Nicotinamide Promotes Apoptosis In Chronic Lymphocytic Leukemia through Activation of the p53/Mir-34a/SIRT1 Tumor Suppressor Network

Abstract Abstract 4627 Nicotinamide (Nam), is the main precursor of nicotinamide adenine dinucleotide (NAD+). It regulates intracellular levels of NAD+ and consequently activities of four classes of NAD+-consuming enzymes, including NADases, mono-ADP-ribosyl transferases (ARTs), poly-ADP-ribose polymerases (PARPs) and sirtuins. Pharmacological doses of Nam inhibit the physiological activation and proliferation of mouse B lymphocytes, suggesting that this agent might affect also human B cell homeostasis. We approaches this issue by comparing the effects of Nam on normal vs. leukemic B lymphocytes. Chronic lymphocytic leukemia (CLL) was selected as disease model, for testing in vitro the therapeutic potential of Nam, due its intrinsic resistance to apoptosis, mediated by an imbalance in the mechanisms regulating cell death, mainly regulated through the activities of NAD+-dependent enzymes. This study shows that pharmacological doses of Nam (5-10 mM) significantly inhibit proliferation and induce apoptosis of CLL cells. At earlier time points, Nam markedly reduces phosphorylation of multiple intracellular substrates, including ERK1/2. Normal B lymphocytes, used as control, were significantly less sensitive to the action of Nam. We hypothesized that these effects could be explained at least in part as a consequence of the inhibitory effects of Nam on NAD+-consuming enzymes. Attention was focused on SIRT1, a deacetylase that plays a critical role in cancer and that acts as a longevity factor. The results demonstrate that Nam exposure inhibits the activity, and also the expression of SIRT1. This effect is apparent only in leukemic cells, where SIRT1 protein levels are significantly higher than in normal B lymphocytes, obtained from spleen or tonsils, markedly less sensitive to Nam effects. The functional block of SIRT1 induced by Nam is followed by activation of p53, transcription of miR-34a and translational repression of SIRT1 mRNA (p53/miR-34a/SIRT1 functional loop). The endpoint is the activation of apoptosis. The same loop is the target of conventional DNA-damaging drugs, such as etoposide. Thus, addition of Nam to conventional DNA-damaging chemotherapeutics agents, leads to an inhibition of SIRT1 through two independent and synergic pathways, resulting in additive effects on apoptosis. In conclusion this work suggests that Nam represents a potentially useful non-chemotherapeutic agent, characterized by a known and established safety profile, to be associated to conventional cytotoxic drugs in the treatment of selected forms of CLL. Disclosures: No relevant conflicts of interest to declare.

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Increased CD39 Expression on CD4+ T-Lymphocytes Has Clinical and Prognostic Significance in Chronic Lymphocytic Leukemia,

Blood ◽

10.1182/blood.v118.21.3895.3895 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3895-3895

Author(s):

Yair Herishanu ◽

Inbal Hazan-Hallevi ◽

Sigi Kay ◽

Varda Deutsch ◽

Aaron Polliack ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

T Lymphocytes ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Prognostic Significance ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Anti Inflammatory

