The Syk\Jak Inhibitor Cerdulatinib (PRT062070) Shows Promising Preclinical Activity in Chronic Lymphocytic Leukemia By Antagonising B Cell Receptor and Microenvironmental Signalling

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1716-1716 ◽  
Author(s):  
Matthew D Blunt ◽  
Jack Parnell ◽  
Marta Larrayoz ◽  
Lindsay Smith ◽  
Rachel Dobson ◽  
...  
Keyword(s):  
B Cell ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Signalling Pathways ◽  
Simultaneous Inhibition ◽  
Induced Apoptosis

Abstract The emergence of B cell receptor (BCR) kinase inhibitors has proved effective for the treatment of a number of B-cell malignancies including chronic lymphocytic leukemia (CLL). BTK and PI3K inhibitors have clear efficacy in suppressing tumor progression but have not been curative. A number of patients have developed resistance to these drugs following mutation of the BTK or PLCγ2 gene. Whilst, other patients are unable to tolerate these drugs due to adverse events or progress whilst on therapy for unknown reasons. Thus the development of novel drugs which are still effective once other BCR-kinases inhibitors become ineffective is of paramount importance. Spleen tyrosine kinase (Syk) is essential for B cell receptor signalling pathways as well as a variety of other surface receptors such as MHCII, FC receptors and integrins, all of which have been shown to play a role in CLL biology. Importantly, Syk inhibition has been shown to overcome resistance to ibrutinib, identifying Syk inhibition as a promising strategy to treat these patients. Furthermore, we have previously shown that IL-4 is found in CLL lymph nodes and can promote resistance to ibrutinib and idelalisib by restoring αIgM induced calcium flux and phosphorylated ERK (ASH 2014, abstract #3299). IL-4 signalling is mediated through the JAK/STAT signalling pathways via JAK1 and JAK3, therefore simultaneous inhibition of both Syk and JAK1/3 may be therapeutically beneficial over BCR kinase inhibitors alone. Cerdulatinib (PRT062070) is a dual JAK/Syk inhibitor in a phase I open label dose escalation study and is currently demonstrating clinical activity in patients with relapsed/refractory B cell malignancies including CLL. Our group has now demonstrated in vitro that cerdulatinib, at plasma concentrations achievable in patients, can induce apoptosis of CLL cells in a concentration and time dependent manner with a mean IC50 of 3µM and 1µM at 48 and 72h respectively, defined by annexin V/PI and cleavage of caspase 3 and poly ADP ribose polymerase (PARP). Apoptosis was caspase dependent since treatment with the pan caspase inhibitor ZVAD.fmk significantly inhibited cerdulatinib induced cell death at 24h. Cerdulatinib induced apoptosis coincided with an increase in pro-apoptotic proteins Noxa and Puma and a decrease in the anti-apoptotic protein Mcl-1. Cerdulatinib significantly inhibited IL-4 induced phosphorylation of STAT6 at 300nM (p=.005), BCR induced phosphorylation of AKTS473 with soluble (p=.008) and bead immobilised (BI) (p=.025) αIgM at 30nM and phosphorylation of AKTT308 with BI αIgM at 300nM (p=.008). Furthermore, in patients with CLL, it is thought that CD40L and IL-4 are key factors, which promote survival of CLL cells in proliferation centres within the lymph node microenvironment. Therefore, we cultured CLL cells with a vehicle control or IL-4\CD40L, prior to treatment with cerdulatinib. Cerdulatinib alone induced similar levels of apoptosis irrespective of IL-4/CD40L treatment, suggesting cerdulatinib may be able to overcome microenvironmental signals and target cells within the lymph node. Next we explored the possibility of augmenting cerdulatinib induced apoptosis by simultaneous inhibition with the Bcl-2\Bcl-XL inhibitor ABT-199. In vitro in the presence of IL-4/CD40L, ABT-199 synergised with cerdulatinib to induce significantly greater cell death than with either agent alone. Therefore these data provide in vitro evidence for the use of cerdulatinib in clinical trials for the treatment of CLL as either a single agent or in combination with other therapies such as ABT-199. Disclosures Strefford: Roche: Research Funding. Davies:Seattle Genetics: Research Funding; Takeda: Honoraria. Coffey:Portola Pharmaceuticals Inc: Employment, Equity Ownership, Research Funding. Steele:Portola Pharmaceuticals: Other: Travel bursary to ASH 2015; Janssen: Other: Travel bursary to EHA 2015.

