lipska ◽  
Agata Filip ◽  
Anna Gumieniczek
Keyword(s):  
Valproic Acid ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Viability ◽  
Antiepileptic Drug ◽  
Drug Combination ◽  
Target Genes ◽  
Histone Deacetylation ◽  
Lymphocytic Leukemia ◽  
Malignant Cells ◽  
The Impact

Malignant cells in chronic lymphocytic leukemia (CLL) show resistance to apoptosis, as well as to chemotherapy, which are related to deletions or mutations of TP53, high expression of MCL1 and BCL2 genes and other abnormalities. Thus, the main goal of the present study was to assess the impact of chlorambucil (CLB) combined with valproic acid (VPA), a known antiepileptic drug and histone deacetylation inhibitor, on apoptosis of the cells isolated from 17 patients with CLL. After incubation with CLB (17.5 µM) and VPA (0.5 mM), percentage of apoptosis, as well as expression of two TP53 target genes (p21 and HDM2) and two genes from Bcl-2 family (BCL2 and MCL1), were tested. As a result, an increased percentage of apoptosis was observed for CLL cells treated with CLB and VPA, and with CLB alone. Under the treatment with the drug combination, the expression of p21 gene was visibly higher than under the treatment with CLB alone. At the same time, the cultures under CLB treatment showed visibly higher expression of BCL2 than the cultures with VPA alone. Thus, the present study strongly suggests further investigations on the CLB and VPA combination in CLL treatment.

scholarly journals Epidemiology Data of Patients with Chronic Lymphocytic Leukemia in West Central Mexico

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5269-5269
Author(s):  
Virgina Campos-Cabrera ◽  
Gregorio Campos-Cabrera ◽  
Salvador Campos-Cabrera ◽  
Alicia Rivera-Trujillo ◽  
Sonia Hernandez-Rodriguez ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Low Frequency ◽  
Epidemiological Data ◽  
Lymphocytic Leukemia ◽  
Female Ratio ◽  
Specific Protocol ◽  
Male Female Ratio ◽  
Epidemiology Data ◽  
International Data

Abstract Background: chronic lymphocytic leukemia (CLL) is a rare disease in Mexican mestizos (Br J Haematol 2015;169:909-911 and Int J Hematol 1999;69:253-255). No significant data on epidemiology is available. Methods: epidemiological data from samples referred to Laboratorios Fatima de Michoacan for flow cytometry immunophenotyping for neoplastic hematological disease. Results: 229 samples were received, 52 were diagnosed as CLL (22.7 %). Male 32 and female 20, ratio 1.6 a 1. Age from 33 to 89 years, average 66; 31 to 40 one, 41 to 50 two, 51 to 60 eleven, 61 to 70 twenty four, 71 to 80 ten, more than 81 four. Expression of CD 38 and ZAP-70 in three; only CD38 in 2, only ZAP-70 in three. Conclusions: similar results in male female ratio and age of presentation are noted as compared with international data. Low frequency of expression in CD38 and ZAP-70 may be due pre-analytic phase in the management of the sample. As a regional group we are trying to have epidemiology data in non-malignant and malignant hematological diseases to form specific protocol treatments. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

CARD11 is a novel target of miR-181b that is upregulated in chronic lymphocytic leukemia

2021 ◽  
Author(s):  
Zhen Kou ◽  
Min Mao ◽  
Hong Liu ◽  
Xiaomin Wang ◽  
Zengsheng Wang ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Target Gene ◽  
Target Genes ◽  
Prognostic Indicator ◽  
Lymphocytic Leukemia ◽  
Poor Survival ◽  
Kaplan Meier ◽  
Poor Prognostic Indicator ◽  
Novel Target

Aim: To investigate the targets of miR-181b in patients with chronic lymphocytic leukemia (CLL). Materials & methods: The bioinformatic softwares were used to indicate the key target genes associated with miR-181b, and the results were verified in CLL patient samples and 293T cells. Results: CARD11 is a potential target gene of miR-181b, an inverse relationship was revealed between the expression of CARD11 and miR-181b in 104 CLL patients, and it was confirmed in vitro with luciferase assays and western blotting. Kaplan–Meier analysis showed that CLL patients with high CARD11 expression demonstrated poor survival. Conclusion: CARD11 is a novel target of miR-181b that is upregulated, which could be a poor prognostic indicator for CLL patients.

