Lumiliximab, An Apoptosis Sensitizer to Multiple CLL Therapeutic Agents

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4209-4209
Author(s):  
Ann Maclaren ◽  
Amy Trauernicht ◽  
Lizbeth Nguyen ◽  
Karen McLachlan
Keyword(s):  
Monoclonal Antibody ◽  
Cell Death ◽  
B Cells ◽  
B Cell ◽  
Single Agent ◽  
Lymphocytic Leukemia ◽  
Lymphoid Tissues ◽  
Lymphoma Cells ◽  
Treatment Regimens ◽  
Apoptotic Pathways

Abstract Chronic lymphocytic leukemia (CLL) is a B cell malignancy characterized by the accumulation of mature phenotype leukemic B cells in blood, spleen and lymphoid tissues. While many patients respond initially to combination chemotherapy regimens, many become chemoresistant and all will ultimately relapse. Recently the addition of novel monoclonal antibody therapies such as rituximab and alemtuzumab to these treatment regimens has provided additional therapeutic benefit to chemorefractory CLL patients and prompted interest in the evaluation of additional B cell surface antigens as targets. Lumiliximab is a primatized monoclonal antibody directed against CD23, a glycoprotein expressed on the majority of CLL cells, and is currently under investigation in patients with relapsed CLL. It was previously demonstrated that the primary mechanism of action of lumiliximab in both CD23+ lymphoma B cells and CLL patient samples is sensitization to apoptotic cell death and that lumiliximab enhances apoptosis in vivo when combined with either fludarabine or rituximab (Pathan et al., Blood, 2008). In the present study we sought to determine whether lumiliximab could enhance the apoptotic activity of a range of CLL therapies which induce cell death via distinct apoptotic pathways. Our studies demonstrate that the addition of lumiliximab in combination with the alkylating agent chlorambucil resulted in a dose-dependent and significant increase in apoptosis of CD23+ lymphoma cells. Lumiliximab also resulted in statistically significantly enhanced apoptosis when combined with alemtuzumab as compared to either single agent alone in both CD23+/CD52+ lymphoma cells and CLL patient samples. Examination of the apoptotic pathways induced by these agents revealed that lumiliximab in combination resulted in more dramatic alterations in downstream effectors of apoptosis such as caspase 3, PARP, and DNA fragmentation. Further studies are ongoing to confirm these observations in xenograft models and to delineate the mechanistic basis of the enhanced apoptotic signaling. These data suggest that the use of lumiliximab in combination with current or emerging CLL therapies could be an effective strategy to augment tumor cell killing and may result in new and more effective treatment regimens for the eradication of CLL.

Overexpression of the CXCR5 chemokine receptor, and its ligand, CXCL13 in B-cell chronic lymphocytic leukemia

Blood ◽  
2007 ◽  
Vol 110 (9) ◽  
pp. 3316-3325 ◽  
Author(s):  
Andrea Bürkle ◽  
Matthias Niedermeier ◽  
Annette Schmitt-Gräff ◽  
William G. Wierda ◽  
Michael J. Keating ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Actin Polymerization ◽  
Serum Levels ◽  
Lymphocytic Leukemia ◽  
Lymphoid Tissues ◽  
Accessory Cells ◽  
Mitogen Activated Protein ◽  
B1 Cells

Abstract CXCL13 is a homeostatic chemokine for lymphocyte homing and positioning within follicles of secondary lymphoid tissues, acting through its cognate receptor, CXCR5. Moreover, the CXCR5-CXCL13 axis plays a unique role in trafficking and homing of B1 cells. Here, we report that chronic lymphocytic leukemia (CLL) B cells express high levels of functional CXCR5. CXCR5 expression levels were similar on CLL B cells and normal CD5+ B cells, and higher compared with normal CD5− B cells, follicular B-helper T cells (TFH cells), or neoplastic B cells from other B-cell neoplasias. Stimulation of CLL cells with CXCL13 induces actin polymerization, CXCR5 endocytosis, chemotaxis, and prolonged activation of p44/42 mitogen-activated protein kinases. Anti-CXCR5 antibodies, pertussis toxin, and wortmannin inhibited chemotaxis to CXCL13, demonstrating the importance of Gi proteins and PI3 kinases for CXCR5 signaling. Moreover, CLL patients had significantly higher CXCL13 serum levels than volunteers, and CXCL13 levels correlated with β2 microglobulin. We detected CXCL13 mRNA expression by nurselike cells, and high levels of CXCL13 protein in supernatants of CLL nurselike cell cultures. By immunohistochemistry, we detected CXCL13+ expression by CD68+ macrophages in situ within CLL lymph nodes. These data suggest that CXCR5 plays a role in CLL cell positioning and cognate interactions between CLL and CXCL13-secreting CD68+ accessory cells in lymphoid tissues.

scholarly journals Expression of Lyt-1 antigen on certain murine B cell lymphomas.

