Transcriptional Mistargeting Of NFAT2 In CLL

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4122-4122
Author(s):  
Melanie Märklin ◽  
Jonas S. Heitmann ◽  
David Worbs ◽  
Alexandra Poljak ◽  
Claude Evouna ◽  
...  
Keyword(s):  
B Cells ◽  
Signaling Pathway ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Resting Cells ◽  
Qrt Pcr ◽  
Mutational Status ◽  
Healthy Donors

Abstract Chronic Lymphocytic Leukemia (CLL) is a hematological malignancy of mature B cells and constitutes the most common leukemia in adults. It is characterized by a progressive accumulation of clonal B cells, which coexpress CD19, CD23 and CD5. The clinical course of CLL can be predicted by serveral prognostic markers like CD38, ZAP70 and cytogenetic abnormalities. While the treatment of CLL has significantly improved during recent years, it remains an essentially incurable disease and the molecular events that lead to its development are still largely elusive. NFAT is a family of highly phosphorylated transcription factors residing in the cytoplasm of resting cells. Upon dephosphorylation NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. NFAT is inactivated by a network of several kinases. Several recent studies have demonstrated that Ca2+/NFAT signaling is involved in the pathogenesis of a wide array of different tumor types including pancreatic adenocarcinoma, breast cancer and Non Hodgkin´s lymphoma. In this study we investigated the significance of the Ca2+/NFAT signaling pathway in B-CLL. For this purpose, we analyzed CLL cell lines (MEC-1, JVM-3) as well as primary blood samples from patients with CLL (n=30). The analyzed patient population exhibited a representative distribution of age, sex, Binet stage, WBC count, cytogenetics and IGVH mutational status. We detected a profound overexpression of NFAT2 mRNA as well as NFAT2 protein in all CLL samples. Using qRT-PCR we found that CD19+CD5+ CLL cells exhibited an at least three fold overexpression of NFAT2 as compared to CD19+ B cells isolated from healthy donors. In one case, NFAT2 expression in CLL cells was 200 times higher than in the corresponding controls. This profound overexpression of NFAT2 in CLL cells could be confirmed on the protein level using Western Blotting and Immunocytochemistry. We could further demonstrate that even under resting conditions significant amounts of NFAT2 protein had translocated to the nucleus in CLL cells, whereas virtually all NFAT2 was in the cytoplasm in healthy B cells. NFAT2 nuclear translocation could be inhibited using pretreatment with Cyclosporin A demonstrating that this process was still calcineurin-dependent in CLL cells. We could further show that nuclear NFAT2 in CLL cells was able to bind DNA using electrophoretic mobility shift assays (EMSA). To assess the transcriptional activity of NFAT2 in human CLL we determined the expression of the apoptosis regulators OX40L, osteopontin and PD-L2, which we previously identified as NFAT2 target genes in a gene expression analysis with CD19+CD5+ CLL cells from TCL1 transgenic mice with intact NFAT2 and NFAT2 deletion, respectively. Interestingly, qRT-PCR revealed a tremendous reduction of all three target genes in the analyzed CLL samples as compared to control B cells from healthy donors. This is particularly remarkable, since in the TCL1 mouse model we observed a similar reduction of the expression of these genes in CLL cells with NFAT2 ablation. In summary, these results provide strong evidence that the Ca2+/NFAT signaling axis is constitutively activated in CD19+CD5+ CLL cells. Our data suggest that the profound overexpression of NFAT2 in CLL cells leads to its targeting to aberrant genetic loci different from its phsiological target genes resulting in a consecutive knock out phenotype with respect to the expression of the apoptosis regulators OX40, osteopontin and PD-L2 in CLL. Further investigation is therefore warranted to decipher the therapeutic potential of modulating the Ca2+/Calcineurin/NFAT signaling pathway in this disease. Disclosures: No relevant conflicts of interest to declare.