Abstract Abstract 3895 Chronic lymphocytic leukemia (CLL) cells depend on their microenvironment for proliferation and survival. Ectonucleotidase CD39 has anti-inflammatory properties as it hydrolyzes pro-inflammatory extra-cellular ATP, generates anti-inflammatory adenosine and also protects regulatory T cells from ATP-induced cell death. In this study we investigated the clinical significance of CD39 expression on CD4+T-cells in 45 patients with CLL as well as its compartmental regulation and explored the possible mechanisms for its induction. Compared to healthy individuals, CD4+CD39+ lymphocytes were increased in the peripheral blood of patients with CLL (4.6%±2.28 vs. 17.3%±12.49, respectively, p=0.004), and correlated with advanced stage of disease (9.72%±5.76, 18.15%±12.03 and 25.90%±16.34, of CD4+ lymphocytes, in patients with Rai stages 0, 1+2 and 3+4, respectively, p=0.019). CD4+CD39+ cells were also higher in patients with CLL who needed therapeutic intervention (untreated; 12.99%±10.63 vs treated; 22.21%±12.88, p=0.01) and in those who were ZAP70+ or had b2-microglobulin levels>3g/L. There were more CD4+CD39+ lymphocytes in the bone marrow compartment (22.25%±16.16) than in the peripheral blood (16.60%±15.84, p=0.009). In-vitro studies showed that CD39 can be induced on CD4+cells by exposure to ATP or indirectly, following B-cell receptor (BCR) engagement (CD4+CD39+ lymphocytes increased by 1.56 fold, in the BCR engaged samples compared to their paired controls; 20.27%±11.3 vs. 13%±9.42, respectively, p=0.0006). Conclusions: Increased CD39 expression on CD4+ T-lymphocytes in CLL associates with an aggressive disease. This may reflect the ability of the leukemic cells to suppress the surrounding immune environment, and contribute to a poorer prognosis. CD39+ may also serve as a future target for the development of novel therapies with immune modulating anti–tumor agents in CLL. Disclosures: No relevant conflicts of interest to declare.

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B-Chronic Lymphocytic Leukemia Autophagyc Cell Death by the Use of Manganese Doped Zinc Oxide Nanoparticles and Photo-Dynamic Therapy.

International Journal of Drug Delivery Technology ◽

10.25258/ijddt.v5i1.8864 ◽

2015 ◽

Vol 5 (1) ◽

Author(s):

Peña Sandra ◽

Marín H. ◽

Rodríguez Felipe ◽

Dreon Marcos ◽

Roque Gustavo ◽

...

Keyword(s):

Zinc Oxide ◽

Chronic Lymphocytic Leukemia ◽

B Lymphocytes ◽

Chemotherapeutic Agents ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Zno Nps ◽

Genetic Abnormalities ◽

Novel Method

B-Chronic Lymphocytic Leukemia (B-CLL) usually follows an adverse, relentless clinical course by slowly developing drug resistance to fludarabine and other chemotherapeutic agents, as well as by acquiring new different genetic abnormalities. As B-CLL cells spontaneously produce high amounts of Reactive Oxygen Species (ROS) having an altered redox state in relation to that of normal B lymphocytes, we decided to probe different metal Zinc nanoparticles (ZnNPs) and quantify the levels of Singlet Oxigen (SO) to see if variations of its intracellular concentrations could execute and accelerate deadly programs in leukemic cells rather than in normal B lymphocytes, when applied with Photodynamic Therapy (PDT). In this way, we developed and tested a variety of metal ZnNPs of which one made of 0.5% Manganese Doped Zinc Oxide (ZnO:Mn) was finally selected for further testing as it had the best fludarabine resistant B-CLL cells in vitro killing activity, specially when combined with PDT. An interesting and rapidly dying process of B-CLL cells, known as autophagy, was always seen under Transmission Electronic Microscopy (TEM) when incubated with these 0.5% Mn doped ZnO NPs. This phenomenon correlated well with those intracellular increases of SO when PDT was administered, and measured by a novel method first described by us. As this therapy seems to be very specific to fludarabine resistant B-CLL cells, producing almost no damage to normal lymphocytes, it could surely contribute in the near future as a new innovative targeted strategy to be delivered in the clinical setting for the definitive benefit of these bad prognostic patients.

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Lymphocyte Function-Associated Antigen 3 (LFA-3): Key Factor of the Interactions Between Nurse-like-Cells and B Leukemic Cells from Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v124.21.1955.1955 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1955-1955

Author(s):

Frederic Boissard ◽

Jean Jacques Fournie ◽

Loic Ysebaert ◽

Mary Poupot

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Lymph Nodes ◽

B Lymphocytes ◽

Adhesion Molecule ◽

Actin Polymerization ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Lymphocyte Function ◽