1981 ◽  
Vol 153 (4) ◽  
pp. 998-1003 ◽  
Author(s):  
L L Lanier ◽  
N L Warner ◽  
J A Ledbetter ◽  
L A Herzenberg
Keyword(s):  
Monoclonal Antibody ◽  
B Cell ◽  
Cell Lineage ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Lymphoid Tissues ◽  
Specific Marker ◽  
Human Homologue ◽  
B Cell Lymphomas ◽  
Cell Sorter

Although the Lyt-1 antigen has previously been considered a T cell-specific marker, recent evidence suggests that a population of Thy-1-, Lyt-1+ cells exists in normal lymphoid tissues. In this study, we have observed that the WEHI-55, WEHI-259, and CH5 B cell lymphomas express high levels of the Lyt-1 antigen, as detected by monoclonal antibodies using the fluorescence activated cell sorter. Three other B cell lymphomas of the 11 examined also gave weak but detectable reactions with the anti Lyt-1 monoclonal antibody. Except for the expression of the Lyt-1 antigen, these lymphomas are typical of cells in the B cell lineage with respect to surface phenotype. The Lyt-1 glycoprotein immunoprecipitated from metabolically labeled WEHI-55 cells is similar in structure to the Lyt-1 glycoprotein on thymocytes. These findings are similar to recent reports that B-type human lymphocytic leukemia cells express the putative human homologue of Lyt-1, the Leu-1 antigen.

Role Of Lyn Kinase In The Pathogenesis Of Chronic Lymphocytic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 668-668
Author(s):  
Phuong-Hien Nguyen ◽  
Nina Reinart ◽  
Michael Hallek
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Lymphocytic Leukemia ◽  
Negative Regulator ◽  
Lymphoid Tissues ◽  
Malignant Cells ◽  
Deficient Mice

Abstract The Src family kinase Lyn is predominantly expressed in B cells and plays a central role in initiating B cell receptor (BCR) signaling. Lyn is associated with BCR complexes and is renowned for its role in B cell activation and proliferation. Active Lyn contributes to positive regulation of signalling through tyrosine phosphorylation of components of the BCR. Intriguingly, Lyn was also shown as a negative regulator of BCR signal transduction. Lyn plays an essential role in negative regulation of signalling through its unique ability to phosphorylate immunoreceptor tyrosine based inhibition motifs (ITIM) in inhibitory cell surface receptors. ITIM phosphorylation induces the recruitment of inhibitory phosphatases such as SHP-1/2 and SHIP-1, which attenuate BCR signalling. Lyn-deficient mice have reduced number of B cells and increased numbers of myeloid progenitors. It was reported that expression and activity of Lyn in human chronic lymphocytic leukemia (CLL) is elevated compared to healthy B cells. Besides, higher levels of Lyn are associated with a shorter treatment-free survival of CLL patients. This rises up a hypothesis about Lyn’s significant role in B cell tumorigenesis, malignant transformation of B cells, and the balance between myeloid cells and B lymphocytes. We generated Eµ-TCL1 transgenic LYN-deficient mice (TCL1+/wtLYN-/-) and monitored them in order to identify the population of malignant B cells and to characterize the development of malignant cells in these mice in comparison with Eµ-TCL1 transgenic mice (TCL1+/wtLYNwt/wt). In comparison to TCL1+/wtLYNwt/wt mice, TCL1+/wtLYN-/- mice show a significantly reduced number of malignant B cells in the peripheral blood, as well as a reduced leukocyte count. Besides, TCL1+/wtLYN-/- mice have significantly decreased infiltration of malignant B cells in lymphoid tissues such as spleen, liver, lymph node and bone marrow. This result is also resembled in a hepato-splenomegaly in the TCL1+/wtLYNwt/wt mice. These mice develop severe splenomegaly and hepatomegaly due to infiltration of malignant cells, while TCL1+/wtLYN-/- mice do not develop hepatomegaly. The non-transgenic LYN-/- control mice develop splenomegaly due to infiltration of myeloid cells. Although TCL1+/wtLYN-/- mice have hindered development of TCL1-induced CLL, preliminary data suggest it is not only due to LYN-deficiency in B cell compartment of these mice. Indeed, B cell of TCL1+/wtLYN-/- mice show enhanced proliferation and better survival ex vivo compared to TCL1+/wtLYNwt/wt mice. Notably, TCL1+/wtLYN-/- mice developed a skewed microenvironment which might contribute to CLL down regulation. LYN-/- microenvironment, particularly in aged mice, does not support engraftment of TCL1-induced leukemic B cell as well as LYNwt/wt mice in our transplantation model. These results point to a complex regulation of Lyn signalling in CLL involving not only leukemic cells but also cells of the micromillieu, that needs further investigation. Disclosures: No relevant conflicts of interest to declare.