Chronic lymphocytic leukemia: revelations from the B-cell receptor

Blood ◽  
2004 ◽  
Vol 103 (12) ◽  
pp. 4389-4395 ◽  
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)

scholarly journals High TCL1 levels are a marker of B-cell receptor pathway responsiveness and adverse outcome in chronic lymphocytic leukemia

Blood ◽  
2009 ◽  
Vol 114 (21) ◽  
pp. 4675-4686 ◽  
Author(s):  
Marco Herling ◽  
Kaushali A. Patel ◽  
Nicole Weit ◽  
Nils Lilienthal ◽  
Michael Hallek ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Clinical Stage ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Advanced Clinical Stage ◽  
Therapy Regimen ◽  
Cell Counts

Abstract Although activation of the B-cell receptor (BCR) signaling pathway is implicated in the pathogenesis of chronic lymphocytic leukemia (CLL), its clinical impact and the molecular correlates of such response are not clearly defined. T-cell leukemia 1 (TCL1), the AKT modulator and proto-oncogene, is differentially expressed in CLL and linked to its pathogenesis based on CD5+ B-cell expansions arising in TCL1-transgenic mice. We studied here the association of TCL1 levels and its intracellular dynamics with the in vitro responses to BCR stimulation in 70 CLL cases. The growth kinetics after BCR engagement correlated strongly with the degree and timing of induced AKT phospho-activation. This signaling intensity was best predicted by TCL1 levels and the kinetics of TCL1-AKT corecruitment to BCR membrane activation complexes, which further included the kinases LYN, SYK, ZAP70, and PKC. High TCL1 levels were also strongly associated with aggressive disease features, such as advanced clinical stage, higher white blood cell counts, and shorter lymphocyte doubling time. Higher TCL1 levels independently predicted an inferior clinical outcome (ie, shorter progression-free survival, P < .001), regardless of therapy regimen, especially for ZAP70+ tumors. We propose TCL1 as a marker of the BCR-responsive CLL subset identifying poor prognostic cases where targeting BCR-associated kinases may be therapeutically useful.

scholarly journals Emerging role of kinase-targeted strategies in chronic lymphocytic leukemia

Hematology ◽  
2012 ◽  
Vol 2012 (1) ◽  
pp. 88-96 ◽  
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.

MEK1/2 Inhibition By MEK162 Is Effective Against Chronic Lymphocytic Leukaemia Cells Under Conditions That Mimic Stimulation of B-Cell Receptor-Mediated Signaling

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3330-3330 ◽  
Author(s):  
Kyle Crassini ◽  
William S Stevenson ◽  
Stephen P. Mulligan ◽  
Oliver Giles Best
Keyword(s):  
Protein Kinase ◽  
B Cell ◽  
Cell Survival ◽  
Research Funding ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Spontaneous Apoptosis ◽  
Viable Cells ◽  
Cells Cultured

