Allosteric Akt Inhibitor MK2206 Synergizes with Bendamustine in Promoting the Apoptosis of Chronic Lymphocytic Leukemia Cells and Selectively Targets B-Cell Receptor Mediated Cytokine Production

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3928-3928
Author(s):  
Wei Ding ◽  
Tait D. Shanafelt ◽  
Connie Lesnick ◽  
Traci Sassoon ◽  
Charla Secreto ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Research Funding ◽  
Cytokine Production ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Leukemic Cells ◽  
Akt Inhibitor ◽  
Akt Activation

Abstract Abstract 3928 Background: Accumulating data support the critical role of PI3K/Akt in CLL B cell receptor (BCR) mediated signal transduction, cell proliferation and survival. In addition recent preclinical and clinical studies indicate that specific PI3K blockade results in robust preclinical and clinical efficacy in CLL. In our model system of CLL B cell-stromal cultures which feature their interaction, platelet derived growth factor (PDGF) present in CLL culture medium drives VEGF production through PI3K/Akt activation in stromal cells (Blood. 2010. 116:2984). Indeed Akt was found to be activated in leukemic cells during the CLL-stroma interaction (Leuk Res. 2008. 32:1565). Therefore, we hypothesized that Akt inhibition should promote CLL B cell apoptosis and abrogate BCR mediated cytokine production. MK2206 is an orally bioavailable highly specific allosteric Akt inhibitor. It has been tested in patients with refractory solid tumors and was demonstrated to be safely administered in a phase I trial. Therefore the goal of this study was to test the preclinical efficacy of MK2206 on both the survival and the BCR mediated cytokine production of CLL leukemic B cells. Methods: Peripheral blood mononuclear cells isolated from CLL patients (n=37) were treated with escalating concentrations of MK2206 (1–16 μM) for 24 hours, 48 hours or 72 hours. The levels of leukemic B cell viability were tested using an (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. The potential impact (antagonistic/additive/synergistic) of Bendamustine in combination with MK2206 was also tested by using the MTT assay. We used the Calcusyn system to calculate the effect of drug interactions. The combination index (CI) as calculated by the program usually indicates synergy when ≤ 0.8 and indicates additive outcomes when between 0.8–1.2. A CI >1.2 indicates antagonism. Downstream signals of Akt activation in CLL B cells were evaluated by testing their expression of Mcl-1, 4EBP1 and p70S6K using immunoblot. The impact of Akt inhibition by MK2206 on cytokine production in response to B-cell receptor ligation with anti-IgM was also tested using a multiplex cytokine analysis (Invitrogen) in a time-course experiment. Results: MK2206 treatment induced concentration- and time-dependent apoptosis in CLL leukemic cells. At 72 hours, the IC50 of MK2206 in the experiments using CLL leukemic cells in vitro is ∼8 mM. MK2206 incubation at 1 or 5 mM cultured with CLL B cells over a 48-hour period abolished of Akt and p70S6K phosphorylation while native PARP was cleaved into the 85 kD polypeptide fragment. However, the expression level of the upstream signal molecule, PI3K, was not changed. Among the CLL patients tested (n = 37), we did not find any difference in sensitivity to MK2206 induced apoptosis based on critical prognostic factors of CD38, ZAP-70, IGHV and del(17p) status. Importantly, we detected synergistic or additive activity between MK2206 and Bendamustine in 11 tested CLL samples when these combinations were used to treat CLL cells in vitro for 72hrs. Thus the median CI value for this group of patients was 0.8 (0.1 – 1.1). Six were found to have CI ≤ 0.8 and five fell within the additive CI values (0.8 – 1.2). Production of immune or chemotactic cytokines (e.g. CCL3, CCL4, MCP-1, IL-1Ra, IL-8 and IL-2R) at 24 hour incubation increased significantly above baseline when CLL cells were stimulated anti-IgM. Akt inhibition with MK2206 selectively abrogated upregulation of CCL3, CCL4, MCP-1 and IL-2R production, but not for IL-8 or IL-1Ra secretion. MK2206 also abolished BCR mediated Akt activation and decreased Erk activation. Conclusion: MK2206, a robust and selective Akt inhibitor, induced significant in vitro apoptosis of CLL B-cells in vitro. Preclinical evidence of a synergistic effect between MK2206 and Bendamustine was also observed independent of prognostic risk. MK2206 abolished BCR mediated Akt activation and selectively abrogates BCR mediated production of cytokines that may promote apoptotic resistance. These findings support the use of MK2206 in treating CLL and indeed we have initiated a phase I/II trial of MK2206 in combination with Bendamustine and Rituximab for relapsed CLL patients(N1087, October 2011). Acknowledgments: This study was funded by the NCI-K23, NCCTG and CLL Global Foundation. Disclosures: Shanafelt: Cephalon: Research Funding; Genentech: Research Funding. Kumar:Genzyme: Research Funding; Novartis: Research Funding; Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Honoraria. Kay:Celgene: Research Funding.

