Surface IgM stimulation induces MEK1/2-dependent MYC expression in chronic lymphocytic leukemia cells

Abstract Although long considered as a disease of failed apoptosis, it is now clear that chronic lymphocytic leukemia (CLL) cells undergo extensive cell division in vivo, especially in progressive disease. Signaling via the B-cell receptor is thought to activate proliferation and survival pathways in CLL cells and also has been linked to poor outcome. Here, we have analyzed the expression of the proto-oncoprotein MYC, an essential positive regulator of the cell cycle, after stimulation of surface IgM (sIgM). MYC expression was rapidly increased after sIgM stimulation in a subset of CLL samples. The ability of sIgM stimulation to increase MYC expression was correlated with sIgM-induced intracellular calcium fluxes. MYC induction was partially dependent on the MEK/ERK signaling pathway, and MYC and phosphorylated ERK1/2 were both expressed within proliferation centers in vivo. Although stimulation of sIgD also resulted in ERK1/2 phosphorylation, responses were relatively short lived compared with sIgM and were associated with significantly reduced MYC induction, suggesting that the kinetics of ERK1/2 activation is a critical determinant of MYC induction. Our results suggest that ERK1/2-dependent induction of MYC is likely to play an important role in antigen-induced CLL cell proliferation.

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Chronic lymphocytic leukemia: revelations from the B-cell receptor

Blood ◽

10.1182/blood-2003-12-4312 ◽

2004 ◽

Vol 103 (12) ◽

pp. 4389-4395 ◽

Cited By ~ 286

Author(s):

Freda K. Stevenson ◽

Federico Caligaris-Cappio

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Receptor ◽

B Cell Receptor ◽

Lymphocytic Leukemia ◽

Cell Of Origin ◽

Regulatory B Cell ◽

V Genes

Abstract The finding that chronic lymphocytic leukemia (CLL) consists of 2 clinical subsets, distinguished by the incidence of somatic mutations in the immunoglobulin (Ig) variable region (V) genes, has clearly linked prognosis to biology. Antigen encounter by the cell of origin is indicated in both subsets by selective but distinct expression of V genes, with evidence for continuing stimulation after transformation. The key to distinctive tumor behavior likely relates to the differential ability of the B-cell receptor (BCR) to respond. Both subsets may be undergoing low-level signaling in vivo, although analysis of blood cells limits knowledge of critical events in the tissue microenvironment. Analysis of signal competence in vitro reveals that unmutated CLL generally continues to respond, whereas mutated CLL is anergized. Differential responsiveness may reflect the increased ability of post-germinal center B cells to be triggered by antigen, leading to long-term anergy. This could minimize cell division in mutated CLL and account for prognostic differences. Unifying features of CLL include low responsiveness, expression of CD25, and production of immunosuppressive cytokines. These properties are reminiscent of regulatory T cells and suggest that the cell of origin of CLL might be a regulatory B cell. Continuing regulatory activity, mediated via autoantigen, could suppress Ig production and lead to disease-associated hypogammaglobulinemia. (Blood. 2004;103:4389-4395)

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Nurture versus Nature: The Microenvironment in Chronic Lymphocytic Leukemia

Hematology ◽

10.1182/asheducation-2011.1.96 ◽

2011 ◽

Vol 2011 (1) ◽

pp. 96-103 ◽

Cited By ~ 101

Author(s):

Jan A. Burger

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Tyrosine Kinase ◽

B Cell ◽

Disease Progression ◽

Drug Testing ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

In Vivo Models

Abstract Intrinsic factors such as genetic lesions, anti-apoptotic proteins, and aberrant signaling networks within leukemia cells have long been the main focus of chronic lymphocytic leukemia (CLL) research. However, over the past decade, it became increasingly clear that external signals from the leukemia microenvironment make pivotal contributions to disease progression in CLL and other B-cell malignancies. Consequently, increasing emphasis is now placed on exploring and targeting the CLL microenvironment. This review highlights critical cellular and molecular pathways of CLL-microenvironment cross-talk. In vitro and in vivo models for studying the CLL microenvironment are discussed, along with their use in searching for therapeutic targets and in drug testing. Clinically, CXCR4 antagonists and small-molecule antagonists of B cell receptor (BCR)-associated kinases (spleen tyrosine kinase [Syk], Bruton's tyrosine kinase [Btk], and PI3Kδ) are the most advanced drugs for targeting specific interactions between CLL cells and the miocroenvironment. Preclinical and first clinical evidence suggests that high-risk CLL patients can particularly benefit from these alternative agents. These findings indicate that interplay between leukemia-inherent and environmental factors, nature and nurture determines disease progression in CLL.