Blocking Antibody

Abstract Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western Countries. This pathology is characterized by an accumulation of monoclonal, non-functional and mature CD5+ CD19+ leukemic B-cells (CLL cells) in lymph nodes, peripheral blood and bone marrow. Despite a high resistance to the in vivo apoptosis, CLL cells die spontaneously in vitro due to a lack in ex vivo conditions of sustaining cells and factors from their microenvironment such as stroma cells (Lagneaux L et al, Blood. 1998; 91:2387-2396), follicular dendritic cells or Nurse-Like-Cells (NLC) (Burger JA et al, Blood. 2000; 96:2655-2663). NLC are derived from CD14+ cells in contact with CLL cells in vitro (Tsukada N et al, Blood. 2002; 99:1030-1037) and were found in lymph nodes of CLL patients (Ysebaert L et al, Leuk Lymphoma. 2011; 52:1404-1406). NLC were shown to have a Tumour Associated Macrophages phenotype and gene expression profile. These cells have been first described to be essential for in vitro CLL cells survival partially through the production of soluble factors such as CXCL12 (Burger JA et al, Blood. 2000; 96:2655-2663), CCL3 and CCL4 (Burger JA et al, Blood. 2009; 113:3050-3058). Thus, other mechanisms are required for CLL cells survival. Indeed, we showed that contact of CLL cells with NLC was necessary to protect CLL cells from the in vitro apoptosis. We then investigated the mechanism of these interactions at a molecular level. We also determined their influences on the in vitro CLL cells survival and on the NLC-induced chemoresistance. We observed close and strong interactions evaluated by the measurement of trogocytosis from NLC to CLL cells. Trogocytosis is an active phenomenon with transfer of membrane fragments from one cell to another. We showed that NLC/CLL cells trogocytosis is dependant to actin polymerization and SRC phosphorylation. To find possible couples of molecules involved in this contact, we compared different transcriptomic data from NLC, monocyte, CLL cells and B lymphocytes. We highlighted potential couples of molecules and confirmed their expression on CLL cells and NLC by flow cytometry analysis. Finally, we obtained 3 couples probably implicated: Lymphocyte Function-Associated Antigen 3 (LFA-3)/CD2, Platelet/Endothelial Cell Adhesion Molecule 1 (PECAM1)/CD38 and Intercellular Adhesion Molecule 1 (ICAM-1)/LFA-1. Antibody blocking strategies revealed that LFA-3, which is up-regulated in CLL cells compared to healthy donors B lymphocytes, was necessary for the interaction between CLL cells/NLC when PECAM1, ICAM-1 and their co-partners were not essential (figure 1). Furthermore, this contact, through LFA-3, induced Akt phosphorylation but not ERK1/2 phosphorylation in CLL cells. Finally, we showed that LFA-3 and its receptor CD2 are necessary to the rescue of CLL cells by NLC (figure 2). To go further, we tested the chemoprotective effect of NLC on CLL cells. We showed that NLC slightly protect CLL cells against bendamustin but not against rituximab, dasatinib or ibrutinib. We hypothesized that the contact through LFA-3 could be involved in this chemoresistance. However, we did not observed a significant effect of the combination of bendamustin and LFA-3-blocking compared to bendamustin alone suggesting that this chemoprotection of CLL cells by NLC involved another pathway. Altogether, our results indicate that overexpression of LFA-3 by CLL cells and its critical implication in the interaction with NLC might be a new therapeutic target in CLL to disturb the interaction of CLL cells with their microenvironment. Figure 1: LFA-3 blocking but not ICAM-1 and PECAM1 decrease trogocytosis from NLC to CLL cells. a) Representative overlay of an experiment of trogocytosis from NLC to CLL cells treated or not by a blocking antibody anti-LFA-3. b) Representative overlay of an experiment of trogocytosis from NLC to CLL cells treated or not by a blocking antibody anti-ICAM-1. c) Representative overlay of an experiment of trogocytosis from NLC to CLL cells treated or not by a blocking antibody anti-PECAM1. Figure 1:. LFA-3 blocking but not ICAM-1 and PECAM1 decrease trogocytosis from NLC to CLL cells. a) Representative overlay of an experiment of trogocytosis from NLC to CLL cells treated or not by a blocking antibody anti-LFA-3. b) Representative overlay of an experiment of trogocytosis from NLC to CLL cells treated or not by a blocking antibody anti-ICAM-1. c) Representative overlay of an experiment of trogocytosis from NLC to CLL cells treated or not by a blocking antibody anti-PECAM1. Figure 2: LFA-3 is critical for the survival of CLL cells in contact with NLC. Figure 2:. LFA-3 is critical for the survival of CLL cells in contact with NLC. Disclosures No relevant conflicts of interest to declare.