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.

scholarly journals B Lymphocyte Chemotaxis Regulated in Association with Microanatomic Localization, Differentiation State, and B Cell Receptor Engagement

1998 ◽  
Vol 187 (5) ◽  
pp. 753-762 ◽  
Author(s):  
Conrad C. Bleul ◽  
Joachim L. Schultze ◽  
Timothy A. Springer
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Messenger Rna ◽  
B Lymphocyte ◽  
Somatic Hypermutation ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Cxc Chemokine

Migration of mature B lymphocytes within secondary lymphoid organs and recirculation between these sites are thought to allow B cells to obtain T cell help, to undergo somatic hypermutation, to differentiate into effector cells, and to home to sites of antibody production. The mechanisms that direct migration of B lymphocytes are unknown, but there is evidence that G protein–coupled receptors, and possibly chemokine receptors, may be involved. Stromal cell– derived factor (SDF)-1α is a CXC chemokine previously characterized as an efficacious chemoattractant for T lymphocytes and monocytes in peripheral blood. Here we show with purified tonsillar B cells that SDF-1α also attracts naive and memory, but not germinal center (GC) B lymphocytes. Furthermore, GC B cells could be converted to respond to SDF-1α by in vitro differentiation into memory B lymphocytes. Conversely, the migratory response in naive and memory B cells was significantly reduced after B cell receptor engagement and CD40 signaling. The receptor for SDF-1, CXC chemokine receptor 4 (CXCR4), was found to be expressed on responsive as well as unresponsive B cell subsets, but was more rapidly downregulated on responsive cells by ligand. Finally, messenger RNA for SDF-1 was detected by in situ hybridization in a layer of cells surrounding the GC. These findings show that responsiveness to the chemoattractant SDF-1α is regulated during B lymphocyte activation, and correlates with positioning of B lymphocytes within a secondary lymphoid organ.

Targeting Early Events of B-Cell Receptor Signaling in Chronic Lymphocytic Leukemia: Suppressed Syk and PLCγ2 Activities Predict Apoptotic Response of Leukemic Cells to Dasatinib

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5023-5023
Author(s):  
Y. Lynn Wang ◽  
Zibo Song ◽  
Pin Lu ◽  
John P. Leonard ◽  
Morton Coleman ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Bcr Signaling

Abstract B cell receptor (BCR) signaling plays an essential role in the pathogenesis of chronic lymphocytic leukemia. In a subset of patients with a poor clinical outcome, BCR ligation leads to increased cell metabolism and cell survival (Cancer Research66, 7158–66, 2006). Based on these findings, we tested whether targeting BCR signaling with dasatinib, an inhibitor of Src kinase, would interfere with the signaling cascade and cause death of CLL B cells. CLL leukemic cells were isolated from 34 patients and were incubated with or without dasatinib at a low dose of 128 nM. Among 34 cases, viability of leukemic cells was reduced by 2% to 90%, with an average of ~50% reduction on day 4 of ex vivo culture. Further study showed that CLL B cells undergo death by apoptosis via the intrinsic pathway which involves the generation of reactive oxygen species. Analysis of the Src family kinases showed that phosphorylation of Src, Lyn and Hck was inhibited by dasatinib not only in those cases that responded to dasatinib with apoptosis, but also in those that did not respond well (<20% apoptosis). Further analysis revealed that suppressed activity of two downstream molecules, Syk and PLC Statistical analysis showed a significant correlation between CLL dasatinib response and their IgVH mutation and ZAP70 status. Cases with worse prognoses by these criteria have a better response to the kinase inhibitor. Lastly, we have also found that ZAP70 positive cases showed a greater degree of PLC

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 A B Cell Receptor with Two Igα Cytoplasmic Domains Supports Development of Mature But Anergic B Cells

2004 ◽  
Vol 199 (6) ◽  
pp. 855-865 ◽  
Author(s):  
Amy Reichlin ◽  
Anna Gazumyan ◽  
Hitoshi Nagaoka ◽  
Kathrin H. Kirsch ◽  
Manfred Kraus ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Cytoplasmic Domain ◽  
B Cell Receptor ◽  
B1a Cells ◽  
Bcr Signaling ◽  
Membrane Bound

B cell receptor (BCR) signaling is mediated through immunoglobulin (Ig)α and Igβ a membrane-bound heterodimer. Igα and Igβ are redundant in their ability to support early B cell development, but their roles in mature B cells have not been defined. To examine the function of Igα–Igβ in mature B cells in vivo we exchanged the cytoplasmic domain of Igα for the cytoplasmic domain of Igβ by gene targeting (Igβc→αc mice). Igβc→αc B cells had lower levels of surface IgM and higher levels of BCR internalization than wild-type B cells. The mutant B cells were able to complete all stages of development and were long lived, but failed to differentiate into B1a cells. In addition, Igβc→αc B cells showed decreased proliferative and Ca2+ responses to BCR stimulation in vitro, and were anergic to T-independent and -dependent antigens in vivo.