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Functional and clinical relevance of VLA-4 (CD49d/CD29) in ibrutinib-treated chronic lymphocytic leukemia

Journal of Experimental Medicine ◽

10.1084/jem.20171288 ◽

2018 ◽

Vol 215 (2) ◽

pp. 681-697 ◽

Cited By ~ 21

Author(s):

Erika Tissino ◽

Dania Benedetti ◽

Sarah E.M. Herman ◽

Elisa ten Hacken ◽

Inhye E. Ahn ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Progression Free Survival ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

Clinical Activity ◽

Independent Manner ◽

Btk Inhibitor

The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib, which antagonizes B cell receptor (BCR) signals, demonstrates remarkable clinical activity in chronic lymphocytic leukemia (CLL). The lymphocytosis experienced by most patients under ibrutinib has previously been attributed to inhibition of BTK-dependent integrin and chemokine cues operating to retain the tumor cells in nodal compartments. Here, we show that the VLA-4 integrin, as expressed by CD49d-positive CLL, can be inside-out activated upon BCR triggering, thus reinforcing the adhesive capacities of CLL cells. In vitro and in vivo ibrutinib treatment, although reducing the constitutive VLA-4 activation and cell adhesion, can be overcome by exogenous BCR triggering in a BTK-independent manner involving PI3K. Clinically, in three independent ibrutinib-treated CLL cohorts, CD49d expression identifies cases with reduced lymphocytosis and inferior nodal response and behaves as independent predictor of shorter progression-free survival, suggesting the retention of CD49d-expressing CLL cells in tissue sites via activated VLA-4. Evaluation of CD49d expression should be incorporated in the characterization of CLL undergoing therapy with BCR inhibitors.

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ZAP-70 Is Not Phosphorylated on the Activating Tyrosine Residue (Tyr319) in Chronic Lymphocytic Leukemia and B-Cell Lymphoma Cells.

Blood ◽

10.1182/blood.v106.11.178.178 ◽

2005 ◽

Vol 106 (11) ◽

pp. 178-178

Author(s):

Stefania Gobessi ◽

Aleksandar Petlickovski ◽

Luca Laurenti ◽

Dimitar G. Efremov

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Progressive Disease ◽

Cell Receptor ◽

Kinase Domain ◽

Lymphocytic Leukemia ◽

Bcr Signaling ◽

Cell Receptor Signaling

Abstract The protein tyrosine kinase ZAP-70 is expressed at high levels in leukemic B-cells from chronic lymphocytic leukemia (CLL) patients with progressive disease and short survival. ZAP-70 is a key component of the proximal T-cell receptor signaling pathway and is highly homologous to Syk, an important B-cell receptor signaling (BCR) molecule. Recent studies indicate that ZAP-70 may participate in BCR signaling as well, but the mechanism of action is still not well understood. In T-cells, upon TCR stimulation ZAP-70 becomes phosphorylated on Tyr319 by the Src-like kinase Lck, which results in the release of the ZAP-70 kinase domain from an autoinhibited state to a fully active conformation. The Tyr319 site in ZAP-70 corresponds to the Tyr352 site in Syk, which is phosphorylated in B-cells following BCR stimulation. We therefore investigated the activation status of ZAP-70 and Syk in BCR stimulated CLL B-cells, using phosphorylation of Tyr319 and Tyr352 as markers of their activation. Analysis of 10 ZAP-70-positive CLL samples by immunoblotting with the phospho-ZAP70Tyr319/SykTyr352 antibody revealed that ZAP-70 is not phosphorylated at this site either before or after BCR stimulation, although in control experiments with Jurkat T-cells ZAP-70 became phosphorylated on Tyr319 upon TCR stimulation. Moreover, the Tyr352 site in Syk was phosphorylated following BCR stimulation in 6 of the 10 CLL B-cell samples. To further investigate the reasons for the unexpected lack of ZAP-70 activation in CLL B-cells, we produced stable transfectants of the BJAB lymphoma B-cell line that expressed ZAP-70 at levels similar to those found in CLL cases with progressive disease. In agreement with the CLL B-cell experiments, the Tyr319 site in ZAP-70 was not phosphorylated either before or after BCR stimulation. Since phosphorylation of Tyr319 is Lck-dependent in T-cells, and this kinase is expressed also in CLL B-cells, we ectopically expressed Lck in the ZAP-70-positive BJAB clones. Again, the Tyr319 site was not phosphorylated, indicating that ZAP-70 does not undergo activation of the kinase domain also in this cellular system. In contrast, BCR crosslinking in BJAB cells induced significant phosphorylation of Tyr352 in Syk, which was further enhanced in the clones that coexpressed ZAP-70. Furthermore, analysis of downstream signaling pathways following BCR stimulation showed stronger and prolonged activation of ERK and to a lesser extent Akt in the ZAP-70 positive clones, whereas no difference was observed in terms of activation of PLC-γ 2, JNK and degradation of the NF-kB inhibitor IkB. These data indicate that ZAP-70 does not undergo full activation in B-cells, but can still enhance activation of certain downstream BCR signaling pathways, possibly by affecting the activity of the related PTK Syk.