scholarly journals Discovery of 4-Anilinoquinolinylchalcone Derivatives as Potential NRF2 Activators

Molecules ◽  
2020 ◽  
Vol 25 (14) ◽  
pp. 3133
Author(s):  
Yu-Tse Kao ◽  
Yi-Siao Chen ◽  
Kai-Wei Tang ◽  
Jin-Ching Lee ◽  
Chih-Hua Tseng ◽  
...  
Keyword(s):  
Target Genes ◽  
Hacat Cell ◽  
Effective Means ◽  
Mrna Level ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Stable Cell Line ◽  
Heme Oxygenase 1 ◽  
Related Factor ◽  
Nrf2 Activator

Activation of nuclear factor erythroid-2-related factor 2 (NRF2) has been proven to be an effective means to prevent the development of cancer, and natural curcumin stands out as a potent NRF2 activator and cancer chemopreventive agent. In this study, we have synthesized a series of 4-anilinoquinolinylchalcone derivatives, and used a NRF2 promoter-driven firefly luciferase reporter stable cell line, the HaCaT/ARE cells, to screen a panel of these compounds. Among them, (E)-3-{4-[(4-acetylphenyl)amino]quinolin-2-yl}-1-(4-fluorophenyl)prop-2-en-1-one (13b) significantly increased NRF2 activity in the HaCaT cell with a half maximal effective concentration (EC50) value of 1.95 μM. Treatment of compound 13b upregulated HaCaT cell NRF2 expression at the protein level. Moreover, the mRNA level of NRF2 target genes, heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC), and glucose-6-phosphate dehydrogenase (G6PD) were significantly increased in HaCaT cells upon the compound 13b treatment. The molecular docking results exhibited that the small molecule 13b is well accommodated by the bound region of Kelch-like ECH-associated protein 1 (Keap1)-Kelch and NRF2 through stable hydrogen bonds and hydrophobic interaction, which contributed to the enhancement of affinity and stability between the ligand and receptor. Compound 13b has been identified as the lead compound for further structural optimization.

Comparative Efficacy of First-Line Chemotherapy-Free Combinations in Chronic Lymphocytic Leukemia (CLL): A Network Meta-Analysis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 25-26
Author(s):  
Philip A Haddad ◽  
Nowell Ganey ◽  
Kevin M. Gallagher
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
English Language ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Meta Analysis ◽  
The Elderly ◽  
Lymphocytic Leukemia ◽  
First Line ◽  
Significant Difference ◽  
Anti Cd20

Introduction: Chronic Lymphocytic Leukemia (CLL) is an incurable B-cell malignancy which disproportionately affects the elderly. Although first-line chemoimmunotherapy (CIT) improved CLL clinical outcomes, recent randomized trials revealed superior outcomes with novel chemotherapy-free combinations (CFC) incorporating anti-CD20 monoclonal antibodies and inhibitors of BTK or Bcl-2. So far, these CFC have not been compared head-to-head. We conducted this network meta-analysis to evaluate their relative efficacy to each other. Methods: A review of the medical literature was conducted using online databases. Inclusion criteria consisted of English language; diagnosis of CLL; trials that explored the efficacy of first-line CFC with Obinutuzumab (O), Rituximab (R), Ibrutinib (IB), Acalabrutinib (ACAL), Venetoclax (VEN) compared to standard CIT that included Chlorambucil (CHLOR) with either R or O, Bendamustine+Rituximab, or Fludarabine+ Cyclophosphamide+R; and phase 3 randomized studies reporting responses, progression, death, and adverse (AE) events. A frequentists network meta-analysis was conducted using netmeta package and random-effects model. Results: Five studies comprising a total of 2,272 participants were included. When O-based CFC data was analyzed, only ACAL-O had a significant lower relative risk (RR) of progression and death (P&D). There were no significant differences with respect to overall response rates (ORR), complete remission (CR), minimal residual disease (MRD), or grade >3 adverse events (Grd3+) among O-based CFC. When R-based CFC data was analyzed, IB and IB-R were not different with respect to RR of P&D, ORR, CR, MRD, or Grd3+. When the data was analyzed as CFC versus combined CIT, only ACAL-O was found to be significantly superior to other O- and R-based CFC with respect to RR of P&D. ORR and Grad3+ rates of O- and R-based CFC were not significantly different. While ACAL-O, IB-O, and VEN-O had superior CR and MRD rates compared to other CFC, there were no significant differences among each other. Conclusions: This network meta-analysis is the first to compare and rank first-line CFC therapies in CLL. It indicates that ACAL-O has a superior profile having the lowest RR of P&D without significant difference in Grd3+ among CFC. Disclosures No relevant conflicts of interest to declare.