Phase I/II study of MEDI-551, a humanized monoclonal antibody targeting CD19, in subjects with relapsed or refractory advanced B-cell malignancies.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 8065-8065 ◽  
Author(s):  
Trishna Goswami ◽  
Andres Forero ◽  
Mehdi Hamadani ◽  
Anne Sonet ◽  
Gregor Verhoef ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Phase I ◽  
B Cell ◽  
Transmembrane Protein ◽  
Single Agent ◽  
Tumor Lysis Syndrome ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
B Cell Malignancies ◽  
Antibody Targeting

8065 Background: Novel B-cell targeting agents, including monoclonal antibodies such as rituximab, are among recent advances in treatment of B-cell malignancies. New approaches are needed for patients progressing after rituximab-based therapies. MEDI-551 is an afucosylated monoclonal antibody targeting CD-19, a B-cell restricted transmembrane protein with enhanced affinity and antibody-dependent cellular cytotoxicity. Methods: Pts with relapsed or refractory follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia, or multiple myeloma received single agent MEDI-551 at dosages ranging from 0.5 mg/kg to 12 mg/kg via intravenous infusion over 28-day cycles; cohorts 1-6 received 0.5, 1, 2, 4, 8, and 12 mg/kg, respectively. Results: 25 pts were enrolled in the phase I portion Jun 2010–Aug 2011. No maximum tolerated dose (MTD) was achieved. Most AEs were grade 1/2 with dose-independent frequency and severity (Table). Six pts had grade 3 toxicities including tumor lysis syndrome, infusion reaction, thrombocytopenia, and neutropenia, or grade 4 neutropenia. No grade 5 AEs were seen. All pts recovered. Three partial responses (PR) and 2 complete responses (CR) were seen in DLBCL and FL pts at 0.5, 4, and 8 mg/kg. Activity included a CR lasting 9 mo. in a FL pt in cohort 1, who is currently being retreated with MEDI-551 on relapse. Conclusions: MEDI-551 demonstrated a safety profile warranting further study and showed no MTD reached at the highest dose studied. Anti-tumor activity is suggested by the responses achieved across dose levels. Phase II is currently enrolling subjects. This study is funded by MedImmune, LLC. [Table: see text]