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Inhibition of Wnt Signaling By Dimethyl Fumarate Results in in Vitro and in Vivo Clearance of Chronic Lymphocytic Leukemia Cells and Has Additive Activity with Ibrutinib

Blood ◽

10.1182/blood.v124.21.4683.4683 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4683-4683 ◽

Cited By ~ 1

Author(s):

Christina C.N. Wu ◽

Fitzgerald Lao ◽

Hongying Li ◽

Laura Rassenti ◽

Thomas J. Kipps ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Wnt Signaling ◽

Dimethyl Fumarate ◽

Lymphocytic Leukemia ◽

Aggressive Disease ◽

Cell Clearance ◽

Survival Pathways ◽

A Cell

Abstract Background: Small molecules that inhibit B cell survival pathways are effective treatments for patients with chronic lymphocytic leukemia (CLL). However, such therapies are not curative, and resistance can develop in some patients. Combination therapies with agents that inhibit several CLL survival pathways may allow for more complete responses, and help prevent treatment resistance. Previous data has shown that the pro-survival Wnt pathway is highly active in CLL and is a negative prognostic factor, and therefore is an attractive target for novel therapies to combine with agents like ibrutinib. Dimethyl fumarate (DMF) is an orally bioavailable fumaric acid ester with immunomodulatory properties, including inhibition of the NF-kB signaling cascade. DMF has been evaluated as a systemic treatment of psoriasis as well as multiple sclerosis. Our group previously observed anti-CLL effects of DMF, mediated in part through oxidative stress. Herein we describe a novel mechanism of action of DMF and ibrutinib, mediated by inhibition of the Wnt signaling pathway. Methods: Effects of DMF and ibrutinib on Wnt signaling were determined using a cell-based LEF/TCF beta-lactamase reporter gene FRET assay. In vitro activity was assessed in primary CLL from patients with indolent and aggressive disease. In vivo activity was evaluated in Rag2-/- gamma chain-/- immunodeficient (RG-KO) mice, which were engrafted with human CLL cells by intraperitoneal injection. DMF and/or ibrutinib were administrated to mice by oral gavage, at clinically used doses and schedules. Results: Both DMF and ibrutinib have an alpha-beta unsaturated ketone that can react with essential free cysteines in the Wnt-driven LEF1 transcription factor. This effect was confirmed by a cell-based reporter gene assay in which DMF inhibited LEF/TCF dependent gene expression at low μM levels. Ibrutinib also inhibited Wnt signaling activity in the same assay. In short term cultures, DMF was cytotoxic to primary CLL cells from patients with both indolent and aggressive disease, at low uM concentrations. The combination of DMF and ibrutinib resulted in a higher degree of CLL cell clearance than achieved by either agent alone (p < 0.05 after multiple comparison adjustments, Dunnet’s method). To evaluate the effect in a preclinical CLL xenograft animal model, we administered DMF by oral lavage to RG-KO mice engrafted with human CLL cells. Doses ranging from 3 to 30 mg/kg BID for 7 days resulted in dose dependent clearance of CLL cells compared to vehicle controls, without observable toxicity to the recipient animals. Moreover, the combination of DMF and ibrutinib resulted in a higher degree of CLL cell clearance than achieved by either agent alone. Preliminary FACS analyses revealed that DMF selectively targets CLL subpopulations of cells with aggressive characteristics, as assessed by CD38 expression. Further molecular analyses of predictive or correlative biomarkers are ongoing. Conclusions: DMF inhibits Wnt signaling, and has single agent activity as a treatment for CLL. The combination of DMF and ibrutinib is more effective than either agent alone, particularly in aggressive disease, and is well tolerated. Clinical trials of DMF in CLL are warranted, and are planned. This work is supported by a Leukemia and Lymphoma Society Specialized Center of Research Grant (7005-14) and by the CLL Research Consortium (5P01CA081534-14). Disclosures No relevant conflicts of interest to declare.