Abstract BACKGROUND Chronic Lymphocytic Leukemia (CLL) is characterised by the clonal expansion of apoptosis resistant B-lymphocytes. However, in vitro and in the absence of pro-survival factors primary CLL cells undergo spontaneous apoptosis. B-cell receptor (BCR) signalling plays a major role in the survival and proliferation of CLL cells, which is highlighted by the clinical efficacy of the Btk and PI3-kinase inhibitors, ibrutinib and idelalisib. Mitogen activated protein kinase (MAPK) is an important mediator of signals downstream of both Btk and PI3-kinase but few studies have shown that inhibitors of MEK1/2, a critical component in the MAPK pathway, have any potential benefit for therapy of CLL. METHODS We sought to investigate the potential of the MEK1/2 inhibitor MEK162 against CLL cells in vitro. To mimic the tonic BCR stimulation experienced in vivo, primary CLL cells were stimulated using an immobilised antibody to IgM or were treated with PMA, a less specific B-cell activator which promotes protein kinase C-dependent MAPK-ERK1/2 signaling. Sensitivity to MEK162 and effects on MAPK-ERK1/2 pathway activity were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and western blot analyses respectively. RESULTS MEK162 treatment of CLL cells cultured in media alone resulted in a modest but significant (P < 0.01) reduction in cell viability; 20µM MEK162 reduced the proportion of viable cells remaining after 48 h to 77.40 +/- 7.81 % relative to vehicle-treated controls. In contrast, BCR stimulation through IgM ligation promoted cell survival 3.2 +/- 1.01 fold and sensitised CLL cells to MEK162; 20µM MEK162 reduced the proportion of viable cells by 56.28 +/- 2.37 % (P < 0.001 relative to control). A similar effect was observed in response to PMA stimulation; cell viability increased 1.78 +/- 0.15 fold and was reduced by 59.55 +/- 10.33 % (P < 0.001 relative to control) following treatment with 20µM MEK162 (Figure 1). At concentrations > 0.05 µM MEK162 was significantly (P < 0.05) more effective against CLL cells stimulated with either anti-IgM or PMA than against cells cultured in media alone. By western blotting we observed low levels of MAPK-ERK1/2 activity in cells cultured in media alone, which we suggest may contribute to the spontaneous apoptosis of these cells and the low degree of sensitivity to MEK162 under these conditions. We confirmed that stimulation with either IgM or PMA results in activation of MAPK-ERK1/2 and show that this response can be effectively blocked using MEK162. The effects of anti-IgM and PMA on cell survival and response to MEK162 were independent of ZAP-70 expression or ATM/TP53 functional status. CONCLUSIONS Our data illustrate the important role of MAPK-ERK1/2 activity in BCR-mediated CLL cell survival and suggest that MEK162 may have potential for CLL therapy. These data highlight the importance of employing appropriate culture conditions in order to make accurate assessments concerning the efficacy of novel agents for the treatment of CLL. Figure 1. Stimulation with IgM or PMA sensitises B-CLL cells to MEK1/2 inhibition by MEK162. Figure 1. Stimulation with IgM or PMA sensitises B-CLL cells to MEK1/2 inhibition by MEK162. Disclosures Mulligan: Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi Aventis: Research Funding; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria.

scholarly journals Emerging role of kinase-targeted strategies in chronic lymphocytic leukemia

Blood ◽  
2012 ◽  
Vol 120 (24) ◽  
pp. 4684-4691 ◽  
Author(s):  
Adrian Wiestner
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Absolute Lymphocyte Count ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Cell Trafficking

AbstractChronic 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.

scholarly journals Development of PROTACs to address clinical limitations associated with BTK-targeted kinase inhibitors

2020 ◽  
Vol 1 (3) ◽  
pp. 131-152 ◽  
Author(s):  
Rachael Arthur ◽  
Beatriz Beatriz Valle-Argos ◽  
Andrew J. Steele ◽  
Graham Packham
Keyword(s):  
B Cell ◽  
Kinase Inhibitors ◽  
Early Stage ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Clinical Responses ◽  
B Cell Receptor Signaling ◽  
Btk Inhibitor ◽  
Emergence Of Resistance

Chronic lymphocytic leukemia is a common form of leukemia and is dependent on growth-promoting signaling via the B-cell receptor. The Bruton tyrosine kinase (BTK) is an important mediator of B-cell receptor signaling and the irreversible BTK inhibitor ibrutinib can trigger dramatic clinical responses in treated patients. However, emergence of resistance and toxicity are major limitations which lead to treatment discontinuation. There remains, therefore, a clear need for new therapeutic options. In this review, we discuss recent progress in the development of BTK-targeted proteolysis targeting chimeras (PROTACs) describing how such agents may provide advantages over ibrutinib and highlighting features of PROTACs that are important for the development of effective BTK degrading agents. Overall, PROTACs appear to be an exciting new approach to target BTK. However, development is at a very early stage and considerable progress is required to refine these agents and optimize their drug-like properties before progression to clinical testing.

scholarly journals Constitutive activation of Lyn kinase enhances BCR responsiveness, but not the development of CLL in Eµ-TCL1 mice

2020 ◽  
Vol 4 (24) ◽  
pp. 6106-6116
Author(s):  
Viktoria Kohlhas ◽  
Michael Hallek ◽  
Phuong-Hien Nguyen
Keyword(s):  
B Cells ◽  
Mouse Model ◽  
B Cell ◽  
Kinase Inhibitors ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Lyn Kinase ◽  
B Mice