Aberrant NFAT2 signaling in chronic lymphocytic leukemia.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 7019-7019
Author(s):  
Jonas S. Heitmann ◽  
Melanie Maerklin ◽  
Alexandra Poljak ◽  
Bettina Hackl ◽  
Juliane Stickel ◽  
...  
Keyword(s):  
B Cells ◽  
Transcriptional Activity ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Therapeutic Potential ◽  
Binding Capacity ◽  
Lymphocytic Leukemia ◽  
Resting Cells ◽  
Mrna Levels ◽  
Qrt Pcr

7019 Background: CLL is a malignancy of mature B cells and constiututes the most common leukemia in adults. It is characterized by a progressive accumulation of clonal B cells, which coexpress CD19, CD23 and CD5. NFAT is a family of highly phosphorylated transcription factors residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. In this study, we investigated the significance of NFAT signaling in B-CLL. Methods: NFAT2 expression and aberrant nuclear translocation in CLL cells (n=30) was assessed by Western Blotting and immunofluorescence. In addition, NFAT2 mRNA levels were measured by qRT-PCR and its DNA binding capacity was assessed using an electrophoretic mobility shift assay. Transcriptional activity of NFAT2 proteins in CLL cells was further analyzed by determining the expression of several well characterized NFAT target genes. Results: We detected a profound overexpression of NFAT2 mRNA and protein in all CLL samples. Using qRT-PCR we found that CD19+CD5+ CLL cells exhibited a significant overexpression of NFAT2 as compared to CD19+ B cells isolated from healthy donors (8-200fold). This overexpression of NFAT2 in CLL cells could also be confirmed on the protein level. We could further demonstrate that even under resting conditions significant amounts of NFAT2 protein had translocated to the nucleus in CLL cells, whereas virtually all NFAT2 was in the cytoplasm in non-malignant B cells. Nuclear NFAT2 in CLL cells was able to bind DNA but its transcriptional activity with respect to several apoptosis-regulating genes (i.e. Spp1, Pdcd1) was severely compromised. Conclusions: These results provide strong evidence that the Ca2+/NFAT signalling axis is constitutively activated in CD5+CD19+ CLL cells. Reduced expression of several apoptosis regulators which are known target genes of NFAT2 links deregulation of this signaling cascade to CLL progression. Further investigation is warranted to investigate the therapeutic potential of modulating Ca2+/Calcineurin/ NFAT signaling in CLL.

Impaired Expression of p66Shc, a Novel Regulator of B-Cell Survival, in Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 801-801
Author(s):  
Cosima T. Baldari ◽  
Nagaja Capitani ◽  
Orso Maria Lucherini ◽  
Elisa Sozzi ◽  
Micol Ferro ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Apoptosis ◽  
Conflicts Of Interest ◽  
Group Analysis ◽  
Lymphocytic Leukemia ◽  
Expression Of Genes ◽  
Mutational Status ◽  
Healthy Donors

Abstract Abstract 801 Intrinsic defects in the apoptotic circuitry underlie to a large extent the extended survival of malignant B cells in chronic lymphocytic leukemia (CLL) and are moreover believed to be responsible for their resistance to chemotherapy. We have recently demonstrated that p66Shc, a member of Shc family of protein adapters, acts as a promoter of apoptosis in T cells. Here we show that p66Shc uncouples the B-cell antigen receptor (BCR) from the Erk and Akt dependent survival pathways, thereby enhancing B-cell apoptosis. Expression of p66Shc was found to be profoundly and consistently impaired in CLL B cells compared to peripheral blood B cells form healthy donors. Moreover, significant differences in p66Shc expression were observed in patients with favorable or unfavorable prognosis, classified on the basis of the mutational status of the IGHV genes, with the lowest expression in the unfavorable prognosis group. Analysis of the expression of genes previously implicated in the apoptosis defects of CLL B cells revealed a selective alteration in the balance of pro- and anti-apoptotic members of the Bcl-2 family in these patients. Reconstitution experiments in CLL B cells, as well as data obtained on B cells from p66Shc-/- mice, showed that p66Shc expression correlates with a bias in the Bcl-2 family towards the pro-apoptotic members. Collectively, the data identify p66Shc as a novel regulator of B cell apoptosis which attenuates survival signals emanating from the BCR and modulates expression of the Bcl-2 family. They moreover provide evidence that the defect in p66Shc expression identified in CLL B cells may be causally related to the imbalance towards the anti-apoptotic Bcl-2 family members observed in these cells. Disclosures: No relevant conflicts of interest to declare.