scholarly journals Statins Potentiate the Cytotoxic Effect of ABT-199 in Diffuse Large B Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3969-3969
Author(s):  
David A. Fruman ◽  
Jong-Hoon Scott Lee ◽  
Thanh-Trang T Vo ◽  
Shruti Bhatt ◽  
Jonathan H. Schatz ◽  
...  
Keyword(s):  
B Cell ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Hematologic Malignancies ◽  
Lymphocytic Leukemia ◽  
Lymphoma Cells ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract BCL-2 is a key pro-survival protein that is highly expressed in many leukemias and lymphomas. ABT-199 (venetoclax) is a small molecule inhibitor of BCL-2 that has demonstrated impressive responses in chronic lymphocytic leukemia (CLL) leading to FDA approval for second line treatment of patients with 17p deletion. However, other hematologic malignancies are less responsive to ABT-199 as a single agent, suggesting that combinations of targeted therapies may be required to elicit more promising responses. We have investigated the potential of combining ABT-199 with HMG-CoA reductase (HMGCR) inhibitors (statins), which have known anti-cancer potential in hematologic malignancies. Using multiple chemically distinct statin compounds, we observed profound synergistic induction of apoptosis when combined with ABT-199 in both human diffuse large B cell lymphoma (DLBCL) as well as acute myeloid leukemia (AML) cell lines. This synergy was also seen in primary murine B lymphoma cells over-expressing MYC and BCL-2. Importantly, addition of exogenous mevalonate completely rescued cells from the combination, confirming on-target efficacy of HMGCR inhibition. Using BH3 profiling, we found that simvastatin significantly primed lymphoma cells for undergoing apoptosis (termed mitochondrial priming). Notably, the degree of priming correlated with its ability to synergize with ABT-199, suggesting that BH3 profiling may be used to predict patient responses. The combination did not synergize to kill normal human peripheral blood mononuclear cells from healthy donors, suggesting that statins may selectively prime cancer cells for apoptosis. Mechanistic studies support the hypothesis that statins synergize with ABT-199 by suppressing protein prenylation, particularly protein geranylgeranylation. In support, the addition of exogenous geranylgeranyl pyrophosphate (GGPP) completely rescued cells from the effects of simvastatin. Furthermore, selective inhibition of protein geranylgeranyl transferase (GGT) increased priming and was sufficient to recapitulate the effects of simvastatin in combination with ABT-199. Statins and GGT inhibitors increased the mitochondrial abundance of a subset of BH3-only pro-apoptotic proteins. Lastly, we have identified Rap1A de-prenylation as a marker of pharmacodynamic response to statins in vivo. Thus, this project highlights a novel combination for use in aggressive lymphomas, establishes its efficacy and tolerability using preclinical models, and provides proof-of-concept to warrant investigation of its clinical potential. Disclosures Letai: AbbVie: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding.

Analysis of PRAME Protein Expression in Normal Lymphoid Tissues and during B Ontogenesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3924-3924
Author(s):  
Lorena L. de Figueiredo-Pontes ◽  
Fabio M. do Nascimento ◽  
Rodrigo S. de Abreu e Lima ◽  
Rodrigo Proto-Siqueira ◽  
Aglair B. Garcia ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Protein Expression ◽  
B Cell ◽  
Mononuclear Cells ◽  
Hematologic Malignancies ◽  
Lymphocytic Leukemia ◽  
Lymphoid Tissues ◽  
Healthy Donors ◽  
Chronic Lymphoproliferative Diseases

Abstract PRAME (Preferentially Expressed Antigen in Melanoma) gene was originally isolated in melanoma. A significant increase in the number of PRAME transcripts has been demonstrated in hematologic malignancies such as acute myeloid and lymphoid leukemias, multiple myeloma and chronic lymphoproliferative diseases. Furthermore, our group generated an anti-PRAME monoclonal antibody (MoAb) and by quantitative flow cytometry has demonstrated that PRAME protein was aberrantly expressed in Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma. However, the expression of this antigen in normal lymphoid tissues and during B cells ontogeneis has not been characterized. To address this question, PRAME protein expression was studied by flow cytometry in peripheral blood (PB, n=15) and bone marrow (BM, n=6) from healthy donors, lymphonodes (n=4) and spleen (n=4) from patients submitted to lymphonode excision or splenectomy for non malignant diseases. First, we determined in which hematopoietic lineage PRAME was expressed by concomitantly staining PB, BM, lymphonode and spleen mononuclear cells (MCs) with anti-PRAME and a panel of MoAbs specific to B(CD19)/ T(CD3)/ NK(CD16/56), monocytic(CD14) and granulocytic(CD33) markers. PRAME was detected exclusively in CD19+ cells. The median percenatge of PRAME positive cells was 5,31% (2,55–12,34%), 13,01% (8,47–38,15%), 12,79% (3,15–23,06%) and 17,5% (12,67–27,43%) in PB, BM, lymphonode and spleen MCs, respectively. Amongst CD19+ cells, we have observed that PRAME was expressed by 42,39% (16,16–75,72%), 16% (13–69,5%), 15,16% (5,49–41,20%) and 48,82%(12,67–58,89%) in PB, BM, lymphonode and spleen, respectively. To establish in which stage of B ontogenesis PRAME was expressed on, cell suspensions stained with anti-CD19 were submitted to positive magnetic separation and labeled with anti-PRAME, CD5, CD27, CD38, CD34, CD10 and IgD MoAbs. PRAME+/CD19+ cells were CD5−, CD27+, CD38+, CD34−, CD10− and IgD+, thus suggesting that PRAME is expressed by the memory B cell compartment of the normal lymphoid tissues. This study defines PRAME as a B cell antigen that may accompany the neoplastic clone proliferation of mature B cell neoplasms. Although PRAME is mainly an embryonic antigen, expressed by carcinomas of immature phenotype, it is expressed by mature B cells in normal and pathological lymphoid tissues. Our findings suggest that maturational events occurring at the germinal center of lymphoid follicles affects PRAME expression.