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NOTCH2 Contributes to Venetoclax Resistance in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2019-128499 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4280-4280

Author(s):

Stefania Fiorcari ◽

Rossana Maffei ◽

Claudio Giacinto Atene ◽

Silvia Martinelli ◽

Leonardo Potenza ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Complete Response ◽

Lymphocytic Leukemia ◽

High Expression ◽

Leukemic Cells ◽

Complete Medium ◽

Intrinsic Resistance ◽

Durable Response ◽

Trisomy 12

BACKGROUND: Chronic lymphocytic leukemia (CLL) has recently experienced an unprecedented revolution thanks to the discovery of crucial pathogenetic mechanisms. Despite considerable therapeutic advancements, the eradication of the disease is not complete and resistance and transformation may occur. Venetoclax is a small-molecule BH3 mimetic that competes for binding in the hydrophobic groove of Bcl-2, a key protein involved in CLL cells survival. Venetoclax was shown to have an excellent antitumor activity in patients with relapsed CLL including those with high risk features. However, venetoclax monotherapy shows an overall response rate of 79% and complete response rates of 16% and leave some open questions mainly related to intrinsic features of CLL patients that may guide a pattern of resistance. A recent work shows that durable response to venetoclax based therapy is more likely in patients having minimal adenopathy, mutated IGHV gene, wild type TP53 and Notch1 genes. AIM OF THE WORK: We hypothesize that specific intrinsic features of CLL cells may contribute to drive possible mechanisms of resistance to venetoclax (ABT-199) treatment. METHODS: Notch2 expression was monitored by western blot in purified CLL cells. Modulation of Notch2 expression in vitro was performed by using siRNA strategy. ABT-199 was used at doses of 0.1 nM or 1 nM. RESULTS: Notch signaling is relevant in CLL pathogenesis. We detected a peculiar high expression of Notch2 in a subgroup of CLL patients carrying trisomy 12. The high expression of Notch2 correlated with high levels of Mcl-1, CD23 and Hes1. Interfering with Notch2 expression in trisomy 12 CLL patients, by siRNA silencing, was able to affect leukemic cells viability, reducing CD23 and Mcl-1 expression. Since Mcl-1 is involved in ABT-199 resistance, we wondered if ABT-199 may have a different effect in CLL cells isolated from patients carrying trisomy 12 comparing to no trisomy 12 cases. After 24h of culture in complete medium, trisomy 12 CLL cells showed a gain in survival rate of 10% during treatment with both 0.1 nM or 1 nM in comparison to no trisomy 12 cases. This advantage in viability reflected the maintenance of Notch2 and Mcl-1 expression in trisomy 12 both at 0.1 nM and 1 nM of ABT-199 compared to control. Conversely, a reduction of Notch2 and consequently Mcl-1 levels were observed in no trisomy 12 cases at both doses of ABT-199. We did not detect any variation in Bcl-2 levels. We wondered if Notch2 expression may reduce the response to ABT-199 in trisomy 12 CLL. To demonstrate this hypothesis, we interfered with Notch2 by silencing strategy. Treatment with Notch2 siRNA decreased the expression of Notch2 and Mcl-1 in combination with ABT-199 as shown in Figure 1A. We also found that Notch2 down-regulation cooperated with ABT-199 decreasing CLL cells viability (Figure 1B). CONCLUSIONS: Collectively, our results show a novel mechanism that may compromise the clinical response to venetoclax in CLL patients. Although the excellent mechanism of action of venetoclax, Bcl-XL and Mcl-1, two major anti-apoptotic proteins of Bcl-2 family not inhibited by venetoclax, are key determinants of both acquired and intrinsic resistance to treatment. The possibility to identify patients with more pronounced expression of Mcl-1 may help to optimize the treatment. The expression of Notch2 identify a subset of CLL patients, mainly harboring trisomy 12 aberration, that through the maintenance of high levels of Mcl-1, may be involved in a reduced response to ABT-199. Disclosures Luppi: Gilead Sci., MSD, Pfizer, Novartis, Abbvie, Sanofi, Daiichi Sankyo, Jazz Pharmaceuticals: Honoraria. Marasca:Janssen and Gilead Sci, Abbvie, Roche and Shire: Honoraria, Research Funding.