Overexpression and Aberrant Nuclear Translocation of NFAT2 in CLL

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3909-3909
Author(s):  
Jonas S. Heitmann ◽  
Melanie Märklin ◽  
Alexandra Poljak ◽  
Bettina S ◽  
Juliane Sarah Stickel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
Western Blotting ◽  
Transcriptional Activity ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Control Cell ◽  
Resting Cells ◽  
Mrna Levels ◽  
Qrt Pcr

Abstract Abstract 3909 Chronic Lymphocytic Leukemia (CLL) is a hematological malignancy of mature B cells and constitutes the most common leukemia in adults. It is characterized by a progressive accumulation of clonal B cells, which coexpress CD19, CD23 and CD5. The clinical course of CLL can be predicted by serveral prognostic markers like CD38, ZAP70 and cytogenetic abnormalities. While the treatment of CLL has significantly improved during recent years, it remains an essentially uncurable disease and the molecular events that lead to its development are still largely ellusive. NFAT is a family of highly phosphorylated transcription factors residing in the cytoplasm of resting cells. Upon dephosphorylation NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. NFAT is inactivated by a network of several kinases. Several recent studies have demonstrated that Ca2+/NFAT signaling is involved in the pathogenesis of a wide array of different tumor types including pancreatic adenocarcinoma, breast cancer and Non Hodgkin′s lymphoma. In this study we investigated the significance of the Ca2+/NFAT signaling pathway in B-CLL. For this purpose, we analyzed CLL cell lines (MEC-1, JVM-3) as well as primary blood samples from patients with CLL (n=30). The analyzed patient population exhibited a representative distribution of age, sex, Binet stage, WBC count, cytogenetics and IGVH mutational status. PBMC were obtained by ficoll density gradient centrifugation and B cells were subsequently isolated using magnetic bead technology. NFAT2 expression and aberrant nuclear translocation was then assessed by Western Blotting and Immunofluorescence staining. In addition, NFAT2 mRNA levels were measured by qRT-PCR and its DNA binding capacity was assessed using an electrophoretic mobility shift assay (EMSA). Transcriptional activity of NFAT2 proteins in CLL cells was further analyzed by determining the expression of several well characterized NFAT target genes by qRT-PCR and Western Blotting. In our analysis, we detected a profound overexpression of NFAT2 mRNA as well as NFAT2 protein in all CLL samples. Using qRT-PCR we found that CD19+CD5+ CLL cells exhibited an at least three fold overexpression of NFAT2 as compared to CD19+ B cells isolated from healthy donors. In one case, NFAT2 expression in CLL cells was 200 times higher than in the corresponding control cell population. This profound overexpression of NFAT2 in CLL cells could be confirmed on the protein level using Western Blotting and Immunocytochemistry. We could further demonstrate that even under resting conditions significant amounts of NFAT2 protein had translocated to the nucleus in CLL cells, whereas virtually all NFAT2 was in the cytoplasm in healthy B cells. NFAT2 nuclear translocation could be inhibited using pretreatment with Cyclosporin A demonstrating that this process was still calcineurin-dependent in CLL cells. We could further show that nuclear NFAT2 in CLL cells was able to bind DNA but that its transcriptional activity with respect to several apoptosis and cell cycle regulating genes was severely compromised when compared with healthy CD19+CD5+ B cells. In summary, these results provide strong evidence that the Ca2+/NFAT signaling axis is con-stitutively activated in CD19+CD5+ CLL cells. Reduced expression of several apoptosis and cell cycle regulating proteins which are known target genes of NFAT2 potentially links deregulation of this signaling cascade to CLL progression. This is in line with our observation that genetic loss of NFAT2 can induce acceleration of CLL in TCL1 mouse model. Further investigation is warranted to decipher the therapeutic potential of modulating Ca2+/Calcineurin/NFAT signaling in 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.