scholarly journals Autoantibody-associated cross-reactive idiotype-bearing human B lymphocytes: distribution and characterization, including Ig VH gene and CD5 antigen expression

Blood ◽  
1991 ◽  
Vol 78 (6) ◽  
pp. 1503-1515 ◽  
Author(s):  
G Inghirami ◽  
DR Foitl ◽  
A Sabichi ◽  
BY Zhu ◽  
DM Knowles
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Cell Subset ◽  
Antigen Expression ◽  
Lymphocytic Leukemia ◽  
Cell Subpopulation ◽  
Lymphoid Tissues ◽  
Vh Gene

Abstract Monoclonal antibodies (MoAbs) specific for autoantibody associated cross-reactive idiotypes (CRIs) frequently recognize the Igs of neoplastic B cells in patients with chronic lymphocytic leukemia (CLL) and/or Waldenstrom's macroglobulinemia. Very little is known regarding the normal B cells expressing CRIs (CRI-positive B cells). Using a variety of MoAbs against CRIs we investigated the distribution and topographic localization of CRI-positive B cells in normal adult human lymphoid tissues. We found that CRI-positive B cells represent a significant B-cell subpopulation expressing surface IgM (greater than 90%), IgG (approximately 5%), or IgA (approximately 2%). CRI-positive B cells are homogeneously distributed throughout all lymphoid tissues, accounting for 10% to 15% of all B lymphocytes, with the exception of the thymus, in which they represent the predominant B cell population. Immunophenotypic studies showed (1) that a small subpopulation (3.7% +/- 0.8%) of CRI-positive B cells are activated in vivo, based on CD25 and CD38 antigen expression; and (2) that approximately 50% of CRI-positive B cells express the 67-Kd pan-T-lymphocyte CD5 antigen, suggesting that the CRI-positive B-cell subset and the recently described CD5-positive B-cell subset are closely related. This hypothesis is supported by the fact that CRI-positive B cells produce oligo or polyreactive Igs, which are a characteristic feature of CD5-positive B cells, and also by the fact that both B-cell subpopulations appear to use similar and restricted Ig VH gene family members.

scholarly journals A Novel Molecular Complex Expressed on Immature B Cells: A Possible Role in T Cell-Independent B Cell Development

1996 ◽  
Vol 5 (1) ◽  
pp. 67-78 ◽  
Author(s):  
Philip J. Griebel ◽  
Paolo Ghia ◽  
Ulf Grawunder ◽  
Giorgio Ferrari
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Molecular Complex ◽  
Cell Development ◽  
B Cell Development ◽  
Lymphoid Tissues

To identify surface molecules that may play a role in regulating ileal Peyer's patch (PP) B cell growth, we generated monoclonal antibodies (mAbs) and then selected them for a unique reactivity with ileal PP B cells. Flow cytometric analysis identified a mAb (SIC4.8R) that labeled 97% of ileal and 50–60% of jejunal PP sIgM+B cells. SIC4.8R also labeled a subpopulation of cortical thymocytes but few B or T cells in other lymphoid tissues, including bone marrow. Immunohistochemistry revealed intense SIC4.8R staining of B cells in the cortex of ileal PP follicles. SIC4.8R also labeled bovine PP B cells, a murine pro-B cell line, and pre-B cells in human bone marrow. Protein chemistry revealed that a structurally similar molecular complex was expressed on sheep ileal PP B cells and thymocytes and murine pro-B cells. Addition of soluble SIC4.8R to cultured ileal PP B cells reduced apoptotic cell death, elevated proliferative responses, partially inhibited anti-Ig-induced cell death, and induced IL-4 responsiveness. In contrast, soluble SIC4.8R had an antiproliferative effect on a mouse pro-B cell line. Finally, SIC4.8R labeling declined following the stimulation of ileal PP B cells with CD40 ligand. In conclusion, the present investigation determined that SIC4.8R identified a novel molecular complex that is expressed at several stages of T cell-independent B cell development in a variety of mammalian species. This observation confirmed that PP B cells are developmentally distinct from other B cell populations in sheep and suggested that the bone marrow may not be a site of B lymphopoiesis in young lambs.