Abnormal Free Light Chain Ratios in Chronic Lymphocytic Leukemia: A New Prognostic Factor?.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1237-1237 ◽  
Author(s):  
Johannes Matschke ◽  
Lewin Eisele ◽  
Ludger Sellmann ◽  
Ulrich Duehrsen ◽  
Jan Duerig ◽  
...  
Keyword(s):  
Prognostic Factor ◽  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Prognostic Significance ◽  
Risk Groups ◽  
Typical Feature ◽  
Lymphocytic Leukemia ◽  
Al Amyloidosis ◽  
Abnormal Ratio

Abstract Abstract 1237 Poster Board I-259 Introduction Free light chains (FLC) have prognostic significance in monoclonal gammopathy of undetermined significance, solitary plasmocytoma of bone, smouldering myeloma, multiple myeloma, Waldenstroms macroglobulinaemia and AL amyloidosis. Although monoclonal protein secretion is a typical feature of plasma cell dyscrasias, it can also be detected in other B cell malignancies including chronic lymphocytic leukemia (CLL). Recent data suggest a significant correlation between abnormal ratio of FLC and outcome. Therefore, we investigated FLC in a large cohort of 120 patients in order to assess the role of FLC in CLL. Methods and Results Plasma samples which had been previously cryopreserved and collected at the time before the initiation of therapy or six months after finishing therapy were used. The levels of FLC were assessed using nephelometric immunoassays (The Binding Site) and quantified nephelometrically with the BNII analyser. A normal FLC range (κlγ) was defined as 0.26-1.65. Moreover, in all cases we evaluated the M band on immunofixation (IF). Abnormal FLC ratios were found in 71 patients (59%) whereas the IF was positive in only 32 cases (27%). In 48 cases the FLC ratio was positive while IF was negative and in only 9 cases the IF was positive while the FLC ratio was normal. In total, 23 patients had both a positive IF and an abnormal FLC ratio. Patients with an abnormal FLC ratio for γ had a significantly shorter treatment-free survival (TFS) than patients with an abnormal ratio for κ or with a normal FLC ratio (median TFS: 34 versus 78 versus 109 months, p=0.042). Evaluation of several disease characteristics in association with FLC of the patients' B-CLL cells showed no significant differences for FLC in the different risk groups (ZAP-70 status, CD38 status, cytogenetics and Binet stage) suggesting no correlation of the FLC with these already established adverse prognostic factors. Conclusion FLC can be detected in a substantial fraction of patients with CLL and the FLC technique improves detection of M-proteins. Moreover, an abnormal FLC ratio is associated with worse outcome, particularly those with a low abnormal FLC ratio. Evaluation of the prognostic significance of abnormal FLC in a larger cohort is currently under way. This data will be presented at the meeting. Future studies are warranted to elucidate the role of FLC as biomarkers of disease and as a prognostic factor for response. Disclosures No relevant conflicts of interest to declare.