Regulation of NOTCH2 Signaling in B-CLL: Involvement of PKC-δ.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1440-1440
Author(s):  
Rainer Hubmann ◽  
Markus Duechler ◽  
Martin Hilgarth ◽  
Susanne Schnabl ◽  
Dita Demirtas ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Fate ◽  
Nuclear Translocation ◽  
Surface Expression ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Mobility Shift ◽  
Mutational Status ◽  
Low Concentrations ◽  
Electrophoretic Mobility Shift Assays

Abstract We have previously shown that NOTCH2 signaling is involved in the overexpression of CD23 in B-cell chronic lymphocytic leukemia (B-CLL) cells (Hubmann et al., BLOOD 2002 May 15;99(10):3742–7). NOTCH2 plays a determining role in the development/homeostasis of CD5+ B1 B-cells and of the related marginal zone (MZ) B2 B-cells, suggesting a potential role for NOTCH2 in B-CLL leukemogenesis. Using electrophoretic mobility shift assays (EMSA) we demonstrate that freshly isolated B-CLL patient samples (n=30) express an activated form of nuclear NOTCH2 (N2IC) irrespective of their prognostic marker profile (ie. IgVH mutational status, CD38 surface expression, cytogenetics). Although the majority of cultured B-CLL samples lose their N2IC activity within one day, DNA-bound N2IC complexes could be maintained by low concentrations of the PKC-stimulating phorbol esther PMA (1ng/ml). This was accompanied by the upregulation of CD23. The effect of PMA on N2IC activation and CD23 expression was abrogated by the PKC-δ inhibitor Rottlerin. Since wild type NOTCH2 signaling is regulated through binding to its ligand followed by γ-secretase mediated cleavage and release of the intracellular domain (N2IC), we induced NOTCH2 signaling by PMA in the presence or absence of the γ-secretase inhibitors (GSI) DAPT and compound E. Results demonstrated that the PMA-induced NOTCH2 activity is resistant to GSI treatment in 24 out of 30 B-CLL cases (80%). This suggests that the leukemic cells from the majority of B-CLL patients express an activated form of N2IC which is independent from γ-secretase cleavage and, thus, do not reqire NOTCH2 ligands for signaling. In conclusion, the results suggest that PKC-δ is involved in the activation/nuclear translocation of N2IC in B-CLL cells. The data may also suggest that deregulated NOTCH2 signaling is an early step in the development of B-CLL and might be critically involved in the aberrant response of the malignant clone to cell fate modulating stimuli acting through PKC.

CD137/4-1BB Is Inducibly Expressed On CLL B Cells through CD40 Signaling.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1266-1266
Author(s):  
Tetsuya Fukuda ◽  
Yukana Nakaima ◽  
Aya Kasubata ◽  
Ken Watanabe ◽  
Takatoshi Koyama ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Nuclear Translocation ◽  
Conflicts Of Interest ◽  
Cytotoxic T Cells ◽  
Cd40 Ligation ◽  
Activated T Cells ◽  
Blocking Antibody ◽  
Healthy Donors

Abstract Abstract 1266 Poster Board I-288 CD137/4-1BB, a TNFR family member is expressed on activated T cells as a co-stimulator. It transduces the signal for cell survival and differentiation and plays a crucial role in CD8 cytotoxic T cells. Recently, an increasing number of reports have indicated its important role on tumor immunity, and the studies of immunotherapy targeting CD137 are on going. Therefore, it is important to know the expression of CD137 and CD137L on malignant cells for the establishment of immunotherapy. As a first step, we examined CD137 expression on PBMCs from healthy donors and CLL patients. When PBMCs from healthy donors were stimulated with PMA and ionomycin, CD137 expression was induced not only on T cells but also on activated B cells. However, when PBMCs from CLL patients were stimulated in the same way, we could not detect CD137 induction on CLL B cells. Since more than 90% of lymphocytes in the patients were CLL B cells, it is conceivable that activated T cells were required to induce CD137 on B cells. To test this hypothesis we next co-cultured CLL cells with T cells activated with anti-CD3/CD28 antibody-coated beads. In this co-culture CD137 was induced on CLL B cells, and this induction was diminished by anti-CD154 blocking antibody. Furthermore, CD137 was inducibly expressed on CLL B cells after co-culture with HeLa cells transfected with CD154 gene. The induction of CD137 mRNA was also clearly detected by RT-PCR after this stimulation. This CD137 induction was more significantly observed on CLL B cells (n=14, MFIR 11.5±6.9) as compared with B cells from healthy donors (n=4, MFIR 3.7±0.6, p=0.001) or non-CLL B cell malignancies (n=9, MFIR 4.2±3.43, p=0.003). Stimulation of CD137 expressed on BJAB transfectants by co-culture with CD137L-transfected CHO cells induced a conspicuous nuclear translocation of p52, a non-canonical NF-κB factor. In agreement with this activation, the expression of survival factor BCL-XL was upregulated. Consequently, the CD137 signal augmented the survival of CD154 stimulated CLL B cells in vitro. CD40 ligation can induce anti-CLL immunity and reduce CLL cells clinically. These data suggest that the inducibly expressed CD137 may diminish the effectiveness. It is possible that the fine adjustment of these co-stimulators can lead more effective immunotherapy. Disclosures No relevant conflicts of interest to declare.