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Stimulation of surface IgM of chronic lymphocytic leukemia cells induces an unfolded protein response dependent on BTK and SYK

Blood ◽

10.1182/blood-2014-04-567198 ◽

2014 ◽

Vol 124 (20) ◽

pp. 3101-3109 ◽

Cited By ~ 20

Author(s):

Sergey Krysov ◽

Andrew J. Steele ◽

Vania Coelho ◽

Adam Linley ◽

Marina Sanchez Hidalgo ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Unfolded Protein Response ◽

Cell Receptor ◽

Immunoglobulin M ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Unfolded Protein ◽

Protein Response ◽

Stimulation Of

Key Points Stimulation of the B-cell receptor of chronic lymphocytic leukemia cells results in activation of an unfolded protein response. Unfolded protein response activation following surface immunoglobulin M stimulation in vitro is dependent on the activity of BTK and SYK.

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Stimulation of the B-cell receptor activates the JAK2/STAT3 signaling pathway in chronic lymphocytic leukemia cells

Blood ◽

10.1182/blood-2013-10-534073 ◽

2014 ◽

Vol 123 (24) ◽

pp. 3797-3802 ◽

Cited By ~ 46

Author(s):

Uri Rozovski ◽

Ji Yuan Wu ◽

David M. Harris ◽

Zhiming Liu ◽

Ping Li ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Receptor ◽

B Cell Receptor ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Stat3 Signaling ◽

Stat3 Signaling Pathway ◽

Key Points ◽

Stimulation Of

Key Points Stimulation of the BCR activates JAK2 and STAT3 in CLL cells. The JAK1/2 inhibitor ruxolitinib induces apoptosis of CLL cells.

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CD4+ T cells sustain aggressive chronic lymphocytic leukemia in Eμ-TCL1 mice through a CD40L-independent mechanism

Blood Advances ◽

10.1182/bloodadvances.2020003795 ◽

2021 ◽

Vol 5 (14) ◽

pp. 2817-2828

Author(s):

Matteo Grioni ◽

Arianna Brevi ◽

Elena Cattaneo ◽

Alessandra Rovida ◽

Jessica Bordini ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Antigen Specificity

Abstract Chronic lymphocytic leukemia (CLL) is caused by the progressive accumulation of mature CD5+ B cells in secondary lymphoid organs. In vitro data suggest that CD4+ T lymphocytes also sustain survival and proliferation of CLL clones through CD40L/CD40 interactions. In vivo data in animal models are conflicting. To clarify this clinically relevant biological issue, we generated genetically modified Eμ-TCL1 mice lacking CD4+ T cells (TCL1+/+AB0), CD40 (TCL1+/+CD40−/−), or CD8+ T cells (TCL1+/+TAP−/−), and we monitored the appearance and progression of a disease that mimics aggressive human CLL by flow cytometry and immunohistochemical analyses. Findings were confirmed by adoptive transfer of leukemic cells into mice lacking CD4+ T cells or CD40L or mice treated with antibodies depleting CD4 T cells or blocking CD40L/CD40 interactions. CLL clones did not proliferate in mice lacking or depleted of CD4+ T cells, thus confirming that CD4+ T cells are essential for CLL development. By contrast, CD8+ T cells exerted an antitumor activity, as indicated by the accelerated disease progression in TCL1+/+TAP−/− mice. Antigen specificity of CD4+ T cells was marginal for CLL development, because CLL clones efficiently proliferated in transgenic mice whose CD4 T cells had a T-cell receptor with CLL-unrelated specificities. Leukemic clones also proliferated when transferred into wild-type mice treated with monoclonal antibodies blocking CD40 or into CD40L−/− mice, and TCL1+/+CD40−/− mice developed frank CLL. Our data demonstrate that CD8+ T cells restrain CLL progression, whereas CD4+ T cells support the growth of leukemic clones in TCL1 mice through CD40-independent and apparently noncognate mechanisms.