Abstract The treatment of chronic lymphocytic leukemia (CLL) has been improved dramatically by inhibitors targeting B-cell receptor (BCR)–associated kinases. The tyrosine kinase Lyn is a key modulator of BCR signaling and shows increased expression and activity in CLL. To evaluate the functional relevance of Lyn for CLL, we generated a conditional knockin mouse model harboring a gain-of-function mutation of the Lyn gene (LynY508F), which was specifically expressed in the B-cell lineage (Lynup-B). Kinase activity profiling revealed an enhanced responsiveness to BCR stimulation in Lynup-B B cells. When crossing Lynup-B mice with Eµ-TCL1 mice (TCL1tg/wt), a transgenic mouse model for CLL, the resulting TCL1tg/wt Lynup-B mice showed no significant change of hepatomegaly, splenomegaly, bone marrow infiltration, or overall survival when compared with TCL1tg/wt mice. Our data also suggested that TCL1 expression has partially masked the effect of the Lynup-B mutation, because the BCR response was only slightly increased in TCL1tg/wt Lynup-B compared with TCL1tg/wt. In contrast, TCL1tg/wt Lynup-B were protected at various degrees against spontaneous apoptosis in vitro and upon treatment with kinase inhibitors targeting the BCR. Collectively, and consistent with our previous data in a Lyn-deficient CLL model, these data lend further suggest that an increased activation of Lyn kinase in B cells does not appear to be a major driver of leukemia progression and the level of increased BCR responsiveness induced by Lynup-B is insufficient to induce clear changes to CLL pathogenesis in vivo.

scholarly journals TAK-243, a Small Molecule Inhibitor of Ubiquitin-Activating Enzyme (UAE), Induces ER Stress and Apoptosis in CLL Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1867-1867
Author(s):  
Scott R Best ◽  
Taylor Rowland ◽  
Cody Paiva ◽  
Nur Bruss ◽  
Stephen E Spurgeon ◽  
...  
Keyword(s):  
B Cells ◽  
Er Stress ◽  
B Cell ◽  
Cell Lines ◽  
Small Molecule ◽  
Research Funding ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Ubiquitin Conjugation