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Aberrant expression of B-lymphocyte stimulator by B chronic lymphocytic leukemia cells: a mechanism for survival

Blood ◽

10.1182/blood-2002-02-0558 ◽

2002 ◽

Vol 100 (8) ◽

pp. 2973-2979 ◽

Cited By ~ 164

Author(s):

Anne J. Novak ◽

Richard J. Bram ◽

Neil E. Kay ◽

Diane F. Jelinek

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

B Lymphocyte ◽

Leukemic Cell ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Aberrant Expression ◽

B Lymphocyte Stimulator

B-cell chronic lymphocytic leukemia (B-CLL) is defined by the accumulation of CD5+ B cells in the periphery and bone marrow. This disease is not characterized by highly proliferative cells but rather by the presence of leukemic cells with significant resistance to apoptosis and, therefore, prolonged survival. B-lymphocyte stimulator (BLyS) is a newly identified tumor necrosis factor (TNF) family member shown to be critical for maintenance of normal B-cell development and homeostasis and it shares significant homology with another TNF superfamily member, APRIL. The striking effects of BLyS on normal B-cell maintenance and survival raises the possibility that it may be involved in pathogenesis and maintenance of hematologic malignancies, including B-CLL. In this study, we investigated the status of APRIL and BLyS expression, as well as their receptors, in this disease. All B-CLL patient cells studied expressed one or more of 3 known receptors for BLyS; however, the pattern of expression was variable. In addition, we demonstrate for the first time that B-CLL cells from a subset of patients aberrantly express BLyS and APRIL mRNA, whereas these molecules were not detectable in normal B cells. Furthermore, we provide in vitro evidence that BLyS protects B-CLL cells from apoptosis and enhances cell survival. Because these molecules are key regulators of B-cell homeostasis and tumor progression, leukemic cell autocrine expression of BLyS and APRIL may be playing an important role in the pathogenesis of this disease.

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High incidence of monoclonal proteins in the serum and urine of chronic lymphocytic leukemia patients

Blood ◽

10.1182/blood.v64.6.1207.1207 ◽

1984 ◽

Vol 64 (6) ◽

pp. 1207-1211 ◽

Cited By ~ 49

Author(s):

MJ Deegan ◽

JP Abraham ◽

M Sawdyk ◽

EJ Van Slyck

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Membrane Immunoglobulin ◽

High Incidence ◽

Secretory Products ◽

Monoclonal Proteins ◽

Chain Type ◽

Serum And Urine

Abstract Chronic lymphocytic leukemia (CLL) is generally considered a nonsecretory B cell immunoproliferative disorder. Conventional electrophoretic and immunoelectrophoretic methods have revealed serum monoclonal proteins in less than 10% of these patients. However, there is increasing experimental evidence from in vitro studies demonstrating that CLL cells may secrete immunoglobulins, particularly free light chains. We examined the serum and urine of 36 consecutive CLL patients for monoclonal proteins using sensitive immunochemical methods (high resolution agarose gel electrophoresis combined with immunofixation). The results obtained were correlated with the Rai stage, quantitative immunoglobulin levels, and lymphocyte membrane immunoglobulin phenotype of the leukemic cells. Twenty-three monoclonal proteins were identified in the serum or urine of 22 patients, an incidence of 61%. Six patients had serum monoclonal proteins, seven had only urinary monoclonal proteins, and nine had monoclonal proteins in serum and urine. In every instance the monoclonal protein was the same light chain type as expressed on the leukemic cells. Our findings suggest that the monoclonal proteins observed in the serum or urine of CLL patients are secretory products of the tumor cells and that their discovery is a function of the sensitivity of the method used for their detection.