CD52 Expression Patterns in Normal and Neoplastic B-Cells: Myeloma Is an Unlikely Target for Single Agent Alemtuzumab Therapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4883-4883
Author(s):  
A. C. Rawstron ◽  
G. Laycock-Brown ◽  
F. E. Davies ◽  
R. G. Owen ◽  
P. Hillmen ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Single Agent ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Normal Controls

Abstract Alemtuzumab is a highly effective monoclonal antibody therapy for some B-cell disorders, and has been suggested as a possible therapeutic agent for treatment of myeloma. Monoclonal antibody therapeutic efficacy is closely associated with the expression level of the therapeutic target, as demonstrated by the lack of efficacy of single-agent rituximab in CLL. However, there are conflicting reports about the expression levels of CD52, the target for alemtuzumab, in plasma cell disorders. The aim of this study is to assess a large series of cases of plasma cell and B-cell disorders, utilising a standard approach to allow comparison of the target molecule. Plasma cells were assessed from patients with myeloma at presentation or relapse (n=106), monoclonal gammopathy of undetermined significance (MGUS, n=34), and from normal controls (n=19). In addition, B-cells were assessed from patients with chronic lymphocytic leukaemia (CLL, n=87), diffuse large B-cell lymphoma (DLBCL, n=10), follicular lymphoma (FCL, n=9), Waldenstroms macroglobulinaemia (WM, n=20), and also from normal bone marrow (n=37). Normal and neoplastic B-cells showed expression of CD52 (>20% of cells above the CD3-control levels) in all patients except for 1/10 DLBL. B-CLL and WM are known to show responses to single-agent alemtuzumab therapy, and these two disorders had the highest levels of expression. In contrast, B-progenitor cells in normal bone marrow are unaffected by alemtuzumab, and proliferate during alemtuzumab treatment in CLL patients. The levels of CD52 expression by normal B-progenitors were 3-fold lower than CLL/WM. In DLBL and FCL, the B-cells showed very similar levels of CD52 expression to normal B-progenitors, on average 2.8-fold lower than CLL. All plasma cells, whether neoplastic (CD19− or CD19+56+) or normal (CD19+56−), showed much lower levels of expression than normal and neoplastic B-cells. Plasma cell CD52 expression was detectable in 68% of normal controls (13/19), 50% of MGUS patients (17/34), and only 43% of myeloma patients (46/106). Expression was uni-modal in all cases. There was significantly lower expression of CD52 by myeloma plasma cells than by their normal counterparts (median 2.4-fold decrease, P=0.03). Neoplastic plasma cell CD52 expression showed a high degree of inter-patient variation, but fewer than 10% of myeloma patients (7/106) had CD52 expression at a similar level to CLL cells. Neoplastic plasma cell CD52 expression was approximately 6-fold lower than that of normal B-progenitors, and nearly 20-fold lower than that of CLL cells. In summary, CD52 expression is not detectable above control levels in a significant proportion of myeloma patients. In cases with detectable CD52 expression, the antigen is at a much lower level than is present on normal B-progenitors, which actively proliferate during alemtuzumab therapy. The risk of immunosuppression due to depletion of residual normal B/T-cells must also be considered. As alemtuzumab efficacy appears to correlate with CD52 expression levels, myeloma is highly unlikely to respond to alemtuzumab as a single agent except in rare cases. However, alemtuzumab is more likely to be effective in the IgM immunosecretory disorders which show strong CD52 expression.