Abstract Introduction: Despite the promise of B-cell receptor-associated kinase inhibitors (BCRi) in CLL, resistance to these agents is inevitable. Ubiquitin-proteasome systems are altered in cancer, leading to destabilization of tumor suppressors, overexpression of proto-oncogenes (e.g., MYC), and impaired DNA repair. Neoplastic B cells exhibit a state of heightened cellular stress and are thereby susceptible to endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). The UPR is activated in CLL cells upon sIgM signaling and inhibited by ibrutinib. Proteasome inhibitors demonstrate clinical activity in certain types of B-cell neoplasia but are inactive in CLL. Here, we investigated an alternative approach to harness the pro-apoptotic UPR in CLL by using TAK-243, a first-in-class small molecule UAE inhibitor. Methods: Peripheral blood cells were obtained from patients with CLL (N=20) and isolated using Ficoll-Hypaque techniques. TAK-243 was obtained from Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Cambridge, MA). TAK-243 efficacy was assessed in CLL cells and 10 DLBCL (diffuse large B-cell lymphoma) cell lines. Results: TAK-243 induced ER stress and the UPR in CLL cells, followed by rapid apoptosis within 2 hours. Following 24-hour incubation, we established an IC50 of ~50 nM (Annexin V+ cells) in CLL cells. By contrast, primary B cells and T cells were less sensitive to TAK-243. Given the importance of tumor microenvironment in CLL cell survival, we evaluated the effect of TAK-243 in a CD40L-expressing stromal co-culture model. Whereas CD40L-stimulated CLL cells were resistant to BCRi, they were fully sensitive to UAE inhibition. TAK-243 had a similar IC50 (~50 nM) across DLBCL cell lines, independent of cell of origin. Treatment with TAK-243 rapidly disrupted ubiquitin conjugation and degradation of proteins controlled by the UPS in CLL cells and DLBCL cell lines. UPR induction occurred within 2 hours, as shown by activation of eIF2α (in both CLL and DLBCL cells) and oligomerization and autophosphorylation of PERK (in DLBCL cells). After 4 hours, neoplastic B cells exhibited late apoptotic phase of the UPR: transcriptional induction of CHOP, GADD34, and NOXA. These events were accompanied by upregulation of pro-apoptotic BH3-only proteins, stabilization of Mcl-1 and, ultimately, cleavage of caspase-3 and PARP. TAK-243 inhibited NFκB pathway, as shown by accumulation of IκBα, a negative pathway regulator. The extent of the UPR in CLL cells varies depending on the initiating signal. For example, B-cell receptor crosslinking induced expression of CHOP and GRP78 in CLL cells, but only weak activation of PERK and no IRE1-dependent processing of XBP1 (Krysov S, et al. Blood. 2014). Targeting UAE in CLL cells induced robust activation of eIF2α, upregulation of CHOP, GADD34 as well as NOXA mRNA, indicating high sensitivity to this pathway. TAK-243 induced a more rapid UPR and exhibited lower IC50 compared with the proteasome inhibitor bortezomib in CLL and DLBCL cells. While both drugs induced autophagy as shown by LC3 processing, only bortezomib treatment led to p62 degradation, suggesting that autophagy was inefficient in response to TAK-243 due to lack of ubiquitin conjugation. Our findings were confirmed in a mouse lymphoma xenograft model. OCI-LY3 cells were inoculated subcutaneously in the right flank of NSG mice and treatment with TAK-243 (10 or 20 mg/kg IV twice weekly) or vehicle control began when tumors reached 10 mm in size. Treatment led to reduced tumor progression, induction of ER stress, and decreased cell proliferation and survival. Conclusions: The UAE inhibitor TAK-243 induces ER stress and promotes apoptosis in CLL cells in vitro and restricts lymphoma growth in vivo. TAK-243 exhibited greater in-vitro cytotoxicity in lymphoma cells compared to bortezomib. Targeting UAE is a novel approach to disrupt the UPS which may hold promise in therapy of CLL and other B-cell malignancies. Disclosures Spurgeon: Bristol Myers Squibb: Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Oncternal: Research Funding; Acerta: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Pharmacyclics: Consultancy, Research Funding; MEI Pharma: Consultancy. Berger:Takeda Pharmaceuticals International Co.: Employment. Danilov:TG Therapeutics: Consultancy; Aptose Biosciences: Research Funding; Astra Zeneca: Consultancy; Genentech: Consultancy, Research Funding; Bayer Oncology: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Gilead Sciences: Consultancy, Research Funding; Takeda Oncology: Research Funding.

Sustained Signaling through the B-Cell Receptor Induces Mcl-1 and Promotes Survival of Chronic Lymphocytic Leukemia B-Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 178-178
Author(s):  
Dimitar G. Efremov ◽  
Aleksandar Petlickovski ◽  
Luca Laurenti ◽  
Xiaoping Li ◽  
Sara Marietti ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Spontaneous Apoptosis ◽  
B Cell Survival ◽  
Bcr Signaling ◽  
Induced Apoptosis