Relationship Between Expression of TLRs and Disease Activity in CLL.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4379-4379
Author(s):  
Wilma Barcellini ◽  
Anna Zaninoni ◽  
Francesca Guia Imperiali ◽  
Gianluigi Reda ◽  
Nicola Orofino ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
B Cells ◽  
Internal Control ◽  
Clinical Course ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Mutational Status ◽  
Tlr4 Gene ◽  
Autoimmune Phenomena

Abstract Abstract 4379 Background Toll-like receptors (TLRs) are major agents of innate immunity and initiators of adaptive immune response by acting as costimulatory signals for B cells and inducing maturation, proliferation and antibody production after pathogen recognition. They are also involved in the self-antigen recognition and could play a role in autoimmune phenomena. It is well known that chronic lymphocytic leukemia (CLL) is characterized by an increased incidence of autoimmune phenomena and immunodeficiency, which can greatly influence the disease outcome leading to a variable clinical course. Aims To evaluate the correlation between the gene expression of TLRs in 41 patients with CLL (mean age±SD 66±18 years, range 42-90, 15 female and 26 male), the clinical course of the disease and the expression of prognostic factors (mutational status of IgVH region, CD38 and ZAP70 expression, and cytogenetic alterations). Methods The gene expression of TLR4, TLR9, and TLR10 was studied. Total RNA was extracted from B cells of patients and controls (pool of 10 healthy donors), cDNA was synthesized, and real-time PCR was performed. For each reaction 50 ng of cDNA were mixed with 1.25μl of TaqMan primer/probe set and 10.25μl of TaqMan Universal Master Mix. The TLR4, TLR9, and TLR10 expression was normalized according to GAPDH as an internal control gene and it was expressed as percentage of control. Results TLR4 gene expression was significantly lower in CLL patients compared to controls (18±3%, mean±SE) while TLR9, and TLR10 gene expression was higher (3457±500% and 2897±440%, respectively). Moreover, considering “progressive” CLL patients (n=20), TLR4 gene expression was significantly lower compared with “indolent” ones (n=21) (10.2±2.2% vs 24.9±4.9%, p=0.01). Patients with unmutated IgVH expression and unfavorable cytogenetic alterations (mostly 11q-) showed even more reduced TLR4 gene expression, but the small numbers did not allow statistical evaluation. TLR4 gene expression was lower in patients with infective diathesis, although not significantly (11.5±2.9% vs 20.0±3.9).The 6 “progressive” CLL patients with autoimmune diseases (hemolytic anemia, Evan's syndrome and phemphigus) showed significantly higher TLR4 gene expression compared with “progressive” patients without autoimmune phenomena (17.7±4.4% vs 6.9±2.1%, p=0.02), possible expression of activated immune system. Conclusion The reduced TLR4 gene expression in CLL patients with “progressive” disease, unmutated IgVH status, and unfavourable cytogenetic alterations is consistent with a reduced ability to a proper immune response to Gram-negative bacterial lipopolysaccarides and consequent increased susceptibility to infections. Disclosures: No relevant conflicts of interest to declare.