Inhibition of JAK2/STAT3 Pathway Leads to Apoptosis in Chronic Lymphocytic Leukemia Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2023-2023 ◽  
Author(s):  
Filippo Severin ◽  
Federica Frezzato ◽  
Veronica Martini ◽  
Flavia Raggi ◽  
Valentina Trimarco ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Viability ◽  
B Cell ◽  
B Lymphocytes ◽  
Sh2 Domain ◽  
Lymphocytic Leukemia ◽  
Lymphoid Tissues ◽  
Stat3 Phosphorylation ◽  
Jak2 Inhibition

Abstract INTRODUCTION Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature clonal CD19+/CD5+/CD23+ B lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. Despite their in vivo prolonged lifespan due to intrinsic defects, CLL leukemic cells rapidly undergo spontaneous apoptosis in vitro, highlighting the need of extrinsic signals delivered by the microenvironment. Several molecules, including those released by mesenchymal stromal cells (MSCs), signal through JAK (Janus kinases)-STAT (Signal Transducers and Activators of Transcription) pathways. We particularly focused on the JAK2/STAT3 axis since Interleukin-6 (IL-6), one of the most abundant cytokines released in the CLL microenvironment, is the key ligand of the receptor triggering this pathway. The deregulation of JAK2/STAT3 axis may lead to aberrant activation of STAT3 and, as a result, to tumor development in hematopoietic cells. METHODS B cells were collected from 12 controls and 46 CLL patients. Purified cells (2x106cells/ml) were cultured, and treated with AG490 (10, 50 and 100μM), AZD1480 (1, 4 and 10μM), Fedratinib (1, 5 and 10μM), and Ruxolitinib (0.313, 2.5 and 10μM) (which are JAK2 inhibitors), and the STAT3 inhibitor Stattic (5, 7.5, and 10μM) for 24, 48 and 72h. Experiments with AG490 and Stattic were performed with/without MSCs. STAT3 expression and phosphorylation were evaluated by Western Blotting (WB) and Flow Cytometry (FC), and its localization was analyzed by confocal microscopy and subcellular fractionation. CLL and normal B cell viability was tested by FC with Annexin V/PI test. RESULTS We demonstrated that STAT3 was highly expressed in malignant B cells with respect to normal B lymphocytes. As far as STAT3 phosphorylation at Tyr705, that is an essential step for STAT3 activation, we demonstrated a constitutive phosphorylation in CLL cells by FC and WB analyses, although in some patients STAT3 Tyr705 phosphorylation is barely detected. We also pointed out that the in vitroincubation of leukemic B cells with AG490 and Stattic and Fedratinib, induces a dose-dependent apoptosis of CLL B cells. However, the tested doses of Ruxolitinib and AZD1480 did not seem to CLL B cell viability but only STAT3 phosphorylation. Both AG490 and Stattic were able to bypass the microenvironmental protection when neoplastic B cells were co-cultured with MSCs. STAT3 Tyr705 localization was analyzed in normal and leukemic B cells by a subcellular protein fractionation. We separated nuclei from cytosol, detecting STAT3 Tyr705 both in the cytosolic and in the nuclear fractions of CLL B cells. We showed that AG490 and Fedratinib treatment on CLL cells can mediate other effects: i) SHP-1 activity is turned on by JAK2 inhibition, decreasing its phosphorylation at Ser591; ii) AG490 administration inactivates protein Lyn, reducing the phosphorylation in its active site at Tyr396. Lyn, a Tyr-kinase, and SH2-domain containing Tyrosine Phosphatase (SHP-1), a Tyr-phosphatase, are both involved in the prolonged lifespan of neoplastic CLL cells. To confirm the link between JAK2 inhibition by AG490 and Lyn dephosphorylation, we added sodium orthovanadate (Na3VO4, 100 μM), a phosphatase inhibitor, to cell culture to restore Lyn activation (resulting from the inactivation of SHP-1 phosphatase); as a result, Lyn Tyr396 phosphorylation was restored. On the contrary, the treatment of CLL cells with Stattic did not induce any change in SHP-1 status with respect to untreated cells since Stattic is effective on STAT3, that is a downstream protein with respect to JAK2. Since Stattic did not affect SHP-1 activation, it does not impact on Lyn activation/phosphorylation. CONCLUSIONS The ability of AG490 and Stattic to induce apoptosis in leukemic B cells bypassing the pro-survival stimuli provided by the tumor microenvironment and the Fedratinib effectiveness at low doses, represents a starting point for the development of new therapeutic strategies in CLL. This study also provides new insights for the investigation of the pathogenesis of CLL focusing the attention on the cross-talk between JAK/STAT and BCR/Lyn axes. Disclosures No relevant conflicts of interest to declare.

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