Abstract The clinical course of chronic lymphocytic leukemia (CLL) differs significantly between patients with mutated (M-CLL) and unmutated (U-CLL) immunoglobulin V genes, implying a role for B-cell receptor (BCR) signaling in the pathogenesis of this disease. BCR stimulation in normal B-cells triggers several crucial signaling pathways, including PI3K/Akt, IKK/NF- κB and the mitogen-activated protein kinases Erk, JNK and p38 MAPK, which can induce proliferation, survival, differentiation or apoptosis, depending on the nature and context of the antigenic stimulation. We have now investigated activation of these downstream signaling pathways, as well as induction of anti-apoptotic proteins and survival of CLL B-cells stimulated with soluble (sol-IgM) and immobilized (imm-IgM) anti-IgM antibodies, which were used to mimic stimulation with soluble and particulate/membrane-bound antigen, respectively. Stimulation with sol-IgM revealed similar activation patterns in the 10 U-CLL and 12 M-CLL cases that partially resembled the pattern described for tolerant B-cells. The response in the U-CLL cases was characterized by transient (<45 minutes) phosphorylation of Akt and Erk, no activation of JNK and p38 MAPK, and activation of IKKβ in 50% of the cases. Most M-CLL cases showed similar activation of Akt and Erk, but lacked activation of IKKβ, whereas three M-CLL cases were completely non-responsive. To investigate the effects on CLL B-cell survival, 14 U-CLL and 19 M-CLL cases were analyzed by Annexin V/PI staining after 48 hours stimulation with sol-IgM. A 10–40% increase in apoptotic cells was observed in the majority of cases from both CLL subsets (p<0.001 with respect to spontaneous apoptosis). Induction of apoptosis was confirmed by analyzing cleavage of the Caspase 3 substrate PARP, and was accompanied by an approximately 50% reduction in the levels of Mcl-1, an antiapoptotic protein implicated in CLL B-cell survival and resistance to chemotherapy. A markedly different response was induced by imm-IgM, which was characterized by activation of IKKβ in all cases and sustained Akt and Erk phosphorylation that persisted over 24 hours. This response resulted in a 2.5 fold mean increase in the levels of Mcl-1, whereas no changes were observed in the levels of Bcl-2 and Bcl-xL. Imm-IgM slightly reduced the percentage of cells undergoing spontaneous apoptosis after 48 hours, but significantly protected from fludarabine- and methylprednisolone-induced apoptosis. To investigate which of the three imm-IgM activated pathways is responsible for induction of Mcl-1 and protection from chemotherapy-induced apoptosis, we incubated CLL B-cells with LY294002, U0126 and BAY-11 (inhibitors of PI3K, ERK and NF- κB, respectively) prior to stimulation with imm-IgM and addition of fludarabine. Induction of Mcl-1 and inhibition of fludarabine-induced PARP cleavage were significantly abrogated only by LY294002, indicating that the PI3K/Akt pathway is the major link between the BCR and apoptosis resistance of CLL B-cells. In conclusion, this study shows that the response of CLL B-cells to BCR stimulation primarily depends on the nature of the antigenic stimulus. Moreover, it shows that only sustained BCR signaling can promote survival of CLL B-cells, and raises the possibility that the distinct clinical and biological behavior of U-CLL and M-CLL is determined by the availability of such stimulation.

B Cell Receptor-ERK1/2 Signal Cancels PAX5-Dependent Repression of BLIMP1 Probably Through PAX5 Phosphorylation: A Mechanism of Antigen-Triggering Plasma Cell Differentiation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1109-1109
Author(s):  
Takahiko Yasuda ◽  
Fumihiko Hayakawa ◽  
Shingo Kurahashi ◽  
Tomoki Naoe
Keyword(s):  
Cell Differentiation ◽  
B Cell ◽  
Plasma Cell ◽  
Transcriptional Repression ◽  
Research Funding ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Luciferase Reporter ◽  
Plasma Cell Differentiation

Abstract Abstract 1109 Plasma cell differentiation is initiated by antigen stimulation of B cell receptor (BCR). Until BCR stimulation, BLIMP1, a master regulator of plasma cell differentiation, is suppressed by PAX5, a key transcriptional repressor to maintain B cell identity. After BCR stimulation, upregulation of BLIMP1 and subsequent suppression of PAX5 by BLIMP1 are observed and thought to be the trigger of plasma cell differentiation; however, the trigger that derepresses BLIMP1 expression is yet to be revealed. Here, we demonstrated PAX5 phosphorylation by ERK1/2, the main component of BCR signal, in vitro and in vivo. The sites of PAX5 phosphorylation were identified by PCR mutagenesis assay. In luciferase reporter assays, transcriptional repression on BLIMP1 promoter by PAX5 was canceled by PAX5 phosphorylation. Furthermore, transcriptional repression by phosphorylation-defective mutant of PAX5 was attenuated by CA-MEK1 co-expression to a significantly lesser extent than that by wild-type PAX5, indicating its resistance to ERK1/2 signal-dependent cancelation of the transcriptional repression (Figure A). Finally, BCR stimulation induced strong ERK1/2 activation, phosphorylation of endogenous PAX5 (Figure B), and upregulation of BLIMP1 mRNA expression in B cells. These phenomena were inhibited by U0126, MEK1 inhibitor. These data imply that PAX5 phosphorylation by BCR signal is the initial event in plasma cell differentiation (Figure C). Disclosures: Naoe: Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.

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