Nucleophosmin-1 and Ribosome-Associated Components Are Constitutively Overexpressed in NOTCH1 Mutated IGHV Unmutated CLL

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3880-3880
Author(s):  
Michele Dal Bo ◽  
Federico Pozzo ◽  
Riccardo Bomben ◽  
Antonella Zucchetto ◽  
Erika Tissino ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Lymphocytic Leukemia ◽  
Amplification Refractory Mutation System ◽  
Qrt Pcr ◽  
Mutational Status ◽  
Frameshift Deletion ◽  
Uptake Assay ◽  
Notch1 Mutations

Abstract Abstract 3880 Background: activating mutations of NOTCH1 have been identified in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, with a higher frequency in unmutated IGHV (IGHV-UM) CLL, chemorefractory CLL and CLL in advanced disease phases. In CLL, all NOTCH1 mutations disrupt the C-terminal PEST domain and cause an accumulation of an active NOTCH1 isoform. Notably, about 80% of NOTCH1 mutations are represented by a CT frameshift deletion at nucleotides 7544–7545 (c.7544–7545delCT). Clinically, the presence of NOTCH1 mutations is an independent predictor of overall survival in CLL and identifies a subset of patients with particularly unfavourable prognosis (Rossi et al, Blood, 119, 521, 2012). Aim: to identify peculiar molecular and biological features of NOTCH1 mutated CLL in the context of IGHV-UM CLL. Methods: the presence of the c.7544–7545delCT NOTCH1 frameshift deletion was investigated by an ad-hoc amplification refractory mutation system (ARMS) PCR set up to obtain an amplicon specific for the NOTCH1 mutated form and a second amplicon as control. The percentage of NOTCH1 DNA in the context of the CLL clone was determined by quantitative real-time PCR (QRT-PCR), calculating the ratio between the amount of the specific NOTCH1 mutated amplicon and the amount of the control amplicon, the latter representing the total amount of NOTCH1 DNA irrespective of its mutational status. Gene expression profile (GEP) was performed by a one-color labeling strategy using the 4×44K Agilent platform. The differential expression of specific genes/proteins was validated by QRT-PCR, western blotting and immunohistochemistry. A BrdU uptake assay was used to evaluate proliferation of CLL cells by CpG/IL2 stimulation. Results: in a cohort of 380 IGHV-UM CLL, the c.7544–7545delCT NOTCH1 mutation was found in 83/380 (21.8%) cases. QRT-PCR revealed a percentage of NOTCH1 mutated DNA ranging from 1 to 37%. CLL cases carrying the c.7544–7545delCT NOTCH1 mutation (NOTCH1-Mut) showed higher NOTCH1 protein expression than CLL cases lacking NOTCH1-Mut employing monoclonal antibodies either recognizing the trans-membrane (mean fold increase=3) or the intra-citoplasmic (mean fold increase=2.1) NOTCH1 domain. A GEP comparing RNA from purified CLL samples of 5 NOTCH1-Mut CLL and 5 CLL lacking NOTCH1-Mut was performed, selecting the 5 NOTCH1-Mut cases among those with the higher percentages of NOTCH1 mutated DNA (percentages of NOTCH1 mutated DNA ranging from 15 to 37%). This approach selected the nucleophosmin 1 gene (NPM1) and genes codifying for several ribosomal proteins (RPS6, RPS10, RPS17, RPS28, RPSA, RPL7A, RPL18) as significantly up regulated in NOTCH1-Mut CLL cases. A higher expression of the above mentioned genes in NOTCH1-Mut CLL was validated in a wider series of 34 cases (18 NOTCH1-Mut cases; NPM1, p=0.03; RPS6, p=0.045; RPS10, p=0.048; RPS17, p=0.048; RPS28, p=0.049; RPSA, p=0.048; RPL7A, p=0.039; RPL18, p=0.041, respectively). Western blot analysis in 8 cases (4 NOTCH1-Mut cases) confirmed a higher NPM1 expression in NOTCH1-Mut cases (range of fold increase from 1.6 to 5.2) also at protein level. Consistently, lymph nodes preparations from NOTCH1-Mut CLL cases revealed a strong NPM1 staining both in nucleoli and cytoplasms. Finally, when stimulated in-vitro with the CpG/IL2 combination, NOTCH1-mut IGHV-UM CLL cells proliferated, as detected by a BrdU uptake assay (>10 fold increase over control), and up-regulated NPM1 both at transcript (mean fold increase=2.02 after 18 hours of CpG exposure, p=0.001) and protein (fold increase of 1.34 after 6 hours of CpG exposure) levels. Conclusion: NPM1 was identified as constitutively overexpressed in NOTCH1-Mut IGHV-UM CLL together with several ribosome-associated components. These findings are suggestive for an increased activity of the ribosomal machinery in NOTCH1-Mut IGHV-UM CLL as part of the molecular processes leading to control of CLL cell growth and survival in this clinically unfavourable disease subset. Disclosures: No relevant conflicts of interest to declare.