Monocytes/Macrophages Are the Major Targets of the CCL3 Chemokine Produced by CD38+CD49d+ Chronic Lymphocytic Leukemia Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2350-2350
Author(s):  
Antonella Zucchetto ◽  
Dania Benedetti ◽  
Claudio Tripodo ◽  
Riccardo Bomben ◽  
Fleur Bossi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Chronic Lymphocytic Leukemia ◽  
T Lymphocytes ◽  
Conflicts Of Interest ◽  
Cell Types ◽  
Lymphocytic Leukemia ◽  
Bone Marrow Biopsies ◽  
Tnf Α ◽  
Significant Difference

Abstract Abstract 2350 Poster Board II-327 Introduction: CD38 and CD49d are associated negative prognosticators in chronic lymphocytic leukemia (CLL). Recent gene expression profiling studies comparing CLL cases expressing low versus high levels of CD38 and CD49d, identified CCL3 as a gene upregulated by CD38+CD49d+ CLL. The release of CCL3 by cultured CLL cells was also demonstrated upon CD38 triggering, and CCL3 protein was found in CLL cells from bone marrow biopsies (BMB) of CD38+ cases (Zucchetto et al., Cancer Res, 2009; 69:4001-9). Given the role of CCL3 as potent chemoattractant for different cell types, we aimed at identifying the major targets of CCL3, as produced by CD38+CD49d+ CLL cells. Methods: CLL infiltrates of BMB were characterized by immunohistochemistry (IHC). Expression of the CCL3 receptors CCR1 and CCR5 by PB CLL subpopulations was evaluated by flow cytometry. T lymphocyte and monocyte migrations were performed by in-vitro transwell chemotaxis assays. Results: IHC analysis of BMB from 16 CLL cases revealed a higher number of infiltrating CD68+ cells in the context of CLL-involved areas of BMB from CD38+CD49d+CCL3+, compared to CD38−CD49d−CCL3− cases (p=0.01). CD3+ lymphocytes were interspersed in the CLL aggregates, but with no significant difference between the two subgroups. Evaluation of CCR1 and CCR5 in PB cell subpopulations from 40 CLL cases expressing or not surface CD38 and CD49d, showed the highest mean fluorescence intensity (MFI) levels for both CCR1 (624±60) and CCR5 (64±9) in the monocytic component, irrespective of CD38 and CD49d expression by CLL cells. Conversely, both CLL cells and residual T lymphocytes showed low MFI levels for CCR1 (19±4 and 14±3) and CCR5 (21±2 and 20±2). High CCR1 and CCR5 expression levels were detected in in-vitro differentiated monocytes from purified PB cells of four CD38+CD49d+ CLL. Accordingly, CCR1 expression was documented in macrophage-like cells in BMB from CD38+CD49d+ CLL. Next, we evaluated the capability of purified monocytes and T lymphocytes from 10 CLL cases to migrate in response to CCL3. In keeping with the strong expression of CCR1, monocytes migrated toward CCL3 at a concentration of 3 ng/mL (migration index, MI= 8.8±0.9, p=0.03), whereas T lymphocytes required a higher CCL3 concentration (100 ng/mL) to display slight migration capability (MI= 1.6±0.2, p=ns). The increased infiltration of macrophages in BMB from CCL3-producing CD38+CD49d+ CLL, prompted us to verify the capability of CCL3-stimulated macrophages to induce the expression by endothelial cells (EC) of the CD49d specific ligand VCAM-1. By using two different EC models (HUVEC and ADMEC), we documented a significant up-regulation of VCAM-1 by EC exposed to conditioned media (CM) collected from cultures of macrophages challenged in-vitro with CCL3 (p=0.002). Notably, increased levels of the pro-inflammatory cytokine TNF-α were detected in CCL3-CM (p=0.006), and neutralization of TNF-α by specific antibodies reverted the capability of CCL3-CM to induce VCAM-1 by EC models. In agreement with these in-vitro data, we found a more prominent meshwork of VCAM-1+ stromal/endothelial cells in lymphoid infiltrates from CD38+CD49d+ CLL compared to CD38−CD49d− cases (p=0.002), and engagement of CD49d by VCAM-1 was able to significantly delay the spontaneous apoptosis observed in cultured CLL cells. Conclusions: CD68+ monocytes/macrophages are likely the main targets for the CLL3 chemokine produced by CD38+CD49d+ CLL cells, and are active in determining, through the release of TNF-α and other yet unidentified cytokines, the overexpression of VCAM-1 by endothelial cells. Experiments aimed at investigating further roles of CD68+ monocytes/macrophage in CLL are currently matter of study. Disclosures: No relevant conflicts of interest to declare.

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