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Emerging role of kinase-targeted strategies in chronic lymphocytic leukemia

Hematology ◽

10.1182/asheducation.v2012.1.88.3801172 ◽

2012 ◽

Vol 2012 (1) ◽

pp. 88-96 ◽

Cited By ~ 26

Author(s):

Adrian Wiestner

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Absolute Lymphocyte Count ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

Cell Trafficking

Abstract Chronic lymphocytic leukemia (CLL) is a malignancy of mature B cells that depend on host factors in the tissue microenvironment for survival and proliferation. In vitro, CLL cells rapidly undergo apoptosis unless microenvironmental factors are provided that support their survival. Signaling pathways activated in the microenvironment in vivo include the B-cell receptor (BCR) and NF-κB pathways. Thus, CLL is a disease “addicted to the host” and is dependent on pathways that promote normal B-cell development, expansion, and survival; this is particularly true in the case of the BCR signaling cascade. Small-molecule inhibitors of kinases that are essential for BCR signal transduction abrogate the stimulating effects of the microenvironment on CLL cells. The orally administered tyrosine kinase inhibitors fostamatinib and ibrutinib and the phosphatidylinositol 3-kinase inhibitor GS-1101 have induced impressive responses in relapsed and refractory CLL patients, mostly with moderate side effects. Reductions in lymphadenopathy and splenomegaly are seen within weeks and are frequently accompanied by a transient rise in absolute lymphocyte count that is asymptomatic and probably the result of changes in CLL cell trafficking. This review discusses the biologic basis for kinase inhibitors as targeted therapy of CLL and summarizes the exciting early clinical experience with these agents.

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Divergence in CD19-Mediated Signaling Unfolds Intra-Clonal Diversity in Chronic Lymphocytic Leukemia Which Correlates with Disease Progression

Blood ◽

10.1182/blood.v120.21.4570.4570 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4570-4570

Author(s):

Yair Herishanu ◽

Sigi Kay ◽

Nili Dezorella ◽

Chava Perry ◽

Varda Deutsch ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Disease Progression ◽

Conflicts Of Interest ◽

Clonal Diversity ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

Cell Fraction ◽

Akt Phosphorylation

Abstract Abstract 4570 Emerging data on intra-clonal diversity imply that this phenomenon may play a role in the clinical outcome of patients with chronic lymphocytic leukemia (CLL), where subsets of the CLL clone responding more robustly to external stimuli may gain a growth and survival advantage. Here we report intra-clonal diversity resolved by responses to CD19 engagement in CLL cells, which can be classified into responding (CD19-R) and non-responding (CD19-N) sub-populations. Engagement of CD19 by anti-CD19 antibody rapidly induced cellular aggregation in the CD19-R CLL cells. The CD19-R CLL cells expressed higher surface levels of CD19 and c-myc mRNA and exhibited distinct morphological features. Both sub-populations reacted to IgM stimulation in a similar manner and exhibited similar levels of Akt phosphorylation, pointing to functional signaling divergence within the B-cell receptor. CD19 unresponsiveness was partially reversible, where CD19-N cells spontaneously recover their signaling capacity following incubation in vitro, pointing to possible in vivo CD19-signaling attenuating mechanisms. This concept was supported by the lower CD19-R occurrence in bone marrow-derived samples compared with cells derived from the peripheral blood of the same patients. CLL patients with more than 18.5% of CD19-R cell fraction had a shorter median time to treatment compared to patients with less than 18.5% of CD19-R cell fraction. Conclusions: Divergence in CD19-mediated signaling unfolds both inter-patient and intra-clonal diversity in CLL. This signaling diversity is associated with physiological implications including the location of the cells and disease progression. Disclosures: No relevant conflicts of interest to declare.

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