CCL3 and CCL4 Plasma Levels Correlate with Established Prognostic Markers in Chronic Lymphocytic Leukemia: Towards a Simple, ELISA-Based Assay for Risk Assessment.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 358-358
Author(s):  
Mariela Sivina ◽  
Elena Hartmann ◽  
Diana Krupnik ◽  
Ruth LaPushin ◽  
Michael Keating ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Plasma Levels ◽  
Prognostic Markers ◽  
Conflicts Of Interest ◽  
High Plasma ◽  
Lymphocytic Leukemia ◽  
White Blood Count ◽  
Mutational Status ◽  
The Mean

Abstract Abstract 358 During the induction of normal immune responses, activated B cells secrete the chemokines CCL3 and CCL4 for recruitment of regulatory T cells (Nat Immunol. 2:1126-32, 2001). This may represent a mechanism enabling cognate interactions between rare antigen-specific T and B cells. We previously reported that CCL3 and CCL4 RNA and protein are induced in CLL cells by co-culture with nurselike cells and after B cell antigen receptor (BCR) cross-linking (Blood 113:3050-8, 2009). We found higher levels of CCL3 and CCL4 in ZAP-70 positive cases, and CCL3 and CCL4 secretion was abrogated by the Syk inhibitor R406. These findings suggest that CCL3/CCL4 secretion by CLL cells correlates with the signaling capacity of the BCR. Also, we previously noticed higher plasma levels of CCL3 and CCL4 in CLL patients, when compared to healthy controls. In this study, we measured CCL3 and CCL4 plasma levels by ELISA in 351 CLL patients, and correlated CCL3 and CCL4 levels with various prognostic markers, including RAI stage, immunoglobulin heavy chain variable region gene (IgVH) mutational status, ZAP-70, β2 microglobulin, CD38, white blood count (WBC), and cytogenetic subgroups. We found that the concentrations of CCL3 and CCL4 were significantly higher in plasma samples from CLL patients who had CLL cells that used unmutated IgVH genes or that expressed ZAP-70 or CD38, or who had an advanced stage of their disease (see Table 1). For example, the mean CCL3 plasma level was 56.4 ± 8.8 pg/ml in patients with CLL cells that used unmutated IgVH (mean ± SEM, n=123) versus 14.5 ± 2.4 in patients with CLL cells that used mutated IgVH (mean ± SEM, n=139, p<0.001). The mean CCL4 level was 171.4 ± 26.3 in patients that had CLL cells that used unmutated IgVH and 92.5 ± 17.5 in patients that had CLL that expressed mutated IgVH. On the other hand, the relative absolute white cell count or presence of chromosomal abnormalities did not correlate with high plasma levels of CCL3 or CCL4. Immunohistochemistry revealed that CCL4 was predominantly expressed in proliferation centers in approximately 50% of the cases. Currently, we are evaluating whether high plasma levels of CCL3 and/or CCL4 are associated with a relatively short time from diagnosis to requiring initial treatment by the IWCLL-working group criteria. Also, we are exploring whether high plasma levels of CCL3 or CCL4 are associated with high proportions of infiltrating T cells in proliferation centers. These studies suggest that patients can be stratified by their relative plasma levels of CCL3 and/or CCL4 and that high plasma-levels of theses chemokines define a characteristic that may be associated with aggressive disease. Disclosures: No relevant conflicts of interest to declare.