Chronic Lymphocytic Leukemia B-Cells Lack or Express Markedly Reduced Levels of the Phosphatase PHLPP, Which Regulates B-Cell Receptor Induced AKT Activation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2080-2080
Author(s):  
Mirza Suljagic ◽  
Luca Laurenti ◽  
Muhammad Alam ◽  
Pablo G Longo ◽  
Sami N Malek ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Akt Pathway ◽  
Immunoblotting Analysis ◽  
Akt Phosphorylation ◽  
Akt Activation

Abstract The PI3K/AKT pathway plays a central role in regulating cellular growth and survival. This pathway is activated by signals derived from various receptors and is tightly regulated through the action of several phosphatases, including SHIP and PTEN, which hydrolyze the PI3K product PIP3, and the recently identified PHLPP, which directly dephosphorylates AKT. Hyperactivation of the PI3K/AKT pathway has been implicated in the pathogenesis of many types of cancer, including chronic lymphocytic leukemia (CLL) and B-cell lymphoma. In addition, gene expression profiling and real-time RT/PCR analysis have recently shown differential expression of PHLPP mRNA in CLL subsets classified according to the presence of the 13q14 abnormality, with many CLL cases demonstrating absent PHLPP expression altogether. These findings prompted us to compare the levels of PHLPP expression in primary CLL B-cells (n=17) with normal tonsillar B-cells (n=4) and various lymphoma cell lines, including the diffuse large B-cell lymphomas (DLBCL) DHL-4, DHL-6, DHL-8, DHL-10, WSU, Toledo, Ly1, Ly3, Ly7 and Ly18, the Burkitt’s lymphoma BJAB and the prolymphocytic leukemia MEC1. Immunoblotting analysis revealed abundant and uniform expression of PHLPP in normal B-cells and in 7 out of 12 investigated lymphoma cell lines. Higher levels were observed in the BJAB, Ly1 and Ly18 cell lines, whereas PHLPP was undetectable in the DLBCL cell lines WSU and Toledo. Remarkably, PHLPP was either not expressed or was expressed at markedly reduced levels in all of the investigated CLL samples, with levels of expression ranging from 0 to 10% of the levels in normal B-cells. In contrast, the levels of expression of the phosphatase SHIP were relatively similar between CLL and normal B-cells. To determine what are the consequences of reduced PHLPP expression on signaling through AKT in malignant B-lymphocytes, we downregulated PHLPP in BJAB and DHL-4 cells by RNA interference. A significant reduction in the levels of PHLPP was achieved in both cell lines, which amounted to 20–40% of the levels in cells transfected with the control siRNA. Immunoblotting analysis of protein extracts from cells transfected with PHLPP and control siRNA did not show a difference in AKT phosphorylation on Ser473 and Thr308, indicating that a reduction in PHLPP expression is not sufficient to augment basal AKT activity. To determine the effects of PHLPP downregulation on agonist-induced AKT activation, we investigated phosphorylation on Ser473 and Thr308 in BJAB and DHL-4 cells stimulated through the B-cell receptor. In both cell lines downregulation of PHLPP resulted in more than a 50% increase in BCR-induced AKT phosphorylation. In contrast, phosphorylation of other signaling molecules that are also activated by BCR crosslinking, such as PLCγ2 and ERK, appeared unaffected by PHLPP downregulation. These data confirm the functional relevance of PHLPP in AKT regulation in B-lymphoid cells and implicate reduced or absent PHLPP expression in CLL B-cells as a potential determinant of BCR-induced AKT signaling in CLL.

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.

THU0045 Epratuzumab, an Antibody Targeting CD22 on B Cells, Induces Phosphoprotein Changes Following B-Cell Receptor Activation in Vitro

2013 ◽  
Vol 72 (Suppl 3) ◽  
pp. A179.1-A179
Author(s):  
S. Lumb ◽  
N. Torbett ◽  
I. Vendrell ◽  
H. Turner ◽  
M. Page ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Receptor Activation ◽  
B Cell Receptor ◽  
Antibody Targeting

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.

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