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Complement alternative pathway activation by chronic lymphocytic leukemia cells: its role in their hepatosplenic localization

Blood ◽

10.1182/blood.v63.2.463.463 ◽

1984 ◽

Vol 63 (2) ◽

pp. 463-467 ◽

Cited By ~ 12

Author(s):

F Praz ◽

G Karsenty ◽

JL Binet ◽

P Lesavre

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Alternative Pathway ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Complement Alternative Pathway ◽

Acetylneuraminic Acid ◽

Pathway Activation ◽

Do So

Abstract Using affinity-purified 125I-F(ab')2 anti-human C3, we have investigated the ability of various leukemic cells to activate complement. Lymphocytes from patients with chronic lymphocytic leukemia (CLL) activated the alternative pathway, but cells from patients with other forms of leukemia or normal lymphocytes did not do so. The amount of C3 deposited on the CLL cells was significantly higher in patients with organomegaly (i.e., splenomegaly and/or hepatomegaly). Activation of complement by CLL cells as assessed by C3 deposition on the membrane occurred both in vivo and in vitro and was not related to the N- acetylneuraminic acid content of the membrane.

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Fibromodulin as a Novel Tumor-Associated Antigen (TAA) in Chronic Lymphocytic Leukemia (CLL) Which Allows Expansion of Specific CD8+ Autologous T Lymphocytes.

Blood ◽

10.1182/blood.v104.11.175.175 ◽

2004 ◽

Vol 104 (11) ◽

pp. 175-175 ◽

Cited By ~ 5

Author(s):

Christine Mayr ◽

Dagmar Bund ◽

Martin Schlee ◽

Andreas Moosmann ◽

Michael Hallek ◽

...

Keyword(s):

T Cells ◽

In Vitro Culture ◽

B Lymphocytes ◽

Cd40 Ligand ◽

Immune Monitoring ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Autologous Tumor ◽

Tumor Associated Antigen

Abstract BACKGROUND: Fibromodulin (FMOD), a collagen binding protein, was shown to be highly overexpressed in CLL cells compared to normal B lymphocytes by gene expression profiling. Therefore FMOD might serve as potential tumor associated antigen (TAA) in CLL, enabling expansion of FMOD-specific T cells. FMOD is physiologically expressed in articular cartilage, tendon and ligament. Furthermore, interactions of FMOD with the growth factor TGF-b were described and it may be a biologically relevant modulator of TGF-b activity. Methods: Unpulsed native CLL cells and CD40 ligand (CD40L)-stimulated CLL cells as antigen presenting cells (APC) were used to expand autologous T cells from 13 patients. RESULTS: In CLL samples derived from 16 patients, high expression of FMOD by real-time RT-PCR was detectable in contrast to normal B lymphocytes. The number of T cells during four weeks of in vitro culture increased 2-fold with native CLL cells as APC and 3.5-fold with CD40L-stimulated CLL cells as APC. The amount of T cells recognizing HLA-A0201 binding FMOD-derived peptides detected by HLA-A2-dimer/peptide staining increased 10-fold during in vitro culture. The T cells expanded were also able to secrete IFN-g upon recognition of the antigen demonstrated by IFN-g-ELISPOT assays. T cells not only recognized HLA-A0201 binding FMOD peptides presented by TAP-deficient T2 cells, but also FMOD overexpressing autologous CLL cells in an HLA-A0201 restricted manner. Neither HLA-A0201 negative CLL cells nor non-malignant cells, i.e. PBMC from healthy donors or tonsilar B cells, were specifically recognized by T cells. When CD40L-stimulated CLL cells were used as APC, which were pulsed with FMOD peptides prior to coincubation with the T cells, significant higher amounts of T cells specifically recognized autologous CLL cells in IFN-g-ELISPOT assays P< 0.018). CONCLUSION: FMOD was shown for the first time to be naturally processed and ( presented as TAA in primary CLL cells. This enables the expansion of autologous tumor-specific T cells which might be applicable in clinical vaccination trials or as a tool for a CLL-specific immune monitoring in the context of vaccination approaches including CD40L-gene modified autologous leukemic cells.