HMGB1 Activates TLR9/RAGE Signalling Pathway and Sustains Chronic Lymphocytic Leukemic Cell in Vitro Survival

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3860-3860
Author(s):  
Amani Al-Malti ◽  
John G. Gribben ◽  
Li Jia
Keyword(s):  
Pattern Recognition ◽  
B Cells ◽  
Cell Survival ◽  
Nuclear Translocation ◽  
Conflicts Of Interest ◽  
Fluorescent Microscopy ◽  
Pattern Recognition Receptors ◽  
Lymphocytic Leukemia ◽  
Advanced Glycosylation End Products

Abstract Abstract 3860 Extracellular HMGB1 (High mobility group box 1 protein) functions as an inflammatory cytokine which stimulates monocytes to produce pro-inflammatory cytokines. HMGB1 is a ubiquitous non-histone nuclear protein and can be passively released by necrotic or late apoptotic cells and actively released by macrophages, monocytes, NK cells, and dendritic cells in response to exogenous and endogenous inflammatory stimuli. As a DNA chaperon, HMGB1 binding to pathogen DNA can be recognized by several pattern recognition receptors, such as Toll-Like Receptor (TLR) 2, TLR4, TLR9 and Receptor for Advanced Glycosylation End products (RAGE) resulting in activation of intracellular survival signaling pathways. It was recently reported that HMGB1-DNA complex binds to RAGE on B-cells and leads to the recruitment of TLR9 and enhances B-cell survival and proliferation. Overexpression of HMGB1 is associated with each of the hallmarks of cancer including unlimited replicative potential, angiogenesis, evading apoptosis, self-sufficiency in growth signals, inflammation, tissue invasion and metastasis. Elevation of serum HMGB1 has been detected in many cancer patients and the levels of serum HMGB1 increases after anti-cancer therapy. However, the potential roles of HMGB1 and the integrity of TLR9/RAGE pathway have not been studied in chronic lymphocytic leukemia (CLL). CLL cells partially undergo spontaneous apoptosis when cells are cultured in vitro and this apoptosis can be decreased by addition of monocytes-derived nurse-like cells (NLCs). We aimed to investigate whether HMGB1 plays a role NLCs preventing apoptosis of CLL cells. The aims of this study were to determine whether HMGB1 is actively released by NLCs and whether CLL cells express pattern recognition receptors for HMGB1. After culture of CLL cells for one week, NLC formation was observed by MTT staining and phase contrast microscopy. HMGB1 and its receptors TLR9 and RAGE were constitutively expressed in all CLL samples studied. HMGB1 was initially expressed in the nucleus of both NLCs and CLL B-cells as detected by immuno-fluorescent microscopy. However, after one week of culture, NLCs started losing nuclear HMGB1 without changing their cellular structure. In NLC surrounding CLL cells, the HMGB1 receptors TLR9 and RAGE co-localized and formed large aggregates, indicating activation of the TLR9/RAGE signal pathway. This was followed by NF-kB (p65) nuclear translocation from its cytosolic localization in fresh CLL cells. To confirm whether the activation of TLR9/RAGE pathway is mediated by HMGB1, recombinant HMGB1 was used to stimulate CLL cells in vitro. Increased HMGB1/RAGE, HMGB1/TLR9 and TLR9/RAGE intracellular aggregations, TLR9 internalization, and NF-kB nuclear translocation, all essential for HMGB1-mediated activation of TLR9/RAGE pathway, were observed after 3 hours of incubation with HMGB1. HMGB1 also promoted CLL cell in vitro colony formation. Blocking HMGB1-induced TLR6/RAGE activation by anti-RAGE antibody reduced NLC-mediated CLL cell survival. In summary, we report, for the first time, that NLCs release HMGB1, activating the TLR9/RAGE pathway in CLL cells. HMGB1 plays an important role in CLL cell survival by activation of TLR9/RAGE pathway and we propose that HMGB1 may be a potential target for treatment of CLL. Disclosures: No relevant conflicts of interest to declare.

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