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ZAP-70 Expression Is Associated with Increased Migration to SDF-1 in Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v108.11.587.587 ◽

2006 ◽

Vol 108 (11) ◽

pp. 587-587

Author(s):

Yuji Miura ◽

Elinor Lee ◽

Federica Gibellini ◽

Therese White ◽

Gerald Marti ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Cxcr4 Expression ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Short Exposure ◽

Mrna Levels ◽

Dependent Manner ◽

Western Blots ◽

Stroma Cell

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature B lymphocytes in the peripheral blood (PB), lymph nodes (LN) and bone marrow (BM). Increasing evidence suggests that CLL cells depend on survival and proliferation signals provided by stroma cells in LN and BM. The chemokine receptor CXCR4 (CD184) and its ligand stromal cell-derived factor-1 (SDF-1) play an important role in trafficking of lymphocytes and may guide CLL cells to stroma cell niches. ZAP70 expression has prognostic value in CLL but the functional consequences of ZAP70 expression remain incompletely defined. Given that ZAP70 has been implicated in CXCR4 signaling its expression could enhance migration to SDF-1 and thereby promote interactions with stroma cells. As measured by flow cytometry, CXCR4 expression on leukemic cells obtained from different anatomic sites differed; cells from the PB (n=24, median 71% above isotype control) expressed CXCR4 more strongly than cells from BM (n=21, median 39%) and from LN (n=9, median 24%). Expression of CD69, an activation marker, followed a reverse pattern with cells from LN and BM typically showing higher expression than cells from PB, albeit with not detectable difference in expression in several patients. In vitro CLL cells from PB migrated in a dose dependent manner to SDF-1, and cells that had migrated down-modulated CXCR4 expression (89% before migration - 54% after migration). After exposure to SDF-1 CXCR4 expression decreased rapidly and remained virtually absent for at least 24 hours. Several mechanisms apparently decrease CXCR4 expression after contact with SDF-1, including internalization (given rapid re-expression of CXCR4 when SDF-1 is washed off after short exposure), protein degradation or inhibition of translation (evidenced by a decrease in total CXCR4 protein on Western blots), and mRNA degradation or transcriptional inhibition (decrease in mRNA levels more than 6 hours from SDF-1 exposure). In vitro migration of ZAP70(+) CLL cells toward SDF-1 through a 5μm membrane (Migration Index [MI] of 12.0, n=5) was significantly increased compared to ZAP70(−) CLL cells (MI of 2.9, n=4, p<0.05). To exclude effects of contaminating cells we repeated these assays with purified CLL cells (negative selection) with similar results. To model the complex interactions of CLL cells with stroma, we cultured PB derived leukemic cells with or without murine marrow stroma cells (S17). CXCR4 expression on CD19+ cells decreased from 90% without S17 to 50% when cultured on S17 cells, consistent with the known SDF-1 secretion by the murine stroma cell line. Conversely, CD69 expression increased from 58% without S17 to 71% with S17 cells. In addition, culturing of CLL cells on an S17 stroma cell layer extended their survival by several weeks when compared to cultures without S17 cells. Our data is consistent with a model in which CLL cells migrate along an SDF-1 gradient to stroma cell niches in BM and LN where they are activated. ZAP70 expression is associated with more effective migration in an SDF-1 gradient and thereby may facilitate access to growth and survival signals which then could contribute to the more progressive nature of ZAP70(+) CLL. The interaction between leukemic cells and stroma may represent a novel target for therapy of patients with CLL.

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

Blood ◽

10.1182/blood.v114.22.2350.2350 ◽

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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